Regulation of Immune Cell Infiltration into the CNS by ......2 MediatorsofInflammation Microglia...
8
Hindawi Publishing Corporation Mediators of Inflammation Volume 2013, Article ID 898165, 8 pages http://dx.doi.org/10.1155/2013/898165 Review Article Regulation of Immune Cell Infiltration into the CNS by Regional Neural Inputs Explained by the Gate Theory Yasunobu Arima, 1 Daisuke Kamimura, 1 Lavannya Sabharwal, 1 Moe Yamada, 1 Hidenori Bando, 1 Hideki Ogura, 1 Toru Atsumi, 1 and Masaaki Murakami 1,2 1 JST-CREST, Graduate School of Frontier Biosciences, Graduate School of Medicine, and WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan 2 Office for University-Industry Collaboration, Osaka University, Science Innovation Center Building A, 4th Floor, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Correspondence should be addressed to Masaaki Murakami; [email protected] Received 6 March 2013; Revised 15 June 2013; Accepted 17 June 2013 Academic Editor: Dennis D. Taub Copyright © 2013 Yasunobu Arima et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. e central nervous system (CNS) is an immune-privileged environment protected by the blood-brain barrier (BBB), which consists of specific endothelial cells that are brought together by tight junctions and tight liner sheets formed by pericytes and astrocytic end- feet. Despite the BBB, various immune and tumor cells can infiltrate the CNS parenchyma, as seen in several autoimmune diseases like multiple sclerosis (MS), cancer metastasis, and virus infections. Aside from a mechanical disruption of the BBB like trauma, how and where these cells enter and accumulate in the CNS from the blood is a matter of debate. Recently, using experimental autoimmune encephalomyelitis (EAE), an animal model of MS, we found a “gateway” at the fiſth lumber cord where pathogenic autoreactive CD4+ T cells can cross the BBB. Interestingly, this gateway is regulated by regional neural stimulations that can be mechanistically explained by the gate theory. In this review, we also discuss this theory and its potential for treating human diseases. 1. Mechanism for BBB Breakdown in Autoimmunity of the CNS e blood-brain barrier (BBB) in blood vessels is known to strictly limit the inflow of substances like proteins and cells from the bloodstream into the CNS (Figure 1), thereby maintaining a homeostatic environment for surrounding neurons and glia cells, a property different from that in peripheral organs. e BBB is formed and maintained by endothelial cells and corresponding tight junctions formed by claudins and occludins in collaboration with pericytes, microglial cells, macrophages, and astrocytes [1, 2]. BBB dysfunction is known to be associated with chronic neurodegenerative disorders, such as Parkinson’s disease and Alzheimer’s disease, and autoimmune diseases in the CNS [3, 4]. An increasing number of studies have shown that one cause of a dysfunctional BBB is inflammatory cytokines. For example, tumor-necrosis factor (TNF), interleukin-(IL-) 1, and IL-17A have all been reported to loosen the BBB [5]. In particular, IL-17A is known to disrupt the BBB in vitro and in vivo. Huppert et al. reported that IL-17A-induced BBB dysfunction involves the formation of reactive oxygen species by NADPH oxidase and xanthine oxidase and that these species lead to the down regulation of tight junction molecules and the activation of the endothelial contractile machinery in vitro [6]. In addition, Kebir et al. reported that treatment with IL-17A increases the protein permeability of human brain endothelial cells and that this permeability is associated with a decrease in the expression of occludin and ZO-1 [7]. A role of IL-17A in BBB disruption has also been found using experimental autoimmune encephalomyelitis (EAE) mice, an animal model of multiple sclerosis (MS), in vivo. In this model, the major source of IL-17A is type-17 helper T (17) cells, particularly autoreactive ones. EAE is significantly suppressed in IL-17A-deficient mice. Instead, these mice exhibit delayed onset, reduced maximum severity
Transcript of Regulation of Immune Cell Infiltration into the CNS by ......2 MediatorsofInflammation Microglia...
Hindawi Publishing CorporationMediators of InflammationVolume 2013 Article ID 898165 8 pageshttpdxdoiorg1011552013898165
Review ArticleRegulation of Immune Cell Infiltration into the CNS byRegional Neural Inputs Explained by the Gate Theory
Yasunobu Arima1 Daisuke Kamimura1 Lavannya Sabharwal1 Moe Yamada1
Hidenori Bando1 Hideki Ogura1 Toru Atsumi1 and Masaaki Murakami12
1 JST-CREST Graduate School of Frontier Biosciences Graduate School of Medicine and WPI Immunology Frontier Research CenterOsaka University Osaka 565-0871 Japan
2Office for University-Industry Collaboration Osaka University Science Innovation Center Building A 4th Floor 2-1 YamadaokaSuita Osaka 565-0871 Japan
Correspondence should be addressed to Masaaki Murakami murakamimoloncmedosaka-uacjp
Received 6 March 2013 Revised 15 June 2013 Accepted 17 June 2013
Academic Editor Dennis D Taub
Copyright copy 2013 Yasunobu Arima et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
The central nervous system (CNS) is an immune-privileged environment protected by the blood-brain barrier (BBB) which consistsof specific endothelial cells that are brought together by tight junctions and tight liner sheets formed by pericytes and astrocytic end-feet Despite the BBB various immune and tumor cells can infiltrate the CNS parenchyma as seen in several autoimmune diseaseslike multiple sclerosis (MS) cancer metastasis and virus infections Aside from a mechanical disruption of the BBB like traumahow and where these cells enter and accumulate in the CNS from the blood is a matter of debate Recently using experimentalautoimmune encephalomyelitis (EAE) an animal model of MS we found a ldquogatewayrdquo at the fifth lumber cord where pathogenicautoreactive CD4+ T cells can cross the BBB Interestingly this gateway is regulated by regional neural stimulations that can bemechanistically explained by the gate theory In this review we also discuss this theory and its potential for treating human diseases
1 Mechanism for BBB Breakdown inAutoimmunity of the CNS
The blood-brain barrier (BBB) in blood vessels is knownto strictly limit the inflow of substances like proteins andcells from the bloodstream into the CNS (Figure 1) therebymaintaining a homeostatic environment for surroundingneurons and glia cells a property different from that inperipheral organs The BBB is formed and maintainedby endothelial cells and corresponding tight junctionsformed by claudins and occludins in collaboration withpericytes microglial cells macrophages and astrocytes [1 2]BBB dysfunction is known to be associated with chronicneurodegenerative disorders such as Parkinsonrsquos disease andAlzheimerrsquos disease and autoimmune diseases in the CNS[3 4] An increasing number of studies have shown that onecause of a dysfunctional BBB is inflammatory cytokines Forexample tumor-necrosis factor 120572 (TNF120572) interleukin-(IL-)
1120573 and IL-17A have all been reported to loosen the BBB [5]In particular IL-17A is known to disrupt the BBB in vitroand in vivo Huppert et al reported that IL-17A-inducedBBB dysfunction involves the formation of reactive oxygenspecies by NADPH oxidase and xanthine oxidase and thatthese species lead to the down regulation of tight junctionmolecules and the activation of the endothelial contractilemachinery in vitro [6] In addition Kebir et al reported thattreatment with IL-17A increases the protein permeability ofhuman brain endothelial cells and that this permeability isassociated with a decrease in the expression of occludin andZO-1 [7] A role of IL-17A in BBB disruption has also beenfound using experimental autoimmune encephalomyelitis(EAE) mice an animal model of multiple sclerosis (MS) invivo In this model the major source of IL-17A is type-17helper T (Th17) cells particularly autoreactive ones EAE issignificantly suppressed in IL-17A-deficient mice Insteadthese mice exhibit delayed onset reduced maximum severity
2 Mediators of Inflammation
Microglia
Neuron
Endothelial cellPericyte
Astrocyte
Blood-brain barrier at venules in the CNS
Immune cellsImmune cells
Tight junctions
Basementmembrane
Normal venules
Figure 1 Venules at the blood-brain barrier in the CNS and other organs Venules at the blood-brain barrier consist of specific endothelialcells that are brought together by tight junctions and tight liner sheets formed by pericytes and astrocytic end-feet These are not present innormal venules (left)
scores ameliorated histological changes and early recovery[8] Additionally an adoptive transfer model in which helperT cells obtained from myelin oligodendrocyte glycoprotein(MOG) immunized mice were infused into naıve recipientsindicated that IL-17A derived fromCD4+T cells is critical forthe induction of EAE [8] In addition MOG-reactive Th17cells obtained from MOG-immunized IL-17A-deficient micewere unable to infiltrate the lumber level of spinal cord in thesame model (see the following for details) [9] Furthermoreit was shown that the adoptive transfer of Th17 cells fromovalbumin-specific T cell receptor transgenic mice whichare unable to recognize CNS antigens does not pass the BBBand migrate into CNS whereas cotransfer of these Th17 cellswith MOG-reactive Th17 cells leads to the accumulation ofboth types of Th17 cells in the CNS (our unpublished dataand [10]) which strongly suggests that antigen recognition ofTh17 cells is required for severe disruption of the BBBAlthough antigen presentation inside the CNS has suggestedthrough observation that the infusion of ovalbumin peptide-loaded antigen-presenting cells into cerebrospinal fluidsinduces an accumulation of ovalbumin-specific Th17 cellsin the CNS [10] the type of antigen-presenting cells andlocation where antigen presentation takes place underphysiological conditions remain elusive Nevertheless theseresults suggest that IL-17A expressing Th17 cells whichrecognize CNS antigens have a major impact on breachingthe BBB in part by decreasing tight junction molecules
2 Neuroimmune Interactions Responsible forInflammation in the CNS
In the previous section we discussed the relationship bet-ween inflammatory cytokines such as IL-17A and disruptionof the BBB This section focuses on the gateway for whichpathogenic CD4+ T cells enter the CNS In patients withMS common early symptoms include vision problems andtingling followed by many neurological signs as the diseaseprogresses It is known that inflammation sites in MS arefound in specific regions of white matter including the brain-stem the optic nerve the cerebellum the long motor andsensory tracts of the spinal cord [11] This fact suggests thatsome CNS regions might be more vulnerable to autoimmuneattacks One hypothesis proposes that chemokine recruit-ment of pathogenic autoreactive T cells is more abundantin these regions Among the many chemokines CCL20 isof particular interest as it attracts Th17 cells that expressCCR6 a receptor for CCL20 Reboldi et al reported thatmicelackingCCR6 are highly resistant to EAE and that the choroidplexus a specialized epithelial structure in the brain known toproduce cerebrospinal fluids expresses CCL20 constitutivelyan effect that acts as an attractant for the first wave ofCCR6+ Th17 cells [12] In that same study however EAEwas induced using the complete Freundrsquos adjuvant which iswidely used to generate active immunization in animals butis also an inducer of systemic inflammation and has manyside effects including fever motor neuron dysfunction such
Mediators of Inflammation 3
lowast
Dorsal
Dorsal
5th lumber cord
Spinalroot
Blood vesselsPathogenic T cells
Spinal cord
vesselDorsal
Ventral
Ventral
3D
Accumulated mononuclearcell numbers
0 50 100 150 200
L6
L5
L4
L3
L2
L1
(a)
(b)
Figure 2 The fifth lumber cord is a gateway into the CNS A cross section of the fifth lumber (L5) cord (a) and actual cell numbers ofmononuclear cells accumulated in each lumber cord segment (b) at a preclinical phase of EAE (5 days after pathogenic Th17 transfer) A 3Dpicture based on ten serial sections of L5 is also shown
as paralysis and apoptosis These side effects could affectthe pathophysiological status of the brain and spinal cordsresulting in different conclusions from the steady state
We recently found a ldquogaterdquo past the BBB in the spinal cordthat autoreactive Th17 cells in the bloodstream can exploitto enter the CNS To make this discovery we first utilizedan adoptive transfer model to induce EAE in which Th17cells obtained fromMOG-immunizedmicewere infused intonaıve recipient mice to maintain CNS quiescence In thisadoptive transfer model we found that MOG-reactive Th17cells preferentially accumulated in the fifth lumber (L5) cordrather than the brain or other levels of the spinal cords atthe earliest phase of EAE (day 5 after T cell transfer) [9](Figure 2) This finding fits well with a typical clinical EAEsign in which the tail is first affected We also found thatblood vessel tracks in L5 are altered due to the formation ofedema in the L5 cord by using a supersensitive MRI (datanot shown) Consistent with these resultsCcl20mRNA levels
were highest in the dorsal venules of L5 compared withthose from other spinal cords and the transfer of CCR6-deficient Th17 cells did not accumulate in the L5 regionInterestingly even in naıve animals without Th17 transfermRNA levels of Ccl20 and many other chemokines werespecifically upregulated in the dorsal venules of L5Thereforedorsal venules in the L5 spinal cord have special properties indiseased as well as healthy conditions
Wepreviously found amechanism for the hyperinductionof inflammatory chemokines and cytokines in nonimmunecells such as fibroblasts endothelial cells and epithelial cellsusing a rheumatoid arthritis modelThemechanism is drivenby a simultaneous activation of two transcription factors NF-120581B and STAT3Thus it was named the ldquoinflammation ampli-fierrdquo because hyperactivation of NF-120581B by activated STAT3induces large amounts of NF-120581B-targeted chemokines andchemotactic factors to promote the recruitment of immunecells (Figure 3) Ccl20 is one such target chemokine and is
4 Mediators of Inflammation
lowastlowastlowast
lowastlowastlowast
IL-17Th17
Inflammationamplifier
Inflammatory chemokines IL-6
Fibroblastsendothelial cells etc
Autoimmune diseaseschronic inflammation graft rejection
IL-7
STAT3
IL-6
0
4
8
12
16
20
Non
e
IL-6
IL-1
7
Chemokine mRNA expression
IL-6
+IL
-17
NF-120581B
Figure 3The inflammation amplifier is defined as amechanism that triggers the hyperinduction of cytokines and chemokines in nonimmunecells and leads to many diseases This induction is induced by the simultaneous activation of NF-120581B and STAT3 IL-7 from nonimmune cellsalso contributes to enhancing the inflammation amplifier by generatingTh17 cells andor sustaining their survivalThe inflammation amplifieris essential for the pathogenesis of F759 arthritis EAE and chronic graft rejection The bar graph on the right indicates typical expressionstatuses of chemokines and IL-6 after inflammation amplifier activation (see the blue bar)
found in vascular endothelial cells Given that chemokineexpressions are elevated in L5 dorsal venules we hypothe-sized a role for the inflammation amplifier The activationstatus of NF-120581B and STAT3 is indeed higher in L5 dorsalvenules than other lumber cords even in naıve healthy miceThe elevated Ccl20mRNA levels at L5 vessels were decreasedin mice devoid of the inflammation amplifier such as IL-6-deficient mice and endothelial cell-specific IL-6 receptor(gp130) deficient mice Even under healthy conditions itis known that some immune cells are present in the CNSsuggesting that there may be a gate to enter the restrictedtissue regardless In this respect it is tempting to speculatethat low-grade activation of the inflammation amplifier at L5dorsal venules creates the gate by inducing certain levels ofchemokines although further studies are required for directevidence that links activation of the inflammation amplifierwith immunity homeostasis in the CNS
