The Role of Different Circulating T Follicular Helper Cell Markers in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic incurable inflammatory autoimmune disease. T follicular helper (Tfh) cells expressing different markers play critical roles in the development of RA. However, their specific mechanisms of action and association with RA clinical parameters are not clear. We therefore performed a cohort study to investigate the effects of different Tfh cell markers on RA pathogenesis. We retrospectively reviewed clinical data from 30 patients diagnosed with RA and 30 healthy controls (HCs) who visited our hospital. Based on X-ray findings, the patients were divided into a joint bone erosion group (n = 17) and a non-erosive joint bone group (n = 13). Using flow cytometry, we determined the frequencies of five peripheral blood CD4+ Tfh cell types characterized by different markers, and examined these cell types for correlations with clinical parameters. RA patients exhibited higher frequencies of CD4+CXCR5+, CD4+CXCR5+ICOS+, CD4+CXCR5+OX40+, and CD4+CXCR5+CD40L+ Tfh cells than HCs. CD4+CXCR5+, CD4+CXCR5+CD40L+, and CD4+CXCR5+OX40+ Tfh cell frequencies positively correlated with disease activity score-28 with erythrocyte sedimentation rate (DAS28-ESR), while those of CD4+CXCR5+ and CD4+CXCR5+CD40L+ Tfh cells were related to rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) antibodies. In RA patients without joint bone erosion, CD4+CXCR5+CD40L+ Tfh cell frequencies were positively correlated with both RF and DAS28-ESR. Serum anti-CCP antibody levels and CD4+CXCR5+ICOS+ Tfh cell frequencies were also positively correlated. Circulating CD4+CXCR5+CD40L+ Tfh cells appear to play critical roles in RA pathogenesis, and restricting CD4+CXCR5+CD40L+ Tfh cells may be a therapeutic strategy for controlling RA.

Interleukin-29 and interleukin-28A induce migration of neutrophils in rheumatoid arthritis.

OBJECTIVES: Type III Interferons, interleukin (IL)-29 and IL-28A, have been implicated in the inflammatory response of rheumatoid arthritis (RA). Increasing evidence suggests an important role of neutrophils in the pathogenesis of RA. However, the underlying mechanism remains unclear. Therefore, we investigated the expression of the receptor of these type III interferons, IL-28R1, on the neutrophils of RA patients, and further explored the roles of IL-29 and IL-28A on neutrophil activity. METHODS: Neutrophils were extracted from peripheral blood of patients who met the diagnostic criteria for RA and healthy controls. The serum levels of IL-29 and IL-28A in RA patients and healthy controls were examined by enzyme-linked immunoassay, and the expression of IL-28R1 on neutrophils was determined by flow cytometry. A transwell assay was performed to determine the chemotactic ability of IL-29 and IL-28A to neutrophils in RA patients. RESULTS: The serum IL-29 but not IL-28A levels were significantly elevated in RA patients, and neither was correlated with RA disease activity. IL-28R1 levels on neutrophils were significantly (p < 0.001) elevated in patients with RA (51.85% (36.10%, 67.03%)) compared with those of healthy controls (4.13% (3.54%, 7.96%)), and IL-29 and IL-28A had a significant chemotactic effect on neutrophils from the peripheral blood of RA patients. CONCLUSION: IL-29 and IL-28A play an important role in regulating neutrophils which participate in the pathogenesis of RA. Therefore, inhibiting IL-29 and IL-28A may be a new therapeutic strategy for RA. Key points • The IL-28R1 levels were increased in neutrophils of RA patients, suggesting its potentially important role in the pathogenesis of RA. • IL-29 and IL-28A induce the migration of neutrophils that participate in the development of RA.

Interleukin-29 regulates T follicular helper cells by repressing BCL6 in rheumatoid arthritis patients.

INTRODUCTION: We aimed to investigate whether Interleukin-29 (IL-29) directly affects T follicular helper (Tfh) cell frequency in rheumatoid arthritis (RA), which are both related to RA-specific antibody responses. METHODS: Here, we explored the effect of IL-29 on Tfh cell production in RA patients using a combination of enzyme-linked immunosorbent assay (ELISA), flow cytometry (FCM), CD4+ T cell culture, western blotting, and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: We reported that serum IL-29 levels, peripheral blood CD4+CXCR5+ Tfh cell frequency, CD4+CXCR5+CD40L+ Tfh cell frequency, and IL-28 receptor (IL-28Rα) and IL-10 receptor (IL-10R2) levels in peripheral blood Tfh cells were higher in RA patients than in healthy controls (HCs). Serum IL-29 levels were positively correlated with peripheral blood CD4+CXCR5+CD40L+ Tfh cell frequency in RA patients, and both parameters also correlated with anti-cyclic citrullinated peptide (anti-CCP) antibodies. Furthermore, we showed that IL-29 may suppress Tfh cell differentiation in RA patients partly via decreased BCL6 level through reduced STAT3 activity. CONCLUSIONS: Taken together, our findings reveal the regulatory effect of IL-29 on Tfh cells, which participate in the pathogenesis of RA and provide new targets for its clinical treatment. Key Points • There is an increase in circulating Tfh cells and IL-29 levels in RA patients, which are correlated to anti-CCP antibodies levels and may be associated with RA pathogenesis. • We show for the first time that IL-29 may contribute to RA by inhibiting Tfh cell production, through decreasing the activity of STAT3 and downregulating the expression of BCL6. • The use of IL-29 biologics in patients with RA inhibits the production of Tfh cells, may prevent progression in patients with RA, and provides new targets for clinical treatment.

