Mast Cell Chymase/Mcpt4 Suppresses the Host Immune Response to Plasmodium yoelii, Limits Malaria-Associated Disruption of Intestinal Barrier Integrity and Reduces Parasite Transmission to Anopheles stephensi

Abstract

An increase in mast cells (MCs) and MCs mediators has been observed in malaria-associated bacteremia, however, the role of these granulocytes in malarial immunity is poorly understood. Herein, we studied the role of mouse MC protease (Mcpt) 4, an ortholog of human MC chymase, in malaria-induced bacteremia using Mcpt4 knockout (Mcpt4^-/-) mice and Mcpt4^+/+ C57BL/6J controls, and the non-lethal mouse parasite Plasmodium yoelii yoelii 17XNL. Significantly lower parasitemia was observed in Mcpt4^-/- mice compared with Mcpt4^+/+ controls by day 10 post infection (PI). Although bacterial 16S DNA levels in blood were not different between groups, increased intestinal permeability to FITC-dextran and altered ileal adherens junction E-cadherin were observed in Mcpt4^-/- mice. Relative to infected Mcpt4^+/+ mice, ileal MC accumulation in Mcpt4^-/- mice occurred two days earlier and IgE levels were higher by days 8-10 PI. Increased levels of circulating myeloperoxidase were observed at 6 and 10 days PI in Mcpt4^+/+ but not Mcpt4^-/- mice, affirming a role for neutrophil activation that was not predictive of parasitemia or bacterial 16S copies in blood. In contrast, early increased plasma levels of TNF-α, IL-12p40 and IL-3 were observed in Mcpt4^-/- mice, while levels of IL-2, IL-10 and MIP1β (CCL4) were increased over the same period in Mcpt4^+/+ mice, suggesting that the host response to infection was skewed toward a type-1 immune response in Mcpt4^-/- mice and type-2 response in Mcpt4^+/+ mice. Spearman analysis revealed an early (day 4 PI) correlation of Mcpt4^-/- parasitemia with TNF-α and IFN-γ, inflammatory cytokines known for their roles in pathogen clearance, a pattern that was observed in Mcpt4^+/+ mice much later (day 10 PI). Transmission success of P. y. yoelii 17XNL to Anopheles stephensi was significantly higher from infected Mcpt4^-/- mice compared with infected Mcpt4^+/+ mice, suggesting that Mcpt4 also impacts transmissibility of sexual stage parasites. Together, these results suggest that early MCs activation and release of Mcpt4 suppresses the host immune response to P. y. yoelii 17XNL, perhaps via degradation of TNF-α and promotion of a type-2 immune response that concordantly protects epithelial barrier integrity, while limiting the systemic response to bacteremia and parasite transmissibility.

Keywords: Anopheles stephensi; Mcpt4; Plasmodium yoelii; bacteremia; chymase; intestinal permeability; malaria; mast cells.

Figure 1

Plasmodium y. yoelii 17XNL parasitemia was decreased, intestinal permeability to FITC-dextran was increased, but bacteremia over time was not different in Mcpt4 ^-/- relative to Mcpt4 ^+/+ mice. (A) Peripheral parasitemia following P. y. yoelii 17XNL infection, (B) intestinal permeability quantified as FITC-dextran in plasma of infected and uninfected mice after oral gavage and (C) bacterial 16S DNA copies/µL of blood in infected and uninfected mice. Each dot represents a single mouse. Parasitemia and bacterial 16S copies were determined for the three replicates (n=11 per group, per time point), while FITC-dextran was determined using the last replicate of mice only (n=3 mice per group per time point). Parasitemia and log 16S copies per μL of blood were analyzed using one-way ANOVA followed by Dunnett’s or Šídák’s multiple comparisons test; lines represent the mean. FITC-dextran data were analyzed with the Kruskal-Wallis test followed by Dunn’s test for multiple comparisons for each time point versus control or between Mcpt4 ^-/- and Mcpt4 ^+/+ mice at specific time points; lines represent the median. P values ≤ 0.05 were considered significant. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001.

Figure 2

Plasmodium y. yoelii 17XNL-infected Mcpt4^-/- mice exhibited earlier infection-associated increases in ileal MCs and higher circulating levels of IgE relative to infected Mcpt4 ^+/+ mice. (A) Numbers of ileal MCs per high-powered field (HPF) from naphthol AS-D chloroacetate esterase (NASDCE) stained sections from uninfected control mice and infected Mcpt4^-/- and Mcpt4 ^+/+ mice. (B) Plasma MC protease 1 (Mcpt1) concentrations as determined by ELISA. (C) Plasma IgE concentrations as determined by ELISA. (D) Representative stained MCs (pink cells indicated by yellow arrows) in ileum of Mcpt4^-/- (left) and Mcpt4 ^+/+ (right) mice at 8 days PI. Number of MCs and IgE levels in plasma were determined for biological replicates 1 and 2 (n=8 mice per group per time point), while plasma Mcpt1 levels were determined for the three replicates (n=11 mice per group per time point). Data were analyzed with the Kruskal-Wallis test followed by Dunn’s multiple comparison for each time point versus control or between Mcpt4^-/- and Mcpt4 ^+/+ mice at specific time points; lines represent the median. P values ≤ 0.05 were considered significant. *P ≤ 0.05, **P ≤ 0.01; ***P ≤ 0.001, ****P ≤ 0.0001.

