Janneke N. Samsom

Interleukin-10 receptor signaling in innate immune cells regulates mucosal immune tolerance and anti-inflammatory macrophage function

Intact interleukin-10 receptor (IL-10R) signaling on effector and T regulatory (Treg) cells are each independently required to maintain immune tolerance. Here we show that IL-10 sensing by innate immune cells, independent of its effects on T cells, was critical for regulating mucosal homeostasis. Following wild-type (WT) CD4(+) T cell transfer, Rag2(-/-)Il10rb(-/-) mice developed severe colitis in association with profound defects in generation and function of Treg cells. Moreover, loss of IL-10R signaling impaired the generation and function of anti-inflammatory intestinal and bone-marrow-derived macrophages and their ability to secrete IL-10. Importantly, transfer of WT but not Il10rb(-/-) anti-inflammatory macrophages ameliorated colitis induction by WT CD4(+) T cells in Rag2(-/-)Il10rb(-/-) mice. Similar alterations in the generation and function of anti-inflammatory macrophages were observed in IL-10R-deficient patients with very early onset inflammatory bowel disease. Collectively, our studies define innate immune IL-10R signaling as a key factor regulating mucosal immune homeostasis in mice and humans.

Cd1d-dependent regulation of bacterial colonization in the intestine of mice

The accumulation of certain species of bacteria in the intestine is involved in both tissue homeostasis and immune-mediated pathologies. The host mechanisms involved in controlling intestinal colonization with commensal bacteria are poorly understood. We observed that under specific pathogen-free or germ-free conditions, intragastric administration of Pseudomonas aeruginosa, E. coli, Staphylococcus aureus, or Lactobacillus gasseri resulted in increased colonization of the small intestine and bacterial translocation in mice lacking Cd1d, an MHC class I-like molecule, compared with WT mice. In contrast, activation of Cd1d-restricted T cells (NKT cells) with alpha-galactosylceramide caused diminished intestinal colonization with the same bacterial strains. We also found prominent differences in the composition of intestinal microbiota, including increased adherent bacteria, in Cd1d-/- mice in comparison to WT mice under specific pathogen-free conditions. Germ-free Cd1d-/- mice exhibited a defect in Paneth cell granule ultrastructure and ability to degranulate after bacterial colonization. In vitro, NKT cells were shown to induce the release of lysozyme from intestinal crypts. Together, these data support a role for Cd1d in regulating intestinal colonization through mechanisms that include the control of Paneth cell function.

The acquisition of tolerance toward cow's milk through probiotic supplementation: a randomized, controlled trial.

BACKGROUND Cows milk allergy (CMA) is the most frequently diagnosed food allergy in infancy. In general, patients have a good prognosis because the majority acquire tolerance within the first years. Interventions have been proposed to accelerate tolerance and reduce morbidity. Probiotic supplementation could be effective through modulation of the immune system. OBJECTIVE We sought to determine whether supplementation with a combination of probiotics (Lactobacillus casei CRL431 and Bifidobacterium lactis Bb-12) accelerates tolerance to cows milk (CM) in infants with CMA. METHODS We performed a double-blind, randomized, placebo-controlled trial in 119 infants with CMA. Infants received CRL431 and Bb-12 supplemented to their standard treatment of extensively hydrolyzed formula for 12 months. Primary outcome was clinical tolerance to CM at 6 and 12 months of treatment. Furthermore, we analyzed T- and B-lymphocyte subsets (CD3(+), CD3(+)CD4(+), CD3(+)CD8(+), and CD20(+)) in peripheral blood at randomization and at 12 months with flow cytometry and examined the presence of viable probiotic strains in fecal samples. RESULTS The cumulative percentage of tolerance to CM at 6 and 12 months was similar in both groups: 56 (77%) in the probiotics group versus 54 (81%) in the placebo group. Infants in the placebo group had higher percentages of CD3(+) and CD3(+)CD4(+) lymphocytes compared with those seen in probiotic-treated infants. Probiotic intake was confirmed because probiotics were isolated from feces more often in treated infants than in the placebo group. CONCLUSION Supplementation of CRL431 and Bb-12 to extensively hydrolyzed formula does not accelerate CM tolerance in infants with CMA.

