B-1a Cells Protect Mice from Sepsis: Critical Role of CREB

Abstract

Bacterial sepsis is a serious life-threatening condition caused by an excessive immune response to infection. B-1 cells differ from conventional B-2 cells by their distinct phenotype and function. A subset of B-1 cells expressing CD5, known as B-1a cells, exhibits innate immune activity. Here we report that B-1a cells play a beneficial role in sepsis by mitigating exaggerated inflammation through a novel mechanism. Using a mouse model of bacterial sepsis, we found that the numbers of B-1a cells in various anatomical locations were significantly decreased. Adoptive transfer of B-1a cells into septic mice significantly attenuated systemic inflammation and improved survival, whereas B-1a cell-deficient CD19^-/- mice were more susceptible to infectious inflammation and mortality. We also demonstrated B-1a cells produced ample amounts of IL-10 which controlled excessive inflammation and the mice treated with IL-10-deficient B-1a cells were not protected against sepsis. Moreover, we identified a novel intracellular signaling molecule, cAMP-response element binding protein (CREB), which serves as a pivotal transcription factor for upregulating IL-10 production by B-1a cells in sepsis through its nuclear translocation and binding to putative responsive elements on IL-10 promoter. Thus, the benefit of B-1a cells in bacterial sepsis is mediated by CREB and the identification of CREB in B-1a cells reveals a potential avenue for treatment in bacterial sepsis.

Figure 1. Status of B-1a cell frequencies and numbers in sepsis

(A) After 20 h of CLP, cells were isolated from PerC, spleen and BM. The cells were then stained with anti-mouse PE-B220, PE-Cy7-CD23, PerCP/Cy5.5-CD5, APC-IgM and Pacific Blue-IgD Abs and then subjected to flow cytometric detection of B-1a cells frequencies. (B-D) Dynamics of B-1a cell frequencies and numbers in PerC, spleen and BM at different time points after CLP operation are shown. Data are expressed as means ± SE (n=6-9 mice/group) and compared by one-way ANOVA and SNK method obtained from three independent experiments (*p<0.05 vs. sham-operated animals). CLP, cecal ligation and puncture; PerC, peritoneal cavity; BM, bone marrow.

Figure 2. Therapeutic potential of adoptive transfer of B-1a cells during sepsis

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, blood was drawn to assess plasma levels of injury markers, (A) ALT, (B) AST, and (C) LDH and pro-inflammatory cytokines (D) IL-6, (E) IL-1β, and chemokine (F) MIP-2. Aside from blood, after 20 h of CLP PerC washouts were collected, spun down, and the supernatants were used to assess (G) IL-6 and (H) IL-1β by ELISA. Data are expressed as means ± SE (n=6-9 mice/group) and compared by one-way ANOVA and SNK method obtained from five independent experiments (*p<0.05 vs. sham-operated group; #p<0.05 vs. PBS-treated CLP mice). Bacterial counts as expressed by CFU in (I) blood and (J) peritoneal washouts are presented. Data are expressed as means ± SE (n=12 mice/group for blood and n=18 mice/group for peritoneal washouts) and compared by Student's t-test (*p<0.05 vs. PBS-treated CLP mice) acquired from five independent experiments. (K) A 10-day period Kaplan-Meier survival curve generated from CLP mice treated with either PBS (n=20 mice), or B-1a cells (5 × 10⁵ cells/mouse) isolated from peritoneal cavities (n=20 mice), or B-2 cells (5 × 10⁵ cells/mouse) isolated from spleens of syngeneic mice (n=14 mice) in 150 μL of sterile PBS via i.p. is shown. *p<0.05 vs. PBS-injected mice, #p<0.05 vs. B-2 cell-treated mice determined by the log-rank test. CLP, cecal ligation puncture; PerC, peritoneal cavity; BM, bone marrow; ALT, alanine aminotransferase; AST, aspartate amino transferase; LDH, lactate dehydrogenase; IL, interleukin; MIP-2, macrophage-inflammatory protein; CFU, colony forming units.

Figure 3. Status of B-1a cell deficient CD19^-/- mice during sepsis

After 20 h of CLP induced in WT and CD19^-/- mice, blood was collected and plasma was subjected to assay for (A) ALT, (B) AST, (C) LDH and (D) IL-6. Data are expressed as means ± SE (n=6-9 mice/group) and compared by one-way ANOVA and SNK method obtained from five independent experiments (*p<0.05 vs. sham-operated group; #p<0.05 vs. WT CLP mice). Bacterial counts in terms of CFU in (E) whole blood and (F) peritoneal lavage fluid are shown. Data are expressed as means ± SE (n=6-12 mice/group) and compared by Student's t test obtained from five independent experiments (*p<0.05 vs. WT CLP mice). (G) Kaplan-Meier survival curve generated from WT and CD19^-/- CLP mice during the 10-day monitoring period is shown. n=21 mice in each group, *p<0.05 vs. WT CLP mice, determined by the log-rank test. CLP, cecal ligation puncture; WT, wild-type; ALT, alanine aminotransferase; AST, aspartate amino transferase; LDH, lactate dehydrogenase; IL, interleukin; CFU, colony forming units.

