Predicting the risk of interstitial lung disease in patients with primary Sjögren's syndrome: Novel nomogram and elevated Th2 cells.

OBJECTIVE: Interstitial lung disease (ILD) is one of the most common pulmonary complications in patients with primary Sjögren's syndrome (pSS). This study was performed to identify immunological risk factors of pSS-associated ILD (pSS-ILD) and to further establish and evaluate of nomograms predicting the risk of ILD in patients with pSS. METHODS: A total of 622 patients with pSS (117 with ILD and 505 without lung involvement) and 166 healthy control subjects (HCs) were ultimately recruited to this retrospective study. Routine examination indicators, tumour markers and lymphocyte (LYMP) subpopulations were extracted. Simple and multiple logistic regressions analyses were performed to screen for independent predictors. Restricted cubic splines were used to examine associations of independent predictors with ILD, and a risk assessment model was constructed. A nomogram prediction model was developed, and receiver operating characteristic (ROC) curve analysis was performed to assess its performance. RESULTS: Univariate and multivariate logistic regression analyses showed that the older age, white blood cell (WBC) count, haemoglobin (HB) level, albumin (ALB) level, CA242 level, and the C-reactive protein (CRP)/LYMP ratio (CLR) were independent predictors of pSS-ILD in a linear manner, these factors were integrated and used to construct a nomogram prediction model. The model had clinical predictive value. In addition, the elevated Th2 cells proportion in pSS patients was significantly positively correlated with lung involvement, while it was negatively correlated with HB and ALB levels. Remarkably, the numbers of Th2 cells were correlated with the CLR in both pSS patients and those with pSS-ILD. CONCLUSIONS: Our novel ILD nomogram could be used to assess the risk of ILD in pSS patients with good discrimination ability. As well as, elevated peripheral blood Th2 cell levels may be related to ILD in patients with pSS.

Short-term and low-dose IL-2 therapy increases the reduced Treg cells in patients with microscopic polyangiitis.

OBJECTIVE: The breakdown of immune tolerance mediated by the reduced regulatory T (Treg) cell contributes to autoimmune diseases, which can be recovered by the short-term and low-dose interleukin 2 (IL-2). However, the role of Treg cells in microscopic polyangiitis (MPA) and the efficacy of short-term and low-dose IL-2 for MPA remain unclear. Therefore, we performed a retrospective study to explore the role of Treg cells and evaluate the efficacy of short-term and low-dose IL-2 therapy in MPA. METHODS: 52 MPA were collected as research objects, and 15 of them voluntarily received short-term and low-dose IL-2 subcutaneous injection combined with conventional therapy. 60 volunteers were recruited as health controls (HC) according to the inclusion and exclusion criteria. The number of circulating CD4 + T cell subsets was detected by flow cytometry. RESULTS: Patients with MPA had reduced circulating Treg cells than HCs (P < 0.001), and the level of Treg cells were reduced in MPA-activity and ANCA-positive group (P = 0.018 and P = 0.008 respectively). The patients with lower Treg cells had the higher incidence of the organ involvement (P = 0.006). The level of Treg cells in MPA was doubled after the short-term and low-dose IL-2 combined with conventional therapy (P = 0.001), and the disease activity indicators such as ESR and CRP were improved (P < 0.05) with no apparent side effects. CONCLUSION: Patients with MPA had reduced circulating Treg cells, especially the MPA-activity and ANCA-positive patients. And the patients with lower Treg cells were more likely to exhibit the organ involvement. Short-term and low-dose IL-2 therapy increased the reduced Treg cells and promoted the remission of the disease at a certain extent with well tolerance.

Analytical and clinical performance of different platforms simultaneously detecting 15 antinuclear antibodies.

BACKGROUND: Antinuclear antibodies (ANAs) are invaluable biomarkers for the diagnosis of autoimmune diseases (AIDs). This study aims to compare the performances of line immunoassay (LIA), multiplex bead-based flow fluorescent immunoassay (MBFFI), and magnetic bar code immunofluorescence assay (MBC-IF) to detect ANA-Profile-15S. METHODS: In total, 184 samples from AID patients and 50 healthy controls (HCs) were collected. Fifteen ANAs (anti-dsDNA, nucleosome, histone, Sm, PCNA, ribosomal-P, SS-A/Ro52, SS-A/Ro60, SS-B/La, centromere B [CENP-B], Scl-70, U1-snRNP, AMA-M2, Jo-1, and Pm/Scl) were subjected to parallel detection by the LIA, MBFFI, and MBC-IF. The consistency between assays was analyzed. The discrepant results were further examined by chemiluminescent immunoassay (CLIA). RESULTS: Anti-SS-A/Ro52 and SS-A/Ro60 autoantibodies were the most common autoantibodies in ANA positive-profiles, and were detected with equal efficiency by the LIA, MBFFI, and MBC-IF (p = 0.101 and p = 0.732, respectively). The three assays showed excellent agreement (consistency range: 66.5%-97.5%), and total consistency was 85.8%. The MBFFI and MBC-IF assays were in good agreement in terms of ANA-Profile-15S determination; the kappa coefficient ranged from 0.59 to 0.95, except for the PCNA and PM-Scl. Of the 262 re-assessed divergent results, 124 (47.33%) were positive on CLIA; the various autoantibodies exhibited variable patterns. More importantly, the ANA-Profile-15S results of the MBFFI and MBC-IF accurately identified patients with AID; the area under the curves ranged from 0.642 to 0.919. CONCLUSIONS: The novel MBFFI and MBC-IF assay performed well in detecting ANA-Profile-15S. The application of MBFFI and MBC-IF play important roles in laboratory diagnosis of AIDs.