Figure 3

Mcpt4^-/- mice exhibited altered patterns of ileal adherens junctions during P. y. yoelii 17XNL infection relative to Mcpt4 ^+/+ mice. Representative images from P. y. yoelii 17XNL-infected Mcpt4^-/- (A) and Mcpt4 ^+/+ (B) mice and uninfected controls. The top, middle and bottom rows contain images of E-cadherin (green) and ZO-1 (red) staining and merged images. Scale bars represent 50 μm length. Quantitation of E-cadherin (C) and ZO-1 (D). Quantitative analysis of fluorescence was performed using NIH ImageJ software. Data are represented as mean ± SD from 10-15 HPF per mouse from three independent biological replicates (n=5 Mcpt4^-/- mice and n=5 Mcpt4 ^+/+ mice per replicate). Data were analyzed with the Kruskal-Wallis test followed by Dunn’s multiple comparison for each time point versus control or between Mcpt4^-/- and Mcpt4 ^+/+ mice at specific time points. P values ≤ 0.05 were considered significant. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Figure 4

Mcpt4^-/- mice exhibited significantly decreased neutrophil activation at 6 and 10 days following P. y. yoelii 17XNL infection. (A) Plasma myeloperoxidase (MPO) detection by ELISA, represented as percentage of positive plasma samples at 4, 6, 8, and 10 days PI. (B) Plasma neutrophil elastase (NE) as determined by ELISA. MPO and NE were determined for the three biological replicates (n=11 mice per group per time point). MPO data were analyzed with Fisher’s exact test and NE data were analyzed with the Kruskal-Wallis test followed by Dunn’s multiple comparison for each time point versus control or between Mcpt4^-/- and Mcpt4 ^+/+ mice at specific time points. P values ≤ 0.05 were considered significant. **P ≤ 0.01; ***P ≤ 0.001, ****P ≤ 0.0001.

Figure 5

Plasma cytokines and chemokines in P. y. yoelii 17XNL-infected Mcpt4^-/- and Mcpt4 ^+/+ mice and uninfected controls. (A–T) The x axis represents the time points in days after infection, and the y axis represents the concentrations of IL-12p40 (A), TNF-α (B), IL-3 (C), IL-2 (D), IL-10 (E), MIP1β (CCL4) (F), IL-6 (G), IFN-γ (H), IL-1β (I), MIP1α (CCL3) (J), IL-4 (K), MCP-1 (CCL2) (L), RANTES (CCL5) (M), GM-CSF (N), IL-9 (O) IL-13 (P) Eotaxin (CCL11) (Q), IL-12p70 (R), IL-17 (S) and KC (CXCL1) (T). Each dot represents a single mouse. Plasma cytokines were determined for the three biological replicates (n=11 mice per group per time point). Data were analyzed with the Kruskal-Wallis test followed by Dunn’s multiple comparison for each time point versus control or between Mcpt4^-/- and Mcpt4 ^+/+ mice at specific time points. P values ≤ 0.05 were considered significant. *P ≤ 0.05, **P ≤ 0.01; ***P ≤ 0.001, **** P ≤ 0.0001.

Figure 6

Ileal cytokines and chemokines in P. y. yoelii 17XNL-infected Mcpt4^-/- and Mcpt4 ^+/+ mice and uninfected controls. (A–L) The x axis represents the time points in days after infection, and the y axis represents the concentrations of IL-12p40 (A), IL-4 (B), IL-12p40 (C), IL-10 (D), KC (CXCL1) (E), RANTES (CCL5) (F), MCP-1 (CCL2) (G), MIP1α (CCL3) (H), MIP1β (CCL4) (I), IL-6 (J), IL-17 (K) and IL-2 (L). Each dot represents a single mouse. Cytokines levels in ileum were determined for the three biological replicates (n=11 mice per group per time point). Data were analyzed with the Kruskal-Wallis test followed by Dunn’s multiple comparison for each time point versus control or between Mcpt4^-/- and Mcpt4 ^+/+ mice at specific time points. P values ≤ 0.05 were considered significant. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Figure 7

Network visualization of Mcpt4^-/- and Mcpt4 ^+/+ mouse phenotypes during P. y. yoelii 17XNL infection. Network visualization of significant correlations ( Figure S3 ) at different times post-infection in Mcpt4^-/- mice (left) and Mcpt4 ^+/+ mice (right). The size of a circular node represents relative fold change (larger circle = larger fold change) of a parameter calculated as average levels in infected mice/average levels in control uninfected mice. The border width represents the degree or number of connections (the higher the number of connected edges, the thicker the edge). Parasitemia and 16S nodes are presented in gray, blue nodes are pro-inflammatory cytokines and chemokines (type-1 immune response), pink nodes are anti-inflammatory or regulatory cytokines and chemokines (type-2 immune response), green nodes are type-17 immunity-related cytokines and chemokines, orange nodes represent cells and cells markers (MCs, Mcpt1, MPO and NE). Gray strokes connect nodes with positive correlations and red strokes reflect negative correlations. Increasing stroke line width reflects increasing Spearman’s correlation value.

Figure 8

Transmission of P. y. yoelii 17XNL from infected Mcpt4^-/- and Mcpt4 ^+/+ mice to Anopheles stephensi. Panels (A, B), respectively, represent the numbers of oocysts per midgut and proportions of infected mosquitoes in three independent biological replicates. Panels (C, D), respectively, illustrate the percentages of mouse erythrocytes infected with asexual stage P. y. yoelii 17XNL and sexual stage gametocytes on the day of mosquito infection (day 3 PI). Parasite transmission was determined in three biological replicates (n=7 mice per group, 30 fed mosquitoes per mouse). The numbers of midgut oocysts in mosquitoes fed on Mcpt4^-/- and Mcpt4 ^+/+ infected mice were analyzed by Mann Whitney test. The percentages of infected mosquitoes (mosquitoes with zero oocysts excluded) were analyzed with Fisher’s exact test. Parasitemia and gametocytemia data were analyzed using unpaired t tests. P values ≤ 0.05 were considered significant. *P ≤ 0.05, **P ≤ 0.01.