Overview of cellular responses during a chronic <i>L. donovani</i> infection.

<p>During an established <i>L. donovani</i> infection, a subset of regulatory DCs in the spleen can produce IL-10 that promotes the expansion of IL-10-producing regulatory T cells (Tr1), as well as inhibiting antimicrobial mechanisms in macrophages and other phagocytic cells (including suppression of ROI and RNI generation). IL-27 produced by regulatory DCs and macrophages, along with T cell–derived IL-21, can drive the differentiation of Th1 cells into Tr1 cells, as well as inhibit Th17 development. IL-10 produced by Tr1 cells can suppress antigen presentation, contributing to T cell dysfunction, as well as down-regulate CD4⁺ T cell IFNγ production. There has been a report that IL-10 can also be produced by Treg cells in the BM of VL patients. Although uptake of infected neutrophils undergoing apoptosis by macrophages contributes to the establishment of <i>L. major</i> infection in mice, no such mechanism has yet been described during <i>L. donovani</i> infection. Abbreviations: N, neutrophil.</p

Overview of cellular responses during an asymptomatic <i>L. donovani</i> infection.

<p>Infected macrophages can produce TNF and IL-1β in response to <i>L. donovani</i> infection as part of the innate immune response. However, DC IL-12 production in response to <i>L. donovani</i> infection is required to drive the differentiation of antigen-specific CD4⁺ T cells into IFNγ- and TNF-producing Th1 cells. These cells can activate infected macrophages and monocytes to produce ROI and RNI that kill intracellular parasites. There are also reports in humans that Th17 and Th22 cells develop in asymptomatic, infected individuals, possibly driven by IL-23 and IL-6. However, the antiparasitic mechanism mediated by these CD4⁺ T cell subsets following <i>L. donovani</i> infection remains unknown. Although parasite-specific antibodies are readily detected in asymptomatic individuals, their role, if any, in control of infection and protection against reinfection is unknown. Abbreviations: MO, monocyte; Mφ, macrophage.</p

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

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

MHV68 suppresses splenic B cell responses during co-infection with <i>Plasmodium</i>.

<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) Absolute numbers of splenic GC B cell populations (B220+ GL7+ CD95+) during <i>P</i>. <i>yoelii</i> XNL and <i>P</i>. <i>chabaudi</i> AS co-infection models with representative gating strategy (Day 12 post <i>P</i>. <i>yoelii</i> or Day 15 post <i>P</i>. <i>chabaudi</i>; <i>Plasmodium</i> vs. co-infected, p<0.05, Mann Whitney U-test). (B) Absolute numbers of splenic plasma cell populations (CD3- B220int CD138+) during <i>P</i>. <i>yoelii</i> XNL AND <i>P</i>. <i>chabaudi</i> AS co-infection models with representative gating strategy (Day 12 post <i>P</i>. <i>yoelii</i> or Day 11 post <i>P</i>. <i>chabaudi</i>; <i>Plasmodium</i> vs. co-infected, p<0.05, Mann Whitney U-test). (C) Spleen section for mock infected, MHV68 infected, <i>P</i>. <i>yoelii</i> XNL infected and MHV68 and <i>P</i>. <i>yoelii</i> XNL co-infected animals at day 8 post infection with <i>P</i>. <i>yoelii</i> XNL (or day 15 post-infection with MHV68). Green: B220-FITC (B cells), Blue: GL7-AF660 (Germinal center B cells) and Red: CD3-AF568 (T cells).</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

The MHV68 M2 gene product is necessary for virus mediated humoral suppression and lethality during <i>Plasmodium</i> co-infection.

