PTEN acts as a metabolic checkpoint molecule in mature B cells to suppress TLR9-mediated inflammation

doi:10.21203/rs.3.rs-2616509/v1

Download PDF

Article

PTEN acts as a metabolic checkpoint molecule in mature B cells to suppress TLR9-mediated inflammation

https://doi.org/10.21203/rs.3.rs-2616509/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Phosphatase and tensin homolog (PTEN) is a negative regulator for PI3K signaling essential for B cell development. To explore the physiological effects of PTEN mutation on peripheral B cells, we generated CD23/cre-PTEN^Flox/Flox (CD23-cKO) mice in this study to avoid the developmental arrest. The mutant mice develop systemic inflammation associated with B cell expansion in the early phase followed with a severe immune cell-infiltration in multiple vital organs. PTEN deficiency leads to an accumulation of PI(3)P, an increase of lysosomal recruitment of TLR9/p38 complex, and an aberrant activation of TLR9/IL-6 axis in B cells. Interestingly, cholesterol biosynthesis pathway is upregulated in mutant cells upon TLR9 engagement. A blockade of cholesterol biosynthesis by targeting SQLE greatly reduces the level of PI(3)P and the interaction between TLR9 and p38, which lowers the level of TLR9-induced IL-6. Thus, PTEN represents a critical metabolic checkpoint that fine-tunes lipid and cholesterol homeostasis to control TLR9-driven inflammation.

Biological sciences/Immunology/Gene regulation in immune cells

Biological sciences/Immunology/Cytokines

B cells

PTEN/TLR9

IL-6

inflammation

Cholesterol

PI(3)P

B cell lymphopoiesis requires precise control to prevent immune diseases. Newly generated B cells in the bone marrow express pre-B cell receptors (pre-BCRs) that mediate positive signals supporting pre-B cell differentiation as well as negative signals driving the elimination of pre-B cells bearing autoreactive heavy chains ^{1 2}. During this transition between immature and mature B cells, large numbers of self-reactive B cells undergo receptor editing to prevent autoreactivity³. Successfully edited immature B cells migrate to the spleen where they differentiate into mature B2 B cells, a population comprised of the follicular (FO) and marginal zone (MZ) B cell subsets. After antigen encounter, FO B cells undergo somatic hypermutation and immunoglobulin (Ig) class switch recombination (CSR) within the germinal center (GC). These cells then differentiate into memory B cells or plasma cells capable of producing high affinity antibodies. In contrast, MZ B cells, as well as B1 B cells derived from fetal liver, produce antibodies of low affinity but do so immediately upon antigen encounter.

PTEN is a 3’-phosphatase that removes 3-phosphoinositide products of PI3Ks ^{4 5 6}. Upon BCR stimulation, activated Class I PI3K catalyzes the formation of PI(3,4,5)P₃, which recruits Akt/PKB from cytosol to plasma membrane, leading to the activation of Akt and the downstream events. By inactivating the PI3K-Akt pathway, PTEN promotes the transcription of recombination-activating genes RAG1/2 required for Ig gene rearrangement and pre-B cell differentiation ^{7 8}. Loss of PTEN at the pre-B cell stage engages a developmental arrest that triggers the apoptosis of autoreactive B cells, indicating that PTEN opposes negative selection at pre-B cell stage ⁹. During maturation, PTEN is required for the elimination of autoreactive B cells by secondary light chain gene recombination ¹⁰. B cell-intrinsic PTEN ablation in mice show reduced FO B cells, but increased MZ B cells and B1a cells, suggesting that PTEN helps to direct the maturation of B cell subsets ^{11 12 13}. PTEN further controls B cell responsiveness and supports the induction of activation-induced cytidine deaminase (AID), which is required for CSR ¹². Thus, PTEN plays multiple important roles throughout B cell development, which is indispensable for induction of a protective adaptive immune response.

Besides producing antibody, B cells interplay innate immune responses via cytokine release. IL-6 produced by B cells exacerbates disease severity in experimental autoimmune encephalomyelitis model via promoting Th17 response ¹⁴. All Toll-like receptors (TLRs) except TLR3 signal through a MyD88-dependent pathway, which in turn activates signaling molecules IRAKs and TRAF6, leading to the activation of transcription factors NF-κB and AP-1 and the production of inflammatory cytokines¹⁵. In B cells, engagement of TLR9 leads to the recruitment of the B cell-specific adaptor BANK1, which promotes p38-phosphorylation and the subsequent activation of MNK1/2 and eIF4E for IL-6 production ¹⁶. In addition, internalized BCR continues to signal through p38 to modulate TLR9 trafficking, leading to B cell hyperresponses ¹⁷. The above studies reveal that B cells engage BCR signaling molecules to operate TLR9 signaling.

Emerging evidence have demonstrated that immune cells frequently engage various metabolic pathways for a proper induction of the differentiation program. Developing B cells engage glycolysis during the transition from pro-B cells pre-B cells and at the immature B cells, the stages required for quick expansion ¹⁸. Newly generated B cells become metabolic quiescence in the resting status ¹⁹. Upon activation through BCR, B cells rapidly increase glycolysis for cell proliferation ^{20 21}. GC B cells further engage fatty acid oxidation to meet energic demand ^{22 23}. Meanwhile, activated B cells via CD40 induce cholesterol biosynthesis pathway, which promotes their antigen presenting ability for allospecific T cells ²⁴. The metabolic reprograming during B cell activation ensures a proper induction of B cell-mediated immune response.

While the above mechanism explains PTEN’s roles during B cell development, it may not apply to mature B cells bearing PTEN mutation. To determine the physiological consequences of PTEN deletion in mature B cells, we examined mice in which conditional knockout (cKO) of PTEN was controlled by CD23-cre (CD23-cKO mice) ²⁵, which mimicked the somatic mutation of PTEN in human. We showed that PTEN deficiency in mature B2 B cells led to an accumulation of PI(3)P, which promoted the lysosomal recruitment of TLR9 signalosome. The resulting hyperactive TLR9/IL-6 loop relied on an active cholesterol biosynthesis pathway. By contrast, B2 B cells from PTEN-deleted mice by mb1-cre (mb1-cKO mice), which had undergone the developmental checkpoints in the bone marrow, did not inherit the inflammatory feature upon TLR9 engagement via downregulation of BCR. The findings reveal that B cell-intrinsic PTEN expression constitutes a metabolic checkpoint in mature B cells that prevents TLR9-mediated inflammation via suppressing lipid and cholesterol biosynthesis. Our study thus provides the molecular insight for targeting B cell-involved inflammatory diseases.

Shortened lifespan of mice with PTEN deletion exclusively in mature B cells.

To study the physiological effects of PTEN deletion specifically in peripheral B cells, we bred PTEN^F/Fmice ²⁶ with CD23-cre mice ²⁵to generate CD23-cre-PTEN^F/Fconditional knock-out(CD23-cKO) mutant animals. In addition, to delete PTEN since pro-B cell stage, we generated mb1-cre-PTEN^F/F(mb1-cKO) mice. We first determined the efficiency of PTEN deletion in immature (IM, B220⁺CD21^lowCD23^low), FO (B220⁺CD21^hiCD23^low), MZ (B220⁺CD21^intCD23^hi) and B1a (B220⁺CD5⁺) B cells in CD23-cKO mice. PTEN deletion efficiency was close to complete (98-100%) in CD23-cKO FO and MZ B cells but occurred in only 46-50% of CD23-cKO IM and B1a B cells (Supplementary Fig. 1a-d). Thus, CD23-cKO mice appeared suitable as a model for studying PTEN-deficient mature B2 B cells.

