Alternatively activated macrophages in intestinal helminth infection: effects on concurrent bacterial colitis

Abstract

The distribution of several pathogenic helminth infections coincides geographically with many devastating microbial diseases, including enteric bacterial infections. To dissect the mechanisms by which helminths modulate the host's response to enteric bacteria and bacteria-mediated intestinal inflammation, we have recently established a coinfection model and shown that coinfection with the helminth Heligmosomoides polygyrus exacerbates colitis induced by infection with the gram-negative bacterial pathogen Citrobacter rodentium. The disease severity of the coinfected mice was correlated with high Citrobacter loads in the gut, translocation of the bacteria into mucosal and systemic immune compartments, delayed bacterial clearance, and a significantly enhanced colonic TNF-alpha response. In the present study, using our in vivo coinfection model as well as in vitro approaches, we test the hypothesis that the phenotypic and functional alterations in macrophages induced by the helminth-driven T cell response may contribute to the observed alterations in the response to C. rodentium. We show that via a STAT6-dependent mechanism H. polygyrus coinfection results in a marked infiltration into the colonic lamina propria of F4/80+ cells that have the phenotype of alternatively activated macrophages. Functional analysis of these macrophages further shows that they are impaired in their killing of internalized bacteria. Yet, these cells produce an enhanced amount of TNF-alpha in response to C. rodentium infection. These results demonstrate that helminth infection can impair host protection against concurrent enteric bacterial infection and promote bacteria-induced intestinal injury through a mechanism that involves the induction of alternatively activated macrophages.

FIGURE 1

Helminth coinfection results in an increased number of macrophages in colonic LP. A–D, BALB/c mice were infected with H. polygyrus (200 L3) and inoculated with C. rodentium (5 × 10⁸ CFU) 7 days later. Uninfected control mice (A) and mice infected with C. rodentium (C.r.) (B), H. polygyrus (H.p.) (C), or both (Co-inf, coinfected) (D) were sacrificed 2 wk afer bacterial infection. Five-micrometer sections of frozen colonic tissue (in OCT) were cut and fixed in ice-cold acetone. After washing with PBS, the sections were blocked with PBS and 1% BSA. The tissue sections were incubated with anti-CD11b-FITC (green) and biotin-labeled anti-F4/80 Ab followed by streptavidin-Cy3. The sections were analyzed by immunofluorescence microscopy. Magnification, ×400. All images were digitized and cropped in Adobe Photoshop LE 5.0 (Adobe Systems). E, The mean number of positive cells detected in each high power field (magnification, ×200) was determined by counting 10 fields from each sample (samples from three mice per group were counted). F, Mice that were coinfected with H. polygyrus and C. rodentium have higher bacterial output in the fecal pellets. The data shown are the number of bacteria recovered from fecal samples of C. rodentium-infected mice and coinfected mice at 3 wk postinfection. The data are represented as the mean ± SEM (n = 5–10 mice at each time point).*, p < 0.05 for a comparison of coinfected group vs every other group. G and H, FACS data show that helminth coinfection enhances the expansion (mean ± SE, n = 3 per group) and activation of macrophages in MLN. MLN cells were collected from noninfected and infected mice and stained for macrophages with FITC-labeled Ab or costained with PE-labeled anti-Mac-1 and FITC-labeled anti MHC II for the determination of activation of the cells. H, FACS data shows MHC II (IA) expression levels on a gated Mac-1 cell population.

FIGURE 2

A primary infection of H. polygyrus (Hp) up-regulates Ym1, Arg1, and Fizz1 expression in macrophages (Mac) of multiple tissues. A–C, Macrophages (were purified from spleen, MLN, and colonic LP of helminth-infected mice at 3–4 wk after infection or from normal control mice. FACS analysis of the purified macrophages revealed that they were >95% F4/80⁺CD11b⁺ cells (data not show). D, Colonic tissues were collected from control and helminth-infected mice (7 days after H. polygyrus (H.p.) infection). Total RNA was isolated from the purified macrophages and colonic tissues. Ym1, Arg1, and Fizz1 expression was determined using realtime PCR. Values are the fold increase compared with baseline obtained from uninfected control mice. The data shown are from one of two experiments performed showing similar results.

FIGURE 3

Detection of alternatively and classically activated macrophages in colonic tissues. Colonic tissues were collected (2 wk after bacterial infection) from various groups. Total RNA was isolated. Gene expression for alternatively activated (Arg1, Fizz1 and Ym1) and classically activated macrophages (iNOS) of colonic tissues was determined using real-time RT-PCR. Values are the fold increase compared with baseline obtained from uninfected control mice. The data shown are from one of two experiments performed showing similar results. H.p., H. polygyrus; C.r., C. rodentium.

