The survival of B cells is compromised in kidney disease

Mice with kidney disease show defects in GC and antibody response

To define how B cells respond in kidney disease, we utilized a clinically relevant mouse model of chemically induced acute kidney disease (Aristolochic acid nephropathy or AAN), as described before^{30,31,32,33,34}. Briefly, C57BL/6 (WT) mice were either intra-peritoneally (i.p.) injected with a single dose of nephrotoxin Aristolochic acid I (AAI), AAII (chemical control for AAI) or PBS (control). Both AAI and AAII are the main components of the Aristolochia and Asarum spp. of plants and exhibit similar mutagenic and genotoxic properties due to their ability to form covalent DNA adducts. The molecular structures of AAI and AAII differ by only one methoxy group, which may have important implications for their toxic effects on kidney epithelial cells. Consequently, mice injected with AAI but not AAII develop renal fibrosis and kidney dysfunction, as evidenced by elevated concentrations of serum blood urea nitrogen (BUN) and uremic toxins^31,32,35.

AAN and control mice were i.p. immunized with a T-dependent model antigen NP-KLH in alum four days after AAI injection, a time point at which AAN mice start developing kidney disease and uremia (Fig. 1A and Fig. S1A, B)³⁰. The mice were assessed for B cell response in the spleen at day 12 post-immunization. AAN mice demonstrated a highly variable but comparable number of B cells in the spleen and bone marrow at day 12 post-immunization (Fig. 1B and Fig. S1C). Interestingly, flow cytometry analysis revealed a marked reduction in the percentage and absolute number of total and NP-specific GC B cells in the AAN spleen and renal lymph nodes (Fig. 1C, D and Fig. S1D). Immunofluorescence staining of spleen sections confirmed smaller GC within the B cell follicles in AAN mice (Fig. 1E). There was no difference in the size of GC between control or AAII-injected spleens. Moreover, AAN mice showed reduced GC response at day 21 post-immunization, indicating that GC formation was indeed diminished and not delayed in kidney disease (Fig. S1E). We observed a negative correlation between the serum BUN level and the percentage of GC B cells in AAN mice at day 12 post-immunization (Fig. S1F). These results indicate that GC formation is impaired in kidney disease during T-dependent immune response.

A Schematic diagram of the experimental design. C57BL/6 (WT) mice were i.p. injected with a single dose of AAI, AAII (7.5 mg/kg b.wt), or PBS (Ctrl). Mice were either immunized with NP-KLH in alum or left non-immunized 4 days post-AAI injection. At 12 days post-immunization, spleens were assessed for B B cells (liveB220⁺) [Non-immun: Ctrl (3), AAI (3), AAII (3); NP-KLH: Ctrl (9), AAI (9), AAII (9)] C total GC B cells (liveB220⁺GL7⁺CD95⁺) [Non-immun: Ctrl (3), AAI (2), AAII (2); NP-KLH: Ctrl (9), AAI (8), AAII (8)], D NP-specific GC B cells (liveB220⁺NP⁺GL7⁺CD95⁺) [NP-KLH: Ctrl (10), AAI (12)] by flow cytometry, and (E) GC formation (n = 3) by immunofluorescence staining of spleen section. Scale bar = 50 μm. Each dot represents individual mice and data are pooled from at least 2–3 independent experiments (B–D). Data expressed as Mean ± SD. Statistical analyses by One-way ANOVA (B, C) and two-sided t-test (D). C Ctrl vs. AAI ****P < 0.0001, AAI vs. AAII ***P = 0.0007 and Ctrl vs. AAI ****P < 0.0001, AAI vs. AAII **P = 0.0060. D *** P = 0.0008. ns: statistically not significant. Source data are provided as a Source Data file.

Impaired isotype switching, affinity maturation, TFh generation, and secondary B cell response in kidney disease

The GCs are important sites for isotype switching and affinity maturation during immune response^6,7. AAN mice exhibited a reduced percentage and absolute number of NP-specific switched IgG1⁺ B cells than control spleens (Fig. 2A and Fig. S2A). The serum was subjected to ELISA to detect IgG1 specific for high-affinity (NP4 or NP7) and low-affinity (NP27) antigens at days 12 and 21 post-immunization. AAI-injected mice showed diminished NP4 or NP7- but not NP27-specific IgG1 titers in the serum and consequently a lower NP4/NP27 ratio (day 12 post-immunization) and NP7/NP27 ratio (day 21 post-immunization) compared to control mice, reflective of a defective affinity maturation (Fig. 2B–D and Fig. S2B). Consistent with this defect, the ELISPOT assay showed a reduced number of NP4-specific IgG1-producing plasma cells in the spleen of AAI-injected mice (Fig. 2E)^6,7. The overall diminished B cell response in AAN mice cannot be attributed to increased glucocorticoids due to kidney disease since serum glucocorticoid levels were comparable between the groups (Fig. S2C). These data suggest that GC formation and affinity maturation were compromised in kidney disease.