Sirolimus therapy restores the PD-1+ICOS+Tfh:CD45RA-Foxp3high activated Tfr cell balance in primary Sjögren's syndrome.

BACKGROUND: Primary Sjögren's syndrome (pSS) is a common chronic autoimmune disease characterized by lymphocytic infiltration of salivary and lacrimal glands. The current study was performed to investigate the roles of follicular helper T (Tfh) and follicular regulatory T (Tfr) subsets in patients with pSS, and to evaluate the effects of sirolimus on these cells. METHODS: Levels of circulating Tfh and Tfr subsets in 58 pSS patients and 26 healthy controls (HC) were determined by flow cytometry. These T cell subsets were also analyzed in 12 patients before and after treatment with sirolimus. Clinical features and correlations with follicular T cells were analyzed systematically. The discriminative ability of the cells and ratios was evaluated based on the area under the receiver operating characteristic curves. RESULTS: Patients with pSS had higher percentage and absolute number of PD-1+ICOS+Tfh cells, while lower percentage and absolute number of Tfr, activated regulatory T (aTreg) cells, and CD45RA-Foxp3high activated Tfr cells. Furthermore, increased number of PD-1+ICOS+Tfh cells was associated with B cells, while decreased numbers of Tfr and their subsets was strongly associated with aTreg cells in pSS patients. Also, the higher proportion of PD-1+ICOS+Tfh cells was positively correlated with higher level of autoantibodies, ESR, IgG, cytokines (IL-2, IL-4, IL-10, IL-17, IFN-γ, TNF-α, IL-21 and sIL-2αR), and disease activity. Unexpectedly, the elevated PD-1+ICOS+Tfh:CD45RA-Foxp3high activated Tfr ratio had the greatest ability to discriminate between pSS and HC, and sirolimus therapy restored the PD-1+ICOS+Tfh cells:CD45RA-Foxp3high activated Tfr ratio, and controlled disease activity. CONCLUSION: The novel ratio of PD-1+ICOS+Tfh to CD45RA-Foxp3high activated Tfr cells can effectively discriminate the pSS patients from controls, and Tfr cell subsets may resemble Treg cell lineages. Furthermore, the PD-1+ICOS+Tfh cells can be used as a biomarker of disease activity and to verify the therapeutic effects of sirolimus in pSS.

Identification of biomarkers by machine learning classifiers to assist diagnose rheumatoid arthritis-associated interstitial lung disease.

BACKGROUND: This study aimed to search for blood biomarkers among the profiles of patients with RA-ILD by using machine learning classifiers and probe correlations between the markers and the characteristics of RA-ILD. METHODS: A total of 153 RA patients were enrolled, including 75 RA-ILD and 78 RA-non-ILD. Routine laboratory data, the levels of tumor markers and autoantibodies, and clinical manifestations were recorded. Univariate analysis, least absolute shrinkage and selection operator (LASSO), random forest (RF), and partial least square (PLS) were performed, and the receiver operating characteristic (ROC) curves were plotted. RESULTS: Univariate analysis showed that, compared to RA-non-ILD, patients with RA-ILD were older (p < 0.001), had higher white blood cell (p = 0.003) and neutrophil counts (p = 0.017), had higher erythrocyte sedimentation rate (p = 0.003) and C-reactive protein (p = 0.003), had higher levels of KL-6 (p < 0.001), D-dimer (p < 0.001), fibrinogen (p < 0.001), fibrinogen degradation products (p < 0.001), lactate dehydrogenase (p < 0.001), hydroxybutyrate dehydrogenase (p < 0.001), carbohydrate antigen (CA) 19-9 (p < 0.001), carcinoembryonic antigen (p = 0.001), and CA242 (p < 0.001), but a significantly lower albumin level (p = 0.003). The areas under the curves (AUCs) of the LASSO, RF, and PLS models attained 0.95 in terms of differentiating patients with RA-ILD from those without. When data from the univariate analysis and the top 10 indicators of the three machine learning models were combined, the most discriminatory markers were age and the KL-6, D-dimer, and CA19-9, with AUCs of 0.814 [95% confidence interval (CI) 0.731-0.880], 0.749 (95% CI 0.660-0.824), 0.749 (95% CI 0.660-0.824), and 0.727 (95% CI 0.637-0.805), respectively. When all four markers were combined, the AUC reached 0.928 (95% CI 0.865-0.968). Notably, neither the KL-6 nor the CA19-9 level correlated with disease activity in RA-ILD group. CONCLUSIONS: The levels of KL-6, D-dimer, and tumor markers greatly aided RA-ILD identification. Machine learning algorithms combined with traditional biostatistical analysis can diagnose patients with RA-ILD and identify biomarkers potentially associated with the disease.

