Elevation in the counts of IL-35-producing B cells infiltrating into lung tissue in mycobacterial infection is associated with the downregulation of Th1/Th17 and upregulation of Foxp3+Treg

IL-35 is an anti-inflammatory cytokine and is thought to be produced by regulatory T (Treg) cells. A previous study found that IL-35 was upregulated in the serum of patients with active tuberculosis (ATB), and IL-35-producing B cells infiltrated to tuberculous granuloma of patients with ATB. Purified B cells from such patients generated more IL-35 after stimulation by antigens of Mycobacterium tuberculosis and secreted more IL-10. However, the function and the underlying mechanisms of IL-35-producing B cells in TB progression have not been investigated. The present study found that the expression of mRNA of IL-35 subsets Ebi3 and p35 was elevated in mononuclear cells from peripheral blood, spleen, bone marrow, and lung tissue in a mouse model infected with Mycobacterium bovis BCG, as tested by real-time polymerase chain reaction. Accordingly, the flow cytometry analysis showed that the counts of a subset of IL-35+ B cells were elevated in the circulating blood and in the spleen, bone marrow, and lung tissue in BCG-infected mice, whereas anti-TB therapy reduced IL-35-producing B cells. Interestingly, BCG infection could drive the infiltration of IL-35-producing B cells into the lung tissue, and the elevated counts of IL-35-producing B cells positively correlated with the bacterial load in the lungs. Importantly, the injection of exogenous IL-35 stimulated the elevation in the counts of IL-35-producing B cells and was associated with the downregulation of Th1/Th17 and upregulation of Foxp3+Treg.The study showed that a subset of IL-35-producing B cells might take part in the downregulation of immune response in mycobacterial infection.

Scientific RepoRtS | (2020) 10:13212 | https://doi.org/10.1038/s41598-020-69984-y www.nature.com/scientificreports/ Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), commonly known as tubercle bacillus, is a major health problem worldwide, with approximately one third of the global population infected and 10 million new cases reported officially to the World Health Organization in 2017 1,2 . The susceptibility to the onset and progression of TB involves not only the damage of infected tissues but also the deregulation of host immune response to the pathogen 3 . Innate immune cells mediating bactericidal effects and subsequent activation of T cell-mediated adaptive immunity play an important role in the clearance of tubercle bacillus 4 5 . Mtb-specific αβ T cells, such as Th1, Th17, and cytolytic CD8 + T cells, play an important role in protective immunity against TB 6,7 , but regulatory T cells are always downregulated in anti-TB immunity 8 . The role of B cells in anti-tuberculosis immunity, however, has long been ignored. Studies suggested that B cells could present antigen for effective priming of T cells and promote humoral immunity in the control of Mtb infection [9][10][11] . Recently, Evidences showed that B cells also played a critical role as regulators to promote anti-TB immunity by producing cytokines [9][10][11] . On the contrary, some recent studies suggested that B cells could also accelerate the progression of infectious diseases [12][13][14][15][16] . In the recent years, a novel subset of B cells, which exert immune-regulatory effects by producing interleukine (IL)-10, transforming growth factor-β (TGF-β), and IL-35, have been identified and classified as regulatory B cells (Bregs). IL-10-producing B cells was first found in chronic intestinal inflammation 17 . They have also been reported in autoimmune diseases 18, allergic diseases 19, graft-versus-host disease 20, and cancer 21 . In M. tuberculosis infections, a subset of CD19 + CD1d + CD5 + Bregs was found associated with active tuberculosis (TB) 22 . Subsequent study 23 revealed that successful anti-TB treatment in human TB induced an increased IL-22 response by reducing the frequencies of CD19 + CD5 + CD1d + Bregs. Notablely, it was found that patients with cavitary TB had significantly higher frequencies of CD19 + CD1d + CD5 + B cells. In a mouse model, B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4 + TEM cells and CD8 + TEM/CD127 + cells 24 . More recently, a study reported that M. tuberculosis mannose-capped lipoarabinomannan induces IL-10-producing B cells and hinders CD4 + Th1 immunity 25 . These findings suggested that IL-10-producing Bregs impaired protective immunity and increased disease