C57BL/6 (WT) mice were i.p. injected with a single dose of AAI, AAII (7.5 mg/kg b.wt), or PBS (Ctrl). Mice were either immunized with NP-KLH in alum or left non-immunized (non-immun) 4 days post-AAI injection. At day 12 post-immunization, spleens were assessed for A NP-specific switched IgG1⁺ B cells (liveIgD⁻IgM⁻CD138⁻Gr1⁻B220⁺NP⁺IgG1⁺) by flow cytometry [Non-immun: Ctrl (3), AAI (3), AAII (3); NP-KLH: Ctrl (9), AAI (8), AAII (7)], B serum NP4 and NP27-specific IgG1 by ELISA [Non-immun: Ctrl (4), AAI (4), AAII (4); NP-KLH Ctrl (12), AAI (8), AAII (10)], and C NP4/NP27-specific IgG1 ratio was determined to measure antibody affinity maturation at day 12 post-immunization [NP-KLH: Ctrl (12), AAI (8), AAII (10)]. D NP7/NP27-specific IgG1 ratio was determined to measure antibody affinity maturation at day 21 post-immunization [NP-KLH: Ctrl (5), AAI (5), AAII (4)]. E NP4-specific antibody-secreting plasma cells generation in the spleen by ELISPOT assay [Non-immun: Ctrl (3), AAI (3), AAII (3); NP-KLH: Ctrl (7), AAI (7), AAII (6)]. F The frequency of TFh (liveCD4⁺CD44⁺CXCR5⁺PD1⁺) cells in the spleen was determined by flow cytometry at day 12 post-immunization [Non-immun: Ctrl (3), AAI (3), AAII (2); NP-KLH: Ctrl (8), AAI (7), AAII (6)]. AAN and control mice were primed with NP-KLH in alum followed by NP-KLH boost 36 days later. Primed only and boosted mice spleens were evaluated for G total GC (liveB220⁺GL7⁺CD95⁺) [Prime only: Ctrl (7), AAI (7), AAII (7); Prime-boost: Ctrl (10), AAI (14), AAII (8)], H NP-specific switched IgG1⁺ B cells (liveIgD⁻IgM⁻CD138⁻ Gr1⁻B220⁺NP⁺IgG1⁺) [Prime only: Ctrl (7), AAI (7), AAII (7); Prime-boost: Ctrl (9), AAI (12), AAII (7)], and I NP4-specific IgG1-producing plasma cells 6 days later [Prime only: Ctrl (7), AAI (7), AAII (7); Prime-boost: Ctrl (9), AAI (12), AAII (7)]. Each dot represents individual mice and data are pooled from at least 2–3 independent experiments (A–I). Data expressed as Mean ± SD. Statistical analyses by One-way ANOVA (A–I). A Ctrl vs. AAI ***P = 0.0007, AAI vs. AAII *P = 0.0252 and Ctrl vs. AAI ****P < 0.0001, AAI vs. AAII ****P < 0.0001. B Ctrl vs. AAI **P = 0.0069, AAI vs. AAII *P = 0.0247. C Ctrl vs. AAI ***P = 0.0004, AAI vs. AAII ***P = 0.0005. D Ctrl vs. AAI *P = 0.0565, AAI vs. AAII **P = 0.0063. E Ctrl vs. AAI *P = 0.020, AAI vs. AAII *P = 0.0299. F Ctrl vs. AAI ***P = 0.0007, AAI vs. AAII *P = 0.0276 and Ctrl vs. AAI **P = 0.0038. G Ctrl vs. AAI ****P < 0.0001, AAI vs. AAII ****P < 0.0001. H Ctrl vs. AAI **P = 0.0038, AAI vs. AAII **P = 0.0073. I Ctrl vs. AAI *P = 0.036, AAI vs. AAII *P = 0.0492. ns: statistically not significant. Source data are provided as a Source Data file.

T-dependent antibody response is initiated and sustained by continuous crosstalk between the activated B and T cells at the boundary of B/T cell zones and within the GC⁸. There was no difference in the frequencies of activated CD4⁺ and CD8⁺ T cells between AAN and control groups at day 12 post-immunization (Fig. S2D, E). However, AAN mice demonstrated a diminished percentage and absolute number of TFh cells compared to control spleens (Fig. 2F and Fig. S2F). This was accompanied by an increase in the number of T regulatory (Treg) cells in the AAI-injected mice (Fig. S2G). Several studies showed that the generation of TFh cells is initiated and driven by IL-6 produced by activated B cells^{36,37,38,39,40,41}. Hence, we measured ex vivo IL-6 production from the splenic B cells isolated from immunized AAN and control mice. The B cells purified from the spleen of AAI-injected mice produced diminished IL-6 than control mice following in vitro LPS stimulation, as assessed by intracellular cytokine staining (Fig. S2H).

We next evaluated secondary B cell response in mice with kidney disease. To achieve this objective, AAI-injected and control mice were primed with NP-KLH in alum followed by a boost with NP-KLH only at day 36 post-immunization. The mice were evaluated for B cell response six days later, a time point at which AAN mice demonstrated kidney dysfunction (Fig. S2I). AAN mice showed diminished secondary total GC formation compared to control mice (Fig. 2G). We also observed diminished NP-specific switched IgG1⁺ B cells and NP4-specific IgG1-producing plasma cells in the spleen of AAN mice (Fig. 2H, I). Overall, these results suggest that mice with kidney disease show impairment in secondary B cell response following prime-boost immunization.

