S100A8 and S100A9 Are Important for Postnatal Development of Gut Microbiota and Immune System in Mice and Infants.

BACKGROUND & AIMS: After birth, the immune system matures via interactions with microbes in the gut. The S100 calcium binding proteins S100A8 and S100A9, and their extracellular complex form, S100A8-A9, are found in high amounts in human breast milk. We studied levels of S100A8-A9 in fecal samples (also called fecal calprotectin) from newborns and during infancy, and their effects on development of the intestinal microbiota and mucosal immune system. METHODS: We collected stool samples (n = 517) from full-term (n = 72) and preterm infants (n = 49) at different timepoints over the first year of life (days 1, 3, 10, 30, 90, 180, and 360). We measured levels of S100A8-A9 by enzyme-linked immunosorbent assay and analyzed fecal microbiomes by 16S sRNA gene sequencing. We also obtained small and large intestine biopsies from 8 adults and 10 newborn infants without inflammatory bowel diseases (controls) and 8 infants with necrotizing enterocolitis and measured levels of S100A8 by immunofluorescence microscopy. Children were followed for 2.5 years and anthropometric data and medical information on infections were collected. We performed studies with newborn C57BL/6J wild-type and S100a9-/- mice (which also lack S100A8). Some mice were fed or given intraperitoneal injections of S100A8 or subcutaneous injections of Staphylococcus aureus. Blood and intestine, mesenterial and celiac lymph nodes were collected; cells and cytokines were measured by flow cytometry and studied in cell culture assays. Colon contents from mice were analyzed by culture-based microbiology assays. RESULTS: Loss of S100A8 and S100A9 in mice altered the phenotypes of colonic lamina propria macrophages, compared with wild-type mice. Intestinal tissues from neonatal S100-knockout mice had reduced levels of CX3CR1 protein, and Il10 and Tgfb1 mRNAs, compared with wild-type mice, and fewer T-regulatory cells. S100-knockout mice weighed 21% more than wild-type mice at age 8 weeks and a higher proportion developed fatal sepsis during the neonatal period. S100-knockout mice had alterations in their fecal microbiomes, with higher abundance of Enterobacteriaceae. Feeding mice S100 at birth prevented the expansion of Enterobacteriaceae, increased numbers of T-regulatory cells and levels of CX3CR1 protein and Il10 mRNA in intestine tissues, and reduced body weight and death from neonatal sepsis. Fecal samples from term infants, but not preterm infants, had significantly higher levels of S100A8-A9 during the first 3 months of life than fecal samples from adults; levels decreased to adult levels after weaning. Fecal samples from infants born by cesarean delivery had lower levels of S100A8-A9 than from infants born by vaginal delivery. S100 proteins were expressed by lamina propria macrophages in intestinal tissues from infants, at higher levels than in intestinal tissues from adults. High fecal levels of S100 proteins, from 30 days to 1 year of age, were associated with higher abundance of Actinobacteria and Bifidobacteriaceae, and lower abundance of Gammaproteobacteria-particularly opportunistic Enterobacteriaceae. A low level of S100 proteins in infants' fecal samples associated with development of sepsis and obesity by age 2 years. CONCLUSION: S100A8 and S100A9 regulate development of the intestinal microbiota and immune system in neonates. Nutritional supplementation with these proteins might aide in development of preterm infants and prevent microbiota-associated disorders in later years.

S100A8 causes a shift toward expression of activatory Fcγ receptors on macrophages via toll-like receptor 4 and regulates Fcγ receptor expression in synovium during chronic experimental arthritis.

OBJECTIVE: The levels of both Fcγ receptor (FcγR) and the alarmins S100A8 and S100A9 are correlated with the development and progression of cartilage destruction during antigen-induced arthritis (AIA). This study was undertaken to study the active involvement of S100A8, S100A9, and S100A8/S100A9 in FcγR regulation in murine macrophages and synovium during AIA. METHODS: Recombinant murine S100A8 (rS100A8) was injected into normal mouse knee joints, and the synovium was isolated for analysis of FcγR messenger RNA (mRNA) expression by reverse transcription-polymerase chain reaction (RT-PCR). Macrophages, including bone marrow macrophages derived from Toll-like receptor 4-deficient (TLR-4(-/-)) mice, and polymorphonuclear cells (PMNs) were stimulated with S100 proteins, and levels of FcγR mRNA and protein were measured using RT-PCR and fluorescence-activated cell sorting analyses. AIA was induced in the knee joints of S100A9-deficient (S100A9(-/-)) mice, compared with wild-type (WT) controls, and the extent of cartilage destruction was determined using immunohistochemical analysis. RESULTS: Intraarticular injection of rS100A8 into the knee joints of normal mice caused a strong up-regulation of mRNA levels of activating FcγRI (64-fold increase) and FcγRIV (256-fold increase) in the synovium. Stimulation of macrophages with rS100A8 led to significant up-regulation of mRNA and protein levels of FcγRI and FcγRIV, but not FcγRIII, while the effects of S100A9 or S100A8/S100A9 complexes were less potent. Stimulation of PMNs (32Dcl3 cell line) with S100 proteins had no effect on FcγR expression. Up-regulation of FcγRI and FcγRIV was abrogated in rS100A8-stimulated macrophages from TLR-4(-/-) mice, indicating that the induction of FcγR expression by S100A8 is mediated by TLR-4. FcγR expression in the inflamed synovium of S100A9(-/-) mice was significantly lower on day 14 after arthritis induction when compared with WT controls, and these findings correlated with reduced severity of matrix metalloproteinase-mediated cartilage destruction. CONCLUSION: S100A8 is a strong promoter of activating FcγRI and FcγRIV in macrophages through the activation of TLR-4, and acts as a regulator of FcγR expression in inflamed synovium in chronic experimental arthritis.