A Decreased Absolute Number of Treg Cells in Patients with Active Rheumatoid Arthritis is Associated with Elevated Serum Osteopontin Levels with Disease Progression.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic and refractory autoimmune disease characterized by synovial inflammation with unknown aetiology. Immune system dysfunction mediated by CD4+ T lymphocytes, which is regulated by the cytokine osteopontin (OPN), plays an important role in the pathogenesis of RA. METHODS: In this study, the levels of peripheral CD4+ T subsets and serum OPN in patients with active RA were measured and analysed to determine the possible pathogenesis of RA and to provide potential therapeutic targets. RESULTS: Serum OPN levels in both patients with active RA and patients with refractory RA were higher than those in healthy controls (HCs). Compared with HCs, the absolute numbers of Th2 cells increased in patients with active RA, while the absolute counts of Th1 and Treg cells decreased. There was no significant difference in CD4+ T subset levels between new-onset and refractory patients. As the condition persisted or deteriorated, a gradual increase in the levels of OPN and gradual declines in the absolute counts of Th1 and Treg cells were observed in patients with active RA. The fewest Th1 and Treg cells and the highest OPN levels were observed in patients with high disease activity. The serum OPN level was only significantly negatively correlated with the absolute counts of Treg cells in the CD4+ T lymphocyte subsets. CONCLUSIONS: Fewer Treg cells with the increase in disease activity may be related to the increased OPN concentration, which may provide new ideas and directions for the targeted immunoregulatory treatment of RA.

Altered levels of circulating CD8+CXCR5+PD-1+T follicular cytotoxic cells in primary Sjögren's syndrome.

BACKGROUND: Circulating CD8+ T-cells expressing the C-X-C chemokine receptor type 5 (CXCR5) (CD8+CXCR5+T), a recently identified follicular cytotoxic T cell subset, are involved in antiviral immunity and autoimmunity, but their abundance and role in the pathogenesis of primary Sjögren syndrome (pSS) are unknown. METHODS: Circulating CD8+CXCR5+T cell and CD8+ regulatory T cells (CD8+Treg) were evaluated in 49 pSS patients (19 patients with pulmonary involvement) and 24 age- and sex-matched healthy controls (HCs) by flow cytometry. Orthogonal partial least squares discriminant analysis (OPLS-DA) was performed, and receiver operating characteristic curves (ROC) were generated to identify characteristic cell subsets. Spearman's correlation analysis was conducted to examine the relationships between CD8+ T cell subsets and clinical features. RESULTS: The proportions and numbers of CD8+CXCR5+, CD8 + CXCR5+ programmed death 1-positive (PD-1+), and CD8+CXCR5-PD-1+T cells were significantly higher, whereas those of CD8+Treg were markedly lower, in pSS patients than HCs. The CD8+CXCR5+PD-1+T cell to CD8+Treg ratio had the greatest discriminatory power for pSS and HCs according to OPLS-DA and ROC analyses. The increased numbers of CD8+CXCR5+T cells and CD8+CXCR5+PD-1+T cells were strongly associated with those of CD4+CXCR5+T and B cells. The proportions and numbers of CD8+CXCR5+PD-1+T cells were increased in pSS patients with lung involvement. CONCLUSIONS: We identified a new CD8+CXCR5+PD-1+T subset, which was increased in abundance in pSS patients, particularly those with lung involvement, compared with HCs. Also, the CD8+CXCR5+PD-1+T to CD8+Treg ratio may be useful for identifying pSS. Our findings suggest that targeting follicular CD8+T cell subsets has therapeutic potential for pSS. Key Points • CD8+CXCR5+ T cells were expanded in the circulation of patients with pSS. • Reduced numbers CD8+Treg cells in pSS patients. • Increased CD8+CXCR5+PD-1+T cells in pSS patients with pulmonary involvement.

Development of Prediction Models for New Integrated Models and a Bioscore System to Identify Bacterial Infections in Systemic Lupus Erythematosus.

