Th17 responses and natural IgM antibodies are related to gut microbiota composition in systemic lupus erythematosus patients.

Intestinal dysbiosis, characterized by a reduced Firmicutes/Bacteroidetes ratio, has been reported in systemic lupus erythematosus (SLE) patients. In this study, in vitro cultures revealed that microbiota isolated from SLE patient stool samples (SLE-M) promoted lymphocyte activation and Th17 differentiation from naïve CD4(+) lymphocytes to a greater extent than healthy control-microbiota. Enrichment of SLE-M with Treg-inducing bacteria showed that a mixture of two Clostridia strains significantly reduced the Th17/Th1 balance, whereas Bifidobacterium bifidum supplementation prevented CD4(+) lymphocyte over-activation, thus supporting a possible therapeutic benefit of probiotics containing Treg-inducer strains in order to restore the Treg/Th17/Th1 imbalance present in SLE. In fact, ex vivo analyses of patient samples showed enlarged Th17 and Foxp3(+) IL-17(+) populations, suggesting a possible Treg-Th17 trans-differentiation. Moreover, analyses of fecal microbiota revealed a negative correlation between IL-17(+) populations and Firmicutes in healthy controls, whereas in SLE this phylum correlated directly with serum levels of IFNγ, a Th1 cytokine slightly reduced in patients. Finally, the frequency of Synergistetes, positively correlated with the Firmicutes/Bacteroidetes ratio in healthy controls, tended to be reduced in patients when anti-dsDNA titers were increased and showed a strong negative correlation with IL-6 serum levels and correlated positively with protective natural IgM antibodies against phosphorylcholine.

DKK1 expression by synovial fibroblasts in very early rheumatoid arthritis associates with lymphocyte adhesion in an in vitro flow co-culture system.

BACKGROUND: Synovial fibroblasts play a key role in joint destruction and regulation of the inflammatory infiltrate in established rheumatoid arthritis (RA). The mechanisms by which this occurs in the earliest stages of RA are largely unknown. We investigated the role of Dickkopf-related protein 1 (DKK1) produced by synovial fibroblasts of patients with very early rheumatoid arthritis (VeRA). METHODS: Fibroblasts were isolated from the disease-modifying anti-rheumatic drug-naive Birmingham early arthritis cohort of patients with new onset of clinically apparent arthritis and inflammatory symptoms of ≤12 weeks' duration, who at follow-up had either resolving arthritis or RA. Endothelial fibroblast co-cultures were formed using porous filters, and lymphocyte adhesion to co-cultures was assessed using phase-contrast microscopy. DKK1 gene expression and secretion were quantified by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: Synovial fibroblasts from patients with VeRA expressed significantly higher levels of DKK1 messenger RNA than those from patients with resolving arthritis. A similar trend was observed for DKK1 protein secretion. In co-culture constructs, more DKK1 tended to be secreted in co-cultures incorporating fibroblasts from VeRA than in co-cultures from non-inflamed joints and resolving arthritis. DKK1 secretion during co-culture positively correlated with lymphocyte adhesion. CONCLUSIONS: Alterations in DKK1 could be involved in the pathogenesis and perpetuation of the inflammatory response in the earliest clinically apparent stages of RA.

IFNα serum levels are associated with endothelial progenitor cells imbalance and disease features in rheumatoid arthritis patients.

