Gammaherpesvirus infection unveils exaggerated germinal center responses in an SR-BI-deficient host

Abstract

Lipid metabolism has emerged as an important regulator of acute viral infections by affecting viral replication and the host immune response. In contrast, the role of host lipid metabolism during chronic viral infection has been less explored. The current study aims to define the role of scavenger receptor class B type I (SR-BI), a primary physiologic receptor for high-density lipoprotein (HDL), during natural gammaherpesvirus infection. Gammaherpesviruses are highly prevalent cancer-associated pathogens that induce and manipulate germinal center responses to establish life-long infection of B cells. Importantly, gammaherpesvirus-infected germinal center B cells are thought to be the target of viral transformation. In this study, we found that global SR-BI deficiency led to increased gammaherpesvirus lytic gene expression in the lungs during the acute stage of infection. Chronic gammaherpesvirus infection of the SR-BI-deficient host was associated with exaggerated germinal center responses and increased differentiation of self-reactive B cells that persisted during the long-term stage of chronic infection. Interestingly, SR-BI-deficient germinal center B cells, although more numerous, failed to support efficient gammaherpesvirus infection. The exaggerated germinal center response was also observed following immunization of the SR-BI-deficient host, unveiling the novel role of SR-BI as a negative regulator of physiological and gammaherpesvirus-driven germinal center responses.

Importance: Lipid metabolism affects diverse acute viral infections. In contrast, less is known about the effect of lipid metabolism on chronic virus infection, including in the context of an intact host. Host lipid homeostasis is maintained via a combination of endogenous lipid synthesis that takes place in most cell types and cellular interaction with exogenous, circulating lipids. This study focuses on defining the interaction between SR-BI, a primary HDL receptor, and natural gammaherpesvirus infection. We found that SR-BI deficiency led to increased expression of lytic gammaherpesvirus genes during acute gammaherpesvirus replication in the lungs. Importantly, chronic gammaherpesvirus infection unveiled the physiological role of SR-BI as a negative regulator of the germinal center response, a B cell differentiation process that is critical for physiological B cell responses and that is manipulated by gammaherpesviruses to establish chronic infection.

Keywords: SR-BI; cholesterol; gammaherpesvirus; germinal center response.

Fig 1

Global SR-BI deficiency leads to elevated MHV68 lytic gene expression but does not affect IFN responses during acute infection. BL6 and SRB1^-/- mice were intranasally infected with 10,000 PFU of MHV68. Lungs were harvested at 7 days post-infection. (A) Lung homogenates were subjected to plaque assay to determine MHV68 lytic titers. (B–F) RNA was isolated from individual lungs at 7 days post-infection and subjected to qRT-PCR analysis for relative mRNA expression of (B) MHV68 orf50, (C) MHV68 orf6, (D) Mx1, (E) Ifih, and (F) Cxcl9. Relative gene expression was normalized to corresponding Gapdh expression levels and further normalized to the average of the BL6 group (set at one and marked by the dotted line). Each symbol represents an individual mouse. Here and throughout all figures, error bars represent the standard error of the mean (SEM). ***P < 0.001, ****P < 0.0001.

Fig 2

Global SR-BI deficiency leads to attenuated MHV68 reactivation in the peritoneal cavity without affecting peritoneal and splenic latent reservoirs and viral reactivation from the splenocytes. BL6 and SR-BI^-/- mice were infected as in Fig. 1 and analyzed at 16 days post-infection. Splenocytes (A, B) and peritoneal cells (C, D) were pooled from 3 to 5 animals/group in each study, and cell suspensions were subjected to limiting dilution assays to define the frequency of MHV68 DNA + cells (A, C) and frequency of ex vivo reactivation (B, D). Data were pooled from the indicated number (N) of independent studies. In the limiting dilution assays, the dotted line is drawn at 63.2%, and the x-coordinate of the intersection of this line with the sigmoid graph represents the inverse of the frequency of positive events.

Fig 3

Global SR-BI deficiency leads to exaggerated MHV68-driven germinal center response. BL6 and SR-BI^-/- mice were infected as in Fig. 1 and analyzed at 16 days post-infection. (A, B). Splenic B cells, defined as CD19⁺ (representative flow plots in panel A), were measured by flow cytometry, with proportion and absolute cell number per spleen shown. (C, D). Splenic germinal center B cells defined as CD19⁺GL7⁺CD95⁺ were measured (representative flow plot in panel C) with proportion and absolute cell number per spleen shown. (E, F). Splenic T follicular helper cells defined as CD3⁺CD4⁺PD-1⁺CXCR5⁺ were measured (representative flow plot in E) with proportion and absolute cell number shown. Each symbol represents an individual animal. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 4

Global SR-BI deficiency leads to increased germinal center response following sheep red blood cells immunization. BL6 and SR-BI^-/- mice were intraperitoneally injected with sheep red blood cells and analyzed 7–9 days post-immunization. Splenic germinal center B cells defined as CD19⁺GL7⁺CD95⁺ were measured (representative flow plot as in Fig. 3C) with proportion (A) and absolute cell number (B) shown. Each symbol represents an individual animal. *P < 0.05.

