Live Imaging of Monocyte Subsets in Immune Complex-Mediated Glomerulonephritis Reveals Distinct Phenotypes and Effector Functions.

BACKGROUND: Immune complexes within glomerular capillary walls cause crescentic GN (CrGN). Monocytes and macrophages are important in mediating CrGN, but little work has been done to phenotype the subpopulations involved and determine their respective contributions to glomerular inflammation. METHODS: Live glomerular imaging using confocal microscopy monitored intravascular monocyte subset behavior during nephrotoxic nephritis (NTN) in a novel WKY-hCD68-GFP monocyte/macrophage reporter rat strain. Flow cytometry and qPCR further analyzed ex vivo the glomerular leukocyte infiltrate during NTN. RESULTS: Non-classical monocytes surveyed the glomerular endothelium via lymphocyte function-associated antigen 1 (LFA-1) in the steady state. During NTN, non-classical monocytes were recruited first, but subsequent recruitment and retention of classical monocytes was associated with glomerular damage. Monocytes recruited to the glomerular vasculature did not undergo transendothelial migration. This finding suggests that inflammation in immune complex-mediated CrGN is predominantly intravascular, driven by dynamic interactions between intravascular blood monocytes and the endothelium. Glomerular endothelium and non-classical monocytes overexpressed a distinct chemokine axis, which may orchestrate inflammatory myeloid cell recruitment and expression of damage mediators. Reduced classical monocyte recruitment in Lewis rats during NTN confirmed a role for CD16 in mediating glomerular damage. CONCLUSIONS: Monocyte subsets with distinct phenotypes and effector functions may be important in driving inflammation in experimental CrGN resulting from immune complexes formed within the glomerular capillary wall. LFA-1-dependent endothelial surveillance by non-classical monocytes may detect immune complexes through CD16, orchestrating the inflammatory response through intravascular retention of classical monocytes, which results in glomerular damage and proteinuria.

Syk Activation in Circulating and Tissue Innate Immune Cells in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

OBJECTIVE: Syk is a cytoplasmic protein tyrosine kinase that plays a role in signaling via B cell and Fc receptors (FcR). FcR engagement and signaling via Syk is thought to be important in antineutrophil cytoplasm antibody (ANCA) IgG-mediated neutrophil activation. This study was undertaken to investigate the role of Syk in ANCA-induced myeloid cell activation and vasculitis pathogenesis. METHODS: Phosphorylation of Syk in myeloid cells from healthy controls and ANCA-associated vasculitis (AAV) patients was analyzed using flow cytometry. The effect of Syk inhibition on myeloperoxidase (MPO)-ANCA IgG activation of cells was investigated using functional assays (interleukin-8 and reactive oxygen species production) and targeted gene analysis with NanoString. Total and phosphorylated Syk at sites of tissue inflammation in patients with AAV was assessed using immunohistochemistry and RNAscope in situ hybridization. RESULTS: We identified increased phosphorylated Syk at critical activatory tyrosine residues in blood neutrophils and monocytes from patients with active AAV compared to patients with disease in remission or healthy controls. Syk was phosphorylated in vitro following MPO-ANCA IgG stimulation, and Syk inhibition was able to prevent ANCA-mediated cellular responses. Using targeted gene expression analysis, we identified up-regulation of FcR- and Syk-dependent signaling pathways following MPO-ANCA IgG stimulation. Finally, we showed that Syk is expressed and phosphorylated in tissue leukocytes at sites of organ inflammation in AAV. CONCLUSION: These findings indicate that Syk plays a critical role in MPO-ANCA IgG-induced myeloid cell responses and that Syk is activated in circulating immune cells and tissue immune cells in AAV; therefore, Syk inhibition may be a potential therapeutic option.

In-silico analysis of myeloid cells across the animal kingdom reveals neutrophil evolution by colony-stimulating factors.

Neutrophils constitute the largest population of phagocytic granulocytes in the blood of mammals. The development and function of neutrophils and monocytes is primarily governed by the granulocyte colony-stimulating factor receptor family (CSF3R/CSF3) and macrophage colony-stimulating factor receptor family (CSF1R/IL34/CSF1) respectively. Using various techniques this study considered how the emergence of receptor:ligand pairings shaped the distribution of blood myeloid cell populations. Comparative gene analysis supported the ancestral pairings of CSF1R/IL34 and CSF3R/CSF3, and the emergence of CSF1 later in lineages after the advent of Jawed/Jawless fish. Further analysis suggested that the emergence of CSF3 lead to reorganisation of granulocyte distribution between amphibian and early reptiles. However, the advent of endothermy likely contributed to the dominance of the neutrophil/heterophil in modern-day mammals and birds. In summary, we show that the emergence of CSF3R/CSF3 was a key factor in the subsequent evolution of the modern-day mammalian neutrophil.