Upon discovering the L5 cord as the entry site ofautoreactive Th17 cells at the initial phase of EAE [9] wesearched for reasons that would make L5 ideal for this gateThe answer came from physiological responses to gravitationstimuli Soleus muscles are constantly stimulated by gravi-tational forces and the dorsal root ganglia of their sensoryneurons are located beside the L5 cord [13] We hypothesizedthat frequent stimulation of the soleus muscles by gravitycould induce activation of the inflammation amplifier viasensory nerves In experiments that had healthy normal
mice suspended from their tails so that only the forelimbscould touch the ground and the hind legs were releasedfrom gravitational forces MOG-reactiveTh17 cells no longeraccumulated at L5 (Figure 4) Instead they accumulated atcervical cords indicating that burdening the arm muscleswith bodyweight opened a new gateway for immune cells [9]Consistent with this observation tail suspension significantlyinhibited Ccl20mRNA expression in L5 dorsal blood vesselsand decreased the expression of the neural activationmarkerc-Fos in L5 dorsal root ganglia In addition when the soleusmuscles of tail-suspended mice were artificially stimulatedby weak electric pulses Ccl20 expression MOG-specificTh17 accumulation and c-Fos levels were restored at L5(Figure 4(b) and not shown)These data strongly suggest thatneural activation by an antigravitational response plays a rolein the activation of the inflammation amplifier leading tothe expression ofmany chemokines includingTh17-attractingCcl20 in L5 dorsal blood venules [9]
What mechanisms do afferent sensory neurons from thesoleus muscle use to regulate the status of blood venulesat L5 Although a precise neural network remains unclearwe have shown sympathetic nerves to be involved Bloodflow speed at L5 dorsal venules became slower when miceare tail suspended while electronic stimulation of the soleusmuscles increases the flow suggesting that automatic nervesincluding sympathetic ones are involved in the response Onthe other hand blood flow speeds in blood vessels other than
Mediators of Inflammation 5
(a)
lowastlowast
lowastlowast
lowastlowast
lowastlowast
0 2 4 6 8
TS ES
minus minus
minus+
+ +
6 h
12 h
18 h
24 h
Relative Ccl20 expression
(b)
Figure 4 Neural stimulation-mediated activation of the inflammation amplifier creates a gateway into the CNS via chemokine productionSchematic illustration of the tail suspensionmodel (a) A string is fastened to the roof of the cage at a height that allows the forelimbs to supportbody weight but prevents the hindlimbs from touching any part of the cage Release from gravitational stimuli caused by tail suspension (TS)results in a decrease of Ccl20 levels at the L5 dorsal venules Electric stimulation (ES) during TS restores the levels in a time-dependentmanner (b)
Sympatheticganglion
Dorsalroot
ganglion
PathogenicT cells
L5L6L4
Inflammation amplifier
5th lumber cord
CCL20
Sympathetic neuron
Sensory neuron
Figure 5 Schematic representation of neural stimulation-mediated activation of the inflammatory amplifier Neural signals from the soleusmuscles caused by gravitational stimulation reache the L5 dorsal root ganglion Subsequent activation of sympathetic nerves alters the status ofendothelial cells in L5 dorsal venules to enhance the inflammation amplifier which leads to the production of chemokines including CCL20Norepinephrine acts as a mediator between the neural signal and activation of the inflammation amplifier The neural network determiningcommunication between soleus muscle-derived sensory neurons and sympathetic neurons that reach L5 is not currently defined (depictedwith a question mark)
6 Mediators of Inflammation
Electric stimulations in muscles
Gravity
TricepsQuadricepsSoleus
Spinal cord
L5 gate L3 gate C5 gate
(1) Regional neural stimulations(1998400) Social stress
(2) Chemokine expression in venules
(3) Gates for immune cells
(1998400998400) Mental stress
(3998400) Gates at specific venules
Figure 6 Each neural stimulation establishes a regional gate of immune cells in the CNS Gravity or electric stimulations to soleus musclescan open the L5 gateway via regional neural stimulations-mediated chemokine expression electronic stimulation of the quadriceps followedby neural stimulation establishes the L3 gateway and gateways in the fifth cervical to fifth thoracic cord vessels were created by electricstimulations to triceps brachii Therefore it is reasonable that neural stimulations induced by stresses like social and mental ones mightestablish gates in some specific sites of the brain
the L5 region such as femoral vessels brain surface vesselsand the portal vein are not affected by tail suspension Fur-thermore treatment with atenolol a 1205731 adrenergic receptorantagonist or prazosin an 1205721 adrenergic receptor antagonistsignificantly suppressesCcl20mRNAexpression NF-120581B acti-vation and MOG-reactive Th17 accumulation at L5 vesselsand also suppresses clinical signs of EAE [9] Consistent withthese in vivo results the addition of norepinephrine to aculture of endothelial cell lines enhances the inflammationamplifier based on IL-6 and Ccl20 expressions Thus anti-gravity responses of the soleus muscles result in sympatheticnerve stimulation which creates a gateway of immune cellsto pass though the CNS via L5 dorsal venules [9] Basedon these findings we propose that MOG-reactive disease-causingTh17 cells make use of the L5 gateway to infiltrate theCNS and induce local inflammation by producing cytokineslike IL-17A which further induces chemokines through theinflammation amplifier and results in chronic inflammationof the CNS (Figure 5) At a later phase of EAE transferredTh17 cells are also found in the brain including regions likethe choroid plexus and cerebellum Whether these Th17 cellsmove within the cerebrospinal fluid to finally reach the brainandor enter the brain directly from the circulation is unclearHowever in the brain of EAE mice with advanced clinicalscores transferred Th17 cells are not uniformly localizedRather they tend to accumulate in specific areas of the brain(our unpublished data) which argues for the involvementof neural activation and subsequent breach of the BBB inthese areas Using this logic we speculate that the relatively
high incidence of vision dysfunction during the initial phasesof MS in humans might be due to the persistent visualstimulation in our everyday lives that activates the opticalnerves and unlocks the gate nearby
Other neuroimmune interactions have been reported byother groups The Kevin Tracey group which is a pioneerin this field has demonstrated that vagus nerve stimulationsuppresses the release of proinflammatory cytokines throughthe nicotinic acetylcholine receptor 1205727 subunit and identifieda subset of T cells producing acetylcholine that can relayneural signals [14ndash16] Acetylcholine is also produced byother immune cells including B cells which have an impacton innate immunity [17] Cao et al reported that micereared in a larger cage with toys and more mice that is anenriched environment are resistant to tumor burden in amanner dependent on sympathetic nerve activation via theBDNFleptin axis [18] Nguyen et al reported a relation-ship between catecholamines alternative macrophages andthermogenesis finding that exposure to cold temperaturesrapidly promotes alternative activation of adipose tissuemacrophages leading to a secretion of catecholamines thatinduces thermogenic gene expressions in brown adiposetissue and lipolysis in white adipose tissues [19] AdditionallyHassan et al showed that behavioral stress promotes prostatecancer development by inhibiting the apoptosis of tumor cellsvia the 1205732-adrenergic receptor [20] Therefore a strategy tomodulate neuroimmune interactions may prove a promisingapproach for therapeutic interventions against many inflam-matory diseases
Mediators of Inflammation 7
ZO-1
ZO-1 ZO-1ZO-1 ZO-1
ZO-1 ZO-1
ZO-2
ZO-2 ZO-2
ZO-2ZO-1
ZO-1
ZO-1 ZO-1
ZO-3 ZO-3
Claudin Claudin
Occludin Occludin
JAM JAM ZO-1
ZO-3 ZO-3
Actin
Actin
Actin
Blood stream
CNS Neuron
Neurotransmitters
Neurotransmitters
PericyteAstrocyte
Tight junction
Neuron
Th17Antigen
Th17
Endo
thel
ial c
ells
stimulation(1) Local neural
immune cellsfollowed by
disruption of BBB
(4) Accumulation of
(6) Accumulation of immune cells in the CNS
(3) Chemokines
(2) Inflammationamplifier
followed by excessive chemokines(5) Enhanced activation of the inflammation amplifier
Figure 7 Schematic representation of the gate theory in the CNSThe gate theory describes how regional neural stimulations direct immunecell infiltration into target organs by crossing gates located at various blood vessels Immune cells in the blood stream cannot enter the targetorgans due to homeostasis of the venules (upper left) Regional neural stimulation secretes neurotransmitters including norepinephrine intothe endothelial cells of the venules (upper right) This stimulation activates the inflammation amplifier in the endothelial cells followed byincreasing local chemokine expression (bottom right) The accumulation of immune cells disrupts the BBB which allows immune cells toenter the CNS (bottom left)
3 The Gate Theory
The dorsal venules of the L5 spinal cord have been found toact as a gateway for MOG-reactive Th17 cells to accumulateinto the CNS in an adoptive transfer model of murine EAEduring steady state Gravity or electric stimulations to soleusmuscles can open the L5 gateway as described above Weextended these findings by applying electric pulses to othermuscle regions Electronic stimulation of the quadriceps orthighmuscles which are known to be regulated by L3 dorsal-root ganglion neurons increased the expression of Ccl20mRNA in L3 cord vessels in mice Similarly chemokinelevels in the fifth cervical to fifth thoracic cord vessels wereupregulated by electric stimulations to epitrochlearistricepsbrachii (upper armmuscles) which are controlled by neuronslocated at the corresponding spinal regions (Figure 6) [9]Based on these findings we proposed ldquothe gate theoryrdquowhich describes how regional neural stimulations directimmune cell infiltration into target organs by crossing gateslocated at various blood venules (Figure 7) Investigationson whether the gate theory can be applied to tissues otherthan the CNS are ongoing The ability to manipulate thesegates at targeted regions in the body is expected to have
significant clinical benefits as closing them should ameliorateautoimmune inflammation in the target organ without anysystemic immune suppression while opening these gatesnear surrounding tumors may enhance cancer immunother-apy effectsWith suchmedical promisemuch effort is neededto identify the precise molecular mechanisms for gating
Conflict of Interests
The authors declare that they have no conflicting financialinterests
Acknowledgments
The authorse thank Dr P Karagiannis (RIKENQBiC OsakaJapan) for carefully reading this paper This work wassupported by KAKENHI (Masaaki Murakami 2439009824659221) Funding program for the JST-CREST program(MasaakiMurakami) Takeda Research Foundation (MasaakiMurakami and Yasunobu Arima) Uehara Foundation (Mas-aaki Murakami) The Naito Foundation (Masaaki Murak-ami) the Waksman Foundation of Japan (Masaaki Murak-ami) Tokyo Biochemical Research Foundation (Masaaki
8 Mediators of Inflammation
Murakami) Osaka Cancer Research Foundation (MasaakiMurakami) Mochida Memorial Foundation (YasunobuArima) Japan Intractable Diseases Research Foundation(Yasunobu Arima) and the Osaka Foundation for the Pro-motion of Clinical Immunology (Masaaki Murakami)
References
[1] N J Abbott L Ronnback and E Hansson ldquoAstrocyte-endo-thelial interactions at the blood-brain barrierrdquo Nature ReviewsNeuroscience vol 7 no 1 pp 41ndash53 2006
[2] E Steed M S Balda and K Matter ldquoDynamics and functionsof tight junctionsrdquo Trends in Cell Biology vol 20 no 3 pp 142ndash149 2010
[3] B V Zlokovic ldquoThe blood-brain barrier in health and chronicneurodegenerative disordersrdquoNeuron vol 57 no 2 pp 178ndash2012008
[4] J Bennett J Basivireddy A Kollar et al ldquoBlood-brain barrierdisruption and enhanced vascular permeability in the multiplesclerosismodel EAErdquo Journal of Neuroimmunology vol 229 no1-2 pp 180ndash191 2010
[5] L Steinman ldquoInflammatory cytokines at the summits of patho-logical signal cascades in brain diseasesrdquo Science Signaling vol6 no 258 p pe3 2013
[6] JHuppert D CloshenA Croxford et al ldquoCellularmechanismsof IL-17-induced blood-brain barrier disruptionrdquo FASEB Jour-nal vol 24 no 4 pp 1023ndash1034 2010
[7] H Kebir K Kreymborg I Ifergan et al ldquoHuman TH17 lym-phocytes promote blood-brain barrier disruption and centralnervous system inflammationrdquo Nature Medicine vol 13 no 10pp 1173ndash1175 2007
[8] Y Komiyama S Nakae T Matsuki et al ldquoIL-17 plays an impor-tant role in the development of experimental autoimmuneencephalomyelitisrdquo Journal of Immunology vol 177 no 1 pp566ndash573 2006
[9] Y Arima M Harada D Kamimura et al ldquoRegional neuralactivation defines a gateway for autoreactive T cells to cross theblood-brain barrierrdquo Cell vol 148 no 3 pp 447ndash457 2012
[10] I Bartholomaus N Kawakami F Odoardi et al ldquoEffector Tcell interactions with meningeal vascular structures in nascentautoimmuneCNS lesionsrdquoNature vol 462 no 7269 pp 94ndash982009
[11] L Steinman ldquoA molecular trio in relapse and remission inmultiple sclerosisrdquoNature Reviews Immunology vol 9 no 6 pp440ndash447 2009
[12] A Reboldi C Coisne D Baumjohann et al ldquoC-C chemokinereceptor 6-regulated entry of TH-17 cells into the CNS throughthe choroid plexus is required for the initiation of EAErdquoNatureImmunology vol 10 no 5 pp 514ndash523 2009
[13] Y Ohira F Kawano J L Stevens X D Wang and A IshiharaldquoLoad-dependent regulation of neuromuscular systemrdquo Journalof Gravitational Physiology vol 11 no 2 pp P127ndashP128 2004
[14] L V Borovikova S Ivanova M Zhang et al ldquoVagus nervestimulation attenuates the systemic inflammatory response toendotoxinrdquo Nature vol 405 no 6785 pp 458ndash462 2000
[15] H Wang M Yu M Ochani et al ldquoNicotinic acetylcholinereceptor 1205727 subunit is an essential regulator of inflammationrdquoNature vol 421 no 6921 pp 384ndash388 2003
[16] M Rosas-Ballina P S Olofsson M Ochani et al ldquoAcety-lcholine-synthesizing T cells relay neural signals in a vagusnerve circuitrdquo Science vol 334 no 6052 pp 98ndash101 2011
[17] C Reardon G S Duncan A Brustle et al ldquoLymphocyte-derived ACh regulates local innate but not adaptive immunityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 4 pp 1410ndash1415 2013
[18] L Cao X Liu E J D Lin et al ldquoEnvironmental and geneticactivation of a brain-adipocyte BDNFleptin axis causes cancerremission and inhibitionrdquo Cell vol 142 no 1 pp 52ndash64 2010
[19] K D Nguyen Y Qiu X Cui et al ldquoAlternatively activatedmacrophages produce catecholamines to sustain adaptive ther-mogenesisrdquo Nature vol 480 no 7375 pp 104ndash108 2011
[20] S Hassan Y Karpova D Baiz et al ldquoBehavioral stress accel-erates prostate cancer development in micerdquo Journal of ClinicalInvestigation vol 123 no 2 pp 874ndash886 2013
2 Mediators of Inflammation
Microglia
Neuron
Endothelial cellPericyte
Astrocyte
Blood-brain barrier at venules in the CNS
Immune cellsImmune cells
Tight junctions
Basementmembrane
Normal venules
Figure 1 Venules at the blood-brain barrier in the CNS and other organs Venules at the blood-brain barrier consist of specific endothelialcells that are brought together by tight junctions and tight liner sheets formed by pericytes and astrocytic end-feet These are not present innormal venules (left)
