Expression of USP25 associates with fibrosis, inflammation and metabolism changes in IgG4-related disease.

IgG4-related disease (IgG4-RD) has complex clinical manifestations ranging from fibrosis and inflammation to deregulated metabolism. The molecular mechanisms underpinning these phenotypes are unclear. In this study, by using IgG4-RD patient peripheral blood mononuclear cells (PBMCs), IgG4-RD cell lines and Usp25 knockout mice, we show that ubiquitin-specific protease 25 (USP25) engages in multiple pathways to regulate fibrotic and inflammatory pathways that are characteristic to IgG4-RD. Reduced USP25 expression in IgG4-RD leads to increased SMAD3 activation, which contributes to fibrosis and induces inflammation through the IL-1ß inflammatory axis. Mechanistically, USP25 prevents ubiquitination of RAC1, thus, downregulation of USP25 leads to ubiquitination and degradation of RAC1. Decreased RAC1 levels result in reduced aldolase A release from the actin cytoskeleton, which then lowers glycolysis. The expression of LYN, a component of the B cell receptor signalosome is also reduced in USP25-deficient B cells, which might result in B cell activation deficiency. Altogether, our results indicate a potential anti-inflammatory and anti-fibrotic role for USP25 and make USP25 a promising diagnostic marker and potential therapeutic target in IgG4-RD.

The association of an elevated Th/Ts ratio and lupus anticoagulant with symptomatic osteonecrosis in systemic lupus erythematosus patients.

Objective: This study aimed to assess the risk factors for symptomatic osteonecrosis (ON) in systemic lupus erythematosus (SLE) and identify clinical characteristics and laboratory markers for predicting symptomatic ON occurrence in SLE patients. Methods: Seventy (6.0%) of 1175 SLE patients diagnosed with symptomatic ON were included in this study. An equal number of SLE patients without symptomatic ON, matched in terms of age and gender, were enrolled in the control group. Clinical symptoms, routine laboratory examinations, lymphocyte subsets, and treatments of these patients were retrospectively reviewed and compared between the two groups. Logistic regression analysis was employed to identify risk factors associated with symptomatic ON in SLE. Results: Among the 70 cases in the symptomatic ON group, 62 (88.6%) patients experienced femoral head necrosis, with bilateral involvement observed in 58 patients. Bone pain was reported in 32 cases (51.6%), and 19 cases (30.6%) presented with multiple symptoms. Univariate analysis revealed significant differences between the two groups in various factors, including disease duration (months), cumulative steroid exposure time, history of thrombosis, neurological involvement, the number of affected organs, myalgia/myasthenia, and the use of medications such as glucocorticoids, immunosuppressants, aspirin, and statins (P<0.05). Moreover, lupus anticoagulant (LA) levels were significantly higher in the symptomatic ON group than in the control group (P<0.05). Furthermore, notable distinctions were observed in peripheral blood immune cells, including an elevated white blood cell count (WBC), a decreased percentage of Ts cells (CD3+CD8+), and an elevated Th/Ts ratio. Logistic regression analysis revealed that a history of thrombosis, LA positivity, and an elevated Th/Ts ratio remained positive factors associated with symptomatic ON (P<0.05). Conclusion: Decreased Ts cells and changes in the T lymphocyte subset play an important regulatory role in the development of symptomatic ON. A history of thrombosis and LA are associated with an increased probability of symptomatic ON in SLE and may serve as potential predictors.

Ultra-high static magnetic fields cause immunosuppression through disrupting B-cell peripheral differentiation and negatively regulating BCR signaling.

To increase the imaging resolution and detection capability, the field strength of static magnetic fields (SMFs) in magnetic resonance imaging (MRI) has significantly increased in the past few decades. However, research on the side effects of high magnetic field is still very inadequate and the effects of SMF above 1 T (Tesla) on B cells have never been reported. Here, we show that 33.0 T ultra-high SMF exposure causes immunosuppression and disrupts B cell differentiation and signaling. 33.0 T SMF treatment resulted in disturbance of B cell peripheral differentiation and antibody secretion and reduced the expression of IgM on B cell membrane, and these might be intensity dependent. In addition, mice exposed to 33.0 T SMF showed inhibition on early activation of B cells, including B cell spreading, B cell receptor clustering and signalosome recruitment, and depression of both positive and negative molecules in the proximal BCR signaling, as well as impaired actin reorganization. Sequencing and gene enrichment analysis showed that SMF stimulation also affects splenic B cells' transcriptome and metabolic pathways. Therefore, in the clinical application of MRI, we should consider the influence of SMF on the immune system and choose the optimal intensity for treatment.