Objectives: Distinguishing flares from bacterial infections in systemic lupus erythematosus (SLE) patients remains a challenge. This study aimed to build a model, using multiple blood cells and plasma indicators, to improve the identification of bacterial infections in SLE. Design: Building PLS-DA/OPLS-DA models and a bioscore system to distinguish bacterial infections from lupus flares in SLE. Setting: Department of Rheumatology of the Second Hospital of Shanxi Medical University. Participants: SLE patients with flares (n = 142) or bacterial infections (n = 106) were recruited in this retrospective study. Outcome: The peripheral blood of these patients was collected by the experimenter to measure the levels of routine examination indicators, immune cells, and cytokines. PLS-DA/OPLS-DA models and a bioscore system were established. Results: Both PLS-DA (R2Y = 0.953, Q2 = 0.931) and OPLS-DA (R2Y = 0.953, Q2 = 0.942) models could clearly identify bacterial infections in SLE. The white blood cell (WBC), neutrophile granulocyte (NEUT), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), procalcitonin (PCT), interleukin-6 (IL-6), IL-10, interferon-γ (IFN-γ), and tumor necrosis factor α (TNF-α) levels were significantly higher in bacteria-infected patients, while regulatory T (Treg) cells obviously decreased. A multivariate analysis using the above 10 dichotomized indicators, based on the cut-off value of their respective ROC curve, was established to screen out the independent predictors and calculate their weights to build a bioscore system, which exhibited a strong diagnosis ability (AUC = 0.842, 95% CI 0.794-0.891). The bioscore system showed that 0 and 100% of SLE patients with scores of 0 and 8-10, respectively, were infected with bacteria. The higher the score, the greater the likelihood of bacterial infections in SLE. Conclusions: The PLS-DA/OPLS-DA models, including the above biomarkers, showed a strong predictive ability for bacterial infections in SLE. Combining WBC, NEUT, CRP, PCT, IL-6, and IFN-γ in a bioscore system may result in faster prediction of bacterial infections in SLE and may guide toward a more appropriate, timely treatment for SLE.

Double-negative T cells are absolutely elevated in patients with antineutrophil cytoplasmic autoantibody-associated vasculitis.

CD3+CD4-CD8- [double-negative (DN)] T cells play vital roles in the pathogenesis of autoimmune disorders. In this study, we investigated the exact level of DN T cells in patients with antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV). Forty patients with active AAV and 19 healthy controls (HCs) were enrolled in this study. Peripheral mononuclear cells were characterised phenotypically via flow cytometry. The potential clinical value of DN T cells was then assessed by the receiver operating characteristic (ROC) curves. The percentage (p < 0.001) and absolute number (p = 0.028) of DN T cells were found to be significantly higher in patients with AAV than in HCs. Relative to HCs, a lower percentage of DN T cells from patients with AAV was of the CD62L+CD45RO+ phenotype (p = 0.024), a higher percentage of these cells was of the CD62L-CD45RO- phenotype (p = 0.043). Patients with AAV had increased percentages of DN T cells expressing interferon (IFN)-γ (p = 0.032), interleukin (IL)-4 (p = 0.039) and IL-17 (p = 0.042). Furthermore, the percentages of IL-17-producing CD4+ T cells and IFN-γ-producing CD8+ T cells were significantly higher in patients with AAV than in HCs (p = 0.014, p = 0.008). Compared with the CD4+ and CD8+ T-cell subsets, DN T cells had the highest fractions of intracellular IL-17 in HCs and patients with AAV (both p < 0.001). In patients with AAV and renal damage, the percentage of DN T cells was expanded relative to that in patients without renal damage (p = 0.016). In addition, conventional methylprednisolone effectively reduced the percentage and overall number of DN T cells in patients with AAV (p = 0.028, p = 0.007). DN T cells represent a T-lymphocyte subset that produces inflammatory cytokines (IFN-γ, IL-4 and IL-17) and is absolutely elevated in patients with AAV. Additional investigations are required to determine their precise role in patients with AAV.

Comprehensive analysis of multiple cytokines and blood parameters for the diagnosis of bacterial infections in rheumatoid arthritis.

