ORIGINAL RESEARCH published: 27 January 2022 doi: 10.3389/fimmu.2022.750068 Pleiotropic Effect of IL-6 Produced by B-Lymphocytes During Early Phases of Adaptive Immune Responses Against TB Infection Irina Linge 1*, Anastasiya Tsareva 1†, Elena Kondratieva 1, Alexander Dyatlov 1†, Juan Hidalgo 2, Ruslan Zvartsev 3 and Alexander Apt 1* 1Laboratory for Immunogenetics, Department of Immunology, Central Institute for Tuberculosis, Moscow, Russia, 2Department of Cellular Biology, Physiology and Immunology, Faculty of Biosciences, Institute of Neurosciences, Universitat Autònoma de Barcelona, Barcelona, Spain, 3 Center for Precision Genome Editing and Genetic Technologies for Edited by: Biomedicine, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia Nathan Peters, University of Calgary, Canada Reviewed by: The role of B cells migrating to the lung and forming follicles during tuberculosis (TB) Giovanni Delogu, inflammation is still the subject of debate. In addition to their antibody production and Catholic University of the antigen-presenting functions, B cells secrete different cytokines and chemokines, thus Sacred Heart, Italy Björn Corleis, participating in complex networks of innate and adaptive immunity. Importantly, lung B- Friedrich-Loeffler-Institute, Germany cells produce high amounts of the pleiotropic gp130 cytokine IL-6. Its role during TB *Correspondence: infection remains controversial, partly due to the fact that IL-6 is produced by different cell Alexander Apt alexapt0151@gmail.com types. To investigate the impact of IL-6 produced by B cells on TB susceptibility and Irina Linge immune responses, we established a mouse strain with specific IL-6 deficiency in B cells iralinge@gmail.com (CD19cre-IL-6fl/fl, B-IL-6KO) on the B6 genetic background. Selective abrogation of IL-6 † Present address: in B cells resulted in shortening the lifespan of TB-infected B-IL-6KO mice compare to the Anastasiya Tsareva, BostonGene, Moscow, Russia wild-type controls. We provide evidence that at the initial TB stages B cells serve as a Alexander Dyatlov, critical source of IL-6. In the lung, the effect of IL-6 deficiency in B cells is associated rather NPO Petrovax Pharm LLC, Moscow, Russia with B and T cell functioning, than with macrophage polarization. TB-infected B-IL-6KO mice displayed diminished sizes of B cells themselves, CD4+IFN-g+, Th17+, and Specialty section: CD4+CXCR5+ follicular T cell populations. The pleiotropic effect of B-cell-derived IL-6 This article was submitted to Microbial Immunology, on T-cells demonstrated in our study bridges two major lymphocyte populations and a section of the journal sheds some light on B- and T-cells interactions during the stage of anti-TB response when Frontiers in Immunology the host switches on a plethora of acquired immune reactions. Received: 30 July 2021 Accepted: 10 January 2022 Keywords: tuberculosis, B-cells, IL-6, lung inflammation, T cells Published: 27 January 2022 Citation: Linge I, Tsareva A, Kondratieva E, INTRODUCTION Dyatlov A, Hidalgo J, Zvartsev R and Apt A (2022) Pleiotropic Effect of IL-6 Tuberculosis (TB) remains a global health problem, affecting about 10 million people and claiming Produced by B-Lymphocytes During Early Phases of Adaptive Immune about 1.2 – 1.5 million lives annually (1). Macrophages and T cells are traditionally considered as Responses Against TB Infection. the main protective cells during anti-TB immune responses (2, 3), while the role of B-cells, whose Front. Immunol. 13:750068. prominent feature is the formation of B-cell follicles (BCFs) in the tuberculous lung in close vicinity doi: 10.3389/fimmu.2022.750068 to TB granuloma, is still the subject of debates (4–6). In addition to antibody production, B cells Frontiers in Immunology | www.frontiersin.org 1 January 2022 | Volume 13 | Article 750068 Linge et al. B-Cell Derived IL-6 in Tuberculosis apparently play many different roles in anti-TB response by MATERIALS AND METHODS producing cytokines/chemokines, serving as antigen-presenting cells, and interacting with other immune cells during lung Mice inflammation [reviewed (4, 6, 7)]. Regarding B-cell cytokine Mice of the C57BL/6JCit (B6) strain (hereafter – WT) were bred production, it was shown in animal models that B cells from TB- under conventional, non-SPF conditions at the Animal Facilities affected lung secrete almost no IL-2, high amounts of IL-6, IL-11, of the Central Institute for Tuberculosis (Moscow, Russia), in and IL-17, and low amounts of IL-10, IFN-g, and TNF-a (8, 9). accordance with the guidelines from the Russian Ministry of Although produced in low amounts, TNF-a from B cells is Health # 755, US Office of Laboratory Animal Welfare (OLAW) involved in the formation of lung BCFs in TB-infected mice (10). Assurance #A5502-11. Water and food were provided ad labium. Both in mice and TB patients, it was demonstrated that B cells Female mice 10-12 weeks of age at the beginning of experiments display a STAT1-centered gene expression signature, produce type were used. All experimental procedures were approved by the 1 interferons (11) and secrete IL-35 (12, 13). Regarding TB Institutional Animal Care and Use Committee (IACUC), pathophysiology, it was emphasized that type 1 interferons protocols 2, 3, 7, 10 of March 6, 2019. polarize macrophages toward the anti-inflammatory M2 Mice