Sara M. Dann

Opposing functions of IKKβ during acute and chronic intestinal inflammation

NF-κB is a key transcriptional regulator of inflammatory responses, but also controls expression of prosurvival genes, whose products protect tissues from damage and may thus act indirectly in an antiinflammatory fashion. The variable importance of these two distinct NF-κB-controlled responses impacts the potential utility of NF-κB inhibition as a treatment strategy for intractable inflammatory conditions, such as inflammatory bowel disease. Here, we show in murine models that inhibition of IKKβ-dependent NF-κB activation exacerbates acute inflammation, but attenuates chronic inflammatory disease in the intestinal tract. Acute ulcerating inflammation is aggravated because of diminished NF-κB-mediated protection against epithelial cell apoptosis and delayed mucosal regeneration secondary to reduced NF-κB-dependent recruitment of inflammatory cells that secrete cytoprotective factors. In contrast, in IL-10-deficient mice, which serve as a model of chronic T cell-dependent colitis, ablation of IKKβ in the intestinal epithelium has no impact, yet IKKβ deficiency in myeloid cells attenuates inflammation and prolongs survival. These results highlight the striking context and tissue dependence of the proinflammatory and antiapoptotic functions of NF-κB. Our findings caution against the therapeutic use of IKKβ/NF-κB inhibitors in acute inflammatory settings dominated by cell loss and ulceration.

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.

Innate immune defenses in the intestinal tract

Purpose of reviewInnate intestinal defenses are important for protection against ingested and commensal microbes. This review highlights recent new insights into innate immune effectors in the intestine. Recent findingsIntestinal epithelial cells, particularly Paneth cells, are the major producers of multiple peptides and proteins with antimicrobial activity in the intestine. The most abundant and diverse of these are the defensins. They are highly microbicidal in vitro and probably important in vivo, yet their physiologic functions remain incompletely understood. Relative defensin deficiency may be a risk factor for Crohns disease and infectious diarrhea. Cathelicidin contributes to mucosal defense against epithelial-adherent bacterial pathogens, and helps to set a threshold for productive infection. Bactericidal/permeability-inducing protein has lipopolysaccharide-neutralizing capacity and kills bacteria when overexpressed in epithelial cells. Resistin-like molecule β is important in mucosal defense against helminths due to its ability to inhibit worm chemotaxis. Antimicrobial lectins, particularly hepatocarcinoma–intestine–pancreas/pancreatic-associated protein, RegIII, and intelectin, can lyse bacteria or interfere with their attachment to epithelial cells. SummaryDiscovery of an expanding set of antimicrobial effectors supports the evolutionary importance of innate intestinal defenses against microbial threats, but also underlines the physiologic and pharmacologic need for a better understanding of the respective functions of these molecules.

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.

Interleukin-15 Activates Human Natural Killer Cells to Clear the Intestinal Protozoan Cryptosporidium

Intracellular protozoans of the genus Cryptosporidium are a major cause of diarrheal illness worldwide, but little is known about the mechanisms that control intestinal infection. We have previously demonstrated interleukin (IL)-15 expression in the intestinal mucosa of seronegative symptomatic volunteers after oral challenge with C. parvum, which suggests a role for IL-15 in the control of acute infection. We hypothesize that IL-15 activates an innate cytolytic cell response that contributes to the clearance of initial C. parvum infection. We report here that IL-15 activates peripheral blood mononuclear cells to lyse Cryptosporidium-infected epithelial cells in a dose-dependent manner. Lysis was due to CD3(-)CD16(+)CD56+ cells (i.e., natural killer [NK] cells). Furthermore, flow cytometry revealed that IL-15 increased expression of the activation receptor NKG2D on NK cells, particularly among the CD16Hi cytolytically active cells. Major histocompatibility complex class I-related molecules A and B (MICA and MICB), ligands for NKG2D, were increased after infection of epithelial cell lines and human ileal tissue. These data suggest that IL-15 has an important role in activating an NK cell-mediated pathway that leads to the elimination of intracellular protozoans from the intestines, which is a previously unrecognized feature of NK cell function.

A novel Calcium Dependent Protein Kinase Inhibitor as a lead compound for treating Cryptosporidiosis

Cryptosporidium parasites infect intestinal cells, causing cryptosporidiosis. Despite its high morbidity and association with stunting in the developing world, current therapies for cryptosporidiosis have limited efficacy. Calcium-dependent protein kinases (CDPKs) are essential enzymes in the biology of protozoan parasites. CDPK1 was cloned from the genome of Cryptosporidium parvum, and potent and specific inhibitors have been developed based on structural studies. In this study, we evaluated the anti-Cryptosporidium activity of a novel CDPK1 inhibitor, 1294, and demonstrated that 1294 significantly reduces parasite infection in vitro, with a half maximal effective concentration of 100 nM. Pharmacokinetic studies revealed that 1294 is well absorbed, with a half-life supporting daily administration. Oral therapy with 1294 eliminated Cryptosporidium parasites from 6 of 7 infected severe combined immunodeficiency-beige mice, and the parasites did not recur in these immunosuppressed mice. Mice treated with 1294 had less epithelial damage, corresponding to less apoptosis. Thus, 1294 is an important lead for the development of drugs for treatment of cryptosporidiosis.

ERK differentially regulates Th17- and Treg-cell development and contributes to the pathogenesis of colitis.

