<i>T. cruzi</i> infection induces MBL expression <i>in vivo</i>.

<p>WT and MyD88^−/− mice were infected with trypomastigotes of the Y strain of <i>T. cruzi</i> and 9 days after infection, MBL-A and MBL-C mRNA accumulation was determined in spleens from infected and uninfected animals by real-time PCR (A). Detection of MBL-C protein was also performed by immunofluorescence microscopy in splenic sections obtained from WT mice infected as above (B). Micrograph shows MBL-C (red) and B220 (blue) staining in both the red (RP) and white pulp (WP). Scale bar, 100 µm. Infected MBL^−/− spleens were included as a control. All panels shown are representative of 2 independent experiments using 3 to 4 mice per group. Bars indicate the standard error of the mean (SEM). Sera from patients with acute, chronic or indeterminate Chagas’ disease were assayed for MBL as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047835#s4" target="_blank">Material and Methods</a> (C). *, Indicates statistically significant differences between acute vs indeterminate Chagas’ disease groups.</p

Enhanced cardiac pathology and fibrosis in MBL^−/− infected with <i>T.</i><i>cruzi</i> Colombiana strain.

<p>WT and MBL^−/− mice were infected with trypomastigotes of the Colombiana strain of <i>T. cruzi</i> and hearts from these animals obtained 5 weeks after infection. Hearts were formalin-fixed, paraffin embedded, stained with H&E or Gomori’s trichrome and scored for pathology and fibrosis, respectively, as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047835#s4" target="_blank">Materials and Methods</a> (A). Right panels show micrographs of Gomori’s trichrome staining for each group. Bars indicate the standard error of the mean (SEM). Uninfected, or WT and MBL^−/− mice infected as in (A) were harvested 5 wks later for determination of hydroxyproline content in total heart (B). Each symbol corresponds to an individual animal, dashed lines represent the mean of each group and solid lines the standard error of the mean (SEM). Heart samples from uninfected controls or from WT and MBL^−/− mice infected as in (A) were obtained and mRNA accumulation of Collagen-1, -3 and -6 determined by real-time PCR (C). The experiments shown are representative of two performed. *, Indicates statistically significant differences between infected WT and MBL^−/− groups.</p

Macrophage populations in livers of <i>S. mansoni</i>-infected IL-4Rα^flox/ΔLysM^Cre mice express <i>Il4r</i>α and alternative activation markers.

<p>IL-4Rα^flox/ΔLysM^Cre mice (open bars) and IL-4Rα^flox/Δ littermate controls (solid bars) were infected percutaneously with 35 cercariae. From mice infected for 9 weeks, CD45+ SiglecF- CD11b+ Ly6G- F4/80+ CD64+ liver leukocytes were sorted and separated based on Ly6C expression with a flow cytometer. Gene expression was measured by qPCR (n = 3; *p<0.05, ***p<0.001). Fold change is displayed relative to gene expression from CD45+ SiglecF- CD11b+ Ly6G- F4/80+ CD64+ Ly6C+ cells sorted from naïve IL-4Rα^flox/Δ littermate control livers. Data shown are mean ±SEM and represent at least two independent experiments.</p

A population of inflammatory IL-4Rα-expressing myeloid cells resists LysM^Cre-mediated deletion.

<p>BALB/c, IL-4Rα^flox/Δ, and IL-4Rα^flox/ΔLysM^Cre mice were injected i.p. with 2 ml thioglycollate 4 d prior to harvest or were left untreated (naïve). Peritoneal cells were harvested from each group, stimulated for 30 min with 20 ng/ml IL-4 (black outline), and compared to unstimulated cells (solid gray). IL-4Rα function was assessed by IL-4-induced phosphorylation of STAT6 using flow cytometry. A, D. Gating strategy for lymphocytes and F4/80^hi CD11b^hi macrophages. Detection of pSTAT6 in lymphocytes (B, E) and macrophages (C, F). Each histogram peak represents an individual mouse (n = 2–6 for 2 independent experiments). G. DNA was isolated from F4/80^hi CD11b^hi macrophages FACS sorted from naïve and thioglycollate-treated IL-4Rα^flox/flox, IL-4Rα^flox/Δ, IL-4Rα^flox/ΔLysM^Cre and IL-4Rα^Δ/Δ mice. Rearrangement of the <i>Il4rα</i> locus was measured using PCR to compare the presence of wild-type (WT) and knockout (KO) <i>Il4rα</i> alleles. Quantification of band intensity is shown in the right panels. Aggregate intensity of WT product plus KO product was normalized to 100 percent for each sample. H. In a separate experiment, CD11b⁺ F4/80⁺ macrophages were sorted from the same naïve or thioglycollate-elicited peritoneal cells. <i>Lyz2</i> gene expression was found to be lower in thioglycollate-elicited macrophages than naïve macrophages (n = 2–5 for 2 independent experiments). Data shown are mean ±SEM and represent two independent experiments (*p<0.05).</p

Normal expression of AAM-associated genes in IL-4Rα^flox/ΔLysM^Cre liver.

<p>IL-4Rα^flox/ΔLysM^Cre mice (open bars) and IL-4Rα^flox/Δ littermate controls (solid bars) were infected percutaneously with 35 cercariae. Expression of selected genes was measured by qPCR in liver tissue 9 weeks (A) and 16 weeks (B) post-infection and normalized to expression in naïve littermate control tissue (n = 7–15; p>0.05 except where noted, **p<0.01). Data shown are mean ±SEM and represent two independent experiments.</p

<i>Lyz2</i>^lo macrophages develop features of AAMs in response to <i>S. mansoni</i> eggs.