Kidney dysfunction and not nephrotoxin impair GC formation and antibody response

To confirm the negative impact of kidney disease on B cell response (Fig. 1), we measured GC formation in AAN and control mice to another T-dependent antigen [2.5% antigen sheep red blood cells (SRBC)] (Fig. 3A and Fig. S3A, B). In line with the NP-KLH immunization, mice with kidney disease showed impaired GC and TFh cell formation following 2.5% SRBC injection (Fig. 3B, C).

A Schematic diagram of the experimental plan. AAN and control mice were i.p. immunized with 2.5% SRBC (n = 8) 4 days post-AAI injection. Seven days later, B total GC (liveB220⁺GL7⁺CD95⁺) [SRBC: Ctrl (8), AAI (8), AAII (8)], and C TFh (liveCD4⁺CD44⁺CXCR5⁺PD1⁺) cells were measured by flow cytometry [SRBC: Ctrl (8), AAI (8), AAII (8)]. AAN mice were either treated with probenecid (PRB + AAI) once on day 0 (relative to AAI injection) or left untreated. Control mice received probenecid only (PRB). Four days later, mice received NP-KLH immunization and assessed for D total GC B cells [PRB (9), AAI (7), PRB + AAI (9)], E TFh cells [PRB (9), AAI (10), PRB + AAI (9)], F NP-specific switched IgG1⁺ B cells (liveIgD⁻IgM⁻CD138⁻ Gr1⁻B220⁺ NP⁺IgG1⁺) by flow cytometry [PRB (9), AAI (7), PRB + AAI (9)], and G serum NP4-specific IgG1 level by ELISA [PRB (8), AAI (6), PRB + AAI (8)]. H Diagram of the experimental design. WT mice were either subjected to 5/6 nephrectomy (5/6 Nx) (n = 7) or sham-operated (Ctrl) (n = 8). Two weeks after surgery, mice were immunized with NP-KLH and assessed for I total GC [Ctrl (8), 5/6 Nx (7)], J TFh cells [Ctrl (8), 5/6 Nx (7)], K NP-specific switched IgG1⁺ B cells [Ctrl (8), 5/6 Nx (6)], L serum NP4 and NP27-specific IgG1 levels [Ctrl (8), 5/6 Nx (7)], and M NP4/NP27- IgG1 ratio to measure affinity maturation [Ctrl (8), 5/6 Nx (7)]. Each dot represents individual mice and data are pooled from at least 2–3 independent experiments (B–G and I–M). Data expressed as Mean ± SD. Statistical analyses by One-way ANOVA (B–G) and two-sided t-test (I–M). B Ctrl vs. AAI **** P < 0.0001, AAI vs. AAII ****P < 0.0001. C Ctrl vs. AAI **P = 0.0027, AAI vs. AAII *P = 0.0160. D PRB vs. AAI **P = 0.0018, AAI vs. PRB + AAI *P = 0.0472. E PRB vs. AAI *P = 0.0112, AAI vs. PRB + AAI **P = 0.0029. F PRB vs. AAI **P = 0.0012, AAI vs. PRB + AAI *P = 0.0415. G PRB vs. AAI * P = 0.0222, AAI vs. PRB + AAI *P = 0.0484. I **P = 0.0096. J *P = 0.0278. K *P = 0.0295. L **P = 0.0028. M **P = 0.0049. ns: statistically not significant. Source data are provided as a Source Data file.

To rule out the possibility of a direct effect of AAI on B cells, NP-KLH immunized AAN and control mice were treated with probenecid, an organic anion transporter inhibitor that prevents damage to kidney tubular epithelial cells and minimizes uremic toxin accumulation without neutralizing AAI, as shown before (Fig. S3C, D)^30,35. Interestingly, probenecid treatment of AAI-injected mice prevented the defect in GC and TFh cell generation (Fig. 3D, E). Moreover, probenecid-treated AAN mice showed an increased number of NP-specific switched IgG1⁺ B cells and NP4-specific serum IgG1 titer than untreated AAN mice, arguing against a direct effect of AAI on B cell response (Fig. 3F, G). To rule out the direct impact of AAI-induced kidney inflammation on B cell response, control, and AAN mice were immunized with NP-KLH ten days post-AAI injection. AAN mice showed a similar reduction in total and NP-specific GC response than control when immunized at a gap of ten days (Fig. S3E).

As an independent approach to confirm the role of kidney dysfunction in impaired B cell response, we assessed GC formation in a surgically induced mouse model of kidney disease following NP-KLH immunization. The model of 5/6 nephrectomy involves excision of the upper and lower poles of one kidney followed by nephrectomy of the converse kidney (Fig. 3H), as previously described³⁰. The kidneys of 5/6 nephrectomized mice demonstrated extensive fibrosis and dysfunction in comparison to sham-operated animals (Fig. S3F, G). The 5/6 nephrectomized animals exhibited impaired GC, TFh and NP-specific switched IgG1⁺ B cell generation compared to sham-operated animals (Fig. 3I–K). Consequently, there was reduced serum NP4-specific antibody formation and defect in antibody affinity maturation in 5/6 nephrectomized mice (Fig. 3L, M). There was no difference in the serum glucocorticoid level between 5/6 nephrectomized and sham-nephrectomized mice (Fig. S3H). These data support the notion that compromised B cell response is due to kidney dysfunction and not AAI’s impact on the immune system per se.