Involvement of MST1/mTORC1/STAT1 activity in the regulation of B-cell receptor signalling by chemokine receptor 2.

BACKGROUND: CCR2 is involved in maintaining immune homeostasis and regulating immune function. This study aims to elucidate the mechanism by which CCR2 regulates B-cell signalling. METHODS: In Ccr2-knockout mice, the development and differentiation of B cells, BCR proximal signals, actin movement and B-cell immune response were determined. Besides, the level of CCR2 in PBMC of SLE patients was analysed by bioinformatics. RESULTS: CCR2 deficiency reduces the proportion and number of follicular B cells, upregulates BCR proximal signalling and enhances the oxidative phosphorylation of B cells. Meanwhile, increased actin filaments aggregation and its associated early-activation events of B cells are also induced by CCR2 deficiency. The MST1/mTORC1/STAT1 axis in B cells is responsible for the regulation of actin remodelling, metabolic activities and transcriptional signalling, specific MST1, mTORC1 or STAT1 inhibitor can rescue the upregulated BCR signalling. Glomerular IgG deposition is obvious in CCR2-deficient mice, accompanied by increased anti-dsDNA IgG level. Additionally, the CCR2 expression in peripheral B cells of SLE patients is decreased than that of healthy controls. CONCLUSIONS: CCR2 can utilise MST1/mTORC1/STAT1 axis to regulate BCR signalling. The interaction between CCR2 and BCR may contribute to exploring the mechanism of autoimmune diseases.

Abelson tyrosine kinase controls BCR signalling and B-cell differentiation by promoting B-cell metabolism.

As a nonreceptor tyrosine kinase, Abelson tyrosine kinase (c-Abl) was first studied in chronic myelogenous leukaemia, and its role in lymphocytes has been well characterised. c-Abl is involved in B-cell development and CD19-associated B-cell antigen receptor (BCR) signalling. Although c-Abl regulates different metabolic pathways, the role of c-Abl is still unknown in B-cell metabolism. In this study, B-cell-specific c-Abl knockout (KO) mice (Mb1Cre+/- c-Ablfl/fl ) were used to investigate how c-Abl regulates B-cell metabolism and BCR signalling. We found that the levels of activation positive BCR signalling proximal molecules, phosphorylated spleen tyrosine kinase (pSYK) and phosphorylated Bruton tyrosine kinase (pBTK), were decreased, while the level of key negative regulator, phosphorylated SH2-containing inositol phosphatase 1 (pSHIP1), was increased in Mb1Cre+/- c-Ablfl/fl mice. Furthermore, we found c-Abl deficiency weakened the B-cell spreading, formation of BCR signalosomes, and the polymerisation of actin during BCR activation, and also impaired the differentiation of germinal center (GC) B-cells both in quiescent condition and after immunisation. Moreover, B-cell mitochondrial respiration and the expression of B-cell metabolism-regulating molecules were downregulated in c-Abl deficiency mice. Overall, c-Abl, which involved in actin remodelling and B-cell metabolism, positively regulates BCR signalling and promotes GC differentiation.

SARS-CoV-2 infection causes immunodeficiency in recovered patients by downregulating CD19 expression in B cells via enhancing B-cell metabolism.

The SARS-CoV-2 infection causes severe immune disruption. However, it is unclear if disrupted immune regulation still exists and pertains in recovered COVID-19 patients. In our study, we have characterized the immune phenotype of B cells from 15 recovered COVID-19 patients, and found that healthy controls and recovered patients had similar B-cell populations before and after BCR stimulation, but the frequencies of PBC in patients were significantly increased when compared to healthy controls before stimulation. However, the percentage of unswitched memory B cells was decreased in recovered patients but not changed in healthy controls upon BCR stimulation. Interestingly, we found that CD19 expression was significantly reduced in almost all the B-cell subsets in recovered patients. Moreover, the BCR signaling and early B-cell response were disrupted upon BCR stimulation. Mechanistically, we found that the reduced CD19 expression was caused by the dysregulation of cell metabolism. In conclusion, we found that SARS-CoV-2 infection causes immunodeficiency in recovered patients by downregulating CD19 expression in B cells via enhancing B-cell metabolism, which may provide a new intervention target to cure COVID-19.

