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. 2015 Mar 9;13(2):229–239. doi: 10.1038/cmi.2015.08

IL-15 temporally reorients IL-10 biased B-1a cells toward IL-12 expression

Amlan Kanti Ghosh 1, Debolina Sinha 1, Subhadeep Mukherjee 1, Ratna Biswas 1, Tapas Biswas 1
PMCID: PMC4786629  PMID: 25748019

Abstract

Interleukin (IL)-15 is known to strongly modulate T-cell function; however, its role in controlling mucosal immunity, including its ability to modulate B-1a cell activity, remains to be elucidated. Here, we show that IL-15 upregulates activation molecules and the costimulatory molecule CD80 on viable B-1a cells. Cell activation was accompanied by the depletion of sialic acid-binding immunoglobulin-like lectin (Siglec)-G, an inhibitor of cell activation that is present on B-1a cells. The IL-15 receptor CD122 was stimulated on B-1a cells by the cytokine showing its direct involvement in IL-15-mediated responses. IL-10 is responsible for the long term survival of B-1a cells in culture, which is initially promoted by IL-15. The upregulation of IL-10 was followed by the appearance of suppressor of cytokine signaling (SOCS)1 in the presence of IL-15 and the loss of IL-10. This resulted in the cells switching to IL-12 expression. This anti-inflammatory to pro-inflammatory shift in the B-1a cell character was independent of the cell-specific marker CD5, which remained highly expressed throughout the in vitro life of the cells. The presence of the immunosuppressive receptor programmed cell death (PD)-1 and its ligand PD-L2 were features of a predominantly IL-10 response. PD-1 and PD-L2 can mediate juxtacrine signaling. However, the abrogation of PD-1 and its ligand was observed when the cells expressed IL-12. This demonstrates an inverse relationship between the receptor and ligand and the pro-inflammatory cytokine. The induction of IgM and IgA, which can play pivotal roles in mucosal immunity, was promoted in the presence of IL-15. Collectively, the data implicate IL-15 as the master cytokine that induces B-1a cells to mount a mucosal immune response.

Keywords: B-1a cells, IgM, innate immunity, interleukin-12 generation, interleukin-15

Introduction

Mucosal effector sites such as the intestinal lamina propria house B-1 cells, which provide the first line of defense against pathogenic microorganisms.1 The B-1 cells include the B-1a (CD11b+CD5+) and B-1b (CD11b+CD5−) cell subsets. Unlike conventional B-2 cells, the B-1a and B-1b sister populations2 can operate independently of T cell participation.3 B-1a cells are predominantly found at the mucosal surfaces of the peritoneal cavity and are the predominant B-cell (approximately 30%) subset of the innate immune system that responds to foreign invaders by producing natural Abs. In contrast, B-1b cells take part in adaptive immunity by developing into IgM-expressing memory cells.4 B-1 cells originate from precursor cells as a separate lineage from the B-2 cell population.5 These cells produce ‘natural' IgM6 and secrete dimeric and polymeric forms of IgA Ab without any stimulation,7 indicating their readiness to respond to pathogens. By virtue of their ability to migrate to the gut-associated lymphatic tissues,8 B-1 cells of the peritoneal cavity constitute 50% of the intestinal IgA expressing plasma cells.9

Interleukin (IL)-15 is a member of the 4-α-helix bundle family of cytokines and is characterized by its ability to sustain T-cell memory and to function as a growth factor for natural killer T cells.10 This pleiotropic cytokine is transpresented by its specific receptor component IL-15Rα on the surface of antigen-presenting cells (APCs), signals through the IL-2Rβ and γc chains in hematopoietic cells, and leads to cellular activation, proliferation and survival.11,12 Although IL-15 mRNA has been found in several cell types including monocytes, macrophages, dendritic cells, fibroblasts, epithelial cells of various origins and skeletal muscle, tight translational control reduces its production to a minimal level. Additionally, the short half-life of this microenvironmental factor, which controls the pro-inflammatory immune response, strictly regulates its availability.13,14

