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

SMAD protein expression data from healthy controls, celiac disease, and environmental enteropathy.

<p>A) Representative western blot example investigating SMAD7; p-SMAD2,3 expression (using ERK1/2 as a normalizing protein) from controls (Ctl), celiac disease (CD), and environmental enteropathy (EE) patients. Note that for p-SMAD2,3, patients 3 and 4 have a double band that controls lacked. This band was likely an artifact and consequently was not considered in the densitometric analyses. B) Elevated SMAD7 densitometry levels in EE compared to controls (Ctl mean±SD = 0.47±0.20 a.u., EE = 1.13±0.25 a.u., p-value<0.05). Corresponds to WB5 (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006224#pntd.0006224.s006" target="_blank">S3 Table</a>). C) Elevated p-SMAD2,3 densitometry levels in EE compared to controls (Ctl mean±SD = 0.38±0.14 a.u., EE = 0.60±0.10 a.u., p-value<0.05). Corresponds to WB2 (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006224#pntd.0006224.s006" target="_blank">S3 Table</a>). D) Elevated p-SMAD2,3 densitometry levels in EE and CD compared to controls, with significant differences only between EE and controls (p-value<0.05) (Ctl mean±SD = 0.34±0.12 a.u., CD = 0.87±0.36 a.u., EE = 0.97±0.11 a.u.). Corresponds to WB1 (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006224#pntd.0006224.s006" target="_blank">S3 Table</a>).</p

TGF-β expression data.

<p>TGF-β expression levels were not significantly different in healthy controls, CD patients, and EE patients (Ctl mean±SD = 162.7±66.3 pg/mL, CD = 190.4±87.8 pg/mL, EE = 193.9±180.4 pg/mL, p-value = 0.793).</p

Immunohistochemical staining of duodenal biopsies from EE subjects for SMAD proteins.

<p>A) Representative immunohistochemical (IHC) photomicrographs at 100x and 400x from an EE duodenal biopsy showing SMAD7 staining in <u>both</u> the epithelium (arrows) and lamina propria (arrowheads). B) Representative IHC photomicrographs at 100x and 400x from an EE duodenal biopsy showing p-SMAD3 staining in <u>only</u> the epithelium (arrows).</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

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

Median LAZ, WLZ and WAZ scores in Pakistani infants over 18 months of follow-up.

<p>Dashed lines show q1 and q3 quartiles. Abbreviations: LAZ = Length-for-Age Z score, WLZ = Weight-for-Length Z score and WAZ = Weight-for-Age Z score.</p

Anti-Flagellin and anti-LPS immunoglobulin concentrations in Pakistani infants.

<p>Closed shapes indicate means±SDs in Pakistani infants over the follow-up period. Open shapes indicate means±SDs values in 36 healthy Boston infants with a mean age of 9.5 months. n = 380 for all immunoglobulins in Pakistani infants at 6 and 9 months of age; n = 36 for all immunoglobulins in Boston infants at 9.5 months of age. **** p< 0.0001, ** p< 0.01 and * p< 0.05 for comparisons of mean biomarker concentrations in Pakistani infants with those of healthy Boston infants (unpaired t tests).</p

Correlation coefficient matrix of immunoglobulins to flagellin and lipolysaccharide with measures of systemic inflammation and enteric inflammation & regeneration.

<p>Correlation coefficient matrix of immunoglobulins to flagellin and lipolysaccharide with measures of systemic inflammation and enteric inflammation & regeneration.</p