scores ameliorated histological changes and early recovery[8] Additionally an adoptive transfer model in which helperT cells obtained from myelin oligodendrocyte glycoprotein(MOG) immunized mice were infused into naıve recipientsindicated that IL-17A derived fromCD4+T cells is critical forthe induction of EAE [8] In addition MOG-reactive Th17cells obtained from MOG-immunized IL-17A-deficient micewere unable to infiltrate the lumber level of spinal cord in thesame model (see the following for details) [9] Furthermoreit was shown that the adoptive transfer of Th17 cells fromovalbumin-specific T cell receptor transgenic mice whichare unable to recognize CNS antigens does not pass the BBBand migrate into CNS whereas cotransfer of these Th17 cellswith MOG-reactive Th17 cells leads to the accumulation ofboth types of Th17 cells in the CNS (our unpublished dataand [10]) which strongly suggests that antigen recognition ofTh17 cells is required for severe disruption of the BBBAlthough antigen presentation inside the CNS has suggestedthrough observation that the infusion of ovalbumin peptide-loaded antigen-presenting cells into cerebrospinal fluidsinduces an accumulation of ovalbumin-specific Th17 cellsin the CNS [10] the type of antigen-presenting cells andlocation where antigen presentation takes place underphysiological conditions remain elusive Nevertheless theseresults suggest that IL-17A expressing Th17 cells whichrecognize CNS antigens have a major impact on breachingthe BBB in part by decreasing tight junction molecules
2 Neuroimmune Interactions Responsible forInflammation in the CNS
In the previous section we discussed the relationship bet-ween inflammatory cytokines such as IL-17A and disruptionof the BBB This section focuses on the gateway for whichpathogenic CD4+ T cells enter the CNS In patients withMS common early symptoms include vision problems andtingling followed by many neurological signs as the diseaseprogresses It is known that inflammation sites in MS arefound in specific regions of white matter including the brain-stem the optic nerve the cerebellum the long motor andsensory tracts of the spinal cord [11] This fact suggests thatsome CNS regions might be more vulnerable to autoimmuneattacks One hypothesis proposes that chemokine recruit-ment of pathogenic autoreactive T cells is more abundantin these regions Among the many chemokines CCL20 isof particular interest as it attracts Th17 cells that expressCCR6 a receptor for CCL20 Reboldi et al reported thatmicelackingCCR6 are highly resistant to EAE and that the choroidplexus a specialized epithelial structure in the brain known toproduce cerebrospinal fluids expresses CCL20 constitutivelyan effect that acts as an attractant for the first wave ofCCR6+ Th17 cells [12] In that same study however EAEwas induced using the complete Freundrsquos adjuvant which iswidely used to generate active immunization in animals butis also an inducer of systemic inflammation and has manyside effects including fever motor neuron dysfunction such
Mediators of Inflammation 3
lowast
Dorsal
Dorsal
5th lumber cord
Spinalroot
Blood vesselsPathogenic T cells
Spinal cord
vesselDorsal
Ventral
Ventral
3D
Accumulated mononuclearcell numbers
0 50 100 150 200
L6
L5
L4
L3
L2
L1
(a)
(b)
Figure 2 The fifth lumber cord is a gateway into the CNS A cross section of the fifth lumber (L5) cord (a) and actual cell numbers ofmononuclear cells accumulated in each lumber cord segment (b) at a preclinical phase of EAE (5 days after pathogenic Th17 transfer) A 3Dpicture based on ten serial sections of L5 is also shown
as paralysis and apoptosis These side effects could affectthe pathophysiological status of the brain and spinal cordsresulting in different conclusions from the steady state
We recently found a ldquogaterdquo past the BBB in the spinal cordthat autoreactive Th17 cells in the bloodstream can exploitto enter the CNS To make this discovery we first utilizedan adoptive transfer model to induce EAE in which Th17cells obtained fromMOG-immunizedmicewere infused intonaıve recipient mice to maintain CNS quiescence In thisadoptive transfer model we found that MOG-reactive Th17cells preferentially accumulated in the fifth lumber (L5) cordrather than the brain or other levels of the spinal cords atthe earliest phase of EAE (day 5 after T cell transfer) [9](Figure 2) This finding fits well with a typical clinical EAEsign in which the tail is first affected We also found thatblood vessel tracks in L5 are altered due to the formation ofedema in the L5 cord by using a supersensitive MRI (datanot shown) Consistent with these resultsCcl20mRNA levels
were highest in the dorsal venules of L5 compared withthose from other spinal cords and the transfer of CCR6-deficient Th17 cells did not accumulate in the L5 regionInterestingly even in naıve animals without Th17 transfermRNA levels of Ccl20 and many other chemokines werespecifically upregulated in the dorsal venules of L5Thereforedorsal venules in the L5 spinal cord have special properties indiseased as well as healthy conditions
Wepreviously found amechanism for the hyperinductionof inflammatory chemokines and cytokines in nonimmunecells such as fibroblasts endothelial cells and epithelial cellsusing a rheumatoid arthritis modelThemechanism is drivenby a simultaneous activation of two transcription factors NF-120581B and STAT3Thus it was named the ldquoinflammation ampli-fierrdquo because hyperactivation of NF-120581B by activated STAT3induces large amounts of NF-120581B-targeted chemokines andchemotactic factors to promote the recruitment of immunecells (Figure 3) Ccl20 is one such target chemokine and is
4 Mediators of Inflammation
lowastlowastlowast
lowastlowastlowast
IL-17Th17
Inflammationamplifier
Inflammatory chemokines IL-6
Fibroblastsendothelial cells etc
Autoimmune diseaseschronic inflammation graft rejection
IL-7
STAT3
IL-6
0
4
8
12
16
20
Non
e
IL-6
IL-1
7
Chemokine mRNA expression
IL-6
+IL
-17
NF-120581B
Figure 3The inflammation amplifier is defined as amechanism that triggers the hyperinduction of cytokines and chemokines in nonimmunecells and leads to many diseases This induction is induced by the simultaneous activation of NF-120581B and STAT3 IL-7 from nonimmune cellsalso contributes to enhancing the inflammation amplifier by generatingTh17 cells andor sustaining their survivalThe inflammation amplifieris essential for the pathogenesis of F759 arthritis EAE and chronic graft rejection The bar graph on the right indicates typical expressionstatuses of chemokines and IL-6 after inflammation amplifier activation (see the blue bar)
found in vascular endothelial cells Given that chemokineexpressions are elevated in L5 dorsal venules we hypothe-sized a role for the inflammation amplifier The activationstatus of NF-120581B and STAT3 is indeed higher in L5 dorsalvenules than other lumber cords even in naıve healthy miceThe elevated Ccl20mRNA levels at L5 vessels were decreasedin mice devoid of the inflammation amplifier such as IL-6-deficient mice and endothelial cell-specific IL-6 receptor(gp130) deficient mice Even under healthy conditions itis known that some immune cells are present in the CNSsuggesting that there may be a gate to enter the restrictedtissue regardless In this respect it is tempting to speculatethat low-grade activation of the inflammation amplifier at L5dorsal venules creates the gate by inducing certain levels ofchemokines although further studies are required for directevidence that links activation of the inflammation amplifierwith immunity homeostasis in the CNS
Upon discovering the L5 cord as the entry site ofautoreactive Th17 cells at the initial phase of EAE [9] wesearched for reasons that would make L5 ideal for this gateThe answer came from physiological responses to gravitationstimuli Soleus muscles are constantly stimulated by gravi-tational forces and the dorsal root ganglia of their sensoryneurons are located beside the L5 cord [13] We hypothesizedthat frequent stimulation of the soleus muscles by gravitycould induce activation of the inflammation amplifier viasensory nerves In experiments that had healthy normal
mice suspended from their tails so that only the forelimbscould touch the ground and the hind legs were releasedfrom gravitational forces MOG-reactiveTh17 cells no longeraccumulated at L5 (Figure 4) Instead they accumulated atcervical cords indicating that burdening the arm muscleswith bodyweight opened a new gateway for immune cells [9]Consistent with this observation tail suspension significantlyinhibited Ccl20mRNA expression in L5 dorsal blood vesselsand decreased the expression of the neural activationmarkerc-Fos in L5 dorsal root ganglia In addition when the soleusmuscles of tail-suspended mice were artificially stimulatedby weak electric pulses Ccl20 expression MOG-specificTh17 accumulation and c-Fos levels were restored at L5(Figure 4(b) and not shown)These data strongly suggest thatneural activation by an antigravitational response plays a rolein the activation of the inflammation amplifier leading tothe expression ofmany chemokines includingTh17-attractingCcl20 in L5 dorsal blood venules [9]
What mechanisms do afferent sensory neurons from thesoleus muscle use to regulate the status of blood venulesat L5 Although a precise neural network remains unclearwe have shown sympathetic nerves to be involved Bloodflow speed at L5 dorsal venules became slower when miceare tail suspended while electronic stimulation of the soleusmuscles increases the flow suggesting that automatic nervesincluding sympathetic ones are involved in the response Onthe other hand blood flow speeds in blood vessels other than
Mediators of Inflammation 5
(a)
lowastlowast
lowastlowast
lowastlowast
lowastlowast
0 2 4 6 8
TS ES
minus minus
minus+
+ +
6 h
12 h
18 h
24 h
Relative Ccl20 expression
(b)
Figure 4 Neural stimulation-mediated activation of the inflammation amplifier creates a gateway into the CNS via chemokine productionSchematic illustration of the tail suspensionmodel (a) A string is fastened to the roof of the cage at a height that allows the forelimbs to supportbody weight but prevents the hindlimbs from touching any part of the cage Release from gravitational stimuli caused by tail suspension (TS)results in a decrease of Ccl20 levels at the L5 dorsal venules Electric stimulation (ES) during TS restores the levels in a time-dependentmanner (b)
Sympatheticganglion
Dorsalroot
ganglion
PathogenicT cells
L5L6L4
Inflammation amplifier
5th lumber cord
CCL20
Sympathetic neuron
Sensory neuron
Figure 5 Schematic representation of neural stimulation-mediated activation of the inflammatory amplifier Neural signals from the soleusmuscles caused by gravitational stimulation reache the L5 dorsal root ganglion Subsequent activation of sympathetic nerves alters the status ofendothelial cells in L5 dorsal venules to enhance the inflammation amplifier which leads to the production of chemokines including CCL20Norepinephrine acts as a mediator between the neural signal and activation of the inflammation amplifier The neural network determiningcommunication between soleus muscle-derived sensory neurons and sympathetic neurons that reach L5 is not currently defined (depictedwith a question mark)
6 Mediators of Inflammation
Electric stimulations in muscles
Gravity
TricepsQuadricepsSoleus
Spinal cord
L5 gate L3 gate C5 gate
(1) Regional neural stimulations(1998400) Social stress
(2) Chemokine expression in venules
(3) Gates for immune cells
(1998400998400) Mental stress
(3998400) Gates at specific venules
Figure 6 Each neural stimulation establishes a regional gate of immune cells in the CNS Gravity or electric stimulations to soleus musclescan open the L5 gateway via regional neural stimulations-mediated chemokine expression electronic stimulation of the quadriceps followedby neural stimulation establishes the L3 gateway and gateways in the fifth cervical to fifth thoracic cord vessels were created by electricstimulations to triceps brachii Therefore it is reasonable that neural stimulations induced by stresses like social and mental ones mightestablish gates in some specific sites of the brain
the L5 region such as femoral vessels brain surface vesselsand the portal vein are not affected by tail suspension Fur-thermore treatment with atenolol a 1205731 adrenergic receptorantagonist or prazosin an 1205721 adrenergic receptor antagonistsignificantly suppressesCcl20mRNAexpression NF-120581B acti-vation and MOG-reactive Th17 accumulation at L5 vesselsand also suppresses clinical signs of EAE [9] Consistent withthese in vivo results the addition of norepinephrine to aculture of endothelial cell lines enhances the inflammationamplifier based on IL-6 and Ccl20 expressions Thus anti-gravity responses of the soleus muscles result in sympatheticnerve stimulation which creates a gateway of immune cellsto pass though the CNS via L5 dorsal venules [9] Basedon these findings we propose that MOG-reactive disease-causingTh17 cells make use of the L5 gateway to infiltrate theCNS and induce local inflammation by producing cytokineslike IL-17A which further induces chemokines through theinflammation amplifier and results in chronic inflammationof the CNS (Figure 5) At a later phase of EAE transferredTh17 cells are also found in the brain including regions likethe choroid plexus and cerebellum Whether these Th17 cellsmove within the cerebrospinal fluid to finally reach the brainandor enter the brain directly from the circulation is unclearHowever in the brain of EAE mice with advanced clinicalscores transferred Th17 cells are not uniformly localizedRather they tend to accumulate in specific areas of the brain(our unpublished data) which argues for the involvementof neural activation and subsequent breach of the BBB inthese areas Using this logic we speculate that the relatively
high incidence of vision dysfunction during the initial phasesof MS in humans might be due to the persistent visualstimulation in our everyday lives that activates the opticalnerves and unlocks the gate nearby
Other neuroimmune interactions have been reported byother groups The Kevin Tracey group which is a pioneerin this field has demonstrated that vagus nerve stimulationsuppresses the release of proinflammatory cytokines throughthe nicotinic acetylcholine receptor 1205727 subunit and identifieda subset of T cells producing acetylcholine that can relayneural signals [14ndash16] Acetylcholine is also produced byother immune cells including B cells which have an impacton innate immunity [17] Cao et al reported that micereared in a larger cage with toys and more mice that is anenriched environment are resistant to tumor burden in amanner dependent on sympathetic nerve activation via theBDNFleptin axis [18] Nguyen et al reported a relation-ship between catecholamines alternative macrophages andthermogenesis finding that exposure to cold temperaturesrapidly promotes alternative activation of adipose tissuemacrophages leading to a secretion of catecholamines thatinduces thermogenic gene expressions in brown adiposetissue and lipolysis in white adipose tissues [19] AdditionallyHassan et al showed that behavioral stress promotes prostatecancer development by inhibiting the apoptosis of tumor cellsvia the 1205732-adrenergic receptor [20] Therefore a strategy tomodulate neuroimmune interactions may prove a promisingapproach for therapeutic interventions against many inflam-matory diseases
Mediators of Inflammation 7
ZO-1
ZO-1 ZO-1ZO-1 ZO-1
ZO-1 ZO-1
ZO-2
ZO-2 ZO-2
ZO-2ZO-1
ZO-1
ZO-1 ZO-1
ZO-3 ZO-3
Claudin Claudin
Occludin Occludin
JAM JAM ZO-1
ZO-3 ZO-3
Actin
Actin
Actin
Blood stream
CNS Neuron
Neurotransmitters
Neurotransmitters
PericyteAstrocyte
Tight junction
Neuron
Th17Antigen
Th17
Endo
thel
ial c
ells
stimulation(1) Local neural
immune cellsfollowed by
disruption of BBB
(4) Accumulation of
(6) Accumulation of immune cells in the CNS
(3) Chemokines
(2) Inflammationamplifier
followed by excessive chemokines(5) Enhanced activation of the inflammation amplifier