The discovery that IL-15 is produced by the intestinal epithelial cells of mammals and is upregulated during several intestinal inflammatory diseases led to an in-depth investigation to decipher the possible role of IL-15 in the interplay between the enterocytes and the intestinal intraepithelial lymphocytes that preserve gastrointestinal homeostasis.15,16 The studies performed to date have indicated that this molecule is an important member of the functional intestinal barrier, particularly in the crosstalk between intestinal intraepithelial lymphocyte T cells and natural killer T cells.16,17,18 Moreover, it has been shown to play a role in the activation of neutrophils and the inhibition of apoptosis.19 Although IL-15 has several important and well-documented roles in regulating T-cell function, including restraining tumorigenesis, the interplay between IL-15 and B-1a cells has received little attention. A few studies have demonstrated that IL-15 stimulates B cell proliferation and immunoglobulin synthesis,20,21 indicating that the role of the cytokine in regulating B-1a cells could be important because these cells are the key players of mucosal immunity.

IL-15 is known to be released by intestinal epithelial cells, an important constituent of the mucosal surface to which B-1 cells home. Thus, we hypothesized that IL-15 regulates B-1a cells because these cells are triggered to produce IgM in the absence of antigens. In this study, we examined the interactions between IL-15 and B-1a cells that resulted in the upregulation of activation and costimulatory molecules. IL-15-mediated activation was associated with switching from an anti-inflammatory cell phenotype to a pro-inflammatory phenotype. This modulation of the cytokine milieu was followed by augmentation of IgM and IgA responses. Priming B-1a cells with IL-15 showed that the cytokine both activates the cells and polarizes them toward a type 1 response. These observations prompted us to postulate that epithelial cell-derived IL-15 can be a key regulator in shaping the naive and unmanipulated B-1a cells for the effective clearance of infection from the gut.

Materials and methods

Reagents

Mouse recombinant IL-2 (rIL-2) (BD Pharmingen, San Diego, CA, USA), rIL-15 (eBioscience, San Diego, CA, USA), E. coli (0111:B4) lipopolysaccharide (LPS) (Sigma-Aldrich, St Louis, MO, USA) and goat F(ab′)2 anti-mouse IgM (Jackson Immunoresearch, West Grove, PA, USA) were commercially obtained for this study.

Mice

C57BL/6 mice were obtained from the National Centre for Laboratory Animal Sciences, National Institute of Nutrition, Jamai-Osmania, Hyderabad, India, and bred and reared in the animal care facility of the National Institute of Cholera and Enteric Diseases, Kolkata, India. The mice were housed in groups of six and given food and water ad libitum. B-1a cells were isolated from the peritoneal cavity of 6–7 week old mice of both sexes. The animal experiments were conducted in accordance with the Animal Ethical Committee guidelines of the National Institute of Cholera and Enteric Diseases, Kolkata, India.

Purification and culture of peritoneal cavity CD19+CD11b+CD5+ B-1a cells

C57BL/6 mice were euthanized and thoroughly cleaned with 70% ethyl alcohol. A small incision was made in the abdomen, and the peritoneal cavity was rinsed several times through the opening with freshly prepared cold sterile phosphate-buffered saline (pH 7.2). The peritoneal washes were passed through 105 µm nylon mesh to generate a single cell suspension. The cells were suspended in phosphate-buffered saline and centrifuged at 400g for 8 min and then depleted of erythrocytes by incubating with 0.15 M NH4Cl containing 10 mM KHCO3 and 0.1 M EDTA (pH 7.2) at 4 °C for 3 min.

The cells were stained at 4 °C in the dark for 30 min with PerCP-conjugated anti-mouse CD19 mAb (BD Pharmingen), PE-conjugated anti-mouse CD11b mAb and APC-conjugated anti-mouse CD5 mAb (eBioscience) for sorting on a FACSAria II cell sorter using FACSDiva software (Becton Dickinson, San Jose, CA, USA) (Supplementary Figure 1). The B-1a cell population obtained by sorting was 98% pure.

