Development of Fatal Intestinal Inflammation in MyD88 Deficient Mice Co-infected with Helminth and Bacterial Enteropathogens

Infections with intestinal helminth and bacterial pathogens, such as enteropathogenic Escherichia coli, continue to be a major global health threat for children. To determine whether and how an intestinal helminth parasite, Heligomosomoides polygyrus, might impact the TLR signaling pathway during the response to a bacterial enteropathogen, MyD88 knockout and wild-type C57BL/6 mice were infected with H. polygyrus, the bacterial enteropathogen Citrobacter rodentium, or both. We found that MyD88 knockout mice co-infected with H. polygyrus and C. rodentium developed more severe intestinal inflammation and elevated mortality compared to the wild-type mice. The enhanced susceptibility to C. rodentium, intestinal injury and mortality of the co-infected MyD88 knockout mice were found to be associated with markedly reduced intestinal phagocyte recruitment, decreased expression of the chemoattractant KC, and a significant increase in bacterial translocation. Moreover, the increase in bacterial infection and disease severity were found to be correlated with a significant downregulation of antimicrobial peptide expression in the intestinal tissue in co-infected MyD88 knockout mice. Our results suggest that the MyD88 signaling pathway plays a critical role for host defense and survival during helminth and enteric bacterial co-infection

p40phox-Deficient Mice Exhibit Impaired Bacterial Clearance and Enhanced Pro-inflammatory Responses during Salmonella enterica serovar Typhimurium Infection

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of acute gastroenteritis in humans. During infection, reactive oxygen species (ROS), generated from NADPH oxidase (a multisubunit enzyme complex), are required for pathogen killing upon phagocytosis and for regulating pro-inflammatory signaling in phagocytic cells. Mutations in subunits forming the NADPH complex may lead to enhanced susceptibility to infection and inflammatory disease. Compared to other NADPH oxidase subunits, the function of p40phox is relatively understudied, particularly in the context of intestinal bacterial infection. In this study, we utilized genetically engineered mice to determine the role of p40phox in the response to S. Typhimurium infection. We show that mice lacking p40phox are more susceptible to oral infection with S. Typhimurium, as demonstrated by significantly enhanced bacterial dissemination to spleen and liver, and development of exacerbated bacterial colitis. Moreover, we demonstrate that the increased infection and disease severity are correlated with markedly increased F4/80+ macrophage and Ly6G+ neutrophil infiltration in the infected tissues, coincident with significantly elevated pro-inflammatory cytokines (IL-1β and TNF-α) and chemoattractant molecules in the infected tissues. Functional analysis of macrophages and neutrophils further shows that p40phox deficiency impairs bacteria- or PMA-induced intracellular ROS production as well as intracellular killing of Salmonella. These observations indicate that the p40phox subunit of NADPH oxidase plays an essential role in suppressing intracellular multiplication of Salmonella in macrophages and in the regulation of both systemic and mucosal inflammatory responses to bacterial infection

Development of fatal intestinal inflammation in MyD88 deficient mice co-infected with helminth and bacterial enteropathogens.

Infections with intestinal helminth and bacterial pathogens, such as enteropathogenic Escherichia coli, continue to be a major global health threat for children. To determine whether and how an intestinal helminth parasite, Heligomosomoides polygyrus, might impact the TLR signaling pathway during the response to a bacterial enteropathogen, MyD88 knockout and wild-type C57BL/6 mice were infected with H. polygyrus, the bacterial enteropathogen Citrobacter rodentium, or both. We found that MyD88 knockout mice co-infected with H. polygyrus and C. rodentium developed more severe intestinal inflammation and elevated mortality compared to the wild-type mice. The enhanced susceptibility to C. rodentium, intestinal injury and mortality of the co-infected MyD88 knockout mice were found to be associated with markedly reduced intestinal phagocyte recruitment, decreased expression of the chemoattractant KC, and a significant increase in bacterial translocation. Moreover, the increase in bacterial infection and disease severity were found to be correlated with a significant downregulation of antimicrobial peptide expression in the intestinal tissue in co-infected MyD88 knockout mice. Our results suggest that the MyD88 signaling pathway plays a critical role for host defense and survival during helminth and enteric bacterial co-infection

