Diet-induced obesity impairs induction of Experimental Autoimmune Encephalomyelitis and is restored by PD-1 blockade Catherine T. Le1,2, Lam Khuat1,2, Cordelia Dunai1,2, Athena Soulika,2 William J. Murphy2,3 1Graduate Group in Immunology, 1,2Department of Dermatology, 3Department of Internal Medicine, Division of Hematology/Oncology, University of California, Davis Abstract Results A Obesity has marked effects on immune function and is associated with increased infection susceptibility and decreased adaptive immune response. We examined the effects of obesity on adaptive autoimmune responses using a diet-induced obesity mouse model. The pathogenesis of experimental autoimmune encephalomyelitis (EAE), a model of autoimmune demyelinating disease of CNS, is mediated by autoreactive Th1 and Th17 CD4 T cell subsets. 8-9-month-old C57BL/6 male mice were fed 60% fat or 10% fat control diet starting at 6-8 weeks old and were immunized with 300 μg of rodent myelin oligodendrocyte glycoprotein (MOG)35-55 peptide in complete Freund’s adjuvant (CFA) containing 5 mg/ml heat-killed M. tuberculosis. Control diet mice exhibited onset of severe clinical symptoms of hind limb paralysis similarly to young wild-type controls, while diet-induced obese mice experienced a significantly delayed onset. Clinical scores correlated with greater number of MOG-specific tetramer-positive CD4 T cells and number of IFN-gamma and IL-17a-expressing CD4 T cells in the CNS of control mice compared to obese mice. Decreased functional cytokine expression and proliferation called into question the exhaustive nature of T cells in obese mice through the upregulation of the PD-1/PD-L1 pathway, a known regulator of EAE. Increased PD-L1-positive CD11c+ DCs were observed in spleen of obese mice compared to control diet-fed mice. We treated EAE-induced mice on control and high-fat diet with anti-PD-1 blockade (29F.1A12) and restored EAE induction in both clinical score and number of pathologic CD4 T cells in the CNS of obese mice. Our findings may indicate an impairment of the adaptive immune system through the PD-1/PD-L1 pathway in obesity, possibly indicating an augmentation of the checkpoint pathway in settings of systemic inflammation.   A B CNS SSC-A hCLIP Tetramer High-fat diet Control diet B MOG38-49 Tetramer Figure 4: Anti-PD-1 blockade restored EAE induction in obese mice. Male C57BL/6 mice fed 60% high-fat diet or 10% control diet for 6 months were immunized for EAE and treated with anti-PD-1 blockade (29F.1A12) or rat IgG on day 0 (500 µg) and every other day until day 10 (250 µg/day). A) EAE clinical score and treatment schema. B) Percentage of mice with score equal to or greater than 3 post-EAE induction. Averages are shown for n=13/group. C CNS D CNS A E CNS Control diet + EAE + rat IgG Background Figure 2: Diet-induced obese mice had delayed and impaired induction of EAE and significantly fewer pathologic CD4 T cells in the CNS on day 18-post induction. Male C57BL/6 mice fed 60% high-fat diet or 10% control diet for 6 months with EAE had decreased number of pathologic T cells on day 18 post-EAE induction. A) Total number of infiltrating MOG38-49 Tetramer+ CD4 T cells in CNS. EAE clinical score. Representative flow gating of MOG38-49 tetramer and hCLIP tetramer staining. C-D) Total number with or without phorbol myristate acetate (PMA) and ionomycin stimulation of C) IFN-g+ and D) IL-17a+ CD4 T cells in CNS. Averages ± SEM are shown for n=6 /group representative of 3-4 experiments. Statistics were generated using Prism software with two-way ANOVA (A) and unpaired two-tailed Student’s t-test (B-D). *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001. Increased adiposity and accumulation of visceral fat results in chronic, low-level inflammation that induces or exacerbates several chronic conditions (Kanneganti et al., Nat Immunol, 2012). Obesity induces increase in CD4+ T cell senescence phenotype with compromised proliferation, reduced IFN-γ production, and upregulation of programmed cell death 1 (PD-1) in visceral fat (Shirakawa et al., JCI, 2016) EAE is negatively regulated by the PD-1/PD-L1/PD-L2 pathway (Salama et al., JEM, 2003, Zhu et al., J Immunol, 2006, Hu et al., Neurosci Bull, 2016, Schreiner et al., Eur J Immunol, 