Figure 7 Schematic representation of the gate theory in the CNSThe gate theory describes how regional neural stimulations direct immunecell infiltration into target organs by crossing gates located at various blood vessels Immune cells in the blood stream cannot enter the targetorgans due to homeostasis of the venules (upper left) Regional neural stimulation secretes neurotransmitters including norepinephrine intothe endothelial cells of the venules (upper right) This stimulation activates the inflammation amplifier in the endothelial cells followed byincreasing local chemokine expression (bottom right) The accumulation of immune cells disrupts the BBB which allows immune cells toenter the CNS (bottom left)
3 The Gate Theory
The dorsal venules of the L5 spinal cord have been found toact as a gateway for MOG-reactive Th17 cells to accumulateinto the CNS in an adoptive transfer model of murine EAEduring steady state Gravity or electric stimulations to soleusmuscles can open the L5 gateway as described above Weextended these findings by applying electric pulses to othermuscle regions Electronic stimulation of the quadriceps orthighmuscles which are known to be regulated by L3 dorsal-root ganglion neurons increased the expression of Ccl20mRNA in L3 cord vessels in mice Similarly chemokinelevels in the fifth cervical to fifth thoracic cord vessels wereupregulated by electric stimulations to epitrochlearistricepsbrachii (upper armmuscles) which are controlled by neuronslocated at the corresponding spinal regions (Figure 6) [9]Based on these findings we proposed ldquothe gate theoryrdquowhich describes how regional neural stimulations directimmune cell infiltration into target organs by crossing gateslocated at various blood venules (Figure 7) Investigationson whether the gate theory can be applied to tissues otherthan the CNS are ongoing The ability to manipulate thesegates at targeted regions in the body is expected to have
significant clinical benefits as closing them should ameliorateautoimmune inflammation in the target organ without anysystemic immune suppression while opening these gatesnear surrounding tumors may enhance cancer immunother-apy effectsWith suchmedical promisemuch effort is neededto identify the precise molecular mechanisms for gating
Conflict of Interests
The authors declare that they have no conflicting financialinterests
Acknowledgments
The authorse thank Dr P Karagiannis (RIKENQBiC OsakaJapan) for carefully reading this paper This work wassupported by KAKENHI (Masaaki Murakami 2439009824659221) Funding program for the JST-CREST program(MasaakiMurakami) Takeda Research Foundation (MasaakiMurakami and Yasunobu Arima) Uehara Foundation (Mas-aaki Murakami) The Naito Foundation (Masaaki Murak-ami) the Waksman Foundation of Japan (Masaaki Murak-ami) Tokyo Biochemical Research Foundation (Masaaki
8 Mediators of Inflammation
Murakami) Osaka Cancer Research Foundation (MasaakiMurakami) Mochida Memorial Foundation (YasunobuArima) Japan Intractable Diseases Research Foundation(Yasunobu Arima) and the Osaka Foundation for the Pro-motion of Clinical Immunology (Masaaki Murakami)
References
[1] N J Abbott L Ronnback and E Hansson ldquoAstrocyte-endo-thelial interactions at the blood-brain barrierrdquo Nature ReviewsNeuroscience vol 7 no 1 pp 41ndash53 2006
[2] E Steed M S Balda and K Matter ldquoDynamics and functionsof tight junctionsrdquo Trends in Cell Biology vol 20 no 3 pp 142ndash149 2010
[3] B V Zlokovic ldquoThe blood-brain barrier in health and chronicneurodegenerative disordersrdquoNeuron vol 57 no 2 pp 178ndash2012008
[4] J Bennett J Basivireddy A Kollar et al ldquoBlood-brain barrierdisruption and enhanced vascular permeability in the multiplesclerosismodel EAErdquo Journal of Neuroimmunology vol 229 no1-2 pp 180ndash191 2010
[5] L Steinman ldquoInflammatory cytokines at the summits of patho-logical signal cascades in brain diseasesrdquo Science Signaling vol6 no 258 p pe3 2013
[6] JHuppert D CloshenA Croxford et al ldquoCellularmechanismsof IL-17-induced blood-brain barrier disruptionrdquo FASEB Jour-nal vol 24 no 4 pp 1023ndash1034 2010
[7] H Kebir K Kreymborg I Ifergan et al ldquoHuman TH17 lym-phocytes promote blood-brain barrier disruption and centralnervous system inflammationrdquo Nature Medicine vol 13 no 10pp 1173ndash1175 2007
[8] Y Komiyama S Nakae T Matsuki et al ldquoIL-17 plays an impor-tant role in the development of experimental autoimmuneencephalomyelitisrdquo Journal of Immunology vol 177 no 1 pp566ndash573 2006
[9] Y Arima M Harada D Kamimura et al ldquoRegional neuralactivation defines a gateway for autoreactive T cells to cross theblood-brain barrierrdquo Cell vol 148 no 3 pp 447ndash457 2012
[10] I Bartholomaus N Kawakami F Odoardi et al ldquoEffector Tcell interactions with meningeal vascular structures in nascentautoimmuneCNS lesionsrdquoNature vol 462 no 7269 pp 94ndash982009
[11] L Steinman ldquoA molecular trio in relapse and remission inmultiple sclerosisrdquoNature Reviews Immunology vol 9 no 6 pp440ndash447 2009
[12] A Reboldi C Coisne D Baumjohann et al ldquoC-C chemokinereceptor 6-regulated entry of TH-17 cells into the CNS throughthe choroid plexus is required for the initiation of EAErdquoNatureImmunology vol 10 no 5 pp 514ndash523 2009
[13] Y Ohira F Kawano J L Stevens X D Wang and A IshiharaldquoLoad-dependent regulation of neuromuscular systemrdquo Journalof Gravitational Physiology vol 11 no 2 pp P127ndashP128 2004
[14] L V Borovikova S Ivanova M Zhang et al ldquoVagus nervestimulation attenuates the systemic inflammatory response toendotoxinrdquo Nature vol 405 no 6785 pp 458ndash462 2000
[15] H Wang M Yu M Ochani et al ldquoNicotinic acetylcholinereceptor 1205727 subunit is an essential regulator of inflammationrdquoNature vol 421 no 6921 pp 384ndash388 2003
[16] M Rosas-Ballina P S Olofsson M Ochani et al ldquoAcety-lcholine-synthesizing T cells relay neural signals in a vagusnerve circuitrdquo Science vol 334 no 6052 pp 98ndash101 2011
[17] C Reardon G S Duncan A Brustle et al ldquoLymphocyte-derived ACh regulates local innate but not adaptive immunityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 4 pp 1410ndash1415 2013
[18] L Cao X Liu E J D Lin et al ldquoEnvironmental and geneticactivation of a brain-adipocyte BDNFleptin axis causes cancerremission and inhibitionrdquo Cell vol 142 no 1 pp 52ndash64 2010
[19] K D Nguyen Y Qiu X Cui et al ldquoAlternatively activatedmacrophages produce catecholamines to sustain adaptive ther-mogenesisrdquo Nature vol 480 no 7375 pp 104ndash108 2011
[20] S Hassan Y Karpova D Baiz et al ldquoBehavioral stress accel-erates prostate cancer development in micerdquo Journal of ClinicalInvestigation vol 123 no 2 pp 874ndash886 2013
Mediators of Inflammation 3
lowast
Dorsal
Dorsal
5th lumber cord
Spinalroot
Blood vesselsPathogenic T cells
Spinal cord
vesselDorsal
Ventral
Ventral
3D
Accumulated mononuclearcell numbers
0 50 100 150 200
L6
L5
L4
L3
L2
L1
(a)
(b)
Figure 2 The fifth lumber cord is a gateway into the CNS A cross section of the fifth lumber (L5) cord (a) and actual cell numbers ofmononuclear cells accumulated in each lumber cord segment (b) at a preclinical phase of EAE (5 days after pathogenic Th17 transfer) A 3Dpicture based on ten serial sections of L5 is also shown
as paralysis and apoptosis These side effects could affectthe pathophysiological status of the brain and spinal cordsresulting in different conclusions from the steady state
We recently found a ldquogaterdquo past the BBB in the spinal cordthat autoreactive Th17 cells in the bloodstream can exploitto enter the CNS To make this discovery we first utilizedan adoptive transfer model to induce EAE in which Th17cells obtained fromMOG-immunizedmicewere infused intonaıve recipient mice to maintain CNS quiescence In thisadoptive transfer model we found that MOG-reactive Th17cells preferentially accumulated in the fifth lumber (L5) cordrather than the brain or other levels of the spinal cords atthe earliest phase of EAE (day 5 after T cell transfer) [9](Figure 2) This finding fits well with a typical clinical EAEsign in which the tail is first affected We also found thatblood vessel tracks in L5 are altered due to the formation ofedema in the L5 cord by using a supersensitive MRI (datanot shown) Consistent with these resultsCcl20mRNA levels
were highest in the dorsal venules of L5 compared withthose from other spinal cords and the transfer of CCR6-deficient Th17 cells did not accumulate in the L5 regionInterestingly even in naıve animals without Th17 transfermRNA levels of Ccl20 and many other chemokines werespecifically upregulated in the dorsal venules of L5Thereforedorsal venules in the L5 spinal cord have special properties indiseased as well as healthy conditions
Wepreviously found amechanism for the hyperinductionof inflammatory chemokines and cytokines in nonimmunecells such as fibroblasts endothelial cells and epithelial cellsusing a rheumatoid arthritis modelThemechanism is drivenby a simultaneous activation of two transcription factors NF-120581B and STAT3Thus it was named the ldquoinflammation ampli-fierrdquo because hyperactivation of NF-120581B by activated STAT3induces large amounts of NF-120581B-targeted chemokines andchemotactic factors to promote the recruitment of immunecells (Figure 3) Ccl20 is one such target chemokine and is
4 Mediators of Inflammation
lowastlowastlowast
lowastlowastlowast
IL-17Th17
Inflammationamplifier
Inflammatory chemokines IL-6
Fibroblastsendothelial cells etc
Autoimmune diseaseschronic inflammation graft rejection
IL-7
STAT3
IL-6
0
4
8
12
16
20
Non
e
IL-6
IL-1
7
Chemokine mRNA expression
IL-6
+IL
-17
NF-120581B
Figure 3The inflammation amplifier is defined as amechanism that triggers the hyperinduction of cytokines and chemokines in nonimmunecells and leads to many diseases This induction is induced by the simultaneous activation of NF-120581B and STAT3 IL-7 from nonimmune cellsalso contributes to enhancing the inflammation amplifier by generatingTh17 cells andor sustaining their survivalThe inflammation amplifieris essential for the pathogenesis of F759 arthritis EAE and chronic graft rejection The bar graph on the right indicates typical expressionstatuses of chemokines and IL-6 after inflammation amplifier activation (see the blue bar)
found in vascular endothelial cells Given that chemokineexpressions are elevated in L5 dorsal venules we hypothe-sized a role for the inflammation amplifier The activationstatus of NF-120581B and STAT3 is indeed higher in L5 dorsalvenules than other lumber cords even in naıve healthy miceThe elevated Ccl20mRNA levels at L5 vessels were decreasedin mice devoid of the inflammation amplifier such as IL-6-deficient mice and endothelial cell-specific IL-6 receptor(gp130) deficient mice Even under healthy conditions itis known that some immune cells are present in the CNSsuggesting that there may be a gate to enter the restrictedtissue regardless In this respect it is tempting to speculatethat low-grade activation of the inflammation amplifier at L5dorsal venules creates the gate by inducing certain levels ofchemokines although further studies are required for directevidence that links activation of the inflammation amplifierwith immunity homeostasis in the CNS
Upon discovering the L5 cord as the entry site ofautoreactive Th17 cells at the initial phase of EAE [9] wesearched for reasons that would make L5 ideal for this gateThe answer came from physiological responses to gravitationstimuli Soleus muscles are constantly stimulated by gravi-tational forces and the dorsal root ganglia of their sensoryneurons are located beside the L5 cord [13] We hypothesizedthat frequent stimulation of the soleus muscles by gravitycould induce activation of the inflammation amplifier viasensory nerves In experiments that had healthy normal
mice suspended from their tails so that only the forelimbscould touch the ground and the hind legs were releasedfrom gravitational forces MOG-reactiveTh17 cells no longeraccumulated at L5 (Figure 4) Instead they accumulated atcervical cords indicating that burdening the arm muscleswith bodyweight opened a new gateway for immune cells [9]Consistent with this observation tail suspension significantlyinhibited Ccl20mRNA expression in L5 dorsal blood vesselsand decreased the expression of the neural activationmarkerc-Fos in L5 dorsal root ganglia In addition when the soleusmuscles of tail-suspended mice were artificially stimulatedby weak electric pulses Ccl20 expression MOG-specificTh17 accumulation and c-Fos levels were restored at L5(Figure 4(b) and not shown)These data strongly suggest thatneural activation by an antigravitational response plays a rolein the activation of the inflammation amplifier leading tothe expression ofmany chemokines includingTh17-attractingCcl20 in L5 dorsal blood venules [9]
What mechanisms do afferent sensory neurons from thesoleus muscle use to regulate the status of blood venulesat L5 Although a precise neural network remains unclearwe have shown sympathetic nerves to be involved Bloodflow speed at L5 dorsal venules became slower when miceare tail suspended while electronic stimulation of the soleusmuscles increases the flow suggesting that automatic nervesincluding sympathetic ones are involved in the response Onthe other hand blood flow speeds in blood vessels other than
Mediators of Inflammation 5
(a)
lowastlowast
lowastlowast
lowastlowast
lowastlowast
0 2 4 6 8
TS ES
minus minus
minus+
+ +
6 h
12 h
18 h
24 h
Relative Ccl20 expression
(b)
Figure 4 Neural stimulation-mediated activation of the inflammation amplifier creates a gateway into the CNS via chemokine productionSchematic illustration of the tail suspensionmodel (a) A string is fastened to the roof of the cage at a height that allows the forelimbs to supportbody weight but prevents the hindlimbs from touching any part of the cage Release from gravitational stimuli caused by tail suspension (TS)results in a decrease of Ccl20 levels at the L5 dorsal venules Electric stimulation (ES) during TS restores the levels in a time-dependentmanner (b)
Sympatheticganglion
Dorsalroot
ganglion
PathogenicT cells
L5L6L4
Inflammation amplifier
5th lumber cord
CCL20
Sympathetic neuron
Sensory neuron
Figure 5 Schematic representation of neural stimulation-mediated activation of the inflammatory amplifier Neural signals from the soleusmuscles caused by gravitational stimulation reache the L5 dorsal root ganglion Subsequent activation of sympathetic nerves alters the status ofendothelial cells in L5 dorsal venules to enhance the inflammation amplifier which leads to the production of chemokines including CCL20Norepinephrine acts as a mediator between the neural signal and activation of the inflammation amplifier The neural network determiningcommunication between soleus muscle-derived sensory neurons and sympathetic neurons that reach L5 is not currently defined (depictedwith a question mark)
6 Mediators of Inflammation
Electric stimulations in muscles
Gravity
TricepsQuadricepsSoleus
Spinal cord
L5 gate L3 gate C5 gate
(1) Regional neural stimulations(1998400) Social stress
(2) Chemokine expression in venules
(3) Gates for immune cells
(1998400998400) Mental stress
(3998400) Gates at specific venules
Figure 6 Each neural stimulation establishes a regional gate of immune cells in the CNS Gravity or electric stimulations to soleus musclescan open the L5 gateway via regional neural stimulations-mediated chemokine expression electronic stimulation of the quadriceps followedby neural stimulation establishes the L3 gateway and gateways in the fifth cervical to fifth thoracic cord vessels were created by electricstimulations to triceps brachii Therefore it is reasonable that neural stimulations induced by stresses like social and mental ones mightestablish gates in some specific sites of the brain