Ubiquitin-specific peptidase 18 regulates the differentiation and function of Treg cells.

Ubiquitin-specific peptidase 18 (USP18) plays an important role in the development of CD11b+ dendritic cells (DCs) and Th17 cells, however, its role in the differentiation of other T cell subsets, especially in regulatory T (Treg) cells, is unknown. In our study, we used Usp18 KO mice to study the loss of USP18 on the impact of Treg cell differentiation and function. We found that USP18 deficiency upregulates the differentiation of Treg cells, which may lead to disrupted homeostasis of peripheral T cells, and downregulates INF-γ, IL-2, IL-17A producing CD4+ T cells and INF-γ producing CD8+ T cells. Mechanistically, we also found that the upregulation of Tregs is due to elevated expression of CD25 in Usp18 KO mice. Finally, we found that the suppressive function of Usp18 KO Tregs is downregulated. Altogether, our study was the first to identify the role of USP18 in Tregs differentiation and its suppressive function, which may provide a new reference for the treatment of Treg function in many autoimmune diseases, and USP18 can be used as a new therapeutic target for precise medical treatment.

CCL2 regulation of MST1-mTOR-STAT1 signaling axis controls BCR signaling and B-cell differentiation.

Chemokines are important regulators of the immune system, inducing specific cellular responses by binding to receptors on immune cells. In SLE patients, decreased expression of CCL2 on mesenchymal stem cells (MSC) prevents inhibition of B-cell proliferation, causing the characteristic autoimmune phenotype. Nevertheless, the intrinsic role of CCL2 on B-cell autoimmunity is unknown. In this study using Ccl2 KO mice, we found that CCL2 deficiency enhanced BCR signaling by upregulating the phosphorylation of the MST1-mTORC1-STAT1 axis, which led to reduced marginal zone (MZ) B cells and increased germinal center (GC) B cells. The abnormal differentiation of MZ and GC B cells were rescued by in vivo inhibition of mTORC1. Additionally, the inhibition of MST1-mTORC1-STAT1 with specific inhibitors in vitro also rescued the BCR signaling upon antigenic stimulation. The deficiency of CCL2 also enhanced the early activation of B cells including B-cell spreading, clustering and signalosome recruitment by upregulating the DOCK8-WASP-actin axis. Our study has revealed the intrinsic role and underlying molecular mechanism of CCL2 in BCR signaling, B-cell differentiation, and humoral response.

Corrigendum: Akt2 Regulates the Differentiation and Function of NKT17 Cells via FoxO-1-ICOS Axis.

[This corrects the article DOI: 10.3389/fimmu.2018.01940.].

DOCK2 couples with LEF-1 to regulate B cell metabolism and memory response.

Dedicator of cytokinesis 2 (DOCK2) is essential for the B cell differentiation, BCR signaling and humoral immune response. However, the role of DOCK2 in the memory response of B cell is unknown. By using two DOCK2 deficient patients, we found that the memory B cells were decreased and the early activation of DOCK2 deficient memory B cells was abolished to the degree of naïve B cells due to the decreased expression of CD19 and CD21 mechanistically. Interestingly the expression of LEF-1, a negative regulator of CD21, was increased in DOCK2 deficient B cells. This was linked to the increased expression of HIF-1α and cell metabolism, which in turn affected the ER structure. Finally, the reduction of memory B cells in DOCK2 patients was due to the increased apoptosis, which might be related with the increased metabolism.

STING couples with PI3K to regulate actin reorganization during BCR activation.

The adaptor protein, STING (stimulator of interferon genes), has been rarely studied in adaptive immunity. We used Sting KO mice and a patient's mutated STING cells to study the effect of STING deficiency on B cell development, differentiation, and BCR signaling. We found that STING deficiency promotes the differentiation of marginal zone B cells. STING is involved in BCR activation and negatively regulates the activation of CD19 and Btk but positively regulates the activation of SHIP. The activation of WASP and accumulation of F-actin were enhanced in Sting KO B cells upon BCR stimulation. Mechanistically, STING uses PI3K mediated by the CD19-Btk axis as a central hub for controlling the actin remodeling that, in turn, offers feedback to BCR signaling. Overall, our study provides a mechanism of how STING regulates BCR signaling via feedback from actin reorganization, which contributes to positive regulation of STING on the humoral immune response.

