A Novel Model of Asymptomatic Plasmodium Parasitemia That Recapitulates Elements of the Human Immune Response to Chronic Infection.

In humans, immunity to Plasmodium sp. generally takes the form of protection from symptomatic malaria (i.e., 'clinical immunity') rather than infection ('sterilizing immunity'). In contrast, mice infected with Plasmodium develop sterilizing immunity, hindering progress in understanding the mechanistic basis of clinical immunity. Here we present a novel model in which mice persistently infected with P. chabaudi exhibit limited clinical symptoms despite sustaining patent parasite burdens for many months. Characterization of immune responses in persistently infected mice revealed development of CD4+ T cell exhaustion, increased production of IL-10, and expansion of B cells with an atypical surface phenotype. Additionally, persistently infected mice displayed a dramatic increase in circulating nonclassical monocytes, a phenomenon that we also observed in humans with both chronic Plasmodium exposure and asymptomatic infection. Following pharmacological clearance of infection, previously persistently infected mice could not control a secondary challenge, indicating that persistent infection disrupts the sterilizing immunity that typically develops in mouse models of acute infection. This study establishes an animal model of asymptomatic, persistent Plasmodium infection that recapitulates several central aspects of the immune response in chronically exposed humans. As such, it provides a novel tool for dissection of immune responses that may prevent development of sterilizing immunity and limit pathology during infection

Blood and tissue pathology in persistently infected mice.

<p>(A) Hematocrit (mean + SEM) was measured on the indicated days (d) post infection (n = 3–5 mice per group, except n = 1 on day 352). Clearing, <i>Ifngr1</i>^<i>-/-</i> mice infected and treated with control antibody as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162132#pone.0162132.g001" target="_blank">Fig 1B</a>. Persistent, <i>Ifngr1</i>^<i>-/-</i> mice infected and treated with α-CD4 antibody as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162132#pone.0162132.g001" target="_blank">Fig 1B</a>. (B) Reticulocytes were enumerated as a percentage of total erythrocytes. 8 d.p.i., <i>Ifngr1</i>^<i>-/-</i> mice infected with <i>P</i>. <i>chabaudi</i> for 8 d. Each dot represents one mouse; means + SEM are shown. **, p < 0.01 by Kruskal-Wallis test with Dunn's post-test. n.s., not significant. (C) Mesenteric lymph nodes (arrows) in cleared and persistently infected mice, 331 d.p.i. (D) Spleens (top) and femurs (bottom) from representative mice, 331 d.p.i. (E) Tissues were excised from the indicated mice 331 d.p.i., processed, and stained with hemotoxylin and eosin. Asterisks mark representative B cell follicles in naive and cleared spleen sections.</p

T cell exhaustion in persistently infected mice.

<p>(A) Gating strategy to identify antigen-experienced CD4⁺ T cells with the proxy markers CD11a and CD49d. (B) Repopulation of antigen-specific T cells in the blood of infected <i>Ifngr1</i>^<i>-/-</i> mice treated 4 d.p.i. (black wedge) as indicated. Means + SEM are shown (n = 5 control and 15 α-CD4-treated mice, pooled from three independent biological replicates). (C) IL-2 and IFN-γ production was measured by intracellular flow cytometry in blood CD4⁺ T cells from naive, acutely infected (6 d.p.i.) or persistently infected <i>Ifngr1</i>^<i>-/-</i> mice. A representative gating strategy is shown. Means + SEM are shown (n = 5 per group). (D) As in (C), except IFNG⁺ CD4⁺ T cells were measured in wild-type mice. (E) Surface expression of PD-1 and LAG-3 was measured on blood CD4⁺ T cells from <i>Ifngr1</i>^<i>-/-</i> mice by flow cytometry. (F) Levels of IFN-γ and IL-10 were measured in plasma. Acute samples were taken 6 d.p.i. Each point represents one mouse; data are pooled from at least three independent experiments and include samples from both wild-type and <i>Ifngr1</i>^<i>-/-</i> mice, collected from 100–250 d.p.i.. Statistical significance in (D and F) was determined by Kruskal-Wallis test with Dunn's post-test. (G) Plasma IFN-γ levels in persistently infected mice, graphed by time post-infection. (H) Persistently infected wild-type mice (n = 4 per group) were treated every 3 d with 300 μg each α-PD-1 and α-LAG-3 or with isotype control antibodies. Black wedges indicate treatment days. *, p < 0.05. **, p < 0.01. ***, p < 0.001. n.s., not significant.</p

A novel model of persistent, patent <i>P</i>. <i>chabaudi</i> infection.