Patients with rheumatoid arthritis (RA) are more susceptible to infections, which elevate the levels of relative cytokines. However, the ability of the cytokines levels to predicate bacterial infections in RA patients remains unclear. Here, we assessed the ability of the combination of serum cytokine levels and blood parameters to diagnose bacterial infections in RA patients. We measured the levels of a panel of serum cytokine and blood parameters in 168 RA patients and 81 healthy individuals. RA patients were divided into the bacterial infection (INFE) group (n = 76) and RA flare without INFE group (n = 92). Bacterial infection was confirmed by microbial culture, imaging, antibiotic response, and typical clinical symptoms. The discriminative ability of the combination of the cytokine levels and inflammatory parameters was assessed using the receiver-operating characteristic (ROC) curves analysis and a novel bioscore system. The levels of interleukin (IL)-6 (p = 0.006), IL-10 (p = 0.019), interferon (IFN)-γ (p = 0.033), CRP (p < 0.001), and ESR (p < 0.001) were higher in patients of the INFE group than in patients with RA flare, and the absolute numbers of CD19+ B cells (p < 0.001) and CD4+ T cells (p = 0.009) were lower. For discriminating bacterial infection, the combination of IL-6, IL-10, IFN-γ, ESR, CRP, CD19+ B cells, and CD4+ T cells, provided an area under the curve (AUC) of 0.827 [(95% confidence interval (CI): 0.760-0.881)], which was profoundly larger than that of IL-6, IL-10, IFN-γ, ESR, CRP, CD19+ B cells, or CD4+ T cells alone. In addition, we also developed a bioscore system based on the combination of these seven biomarkers. Seventeen (100%) patients with a bioscore of 0 were non-infected, while seven (100%) patients with a score of 7 had bacterial infections. The bioscore based on the combination of ESR, CRP, IL-6, IL-10, IFN-γ, CD19+ B cells and CD4+ T cells may be a promising and robust tool to diagnose bacterial infections in RA patients.

Low-dose Interleukin-2: Biology and therapeutic prospects in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic aggressive arthritis that is characterized with systemic inflammation response, the production of abnormal antibodies, and persistent synovitis. One of the key mechanisms underlying the pathogenesis of RA is the imbalance of CD4 + T lymphocyte subsets, from T helper (Th) 17 cells and regulatory T (Treg) cells to T follicular helper (Tfh) cells and T follicular regulatory (Tfr) cells, which can mediate autoimmune inflammatory response to promote the overproduction of cytokines and abnormal antibodies. Although the treatment of RA has greatly changed due to the discovery of biological agents such as anti-TNF, the remission of it is still not satisfactory, thus, it is urgently required new treatment to realize the sustained remission of RA via restoring the immune tolerance. Interleukin-2 (IL-2) has been discovered to be a pleiotropic cytokine to promote inflammatory response and maintain immune tolerance. Low-dose IL-2 therapy is a driver of the imbalance between autoimmunity and immune tolerance towards immune tolerance, which has been tried to treat various autoimmune diseases. Recent researches show that low-dose IL-2 is a promising treatment for RA. In this review, we summarize the advances understandings in the biology of IL-2 and highlight the impact of the IL-2 pathway on the balance of Th17/Treg and Tfh/Tfr aiming to investigate the role of IL-2-mediated immune tolerance in RA and discuss the application and the therapeutic prospect of low-dose IL-2 in the treatment of RA.

Characteristics and reference ranges of CD4+T cell subpopulations among healthy adult Han Chinese in Shanxi Province, North China.

BACKGROUND: Immunophenotyping of blood lymphocytes is an essential tool to evaluate the immune function of patients with immunodeficiency or autoimmunity. Predominately identified CD4+T cell subsets, Th1, Th2, Th17, as well as regulatory T (Treg) cells, play crucial roles in several immunological and pathological conditions. Considering the variations in cell counts among populations and ethnicities, specific CD4+T cell subset reference values need to be locally established to enable meaningful comparisons and accurate data interpretation in clinical and research settings. Therefore, the aim of this study was to establish distributions and reference ranges for blood CD4+T cell subpopulations in age- and sex-balanced healthy adults of a Han Chinese population in Shanxi Province, North China. METHODS: Peripheral blood CD4+T cell subsets were examined in 150 healthy volunteers (75 males, 75 females) aged 20-70 years with a four-color FACSCalibur flow cytometer. RESULTS: Reference value percentages (absolute counts, cells/µl) were defined as 95% of the population for cell types as follows: CD4+T, 23.78-51.07 (360-1127); Th1, 0.43-39.62 (2.64-276.21); Th2, 0.27-3.57 (1.80-27.14); Th17, 0.22-2.62 (1.10-19.54); and Treg, 2.17-7.94 (13.47-64.58). The ranges for the Th1:Th2 and Th17:Treg ratios were 0.59-52.37 and 0.04-0.76, respectively. Notably, a significant increase was observed in the values of Treg cells in older individuals, and the numbers of Treg cells in females also tended to decrease when compared to those in males. Therefore, we established the distribution and reference range of CD4+T cell subsets based on age and sex, demonstrating the lowest values of Treg cells in younger females. CONCLUSIONS: Collectively, our data provide population-, age-, and sex-specific distributions and reference ranges of circulating CD4+T cell subpopulations, which can be adopted to guide clinical decisions and interpretation of immunophenotyping data in the Han Chinese population in Taiyuan, Shanxi Province, China. In addition, the low expression of peripheral Treg cells in younger females may be associated with the predisposition of females to autoimmune diseases.

