Julie Mirpuri

Therapeutic Helminth Infection of Macaques with Idiopathic Chronic Diarrhea Alters the Inflammatory Signature and Mucosal Microbiota of the Colon

Idiopathic chronic diarrhea (ICD) is a leading cause of morbidity amongst rhesus monkeys kept in captivity. Here, we show that exposure of affected animals to the whipworm Trichuris trichiura led to clinical improvement in fecal consistency, accompanied by weight gain, in four out of the five treated monkeys. By flow cytometry analysis of pinch biopsies collected during colonoscopies before and after treatment, we found an induction of a mucosal T(H)2 response following helminth treatment that was associated with a decrease in activated CD4(+) Ki67+ cells. In parallel, expression profiling with oligonucleotide microarrays and real-time PCR analysis revealed reductions in T(H)1-type inflammatory gene expression and increased expression of genes associated with IgE signaling, mast cell activation, eosinophil recruitment, alternative activation of macrophages, and worm expulsion. By quantifying bacterial 16S rRNA in pinch biopsies using real-time PCR analysis, we found reduced bacterial attachment to the intestinal mucosa post-treatment. Finally, deep sequencing of bacterial 16S rRNA revealed changes to the composition of microbial communities attached to the intestinal mucosa following helminth treatment. Thus, the genus Streptophyta of the phylum Cyanobacteria was vastly increased in abundance in three out of five ICD monkeys relative to healthy controls, but was reduced to control levels post-treatment; by contrast, the phylum Tenericutes was expanded post-treatment. These findings suggest that helminth treatment in primates can ameliorate colitis by restoring mucosal barrier functions and reducing overall bacterial attachment, and also by altering the communities of attached bacteria. These results also define ICD in monkeys as a tractable preclinical model for ulcerative colitis in which these effects can be further investigated.

Parasite-induced T H 1 cells and intestinal dysbiosis cooperate in IFN-γ-dependent elimination of Paneth cells

Activation of Toll-like receptors (TLRs) by pathogens triggers cytokine production and T cell activation, immune defense mechanisms that are linked to immunopathology. Here we show that IFN-γ production by CD4+ TH1 cells during mucosal responses to the protozoan parasite Toxoplasma gondii resulted in dysbiosis and the elimination of Paneth cells. Paneth cell death led to loss of antimicrobial peptides and occurred in conjunction with uncontrolled expansion of the Enterobacteriaceae family of Gram-negative bacteria. The expanded intestinal bacteria were required for the parasite-induced intestinal pathology. The investigation of cell type–specific factors regulating TH1 polarization during T. gondii infection identified the T cell–intrinsic TLR pathway as a major regulator of IFN-γ production in CD4+ T cells responsible for Paneth cell death, dysbiosis and intestinal immunopathology.

B Cell-Intrinsic MyD88 Signaling Prevents the Lethal Dissemination of Commensal Bacteria during Colonic Damage

The Toll-like receptor adaptor protein MyD88 is essential for the regulation of intestinal homeostasis in mammals. In this study, we determined that Myd88-deficient mice are susceptible to colonic damage that is induced by dextran sulfate sodium (DSS) administration resulting from uncontrolled dissemination of intestinal commensal bacteria. The DSS-induced mortality of Myd88-deficient mice was completely prevented by antibiotic treatment to deplete commensal bacteria. By using cell type-specific Myd88-deficient mice, we established that B cell-intrinsic MyD88 signaling plays a central role in the resistance to DSS-induced colonic damage via the production of IgM and complement-mediated control of intestinal bacteria. Our results indicate that the lack of intact MyD88 signaling in B cells, coupled with impaired epithelial integrity, enables commensal bacteria to function as highly pathogenic organisms, causing rapid host death.

