CP-25 inhibits the hyperactivation of rheumatic synoviocytes by suppressing the switch in Gαs-Gαi coupling to the ß2-adrenergic receptor.

In essence, the ß2 adrenergic receptor (ß2AR) plays an antiproliferative role by increasing the intracellular cyclic 3',5'-adenosine monophosphate (cAMP) concentration through Gαs coupling, but interestingly, ß2AR antagonists are able to effectively inhibit fibroblast-like synoviocytes (FLSs) proliferation, thus ameliorating experimental RA, indicating that the ß2AR signalling pathway is impaired in RA FLSs via unknown mechanisms. The local epinephrine (Epi) level was found to be much higher in inflammatory joints than in normal joints, and high-level stimulation with Epi or isoproterenol (ISO) directly promoted FLSs proliferation and migration due to impaired ß2AR signalling and cAMP production. By applying inhibitor of receptor internalization, and small interfering RNA (siRNA) of Gαs and Gαi, and by using fluorescence resonance energy transfer and coimmunoprecipitation assays, a switch in Gαs-Gαi coupling to ß2AR was observed in inflammatory FLSs as well as in FLSs with chronic ISO stimulation. This Gαi coupling was then revealed to be initiated by G protein coupled receptor kinase 2 (GRK2) but not ß-arrestin2 or protein kinase A-mediated phosphorylation of ß2AR. Inhibiting the activity of GRK2 with the novel GRK2 inhibitor paeoniflorin-6'-O-benzene sulfonate (CP-25), a derivative of paeoniflorin, or the accepted GRK2 inhibitor paroxetine effectively reversed the switch in Gαs-Gαi coupling to ß2AR during inflammation and restored the intracellular cAMP level in ISO-stimulated FLSs. As expected, CP-25 significantly inhibited the hyperplasia of FLSs in a collagen-induced arthritis (CIA) model (CIA FLSs) and normal FLSs stimulated with ISO and finally ameliorated CIA in rats. Together, our findings revealed the pathological changes in ß2AR signalling in CIA FLSs, determined the underlying mechanisms and identified the pharmacological target of the GRK2 inhibitor CP-25 in treating CIA. Video Abstract.

Attenuation of experimental osteoarthritis with human adipose-derived mesenchymal stem cell therapy: inhibition of the pyroptosis in chondrocytes.

AIM: To explore the role and mechanism of human adipose-derived mesenchymal stem cells (hAD-MSCs) in the treatment of osteoarthritis (OA). METHODS: OA hulth model of Sprague Dawley (SD) rats and 20 ng/ml TNF-α treated chondrocytes were used as models of OA in vivo and in vitro, respectively. hAD-MSCs were administrated in the articular cavity by injection in vivo and co-cultured with chondrocytes using transwell in vitro. Haematoxylin and eosin staining and Safranin-O/Fast green staining were performed to detect tissue destruction and histopathology. Scanning electron microscopy and transmission electron microscopy were used to observe the ultrastructure of chondrocytes. The pyroptosis signaling pathway-related proteins were detected by immunohistochemistry, immunofluorescence, qRT-PCR and Western blot. And small interference technology was used to study the mechanism in depth. RESULTS: hAD-MSCs could delay the development of rat OA, improve the pathological changes of joints, inhibit the expression of NLRP3, Caspase1, GSDMD and TNFR1. In vitro, the expression of pyroptosis signal proteins in chondrocytes was significantly elevated when stimulated with TNF-α, the level of inflammatory factors such as IL-1ß, IL-18 was increased, and the cell morphology was significantly destroyed. While co-cultured with hAD-MSCs, these syndromes were reversed. Knockout of TNFR1 also returned the upregulation of pyroptosis signals which caused by TNF-α. CONCLUSION: These results demonstrated that hAD-MSCs could inhibit pyroptosis signaling pathway of chondrocytes induced by TNF-α, which have raised our understanding of the role of hAD-MSCs as promising therapy for the management of OA.

Chronic ß-adrenergic stress contributes to cardiomyopathy in rodents with collagen-induced arthritis.

