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Intestinal helminth infections elicit Th2-type immunity, which influences host immune responses to additional threats, such as allergens, metabolic disease, and other pathogens. Th2 immunity involves a shift of the CD4 + T-cell population from type-0 to type-2 (Th2) with increased abundance of interleukin (IL)-4 and IL-13. This study sought to investigate if existing gut-restricted intestinal helminth infections impact bacterial-induced acute airway neutrophil recruitment. C57BL/6 mice were divided into four groups: uninfected; helminth-Heligmosomoides polygyrus infected; Pseudomonas aeruginosa infected; and coinfected. Mice infected with H. polygyrus were incubated for 2 weeks, followed by P. aeruginosa intranasal inoculation. Bronchial alveolar lavage, blood, and lung samples were analyzed. Interestingly, infection with gut-restricted helminths resulted in immunological and structural changes in the lung. These changes include increased lung CD4 + T cells, increased Th2 cytokine expression, and airway goblet cell hyperplasia. Furthermore, coinfected mice exhibited significantly more airspace neutrophil infiltration at 6 hours following P. aeruginosa infection and exhibited an improved rate of survival compared with bacterial infected alone. These results suggest that chronic helminth infection of the intestines can influence and enhance acute airway neutrophil responses to P. aeruginosa infection.

Epidemiological studies indicate an inverse correlation between the prevalence of the so-called western diseases, such as obesity and metabolic syndrome, and the exposure to helminths. Obesity, a key risk factor for many chronic health problems, is rising globally and is accompanied by low-grade inflammation in adipose tissues. The precise mechanism by which helminths modulate metabolic syndrome and obesity is not fully understood. We infected high fat diet (HFD)-induced obese mice with the intestinal nematode parasite Heligmosomoides polygyrus and observed that helminth infection resulted in significantly attenuated obesity. Attenuated obesity corresponded with marked upregulation of uncoupling protein 1 (UCP1), a key protein involved in energy expenditure, in adipose tissue, suppression of glucose and triglyceride levels, and alteration in the expression of key genes involved in lipid metabolism. Moreover, the attenuated obesity in infected mice was associated with enhanced helminth-induced Th2/Treg responses and M2 macrophage polarization. Adoptive transfer of helminth-stimulated M2 cells to mice that were not infected with H. polygyrus resulted in a significant amelioration of HFD-induced obesity and increased adipose tissue browning. Thus, our results provide evidence that the helminth-dependent protection against obesity involves the induction of M2 macrophages.

Intestinal epithelial cells absorb nutrients, respond to microbes, function as a barrier and help to coordinate immune responses. Here we report profiling of 53,193 individual epithelial cells from the small intestine and organoids of mice, which enabled the identification and characterization of previously unknown subtypes of intestinal epithelial cell and their gene signatures. We found unexpected diversity in hormone-secreting enteroendocrine cells and constructed the taxonomy of newly identified subtypes, and distinguished between two subtypes of tuft cell, one of which expresses the epithelial cytokine Tslp and the pan-immune marker CD45, which was not previously associated with non-haematopoietic cells. We also characterized the ways in which cell-intrinsic states and the proportions of different cell types respond to bacterial and helminth infections: Salmonella infection caused an increase in the abundance of Paneth cells and enterocytes, and broad activation of an antimicrobial program; Heligmosomoides polygyrus caused an increase in the abundance of goblet and tuft cells. Our survey highlights previously unidentified markers and programs, associates sensory molecules with cell types, and uncovers principles of gut homeostasis and response to pathogens.

Mechanisms mediating adult enteric neurogenesis are largely unknown. Using inflammation-associated neurogenesis models and a transgenic approach, we aimed to understand the cell-source for new neurons in infectious and inflammatory colitis. Dextran sodium sulfate (DSS) and Citrobacter rodentium colitis (CC) was induced in adult mice and colonic neurons were quantified. Sox2GFP and PLP1GFP mice confirmed the cell-type specificity of these markers. Sox2CreER:YFP and PLP1creER:tdT mice were used to determine the fate of these cells after colitis. Sox2 expression was investigated in colonic neurons of human patients with Clostridium difficile or ulcerative colitis. Both DSS and CC led to increased colonic neurons. Following colitis in adult Sox2CreER:YFP mice, YFP initially expressed predominantly by glia becomes expressed by neurons following colitis, without observable DNA replication. Similarly in PLP1CreER:tdT mice, PLP1 cells that co-express S100b but not RET also give rise to neurons following colitis. In human colitis, Sox2-expressing neurons increase from 1-2% to an average 14% in colitis. The new neurons predominantly express calretinin, thus appear to be excitatory. These results suggest that colitis promotes rapid enteric neurogenesis in adult mice and humans through differentiation of Sox2- and PLP1-expressing cells, which represent enteric glia and/or neural progenitors. Further defining neurogenesis will improve understanding and treatment of injury-associated intestinal motility/sensory disorders.