The B-1a cells were suspended in RPMI 1640 medium (Gibco, Grand Island, NY, USA) and the cells were determined to be 97% viable by Trypan blue exclusion using an inverted microscope. The cells were seeded in 96-well round-bottomed tissue culture plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA) at 1×105 cells/well in 200 µl of RPMI 1640 containing 5 U/ml penicillin, 5 µg/ml streptomycin, 0.1% gentamicin, 2% fetal bovine serum (Gibco) and 0.1% insulin–transferrin–selenium (Gibco). The cells were incubated at 37 °C in 5% CO2 for the indicated time periods in either the presence or absence of mouse rIL-2 (10 ng/well), rIL-15 (50 ng/well), LPS (10 ng/well) or goat F(ab′)2 anti-mouse IgM (10 µg/ml).

Cell proliferation and survival assay

The cells were treated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) salt solution (Sigma) for formazan crystal formation to assess the percentage of live cells. After incubation either with or without mouse rIL-15 for the indicated time points, 10 µg (1 mg/ml) of MTT solution were added to each well and incubated at 37 °C for 2 h. The formazan crystals were dissolved in acidic isopropanol and the absorbance was measured at 570 nm in a Varioskan Flash Multimode Reader (ThermoScientific, Waltham, MA, USA).

Intracellular staining of cytokines and SOCS1, -2 and -3

After the incubation of untreated and rIL-15-treated cells, monensin (0.33 µl/ml) (BD Pharmingen) was added 10 h prior to the completion of the incubation to block either IL-10 or IL-12. The blocked cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Pharmingen) and stained with FITC-conjugated anti-mouse Abs against either IL-10 or IL-12 (BD Pharmingen). Similarly, intracellular suppressor of cytokine signaling (SOCS)1, SOCS2 and SOCS3 were detected by fixing and permeabilizing the cells and staining them with either purified anti-mouse SOCS1, SOCS2 or SOCS3 Abs (Cell Signaling Technology, Danvers, MA, USA) and FITC-conjugated anti-rabbit IgG Ab (eBioscience). The statistical analysis was performed by using isotype-matched controls as references.

Reverse transcription polymerase chain reaction (RT-PCR) analysis

The total RNA was isolated from 1×106 B-1a cells treated with either rIL-15 or complete medium alone using RNAqueous-4PCR kit (Ambion Inc., Austin, TX, USA) and reverse transcribed using a Reverse Transcription System kit (Promega, Madison, WI, USA). cDNA served as the template for the amplification of SOCS1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts by PCR in an automated thermal cycler (GeneAmp PCR System 2700; Applied Biosystems, Foster City, CA, USA) using PCR Master Mix (Promega) and the relevant primers. The following specific primers were used for amplification: SOCS1 forward 5′-CTCGAGTAGGATGGTAGCACGCAA-3′ reverse 5′-CATCTTCACGCTGAGCGCGAAGAA-3′ and GAPDH forward 5′- CCATGGAGAAGGCTGGGG-3′ reverse 5′-CAAAGTTGTCATGGATGACC-3′. The control PCRs were performed to ensure that non-specific amplification did not occur. All the PCRs were performed within the linear range of amplification of the corresponding mRNA species. The images were captured using a gel documentation system (Ultra-Violet Products, Upland, CA, USA). The amplified ethidium bromide-stained RT-PCR products were normalized to the relative quantity of GAPDH using Ultra-Violet Products VisionWorksLS 6.8 software (Upland, CA, USA). The changes in mRNA expression were presented as the mean fold induction by IL-15 relative to the untreated controls.

Flow cytometry

The cells were cultured in either complete medium alone or in the presence of rIL-2, rIL-15, LPS or goat F(ab′)2 anti-mouse IgM and then incubated with purified unconjugated anti-mouse CD16/CD32 mAb (Fc Block; BD Pharmingen). The cells were washed with a 20-fold dilution of FACSFlow and stained at 4 °C in the dark for 20 min with one of either FITC-conjugated anti-mouse CD25 (BD Pharmingen), CD40, CD80, CD86, CD122, IgM, IgA, APC-conjugated anti-mouse CD5 (eBioscience) or biotin-conjugated anti-mouse CD69 (BD Pharmingen), programmed cell death (PD)-1 (eBioscience), programmed death-ligand (PD-L)2 mAb. The biotinylated mAbs were visualized using streptavidin-FITC. Sialic-acid-binding immunoglobulin-like lectin (Siglec)-G was detected by staining the cells with purified anti-mouse Siglec-G Ab (Santa Cruz Biotechnology, Dallas, TX, USA) followed by FITC-conjugated anti-goat IgG Ab. Parallel sets of cells were incubated with monoclonal immunoglobulin isotypes to serve as negative controls for nonspecific staining. The cells were fixed in 1% paraformaldehyde and an equal number of cells for each condition were analyzed on a FACSCalibur flow cytometer using CELLQuest software (Becton Dickinson).