Patients with rheumatoid arthritis (RA) have a much higher incidence of cardiac dysfunction, which contributes to the high mortality rate of RA despite anti-arthritic drug therapy. In this study, we investigated dynamic changes in cardiac function in classic animal models of RA and examined the potential effectors of RA-induced heart failure (HF). Collagen-induced arthritis (CIA) models were established in rats and mice. The cardiac function of CIA animals was dynamically monitored using echocardiography and haemodynamics. We showed that cardiac diastolic and systolic dysfunction occurred in CIA animals and persisted after joint inflammation and that serum proinflammatory cytokine (IL-1ß, TNF-α) levels were decreased. We did not find evidence of atherosclerosis (AS) in arthritic animals even though cardiomyopathy was significant. We observed that an impaired cardiac ß1AR-excitation contraction coupling signal was accompanied by sustained increases in blood epinephrine levels in CIA rats. Furthermore, serum epinephrine concentrations were positively correlated with the heart failure biomarker NT-proBNP in RA patients (r2 = +0.53, P < 0.0001). In CIA mice, treatment with the nonselective ßAR blocker carvedilol (2.5 mg·kg-1·d-1, for 4 weeks) or the specific GRK2 inhibitor paroxetine (2.5 mg·kg-1·d-1, for 4 weeks) effectively rescued heart function. We conclude that chronic and persistent ß-adrenergic stress in CIA animals is a significant contributor to cardiomyopathy, which may be a potential target for protecting RA patients against HF.

Mesenchymal stem cells, as glioma exosomal immunosuppressive signal multipliers, enhance MDSCs immunosuppressive activity through the miR-21/SP1/DNMT1 positive feedback loop.

BACKGROUND: The immunosuppressive microenvironment in glioma induces immunotherapy resistance and is associated with poor prognosis. Glioma-associated mesenchymal stem cells (GA-MSCs) play an important role in the formation of the immunosuppressive microenvironment, but the mechanism is still not clear. RESULTS: We found that GA-MSCs promoted the expression of CD73, an ectonucleotidase that drives immunosuppressive microenvironment maintenance by generating adenosine, on myeloid-derived suppressor cells (MDSCs) through immunosuppressive exosomal miR-21 signaling. This process was similar to the immunosuppressive signaling mediated by glioma exosomal miR-21 but more intense. Further study showed that the miR-21/SP1/DNMT1 positive feedback loop in MSCs triggered by glioma exosomal CD44 upregulated MSC exosomal miR-21 expression, amplifying the glioma exosomal immunosuppressive signal. Modified dendritic cell-derived exosomes (Dex) carrying miR-21 inhibitors could target GA-MSCs and reduce CD73 expression on MDSCs, synergizing with anti-PD-1 monoclonal antibody (mAb). CONCLUSIONS: Overall, this work reveals the critical role of MSCs in the glioma microenvironment as signal multipliers to enhance immunosuppressive signaling of glioma exosomes, and disrupting the positive feedback loop in MSCs with modified Dex could improve PD-1 blockade therapy.

EWSR1-induced circNEIL3 promotes glioma progression and exosome-mediated macrophage immunosuppressive polarization via stabilizing IGF2BP3.

BACKGROUND: Gliomas are the most common malignant primary brain tumours with a highly immunosuppressive tumour microenvironment (TME) and poor prognosis. Circular RNAs (circRNA), a newly found type of endogenous noncoding RNA, characterized by high stability, abundance, conservation, have been shown to play an important role in the pathophysiological processes and TME remodelling of various tumours. METHODS: CircRNA sequencing analysis was performed to explore circRNA expression profiles in normal and glioma tissues. The biological function of a novel circRNA, namely, circNEIL3, in glioma development was confirmed both in vitro and in vivo. Mechanistically, RNA pull-down, mass spectrum, RNA immunoprecipitation (RIP), luciferase reporter, and co-immunoprecipitation assays were conducted. RESULTS: We identified circNEIL3, which could be cyclized by EWS RNA-binding protein 1(EWSR1), to be upregulated in glioma tissues and to correlate positively with glioma malignant progression. Functionally, we confirmed that circNEIL3 promotes tumorigenesis and carcinogenic progression of glioma in vitro and in vivo. Mechanistically, circNEIL3 stabilizes IGF2BP3 (insulin-like growth factor 2 mRNA binding protein 3) protein, a known oncogenic protein, by preventing HECTD4-mediated ubiquitination. Moreover, circNEIL3 overexpression glioma cells drives macrophage infiltration into the tumour microenvironment (TME). Finally, circNEIL3 is packaged into exosomes by hnRNPA2B1 and transmitted to infiltrated tumour associated macrophages (TAMs), enabling them to acquire immunosuppressive properties by stabilizing IGF2BP3 and in turn promoting glioma progression. CONCLUSIONS: This work reveals that circNEIL3 plays a nonnegligible multifaceted role in promoting gliomagenesis, malignant progression and macrophage tumour-promoting phenotypes polarization, highlighting that circNEIL3 is a potential prognostic biomarker and therapeutic target in glioma.

