Neutrophil Granulocytes Mediate Cellular Communication Between the Damaged Intestinal Tract and Mesenteric Lymph Nodes at the Onset of Intestinal Graft-Versus-Host Disease

Abstract

Multiple studies have shown the critical role of conditioning-induced damage of the intestinal tract and the intestinal immune system in the onset of graft-versus-host disease (GVHD). However, it is unclear how and where the tissue damage signals are transmitted from the peri-intestinal tissue into the mesenteric lymph nodes (mLNs), where the adaptive T cell response leading to GVHD is very likely initiated. We have recently described a critical role for neutrophil granulocytes (neutrophils) in the initiation of GvHD. We now report that neutrophils are not normally found in mLN, but that neutrophils of recipient origin accumulate in mLN upon conditioning of mice by total body irradiation. This was in parallel to the appearance of bacterial 16S RNA in these LN, supporting the concept of an influx of bacteria or their components via the lymph system, possibly within myeloid cells. Using 3D light sheet fluorescence microcopy (LSFM) of the terminal ileum and the mLN we found neutrophil-containing clusters in the terminal ileum in untreated mice, which enlarged upon irradiation, together with the appearance of infiltrates in the marginal zone of the mLN. Reduction of the bacterial load in the intestinal tract reduced neutrophil recruitment by 90% in the terminal ileum and 70% in the mLN. We then analyzed candidate molecules that could be responsible for the selective recruitment of neutrophils into the ileum/mLN. Using gene-targeted mice and pharmacological inhibitors we could exclude a role for the following molecules on the recipient side: TNF, CCL2 and P2Y2 as well as TLR4 signal transduction, sphingosin-1-phosphate and JAK1/2 signaling. Importantly, we found increased levels of MHC class II and the integrin CD11b on neutrophils residing in the ileum and mLN compared to blood-derived neutrophils. This indicates a different biological function for these LN-residing neutrophils compared to their blood-derived counterparts. One possible function is the direct, MHC II-dependent antigen presentation by neutrophils towards donor T cells. In summary, our data support the concept that neutrophil granulocytes mediate cellular communication between the damaged intestinal tract and mesenteric lymph nodes, where donor T cells are being primed during the early phase of intestinal GVHD.