Sex-specific impact of B cell-derived IL-10 on tuberculosis resistance

Abstract

Introduction: Due to the historical dogma that host defense against intracellular pathogens is primarily mediated by cell-mediated immunity, B cells have long been considered unimportant in providing protection against Mycobacterium tuberculosis (Mtb) and remained understudied for decades. However, emerging evidence highlights the multifaceted role of B cells in tuberculosis (TB) immunity. B cells accumulate at the site of infection in both animal models and human TB patients, suggesting a potential link to protective immunity. Still, the diverse roles of B cells in TB immunity are still being unraveled. In addition to producing antibodies, B cells secrete a wide range of cytokines that can influence the local immune response. In this study, we focused on the relevance of interleukin 10 (IL-10)-secreting B cells in the long-term control of the Mtb Beijing strain HN878.

Methods: B cell-specific IL-10 expression was assessed in IL-10 transcriptional reporter (Vert-X) mice following Mtb infection. To investigate the role of B cell-derived IL-10 in TB immunity, both male and female mice with a targeted knockout of IL-10 in B cells (IL-10^flox/CD19^cre) were infected with Mtb HN878. Disease progression, control of bacterial replication, and immunological changes were monitored throughout the course of infection.

Results: B cells contribute to IL-10 production in the Mtb-infected lung in both sexes, with CD138⁺ plasma cells serving as the primary source of B cell-derived IL-10. Mice lacking B cell-derived IL-10 exhibited increased resistance to aerosol Mtb infection, demonstrated by a delayed onset of clinical symptoms and prolonged survival. Notably, this effect was significantly more pronounced in males compared to females, and was associated with male-specific immune alterations.

Conclusion: Our research highlights a previously unrecognized sex-specific regulatory role of B cell-derived IL-10 during Mtb infection.

Keywords: B cell-derived IL-10; HN878; mouse model; sex differences; tuberculosis.

Figure 1

B cells produce IL-10 after Mtb infection in males and females. (A) Experimental setup. To define IL-10 production in vivo, female and male IL-10 reporter mice (Vert-X) were infected via aerosol with Mtb HN878 and at the indicated time points, single-cell suspensions of lung tissues were analyzed for GFP expression by flow cytometry. (B) shows frequencies of IL10⁺ T cells (as combined CD4⁺CD45⁺ plus CD8⁺CD45⁺ cells). (C) shows frequencies and (D) total B cells numbers (defined as CD138⁺B220^var + CD138^-B220⁺) among IL10⁺CD45⁺ cells. (E) shows UMAP projection of plasma cells (CD138⁺B220^var), germinal center B cells (GL7⁺CD138^-B220⁺) and other B cell subtypes (CD138^-B220⁺) and (G) of IL-10 producing B cell subtypes in male and female lungs at day 84. (F, H) show frequencies of the respective cell subsets at different time points post Mtb infection. (I) shows the frequency of CD138⁺B220^var cells among IL-10⁺ B cells and (J) median fluorescence intensity (MFI) of IL-10 in IL-10 producing CD138⁺B220^var cells. (K) Representative image of a IL-10⁺CD138⁺B220^var cell of a female Vert-X mouse from day 42 p.i. (B, C, D, I, J) Each data point represents one mouse. Data were combined from 2 independent experiments; n = 5-10. Statistical analysis was performed by 2way ANOVA followed by Tukey’s multiple comparisons test. (E, G) Data were generated from 4 female and 5 male mice on day 84 p.i. from 1 representative out of 2 experiments. (F, H) Data are represented as the mean percentage of B cell subtypes to total B cells from 2 independent experiments; n=5-10.

