Gammaherpesvirus Co-infection with Malaria Suppresses Anti-parasitic Humoral Immunity

<div><p>Immunity to non-cerebral severe malaria is estimated to occur within 1-2 infections in areas of endemic transmission for <i>Plasmodium falciparum</i>. Yet, nearly 20% of infected children die annually as a result of severe malaria. Multiple risk factors are postulated to exacerbate malarial disease, one being co-infections with other pathogens. Children living in Sub-Saharan Africa are seropositive for Epstein Barr Virus (EBV) by the age of 6 months. This timing overlaps with the waning of protective maternal antibodies and susceptibility to primary <i>Plasmodium</i> infection. However, the impact of acute EBV infection on the generation of anti-malarial immunity is unknown. Using well established mouse models of infection, we show here that acute, but not latent murine gammaherpesvirus 68 (MHV68) infection suppresses the anti-malarial humoral response to a secondary malaria infection. Importantly, this resulted in the transformation of a non-lethal <i>P</i>. <i>yoelii</i> XNL infection into a lethal one; an outcome that is correlated with a defect in the maintenance of germinal center B cells and T follicular helper (Tfh) cells in the spleen. Furthermore, we have identified the MHV68 M2 protein as an important virus encoded protein that can: (i) suppress anti-MHV68 humoral responses during acute MHV68 infection; and (ii) plays a critical role in the observed suppression of anti-malarial humoral responses in the setting of co-infection. Notably, co-infection with an M2-null mutant MHV68 eliminates lethality of <i>P</i>. <i>yoelii</i> XNL. Collectively, our data demonstrates that an acute gammaherpesvirus infection can negatively impact the development of an anti-malarial immune response. This suggests that acute infection with EBV should be investigated as a risk factor for non-cerebral severe malaria in young children living in areas endemic for <i>Plasmodium</i> transmission.</p></div

M1 expression is associated with lethality in MHV68 infected IFNγR-/- C57Bl/6 mice.

<p>8–12 week old IFNγR-/- C57Bl/6 mice were intranasally infected with 1x10⁵ pfu MHV68 (WT or M1st) or were mock infected. (A) Mice were observed for weight change from starting weight, a loss of 20 percent or greater resulted in sacrifice. (B) Kaplan-Meier curves indicating mouse survival are shown. Significance was determined using log rank test with GraphPad software. <i>P</i> = 0.0027 for WT vs M1st infection.</p

Development of M1 dependent fibrosis in IFNγR-/- C57Bl/6 mice correlates with timing and kinetics of Vβ4⁺ CD8⁺ T cell expansion.

<p>8–12 week old IFNγR-/- C57Bl/6 mice were intranasally infected with 1x10⁵ pfu MHV68 (WT or M1st) or were mock infected and sacrificed at indicated times (n = 1–13 mice/group from one or two independent experiments). Right and accessory lobes were harvested and assessed for hydroxyproline content at days 4, 9, 28, and 90 post-infection. Statistics were performed using Mann-Whitney 2 tailed test to compare WT and M1st (* <i>P</i> = 0.0159, *** <i>P</i> = 0.0008).</p

M1 induced fibrosis in IFNγR-/- C57Bl/6 mice is associated with heightened levels of inflammation in lung tissue.

<p>8–12 week old WT or IFNγR-/- C57Bl/6 mice were intranasally infected with 1000 pfu MHV68 (WT or M1st) or were mock infected and sacrificed at 28 days post infection. Histological analysis was performed on lung tissues stained with hemotoxylin and eosin (H&E) or Masson’s Trichrome (MT). (A) Representative H&E stained sections are shown, scale bar = 100μm. Scores were determined for (A) pathology (mean and std. error are shown) from H&E sections and (B) fibrosis scores from MT sections, correlation was assessed using a Pearson’s Correlation test, showing R = 0.9929 at <i>P</i> = 0.0007, R² = 0.9858. Mock (n = 6), WT (n = 10), and M1st (n = 10).</p

Acute, but not latent, MHV68 infection results in suppressed humoral response.