RhoGDI2 positively regulates the Rho GTPases activation in response to the ß2 outside-in signaling in T cells adhesion and migration on ICAM-1.

Cytoskeletal reorganization driven by Rho GTPases plays a crucial role in the migration of T cells, which are key regulators of immunity. The molecular mechanisms that control actin cytoskeleton remodeling during T cell movement have only partially been clarified as the function of many modulators has not been evaluated in these cells. Here, we report a new function of RhoGDI2 by showing that this protein positively regulates Rho GTPase activation during T cell adhesion and migration. RhoGDI2 knockdown significantly reduced T cell adhesion and migration. Furthermore, RhoGDI2 knockdown decreased the activation of Rac1 and Cdc42, 2 members of Rho GTPases, and the remodeling of the actin cytoskeleton. Upon P-selectin glycoprotein ligand-1 engagement, RhoGDI2 was phosphorylated at Y24 and Y153 by kinases related to ß2 integrin outside-in signaling, Src, c-Abl, and Syk, resulting in the accumulation of RhoGDI2 at the cell membrane. Subsequent phosphorylation of S31 induced the opening of RhoGDI2 and the release of Rho GTPases, whereas phosphorylation of Y153 might promote the activation of Rho GTPases by recruiting Vav1. Moreover, the disruption of lipid rafts with methyl-ß-cyclodextrin blocked the interaction between integrins and RhoGDI2, reducing the level of phosphorylated RhoGDI2 and the activation of downstream Rho GTPases. Based on these observations, RhoGDI2 is a target of intergrin outside-in signaling that activates Rho GTPases during T cell adhesion and migration, and RhoGDI2-mediated signal transduction is based on the lipid rafts integrity.

Effective serum level of etanercept biosimilar and effect of antidrug antibodies on drug levels and clinical efficacy in Chinese patients with ankylosing spondylitis.

OBJECTIVES: To investigate the effective serum level of etanercept biosimilar in Chinese patients with ankylosing spondylitis (AS) who achieve AS Disease Activity Score-C-reactive protein (ASDAS-CRP) < 2.1, and the effect of antidrug antibodies on drug levels and clinical efficacy. METHODS: Our study enrolled 60 patients with AS who were treated with etanercept biosimilar. Serum and clinical data were collected at baseline and treatment weeks 4, 12, and 24. Drug levels and antidrug antibody levels were measured using an enzyme-linked immunosorbent assay while tumour necrosis factor (TNF)-α levels were measured using cytometric bead array. A receiver operating characteristic (ROC) curve was used to analyse effective serum level of etanercept biosimilar. RESULTS: Patients with ASDAS-CRP ≥ 2.1 exhibited significantly lower drug levels than those with ASDAS-CRP < 2.1 did. The cut-off values of effective serum level of patients with AS who achieved ASDAS-CRP < 2.1 at weeks 4, 12, and 24 were 2.32, 2.12, and 2.36 µg/mL, respectively. Patients with drug levels above the cut-off value had lower Bath AS Disease Activity Index (BASDAI) and TNF-α levels. Antidrug antibodies had no effect on the Assessment of Spondylosis Arthritis International Society (ASAS) remission rates, but patients with antidrug antibodies had lower drug levels and higher TNF-α levels. CONCLUSIONS: Detecting serum drug levels and antidrug antibody levels might facilitate estimation of the clinical efficacy and adjustment of medication regimen during etanercept biosimilar therapy in Chinese patients with AS.

Lipid raft­associated ß­adducin participates in neutrophil migration.

