Elizabeth A. Maier

Biomarkers of environmental enteropathy, inflammation, stunting, and impaired growth in children in Northeast Brazil

Critical to the design and assessment of interventions for enteropathy and its developmental consequences in children living in impoverished conditions are non-invasive biomarkers that can detect intestinal damage and predict its effects on growth and development. We therefore assessed fecal, urinary and systemic biomarkers of enteropathy and growth predictors in 375 6–26 month-old children with varying degrees of malnutrition (stunting or wasting) in Northeast Brazil. 301 of these children returned for followup anthropometry after 2-6m. Biomarkers that correlated with stunting included plasma IgA anti-LPS and anti-FliC, zonulin (if >12m old), and intestinal FABP (I-FABP, suggesting prior barrier disruption); and with citrulline, tryptophan and with lower serum amyloid A (SAA) (suggesting impaired defenses). In contrast, subsequent growth was predicted in those with higher fecal MPO or A1AT and also by higher L/M, plasma LPS, I-FABP and SAA (showing intestinal barrier disruption and inflammation). Better growth was predicted in girls with higher plasma citrulline and in boys with higher plasma tryptophan. Interactions were also seen with fecal MPO and neopterin in predicting subsequent growth impairment. Biomarkers clustered into markers of 1) functional intestinal barrier disruption and translocation, 2) structural intestinal barrier disruption and inflammation and 3) systemic inflammation. Principle components pathway analyses also showed that L/M with %L, I-FABP and MPO associate with impaired growth, while also (like MPO) associating with a systemic inflammation cluster of kynurenine, LBP, sCD14, SAA and K/T. Systemic evidence of LPS translocation associated with stunting, while markers of barrier disruption or repair (A1AT and Reg1 with low zonulin) associated with fecal MPO and neopterin. We conclude that key noninvasive biomarkers of intestinal barrier disruption, LPS translocation and of intestinal and systemic inflammation can help elucidate how we recognize, understand, and assess effective interventions for enteropathy and its growth and developmental consequences in children in impoverished settings.

Glutamine and alanyl-glutamine promote crypt expansion and mTOR signaling in murine enteroids

L-glutamine (Gln) is a key metabolic fuel for intestinal epithelial cell proliferation and survival and may be conditionally essential for gut homeostasis during catabolic states. We show that L-alanyl-L-glutamine (Ala-Gln), a stable Gln dipeptide, protects mice against jejunal crypt depletion in the setting of dietary protein and fat deficiency. Separately, we show that murine crypt cultures (enteroids) derived from the jejunum require Gln or Ala-Gln for maximal expansion. Once expanded, enteroids deprived of Gln display a gradual atrophy of cryptlike domains, with decreased epithelial proliferation, but stable proportions of Paneth and goblet cell differentiation, at 24 h. Replenishment of enteroid medium with Gln selectively activates mammalian target of rapamycin (mTOR) signaling pathways, rescues proliferation, and promotes crypt regeneration. Gln deprivation beyond 48 h leads to destabilization of enteroids but persistence of EGFP-Lgr5-positive intestinal stem cells with the capacity to regenerate enteroids upon Gln rescue. Collectively, these findings indicate that Gln deprivation induces a reversible quiescence of intestinal stem cells and provides new insights into nutritional regulation of intestinal epithelial homeostasis.

Enhanced survival following oral and systemic Salmonella enterica serovar Typhimurium infection in polymeric immunoglobulin receptor knockout mice

Background Polymeric immunoglobulin receptor (pIgR) transport of secretory immunoglobulin A (SIgA) to mucosal surfaces is thought to promote gut integrity and immunity to Salmonella enterica serovar Typhimurium (S. Typhimurium), an invasive pathogen in mice. To elucidate potential mechanisms, we assessed intestinal barrier function and both oral and systemic S. Typhimurium virulence in pIgR knockout (KO) and wildtype (WT) mice. Methods In uninfected animals, we harvested jejunal segments for Ussing chamber analyses of transepithelial resistance (TER); mesenteric lymph nodes (mLN) for bacterial culture; and serum and stool for IgA. Separately, we infected mice either orally or intravenously (IV) with S. Typhimurium to compare colonization, tissue dynamics, and inflammation between KOs and WTs. Results Uninfected KOs displayed decreased TER and dramatically increased serum IgA and decreased fecal IgA vs. WT; however, KO mLNs yielded fewer bacterial counts. Remarkably, WTs challenged orally with S. Typhimurium exhibited increased splenomegaly, tissue colonization, and pro-inflammatory cytokines vs. pIgR KOs, which showed increased survival following either oral or IV infection. Conclusions Absence of pIgR compromises gut integrity but does not exacerbate bacterial translocation nor S. Typhimurium infection. These findings raise the possibility that immune adaptation to increased gut permeability and elevated serum IgA in the setting of SIgA deficiency provides compensatory protection against invasive gut pathogens.

Su1368 Undernutrition and Altered Gut Secretory IgA Synergistically Increase Bacterial Burdens in the Mesenteric Lymph Nodes

Background Cystic fibrosis (CF) is a life-limiting disease. Microbial infections, colonization and airway inflammation are the hallmarks of CF lung disease. Similarly, small bowel bacterial overgrowth has been long recognized as a complication of CF. With the recent availability of non-culture-based microbiological techniques, the presence of intestinal dysbiosis in CF has been confirmed. Defects in bacterial host defenses in the CF airways have been speculated and extensively investigated. However, the role of the innate immune system in the intestines of patients with CF remain poorly studied. Human β-defensin 2 (HBD-2) is an antimicrobial protein produced by epithelial surfaces, and upregulated by inflammation. Its expression in the CF intestine is unknown. Methods Faeces from children (aged 0-18 years) with CF and age-matched healthy controls (HC) were collected for faecal HBD-2 and calprotectin. Patients with CF with a pulmonary exacerbation requiring intravenous antibiotics were excluded. Any CF and HC subjects with gastroenteritis, on oral corticosteroids, probiotics and/or non-steroidal anti-inflammatory drugs in the preceding two weeks were also excluded. Results Thirty-three CF patients with mean (SD) age of 7.3 (3.8) years were recruited. Nineteen subjects (58%) were males. Twenty-nine (88%) were PI and 4 (12%) were PS. As a comparison, 33 age-matched HC were enrolled; 14 (42%) were males. There were no significant differences between CF and HC subjects for age (mean (SD) of 7.3 (3.8) vs. 7.0 (4.6) years respectively; P = 0.78) and gender (P = 0.33). All CF patients had detectable faecal HBD-2. There were no difference between faecal HBD-2 in CF and HC (median (IQR): 49.1 (19.7-77.2) vs. 43.4 (26.5-71.9)ng/g; P=0.7), and between PI and PS CF subjects (median (IQR): 49.1 (19.3 77.2) and 46.9 (32.0 104.4) ng/g; P = 0.6). CF subjects with faecal calprotectin ≥50mg/kg and <50mg/kg were also not different (50.5 (19.6-80.2) vs. 43.0 (19.0-70.4); P=0.7). There was no correlation between faecal HBD-2 and calprotectin in CF (r = 0.14; P = 0.4). Conclusions Faecal HBD-2 levels were not increased in children with CF, in inflamed or non-inflamed states. The lack of HBD-2 induction and upregulation under inflammatory conditions may suggest a diminished intestinal innate immune response in CF.