GC B cells from mice with kidney disease show increased apoptosis

To further understand how uremia impacts GC response, flow-sorted NP-specific GC B cells from kidney disease and control mice were subjected to RNA-Seq analysis (Fig. S4A). The principal component analysis plot demonstrated the variation between GC B cells from kidney disease and control mice as well as the similarity between the sample replicates (Fig. S4B). A number of genes were differentially regulated between the GC of mice with kidney disease and the control spleen (Fig. S4C). Fitting with the diminished GC B cell numbers in renal dysfunction, RNA-seq, and Reactome pathway database analysis revealed the downregulation of multiple cell cycle genes in GC B cells from mice with kidney disease (Fig. 4A, B). Accordingly, there was reduced expression of Ki67, a protein that marks all the cells that have entered the cell cycle, in GC B cells during kidney disease (Fig. 4C and Fig. S4D). Using short-pulsed BrdU labeling, we found that similar proportions of GC B cells from AAN and control mice were in the G2M phase, but significantly fewer uremic cells were in the S phase with an increase in G0-G1 than controls (Fig. 4D). Further, transcriptomic and KEGG pathway analysis revealed upregulation of multiple pro-apoptotic and downregulation of anti-apoptotic genes in the GC B cells of AAN mice (Fig. 4E, F). Consequently, NP-specific but not NP-non-specific GC B cells from AAI-injected mice showed increased active Caspase3 expression than control mice (Fig. 4G).

NP-specific GC B cells from the control and AAN spleen (n = 5) were flow sorted on day 12 post-immunization and subjected to RNA-Seq analysis. A Relative expression of significantly differentially expressed genes (q-value < 0.05) related to cell cycle, and B Over-representation pathway analysis of significantly downregulated genes in AAN GC B cells. C MFI of Ki67⁺ GC B cells [NP-KLH: Ctrl (5), AAI (7)]. D Cell cycle status of GC B cells was analyzed at day 12 post-immunization [NP-KLH: Ctrl (15), AAI (15)]. E Relative expression of significantly differentially expressed genes (q-value < 0.05) related to apoptosis and F Over-representation pathway analysis of upregulated genes in GC B cells from AAN mice. G NP⁺ and NP⁻ GC B cells staining for active caspase3 expression (n = 12–14) at day 12 post-immunization [NP-KLH: Ctrl (14), AAI (12)]. Relative expression of significantly differentially expressed genes (q-value < 0.05) related to H Light zone (LZ), and I Dark zone (DZ) GC B cells. J Number of LZ centrocytes (CXCR4^loCD86^hi) and DZ centroblasts (CXCR4^hiCD86^lo) at day 12 post-immunization [NP-KLH: Ctrl (10), AAI (12)]. Each dot represents individual mice and data pooled from at least 2–3 experiments (C, D, G, and J). Data expressed as Mean ± SD. Statistical analyses by two-sided t-test (C, D, G, and J). C *P = 0.0228. D G0-G1 ***P = 0.0001, S ****P < 0.0001. G **P = 0.0023. J ****P < 0.0001 and ***P = 0.0003. Source data are provided as a Source Data file.

GC B cells are classified into centrocytes (CD86^hiCXCR4^lo) and centroblasts (CD86^loCXCR4^hi) based on morphologic criteria^6,7. Within a GC, centrocytes and centroblasts distribute preferentially in a centrocyte-rich light zone (LZ) and a centroblast-rich dark zone (DZ) and migrate extensively between the compartments. Centroblasts undergo vigorous proliferation, somatic hypermutation, and clonal expansion in the DZ⁴². In the LZ, depending on the outcome of BCR and T cell help-based selection, the centrocytes either undergo slow division or apoptosis. Interestingly, several genes identified by transcriptomic analysis to be increased in AAN GC B cells were positive regulators of centrocytes in the LZ (Fig. 4H). On the other hand, control GC B cells exhibited higher expression of genes that positively regulate centroblasts in the DZ (Fig. 4I). While control GC B cells demonstrated the phenotype of centroblasts, AAI-injected total, and NP-specific GC B cells showed increased accumulation in the LZ as centrocytes (Fig. 4J). Taken together, these results highlight a critical role for kidney dysfunction in cell cycle arrest, increased apoptosis, and impaired migratory capabilities of GC B cells.

Hippuric acid drives the loss of mitochondrial membrane potential and increased apoptosis of B cells

We next sought to define the mechanisms by which uremia contributes to increased apoptosis of GC B cells. Our transcriptomic analysis showed upregulation of multiple genes in AAN GC B cells related to loss of mitochondrial membrane potential (Fig. 5A). The loss of mitochondrial membrane potential and enhanced mitochondrial ROS production are the cell-intrinsic factors of apoptosis⁴³. TMRE staining of total GC B cells demonstrated increased loss of mitochondrial membrane potential in mice with kidney dysfunction (Fig. 5B and Fig. S5A). GC B cells from AAI-injected mice also exhibited a moderate increase in mitochondrial ROS production (Fig. 5C and Fig. S5B). These results indicate an essential role of kidney disease in causing loss of mitochondrial membrane potential in GC B cells.

