Clostridium difficile infection (CDI) is the most common cause of hospital-acquired infection in the United States. Host susceptibility and the severity of infection are influenced by disruption of the microbiota and the immune response. However, how the microbiota regulate immune responses to mediate CDI outcome remains unclear. Here, we have investigated the role of the microbiota-linked cytokine IL-25 during infection. Intestinal IL-25 was suppressed during CDI in humans and mice. Restoration of IL-25 reduced CDI-associated mortality and tissue pathology even though equivalent levels of C. difficile bacteria and toxin remained in the gut. IL-25 protection was mediated by gut eosinophils, as demonstrated by an increase in intestinal eosinophils and a loss of IL-25 protection upon eosinophil depletion. These findings support a mechanism whereby the induction of IL-25-mediated eosinophilia can reduce host mortality during active CDI. This work may provide targets for future development of microbial or immune-based therapies.

One example of this relationship is the cytokine interleukin-25 (IL-25), which is dependent on the microbiota, as germ-free and antibiotic-treated mice show decreased IL-25 production (). IL-25 is an inducer of type 2 immune responses and increased levels correlate with decreased IL-23 expression (). IL-25 is capable of inducing type 2 responses characterized by eosinophil, basophil, and mast cell accumulation systemically and at local sites of inflammation (). Although type 2 immunity is typically examined in the context of asthma, allergy, and helminth infection, the consequences of type 2 effector functions are versatile and can mediate pathogenic, protective, or regulatory responses given the environmental contexts (). In human CDI, low eosinophil numbers are a risk factor for persistent diarrhea or death and recurrent disease (). These observations prompt the possibility that microbiota regulation of IL-25 and type 2 immune responses may influence disease severity during CDI. Furthermore, they uncover a potential therapeutic target, which may help to guide future prebiotic and fecal transplant cocktail development to enhance IL-25 and type 2 responses. Since IL-25 is regulated by the microbiota and is expressed inversely to the cytokine IL-23, which is deleterious during CDI, we hypothesize that IL-25 is downregulated during CDI. Increasing its levels might thus reduce disease severity through influencing the immune response.

Identification of an interleukin (IL)-25-dependent cell population that provides IL-4, IL-5, and IL-13 at the onset of helminth expulsion.

In addition to the immune response, the status of the microbiota plays a fundamental role during CDI. The protective capabilities of healthy microbiota to both inhibit and resolve disease is emphasized by the lack of host susceptibility to C. difficile in the presence of intact microbiota and the recently demonstrated efficacy of fecal transplants in preventing relapses (). Despite the central role of both the microbiota and the immune response to regulate disease pathogenesis, the role of the microbiota in influencing the host immune response during CDI is unclear. Crosstalk between the microbiota and the immune system is critical for shaping both the immune response and the microbial composition of the gut.

CDI symptoms range from mild diarrhea to life-threatening pseudomembranous colitis and toxic megacolon. Recent studies indicate that increased inflammatory markers, such as IL-8, are more accurate at predicting poor patient outcome than increased bacterial burden, suggesting that the type and/or intensity of the immune response may control the severity of the disease (). In fact, numerous studies support a dual role for the immune response to CDI. For instance, innate mediators, such as MyD88 signaling, innate lymphoid cells (ILCs), leptin, and IL-22, have been observed to play a protective role during CDI in mice, yet inflammasome-driven IL-23 signaling is deleterious during CDI in mice (). Together these studies support a multifaceted role for the immune response during CDI.

Clostridium difficile infection (CDI) is currently the leading cause of hospital-acquired infection and gastroenteritis-associated deaths in the United States (). As a result, it has been listed as one of three urgent threats by the Centers for Disease Control and Prevention (CDC). Despite therapy, C. difficile causes an estimated 453,000 infections, 83,000 relapses, and 29,300 deaths annually, stressing the need for better treatment and management options (). This gram-positive, spore-forming anaerobic bacterium infects the colon when the normal microbiota have been disrupted, primarily through antibiotic use. Following colonization, the release of chief virulence factors, toxins A and B, causes epithelial cell rounding and death, compromising the integrity of the intestinal barrier. Therapy involves treatment with antibiotics such as vancomycin, fidaxomicin, or metronidazole (). In addition to effectively targeting C. difficile, these antibiotics can inhibit the reestablishment of beneficial endogenous flora, which may in part explain the high numbers of relapses and deaths associated with this disease.