Temporal and spatial interplay of microbiota and intestinal mucosa drive establishment of immune homeostasis in conventionalized mice

During colonization of germfree mice with the total fecal microbial community of their conventionally born and raised siblings (conventionalization), the intestinal mucosal immune system initiates and maintains a balanced immune response. However, the genetic regulation of these balanced, appropriate responses to the microbiota is obscure. Here, combined analysis of germfree and conventionalized mice revealed that the major molecular responses could be detected initiating at day 4 post conventionalization, with a strong induction of innate immune functions followed by stimulation of adaptive immune responses and development and expansion of adaptive immune cells at later stages of conventionalization. This study provides a comprehensive overview of mouse developmental and immune-related cellular pathways and processes that were co-mediated by the commensal microbiota and suggests which mechanisms were involved in this reprogramming. The dynamic, region-dependent mucosal responses to the colonizing microbiota revealed potential transcriptional signatures for the control of intestinal homeostasis in healthy mice, which may help to decipher the genetic basis of pathway dysregulation in human intestinal inflammatory diseases.

Oxazolone-Induced Enterocolitis in Zebrafish Depends on the Composition of the Intestinal Microbiota

BACKGROUND & AIMS The pathogenesis of inflammatory bowel disease involves dysfunctional mucosal immune responses to commensal bacteria in genetically predisposed hosts. Interactions between host cells and bacteria are complicated, making it a challenge to assess their relative contribution to intestinal pathology. We developed a zebrafish model of enterocolitis to study these interactions. METHODS Enterocolitis was induced by intrarectal administration of the hapten oxazolone in adult wild-type and myeloperoxidase-reporter transgenic zebrafish in the presence or absence of antibiotics. Intestinal inflammation was evaluated by histological and flow cytometry analyses and cytokine profiling with quantitative real-time polymerase chain reaction. Changes in the composition of the intestinal microbiota following antibiotic administration were assessed by 16SrRNA sequencing and bacterial load was quantified by culture on nonselective media (colony-forming units). RESULTS In zebrafish, the infiltrate and severity of oxazolone-induced enterocolitis are influenced by the composition of the microbiota. Inflammation is characterized by granulocyte influx; epithelial damage; goblet cell depletion; and increased expression of interleukin-1beta, tumor necrosis factor-alpha, and interleukin-10. Zebrafish given vancomycin had bacterial populations dominated by Fusobacteria and reduced enterocolitis scores, intestinal damage, and percentages of infiltrating neutrophils and eosinophils. In contrast, zebrafish given colistin sulphate had a predominance of proteobacteria and reduced eosinophil and lymphocyte infiltration, but enterocolitis scores were not reduced. CONCLUSIONS In zebrafish with oxazolone-induced enterocolitis, components of the intestinal microbiota affect the severity and composition of the intestinal infiltrate.

Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease

A nonpathogenic virus can promote inflammatory immunity to dietary antigens and may be linked to the development of celiac disease. Viruses compound dietary pathology Reoviruses commonly infect humans and mice asymptomatically. Bouziat et al. found that immune responses to two gut-infecting reoviruses take different paths in mice (see the Perspective by Verdu and Caminero). Both reoviruses invoked protective immune responses, but for one reovirus, when infection happened in the presence of a dietary antigen (such as gluten or ovalbumin), tolerance to the dietary antigen was lost. This was because this strain prevented the formation of tolerogenic T cells. Instead, it promoted T helper 1 immunity to the dietary antigen through interferon regulatory factor 1 signaling. Celiac disease patients also exhibited elevated levels of antibodies against reovirus. Science, this issue p. 44; see also p. 29 Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (TH1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pTreg) conversion and promoting TH1 immunity to dietary antigen. Initiation of TH1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pTreg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD.

Characterization of the Specific Interaction between Sialoadhesin and Sialylated Campylobacter jejuni Lipooligosaccharides