A NP-specific GC B cells from the control and AAN spleen (n = 5) were flow sorted on day 12 post-immunization for RNA-Seq analysis. Relative expression of significantly differentially expressed genes (q-value < 0.05) related to mitochondrial membrane potential. At day 12 post-immunization, B mitochondrial membrane potential [NP = KLH: Ctrl (12), AAI (17)], and C mitoROS production [NP = KLH: Ctrl (12), AAI (15)] were measured in GC B cells by TMRE and MitoSox Red staining, respectively. D Schematic diagram of in vitro B cell assay. E WT resting splenic B cells (n = 5) ± uremic toxins were stimulated with αIgM/αCD40/IL-21 and the number of B220⁺CD138⁺ plasmablasts was determined by flow cytometry at 96 h. WT B cells ± αIgM/αCD40/IL-21 ± uremic toxins and F proliferation (CFSE dilution) of plasmablasts was assessed at 48 h (n = 3), G apoptosis (n = 6), and H mitochondrial membrane potential was measured in plasmablasts at 24 h (n = 4). I HA concentration in the serum of mice was measured by LC-MS/MS [Ctrl (5), AAI (6)]. J WT splenic B cells + αIgM/αCD40/IL-21 ± HA (35 μg/ml) were assessed for the loss of mitochondrial membrane potential in plasmablasts at 24 h. Each dot represents individual mice (B, C, and I) and 5 (E), 3 (F), 6 (G), and 4 (H, J) experiments. Data pooled from 2–3 experiments (B, C, and I). Data expressed as Mean ± SD. Statistical analyses by One-way ANOVA (E–H) and two-sided t-test (B, C, I, and J). B ****P < 0.0001. C **P = 0.0029. E NT vs. GBA ****P < 0.0001, NT vs. IAA **P = 0.0011, NT vs. PC *P = 0.0398, NT vs. PCS *P = 0.0307, NT vs. HA **P = 0.0059, NT vs. Urea **P = 0.0084. G Ctrl vs. HA **P = 0.0019. H Ctrl vs. HA **P = 0.0032. I **P = 0.0037. J ***P = 0.0004. ns: statistically not significant. Source data are provided as a Source Data file.

A previous study has described an in vitro system in which mouse naïve B cells undergo massive expansion and isotype switching and generate GC-like B cells⁴⁴. Although this is a unique and useful system for studying various aspects of GC biology, it truly does not depict in vivo GC reactions within the secondary lymphoid organs. To identify uremic toxin(s), previously linked to immune dysfunction in uremic patients, that could directly inhibit T cell-dependent B cell response, we utilized an in vitro system for B cell activation and their differentiation into plasmablasts^45,46,47,48. We note that AAN mice also showed a reduced number of extrafollicular plasmablasts (day 4 post-immunization) than control mice and lower serum titers of NP27-specific (low affinity) IgM (Fig. S5C, D). The resting splenic B cells were in vitro stimulated with αIgM/αCD40/IL-21 in the presence or absence of different uremic toxins (Fig. 5D). The uremic toxins were added to the culture at concentrations seen in patients with stage 4 and 5 kidney diseases¹². The cells were assessed for plasmablast (liveB220⁺CD138⁺ cells) differentiation by flow cytometry at 4 days post-stimulation (Fig. S5E). Out of total of 9 uremic metabolites tested, only γ-guanidino butyric acid (GBA), indole-3-acetic acid (IAA), para-cresol (PC), para-cresol sulfate (PCS), hippuric acid (HA) and urea (as control) inhibited plasmablast differentiation (Fig. 5E). Since PC is rapidly converted to PCS under in vivo conditions, we have used PCS instead of PC for subsequent experiments.

We selected the above-mentioned five uremic toxins and individually assessed their ability to negatively impact plasmablast proliferation and apoptosis. None of the five toxins inhibited plasmablast proliferation (Fig. 5F and Fig. S5F). Interestingly, only HA showed two-fold higher apoptosis of plasmablasts (Fig. 5G and S5G). The plasmablasts exhibited an increased loss of mitochondrial membrane potential in the presence of HA (Fig. 5H). NP-KLH immunized AAN mice demonstrated an elevated concentration of serum HA (mean value of serum HA is ∼35 μg/ml) than control animals at day 12 post-immunization (Fig. 5I). Resting splenic B cells stimulated with αIgM/αCD40/IL-21 in the presence or absence of 35 μg/ml of HA showed increased loss of mitochondrial membrane potential in plasmablasts than untreated control (Fig. 5J). Collectively, these results identify HA as one of the potential uremic toxins in driving the loss of mitochondrial membrane potential and increased apoptosis of B cells during kidney dysfunction.

