Serum anti-flagellin and anti-lipopolysaccharide immunoglobulins as predictors of linear growth faltering in Pakistani infants at risk for environmental enteric dysfunction

Background: Environmental Enteric Dysfunction (EED) in children from low-income countries has been linked to linear growth declines. There is a critical need to identify sensitive and early EED biomarkers.Objective: Determine whether levels of antibodies against bacterial components flagellin (flic) and lipopolysaccharide (LPS) predict poor growth.Design/Methods: In a prospective birth cohort of 380 children in rural Pakistan blood and stool samples were obtained at ages 6 and 9 months. Linear mixed effects models were used to examine longitudinal associations between quartiles of anti-flic and anti-LPS antibodies and changes in LAZ, WAZ and WLZ scores. Spearman\u27s correlations were measured between anti-flic and anti-LPS immunoglobulins with measures of systemic/enteric inflammation and intestinal regeneration.Results: Anti-LPS IgA correlated significantly with CRP, AGP and Reg1 serum at 6mo and with MPO at 9mo. In multivariate analysis at 6mo of age, higher anti-LPS IgA levels predicted greater declines in LAZ scores over subsequent 18mo (comparing highest to lowest quartile, β (SE) change in LAZ score/year = -0.313 (0.125), p-value = 0.013). Anti-flic Ig A in the two highest quartiles measured at 9mo of age had declines in LAZ of -0.269 (0.126), p = 0.033; and -0.306 (0.129), p = 0.018 respectively, during the subsequent 18mo of life, compared to those in the lowest quartile of anti-flic IgA.Conclusions and Relevance: Elevated anti-flic IgA and anti-LPS IgA antibodies at 6 and 9mo, predict declines in linear growth. Systemic and enteric inflammation correlated with anti-LPS IgA provides mechanistic considerations for potential future interventions

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

<div><p>Background</p><p>Polymeric immunoglobulin receptor (pIgR) transport of secretory immunoglobulin A (SIgA) to mucosal surfaces is thought to promote gut integrity and immunity to <i>Salmonella enterica</i> serovar Typhimurium (<i>S</i>. <i>Typhimurium</i>), an invasive pathogen in mice. To elucidate potential mechanisms, we assessed intestinal barrier function and both oral and systemic S. <i>Typhimurium</i> virulence in pIgR knockout (KO) and wildtype (WT) mice.</p><p>Methods</p><p>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 <i>S</i>. <i>Typhimurium</i> to compare colonization, tissue dynamics, and inflammation between KOs and WTs.</p><p>Results</p><p>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 <i>S</i>. <i>Typhimurium</i> exhibited increased splenomegaly, tissue colonization, and pro-inflammatory cytokines vs. pIgR KOs, which showed increased survival following either oral or IV infection.</p><p>Conclusions</p><p>Absence of pIgR compromises gut integrity but does not exacerbate bacterial translocation nor <i>S</i>. <i>Typhimurium</i> 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.</p></div

Increased survival of pIgR KO following high-dose oral <i>S</i>. <i>Typhimurium</i> challenge.

<p>Mice were gavaged with 10⁹ CFU of <i>S</i>. <i>Typhimurium</i> following a 4-hour fast and followed for 2 weeks. Mean survival for C57BL/6 mice was statistically significantly shorter than the pIgR KO mice (<i>P</i><0.05, log-rank Mantel-Cox test). Data are representative of 3 experiments, with equivalent infectious doses between KO and WT in each experiment.</p

Infant and Maternal Characteristics at six and nine months^*.

<p>Infant and Maternal Characteristics at six and nine months^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193768#t001fn002" target="_blank">*</a>}.</p

Serum pro-inflammatory cytokines TFNα, IL-1β, and IL-6 are significantly lower in pIgR KO mice vs wildtype mice 7 days post oral <i>S</i>. <i>Typhimurium</i> infection.