To understand how eosinophils may be protecting against CDI-associated mortality, we investigated the impact of eosinophil depletion on the colonic intestinal epithelial barrier. Immunohistochemical staining ( Figure 6 A) and scoring ( Figure 6 B) of cecal tissue pathology were analyzed in IL-25-treated ± eosinophil-depleted mice on day 3 post-infection. IL-25 mice lacking eosinophils had increased epithelial destruction and cellular exudate comparable to levels seen in wild-type mice. Additionally, mice lacking eosinophils were the only group to have significantly elevated luminal albumin compared to protected IL-25-treated mice, demonstrating that these mice had the most severe barrier disruption during infection ( Figure 6 C). In line with these findings, IL-25-treated mice lacking eosinophils and PBS control mice had significantly shorter colon length, a measure of more severe colitis, when compared to IL-25-treated mice ( Figure 6 D). These data suggest that the loss of eosinophils permitted the most drastic intestinal tissue damage, despite the presence of IL-25. To rule out the possibility that eosinophils protected mice from mortality by a direct bactericidal function against C. difficile, we quantified C. difficile toxins A and B ( Figure 6 E) and bacterial burden ( Figure 6 F) in the cecal contents on day 3 of infection. We found comparable levels in all groups of mice, indicating that eosinophils do not alter the ability of C. difficile to colonize and produce toxins in the colon. Together, these data signify that eosinophils contribute to IL-25-mediated protection during CDI by protecting host tissue, rather than reducing the capabilities of the pathogen.

(D–F) Colon length (D) and toxin A/B level (E) and C. difficile bacterial burden (F) in cecal contents on day 3 of CDI. Data are from two combined experiments (n = 4–7 mice per group per experiment; mean ± SEM; ∗ p < 0.05 and ∗∗ p < 0.005).

(C) Albumin concentration in the cecal contents on day 3 of CDI. Data are from three combined experiments (n = 2–5 mice per infected groups per experiment; mean ± SEM; ∗ p < 0.05 and ∗∗ p < 0.005).

(A and B) H&E staining (A) and tissue pathology scores (B) of cecal tissue from C57BL/6J mice treated with PBS, rIL-25, or rIL-25 + anti-Siglecf on day 3 of CDI (scale bar, 20 μm). Data represent two combined experiments (n = 4–7 mice per group per experiment; mean ± SEM; ∗ p < 0.05 and ∗∗ p < 0.005).

Lastly, due to mounting evidence supporting a role for eosinophils in promoting IgA responses, we tested whether eosinophils may be protective by increasing antibody levels in the gut (). We measured on day 3 post-infection total IgA ( Figure S6 E) and IgG ( Figure S6 F) levels in the cecal contents of IL-25-treated mice with or without eosinophil depletion. IgG and IgA production were comparable despite the presence or absence of eosinophils, indicating eosinophils do not protect by increasing total IgA or IgG levels during the initial 3 days of CDI infection. Together these data signify that IL-4, mucin, IgA, and IgG responses were likely not responsible for the ability of eosinophils to reduce mortality and morbidity during CDI.

Increased mucin also was observed with IL-25 treatment during CDI. To test whether IL-25 protects via mucus induction, we compared muc2 gene expression ( Figure S6 C) and mucin by PAS histological analysis ( Figure S6 D) in the cecum of IL-25-treated mice with or without eosinophils on day 3 of infection. We did not observe differences in mucin production in the absence of eosinophils. Thus, we concluded that mucin is not likely to contribute to the protective capabilities of eosinophils.

