Intra-granuloma accumulation and inflammatory differentiation of neutrophils underlie mycobacterial ESX-1-mediated immunopathology

The conserved ESX-1 type VII secretion system is a major virulence determinant of pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium marinum . ESX-1 is known to interact with infected macrophages, but its potential roles in regulating other host cells and immunopathology have remained largely unexplored. Using a murine M. marinum infection model we identify neutrophils and Ly6C+MHCII+ monocytes as the main cellular reservoirs for the bacteria. We show that ESX-1 promotes intra-granuloma accumulation of neutrophils and that neutrophils have a previously unrecognized required role in executing ESX-1-mediated pathology. To explore if ESX-1 also regulates the function of recruited neutrophils we performed single cell RNA-seq analysis indicating that ESX-1 drives newly recruited uninfected neutrophils into an inflammatory phenotype via an extrinsic mechanism. In contrast, monocytes restricted the accumulation of neutrophils and immunopathology, demonstrating a major host-protective function for monocytes specifically by suppressing ESX-1-mediated neutrophilic inflammation. iNOS activity was required for the suppressive mechanism and we identified Ly6C+MHCII+ monocytes as the main iNOS-expressing cell type in the infected tissue. These results suggest that ESX-1 mediates immunopathology by driving neutrophil accumulation and phenotypic differentiation in the infected tissue, and they demonstrate an antagonistic interplay between monocytes and neutrophils by which monocytes suppress host-detrimental neutrophilic inflammation. Author Summary The ESX-1 type VII secretion system is required for virulence of pathogenic mycobacteria, including Mycobacterium tuberculosis . ESX-1 interacts with infected macrophages, but its potential roles in regulating other host cells and immunopathology have remained largely unexplored. We demonstrate that ESX-1 promotes immunopathology by driving intra-granuloma accumulation of neutrophils, which upon arrival adopt an inflammatory phenotype in an ESX-1-dependent manner. In contrast, monocytes limited the accumulation of neutrophils and neutrophil-mediated pathology via an iNOS-dependent mechanism, suggesting a major host-protective function for monocytes specifically by restricting ESX-1-mediated neutrophilic inflammation. These findings provide insight into how ESX-1 promotes disease, and they reveal an antagonistic functional relationship between monocytes and neutrophils that may regulate immunopathology not only in mycobacterial infection, but also in other infections as well as in inflammatory conditions and cancer. ### Competing Interest Statement The authors have declared no competing interest.