Proteobacteria-specific IgA regulates maturation of the intestinal microbiota

The intestinal microbiota changes dynamically from birth to adulthood. In this study we identified γ-Proteobacteria as a dominant phylum present in newborn mice that is suppressed in normal adult microbiota. The transition from a neonatal to a mature microbiota was in part regulated by induction of a γ-Proteobacteria-specific IgA response. Neocolonization experiments in germ-free mice further revealed a dominant Proteobacteria-specific IgA response triggered by the immature microbiota. Finally, a role for B cells in the regulation of microbiota maturation was confirmed in IgA-deficient mice. Mice lacking IgA had persistent intestinal colonization with γ-Proteobacteria that resulted in sustained intestinal inflammation and increased susceptibility to neonatal and adult models of intestinal injury. Collectively, these results identify an IgA-dependent mechanism responsible for the maturation of the intestinal microbiota.

TLR-independent neutrophil-derived IFN-γ is important for host resistance to intracellular pathogens

IFN-γ is a major cytokine that is critical for host resistance to a broad range of intracellular pathogens. Production of IFN-γ by natural killer and T cells is initiated by the recognition of pathogens by Toll-like receptors (TLRs). In an experimental model of toxoplasmosis, we have identified the presence of a nonlymphoid source of IFN-γ that was particularly evident in the absence of TLR-mediated recognition of Toxoplasma gondii. Genetically altered mice lacking all lymphoid cells due to deficiencies in Recombination Activating Gene 2 and IL-2Rγc genes also produced IFN-γ in response to the protozoan parasite. Flow-cytometry and morphological examinations of non-NK/non-T IFN-γ+ cells identified neutrophils as the cell type capable of producing IFN-γ. Selective elimination of neutrophils in TLR11−/− mice infected with the parasite resulted in acute susceptibility similar to that observed in IFN-γ–deficient mice. Similarly, Salmonella typhimurium infection of TLR-deficient mice induces the appearance of IFN-γ+ neutrophils. Thus, neutrophils are a crucial source for IFN-γ that is required for TLR-independent host protection against intracellular pathogens.

The NLRP3 inflammasome is critically involved in the development of bronchopulmonary dysplasia

The pathogenesis of bronchopulmonary dysplasia (BPD), a devastating lung disease in preterm infants, includes inflammation, the mechanisms of which are not fully characterized. Here we report that the activation of the NLRP3 inflammasome is associated with the development of BPD. Hyperoxia-exposed neonatal mice have increased caspase-1 activation, IL1β and inflammation, and decreased alveolarization. Nlrp3(-/-) mice have no caspase-1 activity, no IL1β, no inflammatory response and undergo normal alveolarization. Treatment of hyperoxia-exposed mice with either IL1 receptor antagonist to block IL1β or glyburide to block the Nlrp3 inflammasome results in decreased inflammation and increased alveolarization. Ventilated preterm baboons show activation of the NLRP3 inflammasome with increased IL1β:IL1ra ratio. The IL1β:IL1ra ratio in tracheal aspirates from preterm infants with respiratory failure is predictive of the development of BPD. We conclude that early activation of the NLRP3 inflammasome is a key mechanism in the development of BPD, and represents a novel therapeutic target for BPD.

Commensal Escherichia coli Reduces Epithelial Apoptosis through IFN-αA–Mediated Induction of Guanylate Binding Protein-1 in Human and Murine Models of Developing Intestine

Appropriate microbial colonization protects the developing intestine by promoting epithelial barrier function and fostering mucosal tolerance to luminal bacteria. Commensal flora mediate their protective effects through TLR9-dependent activation of cytokines, such as type I IFNs (α, β) and IL-10. Although IFN-β promotes apoptosis, IFN-α activates specific antiapoptotic target genes whose actions preserve epithelial barrier integrity. We have recently identified guanylate binding protein-1 (GBP-1) as an antiapoptotic protein, regulated by both type I and type II IFNs, that promotes intestinal epithelial barrier integrity in mature intestine. However, the mechanisms by which commensal bacteria regulate epithelial apoptosis during colonization of immature intestine and the contributions of GBP-1 are unknown. The healthy newborn intestine is initially colonized with bacterial species present in the maternal gastrointestinal tract, including nonpathogenic Escherichia coli. Therefore, we examined the influence of commensal E. coli on cytokine expression and candidate mediators of apoptosis in preweaned mice. Specifically, enteral exposure of 2 wk-old mice to commensal E. coli for 24 h selectively increased both IFN-αA and GBP-1 mRNA expression and prevented staurosporine-induced epithelial apoptosis. Exogenous IFN-αA treatment also induced GBP-1 expression and protected against staurosporine-induced apoptosis in a GBP-1 dependent manner, both in vitro and ex vivo. These findings identify a role for IFN-αA–mediated GBP-1 expression in the prevention of intestinal epithelial apoptosis by commensal bacteria. Thus IFN-αA mediates the beneficial effects of commensal bacteria and may be a promising therapeutic target to promote barrier integrity and prevent the inappropriate inflammatory responses seen in developing intestine as in necrotizing enterocolitis.