Figure 4. Assessment of IL-10 production by B-1a cells during sepsis and under in vitro condition

(A) After 20 h of CLP, the IL-10 level in plasma was assessed. Data are expressed as means ± SE (n=9 mice/group) and compared by one-way ANOVA and SNK method obtained from three independent experiments (*p<0.05 vs. sham-operated group; #p<0.05 vs. PBS-treated CLP mice). (B) B-1a cells from PerC of WT and TLR4^-/- mice were sort purified by staining the cells with PE-Cy7-B220, PE-CD23, PerCP-Cy5.5-CD5 and Pacific Blue-CD19 Abs. A total of 0.5 × 10⁶ sorted B-1a cells were adoptively transferred into GFP Tg mice via i.p. during CLP operation. After 20 h of CLP, cells from the PerC of septic GFP Tg mice were isolated to perform intracellular staining using anti-mouse APC-IL-10 Ab. The levels of intracellular IL-10 following 20 h of CLP within GFP⁻ B-1a cells are shown. Representative blots obtained from three independent experiments using 5 mice/group are presented. (C) In an in vitro experiment, a total of 1.5 × 10⁵ PerC B-1a cells/ml were stimulated by either LPS (20 μg/ml) or LPS (20 μg/ml) and PMA (50 ng/ml) and ionomycin (100 ng/ml) in combination for 20 h followed by the assessment of IL-10 at the protein level. Data are expressed as means ± SE. The experiment was performed three independent times with n=3-4 wells per group. The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. PBS-treated group; #p<0.05 vs. LPS-treated group). In an in vitro co-culture experiment, 1.5 × 10⁵ PerC macrophages were co-cultured with 1.5 × 10⁵ B-1a cells (1:1) isolated from either WT or IL-10^-/- mice, and then after stimulation with PBS or LPS (20 μg/ml) for 20 h (D) TNF-α and (E) IL-1β levels in the culture supernatant were assessed. Data are expressed as means ± SE obtained from three independent experiment with n=3-4 per group. The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. PBS-treated group; #p<0.05 vs. LPS-treated macrophages; §p<0.05 vs. LPS-treated macrophages and WT B-1a cell co-cultures).

Figure 5. Protective effect of murine B-1a cell secreted IL-10 during sepsis

(A) Sorting of PerC B-1a cells from the WT and IL-10^-/- mice by staining them with FITC-B220, PE-CD23, PerCP-Cy5.5-CD5 and APC-CD19 Abs. (B-E) At the time of CLP, mice were injected with PBS or B-1a cells (5 × 10⁵ cells) isolated from either WT or IL-10^-/- mice in 150 μL of sterile PBS. After 20 h of CLP, blood was drawn and the plasma was assessed for ALT, AST, LDH, and IL-6. Data are expressed as means ± SE (n=5 mice/group) and compared by one-way ANOVA and SNK method obtained from three independent experiments (*p<0.05 vs. sham mice; #p<0.05 vs. PBS-treated CLP mice). (F) A 10-day period Kaplan-Meier survival curve generated from CLP mice treated with either PBS, or PerC B-1a cells of WT or IL-10^-/- mice (5 × 10⁵ cells) in 150 μL of sterile PBS via i.p. is shown. n=19 mice per group, (*p<0.05 vs. WT B-1a cell-injected mice; #p<0.05 vs. IL-10^-/- mice B-1a cells determined by the log-rank test). CLP, cecal ligation puncture; PerC, peritoneal cavity; LPS, lipopolysaccharide; WT, wild-type; PMA, phorbol 12-myristate 13-acetate; TNF-α, tumor necrosis factor-α; ALT, alanine aminotransferase; AST, aspartate amino transferase; LDH, lactate dehydrogenase; IL, interleukin; WT, wild-type.