Previous studies have demonstrated that lipid rafts and ß­adducin serve an important role in leukocyte rolling. In the present study the migratory ability and behavior of neutrophils was demonstrated to rely on the integrity of the lipid raft structure. ß­adducin was demonstrated to have a critical role in neutrophil migration. Knockdown of ß­adducin attenuated the migratory ability of dHL­60 cells and the distribution of ß­adducin in lipid raft structures was changed by N­formylmethionyl­leucyl­phenyl­alanine treatment. Furthermore, the findings demonstrated that the tyrosine phosphorylation of ß­adducin was required for its relocation. The results of the present study suggested that the lipid raft­associated protein ß­adducin may be a novel control point for the excessive infiltration of neutrophils during inflammation.

Elevated Serum Levels of Interleukin-29 Are Associated with Disease Activity in Rheumatoid Arthritis Patients with Anti-Cyclic Citrullinated Peptide Antibodies.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease that may lead to progressive joint destruction. The anti-cyclic citrullinated peptide (anti-CCP) antibody is an essential marker for the diagnosis of RA and has a crucial role in the bone destruction in RA. Recent studies have shown that interleukin (IL)-29, a vital member of type III interferon (IFN) family, could enhance proinflammatory cytokine production and might be involved in the joint destruction in RA. Therefore, in this study, we aimed to examine the role of IL-29 in RA patients with anti-CCP antibodies. The result showed that the serum IL-29 levels were higher in RA patients (n = 68) compared with healthy controls (HC, n = 68, P = 0.019). Correlation analysis demonstrated a significant positive correlation among serum IL-29 level, rheumatoid factor (RF, P < 0.001) and anti-CCP antibodies (P = 0.042). However, when RA patients were divided into two groups according to anti-CCP antibodies, the serum IL-29 levels were significantly higher in anti-CCP-antibodies positive RA patients (n = 54) than those in HC (n = 68) and anti-CCP-antibodies negative RA patients (n = 14). Furthermore, the serum IL-29 levels were positively correlated with the disease activity (P < 0.05) and significantly declined after 6 months of treatment (P < 0.01) in the anti-CCP-antibodies positive RA patients, whereas no significant change was found in the anti-CCP-antibodies negative RA patients (P > 0.05). The findings indicate that IL-29 is a potential biomarker for disease activity in anti-CCP-antibodies positive RA patients.

Lipid raft-associated ß-adducin is required for PSGL-1-mediated neutrophil rolling on P-selectin.

Lipid rafts, a liquid-ordered plasma membrane microdomain, are related to cell-surface receptor function. PSGL-1, a major surface receptor protein for leukocyte, also acts as a signaling receptor in leukocyte rolling. To investigate the role of lipid raft in PSGL-1 signaling in human neutrophils, we quantitatively analyzed lipid raft proteome of human promyelocytic leukemia cell line HL-60 cells and identified a lipid raft-associated protein ß-adducin. PSGL-1 ligation induced dissociation of the raft-associated protein ß-adducin from lipid rafts and actin, as well as phosphorylation of ß-adducin, indicating a transient uncoupling of lipid rafts from the actin cytoskeleton. Knockdown of ß-adducin greatly attenuated HL-60 cells rolling on P-selectin. We also showed that Src kinase is crucial for PSGL-1 ligation-induced ß-adducin phosphorylation and relocation. Taken together, these results show that ß-adducin is a pivotal lipid raft-associated protein in PSGL-1-mediated neutrophil rolling on P-selectin.

PI3K is involved in ß1 integrin clustering by PSGL-1 and promotes ß1 integrin-mediated Jurkat cell adhesion to fibronectin.

P-selectin glycoprotein ligand-1 (PSGL-1) is involved in the initial step of lymphocyte homing by interacting with P- or E-selectins expressed on activated endothelium cells. Besides, it also functions as a receptor to induce signals that increase integrin affinity to ligands and mediate cell adhesion to endothelium. Integrin is required for the second step of lymphocyte homing, whose activation has been reported tightly regulated by inside-out signals triggered by chemokines or the shear-stress generated during lymphocyte rolling on endothelium. However, the relationship between PSGL-1-triggered signals and integrin activation is not clear. In this study, we demonstrated that PSGL-1 ligation induces ß1 integrin-mediated adhesion to fibronectin via regulation of both ß1 subunit clustering and conformation changes. Phosphoinositide 3-kinase (PI3K) is required for PSGL-1-induced ß1 integrin clustering which ultimately regulates ß1 integrin-mediated Jurkat cell adhesion to fibronectin. However, PI3K is not involved in the conformation changes or increases in the total expression of ß1 integrin. Taken together, we found a novel signal pathway, PSGL-1-PI3K-ß1 integrin, demonstrating the cooperation between initial adhesion and subsequent arrest and stable adhesion.

Lipid Raft is required for PSGL-1 ligation induced HL-60 cell adhesion on ICAM-1.