severity. A novel subset of Bregs producing an anti-inflammatory cytokine, IL-35, was reported during Salmonella infections and experimental autoimmune encephalomyelitis (EAE). These cells play a key role in immunosuppression 26,27 . IL-35 is composed of the p35 subunit of IL-12 and the subunit of IL-27 of Epstein-Barr Virus (EBV)-induced gene 3 (EBI3) and appears to be produced exclusively by Treg cells, DC cells, macrophages, human placental trophoblasts, and a variety of tumor tissues [28][29][30] . IL-35 appears to suppress T cell proliferation and differentiation, and induces Treg cells polarization 28,31 . Similar to IL-10 and TGF-β, IL-35 can also ameliorate autoimmune diseases and promote the development of infectious diseases. A previous study showed that patients with active TB exhibited increases in serum IL-35 levels and the mRNA expression of both subunits of IL-35 (p35 and EBI3) in white blood cells and peripheral blood mononuclear cells. Mtb infection was associated with the expression of p35 or EBI3 protein in CD4 + but not in CD8 + T cells. Most p35 + CD4 + T cells and EBI3 + CD4 + T cells expressed the Treg-associated marker CD25 32 . More recently, it was reported that IL-35-producing B cells infiltrated into the tuberculous granuloma of patients with ATB, and the mRNA expression of both subunits of IL-35 (p35 and Ebi3) in purified B cells from patients with ATB was elevated. Furthermore, purified B cells from patients with ATB produced more IL-35 after stimulation by Mtb antigen, and the IL-35-producing B cells expressed the high level of IL-10 33 . However, the function and underlying mechanisms of IL-35-producing B cells in TB progression have not been investigated.

. Mtb-induced T cell immune response involves several conventional (αβ T cells) and unconventional (γδ T cells and CD1restricted cells) T cell subsets
In the present study, a mouse model infected with Mycobacterium bovis bacillus was used to further validate previous findings in patients with TB that Mtb infection could induce the increase in IL-35-producing B cells and their infiltration into TB lesion tissue. In addition, this study examined the effects of IL-35-producing B cells on the bactericidal activity in the mouse model. It also explored the correlation between IL-35-producing B cells and the effector/regulatory T cells in mycobacterial infection.
Mouse model and specimen collection. Wild-type C57BL/6J mice were purchased from the Laboratory Animal Center of Southern Medical University, certified by the Laboratory of Animal Quality of Guangdong Province (license No. SCXK (Guangdong) 2016-0041). All female mice, aged 6-8 weeks, were used and randomized for the experiments. The treatment process and experimental procedures were in accordance with the regulations of the experimental animal management. C57BL/6J mice were divided into BCG infection experimental group and saline control group. The experimental group was infected with BCG via the tail vein at a dose of 2 × 10 6 colony formation unit (CFU)/200 μL, and the control group was injected with 200 μL of saline. The mice were euthanized, and efforts were made to minimize suffering. Blood, spleen, bone marrow, and lung tissues were collected for isolating mononuclear cells at different time points (2, 4, and 8 weeks) after infection for detecting IL-35 subsets. Ebi3 and p35 expression was analyzed by real-time RT-PCR, while the protein expression was analyzed by flow cytometry with intracellular cytokine staining at each time point 35 . Lung tissues were also dissected aseptically for histological and pathological analyses and for determining the bactericidal activity via direct bacterial counting. A total of 10 mice were selected from each group. Attention was paid to the matching of age, weight, and physiological status among the groups. Mycobacterial colony formation unit count. Lung tissues were harvested aseptically at different time points (2, 4, and 8 weeks) after infection. Under sterile conditions, 0.04 g lung tissue was taken from the left lower lobe of the mice. Then, 500 μL of saline was added to the grinder and diluted with an equal volume of 4% NaOH for 10 min. A tenfold serial dilution was performed for quantitative culturing. Aliquots (100 μL) were coated in triplicate on Middlebrook 7H11 agar plates supplemented with 10% oleicacid-albumin-dextrosecatalase (OADC) enrichment for 4-6 weeks of culture in a CO 2 incubator with 5% CO 2 until colonies were large enough to be counted. The mycobacteria viability was quantified by counting CFUs.