<p>C57BL/6 mice showed significantly increased (A) TNFα (<i>P</i><0.0001, Mann-Whitney), (B) IL-1β (<i>P</i><0.005, Mann-Whitney test), and (C) IL-6 (<i>P</i><0.0001, Mann-Whitney) compared to pIgR KO. (D) No difference in IFNγ levels between groups.</p

Intestinal and systemic indicators of <i>S</i>. <i>Typhimurium</i> infection intensity are reduced in pIgR KO mice following oral challenge.

<p>(A) Spleen weight was significantly elevated in C57BL/6 mice 7 days post-oral infection compare to both uninfected C57BL/6 (<i>P</i><0.001) and infected pIgR KO (<i>P</i><0.01, Kruskal-Wallis with Dunn multiple comparison test). (B) Cecum weight was significantly diminished in C57BL/6 mice compared to pIgR KO mice at 7 days post-oral infection (<i>P</i><0.05, Kruskal-Wallis with Dunn multiple comparison test). Data are representative of 3 experiments. Baseline weights of spleen and cecum did not differ in uninfected C57BL/6 and pIgR KO mice.</p

Small intestinal transepithelial resistance is impaired, but translocation of bacteria is diminished, in pIgR KO mice.

<p>Ussing chamber studies of mid-jejunal sections revealed a modest decrease in (A) transepithelial resistance in pIgR knockout mice as compared to C57BL/6 mice (<i>P</i><0.05, by t-test), and no difference in measures of (B) short circuit current, or (C) FITC-dextran flux. Data are representative of 3 experiments. (D) Fewer aerobic bacterial colonies were recovered from homogenized mesenteric lymph nodes of pIgR KO mice compared to C57BL/6 mice (<i>P</i><0.05, Mann-Whitney). Data are representative of 6 experiments.</p

The association of anti-flagellin and anti-lipopolysaccharide immunoglobulin concentrations at 6 and 9 months with annual Z score changes for length using linear mixed effects models.

<p>The association of anti-flagellin and anti-lipopolysaccharide immunoglobulin concentrations at 6 and 9 months with annual Z score changes for length using linear mixed effects models.</p

Impact of enteropathogens on faltering growth in a resource-limited setting

Introduction: Environmental enteropathy is an important contributor to childhood malnutrition in the developing world. Chronic exposure to fecal pathogens leads to alteration in intestinal structure and function, resulting in impaired gut immune function, malabsorption, and growth faltering leading to environmental enteropathy.Methods: A community-based intervention study was carried out on children till 24 months of age in Matiari district, Pakistan. Blood and fecal specimens were collected from the enrolled children aged 3-6 and 9 months. A real-time PCR-based TaqMan array card (TAC) was used to detect enteropathogens.Results: Giardia, Campylobacter spp., enteroaggregative Escherichia coli (EAEC), Enteropathogenic Escherichia coli (EPEC), Enterotoxigenic Escherichia coli (ETEC), and Cryptosporidium spp. were the most prevailing enteropathogens in terms of overall positivity at both time points. Detection of protozoa at enrollment and 9 months was negatively correlated with rate of change in height-for-age Z (ΔHAZ) scores during the first and second years of life. A positive association was found between Giardia, fecal lipocalin (LCN), and alpha 1-Acid Glycoprotein (AGP), while Campylobacter spp. showed positive associations with neopterin (NEO) and myeloperoxidase (MPO).Conclusion: Protozoal colonization is associated with a decline in linear growth velocity during the first 2 years of life in children living in Environmental enteric dysfunction (EED) endemic settings. Mechanistic studies exploring the role of cumulative microbial colonization, their adaptations to undernutrition, and their influence on gut homeostasis are required to understand symptomatic enteropathogen-induced growth faltering

Increased survival of pIgR KO following intravenous <i>S</i>. <i>Typhimurium</i> challenge.

<p>Mice were injected IV via the tail vein with 3.2x10² CFU of <i>S</i>. <i>Typhimurium</i> and followed for 26 days. Mean survival for C57BL/6 mice was statistically significantly shorter than the pIgR KO mice (<i>P</i><0.0001, log-rank Mantel-Cox test). Data are representative of 2 experiments.</p