IL-25 treatment led to enhanced IL-4 production. Since eosinophils were the primary source of IL-4 during CDI and were necessary for IL-25-mediated reduction in mortality, we speculated that IL-4 production may be the mechanism by which eosinophils reduce disease severity. To test this, we compared survival ( Figure S6 A) and morbidity ( Figure S6 B) in PBS control, IL-25-treated, and IL-25 + anti-IL4 monoclonal antibody-treated mice. Neutralization of IL-4 did not influence mortality but did slow disease resolution, suggesting that IL-4 does not play a role in IL-25-mediated enhanced survival during initial disease, but may be important during disease resolution.

IL-25-treated mice lacking eosinophils due to anti-SiglecF depletion experienced increased mortality ( Figure 5 A) and morbidity ( Figure 5 B), demonstrating that eosinophils were an essential downstream effector cell in IL-25-mediated protection. Interestingly, depletion of eosinophils in control mice did not influence mortality, suggesting that a more significant enhancement of eosinophilia to levels seen in IL-25-treated mice may be required for survival benefits. Second, we utilized PHIL mice, transgenic mice that genetically lack eosinophils, to assess the ability of IL-25 to protect in the absence of eosinophils (). PHIL mice and wild-type littermate controls were treated with PBS or IL-25 and assessed for survival rates ( Figure 5 C) and clinical scores ( Figure 5 D) during infection. In agreement with antibody-mediated depletion of eosinophils, PHIL mice could not be rescued from severe disease with IL-25 pretreatment, supporting the necessity of these cells in IL-25-mediated protection. In PHIL experiments, mice were treated with a sub-lethal dose of 10CFUs of C. difficile in order to delineate differences between genotypes. The decreased dose of C. difficile used to challenge mice in PHIL mice experiments likely explains the reduced mortality of wild-type PBS-treated mice when compared to wild-type mice used in other experiments. Interestingly, enhanced disease severity was observed in PHIL mice regardless of IL-25 treatment when compared to wild-type controls, consistent with the importance of eosinophils in CDI. We concluded that eosinophils were the cellular mechanism by which IL-25 protects against CDI mortality and morbidity.

(C and D) C57BL/6J or PHIL mice ± IL-25 treatment and infected with a sub-lethal dose of 10 3 CFUs of C. difficile assessed for survival (C) and clinical scores (D). Data represent two combined experiments (n = 5–10 mice per group; mean ± SEM; p value, ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.0005 compared to IL-25 + anti-SiglecF-infected mice).

(A and B) C57BL/6J mice ± IL-25 treatment given with 20 μg anti-SiglecF or isotype control 1 day prior and 1 day after infection with C. difficile and assessed for survival (A) and clinical scores (B) during infection. Data are representative of two experiments (n = 10 mice per group per experiment; mean ± SEM; ∗ p < 0.05).

IL-25 pretreatment decreased mortality and induced robust eosinophilia during CDI. Furthermore, increased eosinophils in the colon correlated with less severe clinical scores. This led us to hypothesize that eosinophils may be downstream of IL-25-mediated protection. Two distinct models where mice lacked eosinophils were utilized to test the hypothesis. First, PBS- and IL-25-treated mice were treated with either anti-SiglecF, an eosinophil-depleting monoclonal antibody, or an IgG isotype antibody. Anti-SiglecF treatment selectively depleted eosinophils, as demonstrated by a significant decrease in eosinophils ( Figure S5 A), but not neutrophils ( Figure S5 B) in the LP of the colon (). Similarly, total IL-4 ( Figure S5 C) and CD11bIL-4- ( Figure S5 D) expressing cells were significantly reduced with anti-SiglecF treatment. This was expected since eosinophils are the primary producers of IL-4 during CDI.