Enzyme-linked immunosorbent assay (ELISA)

Either IL-10 or IL-12 (p70) present in the culture supernatants at the indicated time points was measured by sandwich ELISA with pairs of Abs for binding and detection using the BD OptEIA ELISA Set according to the manufacturer's instructions (BD Pharmingen). Similarly, the IgM and IgA released by the B-1a cells were quantified using the Ready-Set-Go ELISA kit (eBioscience).

Statistical analysis

The results are expressed as the mean±s.e.m., where applicable, of three independent experiments. The data were analyzed using the two-tailed paired Student's t-test. A P value of <0.05 was considered to be significant, and a P value of <0.005 was considered to be highly significant.

Results

IL-15 induces B-1a cell activation and ablates the Siglec-G receptor

IL-15 induces the expression of activation and costimulatory molecules on APCs such as dendritic cells and macrophages. We investigated whether B-1a cells, an important APC in the gut microenvironment, can be similarly activated. The B-1a cells were sorted and treated with rIL-15, resulting in the proliferation of the viable cells in culture similar to the untreated controls (Figure 1a). The activation molecules CD69, CD25 and CD40 were upregulated 1.5- (P<0.005), 2.1- (P<0.005) and 1.4-fold (P<0.05) respectively, compared to the untreated controls. Anti-IgM Ab was used as the positive control (Figure 1b). Interestingly, the early activation molecule CD69 appeared late on the cells, after 96 h of culture, as found with CD4+ T cells.22 The costimulatory molecule CD80, but not CD86, was upregulated 1.5-fold (P<0.005) by IL-15 treatment compared to the untreated control (Figure 1c), suggesting that B-1a cells can be triggered solely by the cytokine for a mucosal immune response.

Figure 1.

Figure 1

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Analysis of activation and costimulatory molecule expression of viable B-1a cells. (a) The cells were cultured in either the absence or presence of IL-15 and tested at each time point as indicated for viability by the Trypan blue exclusion and MTT assay methods. (b) The cells were cultured with IL-15 (black line), anti-IgM Ab (gray line), compared with the untreated control (shaded) and analyzed by flow cytometry for the expression of CD69 (at 96 h), CD25 (at 24 h) and CD40 (at 24 h). (c) The cells were analyzed by flow cytometry for the expression of CD80 and CD86 (at 48 h) after culturing with either IL-15 (black line), anti-IgM Ab (gray line) or complete medium alone (shaded) (d) The flow cytometric analysis of IL-15 (thick black line) and LPS (thin black line)-induced expression of Siglec-G compared to the untreated control (gray line). The shaded profile indicates the isotype-matched control. The data shown are representative of three independent experiments. The bar diagrams represent the mean±s.e.m. of three separate experiments. *P<0.05, **P<0.005. LPS, lipopolysaccharide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Siglec, sialic acid-binding immunoglobulin-like lectin.

The upregulation of activation markers led us to investigate Siglec-G, a unique inhibitory receptor specifically present on the surface of B-1a cells. The stimulation of the activation markers coincided with the depletion of Siglec-G by 45% (P<0.005) due to IL-15 treatment (Figure 1d). This confirms that the cells are truly activated because the suppression of Siglec-G expression correlates with cell activation.23 The data indicate that IL-15 mediates B-1a cell activation in the absence of the pathogenic stimuli that are associated with Siglec-G downregulation. The treatment of B-1a cells with the pathogen-associated molecule LPS downregulated Siglec-G by 21% (P<0.05). Next, we analyzed whether IL-15 stimulated the IL-15 receptor component reported to receive the transpresented cytokine on the cell surface to induce downstream signaling. The data show a 1.4-fold (P<0.005) increase in the expression of CD122, the common β-chain of the heterotrimeric receptor complex, on the cells upon treatment with the cytokine compared to a 2.2-fold (P<0.05) increase in the presence of the positive control, IL-2 (Figure 2).