Consistent with the previous reports ^{11, 12}, PTEN deficiency in CD23-cKO and mb1-cKO mice led to an accumulation of MZ B cells but a reduction in FO B cells (Fig. 1a, b and Supplementary Fig. 2a, b). In CD23-cKO mice at 8-12 weeks (w) of age, the spleen was enlarged and contained significantly increased B cell numbers (Fig. 1c, d). As expected, peripheral B cells were drastically reduced in mb1-cKO mice, which exhibited a smaller spleen than mb1-control mice (Supplementary Fig. 2c, d). We confirmed that this dramatic decrease in B cells in mb1-cKO mice was due to a severe block in early B cell development ²⁷ (Supplementary Fig. 2e, f). Interestingly, while 85% of mb1-cKO mice survived for longer than one year due to immunodeficiency (Supplementary Fig. 2g), CD23-cKO mice displayed a median survival of only 24w, with the first deaths observed at 12w (Fig. 1e). In sick CD23-cKO mice, splenomegaly, but not lymphadenopathy, was commonly observed (Fig. 1f). Although splenocyte numbers were greatly increased in CD23-cKO mice older than (>) 16w, this enhanced cellularity was not due to an enlarged B cell pool (Fig. 1g), which excluded B cell malignancy as the cause of death of CD23-cKO mice. In CD23-cKO mice of either 8-12w or >16w, serum IgM levels were comparable to those in controls (Fig. 1h). Consistent with the previous finding that B cells lacking PTEN fail to undergo CSR ¹², IgG1 levels in CD23-cKO mice were greatly reduced (Fig. 1h). Notably, levels of anti-nuclear and anti-double stranded DNA (dsDNA) autoantibodies were not elevated in CD23-cKO mice (Fig. 1h).

To determine whether this T cell phenotype was due to unexpected PTEN deletion in thymocytes of CD23-cKO mice, we isolated double-positive (DP) and CD4⁺ and CD8⁺ single-positive (SP) thymocytes from CD23-cKO and control mice at 6w. RT-PCR analysis showed that the PTEN gene was intact in DP and SP thymocytes from both mice (Supplementary Fig. 1e). These findings imply that CD23-cKO mice develop an antibody-independent disorder that leads to premature death.

CD23-cKO mice develop systemic inflammation with increased IL-17

To investigate the premature lethality of CD23-cKO mice, we first determined serum cytokine levels using multiplex cytokine array. In CD23-cKO mice, increased serum levels of IL-17, IL-12 and IL-9 were detected at 8-12w (Fig. 2a and Supplementary Fig. 3), indicating a mixed type of inflammation. Nevertheless, the serum levels of IFNg and TNF-a in mutant mice at 8-12w were reduced (Fig. 2a). Within those cytokine, the increased IL-17 could be further detected in CD23-cKO mice at >16w. Next, because we had also noted that CD23-cKO mice developed respiratory distress syndrome shortly before death, we examined cells in the lungs of sick CD23-cKO mice at >16w by flow cytometry. We detected modest but significant numbers of B cells had infiltrated CD23-cKO lungs at 8-12w(Fig. 2b). However, lung-infiltrating T cells increased dramatically in CD23-cKO mice >16w compared to controls (Supplementary Fig. 4a-c). An aberrant accumulation of CD4⁺ T cells was also confirmed in CD23-cKO livers (Supplementary Fig. 4d, e). Further, the levels of GOT and GPT, the biomarkers indicating liver injury, were anomaly increased in mutant mice >16w (Supplementary Fig. 4f). These results implied that B cell infiltration in vital organs of CD23-cKO mice was an early event occurring prior to T cell infiltration, while T cells gradually expanded in CD23-cKO mice with age.

To explore the nature of peripheral T cells in CD23-cKO mice, we stimulated isolated splenic CD4⁺ T cells in vitro with PMA (P) plus ionomycin (I)for 5h and then subjected these cells to intracellular staining to detect IL-17. In CD23-control co-cultures, 2.24% of CD4⁺ T cells expressed IL-17 after stimulation of P+I. This proportion swelled to 59.6% IL-17⁺ Th17 cells in CD23-cKO co-cultures (Fig. 2c, left). Consistently, stimulation with P+I led to more IL-17 expression by hepatic CD4⁺ T cells from CD23-cKO mice (Fig. 2c, right). In contrast, mb1-cKO splenic CD4⁺ T cells subjected to in vitro stimulation did not differentiate into IL-17⁺ Th17 cells (Supplementary Fig. 2h). The results indicated a Th17-associated inflammation occurred in CD23-cKO mice.

To address whether CD23-cKO B cells released soluble mediator(s) that affected Th17 cell differentiation, we collected the 24h-cultured supernatants from control or CD23-cKO B cells and subjected the supernatants for in vitro Th17 cell differentiation assay with naïve CD4⁺ T cells from wild type mice (Fig.2d). However, none of those B cell supernatants was able to promote Th17 cell differentiation (Fig. 2e, f). We further examined the effect of B cell supernatants on the pathogenic Th17 cell differentiation as previously described ²⁸. To achieve this,IL-23 was added to the co-culture system at day 7 of in vitro Th17 cell differentiation assay. IL-17 produced by T cells was determined at day 10 by FACS (Fig. 2g). The supernatant from CD23-cKO B cells significantly promoted IL-17 production by CD4⁺ T cells while control supernatant did not (Fig. 2h, i). The results confirmed that CD23-cKO B cells released soluble mediator(s), that promoted the pathogenic Th17 cell differentiation in the presence of IL-23. Our findings suggest that PTEN ablation in mature B cells has an unanticipated effect on pathogenic immune cell differentiation, generating increased numbers of inflammatory cells, which lead to multiple organ dysfunction.

Deletion of PTEN in mature B cells leads to a strong induction of TLR9-mediated IL-6

It had been well demonstrated that IL-6 in combination with TGF-β or IL-1β combined IL-23 promotes the differentiation of Th17 cells ²⁹ ³⁰. We next addressed whether the mutant B cells accelerated the systemic inflammation via the production of pro-inflammatory cytokines. We observed that splenic B cells from CD23-cKO mice produced more IL-6 in the resting state than controls (Fig. 3a, b). We screened IL-6 production by CD23-cKO B cells with various stimuli including P+I, TLR4 agonist LPS, TLR9 agonist CpG or the combinations with P+I by intracellular staining, and found that CpG or CpG+P+I induced a great production of IL-6 (Fig. 3c). To address which B cell subsets from CD23-cKO mice caused this phenotype, we sorted FO, MZ and B1a B cells, stimulated those cells with CpG for 24h in 10% FBS/RPMI medium, harvested the cultured supernatants and subjected them to standard IL-6 ELISA assay. Only FO and MZ B cells from CD23-cKO mice owned the capability to produce more IL-6, while B1a B cells from both mice produced nearly no IL-6 upon TLR9-stimulation (Fig. 3d). The increased IL-6 production was not observed in PTEN-deficient B cells upon stimulation with LPS, CL307 or poly (I:C) (Fig. 3c, e). The data suggest that ablation of PTEN by CD23/cre specifically affects TLR9-mediated IL-6 in B2 B cells.

To dissect TLR9/IL-6 signaling in PTEN-deficient matureB cells, we stimulated sorted control and CD23-cKO FO and MZ B cells with CpG in vitro for 24h in a series of cultures with inhibitors that targeted Syk, p38, JNK, and mTOR, respectively. For both FO and MZ B cells, inhibition of p38 led to the most prominent diminution of CpG-induced IL-6 production in both control and CD23-cKO B cells, although the inhibitory effect was not complete (Fig. 3f). Given the role of BANK1 in TLR9/p38 axis ¹⁶, we performed confocal imaging studies and found that the colocalization between BANK1 and TLR9 was strongly induced in CD23-cKO B cells (Fig. 3g and Supplementary Fig. 5a). Remarkably, the increased amount of TLR9-mediated IL-6 was not observed in PTEN^-/-B cells from mb1-cKO mice (Fig. 3h). We noted that 92% of those PTEN^-/- mature B cells from mb1-cKO mice did not express surface IgM or IgD, which was similar to the previous report ¹⁰ (Supplementary Fig. 5b, c). Further, PTEN^-/-B cells from mb1-cKO mice had a defect in IgM-mediated endocytosis (Supplementary Fig. 5d, e). Thus, the low expression of BCR and the defect in IgM-mediated endocytosis in mb1-cKO might explain the difference of TLR9/IL-6 activity between mb1-cKO and CD23-cKO B cells. In brief, our findings imply that loss of PTEN in mature B cells strongly activates TLR9-mediated IL-6 production in a manner dependent on p38. The induction of innate receptor-mediated inflammation in PTEN-deficient B cells is CD23-cKO B cells-restricted. We speculate that PTEN^-/-B cells from mb1-cKO mice which undergo the developmental control are trained to be immune tolerant through downregulating BCR.