the L5 region such as femoral vessels brain surface vesselsand the portal vein are not affected by tail suspension Fur-thermore treatment with atenolol a 1205731 adrenergic receptorantagonist or prazosin an 1205721 adrenergic receptor antagonistsignificantly suppressesCcl20mRNAexpression NF-120581B acti-vation and MOG-reactive Th17 accumulation at L5 vesselsand also suppresses clinical signs of EAE [9] Consistent withthese in vivo results the addition of norepinephrine to aculture of endothelial cell lines enhances the inflammationamplifier based on IL-6 and Ccl20 expressions Thus anti-gravity responses of the soleus muscles result in sympatheticnerve stimulation which creates a gateway of immune cellsto pass though the CNS via L5 dorsal venules [9] Basedon these findings we propose that MOG-reactive disease-causingTh17 cells make use of the L5 gateway to infiltrate theCNS and induce local inflammation by producing cytokineslike IL-17A which further induces chemokines through theinflammation amplifier and results in chronic inflammationof the CNS (Figure 5) At a later phase of EAE transferredTh17 cells are also found in the brain including regions likethe choroid plexus and cerebellum Whether these Th17 cellsmove within the cerebrospinal fluid to finally reach the brainandor enter the brain directly from the circulation is unclearHowever in the brain of EAE mice with advanced clinicalscores transferred Th17 cells are not uniformly localizedRather they tend to accumulate in specific areas of the brain(our unpublished data) which argues for the involvementof neural activation and subsequent breach of the BBB inthese areas Using this logic we speculate that the relatively
high incidence of vision dysfunction during the initial phasesof MS in humans might be due to the persistent visualstimulation in our everyday lives that activates the opticalnerves and unlocks the gate nearby
Other neuroimmune interactions have been reported byother groups The Kevin Tracey group which is a pioneerin this field has demonstrated that vagus nerve stimulationsuppresses the release of proinflammatory cytokines throughthe nicotinic acetylcholine receptor 1205727 subunit and identifieda subset of T cells producing acetylcholine that can relayneural signals [14ndash16] Acetylcholine is also produced byother immune cells including B cells which have an impacton innate immunity [17] Cao et al reported that micereared in a larger cage with toys and more mice that is anenriched environment are resistant to tumor burden in amanner dependent on sympathetic nerve activation via theBDNFleptin axis [18] Nguyen et al reported a relation-ship between catecholamines alternative macrophages andthermogenesis finding that exposure to cold temperaturesrapidly promotes alternative activation of adipose tissuemacrophages leading to a secretion of catecholamines thatinduces thermogenic gene expressions in brown adiposetissue and lipolysis in white adipose tissues [19] AdditionallyHassan et al showed that behavioral stress promotes prostatecancer development by inhibiting the apoptosis of tumor cellsvia the 1205732-adrenergic receptor [20] Therefore a strategy tomodulate neuroimmune interactions may prove a promisingapproach for therapeutic interventions against many inflam-matory diseases
Mediators of Inflammation 7
ZO-1
ZO-1 ZO-1ZO-1 ZO-1
ZO-1 ZO-1
ZO-2
ZO-2 ZO-2
ZO-2ZO-1
ZO-1
ZO-1 ZO-1
ZO-3 ZO-3
Claudin Claudin
Occludin Occludin
JAM JAM ZO-1
ZO-3 ZO-3
Actin
Actin
Actin
Blood stream
CNS Neuron
Neurotransmitters
Neurotransmitters
PericyteAstrocyte
Tight junction
Neuron
Th17Antigen
Th17
Endo
thel
ial c
ells
stimulation(1) Local neural
immune cellsfollowed by
disruption of BBB
(4) Accumulation of
(6) Accumulation of immune cells in the CNS
(3) Chemokines
(2) Inflammationamplifier
followed by excessive chemokines(5) Enhanced activation of the inflammation amplifier
Figure 7 Schematic representation of the gate theory in the CNSThe gate theory describes how regional neural stimulations direct immunecell infiltration into target organs by crossing gates located at various blood vessels Immune cells in the blood stream cannot enter the targetorgans due to homeostasis of the venules (upper left) Regional neural stimulation secretes neurotransmitters including norepinephrine intothe endothelial cells of the venules (upper right) This stimulation activates the inflammation amplifier in the endothelial cells followed byincreasing local chemokine expression (bottom right) The accumulation of immune cells disrupts the BBB which allows immune cells toenter the CNS (bottom left)
3 The Gate Theory
The dorsal venules of the L5 spinal cord have been found toact as a gateway for MOG-reactive Th17 cells to accumulateinto the CNS in an adoptive transfer model of murine EAEduring steady state Gravity or electric stimulations to soleusmuscles can open the L5 gateway as described above Weextended these findings by applying electric pulses to othermuscle regions Electronic stimulation of the quadriceps orthighmuscles which are known to be regulated by L3 dorsal-root ganglion neurons increased the expression of Ccl20mRNA in L3 cord vessels in mice Similarly chemokinelevels in the fifth cervical to fifth thoracic cord vessels wereupregulated by electric stimulations to epitrochlearistricepsbrachii (upper armmuscles) which are controlled by neuronslocated at the corresponding spinal regions (Figure 6) [9]Based on these findings we proposed ldquothe gate theoryrdquowhich describes how regional neural stimulations directimmune cell infiltration into target organs by crossing gateslocated at various blood venules (Figure 7) Investigationson whether the gate theory can be applied to tissues otherthan the CNS are ongoing The ability to manipulate thesegates at targeted regions in the body is expected to have
significant clinical benefits as closing them should ameliorateautoimmune inflammation in the target organ without anysystemic immune suppression while opening these gatesnear surrounding tumors may enhance cancer immunother-apy effectsWith suchmedical promisemuch effort is neededto identify the precise molecular mechanisms for gating
Conflict of Interests
The authors declare that they have no conflicting financialinterests
Acknowledgments
The authorse thank Dr P Karagiannis (RIKENQBiC OsakaJapan) for carefully reading this paper This work wassupported by KAKENHI (Masaaki Murakami 2439009824659221) Funding program for the JST-CREST program(MasaakiMurakami) Takeda Research Foundation (MasaakiMurakami and Yasunobu Arima) Uehara Foundation (Mas-aaki Murakami) The Naito Foundation (Masaaki Murak-ami) the Waksman Foundation of Japan (Masaaki Murak-ami) Tokyo Biochemical Research Foundation (Masaaki
8 Mediators of Inflammation
Murakami) Osaka Cancer Research Foundation (MasaakiMurakami) Mochida Memorial Foundation (YasunobuArima) Japan Intractable Diseases Research Foundation(Yasunobu Arima) and the Osaka Foundation for the Pro-motion of Clinical Immunology (Masaaki Murakami)
References
[1] N J Abbott L Ronnback and E Hansson ldquoAstrocyte-endo-thelial interactions at the blood-brain barrierrdquo Nature ReviewsNeuroscience vol 7 no 1 pp 41ndash53 2006
[2] E Steed M S Balda and K Matter ldquoDynamics and functionsof tight junctionsrdquo Trends in Cell Biology vol 20 no 3 pp 142ndash149 2010
[3] B V Zlokovic ldquoThe blood-brain barrier in health and chronicneurodegenerative disordersrdquoNeuron vol 57 no 2 pp 178ndash2012008
[4] J Bennett J Basivireddy A Kollar et al ldquoBlood-brain barrierdisruption and enhanced vascular permeability in the multiplesclerosismodel EAErdquo Journal of Neuroimmunology vol 229 no1-2 pp 180ndash191 2010
[5] L Steinman ldquoInflammatory cytokines at the summits of patho-logical signal cascades in brain diseasesrdquo Science Signaling vol6 no 258 p pe3 2013
[6] JHuppert D CloshenA Croxford et al ldquoCellularmechanismsof IL-17-induced blood-brain barrier disruptionrdquo FASEB Jour-nal vol 24 no 4 pp 1023ndash1034 2010
[7] H Kebir K Kreymborg I Ifergan et al ldquoHuman TH17 lym-phocytes promote blood-brain barrier disruption and centralnervous system inflammationrdquo Nature Medicine vol 13 no 10pp 1173ndash1175 2007
[8] Y Komiyama S Nakae T Matsuki et al ldquoIL-17 plays an impor-tant role in the development of experimental autoimmuneencephalomyelitisrdquo Journal of Immunology vol 177 no 1 pp566ndash573 2006
[9] Y Arima M Harada D Kamimura et al ldquoRegional neuralactivation defines a gateway for autoreactive T cells to cross theblood-brain barrierrdquo Cell vol 148 no 3 pp 447ndash457 2012
[10] I Bartholomaus N Kawakami F Odoardi et al ldquoEffector Tcell interactions with meningeal vascular structures in nascentautoimmuneCNS lesionsrdquoNature vol 462 no 7269 pp 94ndash982009
[11] L Steinman ldquoA molecular trio in relapse and remission inmultiple sclerosisrdquoNature Reviews Immunology vol 9 no 6 pp440ndash447 2009
[12] A Reboldi C Coisne D Baumjohann et al ldquoC-C chemokinereceptor 6-regulated entry of TH-17 cells into the CNS throughthe choroid plexus is required for the initiation of EAErdquoNatureImmunology vol 10 no 5 pp 514ndash523 2009
[13] Y Ohira F Kawano J L Stevens X D Wang and A IshiharaldquoLoad-dependent regulation of neuromuscular systemrdquo Journalof Gravitational Physiology vol 11 no 2 pp P127ndashP128 2004
[14] L V Borovikova S Ivanova M Zhang et al ldquoVagus nervestimulation attenuates the systemic inflammatory response toendotoxinrdquo Nature vol 405 no 6785 pp 458ndash462 2000
[15] H Wang M Yu M Ochani et al ldquoNicotinic acetylcholinereceptor 1205727 subunit is an essential regulator of inflammationrdquoNature vol 421 no 6921 pp 384ndash388 2003
[16] M Rosas-Ballina P S Olofsson M Ochani et al ldquoAcety-lcholine-synthesizing T cells relay neural signals in a vagusnerve circuitrdquo Science vol 334 no 6052 pp 98ndash101 2011
[17] C Reardon G S Duncan A Brustle et al ldquoLymphocyte-derived ACh regulates local innate but not adaptive immunityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 4 pp 1410ndash1415 2013
[18] L Cao X Liu E J D Lin et al ldquoEnvironmental and geneticactivation of a brain-adipocyte BDNFleptin axis causes cancerremission and inhibitionrdquo Cell vol 142 no 1 pp 52ndash64 2010
[19] K D Nguyen Y Qiu X Cui et al ldquoAlternatively activatedmacrophages produce catecholamines to sustain adaptive ther-mogenesisrdquo Nature vol 480 no 7375 pp 104ndash108 2011
[20] S Hassan Y Karpova D Baiz et al ldquoBehavioral stress accel-erates prostate cancer development in micerdquo Journal of ClinicalInvestigation vol 123 no 2 pp 874ndash886 2013
4 Mediators of Inflammation
lowastlowastlowast
lowastlowastlowast
IL-17Th17
Inflammationamplifier
Inflammatory chemokines IL-6
Fibroblastsendothelial cells etc
Autoimmune diseaseschronic inflammation graft rejection
IL-7
STAT3
IL-6
0
4
8
12
16
20
Non
e
IL-6
IL-1
7
Chemokine mRNA expression
IL-6
+IL
-17
NF-120581B
Figure 3The inflammation amplifier is defined as amechanism that triggers the hyperinduction of cytokines and chemokines in nonimmunecells and leads to many diseases This induction is induced by the simultaneous activation of NF-120581B and STAT3 IL-7 from nonimmune cellsalso contributes to enhancing the inflammation amplifier by generatingTh17 cells andor sustaining their survivalThe inflammation amplifieris essential for the pathogenesis of F759 arthritis EAE and chronic graft rejection The bar graph on the right indicates typical expressionstatuses of chemokines and IL-6 after inflammation amplifier activation (see the blue bar)
found in vascular endothelial cells Given that chemokineexpressions are elevated in L5 dorsal venules we hypothe-sized a role for the inflammation amplifier The activationstatus of NF-120581B and STAT3 is indeed higher in L5 dorsalvenules than other lumber cords even in naıve healthy miceThe elevated Ccl20mRNA levels at L5 vessels were decreasedin mice devoid of the inflammation amplifier such as IL-6-deficient mice and endothelial cell-specific IL-6 receptor(gp130) deficient mice Even under healthy conditions itis known that some immune cells are present in the CNSsuggesting that there may be a gate to enter the restrictedtissue regardless In this respect it is tempting to speculatethat low-grade activation of the inflammation amplifier at L5dorsal venules creates the gate by inducing certain levels ofchemokines although further studies are required for directevidence that links activation of the inflammation amplifierwith immunity homeostasis in the CNS
Upon discovering the L5 cord as the entry site ofautoreactive Th17 cells at the initial phase of EAE [9] wesearched for reasons that would make L5 ideal for this gateThe answer came from physiological responses to gravitationstimuli Soleus muscles are constantly stimulated by gravi-tational forces and the dorsal root ganglia of their sensoryneurons are located beside the L5 cord [13] We hypothesizedthat frequent stimulation of the soleus muscles by gravitycould induce activation of the inflammation amplifier viasensory nerves In experiments that had healthy normal
mice suspended from their tails so that only the forelimbscould touch the ground and the hind legs were releasedfrom gravitational forces MOG-reactiveTh17 cells no longeraccumulated at L5 (Figure 4) Instead they accumulated atcervical cords indicating that burdening the arm muscleswith bodyweight opened a new gateway for immune cells [9]Consistent with this observation tail suspension significantlyinhibited Ccl20mRNA expression in L5 dorsal blood vesselsand decreased the expression of the neural activationmarkerc-Fos in L5 dorsal root ganglia In addition when the soleusmuscles of tail-suspended mice were artificially stimulatedby weak electric pulses Ccl20 expression MOG-specificTh17 accumulation and c-Fos levels were restored at L5(Figure 4(b) and not shown)These data strongly suggest thatneural activation by an antigravitational response plays a rolein the activation of the inflammation amplifier leading tothe expression ofmany chemokines includingTh17-attractingCcl20 in L5 dorsal blood venules [9]
What mechanisms do afferent sensory neurons from thesoleus muscle use to regulate the status of blood venulesat L5 Although a precise neural network remains unclearwe have shown sympathetic nerves to be involved Bloodflow speed at L5 dorsal venules became slower when miceare tail suspended while electronic stimulation of the soleusmuscles increases the flow suggesting that automatic nervesincluding sympathetic ones are involved in the response Onthe other hand blood flow speeds in blood vessels other than
Mediators of Inflammation 5
(a)
lowastlowast
lowastlowast
lowastlowast
lowastlowast
0 2 4 6 8
TS ES
minus minus
minus+
+ +
6 h
12 h
18 h
24 h
Relative Ccl20 expression
(b)
Figure 4 Neural stimulation-mediated activation of the inflammation amplifier creates a gateway into the CNS via chemokine productionSchematic illustration of the tail suspensionmodel (a) A string is fastened to the roof of the cage at a height that allows the forelimbs to supportbody weight but prevents the hindlimbs from touching any part of the cage Release from gravitational stimuli caused by tail suspension (TS)results in a decrease of Ccl20 levels at the L5 dorsal venules Electric stimulation (ES) during TS restores the levels in a time-dependentmanner (b)
Sympatheticganglion
Dorsalroot
ganglion
PathogenicT cells
L5L6L4
Inflammation amplifier
5th lumber cord
CCL20
Sympathetic neuron
Sensory neuron
Figure 5 Schematic representation of neural stimulation-mediated activation of the inflammatory amplifier Neural signals from the soleusmuscles caused by gravitational stimulation reache the L5 dorsal root ganglion Subsequent activation of sympathetic nerves alters the status ofendothelial cells in L5 dorsal venules to enhance the inflammation amplifier which leads to the production of chemokines including CCL20Norepinephrine acts as a mediator between the neural signal and activation of the inflammation amplifier The neural network determiningcommunication between soleus muscle-derived sensory neurons and sympathetic neurons that reach L5 is not currently defined (depictedwith a question mark)