GRK5 Controls SAP97-Dependent Cardiotoxic ß1 Adrenergic Receptor-CaMKII Signaling in Heart Failure.

RATIONALE: Cardiotoxic ß1 adrenergic receptor (ß1AR)-CaMKII (calmodulin-dependent kinase II) signaling is a major and critical feature associated with development of heart failure. SAP97 (synapse-associated protein 97) is a multifunctional scaffold protein that binds directly to the C-terminus of ß1AR and organizes a receptor signalosome. OBJECTIVE: We aim to elucidate the dynamics of ß1AR-SAP97 signalosome and its potential role in chronic cardiotoxic ß1AR-CaMKII signaling that contributes to development of heart failure. METHODS AND RESULTS: The integrity of cardiac ß1AR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine ß1AR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the ß1AR-SAP97 signaling complex is reduced in heart failure. Cardiac-specific deletion of SAP97 yields an aging-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA (protein kinase A)-dependent association of ß1AR with arrestin2 and CaMKII and turns on an Epac (exchange protein directly activated by cAMP)-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 (G-protein receptor kinase-5) is necessary to promote agonist-induced dissociation of SAP97 from ß1AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of ß1AR-SAP97 complex and increases in CaMKII activity in hearts. CONCLUSIONS: These data reveal a critical role of SAP97 in maintaining the integrity of cardiac ß1AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy. Graphical Abstract: A graphical abstract is available for this article.

Down-regulation of A3AR signaling by IL-6-induced GRK2 activation contributes to Th17 cell differentiation.

IL-6-triggered Th17 cell expansion is responsible for the pathogenesis of many immune diseases including rheumatoid arthritis (RA). Traditionally, IL-6 induces Th17 cell differentiation through JAK-STAT3 signaling. In the present work, PKA inhibition reduces in vitro induction of Th17 cells, while IL-6 stimulation of T cells facilitates the internalization of A3AR and increased cAMP production in a GRK2 dependent manner. Inhibition of GRK2 by paroxetine (PAR) or genetic depletion of GRK2 restored A3AR distribution and prevented Th17 cell differentiation. Furthermore, in vivo PAR treatment effectively reduced the splenic Th17 cell proportion in a rat model of collagen-induced arthritis (CIA) which was accompanied by a significant improvement in clinical manifestations. These results indicate that IL-6-induced Th17 cell differentiation not only occurs through JAK-STAT3-RORγt but is also mediated through GRK2-A3AR-cAMP-PKA-CREB/ICER-RORγt. This elucidates the significance of GRK2-controlled cAMP signaling in the differentiation of Th17 cells and its potential application in treating Th17-driven immune diseases such as RA.

TNF-α impairs EP4 signaling through the association of TRAF2-GRK2 in primary fibroblast-like synoviocytes.

Our previous study showed that chronic treatment with tumor necrosis factor-α (TNF-α) decreased cAMP concentration in fibroblast-like synoviocytes (FLSs) of collagen-induced arthritis (CIA) rats. In this study we investigated how TNF-α impairs cAMP homeostasis, particularly clarifying the potential downstream molecules of TNF-α and prostaglandin receptor 4 (EP4) signaling that would interact with each other. Using a cAMP FRET biosensor PM-ICUE3, we demonstrated that TNF-α (20 ng/mL) blocked ONO-4819-triggered EP4 signaling, but not Butaprost-triggered EP2 signaling in normal rat FLSs. We showed that TNF-α (0.02-20 ng/mL) dose-dependently reduced EP4 membrane distribution in normal rat FLS. TNF-α significantly increased TNF receptor 2 (TNFR2) expression and stimulated proliferation in human FLS (hFLS) via ecruiting TNF receptor-associated factor 2 (TRAF2) to cell membrane. More interestingly, we revealed that TRAF2 interacted with G protein-coupled receptor kinase (GRK2) in the cytoplasm of primary hFLS and helped to bring GRK2 to cell membrane in response of TNF-α stimulation, the complex of TRAF2 and GRK2 then separated on the membrane, and translocated GRK2 induced the desensitization and internalization of EP4, leading to reduced production of intracellular cAMP. Silencing of TRAF2 by siRNA substantially diminished TRAF2-GRK2 interaction, blocked the translocation of GRK2, and resulted in upregulated expression of membrane EP4 and intracellular cAMP. In CIA rats, administration of paroxetine to inhibit GRK2 effectively improved the symptoms and clinic parameters with significantly reduced joint synovium inflammation and bone destruction. These results elucidate a novel form of cross-talk between TNFR (a cytokine receptor) and EP4 (a typical G protein-coupled receptor) signaling pathways. The interaction between TRAF2 and GRK2 may become a potential new drug target for the treatment of inflammatory diseases.

