Pleural innate response activator B cells protect against pneumonia via a GM-CSF-IgM axis - PubMed (original) (raw)

. 2014 Jun 2;211(6):1243-56.

doi: 10.1084/jem.20131471. Epub 2014 May 12.

Benjamin G Chousterman 2, Ingo Hilgendorf 2, Clinton S Robbins 2, Igor Theurl 2, Louisa M S Gerhardt 2, Yoshiko Iwamoto 2, Tam D Quach 3, Muhammad Ali 2, John W Chen 2, Thomas L Rothstein 3, Matthias Nahrendorf 2, Ralph Weissleder 4, Filip K Swirski 5

Affiliations

Pleural innate response activator B cells protect against pneumonia via a GM-CSF-IgM axis

Georg F Weber et al. J Exp Med. 2014.

Abstract

Pneumonia is a major cause of mortality worldwide and a serious problem in critical care medicine, but the immunophysiological processes that confer either protection or morbidity are not completely understood. We show that in response to lung infection, B1a B cells migrate from the pleural space to the lung parenchyma to secrete polyreactive emergency immunoglobulin M (IgM). The process requires innate response activator (IRA) B cells, a transitional B1a-derived inflammatory subset which controls IgM production via autocrine granulocyte/macrophage colony-stimulating factor (GM-CSF) signaling. The strategic location of these cells, coupled with the capacity to produce GM-CSF-dependent IgM, ensures effective early frontline defense against bacteria invading the lungs. The study describes a previously unrecognized GM-CSF-IgM axis and positions IRA B cells as orchestrators of protective IgM immunity.

© 2014 Weber et al.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

GM-CSF controls IgM production. (A) In vitro culture of CD19+ serosal B cells. Gating strategy and phenotyping of IRA B cells 2 d after culture in medium or with 10 µg/ml LPS. A representative contour plot of n > 5 is shown. (B) CD131 (Csf2rb) expression on selected cells from WT mice (n = 4). (C) In vitro culture of serosal B1a cells sorted from WT, _Csf2_−/−, and _Csfrb_−/− mice. Data show intracellular IgM and GM-CSF in cells cultured in medium alone or after LPS (10 µg/ml) stimulation after 1 d of culture (n = 3–5). The gate for GM-CSF was set using an isotype control (IgG2a) and the gate for intracellular IgM represents the upper 99% limit of intracellular IgM staining at baseline. (D) In vitro culture of serosal B1a cells sorted from WT, _Csf2_−/−, and Csfrb−/− mice, and B2 cells sorted from WT mice. Percentage of IgM(ic)high cells cultured for 1 d in medium, after stimulation with 10 µg/ml LPS alone, or 10 µg/ml LPS + rGM-CSF, or with a Stat5 inhibitor (n = 3–8, mean ± SD). ns, not significant. (E) IgM ELISA of culture supernatants from the same groups as in D (n = 3–8, mean ± SD). ns, not significant. (F) IgM ELISPOT of cultured B1a cells. A representative ELISPOT of n = 3 is shown. (G) Quantitative RT-PCR analysis of Csf2 and Prdm1 expression in cultured serosal B1a cells from WT, _Csf2_−/−, and _Csf2rb_−/− mice with and without LPS (10 µg/ml) stimulation for 1 d (n = 3). Csf2 expression levels after LPS stimulation is shown relative to WT LPS as mean ± SD. Prdm1 expression after LPS stimulation is shown relative to WT medium as mean ± SD (nd, not detected). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 2.

Figure 2.

Intracellular staining of IgM. (A) Serosal B1a cells were sorted, placed in culture with LPS for 1 d, and stained for surface and intracellular IgM. Data show that the procedure can stain for intracellular IgM and that both surface and intracellular staining can be resolved. Surface IgM staining is followed by intracellular staining. A representative analysis of n > 5 is shown. (B) Validation of IgM production by ELISPOT on sorted B1a cavity cells 1 d after in vitro LPS stimulation. Shown is a representative analysis from n > 5.

Figure 3.

Figure 3.

Identification of IRA B cells after airway challenge. (A) Identification of IRA B cells in the lung 2 d after LPS i.n. challenge. A representative dot plot of n > 5 is shown. (B) Enumeration of IRA B cells in different organs in steady-state and after LPS challenge (n = 4/group). (C) Enumeration as percentage increase (n = 4). Relevant data are presented as mean ± SD and tested by ANOVA; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4.

Figure 4.