Correction to: CX3CR1 positively regulates BCR signaling coupled with cell metabolism via negatively controlling actin remodeling.

In the original published version of the article, the red squares in the figures which indicated the corrections.

CX3CR1 positively regulates BCR signaling coupled with cell metabolism via negatively controlling actin remodeling.

As an important chemokine receptor, the role of CX3CR1 has been studied extensively on the migration of lymphocytes including T and B cells. Although CX3CR1+ B cells have immune suppressor properties, little is known about its role on the regulation of BCR signaling and B cell differentiation as well as the underlying molecular mechanism. We have used CX3CR1 KO mice to study the effect of CX3CR1 deficiency on BCR signaling and B cell differentiation. Interestingly, we found that proximal BCR signaling, such as the activation of CD19, BTK and SHIP was reduced in CX3CR1 KO B cells upon antigenic stimulation. However, the activation of mTORC signaling was enhanced. Mechanistically, we found that the reduced BCR signaling in CX3CR1 KO B cells was due to reduced BCR clustering, which is caused by the enhanced actin accumulation by the plasma membrane via increased activation of WASP. This caused an increased differentiation of MZ B cells in CX3CR1 KO mice and an enhanced generation of plasma cells (PC) and antibodies. Our study shows that CX3CR1 regulates BCR signaling via actin remodeling and affects B cell differentiation and the humoral immune response.

Dedicator of cytokinesis protein 2 couples with lymphoid enhancer-binding factor 1 to regulate expression of CD21 and B-cell differentiation.

BACKGROUND: B-cell receptor (BCR) signaling, combined with CD19 and CD21 signals, imparts specific control of B-cell responses. Dedicator of cytokinesis protein 2 (DOCK2) is critical for the migration and motility of lymphocytes. Although absence of DOCK2 leads to lymphopenia, little is known about the signaling mechanisms and physiologic functions of DOCK2 in B cells. OBJECTIVE: We sought to determine the underlying molecular mechanism of how DOCK2 regulates BCR signaling and peripheral B-cell differentiation. METHODS: In this study we used genetic models for DOCK2, Wiskott-Aldrich syndrome protein (WASP), and lymphoid enhancer-binding factor 1 deficiency to study their interplay in BCR signaling and B-cell differentiation. RESULTS: We found that the absence of DOCK2 led to downregulation of proximal and distal BCR signaling molecules, including CD19, but upregulation of SH2-containing inositol 5 phosphatase 1, a negative signaling molecule. Interestingly, DOCK2 deficiency reduced CD19 and CD21 expression at the mRNA and/or protein levels and was associated with reduced numbers of marginal zone B cells. Additionally, loss of DOCK2 reduced activation of WASP and accelerated degradation of WASP, resulting into reduced actin accumulation and early activation of B cells. Mechanistically, the absence of DOCK2 upregulates the expression of lymphoid enhancer-binding factor 1. These differences were associated with altered humoral responses in the absence of DOCK2. CONCLUSIONS: Overall, our study has provided a novel underlying molecular mechanism of how DOCK2 deficiency regulates surface expression of CD21, which leads to downregulation of CD19-mediated BCR signaling and marginal zone B-cell differentiation.

MARCKS regulates tonic and chronic active B cell receptor signaling.

Tonic or chronic active B-cell receptor (BCR) signaling is essential for the survival of normal or some malignant B cells, respectively. However, the molecular mechanism regulating the strength of these two types of BCR signaling remains unknown. Here, using high-speed high-resolution single-molecule tracking in live cells, we identified that PKCß, STIM1, and IP3R1/2/3 molecules affected the lateral Brownian mobile behavior of BCRs on the plasma membrane of quiescent B cells, which was correlated to the strength of BCR signaling. Further mechanistic studies revealed that these three molecules influenced BCR mobility by regulating the membrane tethering of MARCKS to the inner leaflet of the plasma membrane. Indeed, membrane-untethered or deficiency of MARCKS significantly decreased, while membrane-tethered or overexpression of MARCKS drastically increased the lateral mobility of BCRs. Functional experiments indicated that the membrane-tethered MARCKS suppressed the survival and/or proliferation in both B-cell tumor cells and mouse primary splenic B cells in vitro and in vivo. Mechanistically, we found that membrane-tethered MARCKS increased BCR lateral mobility, and thus decreased BCR nanoclustering by disturbing the interaction between cortical F-actin and the inner leaflet of the plasma membrane, resulting in the suppression of the strength of both tonic and chronic active BCR signaling. Conclusively, MARCKS is a newly identified molecule regulating the strength of BCR signaling by modulating cytoskeleton and plasma membrane interactions, both in the physiological and pathological conditions, suggesting that MARCKS is a putative target for drug design.