with ablation of IL-6 in B cells (hereafter – B-IL-6KO) phenotype (11), whilst IL-35 production by B cells is associated were generated by crossing IL-6flox/flox mice (23) with CD19-Cre with down-regulated Th1/Th17 response and an increase of the knock-in mice (CD19cre/cre), which, together with mice with FoxP3+ Treg population (13). Overall, the physiology of B-cell- systemic IL-6 knockout (IL-6KO) were kindly provided by Prof. secreted cytokines in TB infection is only superficially characterized. S. A. Nedospasov. To generate B-IL-6KO mice, parental IL-6flox/ flox In particular, specific activities of IL-6 produced by B-cells remain and CD19cre/cre strains were mated, and IL-6flox/-CD19cre/- obscure despite its abundance in TB-infected lungs (8, 9). heterozygous littermates were backcrossed to the parental IL- IL-6 is a pleiotropic gp130 cytokine that is involved in several 6flox/flox strain. After PCR genotyping, females bearing the IL- immune reactions and displays pro- and anti-inflammatory 6 fl o x / fl o x CD19 c r e / - genotype were selected for further activity depending upon its cellular sources and the phase of experimentation. Mice were genotyped by the genomic PCR the inflammatory process (14). IL-6 is produced by a variety of using tail DNA. Primers for the CD19-Cre transgene CD19.8 5′- cells, including T- and B-cells, macrophages, and CD11c+ cells. AATGTTGTGCTGCCATGCCTC-3′, CD19.9 5’- GTCTGAA Infection of mice with systemic il6 gene knockout (KO) with a GCATTCCACCGGAA-3’; lck2 5’- AATGTTGCTGGATAG high dose of virulent M. tuberculosis via intravenous (i. v.) route TTTTTACTGC-3’; primers for the IL-6flox transgene F: 5’- demonstrated a markedly elevated susceptibility, decreased CCCACCAAGAACGATAGTCA-3’; R1: 5’- GGTATCCTC IFN-g, and increased IL-4 production compared to the wild TGTGAAGTCTCC-3’; R2: 5’-AGCACTTTATTGGGCTC type (WT) mice (15). On the other hand, aerosol infection in a TATACA-3’; primers for the IL-6KO F: 5’- ACCGCTATGAA similar experimental setting showed much milder differences in GTTCCTCTC-3’; R 5’- CCAACCAACCCTACCTAGA-3’. the disease progression between KO and control mice. In addition, although primary IFN-g production in IL-6KO mice Infection was diminished, mycobacteria-specific memory T cell response Mice were infected with ~102 CFU of virulent M. tuberculosis remained intact (16). Summarizing, a complete abrogation of IL- strain H37Rv (sub-strain Pasteur) using the inhalation Exposure 6-dependent immune reactions during TB course looks rather System (Glas-Col, Terre Haute, IN) exactly as previously detrimental, and in patients with pulmonary TB IL-6 levels in described (24). At different time points post challenge (as sera are elevated compared to healthy donors (17, 18). indicated in Figure 1), mice were euthanized by the thiopental Despite insufficient information available, it is reasonable to (Biochemie GmbH, Vienna, Austria) overdose. assume that the role of IL-6 produced by different cell types in TB pathogenesis is different, as it is firmly established for another CFU Counts and Survival Time key inflammatory cytokine associated with anti-TB response, At indicated time points, spleens were homogenized in 2.0 ml of TNF-a (19). Among scattered observations, it is worth sterile saline and identical lobes of the right lungs from mentioning that M. tuberculosis-infected macrophages produce individual mice were digested in collagenase-DNase 1 mixture IL-6, which inhibits responsiveness to IFN-g of non-infected as described below, except the presence of antibiotics. 10-fold macrophages (20). In the context of the present study it should serial dilutions of 0.2 ml samples were plated on Dubos agar be mentioned that IL-6 has a prominent role in CXCR5+ T (Difco) and incubated at 37°C for 20–22 days before CFU were follicular helper (Tfh) cell differentiation (21), which, in turn, is counted. Survival time was monitored daily starting month 2 important for “correct” co-localization of T cells and post-infection. macrophages within TB foci, providing an optimal anti- mycobacterial response (22). Lung Cell Suspensions Here, using a mouse strain bearing B-cell-specific knockout Two upper right lung lobes from individual mice were mutation in the gene for IL-6 on B6 genetic background, we enzymatically digested exactly as described previously (25). evaluated in the aerosol mouse TB model the contribution of IL- Two bottom right lobes were used for RNA purification. 6 produced by B cells to infection severity/susceptibility in Briefly, blood vessels were washed out by perfusion with 0.02% general and some parameters of cellular immunity in particular. EDTA-PBS through the right ventricle and cut vena cava, lungs Frontiers in Immunology | www.frontiersin.org 2 January 2022 | Volume 13 | Article 750068 Linge et al. B-Cell Derived IL-6 in Tuberculosis BioLegend, dilution 1:100), CD19-AF647 (6D5, BioLegend, dilution 1:400), B220-APC (RA3-6B2, BioLegend, dilution A 1:400), F4/80-AF-488 (BM8, BioLegend, dilution 1:200), Ly6G- PE (1A8, BD Biosciences, dilution 1:200), CD44-FITC (IM7, BioLegend, dilution 1:150), CD62L-PE (MEL-14, BioLegend, dilution 1:200), CD11b-biotine (M1/70, dilution 1:100) with subsequent SAv-PerCP (BioLegend, dilution 1:500) staining. For intracellular staining of the lung cells, BD Fixation/ Permeablization Kit (BD Biosciences) was