Figure 6. Phosphorylation of CREB in IL-10 expressing B-1a cells during CLP and under in vitro stimulation

After 20 h of CLP, PerC washouts were collected and the cells were stained by anti-mouse PE-B220, PE-Cy7-CD23, PerCP-Cy5.5-CD5, Pacific Blue-CD19, APC-IL-10 and FITC-pCREB Abs and subjected to flow cytometry to detect IL-10 and CREB phosphorylation in B-1a cells and compared with that of sham mice. An isotype control Ab for pCREB Ab (goat FITC-IgG) was included while staining the cells with IL-10 Ab to get rid of background staining. (A) Dot blots and histograms representing IL-10 and pCREB expression in B-1a cells in sham and CLP-operated animals, respectively, are shown. (B) Quantitative bar diagrammatic presentation of IL-10 expression in B-1a cells in sham and CLP-operated mice. (C) pCREB expression as measured by MFI in B-1a cells in sham and CLP-operated mice is shown. (D) pCREB expression as measured by MFI in IL-10 expressing or non-expressing B-1a cells in sham and CLP-operated mice is shown. Data are expressed as means ± SE obtained from three independent experiments with n=6 mice per group. The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. sham mice or IL-10 non-expressing B-1a cells from sham mice; #p<0.05 vs. IL-10 non-expressing B-1a cells from CLP mice). (E) A total of 1 × 10⁶ cells isolated from peritoneal cavity were stimulated by either LPS (20 μg/ml) or LPS (20 μg/ml) and PMA (50 ng/ml) and ionomycin (100 ng/ml) in combination for 20 h followed by the surface staining with specific fluorochrome-labeled anti-B220, anti-CD23, anti-CD5 and intracellular staining with anti-pCREB Abs. The samples were then subjected to flow cytometric acquisition and analysis by Flowjo software. Representative histograms of pCREB expression in B-1a cells as indicated by the MFI of isotype control, PBS, LPS and LPS+PMA and ionomycin-treated samples are shown. (F) A quantitative bar diagram of MFI of pCREB expression is presented. Data are expressed as means ± SE obtained from three independent experiments with n=3-4 wells per group. The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. PBS-treated cells). (G, H) B-1a cells (1.5×10⁶ cells/ml) were plated in 48-well cell culture plate and pre-treated with DMSO or CREB inhibitor (20 μM) for 30 minutes, afterward stimulated with either LPS (20 μg/ml) or LPS (20 μg/ml) and PMA (50 ng/ml) and ionomycin (100 ng/ml) in combination for 20 h, followed by the assessment of IL-10 and TNF-α in culture supernatants at protein level. Data are expressed as means ± SE obtained from three independent experiments (n=6 wells/group). The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. PBS- and #p<0.05 vs. DMSO-treated cells). CLP, cecal ligation puncture; PerC, peritoneal cavity; LPS, lipopolysaccharide; TLR4, toll-like receptor 4; PMA, phorbol 12-myristate 13-acetate ; pCREB, phospho cAMP response element binding protein; MFI, mean fluorescence intensity.

Figure 7. Assessment of nuclear translocation of phosphorylated CREB and its binding to the IL-10 promoter in B-1a cells under in vitro stimulation

(A) A total of 2 × 10⁶ cells isolated from peritoneal cavity were stimulated by either LPS (20 μg/ml) or LPS (20 μg/ml) and PMA (50 ng/ml) and ionomycin (100 ng/ml) in combination for 20 h followed by the surface staining with FITC-labeled anti-B220, PE-Cy5-labeled anti-CD5, intracellular staining with BV-480-labeled anti-pCREB Abs and nuclear staining with DAPI. The samples were then subjected to acquisition in ImageStream®X Mark II Imaging Flow Cytometer and analysis by INSPIRE® and IDEAS® software. Representative images of nuclear translocation pCREB in B-1a cells treated with PBS, LPS and LPS+PMA and ionomycin are shown. (B) A total of 1 × 10⁶ B-1a cells sorted from peritoneal cavity of C57BL/6 mice were stimulated by either PBS as control or LPS (20 μg/ml) or LPS (20 μg/ml) and PMA (50 ng/ml) and ionomycin (100 ng/ml) in combination for 20 h. Chromatin was extracted for ChIP assay by sonication, followed by cross-linking and immunoprecipitation using anti-pCREB Ab. Conventional qualitative as well as qRT-PCR was performed and data is presented as gel-electrophoresis blots and fold induction of amplified CREB response element containing DNA in IL-10 promoter normalized against respective input controls. Data are expressed as means ± SE obtained from three independent experiments (n=5 samples/group). Multiple groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. PBS-treated cells). (C) Hypothesis scheme. In B-1a cells, LPS through TLR4-mediated pathway and PMA and Ca²⁺ ionopohre ionomycin up-regulated the phosphorylation of CREB, which in turn served as a pivotal transcription factor to induce IL-10 expression. Increased expression of IL-10 from the B-1a cells finally led to a protection against sepsis. PerC, peritoneal cavity; LPS, lipopolysaccharide; TLR4, toll-like receptor 4; PMA, phorbol 12-myristate 13-acetate; pCREB, phospho cAMP response element binding protein; DAPI, 4′,6-Diamidino-2-Phenylindole, Dihydrochloride; ChIP, chromatin immunoprecipitation.