INTRODUCTION: IFNα has been largely implicated in the ethiopathogenesis of autoimmune diseases but only recently it has been linked to endothelial damage and accelerated atherosclerosis in autoimmunity. In addition, proinflammatory conditions are supposed to be implicated in the cardiovascular status of these patients. Since a role for IFNα in endothelial damage and impaired Endothelial Progenitor Cell (EPC) number and function has been reported in other diseases, we aimed to evaluate the potential associations of IFNα serum levels on EPC populations and cytokine profiles in Rheumatoid Arthritis (RA) patients. METHODS: pre-EPC, EPC and mature EPC (mEPC) populations were quantified by flow cytometry analyzing their differential CD34, CD133 and VEGFR2 expression in blood samples from 120 RA patients, 52 healthy controls (HC), and 83 systemic lupus erythematosus (SLE) patients as disease control. Cytokine serum levels were measured by immunoassays and clinical and immunological data, including cardiovascular (CV) events and CV risk factors, were retrospectively obtained by reviewing clinical records. RESULTS: Long-standing, but not recent onset RA patients displayed a significant depletion of all endothelial progenitor populations, unless high IFNα levels were present. In fact, the IFN(high) RA patient group (n = 40, 33%), showed increased EPC levels, comparable to SLE patients. In addition, high IFNα serum levels were associated with higher disease activity (DAS28), presence of autoantibodies, higher levels of IL-1ß, IL-6, IL-10 and MIP-1α, lower amounts of TGF-ß, and increased mEPC/EPC ratio, thus suggesting higher rates of endothelial damage and an endothelial repair failure. Finally, the relationship between high IFNα levels and occurrence of CV events observed in RA patients seems to support this hypothesis. CONCLUSIONS: IFNα serum marker could be used to identify a group of RA patients with increased disease activity, EPC imbalance, enhanced proinflammatory profile and higher cardiovascular risk, probably due, at least in part, to an impaired endothelial repair.

Relationship between FOXP3 positive populations and cytokine production in systemic lupus erythematosus.

In this work we studied CD4+FOXP3+ populations in systemic lupus erythematosus (SLE) and the relationship with Th cytokine production. We found an increment in CD25-FOXP3+ population in SLE associated with CD4+ downregulation and disease progression. CD25low cells were also upregulated and showed increased percentages of FOXP3+ and CD127-/low cells, supporting the activated status of SLE lymphocytes. Despite the normal levels of CD25highFOXP3+ cells, the negative correlations observed in controls with the frequency of IFNγ, TNFα and IL-10 secreting cells were disrupted in patients, supporting a defective Treg function. Also, CD25high cells showed an altered balance in the production of these cytokines. In addition, CD25highFOXP3+ cells correlated directly with IL-17A and IL-8 but not with TGFß in SLE. The increased proportion of IL-17+ cells among the CD25high subset and the positive correlation between IL-17 levels and Treg cells suggest a trans-differentiation of Treg into Th17 cells in SLE.

Circulating endothelial cells and their progenitors in systemic lupus erythematosus and early rheumatoid arthritis patients.

OBJECTIVE: The aim of this study was to investigate the endothelial progenitor cell population in SLE and early RA patients and its potential relationships with disease features and cytokine serum levels. METHODS: Endothelial progenitor cells (EPCs), mature EPCs (mEPCs) and endothelial cells (ECs) were measured in peripheral blood samples from 83 SLE and 85 early RA patients and 39 healthy controls by flow cytometry on the basis of CD34, VEGF receptor 2 and CD133 expression. Serum levels of IL-1ß, IL-6, IL-8, IL-17, VEGF-A, IFN-α, TGF-ß and GM-CSF were quantified by immunoassays. Clinical and immunological data were obtained by reviewing clinical histories. RESULTS: Circulating EPCs were increased in SLE but not in early RA patients associated with an enhanced CD34(+) bone marrow-progenitor cell release but unrelated to disease features. The amount of mEPCs, however, was significantly higher in SLE patients presenting anti-SSA/SSB antibodies and/or malar rash, whereas the presence of specific autoantibodies was associated with EC counts in early RA and SLE patients. As expected, most cytokines tested were altered in both diseases but, interestingly, IFN-α levels, and to a lesser extent IL-6 and IL-1ß, were associated with CD133 loss and increased mEPC number, whereas VEGF and TGF-ß seem to exert an opposite effect. CONCLUSION: Our results show that high IFN-α levels and/or the presence of disease-specific antibodies may identify a group of SLE patients with increased mEPC and EC counts, and consequently probably defective endothelial repair, thus supporting their use as surrogate biomarkers of endothelial damage and high cardiovascular risk.

Effects of glucocorticoid treatment on CD25⁻FOXP3⁺ population and cytokine-producing cells in rheumatoid arthritis.

