Marginating Transitional B cells Modulate Neutrophils in the Lung During Inflammation and Pneumonia

Pulmonary innate immunity is required for host defense; however, excessive neutrophil inflammation can cause life-threatening acute lung injury. B lymphocytes can be regulatory, yet little is known about peripheral transitional IgM+ B cells in terms of regulatory properties. Using single cell RNA sequencing, we discovered eight IgM+ B cell subsets with unique gene regulatory networks in the lung circulation dominated by transitional type 1 (T1B) and 2 (T2B) B cells. Lung intravital confocal microscopy revealed that T2B marginate in the pulmonary capillaries via CD49e and required CXCL13 and CXCR5. During lung inflammation, marginated T2B dampened excessive neutrophil vascular inflammation via the specialized proresolving molecule lipoxin A4 (LXA4). Exogenous CXCL13 dampened excessive neutrophilic inflammation by increasing marginated B cells and LXA4 recapitulated neutrophil regulation in B-cell deficient mice during inflammation and fungal pneumonia. Thus, the lung microvasculature is enriched in multiple IgM+ B cell subsets with marginating capillary T2B that dampen neutrophil responses. Intact lungs were dissected from 7 mice (all females) and placed in FACS tube with 4 ml of 1X PBS. A gentleMACS™ Dissociator 2.01 was used to mechanically liberate lung cells and cell suspension was filtered through a 70-μm cell strainer. Cell strainer was rinsed with an additional 6 ml of 1X PBS to recover remaining cells. Tissue samples were centrifuged at 2000 rpm for 5 min and supernatant was discarded. To eliminate erythrocyte contamination in single cell suspension, 2 mL of red blood cell (RBC) lysis buffer was added for 3 min. RBC lysis buffer was diluted by adding 10 ml of PBS. The resulting cell suspension was centrifuged at 2000 rpm for 5 min and supernatant containing the medium plus enzyme was discarded. Cells were resuspended in 500 μL of PBS and 12.5 μL of Fc Block was added for 15 min to block Fc receptors before being exposed to anti-CD45-PeCy7 antibody. Following another round of centrifugation at 2000 rpm for 5 min, cell pellet was first stained with 7-AAD viability dye (5μL per 200 μL) for 7 minutes in dark and then with 1 μL of anti-CD45-PeCy7 in 200 μl of final volume for 20 min. Unbound antibodies were rinsed off by centrifuging cells at 2000 rpm for 5 min and discarding the supernatant. Cells were resuspended in 300μL of PBS + 2% BSA then filtered through a 100-μm cell strainer. 7AAD- CD45+ viable immune cells were sorted in PBS + 2% BSA. Percentage of CD45+ cells was determined by flow cytometry and varied between 92-97%. Following centrifugation at 2000 rpm for 10 min, 40,000 CD45+ single cells were resuspended in a 50 μl of PBS + 2% BSA + 1 μl of RNAse inhibitor solution.