Background: Necrotizing enterocolitis (NEC) is an immature intestinal condition resulting in devastating intestinal inflammation due to unknown mechanisms. Evidence has suggested that intestinal maturation attenuates the severity of NEC and Toll-like receptor 4 (TLR4) has been suggested to play a critical role in its pathogenesis. We investigated whether maturational effects of TLR4 expression in immature colon might contribute to the development of NEC., Methods: TLR4 colonocyte expression was detected by immunofluorescence confocal microscopy. Interleukin-6 (IL-6) levels were assayed by an enzyme-linked immunosorbent assay (ELISA)., Results: TLR4 expression was high in fetal colonic epithelium in human and mouse, with earlier gestation having a higher surface/cytoplasm distribution. TLR4 remained high in mouse postnatal day 1 but the surface/cytoplasm distribution was reduced. TLR4 decreased in amount and then was expressed in crypts in the mature human and mouse colon. Hydrocortisone (HC) reduced the surface/cytoplasm distribution of TLR4 in human fetal colon. Elevated IL-6 levels in immature colon after lipopolysaccharide were attenuated by HC in human and mouse., Conclusion: Expression, localization, and signaling of TLR4 in colonic epithelium may be developmentally regulated. HC may accelerate the TLR developmental pathway change to an adult type, which may account for its impact on TLR4 signaling.

The role of microorganisms in the gastrointestinal tract has undergone significant modification in the past few decades with new observations from clinical, epidemiologic, and basic science research. We now know that the perception of these gut microbes as pathogens or even as commensals is somewhat outdated. It is becoming increasingly clear that the gut microbiome plays an important role in a host of activities including digestion, protection from potentially pathogenic organisms, and the regulation and development of the host immune system. The complex interactions between microbes and host combined with recent clinical observations and epidemiologic trends may point to the convergence of two well-supported (though imperfect) hypotheses: the "hygiene hypothesis" and the "fetal programming hypothesis." We are beginning to understand that exposure to microbes before conception, during gestation, and in the neonatal period have profound effects on the developing immune system. Recent observations from a variety of fields help support the expansion of the "fetal programming hypothesis" to a host-microbe corollary that microbe-host interactions at critical windows influence the future immune phenotype, the maintenance of immune health, and the development of immune-mediated disease.

Modulation of the intestinal immune response early in life by administration of probiotic bacteria may be an effective strategy for preventing or attenuating infectious diarrhea. We preinoculated the mice early in life with the probiotic bacteria Lactobacillus acidophilus NCFM (La) at age 2 wk. Dendritic cells (DCs) were collected and purified from mesenteric lymph nodes (MLN) and spleens of the BalbC/ByJ mice. DC isolation and adoptive transfer was used to examine the function of probiotics. We demonstrated that when mice were adoptively transferred with La-primed DCs (t-LaDC) instead of oral consumption with La, there was a similar effect on fecal bacteria counts, IgA levels, and colonic histopathology, as well as cytokine levels in MLN when there was intestinal bacterial infection. The above findings suggest that DCs play a key role in probiotics attenuating Citrobacter rodentium (Cr) colitis. Moreover, the location of La-primed DC hints that there is interaction of DCs and T cells in the digestive system of the host. Up-regulated expression of a surface marker on DCs indicated that inoculation with probiotics will stimulate the function of DCs, thereby further increasing immune response triggered by DC.

Intestinal epithelial cells (IEC) are constantly exposed to bacterial components, such as LPS, without triggering proinflammatory immune responses. This study demonstrates that chronic exposure of human-derived IEC to LPS induces tolerance to an endogenous inflammatory cytokine (IL-1beta) activated IL-8 response that occurs independently of TLR-4/MD-2 signaling. IL-8 production in response to activation by unrelated TNF-alpha and PMA signaling pathways is also inhibited, indicating a broad-spanning tolerance. Quantitative rtPCR and IL-8 promoter-luciferase assays demonstrate that tolerance is regulated at the transcriptional level and occurs independently of IEC cytodifferentiation. By contrast, LPS does not significantly alter other proinflammatory signaling cascades in IEC that function independently of IL-8 production, e.g., IL-6 secretion and PEEC (Hepoxilin A3)-induced neutrophil transepithelial migration in response to invasive Salmonella typhimurium. Human IEC have therefore developed LPS-induced signaling cascades that promote an IL-8 hyporesponsiveness to proinflammatory cytokines while LPS exposure does not compromise the ability of IEC to mount other proinflammatory immune responses to invasive enteropathogens.

Enteropathogenic Escherichia coli (EPEC) is a common pathogen in infantile diarrhea, causing a characteristic histopathologic attaching and effacing (A/E) lesion in the intestinal mucosa. The mouse pathogen Citrobacter rodentium causes a similar A/E lesion in the murine intestine. Like EPEC, C. rodentium infection results in colonic crypt hyperplasia, goblet cell depletion, epithelial proliferation, and mucosal disruption. Using this murine model, we tested the hypothesis that preinoculation of murine gut with Lactobacillus acidophilus early in life can enhance host defense against enteric bacterial infection and attenuate bacteria-mediated colitis. Two-week old BALB/c mice were inoculated with L. acidophilus twice per week for 4 weeks before C. rodentium infection or concomitantly with the exposure to C. rodentium at 6-8 weeks of age. The probiotics were administered twice weekly thereafter. We observed that L. acidophilus inoculation in mice inhibits C. rodentium-induced colitis, which is associated with a decrease in C. rodentium colonization and translocation, an increase in its clearance, and a suppression of colonic myeloperoxidase (MPO) activity. Probiotic treatment also stimulates regulatory cytokine expression in the colon [transforming growth factor beta (TGF-beta), interleukin (IL)-10]. Preinoculation with L. acidophilus is more effective than concomitant use of probiotics in the induction of intestinal IgA secretion and in the downregulation of proinflammatory cytokine expression [tumor necrosis factor alpha (TNF-alpha), IL-6, and IL-12]. These observations suggest that inoculation with probiotics can effectively prevent bacteria-induced colitis by limiting enteric bacteria infection and promoting mucosal protective regulatory immune responses. This study may have ramifications for prevention of infectious diarrhea in human infants and children, particularly in developing countries.