The regulators of BCR signaling during B cell activation.

B lymphocytes produce antibodies under the stimulation of specific antigens, thereby exerting an immune effect. B cells identify antigens by their surface B cell receptor (BCR), which upon stimulation, directs the cell to activate and differentiate into antibody generating plasma cells. Activation of B cells via their BCRs involves signaling pathways that are tightly controlled by various regulators. In this review, we will discuss three major BCR mediated signaling pathways (the PLC-γ2 pathway, PI3K pathway and MAPK pathway) and related regulators, which were roughly divided into positive, negative and mutual-balanced regulators, and the specific regulators of the specific signaling pathway based on regulatory effects.

Akt2 Regulates the Differentiation and Function of NKT17 Cells via FoxO-1-ICOS Axis.

As a critical linker between mTORC1 and mTORC2, Akt is important for the cell metabolism. The role of Akt in the function and development of B and T cells is well characterized, however, the role of Akt for development and function of iNKT cells is unknown. iNKT cells bridge the adaptive and innate immunity, and in this study, we found that the differentiation of NKT17 cells and IL17 production of NKT17 cells were disrupted in Akt2 KO mice. ICOS has been demonstrated to be critical for the differentiation of NKT17 cells and we found that ICOS mRNA and protein expression was reduced in Akt2 KO iNKT cells. As a consequence, phosphorylation of FoxO-1 was downregulated in Akt2 KO thymocytes but the sequestration of FoxO-1 in the nucleus of Akt2 KO iNKT cells was increased. The negative feedback loop between ICOS and FoxO-1 has been demonstrated in CD4+T follicular helper cells. Therefore our study has revealed a new intracellular mechanism in which Akt2 regulates ICOS expression via FoxO-1 and this signaling axis regulates the differentiation and function of NKT17 cells. This study provides a new linker between cell metabolism and function of iNKT cells.

Dock8 regulates BCR signaling and activation of memory B cells via WASP and CD19.

Dock8 deficiency leads to immunodeficiency, and the role of Dock8 in B-cell development and function has been revealed; however, the role of DocK8 on B-cell receptor (BCR) signaling and function of memory B cells remains elusive. In this study, we generated a Dock8 knockout mouse model and collected peripheral blood mononuclear cells from Dock8 patients to study the effect of Dock8 deficiency on the BCR signaling and activation of memory B cells with confocal microscopy and total internal reflection fluorescence microscopy. The activation of key, positive upstream BCR signaling molecules, pCD19 and phosphorylated Brutons tyrosine kinase (pBtk), is reduced. Interestingly, the total protein and activated levels of Wiskott-Aldrich syndrome protein (WASP) are decreased in Dock8-deficient mouse B cells. Our previous research has shown that WASP positively regulates cd19 transcription; furthermore, we found that Dock8 regulates cd19 transcription. What we found in Dock8 patients can be a phenotype copied from Dock8 mice. The early activation of memory B cells from Dock8 patients is disrupted with reduced BCR clustering, B-cell spreading, and signalosome recruitment into the degree of naïve B cells, as well as the transition from naïve B cells to unswitched memory B cells. Overall, our study provides a novel mechanism for Dock8 regulation of BCR signaling by regulating cd19 transcription, as well as the underlying mechanism of noncompetence of memory B cells in Dock8 patients.

The Role of Mst1 in Lymphocyte Homeostasis and Function.

The Hippo pathway is an evolutionarily conserved pathway crucial for regulating tissue size and for limiting cancer development. However, recent work has also uncovered key roles for the mammalian Hippo kinases, Mst1/2, in driving appropriate immune responses by directing T cell migration, morphology, survival, differentiation, and activation. In this review, we discuss the classical signaling pathways orchestrated by the Hippo signaling pathway, and describe how Mst1/2 direct T cell function by mechanisms not seeming to involve the classical Hippo pathway. We also discuss why Mst1/2 might have different functions within organ systems and the immune system. Overall, understanding how Mst1/2 transmit signals to discrete biological processes in different cell types might allow for the development of better drug therapies for the treatments of cancers and immune-related diseases.