PTEN deficiency enhances the lysosomal trafficking of TLR9 signalosome without the engagement of BCR and TLR9

TLR9, which localizes in ER in the steady state, is translocated to lysosomes via endocytic vesicle upon stimulation with CpG ³¹. Ligation of BCR and TLR9 leads to p38 phosphorylation and the colocalization of TLR9 and phosphorylated p38 (p-p38) in autophagosome-like compartments ¹⁷. To address whether and how PTEN deficiency affected the interaction between TLR9 and p-p38, we performed Duolink Proximity Ligation (PLA) assay to explore the lysosomal complex formation in splenic B cells from CD23-control, CD23-cKO, mb1-control and mb1-cKO mice. The intensity of lysosomal marker LAMP1 was strongly enhanced in resting CD23-cKO B cells, but not in mb1-cKO B cells (Fig. 4a, b, c). The signal of TLR9/p-p38 complex, which was comparable between control and CD23-cKO B cells in LAMP-1-negative area, was markedly induced in LAMP-1⁺ cellular compartments in CD23-cKO B cells (Fig. 4a, c and Supplementary Fig. 6a). The increase of lysosomal-localized TLR9/p-p38 complexes caused by PTEN deficiency was not observed in mb1-cKO B cells. Instead, more TLR9/p-p38 complexes were induced in LAMP-1-negative compartments in mb1-cKO B cells than in mb1-controls (Fig. 4b, c and Supplementary Fig. 6b). Our findings suggest that PTEN deficiency accelerates lysosomal trafficking of TLR9/p-p38 complex without BCR and TLR9 engagement in CD23-cKO B cells but not in mb1-cKO B cells. The data further implies that without a functional BCR, TLR9/p-p38 complex is not successfully recruited to lysosome in in mb1-cKO B cells.

TLR9 was colocalized with endosomal PI(3)P in PTEN-deficient B cells in the steady state

As one of PTEN’s substrates, PI(3)P regulates the biosynthesis of phagosome or lysosome, which acts as a hallmark of endosomal system ³² ³³. To address whether PI(3)P was affected in the mutant B cells, we determined the level of PI(3)P in splenic B cells from CD23-cKO and mb1-cKO mice by anti-PI(3)P, and revealed PI(3)P signals by confocal imaging. Loss of PTEN resulted in a great accumulation PI(3)P in CD23-cKO B cells, but not in mb1-cKO B cells (Fig. 5a, b). In addition, PI(3)P was not colocalized with LAMP-1 in all B cells, suggesting these two signals were in distinct cellular compartments. Interestingly, the interaction between TLR9 and PI(3)P in CD23-cKO B cells was significantly increased (Fig. 5c, d and Supplementary Fig. 6c). The findings suggest that loss of PTEN results in an accumulation of PI(3)P, which promotes the endosomal recruitment of TLR9 in resting B cells.

PI(3)P is generated from phosphatidylinositol (PI) catalyzed by class III PI3K PIK3C3/Vps34, or from PI(3,4)P₂ catalyzed by 4’-phosphatase INPP4 (Fig. 5e). To address which pathway contributed to the aberrant IL-6 production mediated by TLR9, we treated sorted FO or MZ B cells from CD23-cKO and control mice with CpG in the presence of PIK3C3 inhibitor SAR405 or INPP4 inhibitor bpV(phen). Treatment with PIK3C3 inhibitor strongly suppressed TLR9-mediated IL-6 in FO B cells from CD23-cKO and control mice, whereas INPP4 inhibitor exerted a minor suppressive effect on IL-6 (Fig. 5f, left). For MZ B cells, treatments with PIK3C3 or INPP4 inhibitors yielded minor effects on TLR9-mediated IL-6 in CD23-cKO cells and no effect on control cells (Fig. 5f, right). The data suggests that loss of PTEN particularly affects TLR9-mediated IL-6 in FO B cells via an abnormal activation of PIK3C3-PI(3)P pathway.

Inhibition of cholesterol biosynthesis by blocking SQLE reduces PI(3)P, TLR9/p38 complex formation, and TLR9-mediated IL-6 in CD23-cKO B cells

To invest more biological processes which were uniquely associated with the inflammatory phenotype of CD23-cKO B cells, we performed single-cell RNA-sequencing (scRNA-seq.) transcriptomic analysis to expose additional genes involved in TLR9/p38-regulated biological processes in CD23-cKO mice. We employed nanowells to capture single cells using the BD Rhapsody platform in combination with oligonucleotide-barcoded antibodies (Ab-seq). Purified splenic B cells from CD23-control, CD23-cKO, mb1-control and mb1-cKO mice were left unstimulated or stimulated with CpG in the absence or presence of the p38 inhibitor SB203580 for 2h, followed by staining with barcoded antibodies recognizing B220, CD19, IgD, CD21, CD23, CD5, CD40, CD80, CD86, CD95, CD138, or MHCII. Within these stainedcells, 16,871 cells containing 12 groups passed quality control measures. Among these, 20,932 genes were detected across all cells. We analyzed scRNA seq. data bygene set enrichment analysis (GSEA) and ingenuity pathway analysis (IPA), and found that cholesterol biosynthesis was the most significant pathway upregulated in FO B cells from CD23-cKO mice (Fig. 6a and Supplementary Fig. 7a). The induction of cholesterol biosynthesis pathway in FO B cells from CD23-cKO mice was also more prominent than those from mb1-cKO mice (Supplementary Fig. 7b). Heatmap analysis showed that genes associated with cholesterol biosynthesis were differentially upregulated in CD23-cKO FO B cells in the resting state and upon CpG-stimulation (Fig. 6b). Namely, nsdhl, ebp, sc5d, and dhrc7 were upregulated in CD23-cKO FO B cells in the resting state. Upon CpG-stimulation, hmgcs1, hmgcr, idi1, sqle, cyp51, msmo were upregulated in CD23-cKO FO B cells (Fig. 6b). We also found that genes including cyp51, sqle, hmgcr, idi1,and hmgcs1 were significantly upregulated in CpG-stimulated CD23-cKO MZ B cells (Supplementary Fig. 7c). The abnormal induction of gene sets in TLR9-stimulated CD23-cKO B cells was to some extent reverted after the treatment with p38 inhibitor, suggesting that TLR9-mediated cholesterol biosynthesis pathway had p38-dependent and p38-independent pathways.

UMAP (Uniform Manifold Approximation and Projection) visualization demonstrated five main clusters of whole B cells. Most FO and MZ B cells were distributed in clusters 1, 2, 3, and 4, while B1a B cells were distributed in clusters 1, 3, 4 and 5 (Supplementary Fig. 8a). In addition, B220⁺bcl6⁺GC B cells were enriched in cluster 4, while B220⁺cd80⁺pdcd1lg2⁺ memory-like B cells in cluster 3, respectively. Only a tiny amount of prdm1⁺plasma cells was detected in cluster 5 of CD23-cKO mice. The Heatmap statistic type of UMAP (UMAP-Heatmap) showed that the upregulated hmgcs1, hmgcr, idi1, sqle, cyp51, and msmo1 mRNAs in CD23-cKO B cells induced by CpG were distributed in all clusters but mainly located in clusters 1 and 3, suggesting that the induction of cholesterol biosynthesis pathway upon TLR9 stimulation happened in heterogeneous B cells (Supplementary Fig. 8b, c). Previous studies have revealed that PI3K-Akt-mTORC1 signaling is essential for the expression of cholesterol biosynthesis pathway through the sterol responsive element binding protein SREBP ³⁴. The UMAP-Heatmap revealed that srebf2, but not srebf1, was highly and steadily expressed in all B cells (Supplementary Fig. 8c). Upon TLR9 engagement, srebf2 levels were further elevated in clusters 1, 3, and 4. The findings imply that the upregulation of cholesterol biosynthesis pathway in CD23-cKO B cells could be mediated by SREBP2.