Characteristics of and reference ranges for peripheral blood lymphocytes and CD4+ T cell subsets in healthy adults in Shanxi Province, North China.

OBJECTIVE: To guide clinical decision making, race-, age- and gender-specific reference ranges for lymphocytes and CD4+ T-cell subsets are required. METHODS: Single platform flow cytometry to determine reference intervals for lymphocyte subpopulations and CD4+ T-cell subsets in 196 healthy Han Chinese adults. RESULTS: The frequencies and absolute numbers of B cells were slightly lower in Han Chinese individuals of the Shanxi region than in individuals from Hong Kong, Germany and Singapore, while percentages and absolute numbers of NK cells were slightly higher compared with individuals from Hong Kong. CD4+/CD8+ T-cell ratios, CD4+ T cell percentages and Th2 cell counts were higher, while frequencies and numbers of CD8+ T cells, numbers of NK cells and percentages of Th1 cells were lower, in females compared with males. CD4+ T cell percentages, CD4+/CD8+ T-cell ratios, numbers of CD8+ T cells and Treg cells, and Th17/Treg cell ratios differed by age. CONCLUSION: We established lymphocyte and CD4+ T-cell subset reference intervals for healthy Han Chinese adults of the Shanxi region. Ethnicity, gender and age affected lymphocyte subset composition.

Increased levels of BPI-ANCA in patients with primary Sjögren's syndrome are associated with lung involvement.

BACKGROUND: The goal of this study was to investigate the association between bactericidal permeability increasing (BPI)-antineutrophil cytoplasmic antibody (ANCA) protein levels and primary Sjogren's syndrome (pSS) with lung involvement, as well as the potential diagnostic performance of BPI-ANCA. METHODS: The levels of BPI-ANCA in pSS patients with (n = 36) and without (n = 85) lung involvement were measured using a commercial ELISA kit. Serological biomarkers and cytokines were measured in these patients as well. Lung involvement was determined by high-resolution computed tomography (HRCT) and/or clinical symptoms. The diagnostic performance of lung involvement was determined by receiver operating characteristic (ROC) curves. RESULTS: The percentage of neutrophils (NEUT%), neutrophil-lymphocyte ratio (NLR), erythrocyte sedimentation rate (ESR), and the levels of BPI-ANCA, C-reactive protein (CRP), interleukin-2 (IL-2) and IL-6 exhibited an upward trend, while the percentage of lymphocytes (LYMP%) and albumin (ALB) level exhibited a downward trend in the lung involvement group. The combination of BPI-ANCA, NEUT% and ALB significantly increased the area under the ROC curve (AUC) to 0.837 (95% confidence interval: 0.742-0.907, sensitivity: 82.14%, specificity: 81.36%, P < 0.001). CONCLUSIONS: Increased BPI-ANCA was found in pSS patients with lung involvement and was associated with inflammation. A combination of BPI-ANCA, NEUT% and ALB had the best AUC, and may serve as anadjunct to distinguish between pSS patients with and without lung involvement.

Sirolimus selectively increases circulating Treg cell numbers and restores the Th17/Treg balance in rheumatoid arthritis patients with low disease activity or in DAS28 remission who previously received conventional disease-modifying anti-rheumatic drugs.

OBJECTIVES: Regulatory T (Treg) cells are crucial players in the prevention of autoimmunity. Mechanistic target of rapamycin (mTOR) signalling negatively controls the development and function of Treg cells. The aim of the present study was to evaluate the effects of rapamycin, under the generic name sirolimus, on CD4+CD25+FoxP3+ Treg cells in rheumatoid arthritis (RA) patients with low disease activity or in DAS28 remission. METHODS: Fifty-five RA patients and 60 healthy controls were enrolled in this study. All patients had previously received conventional disease-modifying anti-rheumatic drugs (DMARDs) and were considered to have a low DAS28 score (≤3.2). Peripheral blood samples and clinical information were obtained at baseline and following 6 and 12 weeks of sirolimus treatment, or after 12 weeks of conventional treatment. Peripheral blood samples were also obtained from the healthy controls. The circulating levels of lymphocyte subpopulations were assessed by flow cytometry. RESULTS: Thirty-five patients received sirolimus and 20 patients continued treatment with conventional DMARDs. The absolute counts and proportions of CD4+CD25+FoxP3+ Treg cells were significantly lower in all RA patients with DAS28 ≤ 3.2 as compared with those in healthy controls. By contrast, the difference in circulating Th17 cell numbers was not significant. Sirolimus administration resulted in elevations in circulating Treg cell numbers and significant reductions in the Th17/Treg cell ratio, whereas the circulating level of Treg cells and the Th17/Treg cell ratio in patients under conventional treatment both showed a tendency of reduction. Furthermore, a greater proportion of patients under sirolimus treatment achieved DAS28-based remission at 12 weeks. CONCLUSIONS: Sirolimus can favourably expand Treg cells in RA patients with DAS28 ≤3.2, consequently restoring a healthy balance of Th17/Treg cells, which might improve the likelihood of long-term and sustained clinical remission and reduce the probability of disease flare-ups in RA.