Helminth-Induced Alterations Of The Gut Microbiota Exacerbate Bacterial Colitis

Infection with the intestinal helminth parasite Heligmosomoides polygyrus exacerbates the colitis caused by the bacterial enteropathogen Citrobacter rodentium. To clarify the underlying mechanism, we analyzed fecal microbiota composition of control and helminth-infected mice and evaluated the functional role of compositional differences by microbiota transplantation experiments. Our results showed that infection of Balb/c mice with H. polygyrus resulted in significant changes in the composition of the gut microbiota, characterized by a marked increase in the abundance of Bacteroidetes and decreases in Firmicutes and Lactobacillales. Recipients of the gut microbiota from helminth-infected wide-type, but not STAT 6-deficient, Balb/c donors had increased fecal pathogen shedding and significant worsening of Citrobacter-induced colitis compared to recipients of microbiota from control donors. Recipients of helminth-altered microbiota also displayed increased regulatory T cells and IL-10 expression. Depletion of CD4+CD25+ T cells and neutralization of IL-10 in recipients of helminth-altered microbiota led to reduced stool C. rodentium numbers and attenuated colitis. These results indicate that alteration of the gut microbiota is a significant contributor to the H. polygyrus-induced exacerbation of C. rodentium colitis. The helminth-induced alteration of the microbiota is Th2-dependent and acts by promoting regulatory T cells that suppress protective responses to bacterial enteropathogens

Helminth co-infection exacerbates <i>C. rodentium-</i>induced colitis and mortality in MyD88 KO mice.

<p>MyD88 knockout and wild-type C57BL/6 mice were infected with <i>H. polygyrus</i> (200 L3) and inoculated with <i>C. rodentium</i> (5×10⁸ CFU) 7 days later. <b>A & C:</b> Body weight changes of wild-type (A) and MyD88 knockout (C) mice that were infected with <i>C. rodentium</i>, <i>H. polygyrus</i>, both <i>H. polygyrus</i> and <i>C. rodentium</i>, and normal control mice during the course of the experiment (8 days) are shown. Data shown are pooled from three independent experiments and are expressed as the body weight change as a percentage of the individual mouse initial body weight ± SE (n = 10–15) at each time point. <b>B &D:</b> Survival curve in wild-type (B) and MyD88 knockout (D) mice. <i>H. polygyrus</i> co-infection results in a significantly increased mortality in MyD88 KO mice.</p

Helminth co-infection results in decreased number of CD11b⁺ cells and antimicrobial peptide expression in colonic tissue.

<p><b>A–C</b>, MyD88 knockout mice were infected with <i>H. polygyrus</i> and inoculated with <i>C. rodentium</i> orally 7 days later. Mice infected with <i>C. rodentium</i> (C.r) (<b>A</b>), <i>H. polygyrus</i> (Hp) (B) and both (Co-inf) (<b>C</b>) were sacrificed. Histological sections of the colon were stained with anti-CD11b-FITC (green) and DAPI (blue), and analyzed by immunofluorescence microscopy. Magnification, ×100. <b>D</b>, Colonic tissue MPO level was determined by ELISA. <b>E–H</b>. Colon tissues were collected from <i>C. rodentium</i>-infected (<b>E</b>: MyD88 knockout; <b>F</b>: C57BL/B6) and co-infected (<b>G</b>: MyD88 knockout and <b>H</b>: C57BL/B6) mice and stained with anti-GR1-FITC (green), anti-F4/80 (red) and DAPI (blue), and analyzed by immunofluorescence microscopy. All images were digitized and cropped in Adobe Photoshop LE 5.0 (Adobe Systems). <b>I</b>–<b>L</b>:Colon tissues were collected from normal control, <i>C. rodentium</i>-infected, <i>H. polygyrus</i> infected, and co-infected wild-type C57BL/6 and MyD88 knockout mice. The expression of Arginase 1, a marker for alternatively active macrophages, was determined in MyD88 knockout mice (<b>I</b>) and B6 mice (<b>J</b>) using quantitative RT-PCR. iNOS expression was determined in MyD88 knockout mice (<b>K</b>) and B6 mice (<b>L</b>). Values are the fold increase compared with baseline obtained from uninfected mice. The data shown are the mean ± the SEM (n = 3–5 mice/group) from one of three experiments performed showing similar results. *p<0.05.</p