Isolation of peripheral blood mononuclear cells and tissue mononuclear cells. Peripheral blood
mononuclear cells (PBMCs) and bone marrow mononuclear cells were prepared as described previously 37,38 . Blood samples or bone marrow irrigating solution was collected in tubes precoated with heparin sodium for anticoagulation. PBMCs or bone marrow mononuclear cells were freshly isolated from blood by standard Ficoll (GE Healthcare) density gradient centrifugation. The cell viability was determined by trypan blue exclusion (> 95% in all experiments). The lung tissue was aseptically dissected. After washing three times with saline to clean the blood, the tissue was homogenized and incubated at room temperature for 30 min with 5 mL of enzymatic hydrolysate [containing 0.05% (w/v) collagenase and 0.01% (w/v) trypsin inhibitor in RPMI1640]. Then, the tissues lysate was gently milled on a 200-mesh filter with a grinding rod. A mouse tissue mononuclear cell separation kit for isolated cells (TBDsciences) was used for lung mononuclear cell (LMC) isolation. Splenic cells were isolated from spleens of mice by gradient centrifugation with Ficoll solution (d = 1.088). The cell viability was determined by trypan blue exclusion (> 95% in all experiments).

Histological analysis and IF staining.
The lung tissue (about 1 cm 3 ) was collected at necropsy and fixed in 10% paraformaldehyde in phosphate-buffered saline (PBS). Randomly selected tissue sections were embedded in paraffin and cut into 5-μm sections on a microtome. The tissue microsections were mounted on glass slides, deparaffinized, and stained with hematoxylin-eosinand carbolfuchsin by the Ziehl-Neelsen method, as reported previously 39 , and then analyzed by a certified pathologist.
For IF staining, the paraffin sections of the lung tissue were dried and dewaxed. The antigen was repaired and blocked with a blocking solution containing 4% BSA and 5% goat serum (Gibco) for 1 h. Then, 50 μL of Scientific RepoRtS | (2020) 10:13212 | https://doi.org/10.1038/s41598-020-69984-y www.nature.com/scientificreports/ pre-mixed primary antibodies (mouse B220/CD45R antibody, R&D; IL-27/IL-35 EBI3 subunit antibody, Novusbio; anti-Mouse P35 FITC) was added to the corresponding tissue blocks and incubated overnight at 4 °C. The concentration of the primary antibodies was diluted according to the recommended folds in the manual. On the next day, the slides were equilibrated at room temperature for 1 h. After washing with PBST, the diluted fluorescently labeled secondary antibody was added dropwise to the tissue sections and incubated at room temperature for 1 h in the darkness. The secondary antibodies anti-rat IgG (H + L) (Alexa Fluor 647 Conjugate, Cell Signaling Technology) and anti-rabbit IgG (H + L), F(ab) 2 Fragment (Alexa Fluor 594 Conjugate, Cell Signaling Technology) were diluted (1:1,000) according to the recommended amount in the instruction manual and rinsed with PBST. Finally, confocal images were captured with a laser scanning confocal microscope LSM800 (Zeiss, Jena, Germany).