Fig 5

MHV68 infection of splenic germinal center B cells is attenuated in SR-BI^-/- mice. BL6 and SR-BI^-/- mice were intranasally infected with 10,000 PFU of MHV68.ORF73βla reporter virus, spleens harvested at 16 days post-infection, and pooled from three animals within each group within each independent experiment. Infected cells were defined as positive for cleaved CCF2 β-lactamase substrate (indicated as CCF2⁺). (A) Representative gating strategy for GL7⁺CD95⁺ germinal center B cells pregated on CD19⁺B220⁺ splenocytes and postgated on population positive for cleaved CCF2 substrate (CCF2⁺). (B) Percent of infected germinal center B cells. In panel B, each symbol represents analysis of splenocytes pooled from 3 to 5 mice/genotype in a single experiment, and the connecting lines represent paired observations within a single study. Results of 3 studies are shown. *P < 0.05.

Fig 6

Global SR-BI deficiency leads to a selective increase in MHV68-driven self-reactive, but not virus-specific antibodies at 16 days post-infection. BL6 and SR-BI^-/- mice were mock- or MHV68-infected as in Fig. 1, sera were collected at 16 days post-infection, and subjected to ELISA to measure levels of total IgG (A), total IgM (B), anti-MHV68 IgG (C), and anti-double stranded DNA IgG (D). Each symbol in panels A and B represents an individual animal. In panel C, sera were pooled from three mock-infected mice and 12-14 MHV68-infected mice for each group. In panel D, sera were pooled from 4 to 5 mock-infected mice and 13-15 MHV68-infected mice per group, with averages and standard error of measurement shown. *P < 0.05; **P < 0.01.

Fig 7

Global SR-BI deficiency does not affect levels of regulatory CD4 T cells but instead leads to increased numbers of MHV68-specific CD8 T cells. BL6 and SR-BI^-/- mice were infected as in Fig. 1 and analyzed at 16 days post-infection. (A, B) Splenic T regulatory cells, defined as CD3⁺CD4⁺FoxP3⁺ (representative flow plots in A), were measured by flow cytometry with proportion and absolute cell number shown in panel B. (C) Proportion and absolute numbers of splenic CD3⁺CD8⁺ T cells. (D, E) Splenic MHV68-specific CD8 T cells defined as CD3⁺CD8⁺CD44^hi, also staining positive with MHV68 orf6 MHC-I tetramer, were measured (representative flow plot in D) with proportion and absolute cell number shown in panel E. Each symbol represents an individual animal. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 8

ApoA-I global deficiency leads to attenuated germinal center response during chronic MHV68 infection. BL6, SR-BI^-/-, and ApoA-I^-/- mice were infected as in Fig. 1 and analyzed at 16 days post-infection. (A) Total serum cholesterol levels. (B, C) Germinal center B cells were identified and quantified as in Fig. 3 with proportion (B) and absolute numbers (C) shown. Each symbol represents an individual animal. (D, E) Frequency of MHV68 DNA + splenocytes and ex vivo reactivation was measured as in Fig. 2. The results were pooled from two independent studies, with splenocytes from three animals/group pooled in each study prior to limiting dilution analyses. *P < 0.05.

Fig 9

Exaggerated MHV68-driven B cell differentiation persists in long-term infected SR-BI^-/- mice. BL6 and SR-BI^-/- mice were infected as in Fig. 1 and analyzed at 35 days post-infection. (A, B) Germinal center B cells were measured as in Fig. 3. (C) Total serum IgG was measured as in Fig. 6. (A through C) Each symbol represents an individual animal. (D, E) Serum IgG titers against dsDNA and MHV68 were measured as in Fig. 6, data were pooled from three mock-infected mice/group and nine MHV68-infected mice/group for each antibody titer. (F, G) Frequency of MHV68 DNA + splenocytes (F) and peritoneal cells (G) was determined as in Fig. 2. Data were pooled from 2 to 3 independent studies.