OBJECTIVES: To investigate CD25(-)FOXP3(+) cells in RA patients and their possible relationship with disease features and response to glucocorticoids (GCs). METHODS: Peripheral blood mononuclear cells were collected from 147 RA patients, 29 healthy controls and 75 SLE patients as disease controls. The proportion of CD4(+)FOXP3(+) cells with negative, low or high CD25 expression and the levels of IL-10-, TNF-α-, IL-17- and IFNγ-producing cells were assessed by flow cytometry. The presence of the high IL-10 genotype (-1082GG), associated with good response to GC, was determined by PCR amplification and hybridization with allele-specific fluorescently labelled probes. Data were related to treatment and clinical parameters. RESULTS: The CD25(-)FOXP3(+) population was significantly increased in RA patients and negatively correlated with DAS-28 and other disease parameters. The IL-10 genotype did not influence the frequency of these cells in controls or the entire RA group; however, GC-treated patient carriers of the high IL-10 genotype presented significantly higher levels of this population in addition to an increased percentage of IL-10-secreting cells and relatively low amounts of TNF-α-, IFN-γ- and IL-17-positive cells. Finally, a prospective study confirmed that genetically high IL-10 producers significantly increase CD25(-)FOXP3(+) cells after 6 months of GC treatment. CONCLUSION: The present study provides the first evidence of increased CD25(-)FOXP3(+) cells in RA patients, which were associated with disease activity and with GC treatment in carriers of the high IL-10 genotype, suggesting that this population plays a role in the clinical response to prednisone in RA.

Glucocorticoids enhance Th17/Th1 imbalance and signal transducer and activator of transcription 3 expression in systemic lupus erythematosus patients.

OBJECTIVE: To investigate the relative amounts of Th17 and Th1 cells present in SLE patients and the possible effects of treatments or disease features on these populations. METHODS: Peripheral blood mononuclear cells were collected from 75 SLE patients and 19 healthy controls and the proportion of Th17 and Th1 populations were assessed by flow cytometry measuring the amount of IL-17 and IFN-γ-producing cells. Gene expression of signal transducers and activators of transcription 3 (STAT3), STAT4, IL-6R and IL-12R were determined in 30 patients and 8 healthy individuals by real-time RT-PCR. Data were related to clinical and immunological parameters and to the treatment followed during the past 3 months. RESULTS: Th17 cells and the Th17/Th1 ratio were significantly increased in SLE patients treated with glucocorticoids compared with healthy individuals, untreated patients or those under other treatments. No association was detected with clinical parameters, but patients with anti-ENA antibodies also displayed increased Th17 responses. Disease activity (SLEDAI) is associated with the Th17/Th1 index only in glucocorticoid-treated patients. In line with these results, gene expression of STAT3 and IL-6R was up-regulated in patients taking these drugs. Accordingly, we found a positive correlation between the Th17/Th1 ratio and STAT3 levels. CONCLUSIONS: The present work provides the first evidence that aberrant Th17/Th1 balance in SLE is linked to the use of glucocorticoids and suggests that the up-regulatory effect of these drugs on the Th17 population could be associated with their ability to increase STAT3 and IL-6R expression. Additionally, anti-ENA positivity could represent a potential biomarker for Th17 bias.

Dexamethasone upregulates FOXP3 expression without increasing regulatory activity.

Recent studies have shown the capacity of corticoids to increase forkhead box p3 (FOXP3) expression, which suggests that these drugs may be able to generate regulatory T cells (Treg). Therefore, corticoids may possibly be employed in protocols to generate or expand Treg cells with the aim of being used in cell transfer therapy. However, given that in humans FOXP3 is not necessarily associated with regulatory function, it is of great importance to ascertain whether FOXP3-expressing cells generated with corticoids are "truly" Treg cells. To this end, we studied the effect of dexamethasone on both human activated lymphocytes and in vitro generated Treg cells as well as regulatory activity of CD4(+)CD25(high) cells from SLE patients users and non users of prednisone. Results show that dexamethasone markedly enhances FOXP3 expression and generates CD25(high) cells with phenotypic characteristics attributable to natural Treg cells. Unexpectedly, in spite of their hyporesponsiveness and enhanced FOXP3 expression, these cells did not exert suppressive activity. Moreover, although dexamethasone was able to enhance FOXP3 expression in in vitro generated Treg cells, once again this effect was not correlated with increased regulatory activity. These results were supported by the fact that CD4(+)CD25(high) cells from steroid-treated SLE patients did not show a higher antiproliferative function than those from non-steroid-treated patients. We conclude that the increment on FOXP3 expression caused by dexamethasone is not connected with regulatory function, supporting the fact that FOXP3 expression in humans is not an exclusive attribute of Treg cells. Subsequently, the use of FOXP3 as a Treg cell marker must be done cautiously, especially in patients with systemic inflammatory diseases or those under corticoid treatment.