HA drives GC B cell apoptosis via GPR109A

A recent study showed that HA binds to GPR109A (encoded by Niacr1 aka Hcar2 gene) in osteoclasts and regulates osteoclastogenesis and bone resorption in mice⁴⁹. GPR109A (aka niacin receptor 1) is a high-affinity GPCR for niacin and gut microbes-derived butyrate and β-hydroxybutyrate^50,51. The GPR109A signaling has been extensively studied in lipolysis, atherogenesis, and intestinal homeostasis^50,51. However, nothing is known about the role of GPR109A in B cells. The RT-qPCR analysis revealed that resting and αIgM/αCD40/IL-21 or lipopolysaccharide (LPS) activated splenic B cells express comparable levels of Niacr1 mRNA (Figs. 6A, S6A, B), which was lower than total splenocytes from WT mice. Moreover, FACS-sorted GC (liveB220⁺GL7⁺CD95⁺), non-GC (liveB220⁺GL7⁻CD95⁻), and total splenocytes from NP-KLH immunized WT mice showed Niacr1 transcript expression at day 12 post-immunization (Figs. 6B, S6C, D). NP-KLH immunized Niacr1^−/− mice served as a negative control. Intriguingly, GPR109A is not expressed in naïve human B cells isolated from blood (Fig. S6E). In contrast to mouse B cells, the GPR109A expression is upregulated in human B cells following in vitro stimulation with αIgM/sCD40L/IL-21 with the highest expression seen in the plasmablasts.

A Resting splenic B cells (n = 5–6) ± αIgM/αCD40/IL-21 or LPS were assessed for Niacr1 mRNA expression by RT-qPCR at 24 h. Total splenocytes from WT and Niacr1^−/− mice (n = 3) were used as positive and negative controls, respectively. B Niacr1 transcript expression was assessed by RT-qPCR on NP-KLH immunized and FACS-sorted non-GC (liveB220⁺GL7⁻CD95⁻), total GC (liveB220⁺GL7⁺CD95⁺) (2 mice pooled together) and total spleen cells from WT and Niacr1^−/− mice (n = 6) at day 12 post-immunization. C Purified total WT B cells from NP-KLH immunized mice (at day 12 post-immunization) were incubated with HA for six hours and assessed for the loss of mitochondrial membrane potential of GC B cells by TMRE staining (B220⁺GL7⁺) cells. B cells ± αIgM/αCD40/IL-21± (D and E) GSK256073/± (F) MMF and mitochondrial membrane potential and apoptosis of plasmablasts were measured. Immunized WT mice were i.p. injected with MMF or left untreated (Ctrl) every alternate day and evaluated for G total GC [Non-immun: Ctrl (4), MMF (4); NP-KLH: Ctrl (7), MMF (11)], H NP-specific GC [Non-immun: Ctrl (4), MMF (4); NP-KLH: Ctrl (7), MMF (11)], I serum NP7 and NP27-specific IgG1 titers and affinity maturation (NP7/NP27-IgG1 ratio) [Non-immun: Ctrl (3), MMF (3); NP-KLH: Ctrl (6), MMF (8)] and J loss of mitochondrial membrane potential of GC B cells [Non-immun: Ctrl (4), MMF (5); NP-KLH: Ctrl (8), MMF (13)]. K Resting splenic B cells (n = 3) from WT and Niacr1^−/− mice were stimulated with αIgM/αCD40/IL-21 in the presence or absence of HA. The B220⁺CD138⁺ cells were analyzed for the loss of mitochondrial membrane potential by TMRE staining 24 h later. Each dot represents individual mice (B and G–J) and individual experiments (A, C–F, and K). Data pooled from 2–3 independent experiments (B and G–J) and individual experiments A (5), C, D (8), E, F (7) and K (3). Data expressed as Mean ± SD. Statistical analyses by One-way ANOVA (A, B, and D–K) and two-sided t-test (C). A Resting vs. WT spleen **P = 0.0015, αIgM/αCD40/IL-21 vs. WT spleen **P = 0.0010, LPS vs WT spleen *P = 0.0523, WT spleen vs. Niacr1^−/− spleen ****P < 0.0001. B Non-GC B cells ***P = 0.0005, GC B cells ***P = 0.0004, Total Spleen ***P = 0.0002. C ***P = 0.0004 and ****P = 0.0005. D ****P < 0.0001. E ****P < 0.0001. F ***P = 0.0008. G ****P < 0.0001. H ***P = 0.0002. I **P = 0.0067 and NP7/NP27: *P = 0.0106. J **P = 0.0015. K **P = 0.0134. ns: statistically not significant. Source data are provided as a Source Data file.

Next, to define the role of HA/GPR109A signaling in GC B cell apoptosis, purified total splenic WT B cells from NP-KLH immunized mice were in vitro incubated with HA for 6 h and assessed for loss of mitochondrial membrane potential. In vitro, HA treatment diminished GC B cell number and induced increased loss of membrane potential in GC B cells, as evidenced by TMRE staining (Fig. 6C).

To analyze the function of GPR109A stimulation during B cell activation, αIgM/αCD40/IL-21 stimulated B cells were incubated in the presence or absence of two different GPR109A agonists [GSK256073 and Monomethyl fumarate (MMF)]^52,53. Interestingly, treatment with GPR109A agonists showed loss of mitochondrial membrane potential and increased apoptosis of plasmablasts in a dose-dependent manner (Fig. 6D–F and Fig. S6F).

Moreover, to assess the impact of GPR109A activation on GC B cells under in vivo conditions, mice were either treated with MMF or left untreated and then assessed for B cell response following immunization. MMF treatment diminished the number of total and NP-specific GC B cells and impaired affinity maturation (Fig. 6G–I). MMF-treated mice exhibited increased loss of mitochondrial membrane potential in GC B cells than the untreated group (Fig. 6J).