6 Mediators of Inflammation
Electric stimulations in muscles
Gravity
TricepsQuadricepsSoleus
Spinal cord
L5 gate L3 gate C5 gate
(1) Regional neural stimulations(1998400) Social stress
(2) Chemokine expression in venules
(3) Gates for immune cells
(1998400998400) Mental stress
(3998400) Gates at specific venules
Figure 6 Each neural stimulation establishes a regional gate of immune cells in the CNS Gravity or electric stimulations to soleus musclescan open the L5 gateway via regional neural stimulations-mediated chemokine expression electronic stimulation of the quadriceps followedby neural stimulation establishes the L3 gateway and gateways in the fifth cervical to fifth thoracic cord vessels were created by electricstimulations to triceps brachii Therefore it is reasonable that neural stimulations induced by stresses like social and mental ones mightestablish gates in some specific sites of the brain
the L5 region such as femoral vessels brain surface vesselsand the portal vein are not affected by tail suspension Fur-thermore treatment with atenolol a 1205731 adrenergic receptorantagonist or prazosin an 1205721 adrenergic receptor antagonistsignificantly suppressesCcl20mRNAexpression NF-120581B acti-vation and MOG-reactive Th17 accumulation at L5 vesselsand also suppresses clinical signs of EAE [9] Consistent withthese in vivo results the addition of norepinephrine to aculture of endothelial cell lines enhances the inflammationamplifier based on IL-6 and Ccl20 expressions Thus anti-gravity responses of the soleus muscles result in sympatheticnerve stimulation which creates a gateway of immune cellsto pass though the CNS via L5 dorsal venules [9] Basedon these findings we propose that MOG-reactive disease-causingTh17 cells make use of the L5 gateway to infiltrate theCNS and induce local inflammation by producing cytokineslike IL-17A which further induces chemokines through theinflammation amplifier and results in chronic inflammationof the CNS (Figure 5) At a later phase of EAE transferredTh17 cells are also found in the brain including regions likethe choroid plexus and cerebellum Whether these Th17 cellsmove within the cerebrospinal fluid to finally reach the brainandor enter the brain directly from the circulation is unclearHowever in the brain of EAE mice with advanced clinicalscores transferred Th17 cells are not uniformly localizedRather they tend to accumulate in specific areas of the brain(our unpublished data) which argues for the involvementof neural activation and subsequent breach of the BBB inthese areas Using this logic we speculate that the relatively
high incidence of vision dysfunction during the initial phasesof MS in humans might be due to the persistent visualstimulation in our everyday lives that activates the opticalnerves and unlocks the gate nearby
Other neuroimmune interactions have been reported byother groups The Kevin Tracey group which is a pioneerin this field has demonstrated that vagus nerve stimulationsuppresses the release of proinflammatory cytokines throughthe nicotinic acetylcholine receptor 1205727 subunit and identifieda subset of T cells producing acetylcholine that can relayneural signals [14ndash16] Acetylcholine is also produced byother immune cells including B cells which have an impacton innate immunity [17] Cao et al reported that micereared in a larger cage with toys and more mice that is anenriched environment are resistant to tumor burden in amanner dependent on sympathetic nerve activation via theBDNFleptin axis [18] Nguyen et al reported a relation-ship between catecholamines alternative macrophages andthermogenesis finding that exposure to cold temperaturesrapidly promotes alternative activation of adipose tissuemacrophages leading to a secretion of catecholamines thatinduces thermogenic gene expressions in brown adiposetissue and lipolysis in white adipose tissues [19] AdditionallyHassan et al showed that behavioral stress promotes prostatecancer development by inhibiting the apoptosis of tumor cellsvia the 1205732-adrenergic receptor [20] Therefore a strategy tomodulate neuroimmune interactions may prove a promisingapproach for therapeutic interventions against many inflam-matory diseases
Mediators of Inflammation 7
ZO-1
ZO-1 ZO-1ZO-1 ZO-1
ZO-1 ZO-1
ZO-2
ZO-2 ZO-2
ZO-2ZO-1
ZO-1
ZO-1 ZO-1
ZO-3 ZO-3
Claudin Claudin
Occludin Occludin
JAM JAM ZO-1
ZO-3 ZO-3
Actin
Actin
Actin
Blood stream
CNS Neuron
Neurotransmitters
Neurotransmitters
PericyteAstrocyte
Tight junction
Neuron
Th17Antigen
Th17
Endo
thel
ial c
ells
stimulation(1) Local neural
immune cellsfollowed by
disruption of BBB
(4) Accumulation of
(6) Accumulation of immune cells in the CNS
(3) Chemokines
(2) Inflammationamplifier
followed by excessive chemokines(5) Enhanced activation of the inflammation amplifier
Figure 7 Schematic representation of the gate theory in the CNSThe gate theory describes how regional neural stimulations direct immunecell infiltration into target organs by crossing gates located at various blood vessels Immune cells in the blood stream cannot enter the targetorgans due to homeostasis of the venules (upper left) Regional neural stimulation secretes neurotransmitters including norepinephrine intothe endothelial cells of the venules (upper right) This stimulation activates the inflammation amplifier in the endothelial cells followed byincreasing local chemokine expression (bottom right) The accumulation of immune cells disrupts the BBB which allows immune cells toenter the CNS (bottom left)
3 The Gate Theory
The dorsal venules of the L5 spinal cord have been found toact as a gateway for MOG-reactive Th17 cells to accumulateinto the CNS in an adoptive transfer model of murine EAEduring steady state Gravity or electric stimulations to soleusmuscles can open the L5 gateway as described above Weextended these findings by applying electric pulses to othermuscle regions Electronic stimulation of the quadriceps orthighmuscles which are known to be regulated by L3 dorsal-root ganglion neurons increased the expression of Ccl20mRNA in L3 cord vessels in mice Similarly chemokinelevels in the fifth cervical to fifth thoracic cord vessels wereupregulated by electric stimulations to epitrochlearistricepsbrachii (upper armmuscles) which are controlled by neuronslocated at the corresponding spinal regions (Figure 6) [9]Based on these findings we proposed ldquothe gate theoryrdquowhich describes how regional neural stimulations directimmune cell infiltration into target organs by crossing gateslocated at various blood venules (Figure 7) Investigationson whether the gate theory can be applied to tissues otherthan the CNS are ongoing The ability to manipulate thesegates at targeted regions in the body is expected to have
significant clinical benefits as closing them should ameliorateautoimmune inflammation in the target organ without anysystemic immune suppression while opening these gatesnear surrounding tumors may enhance cancer immunother-apy effectsWith suchmedical promisemuch effort is neededto identify the precise molecular mechanisms for gating
Conflict of Interests
The authors declare that they have no conflicting financialinterests
Acknowledgments
The authorse thank Dr P Karagiannis (RIKENQBiC OsakaJapan) for carefully reading this paper This work wassupported by KAKENHI (Masaaki Murakami 2439009824659221) Funding program for the JST-CREST program(MasaakiMurakami) Takeda Research Foundation (MasaakiMurakami and Yasunobu Arima) Uehara Foundation (Mas-aaki Murakami) The Naito Foundation (Masaaki Murak-ami) the Waksman Foundation of Japan (Masaaki Murak-ami) Tokyo Biochemical Research Foundation (Masaaki
8 Mediators of Inflammation
Murakami) Osaka Cancer Research Foundation (MasaakiMurakami) Mochida Memorial Foundation (YasunobuArima) Japan Intractable Diseases Research Foundation(Yasunobu Arima) and the Osaka Foundation for the Pro-motion of Clinical Immunology (Masaaki Murakami)
References
[1] N J Abbott L Ronnback and E Hansson ldquoAstrocyte-endo-thelial interactions at the blood-brain barrierrdquo Nature ReviewsNeuroscience vol 7 no 1 pp 41ndash53 2006
[2] E Steed M S Balda and K Matter ldquoDynamics and functionsof tight junctionsrdquo Trends in Cell Biology vol 20 no 3 pp 142ndash149 2010
[3] B V Zlokovic ldquoThe blood-brain barrier in health and chronicneurodegenerative disordersrdquoNeuron vol 57 no 2 pp 178ndash2012008
[4] J Bennett J Basivireddy A Kollar et al ldquoBlood-brain barrierdisruption and enhanced vascular permeability in the multiplesclerosismodel EAErdquo Journal of Neuroimmunology vol 229 no1-2 pp 180ndash191 2010
[5] L Steinman ldquoInflammatory cytokines at the summits of patho-logical signal cascades in brain diseasesrdquo Science Signaling vol6 no 258 p pe3 2013
[6] JHuppert D CloshenA Croxford et al ldquoCellularmechanismsof IL-17-induced blood-brain barrier disruptionrdquo FASEB Jour-nal vol 24 no 4 pp 1023ndash1034 2010
[7] H Kebir K Kreymborg I Ifergan et al ldquoHuman TH17 lym-phocytes promote blood-brain barrier disruption and centralnervous system inflammationrdquo Nature Medicine vol 13 no 10pp 1173ndash1175 2007
[8] Y Komiyama S Nakae T Matsuki et al ldquoIL-17 plays an impor-tant role in the development of experimental autoimmuneencephalomyelitisrdquo Journal of Immunology vol 177 no 1 pp566ndash573 2006
[9] Y Arima M Harada D Kamimura et al ldquoRegional neuralactivation defines a gateway for autoreactive T cells to cross theblood-brain barrierrdquo Cell vol 148 no 3 pp 447ndash457 2012
[10] I Bartholomaus N Kawakami F Odoardi et al ldquoEffector Tcell interactions with meningeal vascular structures in nascentautoimmuneCNS lesionsrdquoNature vol 462 no 7269 pp 94ndash982009
[11] L Steinman ldquoA molecular trio in relapse and remission inmultiple sclerosisrdquoNature Reviews Immunology vol 9 no 6 pp440ndash447 2009
[12] A Reboldi C Coisne D Baumjohann et al ldquoC-C chemokinereceptor 6-regulated entry of TH-17 cells into the CNS throughthe choroid plexus is required for the initiation of EAErdquoNatureImmunology vol 10 no 5 pp 514ndash523 2009
[13] Y Ohira F Kawano J L Stevens X D Wang and A IshiharaldquoLoad-dependent regulation of neuromuscular systemrdquo Journalof Gravitational Physiology vol 11 no 2 pp P127ndashP128 2004
[14] L V Borovikova S Ivanova M Zhang et al ldquoVagus nervestimulation attenuates the systemic inflammatory response toendotoxinrdquo Nature vol 405 no 6785 pp 458ndash462 2000
[15] H Wang M Yu M Ochani et al ldquoNicotinic acetylcholinereceptor 1205727 subunit is an essential regulator of inflammationrdquoNature vol 421 no 6921 pp 384ndash388 2003
[16] M Rosas-Ballina P S Olofsson M Ochani et al ldquoAcety-lcholine-synthesizing T cells relay neural signals in a vagusnerve circuitrdquo Science vol 334 no 6052 pp 98ndash101 2011
[17] C Reardon G S Duncan A Brustle et al ldquoLymphocyte-derived ACh regulates local innate but not adaptive immunityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 4 pp 1410ndash1415 2013
[18] L Cao X Liu E J D Lin et al ldquoEnvironmental and geneticactivation of a brain-adipocyte BDNFleptin axis causes cancerremission and inhibitionrdquo Cell vol 142 no 1 pp 52ndash64 2010
[19] K D Nguyen Y Qiu X Cui et al ldquoAlternatively activatedmacrophages produce catecholamines to sustain adaptive ther-mogenesisrdquo Nature vol 480 no 7375 pp 104ndash108 2011
[20] S Hassan Y Karpova D Baiz et al ldquoBehavioral stress accel-erates prostate cancer development in micerdquo Journal of ClinicalInvestigation vol 123 no 2 pp 874ndash886 2013
Mediators of Inflammation 5
(a)
lowastlowast
lowastlowast
lowastlowast
lowastlowast
0 2 4 6 8
TS ES
minus minus
minus+
+ +
6 h
12 h
18 h
24 h
Relative Ccl20 expression
(b)
Figure 4 Neural stimulation-mediated activation of the inflammation amplifier creates a gateway into the CNS via chemokine productionSchematic illustration of the tail suspensionmodel (a) A string is fastened to the roof of the cage at a height that allows the forelimbs to supportbody weight but prevents the hindlimbs from touching any part of the cage Release from gravitational stimuli caused by tail suspension (TS)results in a decrease of Ccl20 levels at the L5 dorsal venules Electric stimulation (ES) during TS restores the levels in a time-dependentmanner (b)
Sympatheticganglion
Dorsalroot
ganglion
PathogenicT cells
L5L6L4
Inflammation amplifier
5th lumber cord
CCL20
Sympathetic neuron
Sensory neuron
Figure 5 Schematic representation of neural stimulation-mediated activation of the inflammatory amplifier Neural signals from the soleusmuscles caused by gravitational stimulation reache the L5 dorsal root ganglion Subsequent activation of sympathetic nerves alters the status ofendothelial cells in L5 dorsal venules to enhance the inflammation amplifier which leads to the production of chemokines including CCL20Norepinephrine acts as a mediator between the neural signal and activation of the inflammation amplifier The neural network determiningcommunication between soleus muscle-derived sensory neurons and sympathetic neurons that reach L5 is not currently defined (depictedwith a question mark)
6 Mediators of Inflammation
Electric stimulations in muscles
Gravity
TricepsQuadricepsSoleus
Spinal cord
L5 gate L3 gate C5 gate
(1) Regional neural stimulations(1998400) Social stress
(2) Chemokine expression in venules
(3) Gates for immune cells
(1998400998400) Mental stress
(3998400) Gates at specific venules
Figure 6 Each neural stimulation establishes a regional gate of immune cells in the CNS Gravity or electric stimulations to soleus musclescan open the L5 gateway via regional neural stimulations-mediated chemokine expression electronic stimulation of the quadriceps followedby neural stimulation establishes the L3 gateway and gateways in the fifth cervical to fifth thoracic cord vessels were created by electricstimulations to triceps brachii Therefore it is reasonable that neural stimulations induced by stresses like social and mental ones mightestablish gates in some specific sites of the brain
the L5 region such as femoral vessels brain surface vesselsand the portal vein are not affected by tail suspension Fur-thermore treatment with atenolol a 1205731 adrenergic receptorantagonist or prazosin an 1205721 adrenergic receptor antagonistsignificantly suppressesCcl20mRNAexpression NF-120581B acti-vation and MOG-reactive Th17 accumulation at L5 vesselsand also suppresses clinical signs of EAE [9] Consistent withthese in vivo results the addition of norepinephrine to aculture of endothelial cell lines enhances the inflammationamplifier based on IL-6 and Ccl20 expressions Thus anti-gravity responses of the soleus muscles result in sympatheticnerve stimulation which creates a gateway of immune cellsto pass though the CNS via L5 dorsal venules [9] Basedon these findings we propose that MOG-reactive disease-causingTh17 cells make use of the L5 gateway to infiltrate theCNS and induce local inflammation by producing cytokineslike IL-17A which further induces chemokines through theinflammation amplifier and results in chronic inflammationof the CNS (Figure 5) At a later phase of EAE transferredTh17 cells are also found in the brain including regions likethe choroid plexus and cerebellum Whether these Th17 cellsmove within the cerebrospinal fluid to finally reach the brainandor enter the brain directly from the circulation is unclearHowever in the brain of EAE mice with advanced clinicalscores transferred Th17 cells are not uniformly localizedRather they tend to accumulate in specific areas of the brain(our unpublished data) which argues for the involvementof neural activation and subsequent breach of the BBB inthese areas Using this logic we speculate that the relatively