ABSTRACT In Campylobacter jejuni-induced Guillain-Barré syndrome (GBS), molecular mimicry between C. jejuni lipooligosaccharide (LOS) and host gangliosides leads to the production of cross-reactive antibodies directed against the peripheral nerves of the host. Currently, the presence of surface exposed sialylated LOS in C. jejuni is the single known bacterial pathogenesis factor associated with the development of GBS. Using a unique, well-characterized strain collection, we demonstrate that GBS-associated C. jejuni strains bind preferentially to sialoadhesin (Sn, Siglec-1, or CD169), a sialic acid receptor found on a subset of macrophages. In addition, using a whole-cell enzyme-linked immunosorbent assay (ELISA), C. jejuni strains with sialylated LOS bound exclusively to soluble Sn. Mass spectrometry revealed that binding was sialic acid-linkage specific with a preference for α(2,3)-linked sialic acid attached to the terminal galactose of the LOS chain as seen in the gangliosides GD1a, GM1b, and GM3. This molecular interaction was also related to functional consequences as a GBS-associated C. jejuni strain that bound Sn in a whole-cell ELISA adhered to surface-expressed Sn of Sn-transfected CHO cells but was unable to adhere to wild-type CHO cells. Moreover, a sialic acid-negative mutant of the same C. jejuni strain was unable to bind Sn-transfected CHO cells. This is the first report of the preferential binding of GBS-associated C. jejuni strains to the Sn immune receptor (P = 0.014). Moreover, because this binding is dependent on sialylated LOS, the main pathogenic factor in GBS progression, the present findings bring us closer to unraveling the mechanisms that lead to formation of cross-reactive antibodies in GBS disease.

Ontogeny of human hepatic and intestinal transporter gene expression during childhood: age matters.

Many drugs prescribed to children are drug transporter substrates. Drug transporters are membrane-bound proteins that mediate the cellular uptake or efflux of drugs and are important to drug absorption and elimination. Very limited data are available on the effect of age on transporter expression. Our study assessed age-related gene expression of hepatic and intestinal drug transporters. Multidrug resistance protein 2 (MRP2), organic anion transporting polypeptide 1B1 (OATP1B1), and OATP1B3 expression was determined in postmortem liver samples (fetal n = 6, neonatal n = 19, infant n = 7, child n = 2, adult n = 11) and multidrug resistance 1 (MDR1) expression in 61 pediatric liver samples. Intestinal expression of MDR1, MRP2, and OATP2B1 was determined in surgical small bowel samples (neonates n = 15, infants n = 3, adults n = 14). Using real-time reverse-transcription polymerase chain reaction, we measured fetal and pediatric gene expression relative to 18S rRNA (liver) and villin (intestines), and we compared it with adults using the 2−∆∆Ct method. Hepatic expression of MRP2, OATP1B1, and OATP1B3 in all pediatric age groups was significantly lower than in adults. Hepatic MDR1 mRNA expression in fetuses, neonates, and infants was significantly lower than in adults. Neonatal intestinal expressions of MDR1 and MRP2 were comparable to those in adults. Intestinal OATP2B1 expression in neonates was significantly higher than in adults. We provide new data that show organ- and transporter-dependent differences in hepatic and intestinal drug transporter expression in an age-dependent fashion. This suggests that substrate drug absorption mediated by these transporters may be subject to age-related variation in a transporter dependent pattern.

TLR4-Mediated Sensing of Campylobacter jejuni by Dendritic Cells Is Determined by Sialylation

In Guillain-Barré syndrome (GBS), ganglioside mimicry of Campylobacter jejuni lipo-oligosaccharide (LOS) drives the production of cross-reactive Abs to peripheral nerve gangliosides. We determined whether sialic acid residues in C. jejuni LOS modulate dendritic cell (DC) activation and subsequent B cell proliferation as a possible mechanism for the aberrant humoral immune response in GBS. Highly purified sialylated LOS of C. jejuni isolates from three GBS patients induced human DC maturation and secretion of inflammatory cytokines that were inhibited by anti-TLR4 neutralizing Abs. The extent of TLR4 signaling and DC activation was greater with LOS of the wild type isolates than with nonsialylated LOS of the corresponding sialyltransferase gene knockout (cst-II mutant) strains, indicating that sialylation boosts the DC response to C. jejuni LOS. Supernatants of LOS-activated DCs induced B cell proliferation after cross-linking of surface Igs in the absence of T cells. Lower B cell proliferation indices were found with DC supernatants after DC stimulation with cst-II mutant or neuraminidase desialylated LOS. This study showed that sialylation of C. jejuni LOS enhances human DC activation and subsequent B cell proliferation, which may contribute to the development of cross-reactive anti-ganglioside Abs found in GBS patients following C. jejuni infection.

Type 3 innate lymphoid cells maintain intestinal epithelial stem cells after tissue damage

Type 3 lymphoid cells (ILC3s) are required for epithelial activation and proliferation in response to small intestinal tissue damage induced by chemotherapeutics. Multiple ILC3 subsets are activated after intestinal damage, and the absence of ILC3s or their signature cytokine IL-22 results in severely impaired maintenance of intestinal stem cells.