Regulating Th17/Treg Balance Contributes to the Therapeutic Effect of Ziyuglycoside I on Collagen-Induced Arthritis.

To investigate the therapeutic effect and primary pharmacological mechanism of Ziyuglycoside I (Ziyu I) on collagen-induced arthritis (CIA) mice. CIA mice were treated with 5, 10, or 20 mg/kg of Ziyu I or 2 mg/kg of methotrexate (MTX), and clinical manifestations, as well as pathological changes, were observed. T cell viability and subset type were determined, and serum levels of transforming growth factor-beta (TGF-ß) and interleukin-17 (IL-17) were detected. The mRNA expression of retinoid-related orphan receptor-γt (RORγt) and transcription factor forkhead box protein 3 (Foxp3) in mouse spleen lymphocytes was ascertained by the real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). Molecular docking was used to detect whether there was a molecular interaction between Ziyu I and protein kinase B (Akt). The activation of mechanistic target of rapamycin (mTOR) in T cells was verified by Western blotting or immunofluorescence. Ziyu I treatment effectively alleviated arthritis symptoms of CIA mice, including body weight, global score, arthritis index, and a number of swollen joints. Similarly, pathological changes of joints and spleens in arthritic mice were improved. The thymic index, T cell activity, and RORγt production of Ziyu I-treated mice were significantly reduced. Notably, through molecular docking, western blotting, and immunofluorescence data analysis, it was found that Ziyu I could interact directly with Akt to reduce downstream mTOR activation and inhibit helper T cell 17 (Th17) differentiation, thereby regulating Th17/regulatory T cell (Treg) balance and improving arthritis symptoms. Ziyu I effectively improves arthritic symptoms in CIA mice by inhibiting mTOR activation, thereby affecting Th17 differentiation and regulating Th17/Treg balance.

DBA/1 mice display equivalent cardiac function to C57BL/6J mice.

NEW FINDINGS: What is the central question of this study? Do normal adult DBA/1 mice have cardiac function and performance equal to those of C57BL/6J mice? What is the main finding and its importance? Male adult DBA/1 mice show equivalent cardiac function to C57BL/6J mice up to 8 months old. Therefore, cardiac dysfunction could be investigated in an autoimmune diseases model established with DBA/1 mice. ABSTRACT: Cardiovascular mortality has been increasing, and in particular, cardiovascular damage caused by some chronic autoimmune diseases accounts for a large proportion of this. C57BL/6J mice have been used mostly in studies of cardiovascular diseases. However, for purposes of modelling, this strain of mouse has a very low incidence of some chronic immune diseases such as rheumatoid arthritis, to which instead DBA/1 mice are more susceptible. Basic cardiac function differs between mice with different genetic backgrounds. Therefore, we monitored cardiac function and structure of normal male C57BL/6J and DBA/1 mice for six consecutive months. Echocardiography was used to monitor cardiac functions once a month and cardiac systolic function was measured upon isoproterenol challenge at the end of observation. The Excitation-contraction coupling-related proteins were measured by western blotting. Heart tissue sections were subject to haematoxylin-eosin, TUNEL and Alizarin red staining. The results demonstrated that systolic and diastolic function did not vary significantly and both strains were indistinguishable in appearance and structure of hearts. DBA/1 mice showed a good cardiac ß-adrenergic response comparable to C57BL/6J mice with isoproterenol treatment. The phosphorylation of phospholamban at either its protein kinase A or its Ca2+ /calmodulin-dependent protein kinase II site, as well as the activation of troponin I showed no significant difference between strains. These findings suggested that there was no obvious difference in the heart structure and function of normal male DBA/1 mice compared with C57BL/6J mice. The DBA/1 mouse is a strain applicable to investigating autoimmune disease-induced heart dysfunction and exploring potential interventions.