<p>(A) Wild-type (C57BL/6; B6) mice infected with <i>P</i>. <i>chabaudi</i> AS were treated 4 d.p.i. with α-CD4 antibody to deplete CD4⁺ T cells, or with an irrelevant isotype control antibody (n = 3 per group). Parasitemia was monitored by thin blood smear. (B) Parasitemia was monitored in <i>Ifngr1</i>^<i>-/-</i> mice infected and treated with α-CD4 (n = 14) or isotype (n = 3) as in (A). (C) <i>Ifngr1</i>^<i>-/-</i> mice were infected and treated as in (B). CD4⁺ T cells were enumerated in blood by flow cytometry (n = 5 control and 15 α-CD4-treated mice). (D) Infected B6 mice (n = 22) were treated through d 64 with α-CD4 or isotype control antibody. Black wedges indicate antibody administration. Means + SEM are shown. In B-D, data shown are pooled from at least three independent biological replicates.</p

Persistently infected mice display limited clinical symptoms.

<p>Mice of the indicated genotypes were infected and treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162132#pone.0162132.g001" target="_blank">Fig 1B</a> (for <i>Ifngr1</i>^<i>-/-</i>) or <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162132#pone.0162132.g001" target="_blank">Fig 1D</a> (for wild-type) to establish persistent infection. (A) The weights of infected <i>Ifngr1</i>^<i>-/-</i> mice are graphed as a percentage of their value 50 d.p.i. (n = 3 isotype-treated, 14 α-CD4-treated). Black wedge indicates time of antibody treatment. (B) Average (left) and maximum (right) mouse activity was measured by open field test. (C) Body temperature, (D) blood glucose, and (E) plasma ALT activity were measured. Acute measurements were taken from <i>Ifngr1</i>^<i>-/-</i> mice 8 d.p.i.; measurements from persistently infected mice were made 100–150 d.p.i. (B, C, D) or 13 d.p.i. (E). In B-E, each point represents an individual mouse; samples were pooled from at least three independently established cohorts. Significance in B-E was determined by Kruskal-Wallis test with Dunn's post-test. In C-E, significance values represent comparison with naive mice. *, p < 0.05. **, p < 0.01. ***, p < 0.001. n.s., not significant.</p

Previously persistently infected mice are not immune to secondary challenge.

<p>(A) Persistently infected <i>Ifngr1</i>^<i>-/-</i> mice were treated once with a subcurative dose of chloroquine (black wedge). Parasitemia was monitored during and after treatment. (B-G) <i>Ifngr1</i>^<i>-/-</i> mice were infected and treated with α-CD4 antibody 4 d.p.i. to establish persistent infection, or with an isotype control antibody. After 4 months, all mice were treated with pyrimethamine to clear infection and rested for two months. Isotype-treated mice (immune) and previously persistently infected mice (PPI) were then re-infected with <i>P</i>. <i>chabaudi</i>. Additional naive <i>Ifngr1</i>^<i>-/-</i> mice were also infected at this time to serve as non-immune controls. (B) Experimental layout. (C) Parasitemia, (D) body temperature, (E, F) hematocrit and (G) weight were monitored at the indicated timepoints. In (F, G), measurements are expressed as a percentage of their value on the day of re-infection. Data shown are means + SEM taken from one of two independent experiments (n = at least 5 for each group in each experiment). *, p < 0.05; ***, p < 0.001 by Mann-Whitney test. n.s., not significant. In F and G, black asterisks denote significant comparisons between immune and PPI groups; blue asterisks denote significant comparisons between PPI and naive groups.</p

Expansion of nonclassical monocytes in persistent infection and in chronically exposed humans.

<p>(A) Leukocytes were quantified in the blood of cleared and persistently infected mice 100 d.p.i. Means + SEM are shown (n = 4 per group). (B) The abundance of each blood leukocyte subset is expressed as the fold increase in persistently infected mice over cleared mice. The sampling timepoint is indicated on the <i>y</i>-axis. Means + SEM are shown (n = 4 per group). (C) Gating strategy for classical (Ly6C^hi) and nonclassical (Ly6C^lo) monocytes. (D) Blood concentration (mean + SEM) of NCMs in cleared (n = 3) and persistently infected (n = 7) mice. Note different scales on left and right graphs. (E) Frequencies of blood leukocyte subsets. Average values from 3 cleared and 7 persistently infected mice are shown. NK, natural killer cell. NKT, natural killer T cell. CM, classical monocyte. NCM, nonclassical monocyte. (F) Expression of CX3CR1 (left) and CD11c (right) was assessed by flow cytometry on the indicated populations. Each symbol represents one mouse; bars show mean +/- SD. (G) Apoptotic NCMs were quantified in naive and persistently infected mice through flow cytometry. Each point represents one mouse. Means + SEM of four pooled biological replicates are shown. **, p < 0.01 by Mann-Whitney test. (H) Expression of PD-L1 and PD-L2 was assessed on the indicated monocyte populations. Plots from one representative mouse of at least five replicates are shown. (I) Gating strategy for human NCMs. (J) NCM frequencies were assessed in PBMCs by flow cytometry. Low-exposure subjects were from Walakuba, Uganda (EIR = 3.8); high-exposure subjects were from Nagongera (EIR = 125). Each point represents an individual subject. *, p < 0.05 by Wilcoxon. Statistical comparisons to U.S. adults were all significant, but are omitted for clarity.</p