B-1a cells protect mice from sepsis-induced acute lung injury

Abstract

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: Acute lung injury; B-1a cells; IL-10; Inflammation; Neutrophils; Sepsis.

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 × 10⁵ 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

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, and either PBS- or B-1a cell-treated CLP 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

Fig. 3

B-1a cells attenuate MIP-2 and MPO levels in lungs after sepsis. a, b At the time of CLP, mice were treated with either PBS as vehicle or 5 × 10⁵ PerC B-1a cells in 150 μl of PBS by i.p. injection. After 20 h, lung tissue was harvested and mRNA and protein expression of MIP-2 were assessed, respectively. c MPO activity in lungs of sham-operated, and PBS or B-1a cell-treated CLP mice was determined. Data are expressed as means ± SE (n = 9 mice/group from 3 independent experiments) 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; MIP-2, macrophage-inflammatory protein-2; MPO, myeloperoxidase

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

Fig. 5

Treatment with B-1a cells increases IgM levels in the lungs following sepsis. A total of 5 × 10⁵ sorted B-1a cells were delivered into the peritoneal cavity of CLP mice. After 20 h, lung tissue was harvested from sham, PBS-, and B-1a cell-treated mice and assessed IgM levels in total extracted proteins by ELISA. 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). CLP, cecal ligation and puncture; ELISA, enzyme-linked immunosorbent assay

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