used due to the recommendations. Briefly, after 16-17 hours of in vitro B C cultivation in the presence of mycobacterial antigens, cells were harvested, stained first for surface antigens, then fixed with Cytofix/cytoperm buffer for 30 minutes and then stained for intracellular cytokines with IFN-g-PE (BD Biosciences dilution 1:150) and IL-17-PerCp (TC11-18H10.1, BioLegend, dilution 1:150) antibodies. Results are presented as mean ± SD. Lung Pathology and Immunohistochemistry Left lungs were frozen at the regimen of –20°C to –60°C FIGURE 1 | Mice lacking il6 expression in all cells (IL-6KO), or in B-cells (B- temperature gradient in the electronic Cryotome IL-6KO), have a shorter life span after TB infection compared to control (ThermoShandon, UK), and serial 10mm-thick sections were animals. (A) Survival time of mice of the three strains after aerosol infection made across the widest area. For visualization of lung with M. tuberculosis H37Rv, ~100 CFU per mouse. Data from two identical pathology, sections were fixed with ice-cold acetone and experiments were summarized, N = 10-25 mice for groups, log-rank test. (B, C) Mycobacterial counts in lungs and spleens of infected WT and B-IL- stained with hematoxylin-eosin. For IHC, lung cryosections 6KO mice at 3, 7 and 11 weeks post infection. Data from one of two similar were fixed with 1% PFA, blocked with 10% donkey serum, and experiments are displayed as the mean ± SD, 4-5 mice per group per time- incubated with rabbit anti-mouse primary anti-iNOS point. No significant differences between the groups. (NovusBio) pAB, rat anti-mouse primary B220 (IgG2a, clone RA3-6B2, eBioscience), rabbit anti-mouse primary anti-ARG removed, sliced into 1-2 mm3 pieces, and incubated at 37°C for pAB (NovusBio), rabbit anti-mouse primary anti-CD3 (clone 90 min in supplemented RPMI-1640 containing 200 U/ml SP7, ThermoScientific) for 1 h at room temperature. Afterwards, collagenase and 50 U/ml DNase-I (Sigma, MO). Single-cell preparations were incubated with secondary donkey anti-rabbit suspensions from 4 to 5 mice were obtained individually, IgG-Cy3, donkey anti-rat IgG-AF488 or anti-rabbit IgG-AF488 washed twice in HBSS containing 2% FCS and antibiotics, and pAB for 1 h at the room temperature. Slides were preserved using 3 x 105 cells per sample were used for surface phenotyping. 1 x ProLong Gold anti-fade reagent with DAPI (Invitrogen-Life 106 lung cells from each mouse were cultured overnight (16-17 Technologies) before visualization using the Zeiss Axioskop40 hours total) in the absence (control) or in the presence of 10µg/ microscope and AxioCam MRc5 AxioVisio 4 camera (Carl Zeiss, ml mixture of mycobacterial antigens (cultural filtrate was kindly Berlin, Germany). provided by Dr. Vadim Avdienko, CIT) and GolgiPlug (BD Bioscience) for the last 12 hours of incubation for further RNA Purification, cDNA Synthesis and evaluation of intracellular cytokines. Cultural filtrate was Gene Expression Evaluation obtained by culturing M. tuberculosis as a biofilm on the Identical lobes of the right lungs from 4-5 individual mice per surface of the protein-free Sauton’s medium for 4 weeks, group per time point were isolated and immediately put into followed by precipitation of mycobacteria at 3,000g for 20 min, RNA lysis buffer. Total RNA was isolated using the commercial concentration of the protein content in the liquid phase using SV Total RNA Isolation System (Promega). Reverse Amicon cell device, and bringing the final protein concentration transcription of mRNA was performed using oligo(dT) to 1mg/ml with sterile saline. primers, dNTP mix, M-MLV RT and RNasin® (Promega). Quantitative real-time RT-PCR (qPCR) of cDNA was Flow Cytometry performed using qPCRmix-HS SYBR (Evrogen) and cfx-96 Single-cell suspensions were obtained from individual mice and Real-Time PCR Detection System (BioRad). The following cell phenotypes were analyzed by flow cytometry as described primers were used: previously (25) using the FACS Calibur machines (BD hprt: F 5′-GTGATTAGCGATGATGAACCAG-3′, Biosciences). The gating strategy is displayed in Figure S1. The following labeled monoclonal antibodies were used: CD4-PerCp R 5′- CAAGTCTTTCAGTCCTGTCCA-3’; (dilution 1:300), CD4-AF488 (dilution 1:200), or CD4-PE ifng: F 5′-CATAAGCGTCATTGAATCACAC-3′, (dilution 1:200) (all GK1.5, BioLegend), CXCR5-Fitc (L138D7, R 5′-GGTTGTTGACCTCAAACTTGG-3’; Frontiers in Immunology | www.frontiersin.org 3 January 2022 | Volume 13 | Article 750068 Linge et al. B-Cell Derived IL-6 in Tuberculosis il6: F 5′-TCTATACCACTTCACAAGTCG-3′, influence of KO mutations on lung (Figure 1B) and spleen R 5′-TAGGCAAATTTCCTGATTATATCCA-3’; (Figure 1C) CFU counts was observed, suggesting that a more il11: F 5′-ACATGAACTGTGTTTGTCGC-3′, rapid mortality in IL-6-deficient animals was not due to higher mycobacterial loads in their organs. R 5′-ATCGGGTTAGGAGAACAGC-3′; il17a: F 5′-CCAGAATGTGAAGGTCAACC-3′, R 5′-TTCATTGCGGTGGAGAGTC-3’; IL-6 Deficiency in B Cells Alters the Early Mycobacteria-Specific il21: F 5′-AAACTCAAGCCATCAAACCCT-3′, Response of CD4+ T Cells R 5′-AGGAAGGGCATTTAGCTATGTG-3; Production of IFN-g by CD4+ T cells is considered critical for inos: F 5’-GGCAGCCTGTGAGACCTTTG -3’, protective immunity against M. tuberculosis (2). Since there is R 5’-GCATTGGAAGTGAAGCGTTTC -3’; evidence that in IL-6KO mice general production