Using in vivo bio-luminescence imaging and FACS based ...
1
Using in vivo bio-luminescence imaging and FACS based approaches to understand the role of Nfkbid in immunity to Toxoplasma gondii Alex Ahilon-Jeronimo, Scott Souza, Kirk Jensen Introduction Objective Methods/Rationale Results Conclusions Acknowledgments University of California, Merced References CDC - DPDx - Toxoplasmosis. (2017, December 18). Retrieved July 20, 2020, from https://www.cdc.gov/dpdx/toxoplasmosis/index.html Toxoplasma gondii (T-gondii) is one of the most common forms of intracellular parasites in the world, infecting nearly one-third of all humans. The single-celled parasite is the causative pathogen for toxoplasmosis, an orally acquired infection that affects the brain, heart, and skeletal muscle of a host. Toxoplasmosis represents one of many neglected parasitic diseases that are not only a major public health issue but also lack an effective vaccine. 0 10 20 30 0 20 40 60 80 100 Days Percent survival Bumble C57BL/6J Type I RH secondary infection ***P<0.0008 To investigate the effect of Nfkbid in immunity to Toxoplasma gondii Figure 1. Life cycle of Toxoplasma-gondii Figure 2. Survival curves of bumble (nfkbid-/-) and WT (B6) mice against primary (CEP) and secondary (RH) A. Shows that Nfkbid knockout mice survive vaccination at the same rate as the parental wildtype strain (B6). B. mice without Nfkbid are unable to survive high virulence strains of T. gondii. • Ongoing research has been focused on understanding host-pathogen interactions of T-gondii as it relates to the susceptibility of a host to parasitic infections and how the host develops immunity • Host gene Nfkbid was required for secondary immunity against virulent strains of T-gondii • Mice without Nfkbid (bumble) had a lower survival rate and failed to make IgM and little IgG which are key antibodies that recognize and bind to T-gondii • Utilizing In vivo bioluminescence imaging (BLi), mice will be infected with a luciferase-expressing parasite along with Luciferin as the bioluminescent substrate. • Emitted photons per second, photon flux, will be detected by a single photon detection camera. The photon flux will correlate with parasite burden • Mouse pictures will be overlaid with collected photon emission images to demonstrate the concentration of the parasite within each mice. The process will be repeated every 3-4 days for a total of 30 days. • Mice with a single gene altered, B10.A, showed less photon flux compared to B10 mice, which is correlated with a lower parasite burden. This is a proof of concept that changing one gene can dramatically alter a host’s response to T. gondii. The expectation is that there will be some difference between the immune response between B6 and Bumble mice. Our FACS results demonstrate that mice with reduced Nfkbid do produce different amounts and types of antibody's isotypes against the parasite. But, The FACS results do disprove my initial hypothesis that bumble het would have a less relative amount of antibodies than B6 mice. Nevertheless, These results suggest that Nfkbid is controlling the parasite specific-antibody isotypes produced by a host immune response. We expect bumble mice to have greater parasite burden than B6 mice. This conclusion will be supported by In vivo bioluminescence imaging that should display greater photon flux from bumble mice. D1 D4 D8 B10 B10.A • Flow cytometry is the process measuring and analyzing multiple characteristics of single cells or particles as they pass through a beam of light in liquid suspension. • Fluorescence activated cell sorting (FACS) data were analyzed from three different mouse strains; AJ, B6, and heterozygous Bumble. The samples consisted of parasites coated with serum collected from infected mice with T-gondii. The samples were analyzed for the presence of parasite-specific antibodies, detected by antibodies specific to individual mouse isotypes. Color Sample AJ (A) B6 (B) Heterozygous Bumble (H) Naïve (BN) Fluorophores The results of the FACS data analysis demonstrate a difference between the immune response of mice with deficient (heterozygous bumble) and normal Nfkbid (WT) expression. In the experiment each fluorophore is associated with a specific antibody isotope; IgM-PECy7, IgG1-APC, IgG2b-PE, IgG2a-PerCPCy5.5, and IgG3-BV421. B6 samples had relatively less PE, BV421, APC, and PerCP-Cy5-5 fluorescence than heterozygous bumble. The negative control sample, BN, illustrates that this is not a result of any false positive signaling. The results show not only that mice with lower Nfkbid possess less parasite-specific antibody but produce different amounts of specific-antibody isotypes. In this case, B6 mice produced less IgG2b, Igg3, IgM, and IgG3. I’d