<p>(A) Timeline of infection. C57BL/6 mice were infected with 1000 PFU of MHV68 IN at day -60, -30, -15 or -7 and challenged with 10⁵ pRBCs on day 0. Absolute number of (B) splenic GC B cell (B220+ GL7+ CD95+) and plasma cell (CD3- B220int CD138+) populations at day 16 post <i>P</i>. <i>yoelii</i> XNL infection (For GC and PC: Day -7 and Day -15 co-infected vs. <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p<0.05/ Day -30 co-infected vs. <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p>0.05). (C) MHV68 and <i>P</i>. <i>yoelii</i> XNL specific IgG responses at day 16 post <i>P</i>. <i>yoelii</i> XNL infection (Day -7 and Day -15 co-infected vs. <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p<0.05/ Day -30 co-infected vs. <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p>0.05). (D) Global Tfh population (CD4+ PD-1+ CXCR5+), germinal center Tfh (CD4+ GL7+ CXCR5+) and activated/antigen specific Tfh (CD4+ CD44+ PD-1+ CXCR5+) in the spleen at day 16 post <i>P</i>. <i>yoelii</i> XNL infection.</p

CD8⁺ T Cell Response to Gammaherpesvirus Infection Mediates Inflammation and Fibrosis in Interferon Gamma Receptor-Deficient Mice

<div><p>Idiopathic pulmonary fibrosis (IPF), one of the most severe interstitial lung diseases, is a progressive fibrotic disorder of unknown etiology. However, there is growing appreciation for the role of viral infection in disease induction and/or progression. A small animal model of multi-organ fibrosis, which involves murine gammaherpesvirus (MHV68) infection of interferon gamma receptor deficient (IFNγR-/-) mice, has been utilized to model the association of gammaherpesvirus infections and lung fibrosis. Notably, several MHV68 mutants which fail to induce fibrosis have been identified. Our current study aimed to better define the role of the unique MHV68 gene, M1, in development of pulmonary fibrosis. We have previously shown that the M1 gene encodes a secreted protein which possesses superantigen-like function to drive the expansion and activation of Vβ4⁺ CD8⁺ T cells. Here we show that M1-dependent fibrosis is correlated with heightened levels of inflammation in the lung. We observe an M1-dependent cellular infiltrate of innate immune cells with most striking differences at 28 days-post infection. Furthermore, in the absence of M1 protein expression we observed reduced CD8⁺ T cells and MHV68 epitope specific CD8⁺ T cells to the lungs—despite equivalent levels of viral replication between M1 null and wild type MHV68. Notably, backcrossing the IFNγR-/- onto the Balb/c background, which has previously been shown to exhibit weak MHV68-driven Vβ4⁺ CD8⁺ T cell expansion, eliminated MHV68-induced fibrosis—further implicating the activated Vβ4⁺ CD8⁺ T cell population in the induction of fibrosis. We further addressed the role that CD8⁺ T cells play in the induction of fibrosis by depleting CD8⁺ T cells, which protected the mice from fibrotic disease. Taken together these findings are consistent with the hypothesized role of Vβ4⁺ CD8⁺ T cells as mediators of fibrotic disease in IFNγR-/- mice.</p></div

Reduced CD8⁺ and effector CD8⁺ T cells are observed in absence of M1 expression.

<p>8–12 week old IFNγR-/- C57Bl/6 mice were intranasally infected with 1x10⁵ pfu MHV68 (WT or M1st) and sacrificed at 28 days post infection. Whole lungs were harvested and assessed for presence of CD8⁺ T cell populations and effector function (n = 5 mice/group). Mean and std. error are shown for (A) absolute number of lung and CD8⁺ T cells, (B&C) absolute number of tetramer specific and peptide responsive CD8⁺ T cells, with MFI of cytokine expression, (D) absolute number of Vβ4⁺ CD8⁺ T cells and M1 responsive CD8⁺ T cells. Statistics were measured using a Mann-Whitney 2 tailed test (* <i>P</i> = 0.0119, ** <i>P</i> = 0.0079, * <i>P</i> = <0.0004).</p

MHV68 co-infection with the non-lethal <i>P</i>. <i>yoelii</i> XNL in C57BL/6 results in lethal malarial disease and suppressed <i>Plasmodium</i> specific IgG response.