Generation of mixed chimeras. (A) Leukocyte reconstitution in WT/µMT, GM/WT, and GM/µMT mice in the pleural space, blood, and lung 10 wk after irradiation and bone marrow transfer. Data show the relative percentages of myeloid (CD11b+) and nonmyeloid (CD11b−) cells in the three compartments (n = 4, mean ± SD). (B) B cells in the pleural space of a WT mouse. Gating strategy for B1a (CD19+IgMhighCD43+CD5+), B1b (CD19+IgMhighCD43+CD5−), B2 (CD19+IgMlowCD43−CD5−), and B reg (CD19+IgM+CD43−CD5+) B cells is shown. (C) Relative proportions of total B cells and B1a B cells in the pleural space of WT/µMT, GM/WT, and GM/µMT chimeric mice and non-irradiated WT mice in the steady state. The chimeric mice were analyzed 8 wk after bone marrow transfer (n = 3–5, mean ± SD). (D) Enumeration of the various subsets in the three chimeras (n = 3–5, mean ± SD). (E) CD45.2 expression on blood leukocytes in CD45.1+ mice that had been lethally irradiated and reconstituted with CD45.2+ bone marrow cells 10 wk earlier. The plot is representative from that of the three chimeras. (F) Lung H&E in the three sets of chimeric mice 10 wk after bone marrow reconstitution (bars, 200 µm). (G) Turbidity analysis of BAL at 600 nm from WT/µMT, GM/µMT, WTGM (lethal irradiation of WT mouse and 100% reconstitution with _Csf2_−/− BM), and GMWT (lethal irradiation of _Csf2_−/− mouse and 100% reconstitution with WT BM) chimera (n = 3; mean ± SD). (H) BAL of WT/µMT, GM/WT, GM/µMT, WTGM, and GMWT chimeric mice.

Figure 5.

Figure 5.

IRA B cells protect against pneumonia. (A) Clinical score of WT, WT receiving anti-CD116, µMT, WT/µMT, GM/WT, and GM/µMT mice 9 h after infection with E. coli (n = 5–10 mice). (B) Body temperature of the groups above. (C) Bacterial titer in BAL after E. coli infection of WT, WT receiving anti-CD116, µMT, WT/µMT, GM/WT, and GM/µMT mice (n = 5–10 mice). (D) Kaplan-Meier survival curves after S. pneumoniae i.t. infection for WT/µMT, GM/WT, and GM/µMT mice (n = 8–10). Relevant data are presented as mean ± SD and tested by ANOVA; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6.

Figure 6.

Enhanced inflammation but attenuated IgM after E. coli airway infection in the absence of IRA B cells. (A) IgM levels detected by ELISA in serum, BAL, and pleural space 9 h after E. coli infection (n = 6–10 mice). (B) Lung histology after E. coli infection. DAPI: blue; CD19: red; IgM: green; merge: yellow. Representative pictographs of n = 6–10 animals/group are shown (bars: overview, 100 µm; inset, 10 µm). Arrows indicate IgM+CD19+ cells. (C) Analysis of WT/µMT and GM/µMT mice after i.t. E. coli infection. Immunohistochemical staining for neutrophils in lung tissue and enumeration of neutrophils measured by counts of neutrophils per field of view. A representative slide of n = 6–10 is shown (bars, 400 µm). (D) Enumeration of neutrophils in blood and BAL (n = 6–10). (E) Analysis of the phagocytic capacity of neutrophils from the BAL of WT/µMT and GM/µMT mice. Shown are total cell numbers with phagocytosed Pkh26+ bacteria (n = 4). (F) Analysis of BAL levels for IL-1α, IL-6, TNF, and CXCL1 (n = 5–15 mice). Relevant data are presented as mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 7.

Figure 7.

The pleural space is a reservoir of lung-infiltrating B1a B cells. (A) Cartoon depicting the ICAPS model. After skin incision, a small catheter can be navigated through the intercostal space and placed in the pleural space. (B) Preoperative: (i and ii) CT scans before insertion of the catheter. Intraoperative: (iii and iv) CT scans immediately after insertion of the catheter. (v and vi) CT scans after injection of 300 µl Iopamidol iodine CT contrast agent. Postoperative: (vii and viii) CT scans 10 min after the catheter was removed. There were no signs of injection into lung parenchyma, pneumothorax, or leakage (top row of CT scans: axial view; bottom rows of CT scans: sagittal view; arrows denote the tip of the catheter; stars donate the injected Iopamidol iodine CT contrast agent). (C) Intrapleural (i.e., by ICAPS) transfer of GFP+ serosal cells. Cells were transferred into WT mice which were sacrificed 10 min after transfer. Data show transferred cells in the pleural space, BAL, blood, lung, bone marrow, and spleen. (D) Unsorted GFP+ serosal cells were adoptively transferred to the pleural or peritoneal spaces of WT mice that received pulmonary LPS challenge. Data show profile of lung accumulation in recipients 2 d after transfer. A representative experiment of n = 5 is shown. (E) Enumeration of CD19+ and CD19− cells accumulating in the lung and spleen after intrapleural or intraperitoneal transfer of unsorted GFP+ serosal cells. A representative enumeration of n = 5 is shown. (F) Intrapleural adoptive transfer of serosal B1a GFP+ cells. Data show frequency of adoptively transferred (GFP+) cells in the lung, pleural space, and blood 2 d after pulmonary LPS challenge. A representative dot plot from n = 4 is shown. (G) Fluorescence microscopy of lung tissue 2 d after intrapleural adoptive transfer of sorted GFP+ serosal B1a cells (bars: overview, 20 µm; inset, 10 µm).

Figure 8.

Figure 8.