<p>(A) MHV68 specific IgG titers from serum of animals infected with the MR (M2.Marker Rescue) or M2.Stop (ST, M2-null) viruses. Serum was collected and analyzed on days 7, 14 and 21 post infection with either virus (n = 10/ virus) (Day 21, MR vs. M2.Stop, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p<0.05). (B) <i>P</i>. <i>yoelii</i> XNL specific IgG response during <i>P</i>. <i>yoelii</i> XNL co-infection with either the M2.MR or M2.Stop virus. Serum was collected at day 20 post infection with <i>P</i>. <i>yoelii</i> XNL (WT + <i>P</i>. <i>yoelii</i> co-infected vs. <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p<0.05/ WT + <i>P</i>. <i>yoelii</i> co-infected vs. M2.Stop + <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p>0.05). (C) Survival curve during <i>P</i>. <i>yoelii</i> XNL co-infection with either the M2.MR or M2.Stop virus. <u>Note</u>: data representing <i>P</i>. <i>yoelii</i> XNL + MHV68 co-infection is the identical data set to that in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004858#ppat.1004858.g001" target="_blank">Fig 1B</a>. It was added in panel C for comparative purposes. (D) % parasitemia in the periphery during <i>P</i>. <i>yoelii</i> XNL, <i>P</i>. <i>yoelii</i> XNL +MR and <i>P</i>. <i>yoelii</i> XNL + M2.Stop infection.</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

Treg phenotype in UM and SM.

<p>PBMC of 3 AC, 6 UM, and 5 SM patients were stained for CD4, CD25, Foxp3, and CD127, or CD45RO, CD69, CCR7, or TNFRII. (A) Mean fluorescent intensities (MFI) are shown for CD4⁺CD25⁻Foxp3⁻ (solid line) and CD4⁺CD25⁺Foxp3⁺ (dark grey) cells or CD4⁺Foxp3⁺CD127^lo (light grey) cells. Dotted lines represent the isotype controls. One representative donor is shown, and activated T cells with high expression of CD4 were gated out. (B) Pooled TNFRII MFI (3 AC, 6 UM, 5 SM). Horizontal lines show the median (p = 0.005 by Kruskal-Wallis test, and in post-hoc pairwise comparison of UM and SM: p = 0.008, and between SM and AC: p = 0.04). (C) Foxp3 expression is shown for TNFRII positive Treg cells (top panel) and TNFRII negative Treg cells (bottom panel) for one representative UM and one representative SM sample. The dotted line shows isotype control staining. AC, malaria-exposed asymptomatic controls; UM, uncomplicated <i>P. falciparum</i> malaria; SM, severe <i>P. falciparum</i> malaria.</p

In vitro <i>P. falciparum</i> exposure induced Treg cell expansion, TNFRII expression, and enhanced Treg cell activity.

<p>PBMC from healthy malaria-unexposed blood donors (n = 4) were cultured overnight in the presence of <i>P. falciparum</i>–infected red blood cells (pRBC) at pRBC∶PBMC ratios of 2∶1, 1∶2.5, and 1∶10, or uninfected red blood cells (uRBC) at a uRBC∶PBMC ratio of 2∶1 or without RBC. (A) CD4⁺CD25⁺Foxp3⁺CD127^lo Treg cells are represented as percentage of CD4 T cells. (B) TNFRII MFI on CD4⁺ Foxp3⁺CD127^lo Treg cells. (C) sTNFRII (left panel), TNF (middle panel), and IL-10 (right panel) concentration in culture supernatants. (D) Following overnight culture with <i>P. falciparum</i>–infected red blood cells (pRBC) or uninfected red blood cells (uRBC) at a RBC∶PBMC ratio of 1∶2.5, PBMC were sorted into CD4⁺CD25⁻ responder cells and CD4⁺CD25⁺TNFRII⁺ or CD4⁺CD25⁺TNFRII⁻ Tregs and stained intracellularly for Foxp3. Foxp3 expression is shown for CD4⁺CD25⁺TNFRII⁺ or CD4⁺CD25⁺TNFRII⁻ Treg cells (grey line) and CD4⁺CD25⁻ responder cells (dotted line). (E) Sorted cells were tested for suppressive activity. 10⁴ CD4⁺CD25⁻ responder T cells sorted after uRBC exposure were incubated either alone or with TNFRII⁺ or TNFRII⁻ Tregs after uRBC or pRBC exposure at a 1∶1 ratio in 96 well plates pre-coated with 3 µg/mL anti-CD3 antibody (OKT-3) for 3 days. Sorted monocytes were used as APC. Bars show mean cpm+/−SD of triplicate wells.</p