Figure 2

Ablation of B cell derived IL-10 increases resistance of mice to Mtb infection. (A) Experimental setup. Female and male B cell IL-10 KO mice and their B cell IL-10 competent littermates were infected via aerosol with Mtb HN878 and analyzed at the indicated time points. (B) Survival of female (n=9) and male (n=9) B cell IL-10 KO mice and their female (n=5) and male (n=7) B cell IL-10 competent littermates. Survival stratified by sex is shown in (C) for females and (D) for males. CFU of the lung (E) and spleen (F) at day 82 p.i. and day 103 p.i. G) Representative micrographs of acid-fast stained lung sections from moribund female and male littermates and B cell IL-10 KO mice. Bar = 20 µm. Spider chart of cytokines (H) and chemokines (I) measured in lung homogenates from female and male littermates and B cell IL-10 KO mice at day 82 p.i. Concentrations of cytokines/chemokines are shown as pg/ml. IFN-γ (J) and IL-27 (K) in lung homogenates from female (orange) and male (blue) littermates and B cell IL-10 KO mice at day 82 p.i. (L) Correlation of IFN-γ and IL-27 levels as pooled data for littermates and B cell IL-10 KO mice for both sexes. (B–D) Each data point represents one mouse from one experiment. Statistical analysis was performed by log rank test. (E, F) Each data point represents one mouse from one representative experiment out of two (n = 4-7). Statistical analysis was performed by Student’s t-test. (H–L) Each data point represents one mouse from one representative experiment out of two (n = 3-6). Statistical analysis was performed by 2way ANOVA followed by Tukey’s multiple comparisons test. Correlation was calculated using Pearson correlation. LOD, limit of detection.

Figure 3

Ablation of B cell IL-10 increases cellular infiltration into the male lung. Female and male B cell IL-10 KO mice and B cell IL-10 competent littermates were infected via aerosol with Mtb HN878. Lungs were collected at day 82 and PFA-fixed, paraffin embedded tissue sections were stained with H&E. (A) Representative micrographs from one female and male mouse lung per genotype are shown. Bar = 1 mm. (B) Quantitative analysis of the area of lung inflammation shown in (A) segregated by sex and correlations with the levels of IFN-γ (C) and IL-27 (D). (E) Representative micrographs from lungs of one female and male mouse per group stained with antibodies to detect neutrophils (NE⁺) and macrophages (CD68⁺); Bar = 100 µm. (F, G) Quantitative analysis of immune cells as shown in (E). H) PCA of integrated datasets ( Supplementary Table S2 ). Datasets were standardized using the default algorithm in BioVinci and integrated into a PCA plot. (B–D, F–H) Each data point represents one mouse from one representative experiment out of two; n = 3-6. Statistical analysis was performed by 2way ANOVA followed by Tukey’s multiple comparisons test (B F, G). Correlation was calculated using Pearson correlation (C, D).

Figure 4

Ablation of B cell derived IL-10 leads to significant changes in immune response gene transcripts in a sex-specific manner. B cell IL-10 KO mice and their B cell IL-10 competent littermates were infected via aerosol with HN878. Lungs were collected at day 82 and PFA-fixed, paraffin embedded tissue sections were used to extract RNA. Gene expression was analyzed with the nCounter Host Response Panel by Nanostring technology. (A, B) Volcano plots of log2 fold change versus -log10 p-value of the differential gene expression between female (A) and male (B) B cell IL-10 KO mice and their B cell IL-10 competent littermates. Horizontal dot line represents p-value <0.05. Significantly upregulated genes in B cell IL-10 KO mice are represented as blue dots, significantly downregulated genes are red; n = 3/group. (C, D) Selected trend plots of pathway signatures for female and male littermates and B cell IL-10 KO mice. (E–H) Most significantly different pathway signatures are depicted for female and male B cell IL-10 KO mice and their B cell IL-10 competent littermates. Each data point represents one mouse from one experiment; n = 3/group. Mycobacteria-specific IgG (I), IgM (J) and IgA (K) titers were measured in lung homogenates at day 82 p.i. Each data point represents one mouse from one representative experiment out of two (n = 3-6). Statistical analysis was performed by Student’s t-Test.