Figure 2.

Figure 2

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IL-15-mediated upregulation of CD122 on B-1a cells. The cells were cultured for 48 h with IL-15 (black line), IL-2 (gray line) and analyzed by flow cytometry for the expression of CD122 in comparison to the untreated control (shaded). The data shown are representative of three independent experiments. The bar diagrams show the mean±s.e.m. of three separate experiments. *P<0.05.

IL-15 switches B-1a cells from anti-inflammatory to pro-inflammatory cytokine expression

We next studied the modulation of IL-10 in these cells by exogenous IL-15. The enhanced level of the anti-inflammatory cytokine is maintained by CD5, the hallmark of B-1a cells, to ensure self-renewal and hence, longevity in culture in an autocrine fashion.24 Intracellular IL-10 levels were increased 1.6-fold (P<0.005) in B-1a cells by IL-15 after 24 h of culture and were sustained until 48 h (Figure 3a). The cytokine level decreased from 72 h onward with a coincident increase in SOCS1 (Figure 3b), which is known to downregulate the levels of IL-10.25 The blocking of IL-10 induction by SOCS1 (Figure 3c) was supported by the robust expression of SOCS1 mRNA at 96 h (Figure 3d). However, the expression of other closely related members of the SOCS family, SOCS2 and SOCS3, was unaffected by IL-15 treatment (Figure 3e). The intracellular depletion of IL-10 was confirmed by the suppression of cytokine release over time in the presence of IL-15 compared to the untreated control (Figure 3f). Interestingly, the decrease in IL-10 levels was associated with the appearance of intracellular IL-12. Treatment with IL-15 resulted in a 1.8-fold (P<0.005) increase in pro-inflammatory cytokine compared to the untreated control after 96 h of culture (Figure 4a). By ELISA, we could show that 154.5 pg/ml of IL-12 (p70) was secreted (Figure 4b). Collectively, the data indicate that IL-15 modulates the B-1a cell response by curbing the initial amplification of IL-10 through SOCS1 upregulation and facilitating IL-12 production.

Figure 3.

Figure 3

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IL-15-mediated IL-10 and SOCS1 expression by B-1a cells. (a) The intracellular levels of IL-10 as an effect of IL-15 treatment were determined (black lines) compared to the untreated controls (shaded) by flow cytometry at the indicated time points. (b) Similarly, the intracellular expression of SOCS1 in response to IL-15 (black lines) compared to the untreated controls (shaded) is shown. (c) The ratio of IL-10 and SOCS1 expression as an effect of IL-15-treatment are shown. The mean±s.e.m. for each time point from three separate experiments are indicated. (d) The total RNA was extracted from the cells after culturing either with or without IL-15 for 96 h and subjected to RT-PCR using specific primers for SOCS1 and GAPDH. The ethidium bromide-stained PCR products were photographed, and then the images were digitized and analyzed. The PCR products were quantified and expressed as the ratio of the density of each product to the density of the GAPDH band. (e) The flow cytometric analysis of intracellular SOCS2 and SOCS3 levels are shown for the untreated (shaded) and IL-15-treated (black lines) cells. (f) The release of IL-10 in untreated and IL-15-treated culture supernatants was quantified by ELISA at the indicated time points. The bar diagrams represent the mean±s.e.m. of three separate experiments, each performed in triplicate. **P<0.005. The data given are representative of three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SOCS, suppressor of cytokine signaling.

Figure 4.

Figure 4

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IL-15 promotes IL-12 expression by B-1a cells. (a) The cells were cultured for 96 h either with (black line) or without (shaded) IL-15 and the intracellular IL-12 levels were determined by flow cytometry. The results shown are representative of three independent experiments. The bar diagrams show the mean±s.e.m. of three separate experiments. **P<0.005. (b) The release of IL-12 (p70) in untreated and IL-15-treated culture supernatants was quantified by ELISA. The bar diagrams represent the mean±s.e.m. of three separate experiments, each performed in triplicate. *P<0.05, **P<0.005.

