• CSF E1 was trichotomized to create stratified hormone groups based off it’s distribution

• Demographic, clinical and outcome variables were assessed upon enrollment and at 6 months• Glasgow Outcome Score is the main outcome of

interest • Favorable outcome (GOS= 4, 5)• Unfavorable outcome (GOS=1, 2, 3).

• Age, sex, race, body mass index, best in 24 hours Glasgow Coma Scale (GCS), Injury Severity Score (ISS), non-head ISS, hospital length of stay, and mechanism of injury were also analyzed

• Statistical test, Mann-Whitney, Kruskal Wallis, Chi-square tests, covariate adjusted mediation modeling, and Baron Kinney mediation were conducted to analyze potential relationships • Based off inflammatory markers that were

significantly associated with CSF E1 and 6m. GOS, an inflammatory load score was created

• Significant markers, IL6, IL8, IL10, TNFa, sICAM1, and sFAS, were correlated together. Markers that were correlated with all others were included. 3 markers with the greatest correlations from each inflammatory type (chemokine, adhesion molecule, and soluble receptor) were incorporated int the model. The ILS consists of IL6, TNFa, and sICAM1

Department of Physical Medicine & Rehabilitation1, Safar Center for Resuscitation Research2, Clinical & Translational Science Institute3 University of Pittsburgh, Pittsburgh, PA

Christina Lavin1, Leah Vaughan1, Nabil Awan1, Amy Wagner1,2,3

Acute CSF Inflammatory Response post-TBI Mediates Relationship Between CSF Estrone Levels and Global Outcome

Introduction Results Conclusions

References

Acknowledgments

Traumatic Brain Injury (TBI)• After TBI, an acute secondary inflammatory cascade

induces protection and damage that affect neurorecovery• Regulatory crosstalk between cytokines mediate

inflammatory mechanisms needed to mount a neuro-reparative response and mitigate secondary damage following brain injury1

• Reactive astrocytes upregulate aromatase enzyme activity and, in turn, increase production of hormones in the brain.2

• CSF E1 levels influence neuroinflammation after TBI to negatively impact outcome.

• Within the first 3 days of injury, increased CSF E1 expression may be detrimental to neuro-repair and long-term recovery following TBI

• CSF E1 was associated with worse outcome, and higher acute inflammatory load appears to mediate the E1 relationship with poor outcome.

• IL-6 and TNFα increase E1 release alluding to a bi-directional relationship between inflammation and hormone regulation.10

• Understanding hormones and their relationship with neuroinflammation may allow for personalized medicine strategies guided by biomarker levels in order to maximize recovery potential.

• With further characterization of the temporal and interdependent neurodegenerative vs. neuroprotective roles of sex hormones and inflammation, pharmacological drugs can be tailored for administer to patients with TBI to aid in CNS recovery

1. Woodcock T, Morganti-Kossmann MC. The role of markers of inflammation in traumatic brain injury. Front Neurol. 2013;4:18.

2. Gatson JW, Simpkins JW, Yi KD, Idris AH, Minei JP, Wigginton JG. Aromatase Is Increased in Astrocytes in the Presence of Elevated Pressure. Endocrinology. 2011;152(1):207-213. doi:10.1210/en.2010-0724

3. Rakholia MV, Kumar RG, Oh B-M, et al. Systemic Estrone Production and Injury-Induced Sex Hormone Steroidogenesis after Severe Traumatic Brain Injury: A Prognostic Indicator of Traumatic Brain Injury-Related Mortality. J Neurotrauma. August 2018.

4. Toscano V, Sancesario G, Bianchi P, Cicardi C, Casilli D, Giacomini P. Cerebrospinal fluid estrone in pseudotumor cerebri: A change in cerebral steroid hormone metabolism? J Endocrinol Invest. 1991;14(2):81-86. doi:10.1007/BF03350271

5. Garringer JA, Niyonkuru C, McCullough EH, et al. Impact of aromatase genetic variation on hormone levels and global outcome after severe TBI. J Neurotrauma. 2013;30(16):1415-1425.

6. Azcoitia I, Yague JG, Garcia-Segura LM. Estradiol synthesis within the human brain. Neuroscience. 2011;191:139-147.

7. Santarsieri M, Kumar RG, Kochanek PM, Berga S, Wagner AK. Variable neuroendocrine-immune dysfunction in individuals with unfavorable outcome after severe traumatic brain injury. Brain Behav Immun. 2015;45:15-27. doi:10.1016/j.bbi.2014.09.003

8. Capellino S, Straub RH, Cutolo M. Aromatase and regulation of the estrogen-to-androgen ratio in synovial tissue inflammation: common pathway in both sexes. Annals of the New York Academy of Sciences. 2014;1317(1):24-31.

9. Martínez-Chacón G, Brown KA, Docanto MM, et al. IL-10 suppresses TNF-α-induced expression of human aromatase gene in mammary adipose tissue. The FASEB Journal. 2018;32(6):3361-3370.

10. Franczak A, Wojciechowicz B, Kotwica G. Novel aspects of cytokine action in porcine uterus--endometrial and myometrial production of estrone (E1) in the presence of interleukin 1beta (IL1beta), interleukin 6 (IL6) and tumor necrosis factor (TNFalpha)--in vitro study. Folia Biol (Krakow). 2013;61(3-4):253-261.

Methods

CDC R49 CCR323155-03; DODW81XWH-071-0701; NIDILRR 90DP0041

Figure 2: When CSF E1 levels are elevated, there is alsoan increased inflammatory state in the brain. All markerlevels (pg/mL) differed significantly (p<0.05) in low vshigh tertile group comparisons, similarly for middle vshigh groups (with the exception of TNFα and IL-8).