Intracellular cholesterol can be esterified and stored as cholesterolester in the lipid droplets (LDs). Confocal images confirmed that LDs were greatly accumulated in resting CD23-cKO B cells compared to controls (Fig. 6c, d, left panels), but not in mb1-cKO B cells (Supplementary Fig. 6d, e). HMG-CoA reductase (HMGCR) encoded by hmgcr and squalene epoxidase (SQLE, also known as squalene monooxygenase) encoded by sqle are two rate-limiting enzymes for cholesterol synthesis ³⁵ ³⁶. To address how these two enzymes involved in the aberrant production of IL-6 in CD23-cKO B cells, freshly sorted FO and MZ B cells were left unstimulated, or stimulated with CpG in the absence of drugs (DMSO control), or in the presence of lovastatin or NB-598, which targeted HMGCR or SQLE, respectively. IL-6 ELISA assay revealed that the blockade of cholesterol biosynthesis pathway by SQLE inhibitor significantly suppressed TLR9-mediated IL-6 in both FO and MZ B cells from CD23-cKO and control mice (Fig. 6e). Meanwhile, the levels of SQLE mRNA and proteins were not affected in CD23-cKO B cells (Supplementary Fig. 7d, e). Supplement of SQLE inhibitor robustly reduced the amounts of LDs in CD23-cKO B cells to the level comparable to controls, suggesting that the increased LDs in CD23-cKO B cells were derived from an accelerated de novo cholesterol biosynthesis (Fig. 6c, d, right panels). In addition, supplement of SQLE inhibitor significantly reduced the level of PI(3)P in CD23-cKO B cells (Fig. 6e), suggesting that the aberrant cholesterol biosynthesis led to PI(3)P accumulation by PTEN deficiency. We noted that supplement of SQLE inhibitor further suppressed the complex formation of TLR9 and p-p38 in CD23-cKO B cells (Fig. 6f, Supplementary Fig. 7f). Our findings revealed that the aberrant induction of TLR9-mediated IL-6 by PTEN deficiency was attributed partly to the increased cholesterol biosynthesis through SQLE activity. Further, cholesterol biosynthesis beyond the step catalyzed by SQLE promoted PI(3)P accumulation beneficial for the recruitment of TLR9/p-p38 complex.

Inhibition of p38, PIK3C3 and SQLE completely abolishes TLR9-mediated IL-6 in CD23-cKO B cells

A previous study has suggested that the ratio of sterol to squalene affects membrane fluidity/rigidity³⁷. To explore how PTEN deficiency affected the intracellular composition of cholesterol biosynthesis intermediates, we determined the levels of several intermediates of cholesterol biosynthesis pathway including GGPP, squalene, 2,3’-oxidosqualene, lanosterol and zymosterol et al. in CD23-cKO B cells and controls in the resting and upon CpG-stimulation (Fig. 7a, b). Based on the role of lanosterol in modulating membrane fluidity ³⁸, we compared the ratios of lanosterol to squalene between control and CD23-cKO B cells. No matter in the resting state or upon CpG-stimulation, the ratios of lanosterol/squalene in CD23-cKO B cells were higher than those in controls (Fig. 7c), indicating an unfavorable role of squalene for TLR9/IL-6 activation. Interestingly, supplement of squalene led to a suppressive effect on TLR9-mediated IL-6 in control and CD23-cKO B cells (Fig. 7d). The results suggested that PTEN deficiency affected TLR9/IL-6 axis by reducing the intracellular level of squalene through SQLE. Because targeting p38, PIK3C3 or SQLE individually in CD23-cKO B cells only led to a partial reduction of TLR9-mediated IL-6, we lastly addressed the effect of targeting p38, PIK3C3 or SQLE together on TLR9-mediated IL-6. As expectedly, supplement with those inhibitors together completely abolished TLR9-mediated IL-6 in FO B cells from control and CD23-cKO B cells (Fig. 7e). The findings suggested that p38, PIK3C3 and SQLE contributed together to TLR9-mediated IL-6 in wild type and PTEN-deficient B cells (Fig. 8e).

Enforced expression of BCR in mb1-cKO B cells restores the strong induction of TLR9-mediated IL-6

The abnormal TLR9-mediated IL-6 produced by PTEN-deficient B cells was restricted to B cells from CD23-cKO mice, not from mb1-cKO mice. Consistently, the increases of PI(3)P and the assembly of TLR9/p-p38 complex were not observed in B cells from mb1-cKO mice (Table 1). Notably, TLR9 signaling requires BCR-proximal kinase Syk for its activation, which assists in delivering CpG into TLR9-containing endolysosomes ³⁹. Nevertheless, the level of surface IgM as well as IgM-mediated endocytosis in B cells from mb1-cKO mice was nearly absent. We thus speculated that TLR9/IL-6 pathway was inactivated in the peripheral B cells from mb1-cKO mice via downregulating BCR. To address this issue, we bred mb1-cKO mice with HEL-specific BCR-transgenic (HEL^tg) mice to receive HEL^tg mb1-cKO mice (Fig. 8a). Compared to mb1-cKO mice, the expression of surface IgM on IM, FO and MZ B cells in HEL^tg mb1-cKO mice was largely rescued, although the levels remained a bit lower than those from HEL^tg mb1-control mice (Fig. 8b). FO and MZ B cells from HEL^tg mb1-cKO and HEL^tg mb1-control mice were then freshly sorted, left unstimulated/stimulated with CpG, and the supernatants were collected for IL-6 ELSIA. Compared to B cells from control cells, CpG-stimulation in B cells from HEL^tg mb1-cKO mice induced an excess production of IL-6 (Fig. 8c. d). The findings confirmed that the B2 cells in mb1-cKO mice were TLR9-unresponsiveness via downregulating BCR during B cell development, which led mb1-cKO mice becoming immune tolerant. The essential role of BCR for the activation of TLR9/IL-6 was illustrated in Fig. 8e.

Taken together, our study reveals that mutation of PTEN gene in B2 B cells without the developmental control sensitizes TLR9/IL-6 axis, leading to inflammation via the disturbances of lipid and cholesterol homeostasis.

Our study uncovers a key role for PTEN as a gatekeeper preventing the hyperactivation of the TLR9/p38/IL-6 signaling, which controls the inflammatory feature of B cells. CD23-cKO mice develop systemic inflammation with a significant immune cell infiltration in multiple vital organs, which is not observed in mb1-cKO mice. Most of those infiltrated immune cells are CD4⁺ T cells. We assume that the induction of IL-6 produced by PTEN-deficient B cells persistently affect various immune cells, which promote the systemic inflammation eventually. Interestingly, PTEN-deficient B cells are able to induce Th17 cell differentiation in the presence of IL-23, the cytokine produced by dendritic cells or macrophages. It thus suggests that the systemic inflammation in CD23-cKO mice is involved in a chronic and dynamic inflammatory process participated by multiple immune cell types. In agreement with this, an increased level of serum IL-12, which is supposed to be produced by dendritic cells, could be detected in CD23-cKO mice at 8-12w. In contrast, PTEN deletion present during early B cell lymphopoiesis efficiently blocks B cell development, that reduces the generation of mature B cells in mb1-cKO mice. Any residual PTEN-deficient peripheral B cells in mb1-cKO mice downregulate their BCR as well as BCR endocytosis. The capability to produce IL-6 by those PTEN-deficient B cells are comparable to controls, which strongly indicates that downregulation of BCR is helpful to control innate receptor-mediated inflammation.

