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

EphB2 expression on BMDCs can be modulated by ligation with Toll-like receptor ligands.

<p>(A) BMDC were incubated with a Toll Like receptor (TLR)-4 agonist (lipopolysaccharide (LPS) 1μg/ml) and (B) a TLR-9 agonist (CpG1668 1μM) with or without recombinant mouse interferon (IFN)-γ at a concentration of 20ng/ml. <i>EphB2</i> mRNA was quantified by qPCR at different time points post-stimulation. The modulation of transcription for known co-stimulatory molecules CD80 and CD86 as well as the cytokine interleukin (IL)-12p40 in response to TLR stimulation in the same experiments are shown for comparison. (C) The change in EphB2 protein expression at 22 hours post-incubation with LPS and IFN-γ is shown and the mean fluorescence quantified for different conditions. All graphs represent the median value of pooled data across 3 independent BMDC preparations ±SD and data analyzed using One-way ANOVA– Kruskal Wallis test and Dunn’s multiple comparisons post-test. *P<0.05. MFI = Mean Fluorescence Intensity.</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

BMDCs stimulated with EphrinB2 upregulate <i>EphB2</i> mRNA.

<p>Naïve BMDCs were plated onto plate-bound Ephrin-B1-Fc fusion protein (A, B, C) or plate bound Ephrin-B2-Fc fusion protein (D, E, F). The transcription of <i>EphB2</i> with and without the addition of recombinant interferon-γ (IFN-γ) was monitored by qPCR over 24 hours of culture (A, D). The efficiency of the Eph receptor ligation was monitored by assessing tyrosine phosphorylation on DCs by western blot (B, E) and the phosphorylation quantified using densitometry (C, F). All graphs represent the median value of pooled data from 3 independent DC preparations ±SD.</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

Splenic CD11c^hi and bone marrow-derived DCs express EphB2.

<p>(A) Eph receptor expression on the surface of naïve splenic CD11c^hi DCs was detected by incubation with Ephrin-B2-Fc chimeric protein and binding was detected using a biotinylated anti-Fc antibody and streptavidin-APC (SA-APC) using flow cytometry. (B) The expression of EphB2 on BMDCs incubated with anti-EphB2 antibody or isotype control (IgG2a) detected by immunofluorescence. Magnification 40x; Scale bar, 10μm.</p

EphB2 co-localizes with MHC-II on BMDCs.

<p>Two examples are shown: (A) Representative BMDCs stimulated with 1μg/ml lipopolysaccharide (LPS) and 20ng/ml recombinant mouse interferon (IFN)-γ for 22 hours; (B) shows unstimulated cells. EphB2 is shown in red (Northernlights557), MHC-II is shown in green (FITC) and DAPI is shown to demarcate the nuclei of the cells. Magnification 100x; Scale bar, 20μm.</p

Characterization of the Probiotic Yeast <i>Saccharomyces boulardii</i> in the Healthy Mucosal Immune System

<div><p>The probiotic yeast <i>Saccharomyces boulardii</i> has been shown to ameliorate disease severity in the context of many infectious and inflammatory conditions. However, use of <i>S</i>. <i>boulardii</i> as a prophylactic agent or therapeutic delivery vector would require delivery of <i>S</i>. <i>boulardii</i> to a healthy, uninflamed intestine. In contrast to inflamed mucosal tissue, the diverse microbiota, intact epithelial barrier, and fewer inflammatory immune cells within the healthy intestine may all limit the degree to which <i>S</i>. <i>boulardii</i> contacts and influences the host mucosal immune system. Understanding the nature of these interactions is crucial for application of <i>S</i>. <i>boulardii</i> as a prophylactic agent or therapeutic delivery vehicle. In this study, we explore both intrinsic and immunomodulatory properties of <i>S</i>. <i>boulardii</i> in the healthy mucosal immune system. Genomic sequencing and morphological analysis of <i>S</i>. <i>boulardii</i> reveals changes in cell wall components compared to non-probiotic <i>S</i>. <i>cerevisiae</i> that may partially account for probiotic functions of <i>S</i>. <i>boulardii</i>. Flow cytometry and immunohistochemistry demonstrate limited <i>S</i>. <i>boulardii</i> association with murine Peyer’s patches. We also show that although <i>S</i>. <i>boulardii</i> induces a systemic humoral immune response, this response is small in magnitude and not directed against <i>S</i>. <i>boulardii</i> itself. RNA-seq of the draining mesenteric lymph nodes indicates that even repeated administration of <i>S</i>. <i>boulardii</i> induces few transcriptional changes in the healthy intestine. Together these data strongly suggest that interaction between <i>S</i>. <i>boulardii</i> and the mucosal immune system in the healthy intestine is limited, with important implications for future work examining <i>S</i>. <i>boulardii</i> as a prophylactic agent and therapeutic delivery vehicle.</p></div

<i>S</i>. <i>boulardii</i> induces increased total but not antigen specific fecal and serum antibody levels.

<p>(A) Total fecal IgA, serum IgG, and serum IgA levels were determined by ELISA using samples from mice gavaged with vehicle (white bars) or 10⁸ CFU <i>S</i>. <i>boulardii</i> (gray bars) daily for 7, 14, or 28 days (n = 5 mice per group in each of two independent experiments per time point, with error bars showing the standard error of the mean (SEM), *p<0.05, ordinary two-way ANOVA, Sidak multiple comparison test). (B) Percentage of <i>S</i>. <i>boulardii</i> cells positive for IgA or IgG after incubation with serum or fecal samples collected from naïve mice (white bars) or mice gavaged daily with <i>S</i>. <i>boulardii</i> (gray bars) for 7, 14, or 28 days, with gray lines showing the average percentage of stained cells in control samples incubated with secondary antibody only (n = 5 mice per group in each of two independent experiments. Error bars depict SEM.) (C) Representative flow plots depicting the percent of total <i>S</i>. <i>boulardii</i> cells positive for IgA or IgG after incubation with either serum or fecal supernatant.</p

Stimulation of BMDCs from EphB2+/+ and EphB2-/- mice with TLR receptor agonists results in similar levels of secreted cytokine.

<p>BMDCs from EphB2+/+ and EphB2-/- mice were incubated with TLR ligands LPS and CpG1668 with and without the addition of recombinant interferon (IFN)-γ for 20 hours. Interleukin (IL)-12p40 (A), IL-12p70 (B), IL-10 (C) and tumor necrosis factor (TNF)-α (D) were measured in the culture supernatant using Luminex. The graphs are representative of 3 individual experiments and bars represent the mean ±SD of 2 replicate wells plated.</p