hIgDFc-Ig inhibits B cell function by regulating the BCR-Syk-Btk-NF-κB signalling pathway in mice with collagen-induced arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease targeting the synovium. Previous studies have found that IgD may be a potential target for the treatment of RA. We designed a new type of fusion protein, hIgDFc-Ig (DG), to block the binding of IgD to IgD receptor (IgDR). In this study, we found that DG has a significant therapeutic effect in mice with collagen-induced arthritis (CIA). DG improved the claw of irritation symptoms in these mice, inhibited the pathological changes in spleen and joint tissues, and had a moderating effect on B cell subsets at different inflammatory stages. Moreover, DG could also decrease the levels of IgA, IgD, IgM and IgG subtypes of immunoglobulin in the serum of mice with CIA. In vitro, B cell antigen receptor (BCR) knockout Ramos cells were established using the CRISPR/Cas9 technology to further study the activation of BCR signalling by IgD and the effect of DG. We found that the therapeutic effect of DG in mice with CIA may be achieved by inhibiting the activation of BCR signalling by IgD, which may be related to the activation of Igß. In summary, DG may be a potential biological agent for the treatment of RA and it has broad application prospects in the future.

Deficiency of ß-arrestin2 exacerbates inflammatory arthritis by facilitating plasma cell formation.

ß-arrestin2 (ß-arr2) is, a key protein that mediates desensitization and internalization of G protein-coupled receptors and participates in inflammatory and immune responses. Deficiency of ß-arr2 has been found to exacerbate collagen antibody-induced arthritis (CAIA) through unclear mechanisms. In this study we tried to elucidate the molecular mechanisms underlying ß-arr2 depletion-induced exacerbation of CAIA. CAIA was induced in ß-arr2-/- and wild-type (WT) mice by injection of collagen antibodies and LPS. The mice were sacrificed on d 13 after the injection, spleen, thymus and left ankle joints were collected for analysis. Arthritis index (AI) was evaluated every day or every 2 days. We showed that ß-arr2-/- mice with CAIA had a further increase in the percentage of plasma cells in spleen as compared with WT mice with CAIA, which was in accordance with elevated serum IgG1 and IgG2A expression and aggravating clinical performances, pathologic changes in joints and spleen, joint effusion, and joint blood flow. Both LPS stimulation of isolated B lymphocytes in vitro and TNP-LPS challenge in vivo led to significantly higher plasma cell formation and antibodies production in ß-arr2-/- mice as compared with WT mice. LPS treatment induced membrane distribution of toll-like receptor 4 (TLR4) on B lymphocytes, accordingly promoted the nuclear translocation of NF-κB and the transcription of Blimp1. Immunofluorescence analysis confirmed that more TLR4 colocalized with ß-arr2 in B lymphocytes in response to LPS stimulation. Depletion of ß-arr2 restrained TLR4 on B lymphocyte membrane after LPS treatment and further enhanced downstream NF-κB signaling leading to additional increment in plasma cell formation. In summary, ß-arr2 depletion exacerbates CAIA and further increases plasma cell differentiation and antibody production through inhibiting TLR4 endocytosis and aggravating NF-κB signaling.

Non-invasive skin cholesterol testing: a potential proxy for LDL-C and apoB serum measurements.

BACKGROUND: Lipid management is the first line of treatment for decreasing the incidence of cardiovascular events in patients with coronary heart disease (CHD), and a variety of indicators are used to evaluate lipid management. This work analyses the differences in LDL-C and apoB for lipid management evaluation, as well as explores the feasibility of skin cholesterol as a marker that can be measured non-invasively for lipid management. METHODS: The prospective study enrolled 121 patients who had been diagnosed with acute coronary syndrome (ACS) at the department of emergency medicine of the First Affiliated Hospital of the USTC from May 2020 to January 2021, and the patients were grouped into Group I (n=53) and Group II (n=68) according to whether they had comorbid hyperlipidemia and/or diabetes mellitus. All patients were administered 10 mg/day of rosuvastatin and observed for 12 weeks. Lipid management was assessed on the basis of LDL-C and apoB, and linear correlation models were employed to assess the relationship between changes in these well accepted markers to that of changes in skin cholesterol. RESULTS: Out of 121 patients with ACS, 53 patients (43.80 %) had combined hyperlipidemia and/or diabetes mellitus (Group I), while 68 patients (56.20 %) did not (Group II). Cardiovascular events occur at earlier ages in patients with CHD who are comorbid for hyperlipidemia and/or diabetes (P<0.05). LDL-C attainment rate is lower than apoB attainment rate with rosuvastatin therapy (P<0.05), which is mainly attributable to patients with low initial LDL-C. Skin cholesterol reduction correlated with LDL-C reduction. (r=0.501, P<0.001) and apoB reduction (r=0.538, P<0.001). Skin cholesterol reduction continued over all time points measured. CONCLUSIONS: Examination of changes in apoB levels give patients with low initial LDL-C more informative data on lipid management than LDL-C readings. In addition, non-invasive skin cholesterol measurements may have the potential to be used independently for lipid management evaluation.