of IFN-g is arg1: F 5’-GGAATCTGCATGGGCAACCTGTGT-3’, decreased (15, 16), we evaluated how the B-cell-produced IL-6 R 5’-AGGGTCTACGTCTCGCAAGCCA-3’; influences IFN-g production in the lungs. After culturing overnight in vitro in the presence of mycobacterial antigens, fizz: F 5’-TTGCCAATCCAGCTAACTATCCC-3’, IFN-g response in CD4+ T lung cells from infected WT and B-IL- R 5’-CTCCCAAGATCCACAGGCAA-3’; 6KO mice was assessed by flow cytometry (intracellular ym1: F 5’-CTGAAAGACAAGAACACTGAGC-3’, staining). Additionally, we evaluated the overall expression R 5’-ATGGCACTGAACGGGGC-3’; level of the ifng gene in the lung tissue by qPCR. The stat1: F 5’-TGAGACTGTTGGTGAAATTGC-3’, proportion of CD4 + T cells infiltrating lungs increased R 5’-CTTCCGAAATCCTTTAACTGTG-3’ significantly during the course of infection in mice of both strains, and their percentage did not differ between strains at stat3: F 5′-AACTTAATGAAGAGTGCCTTCG-3′, weeks 3 and 7 post-infection (Figure 2A). However, in B-IL- R 5′-TCAACTCAGGAAATTTGACCAG-3’ 6KO mice, at week 3 post-infection the proportion of IFN-g- positive lung CD4+ cells was significantly lower than in control Relative expression levels were calculated by normalizing animals (Figure 2B). In addition, at this time point the levels for genes of interest to that of hprt using the expression level of ifng gene in the lung was also decreased in 2–DDCt method. B-IL-6KO mice (Figure 2C). These differences disappeared by Statistical analysis was performed using GraphPadPrism7 the week 7 post challenge (Figure 2). software. Representative data from one of two identical IL-6 is important for the development and maintenance of a experiments are displayed, except survival curves for which balanced Th17 immune response (26), whose role in TB is combined results from two independent experiments were ambiguous, although several studies suggested the importance summarized. The log-rank test for survival and One- or Two- of Th17 population for the effective immune response against TB way Anova with Tukey post-test for multiple comparison for (27, 28). Given that in mice bearing either genetically disrupted other experiments were used. P < 0.05 was considered il6, or lacking the gp130 receptor in T cells, development of IL- statistically significant. 17-producing Th17 cells is decreased (29), we measured whether IL-6 deficiency in B-cells could affect the mycobacteria-specific Th17 population. Lung cells from mice infected for 3 and 7 weeks RESULTS were cultured overnight in the presence of mycobacterial antigens and IL-17 production by CD4+ T cells was assessed Both Systemic and Conditioned by flow cytometry. Even though the overall frequency of Deficiency in IL-6 Production Increases mycobacteria-specific CD4+IL-17+ cells was lower than 1%, the Severity of TB Infection CD4+IL-17+ population was significantly smaller in B-IL-6KO In early studies, it was shown that in mice with genetically mice at both time points (Figure 2D). However, the expression disrupted il6 gene in all cells (systemic IL-6KO) i. v. challenge level of the il17 gene in the lung was equal in the two mouse with virulent M. tuberculosis resulted in a more rapid mortality strains at week 3 or even slightly increased in B-IL-6KO mice at compared to the wild type mice (15). Similar work utilizing the week 7 post infection (Figure 2E). aerosol infection route reported an increase in the lung IL-6 is crucial for differentiation of follicular CXCR5+ Tfh, mycobacterial counts in IL-6KO mice; however, mortality was which are required for the “correct” co-localization of T cells and not assessed in this study (16). Thus, we evaluated the influence macrophages in the manner optimal for TB control (22). Given of systemic and B-cell-specific IL-6 deficiency on the most that during TB course B cells migrate to the lung and form BCFs prominent features of chronic mycobacterial infections, the life that possess all prominent features of BCFs of secondary lymphoid span of mice, and mycobacterial multiplication in their organs. organs, including the presence of CD4+CXCR5+ Tfh cells (22, 30– As shown in Figure 1A, both systemic and B-cell-restricted IL-6 32), we assumed that the absence of IL-6 in B cells may decrease elimination significantly decreased survival time of the infected the content of these regulatory T-cells during infection. Indeed, animals compared to controls, although selective IL-6 defect in both in lungs and spleens of B-IL-6KO infected mice the B-cells resulted in a less severe phenotype. Unexpectedly, no proportion of CXCR5+ CD4+ Tfh-cells was significantly lower Frontiers in Immunology | www.frontiersin.org 4 January 2022 | Volume 13 | Article 750068 Linge et al. B-Cell Derived IL-6 in Tuberculosis A B C D E F G H I FIGURE 2 | Characteristics of T-cell responses of M. tuberculosis-specific CD4+ T cells from B-IL-6KO and WT mice. (A) Frequency (%) of total CD4+ T cell population in the lung of IL-6B and WT mice, revealed by flow cytometry in 3 and 7 weeks post-infection. (B, D) Single cell lung suspensions were cultivated overnight in the presence or absence (control) of mycobacterial antigens and subsequent intracellular cytokines IFN-g (B) and IL-17 (D) produced by CD4+ T cells were evaluated by flow cytometry. (C, E, I) qPCR analysis of ifng, il17, and il21 mRNAs in lungs at week 3 and 7 post