Although the development of T‐cell subsets is mainly regulated by a master transcriptional regulator and phosphorylation of the STAT protein in response to distinct cytokine stimulation, accumulating data indicate that other signaling pathways are also involved in regulating or fine‐tuning T‐cell lineage commitment. In this report, we investigated the role of ERK, mitogen‐activated protein kinase (MAPK), in Th17 and Treg cell development. We demonstrate that blockade of ERK activation inhibited Th17‐cell development while upregulating Treg cells under Th17 polarization conditions. Inhibition of ERK decreased IL‐6 induction of RAR‐related orphan receptor γt but enhanced TGF‐β induction of Foxp3, and ERK inhibitor‐treated T cells under Th17 conditions possessed suppressive function in vitro because they produced more IL‐10 and TGF‐β and inhibited naïve T‐cell proliferation and IFN‐γ production at levels comparable with that of Treg cells. Furthermore, ERK inhibitor‐treated T cells under Th17 polarization conditions had a decreased potency to induce colitis in vivo. Collectively, our data demonstrated that the ERK pathway differentially regulates Th17‐ and Treg‐cell differentiation, and thus interfering with the ERK pathway could represent a therapeutic treatment for inflammatory bowel diseases and other Th17‐related autoimmune diseases.

GM-CSF-Facilitated Dendritic Cell Recruitment and Survival Govern the Intestinal Mucosal Response to a Mouse Enteric Bacterial Pathogen

Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes dendritic cell (DC) differentiation and survival in vitro. However, its role in host defense at the intestinal mucosa is unknown. We report that infection with the mouse enteric pathogen, Citrobacter rodentium, increased colonic GM-CSF production and CD11c(+) DC recruitment. After infection, GM-CSF(-/-) mice had fewer mucosal CD11c(+) DCs, greater bacterial burden, increased mucosal inflammation and systemic spread of infection, decreased antibody responses, and delayed pathogen clearance. This defective mucosal response was rescued by GM-CSF administration to GM-CSF(-/-) mice and mimicked by CD11c(+) DC depletion in wild-type animals. Diminished mucosal DC numbers in infected GM-CSF(-/-) mice reflected decreased DC recruitment and survival, with the recruitment defect being related to a failure to upregulate epithelial cell production of the DC chemoattractant, CCL22. Thus, GM-CSF produced in the intestinal mucosa acts to enhance host protection against an enteric bacterial pathogen through regulating recruitment and survival of DCs.

Intestinal Immune Response to Human Cryptosporidium sp. Infection

Cryptosporidium is an obligate intracellular protozoan parasite that is a major cause of diarrheal illness worldwide. Cryptosporidium primarily infects the distal small intestine. Immunocompetent hosts control and eliminate the infection, which typically causes acute, self-limited watery diarrhea lasting 5 to 10 days. However, in patients with defects in cellular immune responses (e.g., AIDS, malnutrition, or defects in the CD40-CD154 system), Cryptosporidium frequently causes persistent or chronic diarrhea and may also involve the biliary tract (40). In malnourished children, persistent diarrhea is associated with increased susceptibility to recurrent diarrheal episodes, which can lead to death or chronic nutritional and cognitive sequelae (1, 9, 33). Thus, the host immune response plays a critical role in the control of human cryptosporidiosis. Although extensive studies with various animal models have provided important insight into the host immune response towards Cryptosporidium parvum, the ability of these models to explain the human immune response is limited. The clinical picture in rodents differs from that in humans, as mice do not get diarrhea after infection. Nonhuman primates, although probably the best in vivo model to mimic human disease, are difficult to work with, expensive, and not widely available. Cryptosporidium hominis, the pathogen causing most human cryptosporidiosis, infects only humans and gnotobiotic pigs, thus limiting data from animal models. Most importantly, comparison of animal and human data has shown that the immune response towards Cryptosporidium in humans differs significantly from that in animals; for example, in mice gamma interferon (IFN-γ) production seems to be associated with the innate and primary immune responses (35, 47), whereas in humans it is most probably associated with the memory response towards the parasite (93). Conducting studies to elucidate human mucosal immune responses is difficult. Patients with a natural infection would be the ideal subjects to study, but it is difficult to identify cases. Healthy human volunteers can be studied, but they typically experience a milder illness than malnourished children and AIDS patients. Human intestinal tissue samples can be obtained only by invasive procedures, limiting the numbers of subjects and samples available. Some data can be obtained from in vitro infections, but most of the target cells are immortalized and may not be ideal for studying mechanisms involving apoptosis. Furthermore, the immune cells in the peripheral blood may exhibit properties different from the properties of cells found in the intestinal compartment. Thus, knowledge about the human immune response towards Cryptosporidium infection is far from complete. Still, important recent advances have been made. The goal of this paper is to review the current literature to provide an understanding of the human immune response towards the parasite. We include some relevant data from other models only when the data shed light on studies performed with human cells or tissues.

Recent insights into Clostridium difficile pathogenesis.

Purpose of review Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis in the healthcare setting. An emerging consensus suggests that CDI is caused by pathogenic toxin production, gut microbial dysbiosis and altered host inflammatory responses. The aim of this review is to summarize and highlight recent advances focused on CDI pathogenic mechanisms. Recent findings Potential paradigm shifts relating to the mechanisms of toxin action and inhibition have recently been reported, with new insights into spore germination and surface protein function also gaining traction. Multiomic analysis of microbiome dysbiosis has identified important CDI-associated microbial community shifts that may form the basis of future targeted bacteriotherapy, and functional metabolite biomarkers that require further characterization. Classical innate and adaptive immunity against CDI is rapidly being delineated, with novel innate S-nitrosylation signals also being identified. Summary Studies in patients and animal disease models are shedding new light on the critical roles of the microbiota, metabolome and host responses in primary and recurrent CDI. An improved understanding of the CDI disease pathogenesis will provide the basis for developing new therapies for treating disease and preventing recurrence.