P-selectin glycoprotein ligand-1 (PSGL-1) and integrins are adhesion molecules that play critical roles in host defense and innate immunity. PSGL-1 mediates leukocyte rolling and primes leukocytes for integrin-mediated adhesion. However, the mechanism that PSGL-1 as a rolling receptor in regulating integrin activation has not been well characterized. Here, we investigate the function of lipid raft in regulating PSGL-1 induced ß2 integrin-mediated HL-60 cells adhesion. PSGL-1 ligation with antibody enhances the ß2 integrin activation and ß2 integrin-dependent adhesion to ICAM-1. Importantly, with the treatment of methyl-ß-cyclodextrin (MßCD), we confirm the role of lipid raft in regulating the activation of ß2 integrin. Furthermore, we find that the protein level of PSGL-1 decreased in raft fractions in MßCD treated cells. PSGL-1 ligation induces the recruitment of spleen tyrosine kinase (Syk), a tyrosine kinase and Vav1 (the pivotal downstream effector of Syk signaling pathway involved in cytoskeleton regulation) to lipid raft. Inhibition of Syk activity with pharmacologic inhibitor strongly reduces HL-60 cells adhesion, implicating Syk is crucial for PSGL-1 mediated ß2 integrin activation. Taken together, we report that ligation of PSGL-1 on HL-60 cells activates ß2 integrin, for which lipid raft integrity and Syk activation are responsible. These findings have shed new light on the mechanisms that connect leukocyte initial rolling with subsequent adhesion.

p85-RhoGDI2, a novel complex, is required for PSGL-1-induced ß1 integrin-mediated lymphocyte adhesion to VCAM-1.

P-selectin glycoprotein ligand-1 and ß1 integrin play essential roles in T cell trafficking during inflammation. E-selectin and vascular cell adhesion molecule-1 are their ligands expressed on inflammation-activated endothelium. During the tethering and rolling of lymphocytes on endothelium, P-selectin glycoprotein ligand-1 binds E-selectin and induces signals. Subsequently, ß1 integrin is activated and mediates stable adhesion. However, the intracellular signal pathways from PSGL-1 to ß1 integrin have not yet been fully understood. Here, we find that p85, a regulatory subunit of phosphoinositide 3-kinase, forms a novel complex with Rho-GDP dissociation inhibitor-2, a lymphocyte-specific RhoGTPases dissociation inhibitor. Phosporylations of the p85-bound Rho-GDP dissociation inhibitor-2 on 130 and 153 tyrosine residues by c-Abl and Src were required for the complex to be recruited to P-selectin glycoprotein ligand-1 and thereby regulate ß1 integrin-mediated T cell adhesion to vascular cell adhesion molecule-1. Both shRNAs to Rho-GDP dissociation inhibitor-2 and p85 and over-expression of Rho-GDP dissociation inhibitor-2 Y130F and Y153F significantly reduced the above-mentioned adhesion. Although Rho-GDP dissociation inhibitor-2 in the p85-Rho-GDP dissociation inhibitor-2 complex was also phosphorylated on 24 tyrosine residue by Syk, the phosphorylation is not required for the adhesion. Taken together, we find that specific phosphorylations on 130 and 153 tyrosine residues of p85-bound Rho-GDP dissociation inhibitor-2 are pivotal for P-selectin glycoprotein ligand-1-induced ß1 integrin-mediated lymphocyte adhesion to vascular cell adhesion molecule-1. This will shed new light on the mechanisms that connect leukocyte initial rolling with subsequent adhesion.

PI3K is involved in L-selectin- and PSGL-1-mediated neutrophil rolling on E-selectin via F-actin redistribution and assembly.

L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) are adhesion molecules that play critical roles in neutrophil rolling during inflammation and lymphocyte homing. On the other hand they also function as signaling receptors to induce cytoskeleton changes. The present study is to investigate the signaling kinases responsible for the F-actin changes mediated by L-selectin and PSGL-1 during neutrophil rolling on E-selectin. Western blot analysis demonstrated that PI3K activation, peaking within 5 min, was induced by ligation of L-selectin and PSGL-1 with E-selectin, and that Vav1 (the pivotal downstream effector of PI3K signaling pathway involved in cytoskeleton regulation) was recruited to the membrane and tyrosine-phosphorylated, depending on PI3K. Furthermore, the F-actin redistribution and assembly mediated by ligation with E-selectin were blocked by LY294002, a PI3K specific inhibitor. Additional experiments showed that PI3K activity was involved in neutrophil rolling on E-selectin. However, Syk/Zap70, the well-known upstream kinase of PI3K, was not involved in this event. These data suggest that PI3K is required for the F-actin-based cytoskeleton changes during neutrophil rolling on E-selectin, which may consequently regulate the rolling event.