Pulmonary immunity requires tight regulation, as interstitial inflammation can compromise gas exchange and lead to respiratory failure. Here we found a greater number of aged CD11bhiL-selectinloCXCR4+ polymorphonuclear leukocytes (PMNs) in lung vasculature than in the peripheral circulation. Using pulmonary intravital microscopy, we observed lung PMNs physically interacting with B cells via β2 integrins; this initiated neutrophil apoptosis, which led to macrophage-mediated clearance. Genetic deletion of B cells led to the accumulation of aged PMNs in the lungs without systemic inflammation, which caused pathological fibrotic interstitial lung disease that was attenuated by the adoptive transfer of B cells or depletion of PMNs. Thus, the lungs are an intermediary niche in the PMN lifecycle wherein aged PMNs are regulated by B cells, which restrains their potential to cause pulmonary pathology.
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We thank P. Kubes for mentorship and lab members; L. Zbytnuik, T. Nussbaumer and M. Willson for assisting with reagents and animals; and E. De Heuvel for assistance in processing the histology; and acknowledge the use of the Snyder Mouse Phenomics Resources Laboratory and Live Cell imaging facility (funded by the Snyder Institute) and the flow cytometry core facility. Supported by the Canadian Foundation for Innovation–John R. Evans Leaders fund with matching support from the Alberta Enterprise and Advanced Education Research Capacity Program (infrastructure funding), the Canadian Institutes of Health Research (operating grant RS-342013), the Department of Critical Care Medicine of the University of Calgary (startup funds), the University of Calgary Medical Group (bridge funding), the Cumming School of Medicine Research Enhancement Program and a tier II Canada Research Chair in Pulmonary Immunology, Inflammation and Host Defense (B.G.Y.).
The authors declare no competing financial interests.
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Integrated supplementary information
(a) Pulmonary intravital imaging was used to assess CD19+ (fluorochrome conjugated anti-CD19 antibody), Ly6G+ PMN (fluorochrome conjugated anti-Ly6G antibody) and MHCII+ cells (fluorochrome conjugated anti-MHCII antibody). CD19+ MHCII+ cells were quantified per field of view. Experiments were repeated three times (n = 3). Data is mean ± s.e.m. (b) Flow cytometry was used to assess leukocyte populations. Leukocytes were identified by forward and side scatter. PMN were identified as Ly6G+ and CD19-, while B cells were identified by Ly6G- CD19+. Dead cells were excluded using propidium iodide (PI) staining.
(a) Flow cytometry was used to assess B cells and PMN that were co-incubated in wells that allowed physical contact. MHCII cell surface levels were measured on PMN after B cell co-incubation in wells treated with an isotype control antibody, an irrelevant monoclonal antibody that binds PMN (anti-CD29 monoclonal antibody, clone Hmb1-1, 10 µg/mL) or an anti-CD18 blocking monoclonal antibody (clone GAME-46, 10 µg/ml). Experiments were repeated twice (n = 2). (b) PMN and B cell co-culture experiments were repeated in standard well that allow physical contact. B cells were pre-treated with Eα peptide prior to PMN incubation. MHCII (I-Ab) with properly configured Eα peptide can be detected with the Y-Ae monoclonal antibody. Ova peptide was used as a loading control and will not be detected by Y-Ae. PMN were verified to have acquired MHCII from B cells using a monoclonal antibody that detects I-Ab. Y-Ae antibody stains for B cells and PMN after peptide-loaded B cells are co-cultured with PMN are shown. (n = 3). (c) Single cell suspensions from blood and lung were treated with and without collagenase then cultured in vitro for 4-hours. After the culture, the percentage of MHCII+ annexinV+ early apoptotic PMN were analyzed. Each data point represents an individual experiment. Bars represent s.e.m. One way ANOVA with adjusted p values. * p = 0.02, ** p = 0.009.
(a) Flow cytometry assessed B cell depletion in lung and blood of Rosa-DTR and CD19-DTR mice following DT (25 ng/g of body weight) injected intraperitoneal for four consecutive days. Blood and lung were analyzed for the presence of CD19+ B cells and Ly6G+ PMN. n = 3. (b) B cells were quantified in blood and lung in Rosa-DTR and CD19-DTR mice that received 4 days of DT. Unpaired Student’s t test, ** p = 0.0035 for blood and p = 0.0013 for lung. (c) CD19-DTR mice received DT (25 ng/g of body weight, Monday-Wednesday-Friday) for 3 weeks and received intravenous adoptively transferred C57BL/6 B cells weekly. The numbers of B cells in the lung were quantified using flow cytometry. Each data point represents an individual experiment (n). Bars represent s.e.m.
Circulating PMN were identified by injecting mice with intravenous fluorochrome conjugated anti-CD45 antibody prior to tissue harvesting. Interstitial PMN were identified as Ly6G+ CD45-, while circulating PMN were identified as Ly6G+ CD45+. Alveolar cells, which are not in the circulation, were used to verify that the intravenous anti-CD45 antibody was not leaking from the blood into the tissue. Representative histogram is shown on the left for vascular and tissue pulmonary neutrophils. Bar graph displaying the percentage of CD45- interstitial neutrophils is shown on the right. Each data point represents an individual experiment (n). Bars represent s.e.m.
CD19-DTR or control (Rosa-DTR) mice received DT (25 ng/g of body weight, Monday-Wednesday-Friday) for 3 weeks. Additionally, mice received the PMN depleting antibody (1A8, 250 µg every Monday and Friday) intermittently during the 3-week B cells depletion. Flow cytometry shows PMN counts comparing control mice and B cell depleted mice with or without PMN depletion.
Clodronate (200 μg) was injected into mice intravenously for 24-hours and the percentage of F4/80+ cells were analyzed in bone marrow. The representative dot plots are shown on left and bar graphs showing the percentage are on the right. Unpaired Student’s t test, p = 0.0129, n = 3. (b) The percentage of MHCII+ and (c) MHCII+ annexin V+ early apoptotic PMN circulating in peripheral blood are shown after the clodronate injection. n=3. Bars represent s.e.m. * p = 0.048, ** p = 0.0072.
(a) B cells were quantified in the blood and lung of lymphocyte deficient mice (Rag1-/-). PMN were quantified in the blood and lung of Rag1-/-. Data represent the mean ± s.e.m. For panel a, each data point represents an individual experiment (n). * p = 0.02, ** p = 0.005. (b) CD19-DTR or Rosa-DTR mice received DT (25ng/g of body weight, Monday-Wednesday-Friday) for 3 weeks. Serum was collected at the end of 3 weeks and assayed for total IgG using standard ELISA. n = 4. Bars represent s.e.m. ***p < 0.0005.
Facs assessed monocyte populations from DT-treated Rosa-DTR (Control) and CD19-DTR animals. Lung cells were gated from a CD45+, F4/80-, Ly6G-, CD115+ population. Ly6G+SSC+ neutrophils were gated from CD45+ leukocytes.
Supplementary Figure 9 B cell deletion does not induce the systemic production of inflammatory mediators.
ELISA assessed IFN-γ, GM-CSF, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, MCP-1, TNF-α and KC from (a) lung tissue homogenates, (b) bronchoalveolar lavage (BAL) and (c) serum of Rosa26-DTR and CD19-DTR mice treated with 3 weeks of DT. All mediators were determined using multiplex except KC, which was assessed using a standard ELISA. Data represent the mean ± s.e.m. For panel a-f, each data point represents an individual experiment. Unpaired Student’s t test, *** p < 0.0006.
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