Flow cytometry analysis and intracellular cytokine staining. Flow cytometry (FCM) analysis and intracellular cytokine staining were performed as previously described. PBMCs, LMCs, and mononuclear cells from the spleen and bone marrow were used for detecting IL-35 or IL-10 expression in B cells, while PBMCs and LMCs were used for detecting IFN-γ, IL-17, and Foxp3 expression in CD4 + T cells, by direct intracellular cytokine staining (ICS) without stimulation in vitro 35,40 . PBMCs, LMCs, and mononuclear cells from the spleen and bone marrow were directly stained with anti-mouse-p35-FITC (R&D) and anti-mouse-EBI3-APC (R&D) for detecting IL-35 or stained with anti-mouse-IL-10-PE (JES5-16E3, eBioscience) for detecting IL-10. APC-Anti-mouse-CD4 antibody (eBioscience) and PE-cy7-anti-mouse-IFN-γ antibody (Thermo Fisher Scientific) or PE-anti-mouse-IL-17 antibody (eBioscience) were used for detecting Th1 or Th17. PE-cy7-anti-mouse-CD3 antibody (Biolegend), FITC-anti-mouse-CD4 antibody (Biolegend), APC-anti-mouse-CD25 antibody (eBioscience), and PE-cy5-anti-mouse Foxp3 antibody (eBioscience) were used for detecting Foxp3 + regulatory T cells. ICS was performed as previously described 35 . The cells were washed once with 2% FBS-PBS and stained at room temperature for 25 min with surface marker Abs. For ICS, PBMCs, LMCs, or mononuclear cells of spleen and bone marrow were further washed twice with 2% FBS-PBS, permeabilized with BD FACS permeabilizing solution (BD Biosciences) for 30 min at room temperature, and then stained for another 45 min with cytokine antibodies, followed by two final washes with 2% FBS-PBS buffer and analysis with BD FACSVerse (BD Biosciences) flow cytometry. To ensure specific immune staining in ICS, matched isotype IgG served as negative control for staining cytokines or surface markers.

Statistical analysis.
Flow cytometry data were analyzed by FlowJo software 7.6 (Tree Star Inc., CA, USA).
Statistical analysis was carried out using GraphPad Prism 5 software (GraphPad Software Inc., CA, USA). The difference between the two groups was analyzed using the Student t test. The data of multiple groups were compared by analysis of variance. The correlation was analyzed using Pearson coefficient A P value less than 0.05 was considered statistically significant.
Ethics statement. This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals, and the protocol was approved by the Ethics Committee of Guangdong Medical University (GDMU). The mice were bred under specific pathogen-free conditions in the laboratory animal facility at GDMU. All animal experiments were conducted under isoflurane anesthesia, and all efforts were made to minimize suffering.

Mycobacterial infection induced high mRNA expression of IL-35 subsets Ebi3 and p35 in
PBMCs and immune tissue mononuclear cells. A recent study reported that IL-35 was produced by not only T cells 26,28 but also B cells 27 . A previous study showed that IL-35 was upregulated in serum in patients with active TB and was related to the activation of Treg cells 32 . IL-35 was produced by B cells, with a strong ability to produce IL-10, in the peripheral blood in patients with active TB and in human tuberculous granuloma 33 . For further understanding the roles of IL-35 played in pulmonary tuberculosis, a mouse model was prepared with M. bovis BCG infection (Fig. S1), and mononuclear cells were isolated from peripheral blood, lung, spleen, and bone marrow. Subsequently, the mRNA and protein expression of IL-35 subsets p35 and Ebi3 in these cells was determined by real-time PCR and flow cytometry with ICS. Interestingly, the expression of p35 and Ebi3 genes increased in mononuclear cells in the blood and in each tissue 2, 4, and 8 weeks after BCG infection (Fig. 1), Scientific RepoRtS | (2020) 10:13212 | https://doi.org/10.1038/s41598-020-69984-y www.nature.com/scientificreports/ especially in the circulating blood and lung tissue (Fig. 1). Notably, flow cytometry with ICS analysis also showed that the frequencies of p35 + and Ebi3 + B cells were much higher in BCG-infected mice than in control mice 2, 4, and 8 weeks after BCG infection, which was consistent with the mRNA expression (Fig. 2). Intriguingly, the frequencies of p35 + and Ebi3 + B cells were much higher (about 1.5 times) in the lungs than in the circulating blood, indicating that BCG infection might drive the infiltration of IL-35-producing B cells into the lungs (Fig. 2E, F). Together, these results suggested that a subset of B cells was distributed in immune organs. BCG infection induced the high expression of IL-35 in B cells in the peripheral blood and lung tissue, and also caused the infiltration of B cells in mice.