These observations prompted us to identify the molecular basis of IL-10 induction. PD-1, which acts as an activation-induced inhibitory receptor in T cells, has been shown to govern cellular responses and maintain peripheral tolerance. PD-1 engagement with its ligands, PD-L1 and PD-L2, has also been linked to augmented IL-10 expression.26 In addition to its immunostimulatory effects, IL-15 increased levels of the PD-1 receptor by 1.4-fold (P<0.005) and the PD-L2 ligand by twofold at 16 h of B-1a cell culture (Figure 5a), validating their parallel presence with IL-10 (Figure 3a). IL-15 actually induced 18.6% B-1a cells to express PD-1 and 14.7% cells to express PD-L2 compared to the untreated control at 16 h (Figure 5b). However, at 96 h of culture both PD-1 and PD-L2 levels declined significantly (Figure 5a) indicating they were inversely related to the IL-12-dominated microenvironment (Figure 4a and b). Thus, the time-dependent appearance and disappearance of PD-1 and its ligand PD-L2 under the influence of IL-15 are crucial for the cells to exhibit the anti-inflammatory and pro-inflammatory activity linked with IL-10 and IL-12, respectively. The IL-15 treatment of B-1a cells did not affect CD5 expression up to 96 h (Figure 6), although the cells switched from anti-inflammatory to pro-inflammatory cytokine production. We observed that both untreated and IL-15-treated B-1a cells expressing the marker increased significantly by 47.2% at 96 h of culture, at which time the cells initiated IL-12 expression.

Figure 5.

Figure 5

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The effect of IL-15 on the time dependent expression of PD-1 and PD-L2 on B-1a cells. (a) The expression of PD-1 and its ligand PD-L2 on IL-15-treated (black line) and untreated (shaded) cells at 16 h followed by their abrogation at 96 h. The data given are representative of three independent experiments. The bar diagrams show the mean±s.e.m. of three separate experiments. *P<0.05. (b) The plots represent the percentage of PD-1+ and PD-L2+ B-1a cells at 16 and 96 h (lower right quadrant) over isotype-matched controls. The data represent the results from one out of three independent experiments. PD, programmed cell death.

Figure 6.

Figure 6

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The effect of IL-15 on the time dependent expression of CD5 on B-1a cells. IL-15-treated (black lines) and untreated (shaded) cells were cultured for 4, 24 and 96 h and CD5 expression was analyzed by flow cytometry. The dot plots represent the percentage of CD5-positive B-1a cells at 4, 24 and 96 h (lower right quadrant) compared to the isotype-matched controls. The data represent results from one out of three independent experiments.

IL-15 augmented IgM and IgA expression

Because IL-15 activated the B-1a cells and led to IL-12 expression, we evaluated whether the cytokine could also control the functional capability of the cells by elevating the IgM and IgA responses, the effectors of mucosal immunity. Our results show that IL-15 augmented IgM cell surface expression from day 4 onward. IgM expression peaked at day 7, at levels 1.7-fold (P<0.005) greater than the control (Figure 7a). The quantification of IgM shows that IL-15 treatment induced the release of 48.71 ng/ml of the immunoglobulin at day 4 and 60.2 ng/ml at day 7 over untreated controls (Figure 7b). Although reports from other laboratories have indicated the costimulatory role of the cytokine for IgM synthesis in B cells,20 we found that IL-15 alone is sufficient to induce the release of IgM by B-1a cells. The IgM expression was followed by the upregulation of IgA in the presence of the cytokine (Figure 7a), which was 1.6-fold (P<0.05) higher than the control at day 7. The quantification of IgA shows that 3.65 ng/ml was released at day 4 and 4.1 ng/ml was released at day 7 of culture (Figure 7b), suggesting that IL-15 can elevate both IgM and IgA levels. Thus, the data reveal that IL-15 activates B-1a cells and causes mucosal immune response.

Figure 7.