infection. Expression of each gene was normalized to hprt. (F–H) The amount of CD4+CXCR5+Tfh cells in lungs (F) and frequency within CD4+ population in spleens of infected (G) and non-infected (H) B-IL-6KO and WT animals were assessed by flow cytometry. Results are expressed as mean ± SD, 4-5 mice per group per time-point. The results from one of two similar experiments are displayed. *P < 0.05; **P < 0.01; ****P < 0.0001. than in their WT counterparts (Figures 2F, G). Moreover, even production in the lungs of infected mice. At week 3 post before infection, both total and B-cell-specific IL-6 deficiency challenge, B cells comprise not more than 10 percent of all resulted in a significantly diminished size of this important T- lung cells (Figure 3A). However, in B-IL-6KO mice the total cell population in spleen (Figure 2H, such an experiment is amount of il6 mRNA in the whole lung tissue appeared to be 10- technically infeasible with non-infected lung tissue). In addition, fold lower compared to WT mice, suggesting that B-cells are one at the early stage of infection lung cells of B-IL-6KO mice of the major sources of IL-6 in tuberculous lungs early after expressed significantly less il21 mRNA (Figure 2I). This gene challenge (Figure 3B). This ratio in the il6 gene expression was encodes IL-21, the cytokine intensively produced by Tfh cells retained at least up to week 7 post infection, although the overall (33, 34). expression level significantly decreased by this time point (Figure 3B), despite substantial growth of the lung B-cell B Cells Are an Important Source of IL-6 in content, which also partially depended upon B-cell-provided the Lungs IL-6 (Figure 3A). We limited our IL-6 measurements by the Since IL-6 may originate from many different cell sources, we total mRNA assessment in freshly isolated lung tissue since many next assessed what is the B cell input into the total IL-6 different types of lung cells produce this cytokine and require Frontiers in Immunology | www.frontiersin.org 5 January 2022 | Volume 13 | Article 750068 Linge et al. B-Cell Derived IL-6 in Tuberculosis A B C FIGURE 3 | Lung B-cells are a major source of IL-6 which regulates their accumulation and the expression of IL-6-dependent stat3 and IFN-g-dependent stat1 genes in the lung tissue at the early phase of infection. (A) The total percentile of CD19+ B-cells in the lungs of B-IL-6KO and WT mice at weeks 3 and 7 post infection, revealed by flow cytometry. (B, C) qPCR evaluation of the levels of il6, stat1 and stat3 mRNAs in the lung tissue at weeks 3 and 7 post infection. Gene expression was normalized to that of hprt. Results of one of two similar experiments are displayed as mean ± SD, 4-5 mice per group per time-point. *P < 0.05; **P < 0.01; ****P < 0.0001. different culture conditions for optimal responses. Thus, lung T- T -lymphocytes interact within the inflamed tissue. To this end, cells from mice infected with M. tuberculosis or Pseudomonas we performed the IHC staining of B- and T-cells residing within aeruginosa readily secrete IL-6, TNF-a and IFN-g after the zones of condensed and diffuse inflammation in infected stimulation in vitro with specific antigens in the presence of lungs of the WT and B-IL-6KO mice. As shown in Figures 4B, splenic APC, but the response is markedly inhibited by lung D, within the lymphocyte cuffs surrounding blood vessels and macrophage-produced prostaglandin E2 (our unpublished bronchi B- and T-lymphocytes were located in tight vicinity in observations). However, we expect to resolve these limitations mice of both strains, making possible their physical and by rigorous cell sorting and measure IL-6 production by different functional interactions, e.g., antigen presentation by B-cells. cells at the protein level in our further studies. However, within the zones of diffuse pneumonia such co- An important aspect of IFN-g- and IL-6-mediated signaling is localization (quite often – geminates) was substantially more activation of two transcription factors, Stat-1 and Stat3, frequent in the WT mice (Figure 4E, left). Furthermore, B-cells respectively (35, 36). We have measured the expression levels were practically lacking within the condensed inflammatory foci of corresponding genes and found that at week 3 of infection a in lung parenchyma of B-IL-6KO animals (Figure 4E, right). A decreased level of the ifng and il6 expression is indeed substantial increase of the area of T- and B-cell contacts in the accompanied by the lower expression of stat1 and stat3 in the WT mice possibly provide additional opportunities for the lungs of B-IL-6KO compare to the WT animals (Figure 3C). development of acquired immunity in the lung. Since we obtained a histological evidence that the B-cell- B-Cell-Specific IL-6 Deficiency and produced IL-6 is involved in early lung cells interaction after TB Lung Pathology challenge, we decided to check a possible influence of selective To find out whether the conditioned IL-6 knockout influences IL-6 deficiency on the expression of the gene for another member TB pathogenesis, we assessed lung histopathology in B-IL-6KO of the gp130 cytokine family, IL-11, highly expressed in TB and WT mice at weeks 3 and 7 post infection. There was little susceptible mice (37). This was prompted by our earlier in vivo difference between B-IL-6KO and WT mice