Helminth infection results in enhanced <i>C. rodentium</i> translocation in colonic tissue of MyD88 KO mice.

<p><b>A.</b> Immunofluorescence microscopic analysis of bacterial distribution and localization in the colonic tissues at day 5 post <i>C. rodentium</i> infection. Most of the bacteria were detected in the intestinal epithelial surface in MyD88 knockout mice with bacterial infection alone. An increased bacterial translocation was detected in MyD88 knockout mice with helminth-coinfection. Green: GFP-<i>C. rodentium</i>, Blue: DAPI. Red: CD11c in MLN. B. Numbers of bacteria recovered from fecal samples of <i>C. rodentium</i>-infected and co-infected MyD88 knockout mice at 7 days post-infection. The data shown are represented as the mean ± the SEM (n = 6 to 7 mice). *p<0.05.</p

Helminth co-infection exacerbates <i>C. rodentium-</i>induced colitis and intestinal injury in MyD88 knockout mice.

<p><b>A</b>. Macroscopic examination of colon tissues of MyD88 knockout mice from different treatment groups. Arrows indicate intestinal bleeding. B. Colon tissues were removed from uninfected mice or from mice infected with <i>H. polygyrus</i>, <i>C. rodentium</i>, or both two weeks after bacterial infection, frozen in Tissue Tek OCT compound, and the sections were stained with hematoxylin and eosin. <b>B</b>: Magnification, ×40 and <b>C</b>: Magnification, ×100. Duplicate samples are presented from co-infected and <i>C. rodentium</i>-infected MyD88 knockout mice. Co-inf (chronic): C57BL/6 and MyD88 knockout mice were pre-infected with <i>H. polygyrus</i> for 3 weeks and then co-infected with <i>C. rodentium</i>. Arrows indicate cellular infiltration and loss of intestinal architecture. <b>D</b>. Histopathological score of colonic inflammation in mice infected with <i>C. rodentium</i> or both. The scores were assessed by determination of infiltration of inflammatory cells (score range, 0 to 4), together with the evaluation of cecal tissue damage (score range, 0 to 4). The data shown are pooled from three independent experiments with total (n = 9 to 12 per group). *** p<0.001, **p<0.005.</p

Co-infection with <i>H. polygyrus</i> results in dysregulation of <i>Citrobacter-</i>induced colonic cytokines.

<p>Colon tissues were collected from normal control, <i>C. rodentium</i>-infected, <i>H. polygyrus</i> infected, and co-infected wild-type C57BL/6 and MyD88 knockout mice. Total RNA was isolated. The expression of KC, TNF-α, IL-10 and IL1β was determined using quantitative RT-PCR. Values are the fold increase compared with baseline obtained from uninfected mice. The data shown are the mean ± the SEM (n = 3–5 mice/group) from one of three experiments performed showing similar results. *p<0.05.</p

Co-infection with <i>H. polygyrus</i> results in down-regulation of <i>C. rodentium-</i>induced colonic anti-microbial peptide expression.

<p>Colon tissues were collected from control, <i>C. rodentium</i>-infected, <i>H. polygyrus</i> infected, and co-infected MyD88 knockout and wild-type mice. Total RNA was isolated. IL-22, Reg3γ and Reg3β expression was determined using quantitative RT-PCR. Values are the fold increase compared with baseline obtained from uninfected mice. The data shown are the mean ± the SEM (n = 3–5 mice/group) from one of three experiments performed showing similar results. *p<0.05.</p