Elaine M. Hanson

IL-6-dependent mucosal protection prevents establishment of a microbial niche for attaching/effacing lesion-forming enteric bacterial pathogens

Enteric infections with attaching/effacing lesion-inducing bacterial pathogens are a worldwide health problem. A murine infection model with one such pathogen, Citrobacter rodentium, was used to elucidate the importance of the pleiotropic immune regulator, IL-6, in the pathogenesis of infection. IL-6 was strongly induced in colonic epithelial cells and macrophages upon C. rodentium infection and was required for effective host defense, because mice lacking IL-6 failed to control bacterial numbers 2–3 wk after infection and exhibited increased mortality. IL-6 was not needed for mounting effective T and B cell responses to the pathogens, nor was it important for induction of IFN-γ or TNF-α, cytokines involved in host defense against the bacteria, or the antibacterial effector, NO. Instead, IL-6 played a key role in mucosal protection, since its absence was associated with marked infection-induced apoptosis in the colonic epithelium and subsequent ulcerations. Cell culture studies confirmed that IL-6 protected colon epithelial cells directly against inducible apoptosis, which was accompanied by increased expression of an array of genes encoding antiapoptotic proteins, including Bcl-xL, Mcl-1, cIAP-2, and Bcl-3. Ulcerations appeared to be pathogenetically important, because bacteria localized preferentially to those regions, and chemically induced colonic ulcerations promoted bacterial colonization. Furthermore, blood components likely present in ulcer exudates, particularly alanine, asparagine, and glycine, promoted bacterial growth. Thus, IL-6 is an important regulator of host defense against C. rodentium by protecting the mucosa against ulcerations which can act as a microbial niche for the bacteria.

CXCR2-Dependent Mucosal Neutrophil Influx Protects against Colitis-Associated Diarrhea Caused by an Attaching/Effacing Lesion-Forming Bacterial Pathogen

Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrheal disease in young children, yet symptoms and duration are highly variable for unknown reasons. Citrobacter rodentium, a murine model pathogen that shares important functional features with EPEC, colonizes mice in colon and cecum and causes inflammation, but typically little or no diarrhea. We conducted genome-wide microarray studies to define mechanisms of host defense and disease in C. rodentium infection. A significant fraction of the genes most highly induced in the colon by infection encoded CXC chemokines, particularly CXCL1/2/5 and CXCL9/10, which are ligands for the chemokine receptors CXCR2 and CXCR3, respectively. CD11b+ dendritic cells were the major producers of CXCL1, CXCL5, and CXCL9, while CXCL2 was mainly induced in macrophages. Infection of gene-targeted mice revealed that CXCR3 had a significant but modest role in defense against C. rodentium, whereas CXCR2 had a major and indispensable function. CXCR2 was required for normal mucosal influx of neutrophils, which act as direct antibacterial effectors. Moreover, CXCR2 loss led to severe diarrhea and failure to express critical components of normal ion and fluid transport, including ATPase β2-subunit, CFTR, and DRA. The antidiarrheal functions were unique to CXCR2, since other immune defects leading to increased bacterial load and inflammation did not cause diarrhea. Thus, CXCR2-dependent processes, particularly mucosal neutrophil influx, not only contribute to host defense against C. rodentium, but provide protection against infection-associated diarrhea.

IL-17A promotes protective IgA responses and expression of other potential effectors against the lumen-dwelling enteric parasite Giardia