Sustaining Circulating Regulatory T Cell Subset Contributes to the Therapeutic Effect of Paroxetine on Mice With Diabetic Cardiomyopathy.

BACKGROUND: G protein coupled receptor kinase 2 (GRK2) inhibitor, paroxetine, has been approved to ameliorate diabetic cardiomyopathy (DCM). GRK2 is also involved in regulating T cell functions; the potential modifications of paroxetine on the immune response to DCM is unclear.Methods and Results:DCM mouse was induced by high-fat diet (HFD) feeding. A remarkable reduction in the regulatory T (Treg) cell subset in DCM mouse was found by flow cytometry, with impaired cardiac function evaluated by echocardiography. The inhibited Treg differentiation was attributable to insulin chronic stimulation in a GRK2-PI3K-Akt signaling-dependent manner. The selective GRK2 inhibitor, paroxetine, rescued Treg differentiation in vitro and in vivo. Furthermore, heart function, as well as the activation of excitation-contraction coupling proteins such as phospholamban (PLB) and troponin I (TnI) was effectively promoted in paroxetine-treated DCM mice compared with vehicle-treated DCM mice. Blockade of FoxP3 expression sufficiently inhibited the proportion of Treg cells, abolished the protective effect of paroxetine on heart function as well as PLB and TnI activation in HFD-fed mice. Neither paroxetine nor carvedilol could effectively ameliorate the metabolic disorder of HFD mice. CONCLUSIONS: The impaired systolic heart function of DCM mice was effectively improved by paroxetine therapy, partially through restoring the population of circulating Treg cells by targeting the GRK2-PI3K-Akt pathway.

IgD-Fc-Ig fusion protein, a new biological agent, inhibits T cell function in CIA rats by inhibiting IgD-IgDR-Lck-NF-κB signaling pathways.

IgD-Fc-Ig fusion protein, a new biological agent, is constructed by linking a segment of human IgD-Fc with a segment of human IgG1-Fc, which specifically blocks the IgD-IgDR pathway and selectively inhibits the abnormal proliferation, activation, and differentiation of T cells. In this study we investigated whether IgD-Fc-Ig exerted therapeutic effects in collagen-induced arthritis (CIA) rats. CIA rats were treated with IgD-Fc-Ig (1, 3, and 9 mg/kg) or injected with biological agents etanercept (3 mg/kg) once every 3 days for 40 days. In the PBMCs and spleen lymphocytes of CIA rats, both T and B cells exhibited abnormal proliferation; the percentages of CD3+ total T cells, CD3+CD4+ Th cells, CD3+CD4+CD25+-activated Th cells, Th1(CD4+IFN-γ+), and Th17(CD4+IL-17+) were significantly increased, whereas the Treg (CD4+CD25+Foxp3+) cell percentage was decreased. IgD-Fc-Ig administration dose-dependently decreased the indicators of arthritis; alleviated the histopathology of spleen and joint; reduced serum inflammatory cytokines levels; decreased the percentages of CD3+ total T cells, CD3+CD4+ Th cells, CD3+CD4+CD25+-activated Th cells, Th1 (CD4+IFN-γ+), and Th17(CD4+IL-17+); increased Treg (CD4+CD25+Foxp3+) cell percentage; and down-regulated the expression of key molecules in IgD-IgDR-Lck-NF-κB signaling (p-Lck, p-ZAP70, p-P38, p-NF-κB65). Treatment of normal T cells with IgD (9 µg/mL) in vitro promoted their proliferation. Co-treatment with IgD-Fc-Ig (0.1-10 µg/mL) dose-dependently decreased IgD-stimulated T cell subsets percentages and down-regulated the IgD-IgDR-Lck-NF-κB signaling. In summary, this study demonstrates that IgD-Fc-Ig alleviates CIA and regulates the functions of T cells through inhibiting IgD-IgDR-Lck-NF-κB signaling.

Paeoniflorin-6'-O-benzene sulfonate alleviates collagen-induced arthritis in mice by downregulating BAFF-TRAF2-NF-κB signaling: comparison with biological agents.