Neutrophils are considered the hallmark innate effector cell of human C. difficile infection, but IL-25 signaling is primarily associated with eosinophilia (). Therefore, we sought to identify the ability of IL-25 to modulate neutrophils, eosinophils, and monocytes in LP of the colon on day 3 of CDI. Infection increased the levels of both eosinophils ( Figure 4 A) and neutrophils ( Figure 4 B) compared to antibiotic treatment alone, indicating that both granulocyte subsets were recruited to the LP during infection. IL-25 selectively increased absolute cell numbers and percentages of eosinophils during CDI ( Figure 4 A). In contrast, IL-25 treatment did not influence numbers of neutrophils ( Figure 4 B) or Ly6cand Ly6cmonocytes ( Figure S3 ) during CDI. Increased eosinophilia by both measurements correlated with decreased clinical scores ( Figures 4 C and 4D), implying that eosinophilia may play a role in dampening CDI severity. These data demonstrated that IL-25 pretreatment selectively enhanced eosinophil, but not neutrophil, accumulation during CDI, and they prompted the hypothesis that eosinophils may play a role in IL-25-mediated protection.

(C and D) Absolute number (C) and percentage (D) of live eosinophils in the LP of PBS and IL-25-treated mice plotted against clinical scores on day 3 of CDI. Data are representative of three experiments (n = 4–6 mice per group per experiment).

(A and B) CD45 + CD11b + CD11c mid SiglecF + Ly6g − eosinophils (A) and CD45 + CD11b + Ly6g + Ly6c + neutrophils (B) were isolated from the colonic LP and quantified by flow cytometry for absolute numbers and percentage of live cells on day 3 of CDI. Representative flow plot shows neutrophils and eosinophils as a percentage of live cells gated from CD11b+ cells. Data represent three combined experiments (n = 4–6 mice per group per experiment; mean ± SEM; Student’s two-tailed t test, ∗∗ p < 0.005 and #p < 0.05 from uninfected PBS-treated group).

IL-25 Resulted in the Accumulation of Eosinophils, but Not Neutrophils in the LP of the Colon during CDI

IL-25 also has been demonstrated to enhance mucus production (). Periodic acid-Schiff (PAS) staining ( Figure 3 B) and scoring ( Figure 3 C) of the cecal tissue on day 3 revealed that IL-25 induced mucus production. RNA analysis of cecal tissue by qPCR confirmed increased transcripts for muc2, a gene that encodes a major component of mucin, in IL-25-treated mice ( Figure 3 D). These data prompted the hypothesis that IL-25 may protect the host by bolstering the physical mucus barrier lining the epithelial tissue or by inducing IL-4. From these data, we concluded that IL-25 decreased the deleterious cytokine IL-23 and increased IL-4 and mucin production during CDI.

To evaluate the cellular source of IL-4, we utilized flow cytometry to intracellularly stain and measure IL-4-producing cell populations in the colonic LP on day 3 of infection ( Figure S4 ). In agreement with protein levels, the absolute number of IL-4-producing cells was increased with IL-25 treatment ( Figure S4 A). The majority of IL-4-producing cells were CD11b Figure S4 B). Further examination revealed that IL-4cells were mainly CD11bSiglecF, identifying eosinophils as the primary source of this cytokine during CDI ( Figure S4 C).

The crosstalk between immune responses and the intestinal epithelial is critical to maintaining homeostasis in the gut (). Therefore, we hypothesized that IL-25-mediated epithelial tissue protection during CDI occurred through influences on the immune response. To understand how IL-25 shaped immunity during infection, we evaluated protein levels of inflammatory cytokines in cecal tissue on day 3 of infection ( Figure 3 A). IL-23 is known to have a deleterious role during CDI and also has been indicated to signal inversely of IL-25; thus, we examined whether IL-25 dampened IL-23 levels in the gut (). IL-25 treatment reduced IL-23 protein production in cecal lysates, but had no effect on downstream Th17-like cytokines IL-17 or IL-22 ( Figure 3 A). Therefore, we concluded that the reduction in IL-23 may partly contribute to IL-25-mediated protection, but there are likely additional immune mediators playing a role. Next we evaluated how IL-25 treatment manipulated two canonical type 2 cytokines, IL-4 and IL-13. IL-25 has historically enhanced both cytokines, yet we only observed increased production of IL-4 during CDI ( Figure 3 A). Conversely, we detected decreased IL-13 protein levels in the cecal tissue on day 3. To test if induction of IL-13 by IL-25 occurs earlier than day 3, cecal protein levels also were measured on days 0 and 2 without evidence of increased IL-13 protein with IL-25 treatment ( Figure S3 ). Our studies were done in antibiotic-treated and infected mice, which may explain the lack of IL-25 induction of IL-13 protein. Further investigation into the role of IL-13 and its protein levels in cecal contents and systemically during CDI is needed to establish the relevance of this observation.