To further confirm that HA drives loss of mitochondrial membrane potential in B cells via GPR109A, αIgM/αCD40/IL-21 stimulated splenic B cells from WT and Niacr1^−/− mice were cultured in the presence or absence of HA and evaluated for the loss of mitochondrial membrane potential at 24 h post-stimulation. HA-induced loss of mitochondrial membrane potential in WT plasmablasts, a phenotype reversed in the absence of GPR109A in B cells (Fig. 6K). Collectively, these data indicate that HA-mediated activation of GPR109A contributes to the loss of mitochondrial membrane potential in B cells.

B cell-specific GPR109A expression limits humoral immune response in kidney disease

To test the function of GPR109A in impairing GC B cell response in the context of kidney disease, WT, and Niacr1^−/− mice were subjected to AAN and assessed for B cell response following NP-KLH immunization (Fig. 7A). WT and Niacr1^−/− mice demonstrated comparable kidney dysfunction following AAI injection (Fig. S7A). Both WT and Niacr1^−/− mice showed a similar percentage of the total and NP-specific GC B cells and antibody affinity maturation, indicating that GPR109A is dispensable for GC formation and affinity maturation in the control spleen (Fig. 7B–D). As expected, WT mice subjected to AAN showed a reduced number of GC B cells and a defect in affinity maturation. However, AAI-injected Niacr1^−/− mice showed no impairment in their GC response, as evidenced by a comparable percentage of total and NP-specific GC B cells and antibody affinity maturation.

A Diagram of the experimental design. WT and Niacr1^−/− mice (n = 8–10) were subjected to AAN followed by NP-KLH in alum immunization. B Total GC B cells (liveB220⁺GL7⁺CD95⁺) [NP-KLH: WT Ctrl (10), AAI (8); Niacr1^−/− Ctrl (9), AAI (10)], C NP-specific GC B cells (liveB220⁺NP⁺GL7⁺CD95⁺) by flow cytometry [NP-KLH: WT Ctrl (11), AAI (9); Niacr1^−/− Ctrl (11), AAI (10)], and D antibody affinity maturation as assessed by NP7/NP27-specific serum IgG1 ratio was evaluated at day 12 post-immunization [NP-KLH: WT Ctrl (6), AAI (4); Niacr1^−/− Ctrl (5), AAI (6)]. E Resting splenic B cells from WT or Niacr1^−/− mice were adoptively transferred into control or AAN μMT mice one day before AAI injection. Mice were immunized with NP-KLH in alum four days post-AAI-injection. At day 12 post-immunization, spleens were assessed for F total GC formation [NP-KLH: WT Ctrl (8), AAI (12); Niacr1^−/− Ctrl (10), AAI (10)], G NP-specific GC formation [NP-KLH: WT Ctrl (4), AAI (5); Niacr1^−/− Ctrl (5), AAI (6)], H proliferation [NP-KLH: WT Ctrl (4), AAI (5); Niacr1^−/− Ctrl (5), AAI (6)], I loss of mitochondrial membrane potential [NP-KLH: WT Ctrl (4), AAI (5); Niacr1^−/− Ctrl (5), AAI (6)], J mitochondrial ROS generation by total GC B cells [NP-KLH: WT Ctrl (4), AAI (5); Niacr1^−/− Ctrl (5), AAI (6)], K apoptotic NP-specific GC B cells by flow cytometry [NP-KLH: WT Ctrl (4), AAI (5); Niacr1^−/− Ctrl (5), AAI (6)], and L antibody affinity maturation as assessed by NP7/NP27-specific serum IgG1 ratio [NP-KLH: WT Ctrl (4), AAI (6); Niacr1^−/− Ctrl (4), AAI (5)]. Each dot represents individual mice (B–D and F–L). Data pooled from 2–3 independent experiments (B–D, and F–L). Data expressed as Mean ± SD. Statistical analyses by One-way ANOVA (B–D, and F–L). B WT Ctrl vs AAI ****P < 0.0001, WT AAI vs Niacr1^−/− AAI **P = 0.0017. C WT Ctrl vs AAI **P = 0.005, WT AAI vs Niacr1^−/− AAI *P = 0.04852. D WT Ctrl vs AAI **P = 0.0060. F WT Ctrl vs AAI ****P < 0.0001, WT AAI vs Niacr1^−/− AAI *P = 0.0142. G WT Ctrl vs AAI ****P < 0.0001, WT AAI vs Niacr1^−/− AAI **P = 0.006. H WT Ctrl vs AAI **P = 0.0093, WT AAI vs Niacr1^−/− AAI **P = 0.0018. I WT Ctrl vs AAI *P = 0.0162, WT AAI vs Niacr1^−/− AAI **P = 0.0010. J WT Ctrl vs AAI *P = 0.0243, WT AAI vs Niacr1^−/− AAI *P = 0.0110. K WT Ctrl vs AAI **P = 0.0096, WT AAI vs Niacr1^−/− AAI *P = 0.0115. L WT Ctrl vs AAI *P = 0.0653, WT AAI vs Niacr1^−/− AAI *P = 0.0150. ns: statistically not significant. Source data are provided as a Source Data file.