regarding the observation that IL-11 regulates both self and IL-6 production in dynamics of TB inflammation. As shown in Figure S2, at week infected lungs (38). However, no such dual effect was observed 3 post infection TB foci displayed no regular structure, whilst at for B-cell-specific IL-6: the level of il11 gene expression was week 7 we observed the development of circled granulomata with similar in mice of the two strains (Figure S3). macrophage-occupied centers surrounded by lymphocyte cuffs in mice of both strains. However, synoptic pictures reflecting Macrophage Polarization ratios between the areas occupied by diffuse pneumonia, Infected B-IL-6KO and WT mice did not differ by the sizes of condensed TB foci, and unaffected breathing tissue differed neutrophil (Figure 5A) and macrophage (Figure 5B) between mice of the two strains. As shown in Figures 4A, C populations residing in their lungs. Since there is a growing and Figure S2, in the WT mice zones of diffuse inflammation body of evidence indicating that IL-6 participates in macrophage occupied a larger proportion of the lung tissue compare to the B- polarization toward the anti-inflammatory M2 phenotype (39– IL-6KO mice at week 3 post challenge. As this was an unexpected 42), which is less effective for TB protection (43, 44), we tested a observation, we decided to evaluate how lung-infiltrating B- and few parameters distinguishing M1 and M2 macrophages. Frontiers in Immunology | www.frontiersin.org 6 January 2022 | Volume 13 | Article 750068 Linge et al. B-Cell Derived IL-6 in Tuberculosis A B C D E FIGURE 4 | B-cell-specific IL-6 deficiency affects early lung pathology and co-localization of T- and B-lymphocytes in areas of diffuse inflammation. (A, C) – Representative pictures of lung pathology of WT (A) and B-IL-6KO (C) mice, 3 weeks post challenge. Arrows: blue – lymphocyte cuff near the bronchus; white – condensed inflammatory focus; yellow – diffuse infiltration. (B, D) – B- and T-cell co-localization within the areas indicated by arrows at (A, C) panels. Blue, white and yellow left-side bars correspond to the colors of the arrows at (A, C) panels. The right panels in (B, D) represent digital magnification of the squared areas, and white arrows point at co-localized B (green- stained) and T (red-stained) lymphocytes. (E) – Quantification of T-B cell contacts in diffuse (DA) and condensed areas (CA) of lung inflammation. Mean ± SD, counted per 10 fields. IHC staining of cryosections with a-B220 (for B cells) and a-CD3 (for T cells) antibodies. ** P < 0,01, ***P < 0,001. Between weeks 3 and 7 of infection, in the lungs of B-IL-6KO and strains (Figure 5E). We also tried to assess arginase expression in WT mice we observed a marked (~8-fold) and equal increase in lungs of mice of the two strains by IHC staining. As shown in expression of the inos gene (Figure 5С) encoding inducible NO Figure S5, both in WT and IL-6KO mice arginase-positive cells synthase (iNOS), the enzyme playing a pivotal role in the were present in the inflamed lung tissue in similar quantities. mycobacterial killing in mice (45) and a key marker of the M1 Thus, there was only weak evidence of the involvement of B-cell- macrophage subset. This was supported by IHC staining, derived IL-6 in M2 polarization, however, irrespective to the visualizing high amounts of iNOS in the central zones of TB presence or absence of IL-6 produced by B-cells, M1 pathway lung granulomata in mice of both strains (Figure 5D), whilst at was much more prominent during the early stage of week 3 post challenge iNOS-positive cells were almost TB infection. undetectable in the lung tissue and absent in intact lungs (Figure S4). Surprisingly, whilst in B-IL-6KO lungs the level of arg1 gene expression remained stable, it was significantly down- DISCUSSION regulated in the WT mice between weeks 3 and 7 (Figure 5C), despite a higher il6 gene expression level in their lungs at both IL-6 participates in host defense immune reactions against viral, time points. A more prominent production of arginase instead of parasitic, fungal and bacterial infections and primary inducible NO synthase is often regarded as a property of M2 immunodeficiency diseases involving IL-6 or its signaling macrophages. We observed a stable level of expression of the pathways confirm these observations. Application of IL-6 corresponding arg gene in B-IL-6KO mice, whilst in WT controls inhibitors in a clinical setting demonstrated an increased risk its expression sharply dropped and became significantly lower at of infections, including tuberculosis (46). However, the role of week 7 post challenge (Figure 5E). A suggestively higher IL-6 during TB in humans remains controversial. Few studies expression of the fizz gene involved in the M2 polarization revealed increased IL-6 concentrations in sera of patients with pathway was also observed in B-IL-6KO mice at week 7 post far-advanced pulmonary TB compared to healthy subjects (17) infection, while the expression of ym1 was equal in the two and elevated level of il6 expression in peripheral blood cells from Frontiers in Immunology | www.frontiersin.org 7 January 2022 | Volume 13 | Article 750068 Linge et al. B-Cell Derived IL-6 in Tuberculosis A B C D E FIGURE 5 | Macrophages from infected lungs display general M1-centered polarization only marginally modified