ROCKI Regulates Critical Functions in Macrophages and Neutrophils.

Neutrophils and macrophages are a major cellular component of innate immune response and are recruited rapidly in large numbers to sites of infection. The small GTPase Rho and its downstream effectors, the serine/threonine kinase ROCKI and ROCKII have been implicated in various cellular functions including actin cytoskeleton organization, cell adhesion, and cell motility in non-hematopoietic cells. We show that loss of ROCKI results in a significant reduction in the number of B lymphocytes in the bone marrow (BM). The number of late pre-B cells were significantly reduced in ROCKI−/− bone marrow compared to WT controls (n=4, WT [51%] vs. ROCKI−/− [40%]). No significant difference in the number of pro/pre B-cells or immature B cells were observed in ROCKI−/− BM compared to WT controls (n=4). Surprisingly, recirculating mature B cells were significantly increased in ROCKI−/− BM compared to WT (n=4, WT [13.25%] vs. ROCKI−/− [25.2%]). Consistently, in vitro migration of ROCKI−/− B lymphocytes towards SDF-1was also significantly reduced compared to WT controls. Next, we analyzed the recruitment of macrophages deficient in the expression of ROCKI using a well-studied model of aseptic thioglycolate-induced peritonitis. In spite of comparable expression of F4/80 (WT; 85.63% vs. ROCKI−/−; 88.68%, n=4) and α4β1 and α5β1 integrins (WT; 67.49% & 88.2% vs. ROCKI−/−; 71.82% & 87.09%, respectively, n=4) in WT and ROCKI−/− macrophages, a ∼3.2 fold increase in the migration of macrophages was observed in the peritoneal cavity of ROCKI−/− mice compared to controls (WT; 0.46 X 10⁶ vs. ROCKI−/−; 1.5 × 10⁶, n=3, p<0.01). In vitro, ROCKI deficiency in macrophages resulted in significant increase (∼2.2 fold) in haptotactic transwell migration in response to M-CSF on fibronectin coated wells (n=3, *p<0.0075) as well as an increase in migration in a wound healing assay compared to controls (∼3 fold, n=3, *p<0.005). In addition, ROCKI deficient macrophages also demonstrated a ∼2.5 fold increase in adhesion and spreading on fibronectin (n=3, *p<0.002), although no significant difference in the phagocytosis of sheep red blood cells was observed between WT and ROCKI−/− macrophages (Phagocytic index: WT; 98% vs. ROCKI−/− 97%, n=3, p>.05). Close examination of the cytoskeleton of ROCKI deficient macrophages using confocal microscopy revealed that F-actin was localized more across the entire cell surface in spread cells compared to wildtype controls. Consistently, flow cytometric analysis using Alexa 488-phalloidin staining revealed abundance of F-actin in ROCKI−/− macrophages compared to WT controls (WT; 46.19% vs. ROCKI−/−; 65.23%, n=3, *p<0.05). Furthermore, immunofluorescence imaging of focal adhesion complexes carried out using anti-vinculin antibody revealed more pronounced and increased focal adhesion sites in ROCKI deficient macrophages compared to WT controls. Biochemically, deficiency of ROCKI resulted in increase levels of cyclinD1, a molecule known to play a critical role in macrophage migration. Consistently, deficiency of ROCKI in neutrophils also demonstrated a ∼2.63 fold increase in migration in response to fMLP in vitro and in vivo in response to thioglycolate challenge compared to WT controls (WT; 0.47 X 10⁶, ROCKI−/− vs. 1.6 × 10⁶, *p<0.05). Taken together, our results provide a critical physiologic role for ROCKI in regulating both macrophage and neutrophil functions.