Reply.

Abstract

Potential conflict of interest: Nothing to report. Author names in bold designate shared co‐first authorship. We thank Dr. Ling and his colleagues for their comments on our recent publication. Classification of intestinal mononuclear phagocytes (MNPs) caused some confusion in the past. In particular, most Cx3cr1‐expressing cells that had been considered as dendritic cells (DCs), for example, in the landmark article by Niess et al.1 and in the letter of Dr. Ling, are now commonly regarded as macrophages.1 To analyze the effect of high‐fat diet (HFD) on MNPs, we differentiated between various lamina propria MNP populations, including both DCs and iMΦ based on surface marker expression.3 To study DC subsets, we analyzed the frequency of CD11b+CD11c+MHCII+CD103+ and CD11b–CD11c+ MHCII+CD103+ cells. Both subsets appeared unchanged comparing wild‐type (WT) and Cx3cr1–/– mice. In contrast, we observed a significantly lower abundance of CD11b+CD11c+MHCII+F4/80+ cells in Cx3cr1–/– mice compared to WT mice, a subset of cells that corresponds to CD64+CX3CR1high iMΦ. Consequently, our data suggested that Cx3cr1 deficiency mainly affects iMΦ. Whereas DCs are critical for induction of adaptive immune responses, F4/80+ iMΦ show highest phagocytic capacity of all lamina propria cells and are important for local clearance of bacterial products in the lamina propria.2 Indeed, a reduction of this specific immune cell subset in our study was associated with decreased phagocytic capacity of colonic lamina propria cells. Concomitantly, we measured higher concentrations of endotoxin in portal serum of Cx3cr1–/– mice upon HFD feeding. Accordingly, antibiotic treatment led to a marked improvement of all hepatic hallmarks of nonalcoholic steatohepatitis in Cx3cr1–/– mice, indicating that the pronounced activation of hepatic innate immune response is dependent on the translocation of bacterial products and intestinal barrier dysfunction in Cx3cr1–/– mice. Absence of Cx3cr1 was associated with decreased expression of epithelial tight junctions, a reduced abundance of intestinal macrophages, and increased bacterial translocation. Mechanistically, we observed that depletion of the intestinal microbiota did not only reduce the number of infiltrating macrophages in the liver, but also induced up‐regulation of the restorative markers, CD206 and CD124, on these cells. Therefore, our study suggests that CX3CR1 limits progression of steatohepatitis by maintaining intestinal homeostasis. Future studies are required to investigate the effect of HFD on intestinal adaptive immune response in order to elucidate the role of antigen presentation and mucosal tolerance in maintaining intestinal barrier integrity. A selective depletion of different subsets of MNPs could be an elegant experimental approach to decipher their specific functions for intestinal immune homeostasis. We appreciate this idea; however, we think these experiments are beyond the scope of this study.