FIGURE 4

Helminth-induced increase in macrophage numbers in colonic LP of mice is mediated through a STAT6-dependent mechanism. Uninfected STAT6 KO (A) mice and STAT6 KO mice infected with C. rodentium (C.r.) (B), H. polygyrus (H.p) (C), or both (co-inf, coinfected) (D) were sacrificed 2 wk after bacterial infection. BALB/c mice infected with H. polygyrus (E) and coinfected (F) were included as controls. Magnification, ×400. Five-micrometer sections of frozen colonic tissue (in OCT) were cut, fixed, and stained for CD11b (green) and F4/80 (red) as described in the Fig. 1 legend.

FIGURE 5

H. polygyrus infection fails to induce the development and accumulation of alternatively activated macrophages in the colon of STAT6 KO mice. Colonic tissues were collected from uninfected STAT6 KO mice (Normal), STAT6 KO mice infected with C. rodentium (C.r.), H. polygyrus (H.p.), or both (H.p.-C.r.) (2 wk after bacterial infection). Total RNA was isolated from the colonic tissues. Gene expression for alternatively activated macrophages (Arg1, Fizz1, and Ym1) and classically activated macrophages (iNOS) of colonic tissues was determined using real-time RT-PCR. Values are the fold increase compared with baseline obtained from uninfected control mice. The data shown are from one of two experiments performed showing similar results.

FIGURE 6

H. polygyrus (Hp) infection impairs the bacterial killing capacity of macrophages (Mac). Macrophages were isolated from spleen, MLN (A) and peritoneal cavity (B), incubated overnight in complete DMEM, and then exposed to C. rodentium (or GFP-expressing C. rodentium, ×10 bacteria/cell) for 1 h. After the completion of the infection period, the cells were incubated with gentamicin-containing medium, which kills extracellular bacteria. A, Two and 6 h after antibiotic treatment, the number of viable internalized bacteria recovered in macrophages from spleen and MLN was determined by plating the cell lysates onto LB plates. The data shown is the mean ± SE of triplicate cultures. *, p < 0.05 for a comparison of cells isolated from uninfected mice vs cells obtained from helminth-infected mice. B, Immunofluorescence microscopy data show the number of internalized bacteria in peritoneal macrophages (stained with anti-F4/80 and Cy3) from H. polygyrus (H.p.)-infected and uninfected mice at 2 and 6 h after C. rodentium-GFP infection (p.i., postinfection). Peritoneal macrophages from H. polygyrus-infected mice display an impaired ability to control growth of internalized bacteria compared with cells from uninfected control mice. The data shown are from one of three experiments performed showing similar results.

FIGURE 7

Helminth-induced, alternatively activated macrophages (Mac) (CD206⁺) produce TNF-α in response to C. rodentium (C.r.) exposure. A and B, Macrophages were isolated from MLN of uninfected (Control) and H. polygyrus (Hp)-infected mice, incubated overnight in complete DMEM, and then exposed to C. rodentium for 1 h. Two (A) and 6 h (B) after infection, culture supernatants were collected. TNF-α production of MLN macrophages was measured by ELISA. The data shown are the mean ± SE of triplicate cultures. *, p < 0.05 for a comparison of cells isolated from uninfected mice vs cells obtained from helminth-infected mice. C–F, Colon tissues were prepared as in Fig. 1. The tissues were stained with anti-CD206 Ab (red) and anti-TNF-α (green). The immunofluorescence microscopic analysis showed that C. rodentium infection induced TNF-α production by cells other than CD206⁺ cells (D). H. polygyrus infection results in the induction of TNF-α producing cells that express the alternatively activated phenotype (CD206⁺) (E). Helminth and C. rodentium coinfection (F) resulted in a marked increase in alternatively activated macrophages that produce TNF-α in colon tissues. G, The mean number of TNF-α⁺, CD206⁺, and CD206⁺TNF-α⁺ cells detected in each high power field (×200) by counting five fields from each sample (three mice from each group were counted). Different letters represent significant differences ( p < 0.05). Co-inf, coinfected.

FIGURE 8

Helminth coinfection results in increased colonic inflammation due to impaired ability to control bacterial infection. Colon tissue was prepared from uninfected (A), C. rodentium-infected (B), helminth-and C. rodentium-coinfected (C), and coinfected and antibiotic-treated mice (D). Five-micrometer sections of frozen colonic tissue (in OCT) were cut, fixed, and stained with H&E. The figures shown are representative histology of the distal colon 2 wk after C. rodentium infection.