Not all stimuli through TLRs lead to IL-6 production in B cells. It is noteworthily that PTEN deficiency strongly activates TLR9/IL-6 axis in B2 B cells, suggesting that PTEN deficiency specifically influences TLR9 signaling. In the resting state, TLR9 is localized to the ER. Engagement of TLR9 involves in a sequential endosomal shuttling of TLR9, leading to its translocation from ER, endosome to the lysosome ^{40 41}. Initiation of TLR9 signaling also requires PI(3)P in the endosomal lipid domain, that regulates protein trafficking and lysosome maturation ^{42 32}. Intriguingly, the initiation of TLR4 takes place on the plasma membrane, where PI(4,5)P₂ mediates the binding of signaling molecules ⁴³. Although loss of PTEN can also lead to PI(3,4)P₂ accumulation ⁶, which is potent to promote the formation of PI(3)P through INPP4B ⁴⁴, the treatment in PTEN-deficient B cells with INPP4 inhibitor does not significantly reduce TLR9-mediated IL-6. The observation highlights the essential role of PIK3C3-PI(3)P-TLR9 route for IL-6 production. Through scRNA-seq. analysis, we further discover that the strong activation of TLR9/p38/IL-6 axis caused by PTEN deficiency is involved in the activation of cholesterol biosynthesis pathway. The elevation of LDs in those mutant B cells confirms that PTEN deficiency affects cholesterol homeostasis. The blockade of the second rate-limiting step by SQLE inhibitor, but not the first rate-limiting step, could revert the level of TLR9-induced IL-6 to wild type level, suggesting that SQLE acts as an essential metabolic switch during the onset of inflammation. Further, the supplement of SQLE to block cholesterol biosynthesis pathway strongly suppresses PI(3)P-accumulation and TLR9/p-p38 complex formation, indicating a role of steroid intermediates in the stabilization of PI(3)P and the resulted TLR9 activation. Our data thus reveal that the disturbed lipid as well as cholesterol homeostasis caused by PTEN deficiency strongly activates TLR9 signaling in B cells, leading to the differentiation of inflammatory B cells.

Activated SQLE catalyzes the synthesis 2,3-oxidosqualene, that is subsequently converted to the lanosterol, the first sterol intermediate in cholesterol biosynthesis pathway. Lanosterol has been reported to modulate membrane fluidity and affect immune responses in macrophages ³⁸. In B cells, cholesterol biosynthesis has been reported as a feed-forward mechanism to maintain the integrity of BCRs in lipid rafts ⁴⁵. Our findings imply that the sterol intermediates downstream of SQLE might promote the endosomal trafficking, which stabilize the endosome/lysosome recruitment of TLR9 signaling molecules. Which sterol intermediate regulate endosome/lysosomal fluidity requires further investigation.

Compared to TLR9 signaling in myeloid cells, TLR9 signaling in B cells is uniquely regulated by BCR internalization ¹⁷. In PTEN-deficient B cells, the interactions between TLR9 and PI(3)P as well as the lysosomal localization of TLR9/p38 signalosome are significantly induced, indicating that PTEN deficiency promotes TLR9 activity through accelerating its endosomal and lysosomal trafficking. In mb1-cKO B cells, TLR9/p-p38 complex is greatly accumulated but not colocalized in the lysosome, suggesting that the lysosome maturation without functional BCR is defect in those cells. The observation suggests that downregulation of BCR in mb1-cKO B cells prevents a hyperactivation of TLR9-mediated inflammation, which represents a protective mechanism for immune tolerance.

Cholesterol biosynthesis pathway has been proposed to play a role in the cytokine production and expansion of pathogenic B cells ⁴⁶. Recently, geranylgeranyl pyrophosphate (GGPP), which is converted from famesyl-PP in the non-sterol biosynthesis, has been shown to accelerate IL-10 production in B cells ⁴⁷. Thereby, the formation of GGPP, that takes place between the first and second rate-limiting steps in the cholesterol biosynthesis pathway, seems to direct B cell activation to the anti-inflammatory function. Our study reveals that activation of the second rate-limiting step of cholesterol biosynthesis links B cell activation to TLR9-mediated inflammation. Our study has proposed that somatic mutation of PTEN in peripheral B cells leads to an abnormal TLR9 activity. The findings reveal an essential role of PTEN in the suppression of B2 B cell-mediated inflammation, and highlight that activation of cholesterol biosynthesis pathway contributes to the aberrant induction of TLR9/p38/IL-6 through SQLE. Our study therefore reveals that PTEN is not only a tumor suppressor but also an “inflammation suppressor” that restrains TLR9-mediated inflammation mounted by mature B cells.

Animal studies

PTEN^F/Fmice were obtained from the Jackson Laboratory (JAX stock #006440) ²⁶. The mb1-cre mice ⁴⁸ were obtained from Michael Reth and Roberta Pelanda at the University of Freiburg & Max-Planck Institute for Immunobiology, and CD23-cre mice ²⁵ were obtained from Meinrad Busslinger at the Research Institute of Molecular Pathology, Vienna. All animals were maintained in the Laboratory Animal Center of National Health Research Institute (NHRI). All experiments were approved by the Institutional Animal Care and Use Committee, NHRI.

B cell purification and cell stimulation in vitro

Single-cell suspensions were prepared from mouse spleen by sieving and gentle pipetting through 70-μm nylon mesh filters (Falcon; BD). Non-B cells were depleted by incubation with a cocktail of biotin-conjugated antibodies recognizing NK1.1, Thy1.2, Gr1, DX5, CD11b, CD11c, and Ter119 (BD), followed by enrichment by mixing with IMag^TM Streptavidin Particles Plus beads (557812, BD). Splenic B cells of purity between 94-98% were used in all experiments. For isolation of FO and MZ B cells, purified splenic B cells were suspended in FACS buffer (2%BSA/PBS, 0.1% NaN₃) and sorted by BD Influx™ with a 100-μm nozzle at 4°C. B cells were routinely cultured in 10%FBS/RPMI 1640 complete medium [supplemented with 2 mM L-glutamine (Invitrogen), 100 U penicillin-streptomycin (Invitrogen), 100 mM Hepes (Sigma-Aldrich), 0.055 mM 2-mercaptoethanol (Gibco), and 10% FBS (Hyclone)]. B cells were stimulated with 1 mM CpG ODN1668 (denoted as CpG, tlrl-1668c, InvivoGen), 10 ng ml^-1 CL307 (tlrl-c307, InvivoGen), 10 mg ml^-1 LPS (L2630, Sigma), 5 mg ml^-1 poly(I:C) (tlrl-pic, InvivoGen) for 24 h. The sorted B cells were cultured in the 10%FBS/RPMI complete medium for all experiments. To examine drug effects on IL-6 production, cells were stimulated 1 mM CpG combined with inhibitors including 1 mM BAY61-3606 (HY-76474, MCE), 5 mM SB203580 (tlrl-sb20, InvivoGen), 5 mM SP600125 (S5567, Sigma), 10 mM lovastatin (HY-N0504, MCE), 10 mM NB-598 (HY-16343C, MCE), 10 mM SAR405 (HY-12481, MCE) or 20 nM bpV(phen) (13331, Cayman) for 24h. Cells or the cultured medium were collected and subjected to immunofluorescent experiments or ELISA assays.

BCR endocytosis

Freshly sorted FO and MZ B cells were pre-incubated with APC-conjugated anti-IgM (RMM-1, Biolegend) for 30 min on ice. After washing in ice-cold PBS, labelled cells were incubated at 37°C for 0, 15 or 60 min, respectively. The incubation was terminated by the addition of ice-cold RPMI 1640 (pH 2.5) to strip off surface-bound anti-IgM, followed by the fixation step using the Cytofix™ Fixation Buffer (554655, BD). The engulfed APC signals were determined by flow cytometry. % BCR endocytosis was calculated by [net stripped IgM⁺ (at the time post stimulation, time x) - net stripped IgM⁺ (at time zero)]/[net non-stripped IgM⁺ (at time x) - net stripped IgM⁺ (at time zero)] as previously reported ⁴⁹.