To define the role of B cell-specific GPR109A in impaired GC response, we adoptively transferred resting splenic B cells from WT or Niacr1^−/− mice in control or AAI-injected B cell-deficient μMT mice and assessed for GC response after NP-KLH immunization (Fig. 7E). The recipient μMT mice subjected to AAN and receiving either WT or Niacr1^−/− B cells showed comparable serum BUN level at day 12 post-immunization (Fig. S7B). As expected, μMT mice with kidney disease and receiving WT B cells showed reduced total and NP-specific GC response compared to control μMT spleen (Fig. 7F, G, Fig. S7C). Interestingly, there was no difference in the percentages and absolute numbers of total and NP-specific GC B cells between control and AAN μMT mice receiving Niacr1^−/− B cells after NP-KLH immunization. Moreover, Ki67 staining revealed diminished proliferation of WT total GC B cells in AAN μMT mice, an effect abrogated when GC B cells lack Niacr1 expression (Fig. 7H and Fig. S7D). When evaluated for the loss of mitochondrial membrane potential and mitochondrial ROS generation, Niacr1^−/− but not WT GC B cells showed no increase in the loss of mitochondrial membrane potential and mitochondrial ROS generation in AAN μMT mice in comparison control recipient (Fig. 7I, J and Fig. S7E, F). Consequently, increased Caspase3 activation in NP-specific WT GC B cells was corrected in recipient AAN μMT mice receiving Niacr1^−/− B cells (Fig. 7K). The recipient AAN μMT mice receiving WT B cells demonstrated a defect in antibody affinity maturation compared to the control recipient, an effect not observed in AAN μMT mice receiving Niacr1^−/− B cells (Fig. 7L). These data indicate that uremic toxin(s)-mediated activation of GPR109A in B cells is likely responsible for compromised GC response in kidney disease.

Impaired influenza-specific B and T cell responses in uremia

The patients with kidney disease have a higher risk of death from influenza than those with normal kidney function^26,27. Hence, AAN and control mice have infected with the influenza A virus and assessed for B cell response at 12 days post-infection (p.i) (Fig. 8A). AAI-injected mice showed reduced GC in the spleen than control animals (Fig. 8B). The number of TFh cells in the spleen was also diminished in the AAN mice (Fig. 8C). Accordingly, there was a reduction in the serum anti-PR8HA-specific IgG antibody titer in mice with kidney disease than control animals (Fig. 8D). The inhibitory effect of uremia is not restricted to B cells since AAN mice also exhibited a reduced number of influenza-specific CD8⁺ T cells (Fig. 8E). These results indicate that kidney dysfunction has a negative impact on influenza-specific B and T cell responses following infection.

A Schematic representation of the experimental design. Mice were either injected with AAI or PBS (Ctrl). Four days post-AAI injection, mice were infected with 1000 PFU of mouse-adapted influenza H1N1 strain A/PR/8/34 via oropharyngeal aspiration (n = 7–9) or left sham infected (n = 3). At day 12 p.i., spleen of infected and sham mice was assessed for B GC (liveB220⁺GL7⁺CD95⁺) [Sham: Ctrl (3), AAI (2); Influenza: Ctrl (7), AAI (9)], and C TFh (liveCD4⁺CD44⁺CXCR5⁺PD1⁺) cells by flow cytometry [Sham: Ctrl (3), AAI (2); Influenza: Ctrl (7), AAI (9)], D serum anti-PR8HA IgG titer by HAI assay [Sham: Ctrl (1), AAI (1); Influenza: Ctrl (8), AAI (7)], and E influenza-specific CD8⁺ T cells by tetramer staining [Sham: Ctrl (2), AAI (2); Influenza: Ctrl (7), AAI (7)]. F Influenza A virus infected and sham lungs were subjected to H&E staining and peri-vascular, peri-bronchiolar, and parenchymal inflammation were scored [Sham: Ctrl (1), AAI (1); Influenza: Ctrl (7), AAI (8)]. Scale bar = 50 μm. G Viral load in the lung was quantified by assessing Influenza A virus M1 gene expression by Taqman qPCR assay [Influenza: Ctrl (5), AAI (9)]. Data is normalized to the HPRT gene. Each dot represents individual mice and data are pooled from 2–3 independent experiments (B–G). Data expressed as Mean ± SD. Statistical analyses by One-way ANOVA (B, C, and E), two-sided t-test (F), Mann–Whitney test (G), and Kruskal–Wallis test (D). B ***P = 0.0007. C ***P = 0.0003. D ***P = 0.0002. E ***P = 0.0005. F Peri-vascular *P = 0.020521. G **P = 0.007. Source data are provided as a Source Data file.

To correlate the defect in anti-viral immunity with susceptibility to infection, we compared pulmonary inflammation and viral load between AAN and control mice at day 12 p.i. AAN lungs showed a trend toward diminished peri-vascular, peri-bronchiolar, and parenchymal inflammatory cell influx when compared to control lungs (Fig. 8F). Compromised inflammatory cell infiltration in the AAN lungs resulted in reduced viral clearance, as assessed by qPCR for the M protein gene of the virus (Fig. 8G). Collectively, these results suggest that mice with kidney disease show impaired B and T cell responses leading to uncontrolled viral load in the lungs.