The antibacterial defense against infections depends on the cooperation between distinct phagocytes of the innate immune system, namely macrophages and neutrophils. However, the mechanisms driving this cooperation are incompletely understood. In this study we describe the crosstalk between Ly6C+ and Ly6C− macrophage-subtypes and neutrophils in the context of urinary tract infection (UTI) with uropathogenic E. coli (UPEC). Ly6C− macrophages acted as tissue resident sentinels and attracted circulating phagocytes by chemokines. Ly6C+ macrophages produced tumor necrosis factor (TNF) that licensed Ly6C− macrophages to release preformed CXCL2, which in turn caused matrix metalloproteinases (MMP-9) secretion by neutrophils to enable transepithelial migration.

The urothelium of the urinary bladder represents the first line of defense. However, uropathogenic E. coli (UPEC) damage the urothelium and cause acute bacterial infection. Here, we demonstrate the crosstalk between macrophages and the urothelium stimulating macrophage migration into the urothelium. Using spatial proteomics by MALDI-MSI and LC-MS/MS, a novel algorithm revealed the spatial activation and migration of macrophages. Analysis of the spatial proteome unravelled the coexpression of Myo9b and F4/80 in the infected urothelium, indicating that macrophages have entered the urothelium upon infection. Immunofluorescence microscopy additionally indicated that intraurothelial macrophages phagocytosed UPEC and eliminated neutrophils. Further analysis of the spatial proteome by MALDI-MSI showed strong expression of IL-6 in the urothelium and local inhibition of this molecule reduced macrophage migration into the urothelium and aggravated the infection. After IL-6 inhibition, the expression of matrix metalloproteinases and chemokines, such as CX 3 CL1 was reduced in the urothelium. Accordingly, macrophage migration into the urothelium was diminished in the absence of CX 3 CL1 signaling in Cx 3 cr1 gfp/gfp mice. Conclusively, this study describes the crosstalk between the infected urothelium and macrophages through IL-6-induced CX 3 CL1 expression. Such crosstalk facilitates the relocation of macrophages into the urothelium and reduces bacterial burden in the urinary bladder.Mucosal Immunology (2020) 13:702-714; https://doi.

Discussion on remote activation of Th17 cells by lung myeloid cells during EAE.

Macrophages perform essential functions during bacterial infections, such as phagocytosis of pathogens and elimination of neutrophils to reduce spreading of infection, inflammation and tissue damage. The spatial distribution of macrophages is critical to respond to tissue specific adaptations upon infections. Using a novel algorithm for correlative mass spectrometry imaging and state-of-the-art multiplex microscopy, we report here that macrophages within the urinary bladder are positioned in the connective tissue underneath the urothelium. Invading uropathogenic E.coli induced an IL-6-dependent CX 3 CL1 expression by urothelial cells, facilitating relocation of macrophages from the connective tissue into the urothelium. These cells phagocytosed UPECs and eliminated neutrophils to maintain barrier function of the urothelium, preventing persistent and recurrent urinary tract infection. Bottek et al., 2019 Macrophages maintain barrier function GRAPHICAL ABSTRACT

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