Determination of intracellular cholesterol intermediates

To determine intracellular cholesterol metabolites, B cell pellets (1x10⁷ cells) after PBS washing were suspended in 1 ml isopropanol and snap-frozen in liquid nitrogen. The frozen cells were quickly thawed with a vortex for 30~60s and pelleted by centrifugation at 800 xg for 2 min. The yielded supernatant were collected from two repeated freeze-thaw cycles, transferred to a new tube and evaporated thoroughly to receive pellets. The pellets were solved in 40 ml of isopropanol and subjected to Acquity UPLC-XeVo TQ (Waters) using UPLC-MS-Multiple Reaction Monitoring (MRM) method. Data were analyzed by TargetLynx software (Waters).

ELISA assay

Serum IgM and IgG1 were captured by Goat anti-mouse IgM (1020-1, Southern Biotech) and IgG1 (1070-1, Southern Biotech) antibodies which were coated on the surface of SpectraPlate-384 HB (6007500, PerkinElmer), and then those titers were detected by HRP-conjugated goat anti-mouse IgG (115-035-174, Jackson ImmunoResearch). Serum autoantibodies were determined by using anti-dsDNA ELISA Kit (AKRDD-061, Fujifilm-Wako-Shibayagi inc.) and anti-nuclear antibody ELISA kit (ABIN772680, antibodies-online GmbH). Serum cytokines were detected by using Bio-Plex Pro Mouse Cytokine 23-plex Assay (M60009RDPD, BIO-RAD). The DuoSet ELISA kits to detect IL-6 (DY406-05) were purchase from R&D system. The PI(3)P extraction and the level were detected by using PI(3)P MASS ELISA kit (K-3300, Echelon Biosciences) according to the manufacturer’s instructions.

Flow cytometry

Single cells of 1x10⁶ were washed twice with FACS buffer and maintained in the dark at 4°C throughout experiments. A flow cytometer (CantoII; BD) and FACSDiva software (BD) were used to acquire flow cytometric data, with FlowJo software^TM10 used for analysis. For FACS staining, cells were incubated with anti-mouse antibodies to detect surface markers or intracellular molecules for 15 min on ice, followed by two washes in FACS buffer. Viable cells were gated after staining with 7AAD or Fixable Viability Stain 620 (564996, BD Horizon^TM). For intracellular staining of cytokines, transcription factors or phosphoproteins, cells were fixed and permeabilized using the Transcription Factor Buffer Set (562574 and 563239, BD Pharmingen^TM) prior to incubation with antibodies and according to the manufacturer’s instructions. The following anti-mouse antibodies were purchased from BD: APC Cy7-CD45R/B220 (RA3-6B2, 552094), APC- CD3e (145-2C11, 561826), BV510-CD4 (RM4-5, 563106), FITC or BV421- CD5 (53-7.3, 553020 or 562739), BB515-CD8alpha (53-6.7, 564422), BV510-CD19 (1D3, 562956), PE-CD21/CD35 (7G6, 552957), BB515-CD25 (PC61, 564424), APC-CD43 (S7, 560663), Alexa488-IL6 (MP5-20F3, 561363), Alexa488-IL17A (TC11-18H10, 560220), PE-RORgt (Q31-378, 562607). The following anti-mouse antibodies were purchased from BioLegend: BV510 anti-mouse CD5 (53-7.3, 100627), PE Cy7-CD23 (B3B4, 101613), Pancific Blue-IgD (11-26c.2a, 405711), APC-IgM (RMM-1, 406509).

Generation of UMAP and heatmaps

Gene expression was analyzed using FLOWJO Seqgeq™ (BD Biosciences, version1.6), which contains a plug-in of Uniform Manifold Approximation and Projection (UMAP). The UMAP plot was generated by dimensionality reduction with default parameters, which run on all dimensions of the low dimension representation. The gene expression levels were presented in UMAP plot by using type of heatmap statistic with statistic median value of color map. To generate a heatmap of cytokine gene expression levels, gene expression values were standardized by Z-scores across samples, and graphs were generated using the R package heatmap v1.0.12.

GSEA and IPA analysis

Pathways were analyzed by applying Gene Set Enrichment Analysis (GSEA v4.1.0; http://software.broadinstitute.org/gsea/index.jsp) according to H: hallmark gene set collections of MSigDB v7.4 (Broad Institute, UCSan Diego, USA) ⁵⁰. GSEA ranked all genes based on the significance of differential gene expression between two groups. The enrichment score (ES) was calculated the overrepresented degree of an enriched gene set toward the top (or bottom) of a list of ranked genes. Normalized enriched score (NES) was calculated for the distribution of each enriched gene set in the list of ranked gene sets. The significance of enriched gene sets was identified by the absolute values of NES > 1, nominal P-value ≤ 0.05, and false discovery rate (FDR) ≤ 0.05. Pathways were analyzed by applying the Ingenuity Pathways Knowledge Base of Ingenuity Pathway Analysis (IPA, Ingenuity Systems, Inc. Redwood City, CA) as well. The significance of biological pathways was identified by the fold-change values > 2, Fisher’s Exact T test P-value < 0.05, Z score > 1.5.

Immunofluorescent stain and Duolink^® proximity ligation assay (PLA)

Single splenic B cells were fixed and permeabilized using the Transcription Factor Buffer Set (562574, BD Pharmingen^TM) prior to blocking in Imag-iT^TM FX Signal Enhancer (I36933, Invitrogen). Cells were then incubated with anti-TLR9 (PA5-20203, Thermo Fisher Scientific) or anti-p-p38 Y182 (D3F9, Cell Signaling) at 4°C overnight, followed by anti-rabbit IgG-Alexa488 (A11034, Invitrogen) as the secondary antibody for 1 h, or incubated with anti-BANK1 (F-8, Santa Cruz Biotechnology), anti-PI3P (Z-P003, Echelon Biosciences), and anti-LAMP1 (D2D11, Cell Signaling), which were conjugated with fluorophore using the APEX^TM Alexa Fluor^® 555 Antibody Labeling Kit (A10470, Invitrogen) at 4°C overnight. To stain LDs, cells were incubated with BODIPY™ 493/503 (D3922, Thermo Fisher Scientific) for 1 h. The PLA experiments were performed according to manufacturer’s procedure. Cells were fixed, permeabilized and incubated with anti-TLR9 as described above, anti-PI3P (Z-P003, Echelon Biosciences) and anti-p-p38 Y182 (28B10, Cell signaling) at 4°C overnight. Duolink^® In Situ PLA^® Probe Anti-Rabbit PLUS (DUO92002, Sigma) and Duolink^® In Situ PLA^® Probe Anti-Mouse MINUS (DUO92004, Sigma) were used for detecting the primary antibodies. Duolink^® In Situ Detection Reagent Green (DUO92014, Sigma) were used for ligation and signal amplification. All cells were stained with DAPI, air-dried on slides, and mounted using ProLong^TM Gold Anti-fade Reagent (P36930, Invitrogen). Images were recorded using a Leica TCS SP5 II instrument (Leica Microsystems). The Mander’s coefficient was used as a parameter of colocalization and was calculated by Just Another Colocalization Plugin from Image J software ⁵¹.

Immunoblot assay

For total lysate preparation, cells of 1x10⁷ were lysed by 100 μl 1% DDM lysis buffer [137.5 mM NaCl, 50 mM Tris (pH 7.8), 0.75 mM EDTA (pH 8.0), 1 mM sodium orthovanadate, 10 % glycerol, 1% n-Dodecyl β–D-maltoside (DDM), protease inhibitors freshly added]. The lysates were then fractionated by SDS-PAGE and blotted using standard procedures. Proteins were detected using rabbit anti-SQLE (12544-1-AP, Proteintech) and anti-β-actin (GTX109639, GeneTex). Blots were developed using Immobilon® Western Chemiluminescent HRP Substrate (WBKLS0500, Millipore).

