P16. CD200 and CD200R axis controls sex-specific TLR7 Responses to MHV Infection.

Guruswamy Karnam1, Tomasz P. Rygiel2, Matthijs Raaben4, Guy C. M. Grinwis5, Frank E. Coenjaerts6, Maaike E. Ressing6, Peter J. M. Rottier4, Cornelis A. M. de Haan4, Linde Meyaard3.

Affiliates: 1Department of clinical microbiology, Karolinska Institute, Huddinge, Sweden. 2Department of Immunology, Center of Biostructure Research, Medical University of Warsaw, Warsaw, Poland. 3Laboratory for Translational Immunology, Department of Immunology, University Medical Center Utrecht, Utrecht, The Netherlands; 4Virology division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands, 5Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands, 6Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands

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Background
Immunological checkpoints, such as the inhibitory CD200 receptor (CD200R), play a dual role in balancing the immune system during microbial infection. On the one hand these inhibitory signals prevent excessive immune mediated pathology but on the other hand they may impair clearance of the pathogen. We studied the influence of the inhibitory CD200-CD200R axis on clearance and pathology in mouse hepatitis virus (MHV) infection model. We here report that lack of CD200R signaling has a more profound effect on the beneficial but also on the pathological immune responses to viruses in female mice as compared to male mice, which can be attributed to the capacity of CD200R to inhibit TLR7 responses.

Methods
Wild-type C57BL/6J mice and Cd200−/− mice, which were made and maintained on a full C57BL/6J background, were bred at the Specified Pathogen Free (SPF) unit at the Utrecht University Central animal Laboratory and used between 8 and 10 weeks of age. Mice were injected intraperitoneally with 106 TCID50 of MHV strain A59 expressing the firefly luciferase (FL) reporter gene (MHV-EFLM) [14] in 200 µl PBS. After MHV-EFLM infection (day 0), to measure viral replication mice were imaged at day 2 and day 4 by Bioluminescence imaging. Whole livers were dissected from the mice. The tissues were processed in Lysing Matrix D tubes for RNA isolation. Gene expression levels of IFN-α, IFN-β1, and TLR7 were measured by quantitative PCR using Light Cycler 480 RNA. To measure the interferon concentration in the sera, blood was sampled from naïve mice IFN-α was measured with a mouse interferon alpha ELISA kit (PBL Interferon Source) Liver histopathology of MHV-infected mice were sampled, fixed in 4% neutral buffered formalin, and embedded in paraffin. Total liver sections were examined by light microscopy and foci of hepatocellular necrosis and inflammation were scored in a semi-quantitative manner. Significance was calculated with Mann-Whitney test using GraphPad Prism software.

Results
CD200-deficiency and sex determine the outcome of MHV infection interestingly at both time points we observed a decreased viral spread in female WT mice when compared to males. Moreover, lack of CD200 resulted in a significantly lower level of viral replication in females. WT female mice had significantly lower viral RNA levels than WT male mice. Again, CD200-deficiency greatly decreased virus load in female mice. The number of focal lesions in the liver, typical for MHV was also lower in female mice and again CD200-deficiency had a significant effect on these lesions only in female mice. IFN-α concentration in serum inversely correlated with viral load at day 4. Thus, the combination of female sex and CD200 deficiency results in increased type I IFN production and decreased viral load and pathology upon MHV infection. Finally we found that CD200R-mediated signaling directly inhibits signals transduced via TLR7.

Conclusions
We demonstrate that this particular CD200 and CD200R checkpoint inhibits anti-viral responses that are naturally stronger in females. Release of this checkpoint enlarges these sex differences. Our findings have major implications for therapeutic use of blockers of this pathway, which are currently in clinical trials for the treatment of cancer, as we predict that female patients will have a stronger response to such therapeutics.