IL-17A upregulates P-glycoprotein expression in peripheral blood lymphocytes of patients with rheumatoid arthritis through TAK1.

OBJECTIVES: P-glycoprotein (P-gp) mediated drug efflux is the most essential mechanism of multi-drug resistance (MDR) in rheumatoid arthritis (RA). The study was undertaken to clarify the mechanism whereby IL-17 regulate the P-gp efflux function in peripheral blood lymphocytes of patients with RA. METHODS: Lymphocytes from RA patients and healthy individuals were cultured with IL-17A (0, 10, 100 ng/ml), IL-17A+(5Z)-7-Oxozeaenol (TAK1 inhibitor), and IL-17A+PD98059 (ERK inhibitor), respectively. 24h later, the level of P-gp mRNA expression in peripheral blood lymphocytes was detected by RT-PCR. Meanwhile, the efflux potential of P-gp was assessed by flow cytometry using the fluorescent dye Rhodamine 123, a substrate of P-gp. In order to confirm whether the inhibitors had worked, ERK1/2 and p65, as well as their phosphorylation were detected utilising Western blot analysis. RESULTS: With the exception of the expression of P-gp mRNA between control and IL-17A group, the mRNA expression, as well as the function of P-gp in the different group of healthy individuals was similar, and there was no significant difference (p>0.05). However, as for the RA patients, increased expressions of P-gp mRNA and efflux function were detected in IL-17A group compared with control. Moreover, IL-17A upregulated mRNA level and function of P-gp in a concentrate dependent manner. Upregulated expression of P-gp mRNA and efflux potential of P-gp were inhibited by TAK1 or ERK inhibitors in RA peripheral blood lymphocytes. Among them, TAK1 inhibitor, (5Z) -7-Oxozeaenol, showed a significant difference (p<0.05). Also, the decreased phosphorylation levels of ERK1/2 and p65 were detected with PD98059 and (5Z) -7-Oxozeaenol addition, respectively. CONCLUSIONS: This study showed that inflammatory cytokines IL-17A can upregulate the mRNA expression level and drug efflux function of P-gp on lymphocytes in RA patients through TAK1, in a concentrate dependent manner, contributing to RA drug resistance. Therefore, this may represent a new target for improving the therapeutic reactivity of DMARDs in the long term for RA patients.

Reduced activated regulatory T cells and imbalance of Th17/activated Treg cells marks renal involvement in ANCA-associated vasculitis.

The role of naturally occurring regulatory T cells (Treg) in the control of the immune tolerance of ANCA-associated vasculitis (AAV) has not been well defined. Therefore, we separate the phenotypically heterogeneous Treg cells into different subsets based on the expression of FOXP3 and CD45RA during AAV pathogenesis. Fifty-four AAV patients (38 patients with renal involvement) and 19 healthy controls (HCs) were enrolled in this study. Levels of CD4+T cell subsets and cytokines were detected by flow cytometry. Treg immunesuppression capacity was measured in co-culture experiments. The diagnostic value for Treg subsets was evaluated by the areas under the receiver operating characteristic curves (AUC). Patients with AAV had lower percentages and numbers of activated Treg cells (aTreg, P = 0.044, P = 0.002), while higher levels of total Treg cells (P = 0.001, P = 0.026) with diminished immunosuppression capacity. The proportions of effector memory T-cell subpopulation (P < 0.001) were increased in AAV patients. Interestingly, the AUC of the aTreg improved significantly the diagnostic potential of AAV. Furthermore, the ratio of Th17/aTreg was significantly increased in active and renal vasculitis patient and positive correlation between Th17/Treg subset ratio and creatinine or BUN. In addition, we found that cytokine IL-2 and IL-4 exhibited a downward while IL-6, IL-10, TNF-α, IFN-γ and IL-17A trend upward in AAV patients. Increase in total Treg levels, along with functional deficiency, and decrease in aTreg cells constitute potential novel biomarkers for AAV. And the ratio of Th17/aTreg might serve as an important tool to recognize and monitor AAV patients with renal involvement and disease remission.