Giardia lamblia is a leading protozoan cause of diarrheal disease worldwide. It colonizes the lumen and epithelial surface of the small intestine, but does not invade the mucosa. Acute infection causes only minimal mucosal inflammation. Effective immune defenses exist, yet their identity and mechanisms remain incompletely understood. Interleukin (IL)-17A has emerged as an important cytokine involved in inflammation and antimicrobial defense against bacterial pathogens at mucosal surfaces. In this study, we demonstrate that IL-17A has a crucial function in host defense against Giardia infection. Using murine infection models with G. muris and G. lamblia, we observed marked and selective induction of intestinal IL-17A with peak expression after 2 weeks. Th17 cells in the lamina propria and innate immune cells in the epithelial compartment of the small intestine were responsible for the IL-17A response. Experiments in gene-targeted mice revealed that the cytokine, and its cognate receptor IL-17RA, were required for eradication of the parasite. The actions of the cytokine were mediated by hematopoietic cells, and were required for the transport of IgA into the intestinal lumen, since IL-17A deficiency led to marked reduction of fecal IgA levels, as well as for increased intestinal expression of several other potential effectors, including β-defensin 1 and resistin-like molecule β. In contrast, intestinal hypermotility, another major antigiardial defense mechanism, was not impacted by IL-17A loss. Taken together, these findings demonstrate that IL-17A and IL-17 receptor signaling are essential for intestinal defense against the important lumen-dwelling intestinal parasite Giardia.

Trp53 Deficiency Protects against Acute Intestinal Inflammation

The p53 protein has not only important tumor suppressor activity but also additional immunological and other functions, whose nature and extent are just beginning to be recognized. In this article, we show that p53 has a novel inflammation-promoting action in the intestinal tract, because loss of p53 or the upstream activating kinase, ATM, protects against acute intestinal inflammation in murine models. Mechanistically, deficiency in p53 leads to increased survival of epithelial cells and lamina propria macrophages, higher IL-6 expression owing to enhanced glucose-dependent NF-κB activation, and increased mucosal STAT3 activation. Blockade or loss of IL-6 signaling reverses the protective effects of p53 deficiency. Conversely, IL-6 treatment protects against acute colitis in a manner dependent on STAT3 signaling and induction of cytoprotective factors in epithelial cells. Together, these results indicate that p53 promotes inflammation in the intestinal tract through suppression of epithelium-protective factors, thus significantly expanding the spectrum of physiological and immunological p53 activities unrelated to cancer formation.

Development of Functional Microfold (M) Cells from Intestinal Stem Cells in Primary Human Enteroids

Background & Aims Intestinal microfold (M) cells are specialized epithelial cells that act as gatekeepers of luminal antigens in the intestinal tract. They play a critical role in the intestinal mucosal immune response through transport of viruses, bacteria and other particles and antigens across the epithelium to immune cells within Peyer’s patch regions and other mucosal sites. Recent studies in mice have demonstrated that M cells are generated from Lgr5+ intestinal stem cells (ISCs), and that infection with Salmonella enterica serovar Typhimurium increases M cell formation. However, it is not known whether and how these findings apply to primary human small intestinal epithelium propagated in an in vitro setting. Methods Human intestinal crypts were grown as monolayers with growth factors and treated with recombinant RANKL, and assessed for mRNA transcripts, immunofluorescence and uptake of microparticles and S. Typhimurium. Results Functional M cells were generated by short-term culture of freshly isolated human intestinal crypts in a dose- and time-dependent fashion. RANKL stimulation of the monolayer cultures caused dramatic induction of the M cell-specific markers, SPIB, and Glycoprotein-2 (GP2) in a process primed by canonical WNT signaling. Confocal microscopy demonstrated a pseudopod phenotype of GP2-positive M cells that preferentially take up microparticles. Furthermore, infection of the M cell-enriched cultures with the M cell-tropic enteric pathogen, S. Typhimurium, led to preferential association of the bacteria with M cells, particularly at lower inoculum sizes. Larger inocula caused rapid induction of M cells. Conclusions Human intestinal crypts containing ISCs can be cultured and differentiate into an epithelial layer with functional M cells with characteristic morphological and functional properties. This study is the first to demonstrate that M cells can be induced to form from primary human intestinal epithelium, and that S. Typhimurium preferentially infect these cells in an in vitro setting. We anticipate that this model can be used to generate large numbers of M cells for further functional studies of these key cells of intestinal immune induction and their impact on controlling enteric pathogens and the intestinal microbiome.

