R. G. Ramos1, A. Bhattacharjee2, J. Tian2, B. Firek2, G. Wu2, M. Rogers2, A. Sheppeck2, T. Hand2, M. J. Morowitz1 1University Of Pittsburg,General Surgery,Pittsburgh, PA, USA 2Children’s Hospital Of Pittsburgh Of UPMC,Pediatric General And Thoracic Surgery,Pittsburgh, PA, USA
Introduction: Although enteral formulas are the cornerstone of nutritional therapy in the ICU, little attention has been devoted to their contents or their effects on the immune system or the microbiome. Published results from our lab show that when compared to conventional enteral nutrition (CEN), plant-based enteral nutrition (PBEN) significantly decreases intestinal inflammation and preserves beneficial fiber-degrading anaerobes in the gut. We have also shown that germ-free animals on PBEN are not protected from colitis. With these results in mind, we hypothesized that PBEN improves outcomes in colitis via microbiome-mediated effects on macrophage function.
Methods: Six groups (n=4 per group) of C57BL/6 male mice were fed either CEN or PBEN for 7 days. On day 8, one CEN and one PBEN group began 4% Dextran Sodium Sulfate (DSS) while one CEN and one PBEN group underwent both DSS and macrophage depletion. Macrophage depletion was achieved by intraperitoneal clodronate injection and confirmed by immunohistochemistry. The remaining CEN and PBEN group received clodronate injections without DSS. Weight, stool consistency, and fecal occult blood were tested daily to calculate disease activity index (DAI). A t-test was used to compare mean values. Colonic lamina propria macrophages of animals on PBEN or CEN undergoing DSS and PBEN or CEN without DSS were analyzed with flow cytometery (LSRFortessa and FlowJo software). Surface expression of CD45, Ly6C, MHC-II, CD11b, CX3CR1, and CD11c was used to identify colonic macrophages. These macrophages were subjected to LPS stimulation and IL-10, IL-1, and TNFa were measured. In order to exclude the effects of adaptive immunity, we performed our 4% DSS model on two groups of Rag1 -/- animals (n=4) on either CEN or PBEN.
Results: PBEN but not CEN protected against colitis in mice lacking B and T cells (Rag1-/- ). However, PBEN did not protect against colitis in macrophage depleted animals (p <0.05). Flow cytometry of animals with intact immune function on DSS showed that when compared with CEN fed mice, PBEN animals had greater populations of anti-inflammatory P4 mature macrophages (CD11b hi, CX3CR1 hi, Ly6C lo, MHC-II+), moderately differentiated P3/P5 population (CD11b hi, CX3CR1 int, Ly6C lo, MHC-II+), and a statistically significant increase in IL-10 production.
Conclusion: Our results indicate that PBEN protects against gut inflammation, which is common during critical illness, via effects on both the gut microbiome and intestinal macrophages. It appears that PBEN but not CEN preserves monocyte to macrophage maturation and IL-10 expression in the lamina propria (Figure). Further translational research is required to study the impact of enteral formulas on clinical outcomes in human ICU patients.