(D) Fold change ofMUC2 mRNA in cecal tissue by qPCR relative to gapdh and actin. Data represent three combined experiments (n = 4–6 mice per group per experiment; mean ± SEM; ∗ p < 0.05).

(B and C) Periodic acid-Schiff (PAS) staining of mucins in control and IL-25-treated cecal sections (B, scale bar, 50 μM) and scoring on day 3 of C. difficile infection (C). Data represent two combined experiments (n = 4–6 mice per group per experiment; mean ± SEM; ∗∗ p < 0.005).

(A) ELISA analysis of protein expression of type 17 and type 2 cytokine cecal tissue of C57BL/6J mice on day 3 of C. difficile infection. Data represent two combined experiments (n = 6–8 mice per group per experiment; mean ± SEM; ∗ p < 0.05 and ∗∗ p < 0.005).

Restoration of IL-25 led to significant decreases in mortality ( Figure 2 A) and morbidity ( Figure 2 B), indicating that IL-25 repletion leads to host protection. IL-25 pretreatment also was capable of reducing CDI-associated morbidity in two additional models of CDI, including a spore challenge (strain VPI10643) ( Figures S2 A and S2B) and challenge with a second toxin A and B producing C. difficile strain (strain 630) ( Figures S2 C and S2D). IL-25- and PBS-treated mice surprisingly had similar levels of C. difficile CFUs ( Figure 2 F) and virulence factors, toxins A and B ( Figure 2 E), in the cecal contents, indicating that IL-25 does not protect by influencing the ability of the pathogen to expand or produce toxins in the gut. Immunohistological evaluation of the cecum at day 3 post-infection showed that IL-25 significantly decreased CDI-associated tissue pathology ( Figures 2 C and 2D). IL-25 treatment decreased cellular exudate and inflammatory cell numbers in the LP, but the most profound impact was the prevention of epithelial cell disruption at the intestinal barrier. We concluded that IL-25 reduced disease severity by protecting host tissue and maintaining the integrity of the epithelium, rather than by dampening C. difficile growth or toxin production.

(E and F) Toxin A/B levels (E) and C. difficile bacterial burden in cecal contents (F) on day 3 post C. difficile. Data represent two combined experiments (n = 4–7 mice per group per experiment; mean ± SEM; ∗ p < 0.05 and ∗∗ p < 0.005).

(C and D) Representative H&E-stained cecal sections (C) of mice on day 3 after infection with C. difficile (scale bar, 100 μM) and pathology scores (D) are shown.

(A and B) Survival (A) and clinical scores (B) over the initial 6 days of infection. Data represent four combined experiments (n = 6–10 mice per group per experiment; mean ± SEM; ∗ p < 0.05 and ∗∗∗ p < 0.0005).

C57BL/6J mice were treated with a daily dose of either 0.5 μg recombinant IL-25 protein or PBS daily for 5 days prior to infection with C. difficile.

The microbiota can prevent susceptibility to CDI by outcompeting the pathogen and inducing host factors, yet potentially beneficial influences of the microbiota to modulate the immune response and regulate CDI severity remain unknown (). Our observation of decreased IL-25 protein during CDI suggested that IL-25 regulation of type 2 immunity could be a downstream mechanism of microbiota-mediated protection. To address this question, we tested if repletion of IL-25 could reduce disease severity in the setting of antibiotics and active CDI. Mice were treated with a daily dose of 0.5 μg recombinant IL-25 or PBS daily for 5 days prior to infection ( Figure S1 ). Protection was assessed by mortality and a clinical scoring system of morbidity ().