<p>(A) Timeline of infection. 6–8 week old C57BL/6 mice were infected with 1000 PFU of MHV68 on day -7 followed by infection with 10⁵pRBCs of non-lethal <i>P</i>. <i>yoelii</i> XNL or <i>P</i>. <i>chabaudi</i> AS. Infections consisted of 5 experimental groups: MHV68 + <i>Plasmodium</i>, <i>Plasmodium</i>, MHV68 or mock infected. Each experimental group consisted of n = 5 and was repeated twice. Animals were sacrificed at days 8, 12, 16 and 23 post <i>P</i>. <i>yoelii</i> XNL infection or day 7, 11, 15 and 23 post <i>P</i>. <i>chabaudi</i> AS infection for collection of spleen, lung and blood. (B) Survival analysis of animals co-infected with MHV68 and <i>P</i>. <i>yoelii</i> XNL or <i>P</i>. <i>chabaudi</i> AS. Total IgG and IgM levels in serum in (C) <i>P</i>. <i>yoelii</i> XNL (Day 23 IgG—<i>P</i>. <i>yoelii</i> vs co-infected: p<0.05 Mann Whitney U-test) or (D) <i>P</i>. <i>chabaudi</i> AS co-infection model (Day 11 IgG—<i>P</i>. <i>chabaudi</i> vs co-infected: p<0.05 Mann Whitney U-test). Parasite specific IgG levels in serum during (E) <i>P</i>. <i>yoelii</i> XNL (day 23 post infection, <i>P</i>. <i>yoelii</i> vs co-infected: p<0.05 Mann Whitney U-test) or (F) <i>P</i>. <i>chabaudi</i> AS (day 11 post infection, <i>P</i>. <i>chabaudi</i> vs co-infected: p<0.05 Mann Whitney U-test) co-infection.</p

MHV68 and <i>Plasmodium</i> co-infection results in defective splenic T follicular helper (Tfh) response.

<p>The timeline and experimental set up was identical to that shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004858#ppat.1004858.g001" target="_blank">Fig 1A</a>. (A) Representative flow plots for gating strategies used to define the global Tfh population (CD4+ PD-1+ CXCR5+), germinal center Tfh (CD4+ GL7+ CXCR5+) and activated/antigen specific Tfh (CD4+ CD44+ PD-1+ CXCR5+). (B) Absolute values for all three Tfh subsets are plotted for the <i>P</i>. <i>yoelii</i> XNL (Day 23, all Tfh subsets, <i>P</i>. <i>yoelii</i> vs. co-infected, p<0.05 Mann Whitney U-test) or (C) <i>P</i>. <i>chabaudi</i> co-infection models at multiple time points (Day 23, all Tfh subsets, <i>P</i>. <i>chabaudi</i> vs. co-infected, p<0.05 Mann Whitney U-test).</p

The absence of fibrosis in M1st infected IFNγR-/- C57Bl/6 mice is not due to a failure in profibrotic mediator TGFβ1 expression.

<p>(A) Mink lung epithelial cells (MLEC-clone 32) stably transfected with a plasminogen activator inhibitor-1 fused to a luciferase reporter gene were infected with differing multiplicities of infection and assayed for luciferase activity (a read out of active TGFβ production). 8–12 week old IFNγR-/- C57Bl/6 mice were intranasally infected with 1x10⁵pfu MHV68 (WT or M1st) or mock infected and sacrificed at 28 days post-infection (n = 3–4 mice/group). (B) lung lysates (30μg) were assessed for TGFβ1 expression by western blot (mock lane 1–3, WT lane 4–7, M1st lane 8–11), (C) normalized band density is shown.</p