SRA

Loss of NADPH oxidase activity in phagocytes leads to altered cellular responses and exaggerated inflammation in Chronic Granulomatous Disease (CGD). We sought to assess the effects of Nox2 absence on monocyte-derived macrophages (MoMacs) in gp91phox-/y mice during zymosan-induced peritonitis. MoMacs from CGD and wild type (WT) peritonea were lavaged and characterized over time. Though the numbers harvested from both genotypes were virtually identical, there were marked differences in maturation: newly recruited WT MoMacs rapidly enlarged and matured with loss of Ly6C and gain of MHCII, CD206 and CD36, while MoMacs in CGD remained small and were mostly Ly6C+MHCII-. RNAseq analyses showed few intrinsic differences between genotypes in newly recruited MoMacs, but significant differences over time. WT MoMacs demonstrated changes in metabolism, adhesion and reparative functions, while CGD MoMacs remained inflammatory. Labeling with PKH dye demonstrated that while WT MoMacs were mostly recruited within the first 24 hours and remained in the peritoneum while maturing and enlarging, CGD monocytes continued to stream into the peritoneum for days with many migrating to the diaphragm where they were found in fibrin(ogen) clots surrounding clusters of neutrophils in what appeared to be nascent granulomata. Importantly, these observations appeared to be entirely driven by the milieu: adoptive transfer of CGD MoMacs into inflamed peritonea of WT mice resulted in immunophenotypic maturation and behavior, and conversely, altered maturation/behavior of WT MoMacs was seen after adoptive transfer into inflamed peritonea of CGD mice. These data demonstrate heightened recruitment and fundamental failure of MoMac maturation in CGD. Overall design: Monocytes and monocyte-derived macrophages from C57BL/6 (WT) and gp91 phox -/y (CGD) mice were collected from peritoneal lavage at 3 timepoints after intraperitoneal zymosan injection. Peritoneal lavage cells were FACS sorted to purify monocyte/macropahges with differential expression of the phenotypic markers Ly6C and MHCII, yielding populations of Ly6C+MHCII-, Ly6C+MHCII+, Ly6C-MHCII- or Ly6C-MHCII+ monocyte/macrophages over time. Three replicate cohorts of mice were collected for each treatment condition, resulting in a total of 42 samples analyzed.