0

5

10

15

20

25

30

IL-5(x100)

TNFa (x10)

IL-8(x0.01)

sVCAM1(x10000)

sFAS(x0.01)

IL-10(x0.1)

sICAM1(x1000)

Low CSF E1 Tertile Middle CSF E1 Tertile High CSF E1 Tertile

IL-6(x0.01)

InflammatoryLoad Score

Unfavorable 6 mo.GOS Score

CSF E1 Day 0-3 Mean

Leg BBeta= 1.52084SE= 0.45611p= 0.0011

Leg A: TotalOR= 3.146CI= [1.301, 7.604]p= 0.0109

Leg A: DirectOR= 2.472CI= [1.022, 5.979]p= 0.0446

Leg COR= 1.204CI= [1.046, 1.386]p= 0.0097

Figure 1: Day 0-3 E1 Tertile memberships were assigned based on day 0-3 mean E1 levels for the following percentiles:

• Low: ≤25.27 pg/mL (0-25th)• Middle: 25.27-41.96 pg/mL (25th-75th)• High: >41.96 pg/mL (75th-100th)

5 15 25 35 45 55 65 750

10

20

30

40

50

Day 0-3 Mean Estrone Levels (pg/mL)

Per

cent

Distribution of Day 0-3 Mean E1 Levels

LowTertile

MiddleTertile

HighTertile

Estrone (E1)• Levels of cerebrospinalfluid (CSF) E1 acutely after

TBI has been associated with neuroprotective and neurodegenerative properties• Preclinical studies show CSF E1 to be associated

with neuroprotection2

• Clinical studies show serum E1 to be associated with neurodegeneration3, 4

• Steroidogenesis regulation within the CSF also exemplifies the complex dynamics of sex hormones after TBI5

• In a previous study, acute CSF E1 levels were associated with poor 6-month outcome and dysregulation of steroidogenesis

• In a healthy brain, E1 regulates neural development, synaptic plasticity, and cell survival6

Neuroinflammation• Discrepancies in the neuro-hormone response after

injury may show interactions with systemic pro- and anti-inflammatory cytokines 7• Proinflammatory cytokines increase aromatase

activity therefore regulating systemic steroidogenesis of hormones8

• With the upregulation of TNFα after TBI, it induces expression of the aromatase enzyme within steroidogenesis synthetic cells causing dysregulation from homeostasis9

Table 2: Cohort Demographics

Predictive Factor

OR [95% CI]or

β (SE)p-value Total Effect Direct Effect

Leg A Leg A: ILS-Adjusted

1.032 -0.00080975 (0.01555) 1.033 1.032

[1.003. 1.061] 0.9585 [1.005, 1.062] [1.002, 1.063]

0.0278 0.0201 0.0361

Gender: Male 0.322 -0.76822 (0.75533) 0.333 0.381

vs Female [0.071, 1.447] 0.311 [0.087, 1.283] [0.082, 1.772]

0.1393 0.11 0.21851.204 2.472

[1.046, 1.386] [1.022, 5.979]

0.0097 0.0446AUC or R2 0.732 0.0819 0.723 0.776

Unfavorable 6-month GOS Score

Leg B Leg C

Age

ILS - -

Table 2: Baron Kinney Mediation Analysis

Figure 3: Baron Kenny mediation model; ILS is a partialmediator between day 0-3 mean CSF E1 levels and anunfavorable 6 mo. GOS

Study AimOur study goal was to profile the relationship between CSF E1 and inflammatory cytokines after TBI in order

to understand how CNS hormone physiology influences neuroinflammation after secondary TBI

Study HypothesisWe hypothesize that after TBI, acute CSF E1 shows neurodegeneration due to its interactions with CSF

inflammation to predict global outcome.

Methods• Cohort Size: n=146 adults with severe TBI

• n=135 adults with at least…• One CSF E1 measurement over days 0 to 3• Inflammatory cytokine levels over days 0 to 3

• Inflammatory marker levels were collected using 3 Millipore Luminex Multiplex bead panels: High Sensitivity T cell, Neurodegenerative Markers, and Soluble Receptors

CSF E1 Tertile, ILS, and 6m. GOS Mediation

• Explore the biological mechanism between hormonal and inflammatory regulation and synthesis within the brain to further understand how these biomarkers interaction to attenuate or facilitate in their neurorecovery properties

• Study this relationship systemic hormones and inflammation to learn how these two compartments, (CSF and serum), interact and are regulated after TBI

• Understand how this acute E1 and inflammatory response may predict or be associated with chronic biomarker levels and outcomes

• Explore how the steroidogenic enzyme, aromatase, can be regulated by or regulates inflammatory synthesis and activity

Future Directions

Inflammatory Marker Correlations (Inflammation à GOS)IL6 IL8 IL10 TNFa sICAM1 sFAS

IL6 1 - - - - -- - - - - -

IL8 0.6056 1 - - - -<.0001 - - - - -

IL10 0.5707 0.4479 1 - - -<.0001 <.0001 - - - -

TNFa 0.0445 0.0987 0.01554 1 - -0.6753 0.3521 0.8838 - - -

sICAM1 0.7163 0.4429 0.62661 0.0935 1 -<.0001 <.0001 <.0001 0.378 - -

sFAS 0.5216 0.2693 0.48741 0.08963 0.79007 1<.0001 0.0099 <.0001 0.3982 <.0001 -

Table 1: Significant Inflammatory Marker Correlations to Determine the ILS

Inflammatory Cytokines Day 0-3 Mean by CSF E1 Tertile Groups