high incidence of vision dysfunction during the initial phasesof MS in humans might be due to the persistent visualstimulation in our everyday lives that activates the opticalnerves and unlocks the gate nearby
Other neuroimmune interactions have been reported byother groups The Kevin Tracey group which is a pioneerin this field has demonstrated that vagus nerve stimulationsuppresses the release of proinflammatory cytokines throughthe nicotinic acetylcholine receptor 1205727 subunit and identifieda subset of T cells producing acetylcholine that can relayneural signals [14ndash16] Acetylcholine is also produced byother immune cells including B cells which have an impacton innate immunity [17] Cao et al reported that micereared in a larger cage with toys and more mice that is anenriched environment are resistant to tumor burden in amanner dependent on sympathetic nerve activation via theBDNFleptin axis [18] Nguyen et al reported a relation-ship between catecholamines alternative macrophages andthermogenesis finding that exposure to cold temperaturesrapidly promotes alternative activation of adipose tissuemacrophages leading to a secretion of catecholamines thatinduces thermogenic gene expressions in brown adiposetissue and lipolysis in white adipose tissues [19] AdditionallyHassan et al showed that behavioral stress promotes prostatecancer development by inhibiting the apoptosis of tumor cellsvia the 1205732-adrenergic receptor [20] Therefore a strategy tomodulate neuroimmune interactions may prove a promisingapproach for therapeutic interventions against many inflam-matory diseases
Mediators of Inflammation 7
ZO-1
ZO-1 ZO-1ZO-1 ZO-1
ZO-1 ZO-1
ZO-2
ZO-2 ZO-2
ZO-2ZO-1
ZO-1
ZO-1 ZO-1
ZO-3 ZO-3
Claudin Claudin
Occludin Occludin
JAM JAM ZO-1
ZO-3 ZO-3
Actin
Actin
Actin
Blood stream
CNS Neuron
Neurotransmitters
Neurotransmitters
PericyteAstrocyte
Tight junction
Neuron
Th17Antigen
Th17
Endo
thel
ial c
ells
stimulation(1) Local neural
immune cellsfollowed by
disruption of BBB
(4) Accumulation of
(6) Accumulation of immune cells in the CNS
(3) Chemokines
(2) Inflammationamplifier
followed by excessive chemokines(5) Enhanced activation of the inflammation amplifier
Figure 7 Schematic representation of the gate theory in the CNSThe gate theory describes how regional neural stimulations direct immunecell infiltration into target organs by crossing gates located at various blood vessels Immune cells in the blood stream cannot enter the targetorgans due to homeostasis of the venules (upper left) Regional neural stimulation secretes neurotransmitters including norepinephrine intothe endothelial cells of the venules (upper right) This stimulation activates the inflammation amplifier in the endothelial cells followed byincreasing local chemokine expression (bottom right) The accumulation of immune cells disrupts the BBB which allows immune cells toenter the CNS (bottom left)
3 The Gate Theory
The dorsal venules of the L5 spinal cord have been found toact as a gateway for MOG-reactive Th17 cells to accumulateinto the CNS in an adoptive transfer model of murine EAEduring steady state Gravity or electric stimulations to soleusmuscles can open the L5 gateway as described above Weextended these findings by applying electric pulses to othermuscle regions Electronic stimulation of the quadriceps orthighmuscles which are known to be regulated by L3 dorsal-root ganglion neurons increased the expression of Ccl20mRNA in L3 cord vessels in mice Similarly chemokinelevels in the fifth cervical to fifth thoracic cord vessels wereupregulated by electric stimulations to epitrochlearistricepsbrachii (upper armmuscles) which are controlled by neuronslocated at the corresponding spinal regions (Figure 6) [9]Based on these findings we proposed ldquothe gate theoryrdquowhich describes how regional neural stimulations directimmune cell infiltration into target organs by crossing gateslocated at various blood venules (Figure 7) Investigationson whether the gate theory can be applied to tissues otherthan the CNS are ongoing The ability to manipulate thesegates at targeted regions in the body is expected to have
significant clinical benefits as closing them should ameliorateautoimmune inflammation in the target organ without anysystemic immune suppression while opening these gatesnear surrounding tumors may enhance cancer immunother-apy effectsWith suchmedical promisemuch effort is neededto identify the precise molecular mechanisms for gating
Conflict of Interests
The authors declare that they have no conflicting financialinterests
Acknowledgments
The authorse thank Dr P Karagiannis (RIKENQBiC OsakaJapan) for carefully reading this paper This work wassupported by KAKENHI (Masaaki Murakami 2439009824659221) Funding program for the JST-CREST program(MasaakiMurakami) Takeda Research Foundation (MasaakiMurakami and Yasunobu Arima) Uehara Foundation (Mas-aaki Murakami) The Naito Foundation (Masaaki Murak-ami) the Waksman Foundation of Japan (Masaaki Murak-ami) Tokyo Biochemical Research Foundation (Masaaki
8 Mediators of Inflammation
Murakami) Osaka Cancer Research Foundation (MasaakiMurakami) Mochida Memorial Foundation (YasunobuArima) Japan Intractable Diseases Research Foundation(Yasunobu Arima) and the Osaka Foundation for the Pro-motion of Clinical Immunology (Masaaki Murakami)
References
[1] N J Abbott L Ronnback and E Hansson ldquoAstrocyte-endo-thelial interactions at the blood-brain barrierrdquo Nature ReviewsNeuroscience vol 7 no 1 pp 41ndash53 2006
[2] E Steed M S Balda and K Matter ldquoDynamics and functionsof tight junctionsrdquo Trends in Cell Biology vol 20 no 3 pp 142ndash149 2010
[3] B V Zlokovic ldquoThe blood-brain barrier in health and chronicneurodegenerative disordersrdquoNeuron vol 57 no 2 pp 178ndash2012008
[4] J Bennett J Basivireddy A Kollar et al ldquoBlood-brain barrierdisruption and enhanced vascular permeability in the multiplesclerosismodel EAErdquo Journal of Neuroimmunology vol 229 no1-2 pp 180ndash191 2010
[5] L Steinman ldquoInflammatory cytokines at the summits of patho-logical signal cascades in brain diseasesrdquo Science Signaling vol6 no 258 p pe3 2013
[6] JHuppert D CloshenA Croxford et al ldquoCellularmechanismsof IL-17-induced blood-brain barrier disruptionrdquo FASEB Jour-nal vol 24 no 4 pp 1023ndash1034 2010
[7] H Kebir K Kreymborg I Ifergan et al ldquoHuman TH17 lym-phocytes promote blood-brain barrier disruption and centralnervous system inflammationrdquo Nature Medicine vol 13 no 10pp 1173ndash1175 2007
[8] Y Komiyama S Nakae T Matsuki et al ldquoIL-17 plays an impor-tant role in the development of experimental autoimmuneencephalomyelitisrdquo Journal of Immunology vol 177 no 1 pp566ndash573 2006
[9] Y Arima M Harada D Kamimura et al ldquoRegional neuralactivation defines a gateway for autoreactive T cells to cross theblood-brain barrierrdquo Cell vol 148 no 3 pp 447ndash457 2012
[10] I Bartholomaus N Kawakami F Odoardi et al ldquoEffector Tcell interactions with meningeal vascular structures in nascentautoimmuneCNS lesionsrdquoNature vol 462 no 7269 pp 94ndash982009
[11] L Steinman ldquoA molecular trio in relapse and remission inmultiple sclerosisrdquoNature Reviews Immunology vol 9 no 6 pp440ndash447 2009
[12] A Reboldi C Coisne D Baumjohann et al ldquoC-C chemokinereceptor 6-regulated entry of TH-17 cells into the CNS throughthe choroid plexus is required for the initiation of EAErdquoNatureImmunology vol 10 no 5 pp 514ndash523 2009
[13] Y Ohira F Kawano J L Stevens X D Wang and A IshiharaldquoLoad-dependent regulation of neuromuscular systemrdquo Journalof Gravitational Physiology vol 11 no 2 pp P127ndashP128 2004
[14] L V Borovikova S Ivanova M Zhang et al ldquoVagus nervestimulation attenuates the systemic inflammatory response toendotoxinrdquo Nature vol 405 no 6785 pp 458ndash462 2000
[15] H Wang M Yu M Ochani et al ldquoNicotinic acetylcholinereceptor 1205727 subunit is an essential regulator of inflammationrdquoNature vol 421 no 6921 pp 384ndash388 2003
[16] M Rosas-Ballina P S Olofsson M Ochani et al ldquoAcety-lcholine-synthesizing T cells relay neural signals in a vagusnerve circuitrdquo Science vol 334 no 6052 pp 98ndash101 2011
[17] C Reardon G S Duncan A Brustle et al ldquoLymphocyte-derived ACh regulates local innate but not adaptive immunityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 4 pp 1410ndash1415 2013
[18] L Cao X Liu E J D Lin et al ldquoEnvironmental and geneticactivation of a brain-adipocyte BDNFleptin axis causes cancerremission and inhibitionrdquo Cell vol 142 no 1 pp 52ndash64 2010
[19] K D Nguyen Y Qiu X Cui et al ldquoAlternatively activatedmacrophages produce catecholamines to sustain adaptive ther-mogenesisrdquo Nature vol 480 no 7375 pp 104ndash108 2011
[20] S Hassan Y Karpova D Baiz et al ldquoBehavioral stress accel-erates prostate cancer development in micerdquo Journal of ClinicalInvestigation vol 123 no 2 pp 874ndash886 2013
6 Mediators of Inflammation
Electric stimulations in muscles
Gravity
TricepsQuadricepsSoleus
Spinal cord
L5 gate L3 gate C5 gate
(1) Regional neural stimulations(1998400) Social stress
(2) Chemokine expression in venules
(3) Gates for immune cells
(1998400998400) Mental stress
(3998400) Gates at specific venules
Figure 6 Each neural stimulation establishes a regional gate of immune cells in the CNS Gravity or electric stimulations to soleus musclescan open the L5 gateway via regional neural stimulations-mediated chemokine expression electronic stimulation of the quadriceps followedby neural stimulation establishes the L3 gateway and gateways in the fifth cervical to fifth thoracic cord vessels were created by electricstimulations to triceps brachii Therefore it is reasonable that neural stimulations induced by stresses like social and mental ones mightestablish gates in some specific sites of the brain
the L5 region such as femoral vessels brain surface vesselsand the portal vein are not affected by tail suspension Fur-thermore treatment with atenolol a 1205731 adrenergic receptorantagonist or prazosin an 1205721 adrenergic receptor antagonistsignificantly suppressesCcl20mRNAexpression NF-120581B acti-vation and MOG-reactive Th17 accumulation at L5 vesselsand also suppresses clinical signs of EAE [9] Consistent withthese in vivo results the addition of norepinephrine to aculture of endothelial cell lines enhances the inflammationamplifier based on IL-6 and Ccl20 expressions Thus anti-gravity responses of the soleus muscles result in sympatheticnerve stimulation which creates a gateway of immune cellsto pass though the CNS via L5 dorsal venules [9] Basedon these findings we propose that MOG-reactive disease-causingTh17 cells make use of the L5 gateway to infiltrate theCNS and induce local inflammation by producing cytokineslike IL-17A which further induces chemokines through theinflammation amplifier and results in chronic inflammationof the CNS (Figure 5) At a later phase of EAE transferredTh17 cells are also found in the brain including regions likethe choroid plexus and cerebellum Whether these Th17 cellsmove within the cerebrospinal fluid to finally reach the brainandor enter the brain directly from the circulation is unclearHowever in the brain of EAE mice with advanced clinicalscores transferred Th17 cells are not uniformly localizedRather they tend to accumulate in specific areas of the brain(our unpublished data) which argues for the involvementof neural activation and subsequent breach of the BBB inthese areas Using this logic we speculate that the relatively
high incidence of vision dysfunction during the initial phasesof MS in humans might be due to the persistent visualstimulation in our everyday lives that activates the opticalnerves and unlocks the gate nearby
Other neuroimmune interactions have been reported byother groups The Kevin Tracey group which is a pioneerin this field has demonstrated that vagus nerve stimulationsuppresses the release of proinflammatory cytokines throughthe nicotinic acetylcholine receptor 1205727 subunit and identifieda subset of T cells producing acetylcholine that can relayneural signals [14ndash16] Acetylcholine is also produced byother immune cells including B cells which have an impacton innate immunity [17] Cao et al reported that micereared in a larger cage with toys and more mice that is anenriched environment are resistant to tumor burden in amanner dependent on sympathetic nerve activation via theBDNFleptin axis [18] Nguyen et al reported a relation-ship between catecholamines alternative macrophages andthermogenesis finding that exposure to cold temperaturesrapidly promotes alternative activation of adipose tissuemacrophages leading to a secretion of catecholamines thatinduces thermogenic gene expressions in brown adiposetissue and lipolysis in white adipose tissues [19] AdditionallyHassan et al showed that behavioral stress promotes prostatecancer development by inhibiting the apoptosis of tumor cellsvia the 1205732-adrenergic receptor [20] Therefore a strategy tomodulate neuroimmune interactions may prove a promisingapproach for therapeutic interventions against many inflam-matory diseases
Mediators of Inflammation 7
ZO-1
ZO-1 ZO-1ZO-1 ZO-1
ZO-1 ZO-1
ZO-2
ZO-2 ZO-2
ZO-2ZO-1
ZO-1
ZO-1 ZO-1
ZO-3 ZO-3
Claudin Claudin
Occludin Occludin
JAM JAM ZO-1
ZO-3 ZO-3
Actin
Actin
Actin
Blood stream
CNS Neuron
Neurotransmitters
Neurotransmitters
PericyteAstrocyte
Tight junction
Neuron
Th17Antigen
Th17
Endo
thel
ial c
ells
stimulation(1) Local neural
immune cellsfollowed by
disruption of BBB
(4) Accumulation of
(6) Accumulation of immune cells in the CNS
(3) Chemokines
(2) Inflammationamplifier
followed by excessive chemokines(5) Enhanced activation of the inflammation amplifier
Figure 7 Schematic representation of the gate theory in the CNSThe gate theory describes how regional neural stimulations direct immunecell infiltration into target organs by crossing gates located at various blood vessels Immune cells in the blood stream cannot enter the targetorgans due to homeostasis of the venules (upper left) Regional neural stimulation secretes neurotransmitters including norepinephrine intothe endothelial cells of the venules (upper right) This stimulation activates the inflammation amplifier in the endothelial cells followed byincreasing local chemokine expression (bottom right) The accumulation of immune cells disrupts the BBB which allows immune cells toenter the CNS (bottom left)
3 The Gate Theory
The dorsal venules of the L5 spinal cord have been found toact as a gateway for MOG-reactive Th17 cells to accumulateinto the CNS in an adoptive transfer model of murine EAEduring steady state Gravity or electric stimulations to soleusmuscles can open the L5 gateway as described above Weextended these findings by applying electric pulses to othermuscle regions Electronic stimulation of the quadriceps orthighmuscles which are known to be regulated by L3 dorsal-root ganglion neurons increased the expression of Ccl20mRNA in L3 cord vessels in mice Similarly chemokinelevels in the fifth cervical to fifth thoracic cord vessels wereupregulated by electric stimulations to epitrochlearistricepsbrachii (upper armmuscles) which are controlled by neuronslocated at the corresponding spinal regions (Figure 6) [9]Based on these findings we proposed ldquothe gate theoryrdquowhich describes how regional neural stimulations directimmune cell infiltration into target organs by crossing gateslocated at various blood venules (Figure 7) Investigationson whether the gate theory can be applied to tissues otherthan the CNS are ongoing The ability to manipulate thesegates at targeted regions in the body is expected to have
significant clinical benefits as closing them should ameliorateautoimmune inflammation in the target organ without anysystemic immune suppression while opening these gatesnear surrounding tumors may enhance cancer immunother-apy effectsWith suchmedical promisemuch effort is neededto identify the precise molecular mechanisms for gating
Conflict of Interests
The authors declare that they have no conflicting financialinterests