Lactobacillus rhamnosus (LGG) Regulates IL-10 Signaling in the Developing Murine Colon through Upregulation of the IL-10R2 Receptor Subunit

The intestinal microflora is critical for normal development, with aberrant colonization increasing the risk for necrotizing enterocolitis (NEC). In contrast, probiotic bacteria have been shown to decrease its incidence. Multiple pro- and anti-inflammatory cytokines have been identified as markers of intestinal inflammation, both in human patients with NEC and in models of immature intestine. Specifically, IL-10 signaling attenuates intestinal responses to gut dysbiosis, and disruption of this pathway exacerbates inflammation in murine models of NEC. However, the effects of probiotics on IL-10 and its signaling pathway, remain poorly defined. Real-time PCR profiling revealed developmental regulation of MIP-2, TNF-α, IL-12, IL-10 and the IL-10R2 subunit of the IL-10 receptor in immature murine colon, while the expression of IL-6 and IL-18 was independent of postnatal age. Enteral administration of the probiotic Lactobacillus rhamnosus GG (LGG) down-regulated the expression of TNF-α and MIP-2 and yet failed to alter IL-10 mRNA and protein expression. LGG did however induce mRNA expression of the IL-10R2 subunit of the IL-10 receptor. IL-10 receptor activation has been associated with signal transducer and activator of transcription (STAT) 3-dependent induction of members of the suppressors of cytokine signaling (SOCS) family. In 2 week-old mice, LGG also induced STAT3 phosphorylation, increased colonic expression of SOCS-3, and attenuated colonic production of MIP-2 and TNF-α. These LGG-dependent changes in phosphoSTAT3, SOCS3, MIP-2 and TNF-α were all inhibited by antibody-mediated blockade of the IL-10 receptor. Thus LGG decreased baseline proinflammatory cytokine expression in the developing colon through upregulation of IL-10 receptor-mediated signaling, most likely due to the combined induction of phospho-STAT3 and SOCS3. Furthermore, LGG-dependent increases in IL-10R2 were associated with reductions in TNF-α, MIP-2 and disease severity in a murine model of intestinal injury in the immature colon.

Issues of Prematurity and HIV Infection

Prematurity and HIV present a complex challenge, with biologic underpinnings that are often confounded by a myriad of other factors that coexist in this high-risk population. Furthermore, many of the current management options designed to reduce mother-to-infant transmission, including antiretroviral therapy and cesarean birth, may each have an independent effect on prematurity. These issues notwithstanding, knowledge gained from randomized controlled trials and epidemiologic studies has made a significant impact on the approach to this challenging public health problem worldwide. This article discusses the significance, contribution, and management of perinatal transmission of HIV in prematurity.

IL-6 signaling SOCS critical for IL-12 host response to Toxoplasma gondii

SOCS are a family of proteins that play an important role in the negative regulation of the cytokine-JAK-STAT pathway. Socs3 deletion results in prolonged IL-6 signaling measured by STAT3 phosphorylation. A role for STAT3 and SOCS3 in the context of Toxoplasma gondii infection is of particular importance, because STAT3 appears to be a key target of T. gondii virulence factors. By utilizing LysM-cre Socs3(fl/fl) mice, the Hunter laboratory recently established that macrophage-specific SOCS3 knockout mice have enhanced susceptibility to infection with T. gondii. The authors demonstrated that lack of SOCS3-mediated control of IL-6 signaling results in acute susceptibility to T. gondii due to impaired IL-12 production by inflammatory monocytes, macrophages and neutrophils. This article further explores these findings and their implications in the field of host resistance to microbial pathogens.