IL-25 expression was evaluated by both immunohistochemical staining of the cecum and total protein in cecal lysates. IL-25 expression by both analyses was suppressed by antibiotics but further diminished on day 3 of CDI ( Figures 1 C and 1D). These data suggest that the environment created by CDI not only sustains but also further decreases IL-25 protein levels compared to antibiotic treatment alone. Separation of the colonic epithelial from the lamina propria (LP) indicated that IL-25 protein was found primarily in epithelial cells ( Figure 1 E). Additionally, epithelial cell-specific IL-25 was similarly reduced from antibiotic-treated levels on day 3 of CDI ( Figure 1 F). In contrast, IL-25 protein was observed in both epithelial cell and cells infiltrating the LP in human biopsies, suggesting IL-25 expression might differ between humans and mice. This observation requires further investigation; but, regardless of human and mouse cell differences in IL-25 expression, IL-25 protein expression was decreased during CDI in both human and mice. Overall, these data indicate that epithelial cells in mice are the primary source of IL-25 protein and that CDI significantly decreases IL-25 levels from antibiotic treatment alone.

We wished to understand if the reduction in IL-25 expression during human CDI was due to antibiotic treatments that CDI patients were likely receiving or the infection itself. IL-25 protein was measured in the cecum of C57BL/6J mice that were untreated; given only antibiotics; or on days 1, 2, and 3 post-C. difficile infection. Our infection model consists of antibiotic treatment in order to render mice susceptible to infection, followed by gavage with ∼10–10colony-forming units (CFUs) of vegetative C. difficile (strain VPI10643) ().

The presence of healthy microbiota has been shown to both prevent susceptibility to and resolve active CDI. The expression of the type 2 cytokine IL-25 is dependent on the microbiota, as demonstrated by its diminished expression in antibiotic-treated and germ-free mice (). Therefore, we hypothesized that IL-25 protein expression is decreased during CDI. To evaluate IL-25 protein regulation during human CDI, we stained colon biopsies of CDI-negative (−) and CDI-positive (+) patients ( Figure 1 A; Table S1 ), and we scored for IL-25 staining ( Figure 1 B). Significant reductions in IL-25 expression were observed in CDI patients when compared to controls.

(E and F) Lamina propria (LP) and epithelial cells (ECs) in the colon were separated and analyzed for IL-25 protein from untreated, antibiotic only, and day 3 post-C. difficile-infected mice. (E) Data represent IL-25 protein from combined time points. (F) Data represent IL-25 protein in the epithelium alone on each time point (n = 4–10 per group; mean ± SEM; ∗ p value < 0.05).

(D) IL-25 protein in mouse cecal tissue measured by ELISA. Data represent two combined experiments (n = 5–8 mice per group per experiment; mean ± SEM; p value from antibiotic-treated mice, ∗ p < 0.05; p value from untreated mice, #p < 0.05, ##p < 0.005, and ###p < 0.0005).

(C) Representative immunohistochemical staining for IL-25 in ceca of C57BL/6J mice that were untreated (UT), antibiotic treated (ABX), or infected with C. difficile is shown (scale bar, 100 μM).

Discussion

This work demonstrates that repletion of IL-25 protected from CDI-associated mortality and morbidity through the action of gut eosinophils. We discovered that IL-25, a cytokine regulated by the microbiota, was repressed in the colon of humans and mice with CDI. Restoration of IL-25 reduced disease severity, despite the presence of equivalent levels of C. difficile and toxins in the gut lumen. IL-25 treatment reduced mortality and morbidity and enhanced integrity of gut epithelial barrier in an eosinophil-dependent manner. Therefore, this work demonstrates a mechanism by which a microbiota-regulated cytokine can induce an innate eosinophilic response that protects the host epithelium and reduces mortality during CDI.