Immunohistochemistry (IHC)

Mouse lungs and livers were fixed in excess 10% neutral-buffered formalin at 4°C with shaking overnight. Fixed lungs and livers were washed in ice-cold PBS, embedded in paraffin, and sectioned with a microtome set to 5 μm. Tissue sections were mounted on slides coated with amino-propyl-tri-ethoxy-silane and dried overnight. Dried slides were deparaffinized in xylene and rehydrated by immersing in an ethanol series from 100% to 70%, followed by washing in PBS containing 0.1% Tween-20. Antigen was retrieved by pressure-cooking slides in 10 mM sodium citrate. Slides were cooled and incubated with 0.3% hydrogen peroxidase in methanol. For blocking, sections were incubated in 1% BSA in PBS for 1h at room temperature. CD4⁺ T cells in sections were detectedby incubation with rabbit anti-mouse CD4 (D7D2Z, Cell Signaling) overnight at 4°C, followed by incubation with secondary antibodies and the ImmPRESSPolymer Detection Kit (MP-7801, Vector Labs) according to the manufacturer’s instructions. Slides were counterstained with hematoxylin, mounted, and scanned using an image capture device (Leica SCN 400) at 40x magnification.

Single-cell RNA-sequencing (scRNA-seq.)

Purified B cells were left unstimulated or stimulated for 2 hr at 37°C with 1 µM CPG OND1668, or 1 µM CPG plus 5 µM p38 inhibitor SB203580. Viable single cells were harvested for single-cell analysis using the BD Rhapsody system (633802, BD Rhapsody™ WTA Reagent Kit 4 pack). Cells were labeled by incubation for 1hr on ice with BD™ Abseq antibody-oligo-conjugates recognizing B220 (RA3-6B2, 940110), CD5 (53-7.3, 940147), CD19 (1D3, 940111), CD21/CD35 (7G6, 940156), CD23 (B3B4, 940161), CD40 (3/23, 940143), CD80 (16-10A1, 940141), CD86 (GL1, 940129), CD95 (Jo2, 940134), CD138 (281-2, 940137), IgD (11-26c.2a, 940153) or MHCII (M5/114.15.2, 940123). Labeled cells were transferred into nano-well cartridges loaded with Cell Capture Beads (BD Rhapsody™) following the manufacturer’s protocol. The single-cell RNA library was then established according to the manufacturer’s instructions. The library containing 7754.72 molecule counts per gene per cell were recorded as a Fastqs file and uploaded to the Seven Bridges platform for data demultiplexed analysis (https://www.sevenbridges.com).

T cell purification and Th17 differentiation in vitro

Single-cell suspensions were prepared from mouse lymph nodes by sieving and gentle pipetting through 70-μm nylon mesh filters (Falcon; BD). Non-naïve CD4⁺ T cells were depleted by incubation with a cocktail of biotin-conjugated antibodies recognizing B220, CD8, NK1.1, DX5, CD44, CD25, Gr1, CD11b, CD11c, and Ter119 (BD), followed by enrichment by mixing with IMag^TM Streptavidin Particles Plus beads (557812, BD). The purity was between 94-98%. Pure naive CD4⁺ T cells were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine (Invitrogen), 100 U penicillin-streptomycin (Invitrogen), 100 mM Hepes (Sigma-Aldrich), 0.055 mM 2-mercaptoethanol (Gibco), and 10% FBS (Hyclone) for the below experiments. To quickly induce IL-17 production, CD4⁺ T cells were stimulated with 50 ng ml^-1 PMA (P1585, Sigma) and 500 ng ml^-1 Ionomycin (I9657, Sigma) in the presences of Brefedin A (1:1000, 555029, BD) and Monensin (1:1500, 554724, BD) for 5 h. The cells were then subjected to anti-IL-17 (TC11-18H10, 560220, BD) intracellular staining following the standard protocol. For Th17 differentiation assay in vitro, 1x10⁶ naive CD4⁺ T cells were cultured in 10%FBS/RPMI complete medium in 24-well plates precoated with 5 μg ml^-1anti-CD3e (100331, Biolegend) and 2 mg ml^-1 anti-CD28 (553294, BD) with the presence of 20 ng ml^-1 IL-2 (402-ML, R&D Systems) for 72 h. At day 3, B cell supernatants of 24 h-culture, together with 10 μg ml^-1 anti-IL-4 (504102, Biolegend) and 10 μg ml^-1anti-IFNγ (551216, BD), were added to T cell culture. In the meanwhile, 10 ng ml^-1TGF-β (7666-MB-005, R&D Systems) and 20 ng ml^-1IL-6 (406-ML-005, R&D Systems) were supplied to an independent T cell-culture as positive control. After the additional culture for 96 h, T cells were harvested and subjected to the IL-17 intracellular staining. To induce pathogenic Th17 cells, 1x10⁶naive CD4⁺ T cells were cultured as described above for 7 days. At day 7, 1 μg ml^-1soluble anti-CD3e, anti-CD28 and 20 ng ml^-1IL-23 (1887-ML-010, R&D Systems) were supplied to T cell-culture. After an additional culture for 72h, T cells were harvested and subjected to IL-17 intracellular staining.

Quantification and statistical analysis

Results were represented as mean ± S.E.M.. Each representative data was shown from at least two independent repeats. Statistical analysis was conducted by using GraphPad Prism v7.0 (GraphPad Software). Statistical differences were determined by unpaired two-tailed t-test with p-values ≤0.05.