Acknowledgments
The authorse thank Dr P Karagiannis (RIKENQBiC OsakaJapan) for carefully reading this paper This work wassupported by KAKENHI (Masaaki Murakami 2439009824659221) Funding program for the JST-CREST program(MasaakiMurakami) Takeda Research Foundation (MasaakiMurakami and Yasunobu Arima) Uehara Foundation (Mas-aaki Murakami) The Naito Foundation (Masaaki Murak-ami) the Waksman Foundation of Japan (Masaaki Murak-ami) Tokyo Biochemical Research Foundation (Masaaki
8 Mediators of Inflammation
Murakami) Osaka Cancer Research Foundation (MasaakiMurakami) Mochida Memorial Foundation (YasunobuArima) Japan Intractable Diseases Research Foundation(Yasunobu Arima) and the Osaka Foundation for the Pro-motion of Clinical Immunology (Masaaki Murakami)
References
[1] N J Abbott L Ronnback and E Hansson ldquoAstrocyte-endo-thelial interactions at the blood-brain barrierrdquo Nature ReviewsNeuroscience vol 7 no 1 pp 41ndash53 2006
[2] E Steed M S Balda and K Matter ldquoDynamics and functionsof tight junctionsrdquo Trends in Cell Biology vol 20 no 3 pp 142ndash149 2010
[3] B V Zlokovic ldquoThe blood-brain barrier in health and chronicneurodegenerative disordersrdquoNeuron vol 57 no 2 pp 178ndash2012008
[4] J Bennett J Basivireddy A Kollar et al ldquoBlood-brain barrierdisruption and enhanced vascular permeability in the multiplesclerosismodel EAErdquo Journal of Neuroimmunology vol 229 no1-2 pp 180ndash191 2010
[5] L Steinman ldquoInflammatory cytokines at the summits of patho-logical signal cascades in brain diseasesrdquo Science Signaling vol6 no 258 p pe3 2013
[6] JHuppert D CloshenA Croxford et al ldquoCellularmechanismsof IL-17-induced blood-brain barrier disruptionrdquo FASEB Jour-nal vol 24 no 4 pp 1023ndash1034 2010
[7] H Kebir K Kreymborg I Ifergan et al ldquoHuman TH17 lym-phocytes promote blood-brain barrier disruption and centralnervous system inflammationrdquo Nature Medicine vol 13 no 10pp 1173ndash1175 2007
[8] Y Komiyama S Nakae T Matsuki et al ldquoIL-17 plays an impor-tant role in the development of experimental autoimmuneencephalomyelitisrdquo Journal of Immunology vol 177 no 1 pp566ndash573 2006
[9] Y Arima M Harada D Kamimura et al ldquoRegional neuralactivation defines a gateway for autoreactive T cells to cross theblood-brain barrierrdquo Cell vol 148 no 3 pp 447ndash457 2012
[10] I Bartholomaus N Kawakami F Odoardi et al ldquoEffector Tcell interactions with meningeal vascular structures in nascentautoimmuneCNS lesionsrdquoNature vol 462 no 7269 pp 94ndash982009
[11] L Steinman ldquoA molecular trio in relapse and remission inmultiple sclerosisrdquoNature Reviews Immunology vol 9 no 6 pp440ndash447 2009
[12] A Reboldi C Coisne D Baumjohann et al ldquoC-C chemokinereceptor 6-regulated entry of TH-17 cells into the CNS throughthe choroid plexus is required for the initiation of EAErdquoNatureImmunology vol 10 no 5 pp 514ndash523 2009
[13] Y Ohira F Kawano J L Stevens X D Wang and A IshiharaldquoLoad-dependent regulation of neuromuscular systemrdquo Journalof Gravitational Physiology vol 11 no 2 pp P127ndashP128 2004
[14] L V Borovikova S Ivanova M Zhang et al ldquoVagus nervestimulation attenuates the systemic inflammatory response toendotoxinrdquo Nature vol 405 no 6785 pp 458ndash462 2000
[15] H Wang M Yu M Ochani et al ldquoNicotinic acetylcholinereceptor 1205727 subunit is an essential regulator of inflammationrdquoNature vol 421 no 6921 pp 384ndash388 2003
[16] M Rosas-Ballina P S Olofsson M Ochani et al ldquoAcety-lcholine-synthesizing T cells relay neural signals in a vagusnerve circuitrdquo Science vol 334 no 6052 pp 98ndash101 2011
[17] C Reardon G S Duncan A Brustle et al ldquoLymphocyte-derived ACh regulates local innate but not adaptive immunityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 4 pp 1410ndash1415 2013
[18] L Cao X Liu E J D Lin et al ldquoEnvironmental and geneticactivation of a brain-adipocyte BDNFleptin axis causes cancerremission and inhibitionrdquo Cell vol 142 no 1 pp 52ndash64 2010
[19] K D Nguyen Y Qiu X Cui et al ldquoAlternatively activatedmacrophages produce catecholamines to sustain adaptive ther-mogenesisrdquo Nature vol 480 no 7375 pp 104ndash108 2011
[20] S Hassan Y Karpova D Baiz et al ldquoBehavioral stress accel-erates prostate cancer development in micerdquo Journal of ClinicalInvestigation vol 123 no 2 pp 874ndash886 2013
Mediators of Inflammation 7
ZO-1
ZO-1 ZO-1ZO-1 ZO-1
ZO-1 ZO-1
ZO-2
ZO-2 ZO-2
ZO-2ZO-1
ZO-1
ZO-1 ZO-1
ZO-3 ZO-3
Claudin Claudin
Occludin Occludin
JAM JAM ZO-1
ZO-3 ZO-3
Actin
Actin
Actin
Blood stream
CNS Neuron
Neurotransmitters
Neurotransmitters
PericyteAstrocyte
Tight junction
Neuron
Th17Antigen
Th17
Endo
thel
ial c
ells
stimulation(1) Local neural
immune cellsfollowed by
disruption of BBB
(4) Accumulation of
(6) Accumulation of immune cells in the CNS
(3) Chemokines
(2) Inflammationamplifier
followed by excessive chemokines(5) Enhanced activation of the inflammation amplifier
Figure 7 Schematic representation of the gate theory in the CNSThe gate theory describes how regional neural stimulations direct immunecell infiltration into target organs by crossing gates located at various blood vessels Immune cells in the blood stream cannot enter the targetorgans due to homeostasis of the venules (upper left) Regional neural stimulation secretes neurotransmitters including norepinephrine intothe endothelial cells of the venules (upper right) This stimulation activates the inflammation amplifier in the endothelial cells followed byincreasing local chemokine expression (bottom right) The accumulation of immune cells disrupts the BBB which allows immune cells toenter the CNS (bottom left)
3 The Gate Theory
The dorsal venules of the L5 spinal cord have been found toact as a gateway for MOG-reactive Th17 cells to accumulateinto the CNS in an adoptive transfer model of murine EAEduring steady state Gravity or electric stimulations to soleusmuscles can open the L5 gateway as described above Weextended these findings by applying electric pulses to othermuscle regions Electronic stimulation of the quadriceps orthighmuscles which are known to be regulated by L3 dorsal-root ganglion neurons increased the expression of Ccl20mRNA in L3 cord vessels in mice Similarly chemokinelevels in the fifth cervical to fifth thoracic cord vessels wereupregulated by electric stimulations to epitrochlearistricepsbrachii (upper armmuscles) which are controlled by neuronslocated at the corresponding spinal regions (Figure 6) [9]Based on these findings we proposed ldquothe gate theoryrdquowhich describes how regional neural stimulations directimmune cell infiltration into target organs by crossing gateslocated at various blood venules (Figure 7) Investigationson whether the gate theory can be applied to tissues otherthan the CNS are ongoing The ability to manipulate thesegates at targeted regions in the body is expected to have
significant clinical benefits as closing them should ameliorateautoimmune inflammation in the target organ without anysystemic immune suppression while opening these gatesnear surrounding tumors may enhance cancer immunother-apy effectsWith suchmedical promisemuch effort is neededto identify the precise molecular mechanisms for gating
Conflict of Interests
The authors declare that they have no conflicting financialinterests
Acknowledgments
The authorse thank Dr P Karagiannis (RIKENQBiC OsakaJapan) for carefully reading this paper This work wassupported by KAKENHI (Masaaki Murakami 2439009824659221) Funding program for the JST-CREST program(MasaakiMurakami) Takeda Research Foundation (MasaakiMurakami and Yasunobu Arima) Uehara Foundation (Mas-aaki Murakami) The Naito Foundation (Masaaki Murak-ami) the Waksman Foundation of Japan (Masaaki Murak-ami) Tokyo Biochemical Research Foundation (Masaaki
8 Mediators of Inflammation
Murakami) Osaka Cancer Research Foundation (MasaakiMurakami) Mochida Memorial Foundation (YasunobuArima) Japan Intractable Diseases Research Foundation(Yasunobu Arima) and the Osaka Foundation for the Pro-motion of Clinical Immunology (Masaaki Murakami)
References
[1] N J Abbott L Ronnback and E Hansson ldquoAstrocyte-endo-thelial interactions at the blood-brain barrierrdquo Nature ReviewsNeuroscience vol 7 no 1 pp 41ndash53 2006
[2] E Steed M S Balda and K Matter ldquoDynamics and functionsof tight junctionsrdquo Trends in Cell Biology vol 20 no 3 pp 142ndash149 2010
[3] B V Zlokovic ldquoThe blood-brain barrier in health and chronicneurodegenerative disordersrdquoNeuron vol 57 no 2 pp 178ndash2012008
[4] J Bennett J Basivireddy A Kollar et al ldquoBlood-brain barrierdisruption and enhanced vascular permeability in the multiplesclerosismodel EAErdquo Journal of Neuroimmunology vol 229 no1-2 pp 180ndash191 2010
[5] L Steinman ldquoInflammatory cytokines at the summits of patho-logical signal cascades in brain diseasesrdquo Science Signaling vol6 no 258 p pe3 2013
[6] JHuppert D CloshenA Croxford et al ldquoCellularmechanismsof IL-17-induced blood-brain barrier disruptionrdquo FASEB Jour-nal vol 24 no 4 pp 1023ndash1034 2010
[7] H Kebir K Kreymborg I Ifergan et al ldquoHuman TH17 lym-phocytes promote blood-brain barrier disruption and centralnervous system inflammationrdquo Nature Medicine vol 13 no 10pp 1173ndash1175 2007
[8] Y Komiyama S Nakae T Matsuki et al ldquoIL-17 plays an impor-tant role in the development of experimental autoimmuneencephalomyelitisrdquo Journal of Immunology vol 177 no 1 pp566ndash573 2006
[9] Y Arima M Harada D Kamimura et al ldquoRegional neuralactivation defines a gateway for autoreactive T cells to cross theblood-brain barrierrdquo Cell vol 148 no 3 pp 447ndash457 2012
[10] I Bartholomaus N Kawakami F Odoardi et al ldquoEffector Tcell interactions with meningeal vascular structures in nascentautoimmuneCNS lesionsrdquoNature vol 462 no 7269 pp 94ndash982009
[11] L Steinman ldquoA molecular trio in relapse and remission inmultiple sclerosisrdquoNature Reviews Immunology vol 9 no 6 pp440ndash447 2009
[12] A Reboldi C Coisne D Baumjohann et al ldquoC-C chemokinereceptor 6-regulated entry of TH-17 cells into the CNS throughthe choroid plexus is required for the initiation of EAErdquoNatureImmunology vol 10 no 5 pp 514ndash523 2009
[13] Y Ohira F Kawano J L Stevens X D Wang and A IshiharaldquoLoad-dependent regulation of neuromuscular systemrdquo Journalof Gravitational Physiology vol 11 no 2 pp P127ndashP128 2004
[14] L V Borovikova S Ivanova M Zhang et al ldquoVagus nervestimulation attenuates the systemic inflammatory response toendotoxinrdquo Nature vol 405 no 6785 pp 458ndash462 2000
[15] H Wang M Yu M Ochani et al ldquoNicotinic acetylcholinereceptor 1205727 subunit is an essential regulator of inflammationrdquoNature vol 421 no 6921 pp 384ndash388 2003
[16] M Rosas-Ballina P S Olofsson M Ochani et al ldquoAcety-lcholine-synthesizing T cells relay neural signals in a vagusnerve circuitrdquo Science vol 334 no 6052 pp 98ndash101 2011
[17] C Reardon G S Duncan A Brustle et al ldquoLymphocyte-derived ACh regulates local innate but not adaptive immunityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 4 pp 1410ndash1415 2013
[18] L Cao X Liu E J D Lin et al ldquoEnvironmental and geneticactivation of a brain-adipocyte BDNFleptin axis causes cancerremission and inhibitionrdquo Cell vol 142 no 1 pp 52ndash64 2010
[19] K D Nguyen Y Qiu X Cui et al ldquoAlternatively activatedmacrophages produce catecholamines to sustain adaptive ther-mogenesisrdquo Nature vol 480 no 7375 pp 104ndash108 2011
[20] S Hassan Y Karpova D Baiz et al ldquoBehavioral stress accel-erates prostate cancer development in micerdquo Journal of ClinicalInvestigation vol 123 no 2 pp 874ndash886 2013
8 Mediators of Inflammation
Murakami) Osaka Cancer Research Foundation (MasaakiMurakami) Mochida Memorial Foundation (YasunobuArima) Japan Intractable Diseases Research Foundation(Yasunobu Arima) and the Osaka Foundation for the Pro-motion of Clinical Immunology (Masaaki Murakami)
References
[1] N J Abbott L Ronnback and E Hansson ldquoAstrocyte-endo-thelial interactions at the blood-brain barrierrdquo Nature ReviewsNeuroscience vol 7 no 1 pp 41ndash53 2006
[2] E Steed M S Balda and K Matter ldquoDynamics and functionsof tight junctionsrdquo Trends in Cell Biology vol 20 no 3 pp 142ndash149 2010
[3] B V Zlokovic ldquoThe blood-brain barrier in health and chronicneurodegenerative disordersrdquoNeuron vol 57 no 2 pp 178ndash2012008
[4] J Bennett J Basivireddy A Kollar et al ldquoBlood-brain barrierdisruption and enhanced vascular permeability in the multiplesclerosismodel EAErdquo Journal of Neuroimmunology vol 229 no1-2 pp 180ndash191 2010
[5] L Steinman ldquoInflammatory cytokines at the summits of patho-logical signal cascades in brain diseasesrdquo Science Signaling vol6 no 258 p pe3 2013
[6] JHuppert D CloshenA Croxford et al ldquoCellularmechanismsof IL-17-induced blood-brain barrier disruptionrdquo FASEB Jour-nal vol 24 no 4 pp 1023ndash1034 2010
[7] H Kebir K Kreymborg I Ifergan et al ldquoHuman TH17 lym-phocytes promote blood-brain barrier disruption and centralnervous system inflammationrdquo Nature Medicine vol 13 no 10pp 1173ndash1175 2007
[8] Y Komiyama S Nakae T Matsuki et al ldquoIL-17 plays an impor-tant role in the development of experimental autoimmuneencephalomyelitisrdquo Journal of Immunology vol 177 no 1 pp566ndash573 2006
[9] Y Arima M Harada D Kamimura et al ldquoRegional neuralactivation defines a gateway for autoreactive T cells to cross theblood-brain barrierrdquo Cell vol 148 no 3 pp 447ndash457 2012
[10] I Bartholomaus N Kawakami F Odoardi et al ldquoEffector Tcell interactions with meningeal vascular structures in nascentautoimmuneCNS lesionsrdquoNature vol 462 no 7269 pp 94ndash982009
[11] L Steinman ldquoA molecular trio in relapse and remission inmultiple sclerosisrdquoNature Reviews Immunology vol 9 no 6 pp440ndash447 2009
[12] A Reboldi C Coisne D Baumjohann et al ldquoC-C chemokinereceptor 6-regulated entry of TH-17 cells into the CNS throughthe choroid plexus is required for the initiation of EAErdquoNatureImmunology vol 10 no 5 pp 514ndash523 2009
[13] Y Ohira F Kawano J L Stevens X D Wang and A IshiharaldquoLoad-dependent regulation of neuromuscular systemrdquo Journalof Gravitational Physiology vol 11 no 2 pp P127ndashP128 2004
[14] L V Borovikova S Ivanova M Zhang et al ldquoVagus nervestimulation attenuates the systemic inflammatory response toendotoxinrdquo Nature vol 405 no 6785 pp 458ndash462 2000
[15] H Wang M Yu M Ochani et al ldquoNicotinic acetylcholinereceptor 1205727 subunit is an essential regulator of inflammationrdquoNature vol 421 no 6921 pp 384ndash388 2003
[16] M Rosas-Ballina P S Olofsson M Ochani et al ldquoAcety-lcholine-synthesizing T cells relay neural signals in a vagusnerve circuitrdquo Science vol 334 no 6052 pp 98ndash101 2011
[17] C Reardon G S Duncan A Brustle et al ldquoLymphocyte-derived ACh regulates local innate but not adaptive immunityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 4 pp 1410ndash1415 2013
[18] L Cao X Liu E J D Lin et al ldquoEnvironmental and geneticactivation of a brain-adipocyte BDNFleptin axis causes cancerremission and inhibitionrdquo Cell vol 142 no 1 pp 52ndash64 2010
[19] K D Nguyen Y Qiu X Cui et al ldquoAlternatively activatedmacrophages produce catecholamines to sustain adaptive ther-mogenesisrdquo Nature vol 480 no 7375 pp 104ndash108 2011
[20] S Hassan Y Karpova D Baiz et al ldquoBehavioral stress accel-erates prostate cancer development in micerdquo Journal of ClinicalInvestigation vol 123 no 2 pp 874ndash886 2013
![Impaired Fracture Healing after Hemorrhagic Shock · 2 MediatorsofInflammation insult[6,7],whereasothersobservedanegativeinfluenceof trauma-hemorrhageonfracturehealing[8].However,these](https://static.fdocuments.us/doc/165x107/5f2172b589ad1a749f3c64bf/impaired-fracture-healing-after-hemorrhagic-shock-2-mediatorsofinflammation-insult67whereasothersobservedanegativeinfluenceof.jpg)