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Gold J.A. Interleukin 5 is protective during sepsis in an eosinophil-independent manner. Eosinophils were identified as the effector cell by which IL-25 signaling protected against CDI-associated mortality. While previously shown to be protective against gut helminth infection, the role of eosinophils in CDI was unanticipated. In human CDI, peripheral eosinophils have been associated with protection from persistent diarrhea and death, which supports our finding in mice of their protective role (). Currently, eosinophils remain heavily examined in the context of allergy, asthma, and parasitic infection, while our understanding of their role in the broader context of bacterial infections remains incomplete. Although there has been evidence of eosinophils having antibacterial capabilities in vitro, in vivo correlatives of their role in bacterial infections are limited (). Since eosinophils did not reduce the burden of the pathogen, it is likely that their action occurred downstream and involved maintaining the intestinal barrier.

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Berek C. Eosinophils promote generation and maintenance of immunoglobulin-A-expressing plasma cells and contribute to gut immune homeostasis. Eosinophils have several effector functions that may be beneficial to protecting host tissue during CDI. First, eosinophils may protect the host by regulating immune responses to promote a balanced inflammatory environment that effectively combats the pathogen but prevents off-target host tissue destruction. This is plausible, as the immune response has a multifaceted role during disease and different immune mediators play a protective or pathogenic role during CDI (). Eosinophils previously have been demonstrated to promote a beneficial immune response in the colon. For instance, in a model of dextran sodium sulfate (DSS)-induced colitis, eosinophils reduced intestinal pathology by dampening inflammatory mediators in the colon via the lipid mediator protectin D1 (). Likewise, recent reports indicate that eosinophils specific to the LP are capable of inducing the development of regulatory T cells (Treg) and play an important role in maintaining gut homeostasis by promoting IgA responses ().

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Rothenberg M.E. Eosinophils in mucosal immune responses. In our model, it is possible that the environment in the colon created by enhanced eosinophils may functionally influence other immune mediators to result in a balanced immune response that is beneficial to host outcome. This hypothesis is supported by our results that IL-25 can selectively reduce deleterious IL-23, but does not influence downstream cytokine IL-22, which has been demonstrated to have a protective role during CDI (). Eosinophils also may protect host tissues through their well-documented ability to remodel and repair host tissue, limiting pathogen or commensal entry into the LP. Likewise, eosinophils may protect the host by facilitating rapid wound healing responses after disruption by the pathogen (). Thus, IL-25-mediated eosinophilia may protect against CDI-associated mortality by creating a balance between proinflammatory and tolerogenic immune responses and/or by inducing tissue remodeling and repair pathways to strengthen the epithelial barrier.

Finally, it remains unclear whether the ability of eosinophils to reduce mortality is specific to the IL-25 signal, or if other cytokines and chemokines that promote eosinophilia also are capable of protecting the host. Our data demonstrate that, in wild-type infection where IL-25 signal is diminished, depletion of eosinophils does not influence host mortality, suggesting that the eosinophils recruited during CDI in the absence of IL-25 treatment are not sufficient to reduce the severity of disease. It is possible that robust eosinophilia to levels higher than those seen in wild-type infection is necessary to reduce mortality and that any eosinophilia-promoting cytokine is capable of protecting the host. Alternatively, it is conceivable that IL-25 functions not only to support eosinophilia in the gut but directly or indirectly primes eosinophils to function in a manner that is protective toward host tissue. Further examination is required to understand how IL-25 influences eosinophils to mediate protection during CDI.

Overall, our study identifies IL-25 as a component of the immune response that is regulated by healthy microbiota and reduces pathology associated with CDI. We identified an essential role for eosinophils in this process. Enhanced mortality, relapse rates, and increased prevalence of CDI in the United States stress the need for better therapies and management strategies. Modulating the innate immune response to reduce CDI-associated pathology may offer advantages to currently inadequate antibiotic therapies, and, by acting downstream of the microbiota, it may complement microbial-based therapeutic development.