BCG infection-driven infiltration of IL-35-producing B cells into lung tissue.
The results suggested that the frequency of IL-35-producing B cells in the lung tissue was about 1.5 times higher as that in PBMCs after BCG infection for 4 weeks. Then, whether mycobacterial infection could induce the infiltration of IL-35-producing B cells into lung tissue was analyzed in this study. FCM results showed that the frequencies of p35 + and Ebi3 + B cells in the lungs were much higher than those in the circulating blood and in the spleen and bone marrow. The frequency of p35 + Ebi3 + B cells reached a peak level 4 weeks after BCG infection. Even after 8 weeks, it remained at a high level (Fig. 3A-C). Furthermore, IF staining with confocal analysis was performed on lung tissue sections of BCG-infected mice to determine the effects of mycobacterial infection on the alterations of IL-35-producing B cells. The surface markers of B cells B220 and two subunit markers of IL-35 (Ebi3 and p35) were stained with different fluorescently labeled antibodies. The results visualized that more Ebi3 + p35 + B220 + B cells appeared in the lung tissue in BCG-infected mice than in control (Fig. 3D).
Taken together, these results suggested that BCG infection could drive the infiltration of IL-35-producing B cells into the lung tissue in mice.

Anti-TB therapy reduced the counts of IL-35-producing B cells in BCG-infected mice.
The present study sought to determine whether treatment with anti-TB drugs could alter IL-35-producing B cells so as to validate the effect of mycobacterial infection on IL-35-producing B cells from the opposite side. Along with the anti-TB drug rifampicin (RIF) and isoniazid (INH) treatment, populations of IL-35-producing B cells reduced not only in PBMCs but also in LMCs compared with mice without anti-TB treatment (Fig. 4). This result further confirmed that mycobacterial infection indeed induced an increase in the counts of IL-35-producing B cells.

Correlation of the counts of IL-35-producing B cells with bacterial load in the lungs in
BCG-infected mice. Previous reports indicated that IL-35-producing B cells possessed negative immune regulatory effects, for example, the downregulation of the development of autoimmune diseases 27 . However, the relationship between IL-35-producing B cells and the bacterium-clearing function in the lungs in pulmonary tuberculosis has not been reported yet. The mycobacterial colony formation unit count method was used in this study to calculate the bacilli number in the lung tissue from BCG-infected mice to determine whether IL-35-producing B cells affected the bactericidal function in vivo. The bacterial numbers were different 2, 4, and  26 . A previous study indicated that IL-35-producing B cells in patients with active TB possessed a stronger ability to produce more IL-10 33 . This study also aimed to determine whether the mycobacterial infection in mice could induce IL-10 production in IL-35-producing B cells. Direct ICS with flow cytometry was used to test the frequencies of IL-10-positive B cells in PBMCs and LMCs from BCG-infected mice. The results showed that the frequency of IL-10-positive B cells in LMCs were higher than those in PBMCs both in BCG-infected mice and in controls (Fig. 6A,B). The frequencies of IL-10-positive B cells in BCG-infected mice, in both the blood and lung tissue, were much higher than that in controls (Fig. 6C). Notably, IL-35-producing B cells in the blood, not only in controls but also in BCG-infected mice, possessed strong ability to produce IL-10, displaying that the frequencies of IL-10-positive B cells in IL-35-producing B cells were much higher than that in IL-35-negative B cells both in BCG-infected mice and in controls (Fig. 6D,E). Importantly, BCG infection induced more IL-10 production in IL-35-producing B cells than in controls (Fig. 6F). These results indicated that BCG infection induced a high level of IL-10 production in IL-35-producing B cells.  www.nature.com/scientificreports/ than those in BCG-infected mice without exogenous IL-35 treatment (Fig. 7A,B). Consistent with the previous results, paired comparison analysis further confirmed that the percentage of IL-35-producing B cells was higher in the lung tissue than in the circulating blood (Fig. 7C-E), and exogenous IL-35 treatment promoted the infiltration of IL-35-producing B cells into the lung tissue (Fig. 7E). Exogenous IL-35 treatment obviously impacted Th1/Th17, despite BCG infection-induced elevation in the percentages of anti-tuberculosis effector cells Th1/ Th17, displaying that the frequencies of IFN-γ/IL-17-positive cells in CD4 + T cells increased in BCG-infected mice compared with controls (Fig. 8A,B and E,F, respectively). These findings implied that the frequencies of  The P value is shown in each column, *P < 0.05; **P < 0.01; and ***P < 0.001, or in each graph directly.