2008). High-fat diet for 10 weeks has been observed to accelerate EAE (Winer et al., Eur J Immunol, 2009, Endo et al., Cell Rep, 2015) It is not yet understood how obesity and aging interact to effect T cell exhaustion or priming through PD-1/PD-L1 parameters. High-fat diet + EAE + rat IgG High-fat diet + EAE + anti-PD-1 B High-fat diet + EAE + rat IgG A Control diet SSC-A High-fat diet B CNS PD-1 Spleen C D MHC II PD-L1 SSC-A SSC-A IFN-g High-fat diet + EAE + anti-PD-1 C D CNS CNS Ki67 Results Figure 5: Anti-PD-1 blockade increased the number of pathologic CD4 T cells infiltrating in the CNS of obese mice on day 16 post-EAE induction. Male C57BL/6 mice fed 60% high-fat diet or 10% control diet for 6 months were immunized for EAE and treated with anti-PD-1 blockade (29F.1A12) or rat IgG. A) Total MOG38-49 Tetramer+ CD4 T cells in CNS. B) Representative flow plots of IFN-g on CD4 T cells in CNS. C) Total number IFN-g+ CD4 T cells in CNS. D) IFN-g MFI of IFN-g+ CD4 T cells in CNS. E) Representative flow plot of Ki67+ gating on CD4 T cells in CNS. Averages ±SEM are shown for n=5-8/group representative of 2 experiments. Statistics were generated using Prism software with one-way ANOVA with Dunn’s (A) and unpaired two-tailed Student’s t-test (C-D). *=P<0.05, **=P<0.01. A B Conclusions Diet-induced obesity delays induction of Experimental Autoimmune Encephalomyelitis (EAE) compared to control diet-fed mice. The delay correlates with decreased number of pathologic T cells in the CNS. Obese mice have PD-L1 upregulation on DCs expressing low levels of MHC II and decreased activation of DC in secondary lymphoid tissue. PD-1 blockade restores induction of EAE in diet-induced obese mice. This restoration is correlated with increased number of MOG-specific, cytokine-producing, and Ki67+ CD4 T cells in the CNS in anti-PD-1-treated obese mice. Results indicate an upregulation of PD-1/PD-L1 pathway in obesity with negative impact on T cell priming in secondary lymphoid tissue. D C Score Symptoms 0.00 No detectable signs 0.50 Distal limp tail or light waddling 1.00 Limp tail or waddling gait 1.50 Limp tail and waddling gait 2.00 Single limb paresis 3.00 Double limb paresis 3.25 Severe double limb paresis 3.50 Single limb paralysis and paresis of second limb 4.00 Full paralysis of 2 limbs 4.50 Moribund 5.00 Death 10% HFD 60% HFD 6-8 weeks old 8 months old 300 μg MOG35-55 peptide in CFA (Day 0) 200 ng of pertussis toxin (Day 0, 2) Figure 3: PD-L1 upregulation on dendritic cells was observed in spleen of obese mice, while PD-1 was not upregulated on pathologic T cells on day 16 post-EAE induction. C57BL/6 male mice were fed ad libitum 60% high-fat diet or 10% control diet for 6 months and were induced with EAE to examine PD-1 levels on T cells on day 16-post induction. A) Representative flow plot of PD-1+ gating on CD4 T cells in CNS. PD-1+ percentage of MOG38-49-specific CD4 T cells. B) PD-1 median fluorescence intensity (MFI) on PD-1+ MOG38-49-specific CD4 T cells. C) Representative flow plot of MHC II low and MHC II high gating on CD11c+ DCs. Frequency of MHC II low and high CD11c+ DC in spleen. D) Representative flow plot of PD-L1+ gating on MHC II low CD11c+ DCs. Frequency of PD-L1 on MHC II low and high CD11c+ DC in spleen. Averages ±SEM are shown for n=5/group representative of 3 experiments. Statistics were generated using Prism software with unpaired two-tailed Student’s t-test (A-D). *=P<0.05, **=P<0.01. Figure 1: Mice placed on 60% high-fat diet gain significantly greater adiposity and weight compared to mice placed on 10% high-fat control diet. A) Extensive visceral and subcutaneous fat deposition in ad-lib fed 10% high-fat diet vs 60% high-fat diet e via MRI. B) Comparison of weight (g) of male C57BL/6 mice included in this study for 6 months of diet. C) Schema of experimental design. Male C57BL/6 mice were placed on diet for 6 months then immunized with 300 µg of MOG35-55 peptide in CFA on Day 0 and 200 ng of Pertussis toxin on Day 0 and 2. D) EAE clinical score. Statistics were generated using Prism software with unpaired two-tailed Student’s t-test (B) to compare groups. **** = P≤0.0001. Acknowledgements This work has been supported by the UC Davis CTSC TL1 TR001861, NIH RO1 CA214048, and NIH RO1 CA095572.