Paeoniflorin-6'-O-benzene sulfonate (CP-25) is a new ester derivative of paeoniflorin with improved lipid solubility and oral bioavailability, as well as better anti-inflammatory activity than its parent compound. In this study we explored whether CP-25 exerted therapeutic effects in collagen-induced arthritis (CIA) mice through regulating B-cell activating factor (BAFF)-BAFF receptors-mediated signaling pathways. CIA mice were given CP-25 or injected with biological agents rituximab or etanercept for 40 days. In CIA mice, we found that T cells and B cells exhibited abnormal proliferation; the percentages of CD19+ total B cells, CD19+CD27+-activated B cells, CD19+BAFFR+ and CD19+TACI+ cells were significantly increased in PBMCs and spleen lymphocytes. CP-25 suppressed the indicators of arthritis, alleviated histopathology, accompanied by reduced BAFF and BAFF receptors expressions, inhibited serum immunoglobulin levels, decreased the B-cell subsets percentages, and prevented the expressions of key molecules in NF-κB signaling. Furthermore, we showed that treatment with CP-25 reduced CD19+TRAF2+ cell expressions stimulated by BAFF and decreased TRAF2 overexpression in HEK293 cells in vitro. Thus, CP-25 restored the abnormal T cells proliferation and B-cell percentages to the normal levels, and normalized the elevated levels of IgA, IgG2a and key proteins in NF-κB signaling. In comparison, rituximab and etanercept displayed stronger anti-inflammatory activities than CP-25; they suppressed the elevated inflammatory indexes to below the normal levels in CIA mice. In summary, our results provide evidence that CP-25 alleviates CIA and regulates the functions of B cells through BAFF-TRAF2-NF-κB signaling. CP-25 would be a soft immunomodulatory drug with anti-inflammatory effect.

Ginsenoside compound-K inhibits the activity of B cells through inducing IgD-B cell receptor endocytosis in mice with collagen-induced arthritis.

Previously, ginsenoside metabolite compound K (C-K) was able to reduce B cell proliferation and serum anti-type II collagen (anti-CII) antibody to normal levels in mice with collagen-induced arthritis (CIA); however, the mechanism by which C-K restores B cell balance is unclear. In the present work, C-K treatment not only alleviated the polyarthritis index, swollen joint count, pathological scores of spleen and joints, spleen index, B cell proliferation and the level of serum antibodies (IgG1, IgG2a and anti-collagen II), but C-K treatment also restored B cell subsets including regulatory B cells, plasma cells, memory B cells, mature B cells, and follicular B cells in CIA mice. Interestingly, C-K did not change the expression level of immunoglobulin D-type B-cell receptor (IgD-BCR) but promoted IgD-BCR endocytosis. C-K treatment enhanced ß-arrestin1 expression, facilitating the colocalization between IgD and ß-arrestin1, as well as colocalization between IgD and adaptor protein 2 (AP2). Inhibition of the ß-arrestin1-AP2 interaction with barbadin significantly reduced the ability of C-K to attenuate IgD-BCR plasma membrane localization. These results taken together depict that C-K ameliorates CIA in part by inhibiting B cell activation through the triggering of IgD-BCR internalization in a ß-arrestin1-AP2 dependent manner.

Inhibiting Insulin-Mediated ß2-Adrenergic Receptor Activation Prevents Diabetes-Associated Cardiac Dysfunction.

BACKGROUND: Type 2 diabetes mellitus (DM) and obesity independently increase the risk of heart failure by incompletely understood mechanisms. We propose that hyperinsulinemia might promote adverse consequences in the hearts of subjects with type-2 DM and obesity. METHODS: High-fat diet feeding was used to induce obesity and DM in wild-type mice or mice lacking ß2-adrenergic receptor (ß2AR) or ß-arrestin2. Wild-type mice fed with high-fat diet were treated with a ß-blocker carvedilol or a GRK2 (G-protein-coupled receptor kinase 2) inhibitor. We examined signaling and cardiac contractile function. RESULTS: High-fat diet feeding selectively increases the expression of phosphodiesterase 4D (PDE4D) in mouse hearts, in concert with reduced protein kinase A phosphorylation of phospholamban, which contributes to systolic and diastolic dysfunction. The expression of PDE4D is also elevated in human hearts with DM. The induction of PDE4D expression is mediated by an insulin receptor, insulin receptor substrate, and GRK2 and ß-arrestin2-dependent transactivation of a ß2AR-extracellular regulated protein kinase signaling cascade. Thus, pharmacological inhibition of ß2AR or GRK2, or genetic deletion of ß2AR or ß-arrestin2, all significantly attenuate insulin-induced phosphorylation of extracellular regulated protein kinase and PDE4D induction to prevent DM-related contractile dysfunction. CONCLUSIONS: These studies elucidate a novel mechanism by which hyperinsulinemia contributes to heart failure by increasing PDE4D expression and identify ß2AR or GRK2 as plausible therapeutic targets for preventing or treating heart failure in subjects with type 2 DM.

Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure.

RATIONALE: In heart failure, myofilament proteins display abnormal phosphorylation, which contributes to contractile dysfunction. The mechanisms underlying the dysregulation of protein phosphorylation on myofilaments is not clear. OBJECTIVE: This study aims to understand the mechanisms underlying altered phosphorylation of myofilament proteins in heart failure. METHODS AND RESULTS: We generate a novel genetically encoded protein kinase A (PKA) biosensor anchored onto the myofilaments in rabbit cardiac myocytes to examine PKA activity at the myofilaments in responses to adrenergic stimulation. We show that PKA activity is shifted from the sarcolemma to the myofilaments in hypertrophic failing rabbit myocytes. In particular, the increased PKA activity on the myofilaments is because of an enhanced ß2 adrenergic receptor signal selectively directed to the myofilaments together with a reduced phosphodiesterase activity associated with the myofibrils. Mechanistically, the enhanced PKA activity on the myofilaments is associated with downregulation of caveolin-3 in the hypertrophic failing rabbit myocytes. Reintroduction of caveolin-3 in the failing myocytes is able to normalize the distribution of ß2 adrenergic receptor signal by preventing PKA signal access to the myofilaments and to restore contractile response to adrenergic stimulation. CONCLUSIONS: In hypertrophic rabbit myocytes, selectively enhanced ß2 adrenergic receptor signaling toward the myofilaments contributes to elevated PKA activity and PKA phosphorylation of myofilament proteins. Reintroduction of caveolin-3 is able to confine ß2 adrenergic receptor signaling and restore myocyte contractility in response to ß adrenergic stimulation.

High-fat diet induces protein kinase A and G-protein receptor kinase phosphorylation of ß2 -adrenergic receptor and impairs cardiac adrenergic reserve in animal hearts.

KEY POINTS: Patients with diabetes show a blunted cardiac inotropic response to ß-adrenergic stimulation despite normal cardiac contractile reserve. Acute insulin stimulation impairs ß-adrenergically induced contractile function in isolated cardiomyocytes and Langendorff-perfused hearts. In this study, we aimed to examine the potential effects of hyperinsulinaemia associated with high-fat diet (HFD) feeding on the cardiac ß2 -adrenergic receptor signalling and the impacts on cardiac contractile function. We showed that 8 weeks of HFD feeding leads to reductions in cardiac functional reserve in response to ß-adrenergic stimulation without significant alteration of cardiac structure and function, which is associated with significant changes in ß2 -adrenergic receptor phosphorylation at protein kinase A and G-protein receptor kinase sites in the myocardium. The results suggest that clinical intervention might be applied to subjects in early diabetes without cardiac symptoms to prevent further cardiac complications. ABSTRACT: Patients with diabetes display reduced exercise capability and impaired cardiac contractile reserve in response to adrenergic stimulation. We have recently uncovered an insulin receptor and adrenergic receptor signal network in the heart. The aim of this study was to understand the impacts of high-fat diet (HFD) on the insulin-adrenergic receptor signal network in hearts. After 8 weeks of HFD feeding, mice exhibited diabetes, with elevated insulin and glucose concentrations associated with body weight gain. Mice fed an HFD had normal cardiac structure and function. However, the HFD-fed mice displayed a significant elevation of phosphorylation of the ß2 -adrenergic receptor (ß2 AR) at both the protein kinase A site serine 261/262 and the G-protein-coupled receptor kinase site serine 355/356 and impaired adrenergic reserve when compared with mice fed on normal chow. Isolated myocytes from HFD-fed mice also displayed a reduced contractile response to adrenergic stimulation when compared with those of control mice fed normal chow. Genetic deletion of the ß2 AR led to a normalized adrenergic response and preserved cardiac contractile reserve in HFD-fed mice. Together, these data indicate that HFD promotes phosphorylation of the ß2 AR, contributing to impairment of cardiac contractile reserve before cardiac structural and functional remodelling, suggesting that early intervention in the insulin-adrenergic signalling network might be effective in prevention of cardiac complications in diabetes.