Pleural B1a B cells accumulating in the lung produce opsonizing IgM that is sufficient to confer survival. (A) Intracellular IgM (IgM (ic)) reservoirs in steady-state pleural GFP+ B1a cells (left dot plot) and in GFP+ B1a cells that had infiltrated LPS-challenged lungs after intrapleural transfer (right dot plot). The dotted line represents the upper 99% limit of intracellular IgM staining in steady-state cells. Representative analysis from n = 4 is shown. (B) Mean fluorescence intensity (MFI) of IgM (ic) from A (n = 4). (C) IgM ELISPOT analysis of cells as in A. Representative analysis from n = 2 experiments is shown. (D) Kaplan-Meier survival curves in response to E. coli infection in WT mice; µMT mice; and µMT mice that received WT pleural B cells in the pleural space, _Csf2_−/− pleural cells in the pleural space, and WT blood cells into the blood at the time of infection (n = 10 mice). (E) Opsonization of bacteria with IgM. Data show Pkh26-labeled E. coli retrieved from the BAL of either µMT mice or µMT mice spiked with pleural WT B cells in the pleural space. Bacteria (i.t.) and cell transfer (i.pls.) were conducted 6 h before BAL. An antibody against IgM shows opsonization of labeled bacteria. A representative analysis of n = 3 is shown. (F) IgM is polyclonal. WT mice received PBS or LPS. 6 h later, BAL was collected, and capacity of IgM to bind to S. aureus and P. aeruginosa was measured. Data show binding relative to a commercially available polyclonal IgM (n = 3). (G) IgM ELISA of pleural fluid and BAL after E. coli infection. µMT mice received WT or _Csf2_−/− pleural B1a cells into the pleural space at the time of infection (n = 3). Relevant data are presented as mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 9.

Figure 9.

IRA B cells and secreted IgM are required for protection against pneumonia. (A) Postsort analysis of sorted WT, sIgM−/−, _Csf2_−/− serosal B cells, or WT serosal non–B cells. Representative plots are shown of n > 10. (B) GM/µMT (i.e., IRA B cell KO) mice received intrapleural transfer of WT, sIgM−/−, Csf2−/− serosal B cells, and WT non–B cells (n = 6–15 mice). 6 h later, mice were infected i.t. with E. coli. 9 h later, clinical score, body temperature, bacterial titer in the BAL, and IgM in serum were measured. Data are presented as mean ± SD and tested by ANOVA. (C) Kaplan-Meier Survival Curve of GM/µMT mice infected with E. coli receiving either PBS or polyclonal IgM i.t. (n = 10). Relevant data are presented as mean ± SD; ***, P < 0.001.

Figure 10.

Figure 10.

IRA B cells in humans. (A) Human pleural B cells were placed in vitro for 2 d and stimulated with anti-Ig and IL-2. Data show the appearance of GM-CSF–producing, IRA-like B cells. (B) Fluorescence-minus-one (FMO) and isotype controls of stimulated human B cells. (C) Total B cells were collected from human pleural space, cord blood, and peripheral blood and cultured for 2 d either in medium or with anti-Ig and IL-2 stimulation. Data show quantity of IRA-like B cells appearing in each condition. (D) Total B cells from the pleural space were cultured as in C, and with anti–GM-CSF, anti-CD116, or a Stat5 inhibitor. Data show quantity of IRA-like B cells appearing in each condition (n = 4–15). Relevant data are presented as mean ± SD. ***, P < 0.001. (E) Model for the function of IRA B cells in mouse pneumonia. During airway infection pleural space B1a cells recognize bacteria or its components (1). This leads to the generation (2) of IRA B cells which produce GM-CSF and express the GM-CSF receptor. GM-CSF acts on its receptor in an autocrine (3) manner. The signaling orchestrates generation of IgM-producing cells (4) which relocate to the lung (5). IgM derived from pleural space B cells is essential to the control of bacteremia (6).

References

    1. Ansel K.M., Harris R.B., Cyster J.G. 2002. CXCL13 is required for B1 cell homing, natural antibody production, and body cavity immunity. Immunity. 16:67–76 10.1016/S1074-7613(01)00257-6 - DOI - PubMed
    1. Baumgarth N. 2011. The double life of a B-1 cell: self-reactivity selects for protective effector functions. Nat. Rev. Immunol. 11:34–46 10.1038/nri2901 - DOI - PubMed
    1. Baumgarth N., Herman O.C., Jager G.C., Brown L.E., Herzenberg L.A., Chen J. 2000. B-1 and B-2 cell–derived immunoglobulin M antibodies are nonredundant components of the protective response to influenza virus infection. J. Exp. Med. 192:271–280 10.1084/jem.192.2.271 - DOI - PMC - PubMed
    1. Bezbradica J.S., Gordy L.E., Stanic A.K., Dragovic S., Hill T., Hawiger J., Unutmaz D., Van Kaer L., Joyce S. 2006. Granulocyte-macrophage colony-stimulating factor regulates effector differentiation of invariant natural killer T cells during thymic ontogeny. Immunity. 25:487–497 10.1016/j.immuni.2006.06.017 - DOI - PubMed
    1. Bo L., Wang F., Zhu J., Li J., Deng X. 2011. Granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) for sepsis: a meta-analysis. Crit. Care. 15:R58 10.1186/cc10031 - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources