Background: Sepsis morbidity and mortality are aggravated by acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Mouse B-1a cells are a phenotypically and functionally unique sub-population of B cells, providing immediate protection against infection by releasing natural antibodies and immunomodulatory molecules. We hypothesize that B-1a cells ameliorate sepsis-induced ALI. Methods: Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). PBS or B-1a cells were adoptively transferred into the septic mice intraperitoneally. After 20 h of CLP, lungs were harvested and assessed by PCR and ELISA for pro-inflammatory cytokines (IL-6, IL-1β) and chemokine (MIP-2) expression, by histology for injury, by TUNEL and cleaved caspase-3 for apoptosis, and by myeloperoxidase (MPO) assay for neutrophil infiltration. Results: We found that septic mice adoptively transferred with B-1a cells significantly decreased the mRNA and protein levels of IL-6, IL-1β and MIP-2 in the lungs compared to PBS-treated mice. Mice treated with B-1a cells showed dramatic improvement in lung injury compared to PBS-treated mice after sepsis. We found apoptosis in the lungs was significantly inhibited in B-1a cell injected mice compared to PBS-treated mice after sepsis. B-1a cell treatment significantly down-regulated MPO levels in the lungs compared to PBS-treated mice in sepsis. The −/− protective outcomes of B-1a cells in ALI was further confirmed by using B-1a cell deficient CD19 mice, which showed significant increase in the lung injury scores following sepsis as compared to WT mice. Conclusions: Our results demonstrate a novel therapeutic potential of B-1a cells to treat sepsis-induced ALI. Keywords: B-1a cells, Sepsis, Acute lung injury, Inflammation, Neutrophils, IL-10 Background 2014). The pathophysiology of sepsis-induced ALI is less Based on the Third International Consensus Definitions well understood. Antibiotics and supportive measures for Sepsis and Septic Shock (Sepsis-3), sepsis is defined are the only treatments available for patients with sepsis as “life-threatening organ dysfunction caused by a dys- and ALI, and these measures have limited impact on the regulated host response to infection” (Singer et al. 2016). high mortality rates of sepsis. In the United States, there are approximately 1 million Immune cells recognize pathogen-associated molecu- cases of sepsis annually, with a mortality rate up to 40% lar patterns (PAMPs) via their toll-like receptors (TLRs) (Vincent et al. 2014; Martin et al. 2006; Aziz et al. 2013). to exaggerate “cytokine storm”, which trigger inflamma- The lungs are particularly susceptible to injury during tion and impair tissue function during sepsis (Aziz et al. sepsis, and more than 50% of patients with sepsis 2013; Barton and Medzhitov 2003; Foster and Medzhitov develop acute lung injury (ALI) or acute respiratory dis- 2009). Neutrophil infiltration in lungs is a major patho- tress syndrome (ARDS) (Sevransky et al. 2009; Gu et al. physiological hallmark of ALI. Uncontrolled migration of neutrophils into lungs leads to exaggerated produc- tion of cytokines, chemokines, myeloperoxidase (MPO), * Correspondence: email@example.com reactive oxygen species (ROS), nitric oxide (NO), and Center for Immunology and Inflammation, The Feinstein Institute for neutrophil extracellular traps (NETs) causing Medical Research, 350 Community Dr, Manhasset, NY 11030, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Aziz et al. Molecular Medicine (2018) 24:26 Page 2 of 12 unrestrained inflammation, lung dysfunction and death 2000; Choi and Baumgarth 2008). During influenza virus (Aziz et al. 2013; Grommes and Soehnlein 2011; Abra- infection B-1a cells migrate from serosal cavities to the ham 2003; Lee and Downey 2001; Brinkmann et al. lungs, where they secrete natural Abs and other immu- 2004; Kaplan and Radic 2012; Delgado-Rizo et al. 2017). nomodulatory molecules to protect rodents against Thus, regulating the exaggerated function of neutrophils influenza virus infection (Baumgarth et al. 2000; Choi and their uncontrolled infiltration into lungs serves as and Baumgarth 2008). Consistently, in various animal an effective therapeutic tool in ALI. The early onset of models of ALI initiated by direct instillation of LPS, E. pro-inflammatory cytokine storm, often contributing to coli or S. pneumoniae, B-1a cells were shown to migrate the lung injury in sepsis, can be reversed by the actions from the pleural cavity to the lung parenchymal tissues, of anti-inflammatory cytokines such as interleukin where they secrete GM-CSF and IgM to protect rodents (IL)-10 (Cinel and Opal 2009; Kono et al. 2006). We re- against ALI (Weber et al. 2014). A recent study has cently demonstrated the beneficial role of IL-10 produ- demonstrated that due to the loss of function of natural cing B-1a cells in sepsis by controlling the systemic IgM as secreted from the B-1a cells could be the cause levels of pro-inflammatory cytokines, chemokines and of poor prognostic outcomes of lung infection in aged bacterial loads (Aziz et al. 2017), while their role in ALI animals (Holodick et al. 2016). The beneficial role of remained unknown. Elucidation of the novel role of B-1a cells in lungs was shown in virus and bacterial B-1a cells in lungs during sepsis will not only improve infections, as well as in young over old mice with S. our understanding of ALI pathophysiology, but also help pneumoniae infection, indicating that these cells play a us to develop effective therapeutics against ALI. pivotal role in lung diseases. Nonetheless, their role in In mouse, B cells consist of various subpopulations, sepsis-induced ALI remains unknown. which include follicular (FO), marginal zone (MZ) and In the current study, we aimed to study the role of B-1 B cells (Aziz et al. 2015). The role of FO and MZ B B-1a cells in ALI during sepsis. Our study for the first cells collectively known as B-2 cells in the early immune time revealed the protective role of B-1a cells against response and inflammatory cytokine production during sepsis-induced ALI by controlling exaggerated inflam- sepsis has been demonstrated previously (Kelly-Scumpia mation and infiltration of neutrophils in lungs. Thus, et al. 2011; Honda et al. 2016). B-1 cells comprising a B-1a cells could represent a promising therapeutic in minor portion of the total B cells in mice display unique sepsis-induced ALI. features in terms of their phenotype, localization, development, signaling and function (Aziz et al. 2015; Methods Martin and Kearney 2001; Kantor et al. 1992). The Animals lo hi surface phenotype of murine B-1 cells is B220 , IgM , Wild-type (WT) C57BL/6 mice obtained from Taconic lo − hi + tm1(cre)cgn −/− IgD , CD23 , CD19 and CD43 (Aziz et al. 2017; Aziz (Albany, NY) and B6.129P2(C)CD19 /J (CD19 ) et al. 2015; Kantor et al. 1992). B-1 cells can be further mice obtained from The Jackson Laboratory were housed divided into B-1a and B-1b cells, depending on their sur- in a temperature and light controlled room and fed a face expression of CD5 (Kantor et al. 1992; Berland and standard laboratory diet. For all experiments male 8- to Wortis 2002). B-1a cells are predominantly localized in 10-week-old 21–28 g of body weight (BW) mice were the peritoneal cavity; however, a small portion of B-1a used. Animals were randomly assigned to sham, vehicle cells can also be found in the respiratory tract, intestinal control and B-1a cell treatment groups. Number of ani- tissues, lymph nodes, spleen and bone marrow (Aziz et mals estimated in each group was based on our previous al. 2015; Yenson and Baumgarth 2014). B1a cells can se- study on an animal model of sepsis (Aziz et al. 2017). All crete large amounts of natural IgM and IgA that are animal protocols were approved by our Institutional capable of recognition and clearance of invading patho- Animal Care and Use Committee. gens (Aziz et al. 2015; Grönwall et al. 2012; Vas et al. 2012). Natural antibodies have antigen specificity for a Murine model of polymicrobial sepsis number of microbial epitopes such as phospholipids and Mice were anesthetized with 2% isofluorane inhalation lipopolysaccharides (LPS) (Grönwall et al. 2012;Vas et and underwent cecal ligation and puncture (CLP). A al. 2012). Murine B-1a cells are known to produce ample 2-cm incision was made to the abdominal wall, and the amount of IL-10 and granulocyte macrophage colony cecum was exposed and ligated 0.5 cm from the tip with stimulating factor (GM-CSF), which attenuate excessive 4–0 silk suture. A 22-gauge needle was used to make inflammation during sepsis (Aziz et al. 2017; Aziz et al. one puncture through and through to the distal cecum, 2015; Rauch et al. 2012). extruding a small amount of fecal contents. The cecum Recent findings demonstrate an active role of B-1a was replaced into the abdominal cavity, and the exposed cells for protection against lung infection caused by in- abdominal wall was closed in two layers with running fluenza virus (Baumgarth et al. 1999; Baumgarth et al. 4–0 silk suture. In sham-operated mice only laparotomy Aziz et al. Molecular Medicine (2018) 24:26 Page 3 of 12 was performed, but their cecum was not ligated and ELISA punctured. Animals were resuscitated with 1 ml of nor- The lung tissue was crushed in liquid nitrogen, and mal saline subcutaneously. In another experiment, WT approximately 50 mg of powdered tissues were dissolved −/− and CD19 mice were subjected to either sham or CLP in 500 μloflysis buffer (10mM Hepes,pH 7.4, 5 mM operation following the above CLP protocol. MgCl , 1 mM DTT, 1% Triton X-100, and 2 mM each of EDTA and EGTA), and subjected to sonication on ice. Adoptive transfer of murine B-1a cells Protein concentration was determined by the BioRad pro- Murine B-1a cells in peritoneal washouts were stained tein assay reagent (Hercules, CA). Equal amounts (50 μg) with FITC-B220 (clone RA3-6B2), Pacific Blue-CD23 of proteins were loaded into respective enzyme-linked im- (clone B3B4), and PE-Cy5-CD5 (clone 53-7.3) obtained munosorbent assay (ELISA) wells for assessment of IL-6, from BD Biosciences (San Diego, CA). B-1a cells with IL-1β,TNF-α,IFNγ, IL-10 and MIP-2 by using the kits ob- − lo int phenotype, CD23 B220 CD5 were sort-purified using tained from BD Biosciences, and IgM by using the kit a BD Biosciences Influx instrument. Post-sort analysis from Bethyl Laboratories, Inc., Montgomery, TX. showed PerC B-1a cells to be ≥98% pure. Sort-purified B-1a cells were washed with PBS and then suspended in Lung tissue histology PBS for adoptive transfer into septic mice through intra- Formalin fixed and paraffin embedded lung tissue blocks peritoneal (i.p.) injection. At the time of CLP operation, were sectioned at 5 μm thickness and placed on glass 5×10 B-1a cells suspended in 150 μl of PBS were slides. Lung tissue sections were stained with hematoxylin delivered into the peritoneal cavity and the abdominal & eosin (H&E) and observed under a light microscope. wound was closed with running 4–0 silk suture. As vehicle Morphological changes were scored as nil (0), mild (1), negative control, 150 μl of PBS was injected into the abdo- moderate (2), or severe (3) injury based on the presence of men of CLP-operated mice. The animals were allowed food exudates, hyperemia or congestion, infiltration of neutro- and water ad libitum, and at 20 h after CLP operation and phils, alveolar hemorrhage, presence of debris, and cellular B-1a cell transfer the animals were euthanized and lungs hyperplasia, in a blinded fashion (Aziz et al. 2012;Hirano were collected for various ex vivo analyses. et al. 2015). The sums of scores of different animals were averaged and plotted on a bar graph. Quantitative real-time PCR assay Total RNA was extracted from lung tissues using TRIzol Myeloperoxidase assay reagent (Invitrogen; Carlsbad, CA) and reverse-transcribed A total of 50–100 mg of liquid nitrogen-based powered lung into cDNA using reverse transcriptase enzyme (Applied tissues were homogenized in KPO buffer containing 0.5% Biosystems; Foster City, CA). The PCR reaction was per- hexa-decyl-trimethyl-ammonium bromide (Sigma-Aldrich, formed in 20 μl of final volume containing 0.08 μMoffor- St. Louis, MO) using a sonicator with the samples placed in ward and reverse primer, 2 μlof10–20×dilutedoriginal ice. After centrifuging, the supernatant was diluted in reac- cDNA, and 10 μl SYBR Green PCR Master Mix (Applied tion solution which contains O-Dianisidine dihydrochloride Biosystems) using Applied Biosystems 7300 real-time PCR (Sigma-Aldrich) and H O (ThermoFisher Scientific, 2 2 machine. Mouse β-actin served as an internal control gene Waltham, MA) as substrate. Rate of change in optical for normalization. Relative expression of mRNA was repre- density (ΔOD) between 1 and 4 min was measured at sented as fold change in comparison to the sham group. 460 nm to calculate myeloperoxidase (MPO) activity (Aziz The sense and anti-sense primer sequences of mouse genes et al. 2012). are, IL-6 (NM_031168): 5’-CCGGAGAGGAGACTTCACA G-3′ and 5′-GGAAATTGGGGTAGGAAGGA-3′;IL-1β TUNEL assay (NM_008361): 5’-CAGGATGAGGACATGAGCACC-3′ The presence of apoptotic cells in lung tissue sections and 5’-CTCTGCAGACTCAAACTCCAC-3′;tumor ne- was determined using a terminal deoxynucleotide trans- crosis factor-α (TNF-α)(NM_013693.2): 5′-AGACCCTCA ferase dUTP nick end labeling (TUNEL) assay kit (Roche CACTCAGATCATCTTC-3′ and 5′-TTGCTACGA Diagnostics, Indianapolis, IN). Briefly, lung tissues were CGTGGGCTACA-3′;interferon γ (IFNγ) (NM_008337): fixed in 10% phosphate buffered formalin and were then 5’-GGCTTTGCAGCTCTTCCTC-3′ and 5’-CCAG embedded into paraffin and sectioned at 5 μm following TTCCTCCAGATATCCAA-3′; IL-10 (NM_010548): standard histology procedures. Lung sections were 5’-CAGCCGGGAAGACAATAA CT-3′ and 5’-GCAT dewaxed, rehydrated and equilibrated in Tris buffered TAAGGAGTCGGTTAGCA-3′;MIP-2 (NM_009140): saline (TBS). The sections were then digested with 5’-CCCTGGTTC AGAAAATCATCCA-3′ and 5’-GCTC 20 μg/mL proteinase K for 20 min at room CTCCTTTCCAGGTCAGT-3′; β-actin (NM_007393): temperature. The lung tissue sections were then washed 5’-CGTGAAAAGATGACCCAGATCA-3′ and 5’-TGGT and incubated with a cocktail containing terminal deoxy- ACGACCAGAGGCATACAG-3′. nucleotidyl transferase enzyme and fluorescence labeled Aziz et al. Molecular Medicine (2018) 24:26 Page 4 of 12 nucleotides and examined under a fluorescence micro- rate of changes of fluorescence intensity at 37 °C were mea- scope (Nikon Eclipse Ti-S, Melville, NY). sured at 370 nm (excitation wavelength) and 450 nm (emis- sion wavelength) in a fluorometer (Synergy H1, BioTek, Caspase-3 enzyme activity assay Winooski, VT). The caspase-3 enzyme activity was The caspase-3 enzyme activity in lung tissues was assessed expressed as mM AMC/min/g of protein (Aziz et al. 2012). by a fluorimetric assay system kit (Sigma, Saint Louis, MO). Lung tissues were homogenized in liquid nitrogen, and ap- Statistical analysis proximately 50 mg of powdered tissues were dissolved in Figure preparation and data analyses were performed by 500 μl of lysis buffer, which contains a cocktail of 10 mM using SigmaPlot 12.5 software (Systat Software Inc., San Hepes, pH 7.4, 5 mM MgCl ,1mM DTT, 1% Triton Jose, CA). Data represented in the figures are expressed as X-100, and 2 mM each of EDTA and EGTA, and then sub- mean ± standard error (SE). One way analysis of variance jected to sonication by placing the samples in ice. Protein (ANOVA) was used for comparison among multiple concentration was measured by the Bio Rad protein assay groups and the significance was determined by the reagent (Hercules). Equal amounts of proteins in a 5 μlvol- Student-Newman-Keuls (SNK) test. Paired two-tailed umewereadded to the100 μl assay buffer (20 mM Hepes, Student’s t-test was applied for two-group comparisons. pH 7.4, 5 mM DTT, 2 mM EDTA, and 0.1% CHAPS) con- Significance was determined as p ≤ 0.05 between taining 10 μM DEVD-AMC substrate molecule and the experimental groups. 150 # Cells (PerC) B-1a cells FSC FSC B220 # Sham PBS B-1a Cells CLP d f h 25 6 2 6 * * 1.5 4 4 0.5 0 0 0 e g i 600 450 450 * * 500 * 400 300 300 200 150 0 0 0 Sham PBS B-1a Cells Sham PBS B-1a Cells Sham PBS B-1a Cells Sham PBS B-1a Cells CLP CLP CLP CLP Fig. 1 Adoptive transfer of B-1a cells attenuates lung inflammation. a Peritoneal washout cells isolated from healthy mice were stained with anti- mouse Pacific Blue-CD23, FITC-B220 and PE-Cy5 Abs and subjected to sort purification by using a flow cytometry-based cell sorting system. A total of 5 × 10 B-1a cells suspended in 150 μl of PBS were delivered into the peritoneal cavity of CLP mice. After 20 h, lung tissue was harvested and mRNA and protein expression of b, c IL-6, d, e IL-1β, f, g TNF-α, h, i IFNγ and j, k IL-10 were assessed, respectively. Data are expressed as * # means ± SE (n = 9 mice/group) and compared by one-way ANOVA and SNK method ( p < 0.05 vs. sham mice; p < 0.05 vs. PBS-treated CLP mice). CLP, cecal ligation and puncture; IL, interleukin IL-1β (pg/mg protein) C57BL/6 IL-1β mRNA Folds SSC TNF-α (pg/mg protein) TNF-α mRNA Folds CD23 CD5 IFNγ (pg/mg protein) IFNγ mRNA Folds C57BL/6 (CLP) IL-6 (pg/mg protein) IL-6 mRNA Folds IL-10 (pg/mg protein) IL-10 mRNA Folds Aziz et al. Molecular Medicine (2018) 24:26 Page 5 of 12 Results could not find significant increase of the expression of IFNγ B-1a cells attenuate the expression of pro-inflammatory at both mRNA and protein levels in lungs at 20 h of CLP, cytokines in the lungs during sepsis which could be due to the fact that its up-regulation might Peritoneal B-1a cells were sort-purified based on occur at earlier time point after CLP operation, and there- − lo int CD23 B220 CD5 surface phenotype from healthy mice fore 20 h after CLP was too late to determine its and then injected into mice immediately after CLP up-regulation in lung tissues (Fig. 1h, i). Similar to the pat- operation (Fig. 1a). At 20 h after CLP operation, lungs were terns of expression of pro-inflammatory cytokines, we found harvested to assess the expression of pro- and significant up-regulation of IL-10 expression at mRNA and anti-inflammatory cytokines. Expression of IL-6 and IL-1β proteinlevelsinthe lung tissuesfollowing CLPoperation as in lung tissue from CLP mice was significantly up-regulated compared to sham-operated mice (Fig. 1j, k). We noticed a compared to sham-operated mice, while the adoptive trans- trend towards decreasing the expression of IL-10 in lungs of fer of B-1a cells significantly down-regulated expression of B-1a cell-treated CLP mice as compared to vehicle-treated IL-6 and IL-1β by 51 and 54%, respectively at the mRNA CLP mice, reflecting the remission of inflammation after and 55 and 51%, respectively at the protein level (Fig. 1b-e). B-1a cell treatment in septic mice (Fig. 1j, k). We found significant up-regulation of the expression of TNF-α at mRNA and protein levels in the lung tissues of Treatment of septic mice with B-1a cells attenuates lung CLP mice, while there was a trend towards down-regulation injury scores of TNF-α expression in lungs of B-1a cell-treated CLP mice Histological images of lung tissue showed decreased as compared to vehicle-treated CLP mice (Fig. 1f, g). We levels of alveolar congestion, exudate, interstitial and Fig. 2 Treatment with B-1a cells improves the histopathological score of lung tissue damage in sepsis. a Lung tissue was collected after 20 h from sham-operated, andeitherPBS-orB-1acell-treatedCLP mice and stained with H&E. Each slide was observed under light microscopy at × 100 original magnification in a blinded fashion. Representative images for each group are shown. Scale bar, 100 μm. b Histological injury scores of the lungs in different groups were quantified as described in Materials and Methods. Data from three independent experiments are expressed as means ± SE (n = 6 mice/group) and compared by one-way ANOVA and SNK method ( p <0.05 vs. shams; p < 0.05 vs. PBS-treated CLP mice). CLP, cecal ligation and puncture; H&E, hematoxylin and eosin Aziz et al. Molecular Medicine (2018) 24:26 Page 6 of 12 alveolar cellular infiltrates, intra-alveolar capillary hem- orrhages, and damage of epithelial architecture, in B-1a cell-treated CLP mice as compared to PBS-treated CLP mice (Fig. 2a). These histological changes were reflected in a significant decrease in lung tissue injury score in B-1a cell-treated mice compared to PBS-treated CLP mice by a mean value of 54% (Fig. 2b). On the other hand, the sham-operated mouse lungs showed normal histological architecture. B-1a cells attenuate chemokine and MPO levels in the lungs of septic mice Chemokines such as MIP-2 play a pivotal role in the in- filtration of neutrophils in lungs during sepsis (Aziz et al. 2013; Abraham 2003). In lung tissue following sepsis, we noticed significant up-regulation of MIP-2 expression compared to sham mice, while the mice treated with B-1a cells significantly reduced the expression of MIP-2 by 49 and 46%, respectively at the mRNA and protein levels compared to PBS-treated CLP mice (Fig. 3a, b). The neutrophil infiltration in lungs as measured by the amount of MPO showed significant inhibition in B-1a cell-treated mice by 41% as compared to PBS-treated mice during CLP (Fig. 3c). Treatment with B-1a cells attenuates apoptosis in the lung during sepsis Sepsis resulted in a significant increase in the number of apoptotic cells in lungs (Aziz et al. 2013; Aziz et al. 2012). Here, we noticed that the septic mice treated with B-1a cells experienced a significant decrease in the num- bers of apoptotic cells by 56% compared to PBS-treated septic mice (Fig. 4a, b). Furthermore, following sepsis we noticed a significant increase of the activation of caspase-3, the rate-limiting enzyme for apoptosis in the lungs, compared to sham-operated mice. However, the treatment of septic mice with B-1a cells significantly re- duced the level of active caspase-3 by mean values of 52%, compared to PBS-treated septic mice (Fig. 4c). Fig. 3 B-1a cells attenuate MIP-2 and MPO levels in lungs after sepsis. Treatment with B-1a cells restores IgM levels in lung a, b At the time of CLP, mice were treated with either PBS as vehicle tissues during sepsis or 5 × 10 PerC B-1a cells in 150 μl of PBS by i.p. injection. After 20 h, About 80% of the IgM present in the blood are natural lung tissue was harvested and mRNA and protein expression of MIP-2 IgM which comes from the B-1a cells and its levels are were assessed, respectively. c MPO activity in lungs of sham-operated, high at steady-state (Aziz et al. 2015). We previously and PBS or B-1a cell-treated CLP mice was determined. Data are expressed as means ± SE (n = 9 mice/group from 3 independent showed that during sepsis the circulatory (blood) level of experiments) and compared by one-way ANOVA and SNK method ( p IgM were decreased during sepsis, while after adoptive < 0.05 vs. shams; p < 0.05 vs. PBS-treated CLP mice). CLP, cecal ligation transfer of B-1a cells in the septic mice increased the and puncture; MIP-2, macrophage-inflammatory protein-2; level of IgM in the blood (Aziz et al. 2017). In addition MPO, myeloperoxidase to this, within the peritoneal cavity (local infectious foci) the IgM levels were also increased following treatment of septic mice with B-1a cells (Aziz et al. 2017). To know whether or not IgM is present in the lungs and their levels are altered during sepsis, we assessed IgM levels Aziz et al. Molecular Medicine (2018) 24:26 Page 7 of 12 Fig. 4 Treatment with B-1a cells attenuates apoptosis in lungs after sepsis. After 20 h of CLP, lung tissues were collected from PBS or B-1a cell treated mice. a Lung tissue sections were prepared for TUNEL staining shown in green, and for nuclear staining using PI shown in red. Representative images at × 100 original magnification are shown. Scale bar, 100 μm. b TUNEL positive apoptotic cells were counted at 18 random fields in a blinded fashion, and the average numbers of cells per field are shown. c Cleaved Caspase-3 activity in total lung tissues of sham-operated, and PBS or B-1a cell-treated CLP mice was determined. Data are expressed as means ± SE (n = 6 mice/group) and compared by * # one-way ANOVA and SNK method ( p < 0.05 vs. shams; p < 0.05 vs. PBS-treated CLP mice). CLP, cecal ligation and puncture; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; PI, propidium iodide in the lung tissues in sham and CLP-operated vehicle- deficiency of B-1a cells would exacerbate lung injury or B-1a cell-treated mice. We found that during sepsis during sepsis. Following CLP, histological images of the IgM levels in the lungs were significantly decreased as lung tissues showed increased levels of alveolar conges- compared to sham mice, while treatment of CLP mice tion, exudate, interstitial and alveolar cellular infiltrates, with B-1a cells significantly increased the level of IgM in intra-alveolar capillary hemorrhages, and extensive dam- −/− the lungs (Fig. 5). Therefore, the B-1a cell-mediated pro- age of epithelial architecture in CD19 mice as com- tection against sepsis-induced ALI could be mediated pared to WT mice (Fig. 6a). These histological changes through both systemic and local increase of IgM. were reflected in a significant increase in lung tissue in- −/− jury score in CD19 mice compared to WT mice by a −/− Deficiency of B-1a cells in CD19 mice exacerbates lung mean value of 54% after CLP (Fig. 6b). injury B cells express the co-receptor CD19, which serves as a Discussion positive regulator of B cell receptor (BCR) signaling and B-1a cells are part of innate immune system and exhibit is critical for B cell development and activation (Aziz et unique phenotypic, developmental, localizations, signal- al. 2017; Aziz et al. 2015; Haas et al. 2005). It has been ing and functional characteristics that differ from the shown that transgenic mice over expressing CD19 gen- conventional B-2 cells (Aziz et al. 2015). B-1a cells erate excess B-1a cells which provide protection against are innate-like, while B-2 cells are adaptive-type infection, while CD19-deficient mice lack B-1a cells and immune-reactive lymphoid cells. B-1a cells spontan- are susceptible to infection (Haas et al. 2005). We exam- eously secrete germline-like, polyreactive natural anti- −/− ined CD19 mice to determine whether or not the body(IgM),which acts as afirst line of defenseby Aziz et al. Molecular Medicine (2018) 24:26 Page 8 of 12 neutralizing a wide range of pathogens (Aziz et al. 2015; Grönwall et al. 2012). B-1a cells are known to produce several immunomodulatory molecules either spontan- eously or in the presence of stimulation, which attenuate infectious and inflammatory diseases including influenza, pneumonia, atherosclerosis, inflammatory bowel disease, autoimmunity, obesity and diabetes mellitus [reviewed in (Aziz et al. 2015)]. Recently, the beneficial role of B-1a cells in sepsis has been reported (Aziz et al. 2017; Rauch et al. 2012), and this was shown to be mediated through the control of excessive systemic inflammation and bacter- ial burdens. Nonetheless, the role of B-1a cells in mitigat- ing inflammation and injuries to the remote organs especially lungs, during sepsis was not known. In the Fig. 5 Treatment with B-1a cells increases IgM levels in the lungs current study, we primarily focused on the role of B-1a following sepsis. A total of 5 × 10 sorted B-1a cells were delivered cells in attenuating ALI during sepsis. into the peritoneal cavity of CLP mice. After 20 h, lung tissue was Using a mouse model of sepsis, we previously showed harvested from sham, PBS-, and B-1a cell-treated mice and assessed that the numbers of B-1a cells in peritoneal cavity, IgM levels in total extracted proteins by ELISA. Data are expressed as spleen and bone-marrow were significantly decreased means ± SE (n = 9 mice/group) and compared by one-way ANOVA and SNK method ( p < 0.05 vs. sham mice). CLP, cecal ligation and (Aziz et al. 2017). Adoptive transfer of syngeneic B-1a puncture; ELISA, enzyme-linked immunosorbent assay cells into septic mice significantly attenuated systemic inflammatory and injury parameters as well as bacterial burden in the blood and peritoneal cavity (Aziz et al. −/− Fig. 6 Deficiency of B-1a cells exaggerates lung injury during sepsis. a After 20 h of CLP induced in WT and CD19 mice, lung tissues were harvested and stained with H&E. The slides were observed under light microscopy at × 100 original magnification in a blinded fashion. −/− Representative images for each group are shown. Scale bar, 100 μm. b Histological injury scores of the lungs in WT and CD19 mice were quantified as described in Materials and Methods. Data obtained from three independent experiments are expressed as means ± SE (n = 6 mice/ * # group) and compared by one-way ANOVA and SNK method ( p < 0.05 vs. shams; p < 0.05 vs. WT CLP mice). CLP, cecal ligation and puncture; H&E, hematoxylin and eosin Aziz et al. Molecular Medicine (2018) 24:26 Page 9 of 12 2017). In the current study, we found that the adoptive B-1a cells can serve as antigen presenting cells, providing transfer of murine B-1a cells into septic mice signifi- effective signaling to T-cells via CD80 and CD86 mole- cantly attenuated the expression of pro-inflammatory cules, which are expressed on B-1a cells (Aziz et al. 2015). cytokines IL-6 and IL-1β in the lungs. We also found Therefore, in parallel to study the crosstalk effect between overall improvement of lung injury scores in B-1a B-1a cells and macrophages, it would be of interest for cell-treated mice during sepsis. The attenuation of future studies to elucidate the novel role of B-1a cells on sepsis-induced lung injury was correlated with reduced T cells in the lungs during sepsis. levels of chemokine expression, neutrophil infiltration as GM-CSF is mainly produced by the innate response assessed by MPO, and cellular apoptosis through the activator (IRA) B cells (Rauch et al. 2012). Our current down-regulation of caspase-3 activity. We previously study focused on the effect of IL-10- and IgM-producing −/− demonstrated B-1a cell–deficient CD19 mice were B-1a cells in sepsis-induced ALI. In our previous study, more susceptible to infectious inflammation, thereby we demonstrated that the septic mice treated with −/− causing an increased mortality rate in sepsis (Aziz et al. IL-10 B-1a cells did not show protection against −/− 2017). Here, we also found that the CD19 mice sepsis (Aziz et al. 2017), thus pointing to the role of showed significantly increased levels of lung injury B-1a cell-secreted IL-10 to exert beneficial role in sepsis. scores as compared to WT mice after sepsis, thus sug- We also demonstrated that the levels of GM-CSF in −/− gesting the pivotal beneficial role of B-1a cells to protect B-1a cells between WT and IL-10 mice strains follow- mice from ALI during sepsis. The improvement of sys- ing sepsis were remained same (Aziz et al. 2017), indi- temic inflammation and lung injury and inflammation cating that the lack of IL-10 in B-1a cells could be after administration of B-1a cells in septic animals can detrimental in sepsis without affecting the levels of be better reflected in their survival outcomes. In our GM-CSF. Future studies focusing on the role of previous study, we demonstrated significant improve- GM-CSF producing IRA B cells will help reveal the ment of the survival outcome in B-1a cell-treated mice importance of IRA B cells in sepsis-induced ALI. over that of PBS-treated mice with sepsis (Aziz et al. In sepsis, irresistible migration of neutrophils into the −/− 2017). By contrast, the B-1a cell deficient CD19 mice lungs leads to endothelial cell injury and sustained in- had significantly reduced rate of survival as compared to flammation (Aziz et al. 2013; Aziz et al. 2012; Hirano et the WT mice during sepsis (Aziz et al. 2017). al. 2015; Hirano et al. 2016). The patients with ARDS The crosstalk effect between B-1a cells and macro- represent huge infiltration of neutrophils in the lung phages has been demonstrated in previous reports (Thies tissues which correlates with the severity of lung injury et al. 2013; Barbeiro et al. 2011). B-1a cells produce IL-10 as a result of releasing ample amounts of proteolytic in response to LPS stimulation (Aziz et al. 2017; Barbeiro enzymes and pro-inflammatory mediators from the infil- et al. 2011). In B-1a cells and macrophages co-cultures, trated neutrophils into the lung tissue beds (Abraham production of pro-inflammatory cytokines was lower and 2003; Williams and Chambers 2014). Thus, it is sug- the production of anti-inflammatory cytokine IL-10 was gested that the regulation of neutrophil infiltration into higher than in macrophage monocultures (Barbeiro et al. the lungs could be an effective therapeutic approach in −/− 2011). Interestingly, co-culture of IL-10 B-1a cells and septic-induced ALI. Here, in the current study, we WT macrophages did not reduce the levels of the noticed dramatic reduction of neutrophil infiltration in pro-inflammatory cytokines (Aziz et al. 2017), indicating the lungs as measured by MPO and chemokine MIP-2 the pivotal regulatory role of B-1a cells in controlling in- levels which ultimately led to diminished lung tissue in- flammation. Beside these in vitro findings, we demon- jury in the B-1a cell-treated mice. Although the direct strated the beneficial role of B-1a cells during sepsis roles of B-1a cells on macrophages and T cells had been through the production of anti-inflammatory cytokine delineated previously, the effect of B-1a cells on neutro- IL-10 (Aziz et al. 2017). Lungs contain resident alveolar phils is largely unknown. Elucidation of the direct role of macrophages which during sepsis become activated to B-1a cells on neutrophils will provide additional insights produce excessive amounts of pro-inflammatory cytokines into the pathophysiology of ALI in sepsis. and chemokines (Aziz et al. 2012; Moldoveanu et al. In the context of lung injury and inflammation caused 2009). However, we noticed significant decreases in the by viral and bacterial infections, several reports have expression of pro-inflammatory cytokines IL-6 and IL-1β already demonstrated the beneficial role of B-1a cells in and chemokine MIP-2 in the lungs of B-1a cell-treated protecting mice from lung injury, mainly mediated mice during sepsis. Since B-1a cells are known to produce through the release of natural IgM (Baumgarth et al. excessive amounts of anti-inflammatory cytokine IL-10, it 1999; Baumgarth et al. 2000; Weber et al. 2014). Natural is therefore understandable that the B-1a cells could IgM secreted from B-1a cells eliminates invading patho- temper the pro-inflammatory responses of alveolar macro- gens and also scavenges dying cells, which in turn can phages and thus protect mice from ALI during sepsis. attenuate inflammation and tissue injury (Grönwall et al. Aziz et al. Molecular Medicine (2018) 24:26 Page 10 of 12 2012; Vas et al. 2012). On the other hand, mice lacking et al. 2013; Bosmann and Ward 2013; Rittirsch et al. natural IgM are prone to develop autoimmune diseases 2008). Therefore, in order to obtain optimal inhibition because of the failure to neutralize/remove antigens and of pro-inflammatory cytokines and chemokines by the apoptotic cells to maintain homeostasis (Aziz et al. 2015; treatment of B-1a cells, we chose time of treatment at Boes et al. 2000). In the current study, we noticed sig- CLP induction instead of a later time point. We nificant reduction in the number of apoptotic cells in delivered the B-1a cells into the septic mice through the the lungs following B-1a cell treatment in septic mice. intraperitoneal route; however, administration of B-1a Although here we did not assess the phagocytic clear- cells intravenously would help shift this laboratory ance of apoptotic cells by professional phagocytes, we strategy to bedside approaches. found that the septic mice treated with B-1a cells In the present study, we used C57BL/6 WT mice, also showed reduced levels of caspase-3 activity, indicating known as B6 mice obtained from the Taconic to inhibition of cellular apoptosis by B-1a cell treatment. It compare the outcomes of sepsis-induced ALI with B6 −/− has been demonstrated that endothelial cell pyroptosis, a background B-1a cell deficient CD19 mice obtained form of cell death, may result in sepsis-induced ALI from the Jackson lab. Our previous studies on B6 back- through the activation of caspases (Cheng et al. 2017; ground of mice of Taconic and Jackson lab showed simi- Aziz et al. 2014). Since the pyroptotic cells also undergo lar outcomes in their survival in CLP-induced sepsis DNA fragmentation and, like apoptotic cells show posi- (Giangola et al. 2013; Qiang et al. 2013). However, since tive TUNEL staining (Mariathasan et al. 2005), our the immune responses of mice may vary among various TUNEL assay data in lung tissues pointed to the possi- strains and vendors (Otto et al. 2016), we consider this bility of decreased pyroptosis of lung cells following as one of our limitations in experimental designing. −/− treatment of septic mice with B-1a cells. Further studies Further studies using control WT mice and CD19 mice by staining the lung tissue sections with endothelial cell from the same vendor will strengthen our present finding marker CD31 Ab, TUNEL and caspase-1 Ab will help of the beneficial effect of B-1a cells on ALI during sepsis. confirm the status of endothelial cell pyroptosis in lungs during sepsis, and also demonstrate the inhibitory effect Conclusions of B-1a cells for endothelial cell pyroptosis during sepsis. We identified the beneficial role of murine B-1a cells in During influenza virus infection, the therapeutic po- sepsis-induced ALI through the mitigation of inflamma- tential of murine B-1a cells was mainly generated by tion and injury to the lungs. Recently, a B cell popula- their enrichment at the lungs as a result of their trans- tion in human has been identified which represents location from serosal cavities where they are generally functional characteristics that match with murine B-1a localized at the steady-state condition (30). Following cells, including autonomous production of natural IgM, their translocation into lungs, B-1a cells autonomously constitutive basal expression of intracellular signal secrete natural Abs and other immunomodulatory mole- transduction molecules, and effective stimulation of T cules to protect hosts against influenza virus infection lymphocytes (Aziz et al. 2015; Griffin et al. 2011; (Aziz et al. 2015; Baumgarth et al. 1999; Baumgarth et Rothstein et al. 2013). Our current study demonstrating al. 2000). In line with this fact, Weber, et al. showed the role of mouse B-1a cells in sepsis-induced ALI fur- B-1a cells migrate from the pleural cavity to the intersti- ther focuses on identifying valuable lessons that may be tial lung tissues, where they produce ample amount of applicable to human B-1a cells. GM-CSF and natural Abs to protect the host from endo- toxin or S. pneumoniae-induced ALI in mice (Weber et Abbreviations al. 2014). In the current study utilizing murine model of ALI: Acute lung injury; ARDS: Acute respiratory distress syndrome; BCR: B-cell sepsis, B-1a cells could be enriched into the lungs as a receptor; CLP: Cecal ligation and puncture; ELISA: Enzyme-linked result of their translocation from the site of origin to immunosorbent assay; FO: Follicular; GM-CSF: Granulocyte-macrophage colony-stimulating factor; LPS: Lipopolysaccharides; MIP-2: Macrophage- protect mice against lung inflammation. inflammatory protein-2; MPO: Myeloperoxidase; MZ: Marginal zone; In the current study, we injected septic mice with B-1a NETs: Neutrophil extracellular traps; NO: Nitric oxide; PAMP: Pathogen- cells at the time of CLP operation, the post-treatment of associated molecular pattern; PBS: Phosphate-buffered saline; ROS: Reactive oxygen species; TLR: Toll-like receptor; TUNEL: Terminal deoxynucleotide septic mice with B-1a cells would help advance our transferase dUTP nick end labeling current therapeutic strategy towards more clinically rele- vant circumstances. We basically chose to treat mice Acknowledgements with B-1a cells immediately after CLP rather than We thank the NIH for supporting the study. post-surgery because most of the pro-inflammatory cyto- kines and chemokines are expressed early/hyperdynamic Funding phase in sepsis, reaching maximum levels around 10– This study was supported by the National Institutes of Health (NIH) grants 12 h after CLP and then returns to normal levels (Aziz R35GM118337, R01GM053008 and R01GM057468 to PW and R01AI029690 to TLR. Aziz et al. Molecular Medicine (2018) 24:26 Page 11 of 12 Availability of data and materials Brinkmann V, et al. Neutrophil extracellular traps kill bacteria. Science. 2004;303: All data generated or analyzed during this study are included in this 1532–5. published article. Cheng KT, et al. Caspase-11-mediated endothelial pyroptosis underlies endotoxemia-induced lung injury. J Clin Invest. 2017;127:4124–35. Authors’ contributions Choi YS, Baumgarth N. Dual role for B-1a cells in immunity to influenza virus PW conceived the idea of the project. MA, TLR and PW designed the infection. J Exp Med. 2008;205:3053–64. experiments. MA, NEH, YO, MZ and MO performed the experiments. MA, MO Cinel I, Opal SM. Molecular biology of inflammation and sepsis: a primer. Crit and YO performed CLP and measured lung parameters. MZ performed lung Care Med. 2009;37:291–304. −/− IHC. NEH sorted murine PerC B-1a cells and maintained CD19 mice Delgado-Rizo V, et al. Neutrophil extracellular traps and its implications in breeders. MA analyzed the data and wrote the manuscript. TLR and PW inflammation: an overview. Front Immunol. 2017;8:81. reviewed and edited the manuscript. All authors read and approved the final Foster SL, Medzhitov R. Gene-specific control of the TLR-induced inflammatory manuscript. response. Clin Immunol. 2009;130:7–15. Giangola MD, et al. Growth arrest-specific protein 6 attenuates neutrophil Ethics approval migration and acute lung injury in sepsis. Shock. 2013;40:485–91. All animal protocols were approved by our Institutional Animal Care and Use Griffin DO, Holodick NE, Rothstein TL. Human B1 cells in umbilical cord and adult Committee of the Feinstein Institute for Medical Research. peripheral blood express the novel phenotype CD20+ CD27+ CD43+ CD70. J Exp Med. 2011;208:67–80. Consent for publication Grommes J, Soehnlein O. Contribution of neutrophils to acute lung injury. Mol All authors have contributed to, read and approved the final version of this Med. 2011;17:293–307. manuscript for submission and publication in the journal Molecular Medicine. Grönwall C, Vas J, Silverman GJ. Protective roles of natural IgM antibodies. Front Immunol. 2012;3:66. Competing interests Gu WJ, Wan YD, Tie HT, Kan QC, Sun TW. Risk of acute lung injury/acute The authors declare that they have no competing interests. respiratory distress syndrome in critically ill adult patients with pre-existing diabetes: a meta-analysis. PLoS One. 2014;9:e90426. Haas KM, Poe JC, Steeber DA, Tedder TF. B-1a and B-1b cells exhibit distinct Publisher’sNote developmental requirements and have unique functional roles in innate and Springer Nature remains neutral with regard to jurisdictional claims in adaptive immunity to S. pneumoniae. Immunity. 2005;23:7–18. published maps and institutional affiliations. Hirano Y, Aziz M, Wang P. Role of reverse transendothelial migration of neutrophils in inflammation. Biol Chem. 2016;397:497–506. Author details 1 Hirano Y, et al. Neutralization of osteopontin attenuates neutrophil migration in Center for Immunology and Inflammation, The Feinstein Institute for sepsis-induced acute lung injury. Crit Care. 2015;19:53. Medical Research, 350 Community Dr, Manhasset, NY 11030, USA. 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Published: May 29, 2018