Exogenous IL-35-induced elevation in the frequencies of IL-35-producing B cells and their infil-
Scientific RepoRtS | (2020) 10:13212 | https://doi.org/10.1038/s41598-020-69984-y www.nature.com/scientificreports/ IFN-γ-or IL-17-positive cells in CD4 + T cells were much lower in BCG + IL-35-treated group than in the BCG only-treated group (Fig. 8A,B and E,F, respectively). Interestingly, exogenous IL-35 treatment obviously induced elevation in the frequency of Treg cells in the peripheral blood and lung tissue, indicating that the frequency of Treg cells was much higher in the BCG + IL-35-treated group than in the BCG only-treated group (Fig. 8I,J). In addition, comparison of exogenous IL-35 treatment-driven Treg infiltration in the lung tissue showed that the frequency of Treg cells in the lung tissue was much higher than that in the blood (Fig. 8I,J). Furthermore, exogenous IL-35 treatment likely impacted Th1/Th17 and Treg via inducing the elevation in the counts of IL-35-producing B cells because the frequency of IL-35-producing B cells negatively correlated with the percentage of IFN-γ-positive or IL-17-positive cells in CD4 + T cells (Fig. 8C, D and G, H, respectively) and positively correlated with Foxp3 + Treg (Fig. 8K,L). Thus, these data suggested that IL-35 could trigger the elevation in the counts of IL-35-producing B cells, promote their infiltration into lung tissue, and downregulate anti-tuberculosis effector cell Th1/Th17 infiltration. Further, it drove Foxp3 + Treg elevation and infiltration into lung tissue.

Discussion
This study was performed based on a recent report about the patients with active TB who had IL-35-producing B cells with a stronger ability to secrete IL-10. The findings were as follows: (1) M. bovis BCG infection elevated the percentages of IL-35-producing B cells, a novel subset of regulatory B cells, and promoted their infiltration into the lung tissue. Also, anti-TB chemotherapy control of BCG infections could reverse the counts of IL-35-producing B cells in the peripheral blood and lung tissue. Elevated counts of IL-35-producing B cells negatively correlated with the bactericidal ability in mycobacterial infection. (2) Exogenous IL-35 treatment could induce the elevation in the counts of IL-35-producing B cells and promote their infiltration into the lung tissue, further downregulating the infiltration of anti-tuberculosis effector cell Th1/Th17 and upregulating the infiltration of Foxp3 + regulatory T cells into the lung tissue.