Figure 7

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IL-15-induced expression of IgM and IgA. (a) The B-1a cells were cultured with IL-15 (black line), anti-IgM Ab (gray line) and compared with the untreated control (shaded) as indicated and analyzed for cell surface expression of IgM and IgA. The results shown are representative of three independent experiments. The bar diagrams show the mean±s.e.m. of three separate experiments. *P<0.05, **P<0.005. (b) The B-1a cells were cultured with either complete medium alone (open bar) or IL-15 (solid bar). The levels of IgM and IgA in the cell-free supernatants were quantified by ELISA at the indicated time points. The data represent the mean±s.e.m. of three independent experiments, each performed in triplicate.

Discussion

IL-15 is known to promote the differentiation and proliferation of naïve as well as activated B lymphocytes.20,21 The components of the IL-15 receptor are expressed by B cells and mediate the subsequent signaling events.27 IL-15 in combination with recombinant CD40L strongly induces IgG1, IgM and IgA in cultured B cells.20 In the context of cell death, the cytokine has proven to be essential for inhibiting Fas-mediated apoptosis and inducing Ab production partially independently of CD4 help.28,29 Additionally, IL-15 prevents the apoptosis of activated human B cells induced by either anti-Fas, anti-IgM or anti-IgM Ab and dexamethasone suggesting that it is a critical modulator of B-cell activity.30 On the other hand, in vivo reports indicate that IL-15 controls B-cell homeostasis indirectly via the natural killer cell-derived interferon-γ that checks cell proliferation.31 However, IL-15−/− and IL-15Rα−/− mice display similar B-cell numbers compared to normal IL-15 sufficient mice,14 indicating that the cytokine is dispensable for B lymphocytes. This finding discouraged further investigations. Thus, it remains extremely important to determine how B cells are controlled by the cytokine, which is the central focus of this study. The present work shows that IL-15 solely regulates B-1a cell activation and orchestrates aspects of mucosal immunity, such as IgM production, without antigenic mediation. Because this important cytokine is often released by intestinal epithelial cells, this work also implies that B-1a cells, which frequently migrate to the gut-associated lymphatic tissues, are governed by IL-15.

In this study, we first determined that IL-15 stimulates the activation and costimulatory molecules of B-1a cells, suggesting that the primed cells will be responsive to antigenic challenges. The CD19+CD11b+CD5+ B-1a cell population that interacted with the cytokine maximally expressed the activation marker CD25 and the costimulatory molecule CD80. The data show that these cells engage in direct crosstalk with IL-15; the strong influence of IL-15 on these cells coincides with an increase in the expression of the IL-15 receptor CD122. Siglec-G belongs to a superfamily of immunoglobulin-like lectins with the ability to recognize sialic acid-containing structures, functions as a negative regulator of B-1 cells and inhibits BCR-mediated signaling.23 Siglec-G−/− mice have increased numbers of B-1a cells with a lower apoptosis rate,32 indicating that it has a negative impact on cell activation. We show for the first time that the downregulation of Siglec-G correlates with the degree of IL-15 mediated activation in B-1a cells.

The decrease of Siglec-G corresponds to cellular proliferation,33 a phenomenon that occurs downstream of IL-15 stimulation in B lymphocytes. This correlation led us to investigate whether IL-15 modulates the levels of IL-10 in B-1a cells because IL-10 must remain at an elevated level to allow the cells to survive long term in vitro culture via the expression of the signature cell marker CD5.24 Initially, IL-10 was elevated by IL-15 in the absence of SOCS1. Surprisingly, the cells not only decreased their expression of the anti-inflammatory cytokine with the increase in SOCS1 but also tended toward a proinflammatory response, as evidenced by the expression of the key type 1 cytokine IL-12. SOCS1 has been implicated in the IL-15 driven response of antigen-naive CD8 T cells and CD8+CD44hi memory T lymphocytes.34,35 Consistent with these reports, our study adds to the novel role of SOCS1 in regulating IL-15 mediated signaling in B-1a cells. The switching of B-1a cells from IL-10 to IL-12 production shows the modulatory role of IL-15 in driving the cells toward type 1 polarization.