Attenuation of Intestinal Inflammation in Interleukin-10-Deficient Mice Infected with Citrobacter rodentium

ABSTRACT Interleukin-10 (IL-10) curtails immune responses to microbial infection and autoantigens and contributes to intestinal immune homeostasis, yet administration of IL-10 has not been effective at attenuating chronic intestinal inflammatory conditions, suggesting that its immune functions may be context dependent. To gain a broader understanding of the importance of IL-10 in controlling mucosal immune responses to infectious challenges, we employed the murine attaching and effacing pathogen Citrobacter rodentium, which colonizes primarily the surfaces of the cecum and colon and causes transient mucosal inflammation driven by Th17 and Th1 T helper cells. Infection induced macrophage and dendritic cell production of IL-10, which diminished antibacterial host defenses, because IL-10-deficient mice cleared infection faster than wild-type controls. In parallel, the mice had less acute infection-associated colitis and resolved it more rapidly than controls. Importantly, transient C. rodentium infection protected IL-10-deficient mice against the later development of spontaneous colitis that normally occurs with aging in these mice. Genome-wide expression studies revealed that IL-10 deficiency was associated with downregulation of proinflammatory pathways but increased expression of the anti-inflammatory cytokine IL-27 in response to infection. IL-27 was found to suppress in vitro Th17 and, to a lesser degree, Th1 differentiation independent of IL-10. Furthermore, neutralization of IL-27 resulted in more severe colitis in infected IL-10-deficient mice. Together, these findings indicate that IL-10 is dispensable for resolving C. rodentium-associated colitis and further suggest that IL-27 may be a critical factor for controlling intestinal inflammation and Th17 and Th1 development by IL-10-independent mechanisms.

Coordinate Down-regulation of Adenylyl Cyclase Isoforms and the Stimulatory G Protein (Gs) in Intestinal Epithelial Cell Differentiation

The intestinal epithelium is dynamic, with proliferation of undifferentiated crypt cells balanced by terminal differentiation and cell death at the colon surface or small intestinal villus tips. Cyclic AMP, induced by agonists such as prostaglandin E2 and vasoactive intestinal polypeptide, promotes proliferation and ion secretion and suppresses apoptosis in intestinal epithelial cells. Here, we show that cell differentiation in a model intestinal epithelium leads to attenuation of cAMP production in response to G protein-coupled receptor and receptor-independent agonists. Concomitantly, key components of the cAMP cascade, the α subunit of the stimulatory G protein, Gs, and adenylyl cyclase (AC) isoforms 3, 4, 6, and 7 are down-regulated. By contrast, AC1, AC2, AC8, and AC9, and the receptors for prostaglandin E2 and vasoactive intestinal polypeptide, are not expressed or not affected by differentiation. We confirmed key findings in normal murine colon epithelium, in which the major AC isoforms and Gsα are markedly down-regulated in differentiated surface cells. Suppression of AC isoforms and Gsα is functionally important, because their constitutive expression completely reverses differentiation-induced cAMP attenuation. Thus, down-regulation of AC isoforms and Gsα is an integral part of the intestinal epithelial differentiation program, perhaps serving to release cells from cAMP-promoted anti-apoptosis as a prerequisite for cell death upon terminal differentiation.

Indispensable functions of ABL and PDGF receptor kinases in epithelial adherence of attaching/effacing pathogens under physiological conditions

Enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium are attaching-and-effacing (A/E) pathogens that cause intestinal inflammation and diarrhea. The bacteria adhere to the intestinal epithelium, destroy microvilli, and induce actin-filled membranous pedestals but do not invade the mucosa. Adherence leads to activation of several host cell kinases, including FYN, n-SRC, YES, ABL, and ARG, phosphorylation of the bacterial translocated intimin receptor, and actin polymerization and pedestal formation in cultured cells. However, marked functional redundancy appears to exist between kinases, and their physiological importance in A/E pathogen infections has remained unclear. To address this question, we employed a novel dynamic in vitro infection model that mimics transient and short-term interactions in the intestinal tract. Screening of a kinase inhibitor library and RNA interference experiments in vitro revealed that ABL and platelet-derived growth factor (PDGF) receptor (PDGFR) kinases, as well as p38 MAP kinase, have unique, indispensable roles in early attachment of EPEC to epithelial cells under dynamic infection conditions. Studies with mutant EPEC showed that the attachment functions of ABL and PDGFR were independent of the intimin receptor but required bacterial bundle-forming pili. Furthermore, inhibition of ABL and PDGFR with imatinib protected against infection of mice with modest loads of C. rodentium, whereas the kinases were dispensable for high inocula or late after infection. These results indicate that ABL and PDGFR have indispensable roles in early A/E pathogen attachment to intestinal epithelial cells and for in vivo infection with limiting inocula but are not required for late intimate bacterial attachment or high inoculum infections.

Visualizing the enteric nervous system using genetically engineered double reporter mice: Comparison with immunofluorescence

Background and aims The enteric nervous system (ENS) plays a crucial role in the control of gastrointestinal motility, secretion and absorption functions. Immunohistochemistry has been widely used to visualize neurons of the ENS for more than two decades. Genetically engineered mice that report specific proteins can also be used to visualize neurons of the ENS. The goal of our study was to develop a mouse that expresses fluorescent neuronal nitric oxide synthase (nNOS) and choline acetyltransferase (ChAT), the two proteins expressed in 95% of the ENS neurons. We compared ENS neurons visualized in the reporter mouse with the wild type mouse stained using classical immunostaining techniques. Methods Mice hemizygous for ChAT-ChR2-YFP BAC transgene with expression of the mhChR2:YFP fusion protein directed by ChAT promoter/enhancer regions on the BAC transgene were purchased commercially. The Cre/LoxP technique of somatic recombination was used to construct mice with nNOS positive neurons. The two mice were crossbred and tissues were harvested and examined using fluorescent microscopy. Immunostaining was performed in the wild type mice, using antibodies to nNOS, ChAT, Hu and PGP 9.5. Results Greater than 95% of the ENS neurons were positive for either nNOS or ChAT or both. The nNOS and ChAT neurons and their processes in the ENS were well visualized in all the regions of the GI tract, i.e., esophagus, small intestine and colon. The number of nNOS and ChAT neurons was approximately same in the reporter mouse and immunostaining method in the wild type mouse. The nNOS fluorescence in the reporter mouse was seen in both cytoplasm as well as nucleus but in the immunostained specimens it was seen only in the cytoplasm. Conclusion We propose that the genetically engineered double reporter mouse for ChAT and nNOS proteins is a powerful tool to study of the effects of various diseases on the ENS without the need for immunostaining.

Giardia Infection of the Small Intestine Induces Chronic Colitis in Genetically Susceptible Hosts

The lumen-dwelling protozoan Giardia is an important parasitic cause of diarrheal disease worldwide. Infection can persist over extended periods with minimal intestinal inflammation, suggesting that Giardia may attenuate host responses to ensure its survival, although clearance eventually occurs in most cases. IL-10 is an anti-inflammatory regulator critical for intestinal homeostasis and controlling host responses to bacterial exposure, yet its potential role in coordinating antiprotozoal host defense in the intestine is not known. In this study, we found that murine infection with the natural enteric pathogen Giardia muris induced a transient IL-10 response after 2–4 wk at the primary site of infection in the upper small intestine, but parasite colonization and eradication were not affected by the absence of the cytokine in gene-targeted mice. However, IL-10 was critical for controlling infection-associated immunological sequelae in the colon because severe and persistent diarrhea and colitis were observed in IL-10–deficient mice within 1–2 wk postinfection but not in uninfected littermate controls. Inflammation was characterized by epithelial hyperplasia, neutrophil and macrophage expansion, and Th1 induction and could be prevented by blockade of IL-12/IL-23 p40 but not depletion of CD11c+ dendritic cells. Furthermore, the intestinal microbiota underwent characteristic shifts in composition and was required for disease because antibiotics and loss of TLR signaling in MyD88-deficient mice protected against colitis. Together, our data suggest that transient infection by a luminal and seemingly noninflammatory pathogen can trigger sustained colitis in genetically susceptible hosts, which has broader implications for understanding postinfectious syndromes and other chronic intestinal inflammatory conditions.