Cytokines and regulatory T cells in rheumatoid arthritis and their relationship with response to corticosteroids.

OBJECTIVE: To analyze circulating cytokines and regulatory T cells (Treg) in patients with rheumatoid arthritis (RA) of different durations, and their association with functional interleukin 10 (IL-10) and tumor necrosis factor-α (TNF-α) genotypes in patients treated with corticosteroids. METHODS: Serum levels of IL-6, IL-10, IL-17, IL-18, TNF-α, and transforming growth factor-ß (TGF-ß) were quantified in 196 patients and 61 healthy controls. Percentage of CD4+CD25high cells was determined by flow cytometry and Foxp3 expression by real-time reverse-transcription polymerase chain reaction. Data were related to clinical measurements and presence of the genotype -1082GG IL-10/-308GG TNF-α, previously associated with good response to corticosteroids. RESULTS: Levels of TNF-α, IL-6, and IL-18 were significantly higher in patients compared to controls, while TGF-ß and IL-10 were lower. Serum samples of patients at disease onset (n = 32) had increased IL-6 and decreased TGF-ß, but there were no differences in other cytokines. These patients also presented a higher percentage of CD4+CD25high cells than those with established disease, although no significant differences were detected in Foxp3. Patients under corticosteroid treatment who were carriers of the good responder genotype had higher levels of TGF-ß, Foxp3, and Treg compared to patients with other genotypes, while relatively lower levels of TNF-α and IL-17 were observed. CONCLUSION: Patients at onset of RA present fewer alterations in cytokine levels and Treg than those with longer disease duration, supporting the role of disease progression in subsequent changes. The antiinflammatory balance observed in high IL-10/low TNF-α patients treated with prednisone supports the use of these genetic polymorphisms as predictors of response to corticosteroid therapy.

Interleukin 10 and tumor necrosis factor-alpha genotypes in rheumatoid arthritis--association with clinical response to glucocorticoids.

OBJECTIVE: There are dysregulated levels of interleukin 10 (IL-10) and tumor necrosis factor-alpha (TNF-alpha) in rheumatoid arthritis (RA), and their role in the disease is controversial. We analyzed the association of functional polymorphisms of IL-10 and TNF-alpha with susceptibility and disease characteristics at the time of diagnosis, and we also evaluated their possible use as predictors of clinical response to treatments. METHODS: Patients with recent-onset RA (n = 162) and healthy controls (n = 373) were genotyped for -1082 IL-10 and -308 TNF-alpha polymorphisms and data were related to clinical and immunological measurements of patients at the time of diagnosis. Response to treatment after 6 months was determined in 125 patients by the absolute change in Disease Activity Score (DAS28) and the American College of Rheumatology criteria for improvement. RESULTS: We found a reduced frequency of the low IL-10 producer genotype (-1082AA) in patients with RA compared to controls (26.5% vs 38.9%; p = 0.006), while it is a risk factor for anticyclic citrullinated peptide antibodies (anti-CCP) positivity (p = 0.028). Evaluation of clinical response to treatments indicated that carriage of the high IL-10 genotype was associated with a favorable outcome (p = 0.009), specifically to prednisone therapy (p = 0.0003). No significant effects were observed with TNF-alpha polymorphism alone; however, in combination with the IL-10 genotype, it increased the strength of these associations. CONCLUSION: Results show an association between the low IL-10 producer genotype and protection from RA; nevertheless, when other specific genetic and/or environmental factors trigger onset of RA, this genotype may predispose to development of anti-CCP+ RA disease with reduced response to prednisone treatment.