like to thank my mentors, Kirk Jensen and Scott Souza for their help and support over the summer. I would also like to thank all the members of UROC and SURI for the opportunity of performing research at UC Merced. Type III CEP primary infection Bumble (Nfkbid -/-) C57BL/6J (Nfkbid +/+) Percent survival 0 10 20 30 0 20 40 60 80 100 Days Figure 4. A) In vivo bioluminescence workflow B) Example luciferase assay of B10 and B10.A mice at 1, 4 and 8 day time points. Figure 3. Gating strategy of serum coated GFP+ parasites Figure 5. A. Associated antibody isotype by histogram for each dilution (IgG1 is APC, IgG2a is PerCPCy5.5, IgG2b is PE, IgG3 is BV421, and IgM is PECy7) for AJ, B6, Heterozygous bumble (H) and negative control (BN2) B. Cumulative parasite-specific antibody MFI In vivo bioluminescence imaging I hypothesize that bumble (Nfkbid knockout) will greater parasite burden than WT B6 mice. Antibody Reactivity I hypothesize that mice with lower Nfkbid will possess less-parasite specific antibodies than mice with normal Nfkbid expression. Isolating Parasite- GFP+ events Finding parasite by size Removing doublets Infect mouse with bioluminescent parasite Inject bioluminescent substrate into mouse Measure photon flux A B Background IgG2b IgM IgG3 IgG2a IgG1 A 1 10 100 1000 10000 100000 Average No Serum Average B6 Average AJ Average Het Comp-APC-A 1 10 100 1000 10000 100000 1000000 Average No Serum Average B6 Average AJ Average Het Comp-BV421-A 1 10 100 1000 10000 100000 Average No Serum Average B6 Average AJ Average Het Comp-PE-A 1 10 100 1000 10000 Average No Serum Average B6 Average AJ Average Het Comp-PE-Cy7-A 1 10 100 1000 10000 100000 Average No Serum Average B6 Average AJ Average Het Comp-PerCP-Cy5-5-A B
Transcript of Using in vivo bio-luminescence imaging and FACS based ...
Using in vivo bio-luminescence imaging and FACS based approaches to understand the role of Nfkbid in immunity to
Toxoplasma gondiiAlex Ahilon-Jeronimo, Scott Souza, Kirk Jensen
Introduction
Objective
Methods/Rationale Results
Conclusions
Acknowledgments
University of California, Merced
ReferencesCDC - DPDx - Toxoplasmosis. (2017, December 18). Retrieved July 20, 2020, from https://www.cdc.gov/dpdx/toxoplasmosis/index.html
Toxoplasma gondii (T-gondii) is one of the most common forms of intracellularparasites in the world, infecting nearly one-third of all humans. The single-celledparasite is the causative pathogen for toxoplasmosis, an orally acquired infection thataffects the brain, heart, and skeletal muscle of a host. Toxoplasmosis represents oneof many neglected parasitic diseases that are not only a major public health issue butalso lack an effective vaccine.
C
0 10 20 300
20
40
60
80
100
Days
Perc
ent s
urvi
val
BumbleC57BL/6J
Type I RH secondary infection
***P<0.0008
DType I RH burden (GFP+) on Day 7
C57BL/6J Bumble0.00
0.02
0.04
0.06
0.08
% G
FP+
of a
ll ev
ents
(PI-)
**P<0.002
BA
Days
Type I GT1 secondary infectionType III CEP primary infection
Bumble (Nfkbid -/-)C57BL/6J (Nfkbid +/+)
Perc
ent s
urvi
val
Perc
ent s
urvi
val
0 10 20 300
20
40
60
80
100
Days0 5 10 15 20
0
20
40
60
80
100
*P<0.02
BumbleC57BL/6J
To investigate the effect of Nfkbid in immunity to Toxoplasma gondii
Figure 1. Life cycle of Toxoplasma-gondii
Figure 2. Survival curves of bumble (nfkbid-/-) and WT (B6) mice against primary (CEP) and secondary (RH) A. Shows that Nfkbid knockout mice survive vaccination at the same rate as the parental wildtype strain (B6). B. mice without Nfkbid are unable to survive high virulence strains of T. gondii.
• Ongoing research has been focused on understanding host-pathogen interactions of T-gondii as it relates to the susceptibility of a host to parasitic infections and how the host develops immunity
• Host gene Nfkbid was required for secondary immunity against virulent strains of T-gondii
• Mice without Nfkbid (bumble) had a lower survival rate and failed to make IgM and little IgG which are key antibodies that recognize and bind to T-gondii
• Utilizing In vivo bioluminescence imaging (BLi), mice will be infected with a luciferase-expressing parasite along with Luciferin as the bioluminescent substrate.
• Emitted photons per second, photon flux, will be detected by a single photon detection camera. The photon flux will correlate with parasite burden
• Mouse pictures will be overlaid with collected photon emission images to demonstrate the concentration of the parasite within each mice. The process will be repeated every 3-4 days for a total of 30 days.
• Mice with a single gene altered, B10.A, showed less photon flux compared to B10 mice, which is correlated with a lower parasite burden. This is a proof of concept that changing one gene can dramatically alter a host’s response to T. gondii.