T-Follicular Regulatory Cells: Potential Therapeutic Targets in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an incurable aggressive chronic inflammatory joint disease with a worldwide prevalence. High levels of autoantibodies and chronic inflammation may be involved in the pathology. Notably, T follicular regulatory (Tfr) cells are critical mediators of T follicular helper (Tfh) cell generation and antibody production in the germinal center (GC) reaction. Changes in the number and function of Tfr cells may lead to dysregulation of the GC reaction and the production of aberrant autoantibodies. Regulation of the function and number of Tfr cells could be an effective strategy for precisely controlling antibody production, reestablishing immune homeostasis, and thereby improving the outcome of RA. This review summarizes advances in our understanding of the biology and functions of Tfr cells. The involvement of Tfr cells and other immune cell subsets in RA is also discussed. Furthermore, we highlight the potential therapeutic targets related to Tfr cells and restoring the Tfr/Tfh balance via cytokines, microRNAs, the mammalian target of rapamycin (mTOR) signaling pathway, and the gut microbiota, which will facilitate further research on RA and other immune-mediated diseases.

Low-Dose Sirolimus Immunoregulation Therapy in Patients with Active Rheumatoid Arthritis: A 24-Week Follow-Up of the Randomized, Open-Label, Parallel-Controlled Trial.

BACKGROUND: We have reported previously the insufficient absolute number or functional defects of regulatory T cells (Tregs) in patients with rheumatoid arthritis (RA), challenging conventional unspecific immunosuppressive therapy. Sirolimus, a mTOR inhibitor, is reported to allow growth of functional Tregs; here, we investigated the efficacy of low-dose sirolimus combined with conventional immunosuppressants (sirolimus immunoregulation therapy) for RA treatment with lower side effects and better tolerance. METHODS: In this nonblinded and parallel-group trial, we randomly assigned 62 patients to receive conventional glucocorticoids and immunosuppressants with or without sirolimus at a dosage of 0.5 mg on alternate days for 24 weeks in a 2 : 1 ratio. The demographic features, clinical manifestations, and laboratory indicators including peripheral blood lymphocyte subgroups and CD4+T subsets were compared before and after the treatment. RESULTS: Finally, 37 patients in the sirolimus group and 18 in the conventional treated group completed the 6-month study. By 24 weeks, the patients with sirolimus experienced significant reduction in disease activity indicators including DAS28, ESR, and the number of tender joints and swollen joints (p < 0.001). Notably, they had a higher level of Tregs as compared with those with conventional therapy alone (p < 0.05), indicating that sirolimus could partly restore the reduced Tregs. Concomitantly, their usage of immunosuppressants for controlling disease activity was decreased as compared with the conventional group with no difference in blood routine, and liver and renal functions both before and after the treatment of sirolimus and between the two groups (p > 0.05). CONCLUSIONS: Low-dose sirolimus immunoregulatory therapy selectively upregulated Tregs and partly replaced the usage of immunosuppressants to control disease activity without overtreatment and evaluable side effect. Further study is required using a large sample of RA patients treated with sirolimus for a longer period. This trial is registered at the Chinese Clinical Trial Registry (http://www.chictr.org.cn/showproj.aspx?proj=17245).

Absolute reduction of regulatory T cells and regulatory effect of short-term and low-dose IL-2 in polymyositis or dermatomyositis.

AIM: The study aimed to investigate the changes in peripherallymphocyte and CD4+T subsets and to observe the regulatory effect of low-dose interleukin-2 (ld-IL2) on these cells in polymyositis or dermatomyositis (PM/DM). METHODS: Lymphocyte subsets (CD3+T, CD4+T, CD8+T, B and natural killer (NK) cells), CD4+T subsets (Th1, Th2, Th17 and regulatory T (Treg) cells) and multiple cytokines of 71 patients after admission and treatment were measured by flow cytometry, as well as these indicators in 30 healthy controls (HCs). In DM, 35 cases were administrated with ld-IL2 combined with conventional therapy, the remaining 26 patients received conventional therapy only. RESULTS: The numbers of CD3+T and CD4+T cells in PM/DM were markedly decreased. Meanwhile, the absolute number and percentage of peripheral Treg cells in PM/DM, as well as Th1 cells in DM, were significantly lower than those in HCs (P < 0.05), but Th2 and Th17 cells had no significant difference. The ratio of Th17/Treg in PM (P = 0.031) and in DM (P = 0.003) were obviously higher than that in HCs. The deficiency of Treg cells was associated with the occurrence of interstitial lung disease (ILD) in myositis patients. Meanwhile, reduced production of IL-2 was also observed in PM/DM (P < 0.001). ld-IL2 combination therapy could significantly increase the numbers of CD4+T subsets in DM, especially Treg cells (expanded 2.5 times). CONCLUSIONS: The decline of peripheral Treg cells and serum IL-2 were found in PM/DM. ld-IL2 combination therapy could significantly increase the number of Treg cells.