More recently, the counts of IL-35-producing B cells were found to increase in the peripheral blood and tuberculosis granuloma in patients with active TB 33 . IL-35 + CD20 + B cells were found within the tuberculous granuloma of ATB patients by Immunohistochemical (IHC) and immunofluorescense (IF) staining and the Figure 6. BCG infection increased IL-10 production in IL-35-producing B cells. The production of IL-10 in B cells was determined by intracellular cytokine flow cytometry. PBMCs or LMC was extracted from BCGinfected mice (n = 10). Then, the frequencies of IL-10-producing in total B cells or in CD19 + p35 + _B cells were analyzed by flow cytometry. The gating strategy is shown in Figure S3 27,43 . Furthermore, not only the heterodimer of IL-35 but its subunit p35 or Ebi3 possesses the anti-inflammatory capacity [44][45][46] . Recombinant IL-35 can stimulate Breg cells to secrete IL-10 and IL-35 26,27 . Interestingly, Recombinant IL-35 stimulation can dampene cytotoxicity and interferon-γ production in both direct and indirect contact co-culture systems in viral hepatitis-induced acute-on-chronic liver failure 47 . For further understanding the role of IL-35-producing B cells in mycobacterial infection, exogenous recombinant IL-35 was used in this study to treat BCG-infected mice, and its effects were validated. Treatment with exogenous IL-35 could induce more IL-35-producing B cells in the peripheral blood and lung tissue. Moreover, about 22% of B cells co-expressing p35 and Ebi3 existed in the lung tissue in BCG-infected mice treated with IL-35, which was much higher than that in mice without IL-35 treatment (about 14%). This suggested that exogenous IL-35 treatment not only caused the elevation in the counts of IL-35-producing B cells but also promoted their infiltration into the lung tissue. These results were also consistent with the recent finding of the prominent infiltration of IL-35-producing B cells into the TB granuloma of patients with ATB. These results might confirm, to some extent, that IL-35-producing B cells possessed autocrine or paracrine characteristics to induce the proliferation of IL-35-producing B cells.
Then, experiments were conducted to analyze the relationship between IL-35-producing B cells and T cells. Concomitant with the elevation in the counts of IL-35-producing B cells in the peripheral blood and lung tissue, the frequencies of IFN-γ-producing CD4 + T cells (Th1) and IL-17-producing CD4 + T cells (Th17) decreased. Reciprocally, the frequency of Foxp3 + Treg cells increased in the peripheral blood and lung tissue. In addition, the frequency of IL-35-producing B cells displayed a negative correlation withTh1/Th17, but a positive correlation with Treg.
The present study showed that the frequencies of IL-10-positive B cells in BCG-infected mice were much higher than those in controls and the frequencies of IL-10-positive B cells in IL-35-producing B cells were much higher than those in IL-35-negative B cells both in BCG-infected mice and in controls (Fig. 6D,E). Of note, BCG infection caused more IL-10 production in IL-35-producing B cells than in controls, displaying that the frequency of IL-10-positive B cells in IL-35-producing B cells was much higher than that in controls (Fig. 6F). IL-10-producing Breg cells constitute a major population of B cells that is associated with the development of autoimmune diseases and cancers [17][18][19][20][21] . A previous study also found that IL-35 negatively regulated the development of autoimmune diseases by inducing the generation and expansion of IL-10-producing B cells 27 . During www.nature.com/scientificreports/ Salmonella infection and EAE, IL-35-and IL-10-producing B cells were the main source of B-cell-derived IL-35 and IL-10 26 . Prominent IL-10 + B cells have been found in patients with active TB 48, and M. tuberculosis mannosecapped lipoarabinomannan induces IL-10-producing B cells and hinders CD4 + Th1 immunity 25 . A previous study demonstrated that IL-35-producing B cells from patients with active TB possessed a stronger ability to produce IL-10 33 . Thus, the data further suggest that BCG infection induced high levels of IL-10 production in IL-35-producing B cells which might be the mechanism underlying the downregulation of immunity against mycobacterial infection. The present study expanded previously undescribed findings in a Mycobacterium-infected mouse model to confirm the results of a recent study on patients with active TB showing the elevation in the counts of IL-35-producing B cells in the blood and their infiltration into TB lesion tissue. The findings also extended the observations that elevated counts of IL-35-producing B cells downregulated the bactericidal activity in mycobacterial infection in vivo. The underlying mechanisms might be associated with the autocrine or paracrine characteristic of IL-35-producing B cells to secrete high levels of IL-35 and IL-10, and also their tendency to inhibit the function of effector Th1/Th17 cells and promote the function of regulatory Foxp3 + Treg cells. However, the underlying mechanisms discussed in the present study were deduced from the association between IL-35-producing B cells and effector/regulatory anti-tuberculosis T cells. Further studies should be performed with knockout mice or using siRNA assay to silence the expression of IL-35, so as to clarify whether the absence of IL-35-producing B cells could be important to TB progression.