N. J. Klingensmith1, K. Fay1, C. Chen1, J. Lyons1, C. M. Coopersmith1 1Emory University School Of Medicine,Surgery,Atlanta, GA, USA
Background – Sepsis is associated with intestinal hyperpermeability which carries significant mortality. This permeability is augmented in mice lacking Junctional Adhesion Molecule A (JAM-A), an enterocyte tight junction protein. Our unpublished data reveal JAM-A-/- mice paradoxically have decreased systemic bacterial burden and improved sepsis survival following cecal ligation and puncture (CLP). At baseline, the intestinal epithelium, local immune system, and gut microbiome all interact to maintain homeostasis. How the immune system and microbiome respond to intestinal hyperpermeability in septic JAM-A-/- animals may provide a mechanism for their improved sepsis survival.
Methods – 6-12 week old male and female C57BL/6 (WT) and JAM-A-/- mice underwent CLP to induce sepsis and were sacrificed 24 hours later for functional studies. Mesenteric lymph nodes (MLN) and small bowel Peyer’s Patches (PP) were collected and B220+ cells were stained and counted by flow cytometry. Serum and small bowel homogenate IgA were measured by ELISA. Intestinal microbiome taxonomy were identified from fecal samples by 16S rRNA amplicon pyrosequencing followed by QIIME pipeline analysis. Statistical analysis was performed using a Student’s t-test or Mann-Whittney test depending on Gaussian distribution and a p-value of <0.05 was considered to be statistically significant.
Results- Septic JAM-A-/- mice had a greater frequency of B cells compared to septic WT controls in their MLN (mean frequency, 25.2±2.3% vs 17.2±1.4%, p=0.009, n=10) and PP (mean frequency, 19.0±1.7% vs 12.8±2.2%, p=0.04, n=7). IgA within the intestine, a product of B cells, was elevated in septic JAM-A-/- animals compared to septic WT controls (mean, 241.7±37.9 vs 131.8±19.8 ng/mg of gut tissue, p=0.02, n=7-10) suggesting prior antigenic exposure. Furthermore, serum IgA concentration in septic JAM-A-/- mice was increased compared to septic WT mice (mean, 178.1±43.9 vs 39.5±6.9 ng/mL, p=0.005, n=11) providing a potential mechanism for decreased systemic bacterial burden in septic JAM-A-/- mice. Principle coordinate analysis of weighted and unweighted UniFrac distances of 16S rRNA sequencing revealed separation between septic JAM-A-/- mice and septic WT animals (p<0.001, n=5-7).
Conclusion – JAM-A-/- mice have greater populations of B cells in their MLN and small bowel PP that produce more IgA. The increased gut IgA may result in a shift toward less pathogenic microflora which are potentially cleared more easily in the event of systemic contamination, such as in the setting of sepsis. Greater antigen presentation, local immune priming, and alteration of the microflora may be the source of improved sepsis survival in JAM-A-/- mice.