Data Availability

Raw single cell seq. data derived from mice that support the findings of this study have been deposited at NIH GEO database with the access codes GSE179715. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Keenan, R.A. et al. Censoring of autoreactive B cell development by the pre-B cell receptor. Science. 321, 696–699 (2008).
Herzog, S., Reth, M. & Jumaa, H. Regulation of B-cell proliferation and differentiation by pre-B-cell receptor signalling. Nat. Rev. Immunol. 9, 195–205 (2009).
Wardemann, H. et al. Predominant autoantibody production by early human B cell precursors. Science. 301, 1374–1377 (2003).
Burman, C. & Ktistakis, N.T. Regulation of autophagy by phosphatidylinositol 3-phosphate. FEBS Lett 584, 1302–1312 (2010).
Hollander, M.C., Blumenthal, G.M. & Dennis, P.A. PTEN loss in the continuum of common cancers, rare syndromes and mouse models. Nat. Rev. Cancer 11, 289–301 (2011).
Malek, M. et al. PTEN regulates PI(3,4)P2 signaling downstream of class I PI3K. Mol. Cell 68, 566–580.e510 (2017).
Amin, R.H. & Schlissel, M.S. Foxo1 directly regulates the transcription of recombination-activating genes during B cell development. Nat. Immunol. 9, 613–622 (2008).
Dengler, H.S. et al. Distinct functions for the transcription factor Foxo1 at various stages of B cell differentiation. Nat. Immunol. 9, 1388–1398 (2008).
Shojaee, S. et al. PTEN opposes negative selection and enables oncogenic transformation of pre-B cells. Nat. Med. 22, 379–387 (2016).
Setz, C.S. et al. Pten controls B-cell responsiveness and germinal center reaction by regulating the expression of IgD BCR. Embo j 38 (2019).
Anzelon, A.N., Wu, H. & Rickert, R.C. Pten inactivation alters peripheral B lymphocyte fate and reconstitutes CD19 function. Nat. Immunol. 4, 287–294 (2003).
Suzuki, A. et al. Critical roles of Pten in B cell homeostasis and immunoglobulin class switch recombination. J. Exp. Med. 197, 657–667 (2003).
Matsushita, T. et al. A novel splenic B1 regulatory cell subset suppresses allergic disease through phosphatidylinositol 3-kinase–Akt pathway activation. J. Allergy Clin. Immunol. 138, 1170–1182.e1179 (2016).
Barr, T.A. et al. B cell depletion therapy ameliorates autoimmune disease through ablation of IL-6-producing B cells. J. Exp. Med. 209, 1001–1010 (2012).
Yeh, D.W., Huang, L.R., Chen, Y.W., Huang, C.F. & Chuang, T.H. Interplay between Inflammation and Stemness in Cancer Cells: The Role of Toll-Like Receptor Signaling. J. Immunol. Res. 2016, 4368101 (2016).
Wu, Y.-Y., Kumar, R., Haque, M.S., Castillejo-López, C. & Alarcón-Riquelme, M.E. BANK1 controls CpG-induced IL-6 secretion via a p38 and MNK1/2/eIF4E translation initiation pathway. J. Immunol. 191, 6110–6116 (2013).
Chaturvedi, A., Dorward, D. & Pierce, S.K. The B Cell Receptor Governs the Subcellular Location of Toll-like Receptor 9 Leading to Hyperresponses to DNA-Containing Antigens. Immunity 28, 799–809 (2008).
Kojima, H. et al. Differentiation stage-specific requirement in hypoxia-inducible factor-1alpha-regulated glycolytic pathway during murine B cell development in bone marrow. J Immunol 184, 154–163 (2010).
Farmer, J.R. et al. Induction of metabolic quiescence defines the transitional to follicular B cell switch. Sci Signal 12 (2019).
Doughty, C.A. et al. Antigen receptor-mediated changes in glucose metabolism in B lymphocytes: role of phosphatidylinositol 3-kinase signaling in the glycolytic control of growth. Blood 107, 4458–4465 (2006).
Caro-Maldonado, A. et al. Metabolic reprogramming is required for antibody production that is suppressed in anergic but exaggerated in chronically BAFF-exposed B cells. J Immunol 192, 3626–3636 (2014).
Waters, L.R., Ahsan, F.M., Wolf, D.M., Shirihai, O. & Teitell, M.A. Initial B Cell Activation Induces Metabolic Reprogramming and Mitochondrial Remodeling. iScience 5, 99–109 (2018).
Weisel, F.J. et al. Germinal center B cells selectively oxidize fatty acids for energy while conducting minimal glycolysis. Nat Immunol 21, 331–342 (2020).
Shimabukuro-Vornhagen, A. et al. Inhibition of protein geranylgeranylation specifically interferes with CD40-dependent B cell activation, resulting in a reduced capacity to induce T cell immunity. J Immunol 193, 5294–5305 (2014).
Kwon, K. et al. Instructive role of the transcription factor E2A in early B lymphopoiesis and germinal center B cell development. Immunity 28, 751–762 (2008).
Lesche, R. et al. Cre/loxP-mediated inactivation of the murine Pten tumor suppressor gene. Genesis 32, 148–149 (2002).
Lai, M. et al. Regulation of B-cell development and tolerance by different members of the miR-17∼92 family microRNAs. Nat. Commun. 7, 12207–12207 (2016).
Lee, Y. et al. Induction and molecular signature of pathogenic TH17 cells. Nat Immunol 13, 991–999 (2012).
McGeachy, M.J. et al. TGF-beta and IL-6 drive the production of IL-17 and IL-10 by T cells and restrain T(H)-17 cell-mediated pathology. Nat Immunol 8, 1390–1397 (2007).
Revu, S. et al. IL-23 and IL-1β Drive Human Th17 Cell Differentiation and Metabolic Reprogramming in Absence of CD28 Costimulation. Cell Rep 22, 2642–2653 (2018).
Leifer, C.A. et al. TLR9 is localized in the endoplasmic reticulum prior to stimulation. J Immunol 173, 1179–1183 (2004).
Jeschke, A. et al. Phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate regulate phagolysosome biogenesis. Proc Natl Acad Sci U S A 112, 4636–4641 (2015).
Wallroth, A. & Haucke, V. Phosphoinositide conversion in endocytosis and the endolysosomal system. J Biol Chem 293, 1526–1535 (2018).
Porstmann, T. et al. SREBP activity is regulated by mTORC1 and contributes to Akt-dependent cell growth. Cell Metab 8, 224–236 (2008).
Rodwell, V.W., Nordstrom, J.L. & Mitschelen, J.J. Regulation of HMG-CoA Reductase. In: Paoletti, R. & Kritchevsky, D. (eds). Advances in Lipid Research, vol. 14. Elsevier, 1976, pp 1–74.
Gill, S., Stevenson, J., Kristiana, I. & Brown, A.J. Cholesterol-dependent degradation of squalene monooxygenase, a control point in cholesterol synthesis beyond HMG-CoA reductase. Cell Metab 13, 260–273 (2011).
Spanova, M. et al. Influence of squalene on lipid particle/droplet and membrane organization in the yeast Saccharomyces cerevisiae. Biochim Biophys Acta 1821, 647–653 (2012).
Araldi, E. et al. Lanosterol Modulates TLR4-Mediated Innate Immune Responses in Macrophages. Cell Rep 19, 2743–2755 (2017).
Kremlitzka, M., Mácsik-Valent, B. & Erdei, A. Syk is indispensable for CpG-induced activation and differentiation of human B cells. Cell. Mol. Life Sci. 72, 2223–2236 (2015).
Latz, E. et al. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat Immunol 5, 190–198 (2004).
Kim, Y.M., Brinkmann, M.M., Paquet, M.E. & Ploegh, H.L. UNC93B1 delivers nucleotide-sensing toll-like receptors to endolysosomes. Nature 452, 234–238 (2008).
Bonham, K.S. et al. A promiscuous lipid-binding protein diversifies the subcellular sites of toll-like receptor signal transduction. Cell 156, 705–716 (2014).
Park, B.S. et al. The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. Nature 458, 1191–1195 (2009).
Gewinner, C. et al. Evidence that inositol polyphosphate 4-phosphatase type II is a tumor suppressor that inhibits PI3K signaling. Cancer Cell 16, 115–125 (2009).
Chen, L. et al. SYK inhibition modulates distinct PI3K/AKT- dependent survival pathways and cholesterol biosynthesis in diffuse large B cell lymphomas. Cancer Cell 23, 826–838 (2013).
Ramesh, A. et al. A pathogenic and clonally expanded B cell transcriptome in active multiple sclerosis. Proc. Natl. Acad. Sci. USA 117, 22932–22943 (2020).
Bibby, J.A. et al. Cholesterol metabolism drives regulatory B cell IL-10 through provision of geranylgeranyl pyrophosphate. Nat. Commun. 11, 3412 (2020).
Hobeika, E. et al. Testing gene function early in the B cell lineage in mb1-cre mice. Proc. Natl. Acad. Sci. USA 103, 13789–13794 (2006).
Akkaya, M. et al. Toll-like receptor 9 antagonizes antibody affinity maturation. Nat. Immunol. 19, 255–266 (2018).
Subramanian, A. et al. Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles. Proceedings of the National Academy of Sciences 102, 15545 (2005).
Bolte, S. & Cordelières, F.P. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213–232 (2006).

Table 1

A comparison of phenotypes between PTEN-deficient B cells from CD23-cKO and mb1-cKO mice.
PTEN-deficient B cells	CD23-cKO mice	mb-1-cKO mice
TLR9-mediated IL-6	increased	unchanged
Lysosomal localization of TLR9-p38 complex	increased	unchanged
PI(3)P level	increased	unchanged
Cholesterol biosynthesis (mRNA)	increased	weakly induced
Lipid droplets	increased	reduced
Surface IgM	normal	nearly absent
IgM-mediated endocytosis	normal	absent

There is NO Competing Interest.

SupplementalFiguresandLegends20230222.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

PTEN acts as a metabolic checkpoint molecule in mature B cells to suppress TLR9-mediated inflammation

Status:

Version 1

Abstract

Figures

Introduction

Results

Shortened lifespan of mice with PTEN deletion exclusively in mature B cells.

CD23-cKO mice develop systemic inflammation with increased IL-17

Deletion of PTEN in mature B cells leads to a strong induction of TLR9-mediated IL-6

PTEN deficiency enhances the lysosomal trafficking of TLR9 signalosome without the engagement of BCR and TLR9

TLR9 was colocalized with endosomal PI(3)P in PTEN-deficient B cells in the steady state

Discussion

Methods

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1