by the absence of B-cell-derived IL-6. The content of Ly6G+ neutrophils (A) and F4/80+ macrophages (B) in the lungs of WT and B-IL-6KO mice, revealed by flow cytometry. (C) Elevation in the inos gene expression between weeks 3 and 7 post challenge, and (D) immune histochemical staining of iNOS-positive cells within TB granulomata at week 7 post infection. (E) qPCR measurements of arg1, fizz, and ym1 RNAs in the lung tissue at weeks 3 and 7 post infection. See the legend for Figure 3. *P < 0,05, **P < 0,01, ***P < 0,001, ****P < 0,0001. TB patients (18). Other research teams reported on either a Th17 induction. In the present study, we demonstrate that selective decrease in IL-6 levels in supernatants of the whole blood elimination of IL-6 from B-cells increases susceptibility to TB in cultures from patients with active compare to latent TB (47) or terms of the post-infection life span (Figure 1). However, no the lack of significant differences between these cohorts (48, 49). increase in mycobacterial CFU content in organs compare to IL-6 production by many different cells may potentially have control animals was found, in parallel with similar earlier different effects on inflammation, which requires the application of investigations (22, 29). The fact that susceptibility to TB, as mouse strains with cell type-specific KO mutations for il6 or its measured by mortality curves or CFU counts, not always receptor genes for experimental dissection of the effects of IL-6 correlate was reported previously (51). By now, there are no from diverse cell sources on different cells. Thus, TB infection in unambiguous explanations for the lack of correlation between mice with conditional gp130 KO in macrophages and neutrophils CFU and mortality data. was characterized by elevated levels of pro-inflammatory cytokines As mentioned above, B cells migrate into TB-infected lung and enhanced Th1 and Th17 immune responses, along with an tissue and form BCFs in close vicinity of granulomata. CD4+ T increased iNOS expression (50). However, these serious shifts in cells also migrate into the lung and are readily observed within immune responses have not enhanced the control of mycobacterial BCFs where they directly contact B-cells and proliferate (9, 52). growth, probably since gp130 is a common receptor for other Thus, we assessed whether the lack of IL-6 in B cells influences the cytokines, e. g., IL-11 and IL-27, and too many bystander establishment of mycobacteria-specific T cell responses. It regulatory pathways were altered in this experimental setting. An appeared that IL-6 deficiency in B cells leads to a general down- analogous approach was applied recently in the study of Ritter and regulation of the ifng gene expression in the lung and a decreased coauthors (29), who used CD4cregp130loxP/loxP mice for studying frequency of mycobacteria-specific CD4+IFN-g+ T cells at the early the role of IL-6 in Th17 induction during TB when signaling from stage of infection (Figure 2B). Interestingly, the frequency of IL-6 was abrogated in CD4+ T cells. Their data showed that gp130- CD4 + IL-17 + cells was also decreased in B-IL-6KO mice provided signaling in these cells was not absolutely essential for (Figure 2D). These results are in agreement with observations Frontiers in Immunology | www.frontiersin.org 8 January 2022 | Volume 13 | Article 750068 Linge et al. B-Cell Derived IL-6 in Tuberculosis made by Ritter et al. (28), who showed that the numbers of plethora of acquired immune reactions (2). More experimentation mycobacteria-specific Th17 cells in CD4cregp130loxP/loxP mice is needed to clarify its possible role during the late phases of were decreased. This suggests that both IL-6 from B cells and infection, on the background of severe lung pathology and IL-6/gp130 signaling in CD4 + T-cells are important for dysfunction. Nevertheless, pleiotropic effects of B-cell-derived IL- Th17 differentiation. 6 demonstrated in our study provide some explanations of how B- BCFs in the tuberculous lung possess all prominent features of and T-cells interact during the early phase of anti-TB responses. BCFs of secondary lymphoid organs, including the presence of CXCR5+ Tfh cells (22, 53). Since Tfh differentiation requires both IL-6 and IL-21 (21, 54), and the number of “TB BCFs” is reduced in DATA AVAILABILITY STATEMENT mice with systemic IL-6 knockout (22), we evaluated whether IL-6 exactly from B cells has an impact on Tfh content. As shown in The raw data supporting the conclusions of this article will be Figure 2, not only the content of CXCR5+ Tfh cells was decreased made available by the authors, without undue reservation. in infected B-IL-6KO compared to control mice, but in spleens of non-infected IL-6KO and B-IL-6KO animals, the numbers of CXCR5+ Tfh cells also were lower than in controls. This suggests ETHICS STATEMENT that IL-6 from B cells is required for general differentiation and maintenance of CXCR5+ Tfh cells. In addition, at week 3 post The animal study was reviewed and approved by Institutional infection the expression level of the il21 gene was lower in B-IL- Animal Care and Use Committee (IACUC), protocols 2, 3, 7, 10 6KO mice (Figure 2). Lymphocyte sources of IL-6, which triggers of March 6, 2019. IL-21 production by CD4+ T cells (55), include B-cells, NK cells, and CD8+ T cells (56). Our results show that IL-6 from B cells is an important factor for local Tfh formation and IL-21 production during the early phase of infection. AUTHOR CONTRIBUTIONS It is worth mentioning that along with the infection IL, AT, AD, and EK performed experiments. RZ assisted for progression (weeks 7 and 11) we observed a gradual decrease CD19cre-IL-6flox/flox mouse strain establishment and creation in the influence of IL-6 from B-cells onto T cell response against of primers for qPCR. JH established IL-6flox mouse strain. IL mycobacteria. Given that at week 3 post infection, despite an and AA designed experiments, discussed results and wrote the equal content of B-cells, the overall level of il6 expression in the manuscript. All authors contributed to the article and approved lung is ~10 fold lower in B-IL-6KO mice compared to controls, the submitted version. we may conclude that B-cells serve as a major source of IL-6 in the lung exactly at the early phase of infection. This correlates with the results obtained in nonhuman primates, showing that the depletion of B-cells prior to infection results in a decrease in FUNDING general lung IL-6 production (8). Our observation that IL-6 of the B-cell origin is requisite for B- and T-cell co-localization in This work was supported by the RFBR grant 19-04-00058 (to the lung zones distant from blood vessels and bronchi (Figure 4) AA). Maintenance of IL-6fl/fl and CD19-Cre mice was also suggests that at the early phase of infection IL-6 from B-cells supported by the grant 075-15-2019-1660 from the Ministry of plays an important role in the development of acquired anti-TB Science and Higher Education of the Russian Federation. JH immunity in the lung. However, it is fairly possible that when acknowledges Grant RTI2018-101105-B-100 from Ministerio de immune response is already activated and the overall IL-6 level Economı́ a y Competitividad and European Regional decreases, other sources of this cytokine are sufficient for Development Fund. The revision of the manuscript was supporting the ongoing antigen-specific T cell response and supported by Russian Science Foundation 22-25-00308 (to IL). may replace B-cell-produced IL-6. For example, it was reported in the context of TB at the murine type 2 diabetes background that NK-CD11c+ cells were the main source of IL-6 at month 6 ACKNOWLEDGMENTS post challenge (57). Lung B-cells mostly reside in follicles and interact with limited numbers of co-localized T-cells, whilst a We thank Prof. S.A. Nedospasov for providing IL-6flox, CD19cre larger part of the T-cell population is diffusely distributed in the and IL-6KO mice and Dr. V. Avdienko for providing lung tissue, where IL-6 from macrophages and CD11c+ antigen- mycobacterial cultural filtrate. presenting cells may serve as an alternative source. In addition, there is evidence that IL-6 production by T-cells themselves is supported by a positive autocrine regulatory loop (58). SUPPLEMENTARY MATERIAL Taken together, our results clearly indicate that the most prominent role of B-cell-derived IL-6 in anti-TB response The Supplementary Material for this article can be found online concerns the early phase of infection – more precisely, the third at: https://www.frontiersin.org/articles/10.3389/fimmu.2022. week of its development – when the host starts switching on a 750068/full#supplementary-material Frontiers in Immunology | www.frontiersin.org 9 January 2022 | Volume 13 | Article 750068 Linge et al. B-Cell Derived IL-6 in Tuberculosis REFERENCES 20. Nagabhushanam V, Solache A, Ting LM, Escaron CJ, Zhang JY, Ernst JD. 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Publisher’s Note: All claims expressed in this article are solely those of the authors Combined Analysis of IFN–g, IL–2, IL–5, IL–10, IL–1RA and MCP–1 in QFT and do not necessarily represent those of their affiliated organizations, or those of Supernatant Is Useful for Distinguishing Active Tuberculosis From Latent the publisher, the editors and the reviewers. Any product that may be evaluated in Infection. PloS One (2016) 11:e0152483. doi: 10.1371/JOURNAL. this article, or claim that may be made by its manufacturer, is not guaranteed or PONE.0152483 endorsed by the publisher. 49. Manna MP, Orlando V, Donni PL, Sireci G, di Carlo P, Cascio A, et al. Identification of Plasma Biomarkers for Discrimination Between Tuberculosis Copyright © 2022 Linge, Tsareva, Kondratieva, Dyatlov, Hidalgo, Zvartsev and Apt. Infection/Disease and Pulmonary non Tuberculosis Disease. PloS One (2018) This is an open-access article distributed under the terms of the Creative Commons 13:e0192664. doi: 10.1371/JOURNAL.PONE.0192664 Attribution License (CC BY). The use, distribution or reproduction in other forums is 50. Sodenkamp J, Behrends J, Förster I, Müller W, Ehlers S, Hölscher C. Gp130 on permitted, provided the original author(s) and the copyright owner(s) are credited and Macrophages/Granulocytes Modulates Inflammation During Experimental that the original publication in this journal is cited, in accordance with accepted Tuberculosis. Eur J Cell Biol (2011) 90:505–14. doi: 10.1016/J.EJCB. academic practice. No use, distribution or reproduction is permitted which does not 2010.10.010 comply with these terms. Frontiers in Immunology | www.frontiersin.org 11 January 2022 | Volume 13 | Article 750068