The expectation is that there will be some difference between the immune responsebetween B6 and Bumble mice. Our FACS results demonstrate that mice with reducedNfkbid do produce different amounts and types of antibody's isotypes against theparasite. But, The FACS results do disprove my initial hypothesis that bumble hetwould have a less relative amount of antibodies than B6 mice. Nevertheless, Theseresults suggest that Nfkbid is controlling the parasite specific-antibody isotypesproduced by a host immune response. We expect bumble mice to have greaterparasite burden than B6 mice. This conclusion will be supported by In vivobioluminescence imaging that should display greater photon flux from bumble mice.
D1 D4 D8
B10
B10.A
• Flow cytometry is the process measuring and analyzing multiple characteristics of single cells or particles as they pass through a beam of light in liquid suspension.
• Fluorescence activated cell sorting (FACS) data were analyzed from three different mouse strains; AJ, B6, and heterozygous Bumble. The samples consisted of parasites coated with serum collected from infected mice with T-gondii. The samples were analyzed for the presence of parasite-specific antibodies, detected by antibodies specific to individual mouse isotypes.
Color Sample
AJ (A)
B6 (B)
Heterozygous Bumble (H)
Naïve (BN)
Fluorophores
The results of the FACS data analysis demonstrate a difference between the immuneresponse of mice with deficient (heterozygous bumble) and normal Nfkbid (WT)expression. In the experiment each fluorophore is associated with a specific antibodyisotope; IgM-PECy7, IgG1-APC, IgG2b-PE, IgG2a-PerCPCy5.5, and IgG3-BV421. B6samples had relatively less PE, BV421, APC, and PerCP-Cy5-5 fluorescence thanheterozygous bumble. The negative control sample, BN, illustrates that this is not aresult of any false positive signaling. The results show not only that mice with lowerNfkbid possess less parasite-specific antibody but produce different amounts ofspecific-antibody isotypes. In this case, B6 mice produced less IgG2b, Igg3, IgM, andIgG3.
I’d like to thank my mentors, Kirk Jensen and Scott Souza for their help andsupport over the summer. I would also like to thank all the members of UROCand SURI for the opportunity of performing research at UC Merced.
C
0 10 20 300
20
40
60
80
100
Days
Perc
ent s
urvi
val
BumbleC57BL/6J
Type I RH secondary infection
***P<0.0008
DType I RH burden (GFP+) on Day 7
C57BL/6J Bumble0.00
0.02
0.04
0.06
0.08
% G
FP+
of a
ll ev
ents
(PI-)
**P<0.002
BA
Days
Type I GT1 secondary infectionType III CEP primary infection
Bumble (Nfkbid -/-)C57BL/6J (Nfkbid +/+)
Perc
ent s
urvi
val
Perc
ent s
urvi
val
0 10 20 300
20
40
60
80
100
Days0 5 10 15 20
0
20
40
60
80
100
*P<0.02
BumbleC57BL/6J
Figure 4. A) In vivo bioluminescence workflow B) Example luciferase assay of B10 and B10.A mice at 1, 4 and 8 day time points.
Figure 3. Gating strategy of serum coated GFP+ parasites
Figure 5. A. Associated antibody isotype by histogram for each dilution (IgG1 is APC, IgG2a is PerCPCy5.5, IgG2b is PE, IgG3 is BV421, and IgM is PECy7) for AJ, B6, Heterozygous bumble (H) and negative control (BN2) B. Cumulative parasite-specific antibody MFI
In vivo bioluminescence imagingI hypothesize that bumble (Nfkbid knockout) will greater parasite burden than WT
B6 mice.
Antibody ReactivityI hypothesize that mice with lower Nfkbid will possess less-parasite specific
antibodies than mice with normal Nfkbid expression.
Isolating Parasite-GFP+ events
Finding parasite by size
Removingdoublets
Infect mouse with bioluminescent parasite
Inject bioluminescent substrate into mouse Measure photon flux
A B
Background
IgG2b IgM IgG3 IgG2a IgG1A
1
10
100
1000
10000
100000
AverageNo
Serum
AverageB6
AverageAJ
AverageHet
Comp-APC-A
1
10
100
1000
10000
100000
1000000
AverageNo
Serum
AverageB6
AverageAJ
AverageHet
Comp-BV421-A
1
10
100
1000
10000
100000
AverageNo
Serum
AverageB6
AverageAJ
AverageHet
Comp-PE-A
1
10
100
1000
10000
AverageNo
Serum
AverageB6
AverageAJ
AverageHet
Comp-PE-Cy7-A
1
10
100
1000
10000
100000
AverageNo
Serum
AverageB6
AverageAJ
AverageHet
Comp-PerCP-Cy5-5-AB