<p>IL-4Rα^flox/ΔLysMCre mice (open bars) and littermate controls (solid bars) were left untreated (naïve), challenged with 5000 <i>S. mansoni</i> eggs i.p. 4 days before harvest (1^o), 18 days before harvest (1^o-rested), or challenged on both 18 days and 4 days before harvest (1^o-rechallenged). A. Total peritoneal cells were sorted for F4/80^hi CD11b^hi cells at a purity of >90%. B. Representative 20× images of sorted F4/80^hi CD11b^hi macrophages after cytospin and hematoylin and eosin staining. C,D. The sorted cells were assayed for <i>Il4rα</i> and <i>Lyz2</i> gene expression (C), and gene expression of markers of alternative activation (D). Fold change in gene expression is shown relative to the expression levels in sorted F4/80^hi CD11b^hi cells from naïve littermate controls. E. Surface expression of mannose receptor measured by flow cytometry on unsorted F4/80^hi CD11b^hi peritoneal cells from the same treatment groups. F. Arginase activity in sorted macrophages. (n = 3–6, ns = not significant) Data shown are mean ±SEM and represent at least two independent experiments.</p

Inflammation but not fibrosis is exacerbated in chronically infected IL-4Rα^flox/ΔLysM^Cre mice.

<p>IL-4Rα^flox/ΔLysM^Cre mice and IL-4Rα^flox/Δ littermate controls were infected percutaneously with 35 <i>S. mansoni</i> cercariae. A–C. Representative 10× images of granuloma formation 9 weeks and 16 weeks post-infection from (A) hematoxylin and eosin-stained sections of intestinal tissue, (B) Giemsa-stained sections of liver tissue, or (C) picrosirius red-stained sections of liver tissue. D. Liver granuloma size in IL-4Rα^flox/ΔLysM^Cre (open bars) and IL-4Rα^flox/Δ littermate control (solid bars) mice. E. Liver fibrosis was assessed by hydroxyproline content, normalized to mass or worm pairs recovered by perfusion of infected mice through the portal vein. Data shown are mean ±SEM and represent two independent experiments (n = 15, ns = not significant).</p

Surviving chronic <i>S. mansoni</i> infection depends on <i>Il4rα</i> allele, not LysM^Cre expression.

<p>IL-4Rα^flox/ΔLysM^Cre mice (open circles and bars), IL-4Rα^flox/Δ littermate controls (solid circles and bars), IL-4Rα^flox/flox mice (solid squares), and IL-4Rα^Δ/Δ (open triangles) were infected percutaneously with 35 <i>Schistosoma mansoni</i> cercariae. A. Survival kinetics through 16 weeks (n = 10–20 per group). B. Th1 response. 9 or 16 weeks post-infection, liver leukocytes were isolated, restimulated with phorbol myristate acetate/ionomycin, stained for IFN-γ, and analyzed by flow cytometry (n = 7–15, ns = not significant). C. Hepatotoxicity. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assayed in serum 9 or 16 weeks after infection (n = 7–8 mice, ns = not significant). Data shown are mean ±SEM and represent at least two independent experiments.</p

Normal cytokine response in <i>S. mansoni</i>-infected IL-4Rα^flox/ΔLysM^Cre mice.

<p>IL-4Rα^flox/ΔLysM^Cre mice and IL-4Rα^flox/Δ littermate controls were infected percutaneously with 35 <i>S. mansoni</i> cercariae. A. Serum IL-13Rα2 levels were measured by ELISA 9 or 16 weeks post-infection. The open and solid portions of each bar correspond to unbound IL-13Rα2 and IL-13Rα2 bound to IL-13, respectively. B. Tissue cytokine levels. Expression of <i>il12p40</i> and <i>il10</i> was quantified by qPCR from liver tissue snips of IL-4Rα^flox/ΔLysM^Cre mice (open bars) or IL-4Rα^flox/Δ littermate controls (solid bars). C. Th2 response. Liver leukocytes were isolated from IL-4Rα^flox/ΔLysM^Cre mice (open bars) or IL-4Rα^flox/Δ littermate controls (solid bars), stimulated with phorbol myristate acetate/ionomycin, and analyzed by flow cytometry. The percentage of CD4⁺ leukocytes expressing intracellular IL-4 and IL-13 are shown. (n = 7-15 for each experiment, ns = not significant). Data shown are mean ±SEM and represent two independent experiments.</p

Distinct populations of alternatively activated macrophages control inflammation and fibrosis.

<p>Diagram showing the roles of various subsets of alternatively activated macrophages in the pathogenesis of schistosomiasis. In IL-4Rα^flox/ΔLysM^Cre mice, LysM^Cre results in the elimination of only mature IL-4Rα⁺Arg1⁺Lyz2^hi AAMs. The loss of this subset of AAMs results in the failure to downmodulate granulomatous inflammation in acute and chronic schistosomiasis. However, a substantial population of inflammatory IL-4Rα⁺Lyz2^lo AAMs is preserved in these mice that expresses Arg1 in response in response to IL-4 and IL-13 and slows the progression of fibrosis. In Arg1^flox/floxTie2^Cre mice, both populations of AAMs are defective because of the loss of Arg1 activity, leading to exacerbation of both egg-induced inflammation and fibrosis at both the acute and chronic stage of infection (far right panel). In comparison, Arg1^flox/ΔLysM^Cre primarily deletes Arg1 in the mature Lyz2^hi population, leading to a much more modest increase in fibrosis and granulomatous inflammation, which only reaches significance at the chronic stage of infection with <i>S. mansoni</i>. Wild-type IL-4Rα^flox/Δmice have both mature resident and inflammatory AAMs expressing IL-4Rα and Arg1 so both forms of pathology (inflammation and fibrosis) are substantially controlled in these mice (far left panel).</p