Chronic lymphocytic leukemia is characterized by the accumulation of CD5+ B cells, and IL-10 is produced in a CD5-dependent manner to ensure the self-renewal of the malignant cells.24 In contrast, IL-15 did not affect the level of CD5 on B-1a cells either at 24 h when IL-10 was high or at 96 h when IL-12 alone was expressed. These data reveal an important aspect of IL-15: it can alter the constitutive cytokine milieu of B-1a cells without affecting the key marker CD5.

Next, we identified the molecular players associated with IL-10 in the IL-15-stimulated cells. Dysregulated T and B cell numbers and functions in PD-1−/− mice highlight the pivotal role of the PD-1/PD-L pathway in lymphocyte homeostasis.36,37,38 In general, PD-1 is basally present on T and B cells and is ligated by either PD-L1 or PD-L2 on APCs. Of the two ligands, we were struck by the tissue-specific inducible expression of PD-L2, particularly in the peritoneal cavity B-1 B cell subset.39 As evidence of juxtacrine signaling, where cells signal by direct contact with a membrane-associated form of the ligand and a cognate receptor on an adjacent cell, it is reported that expressed PD-1 receptor and both the ligands fine-tune T-T cell responses in human T cells.22 Here, we show that IL-15 induced the expression of the immunosuppressive receptor PD-1 and its ligand PD-L2 on the same cells as this work involved only B-1a cells. Moreover, we report the novel finding that IL-15 initially induces production of the anti-inflammatory IL-10 in parallel with PD-1/PD-L2. This was followed by the abrogation of IL-10 along with the receptor and its ligand, allowing the cells to become IL-12-positive. The data show pro-inflammatory responses become dominant following the depletion of PD-1/PD-L2. Reports from other laboratories indicate that IL-15 mediated PD-1 and IL-10 overexpression in CD8+ T cells hampers anti-tumor therapy and support our data.40 This counter regulation of downregulating IL-10 and initiating inflammation is significant in the context of gut immunity.

We show that the IL-15 mediated activation of B-1a cells promoted a proinflammatory response. Thus, we analyzed whether the cells were further responsive to express Ab as these cells are reported to produce IgM without any antigenic provocation. A report suggested that IL-15 promoted IgA synthesis at definite mucosal sites, but its impact on ‘natural' IgM production by B-1 cells remains unresolved.21 The immunization of animals with LPS of Francisella tularensis live vaccine strain (Ft LVS) showed protection against attenuated Ft LVS in which opsonizing IgM specifically derived from B-1a cells had played an important role.41 Our data show that IL-15, which is expressed by enterocytes and found at the mucosal surfaces of the intestinal epithelium, directly augments IgM and IgA production. The upregulation of IgM by the cytokine suggests that IL-15 and B-1a cell function in close association to maintain the levels of IgM, a characteristic feature of these cells. Additionally IL-15 enhances IgA, the signature molecule of the mucosal immune system. The stimulation of B-1a cells by IL-15 could be particularly important in the context of the human umbilical cord and adult peripheral blood-derived spontaneous IgM secreting B-1 cells that decline with age.42

These features indicate that IL-15 has a dominant effect on the pathogenesis of inflammation and is an important member of the mucosal ‘immunological niche'.43 Collectively, our data show that IL-15 directly controls B-1a cells by modulating their responses to proinflammatory signals. Understanding the pivotal role of IL-15 in B-1a cells will reveal how these cells are triggered to restrain the infections that are a consistent threat to the gut.

Acknowledgments

This work was supported by the Indian Council of Medical Research (intramural project number IM/TB/12-13/6), the Government of India, New Delhi-110 029, India. AKG and DS are recipients of Senior Research Fellowships from the University Grants Commission (F. No. 2-77/98 (SA-I)), New Delhi-110 002. SM is the recipient of a Senior Research Fellowship from the Council of Scientific and Industrial Research (National Eligibility Test Fellowship No. 09/482(0049)/2009-EMR-I), New Delhi-110 012. RB is the recipient of a Research Scientist position of the project BT/PR8480/MED/29/729/2013 from the Department of Biotechnology, New Delhi 110 003, India.

Footnotes

Supplementary Information accompanies the paper on Cellular & Molecular Immunology's website. (http://www.nature.com/cmi).

Supplementary Information

Supplementary information
Click here for additional data file. (210.2KB, pdf)

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