Proteome-wide Mendelian randomization identifies causal links between blood proteins and severe COVID-19.

In November 2021, the COVID-19 pandemic death toll surpassed five million individuals. We applied Mendelian randomization including >3,000 blood proteins as exposures to identify potential biomarkers that may indicate risk for hospitalization or need for respiratory support or death due to COVID-19, respectively. After multiple testing correction, using genetic instruments and under the assumptions of Mendelian Randomization, our results were consistent with higher blood levels of five proteins GCNT4, CD207, RAB14, C1GALT1C1, and ABO being causally associated with an increased risk of hospitalization or respiratory support/death due to COVID-19 (ORs = 1.12-1.35). Higher levels of FAAH2 were solely associated with an increased risk of hospitalization (OR = 1.19). On the contrary, higher levels of SELL, SELE, and PECAM-1 decrease risk of hospitalization or need for respiratory support/death (ORs = 0.80-0.91). Higher levels of LCTL, SFTPD, KEL, and ATP2A3 were solely associated with a decreased risk of hospitalization (ORs = 0.86-0.93), whilst higher levels of ICAM-1 were solely associated with a decreased risk of respiratory support/death of COVID-19 (OR = 0.84). Our findings implicate blood group markers and binding proteins in both hospitalization and need for respiratory support/death. They, additionally, suggest that higher levels of endocannabinoid enzymes may increase the risk of hospitalization. Our research replicates findings of blood markers previously associated with COVID-19 and prioritises additional blood markers for risk prediction of severe forms of COVID-19. Furthermore, we pinpoint druggable targets potentially implicated in disease pathology.

Comparative analysis of centrally mediated and inflammatory pain experiences amongst patients diagnosed with rheumatoid arthritis: A multimethods study.

BACKGROUND: The identification of pain originating from distinct biological processes may lead to individualised pain treatment. In this study, we aimed to explore the pain experiences of patients with rheumatoid arthritis (RA), differentiating between those predominantly exhibiting features of peripheral inflammatory versus centrally mediated pain. METHODS: Through a multimethods approach we (i) quantitatively analysed the differences in pain descriptors between patients diagnosed with RA experiencing peripheral inflammatory and centrally mediated pain, utilising the Short Form-McGill Pain Questionnaire which includes the pain visual analogue scale (VAS) and (ii) qualitatively explored their subjective pain experiences grounded in the biopsychosocial model, commonly applied in chronic pain. RESULTS: Participants with centrally mediated pain reported higher pain scores on the VAS, used a wider range of pain descriptors, and a higher proportion selected each descriptor compared to those with inflammatory pain (p < .001). The qualitative analysis revealed the centrally mediated pain group's experiences were overwhelming and relentless, struggling to precisely articulate the nature of their pain. In contrast, individuals with inflammatory pain expressed their pain in more tangible terms and shared their adaptive and coping strategies. Importantly, both groups revealed the substantial psychological, functional and social impacts of their pain, highlighting the often 'invisible' and misunderstood nature of their symptoms. CONCLUSION: This study has gained a deeper insight into the pain experiences of patients living with RA, particularly in differentiating between centrally mediated and inflammatory types of pain, potentially facilitating a more individualised approach to pain treatment. PATIENT CONTRIBUTION: Patients actively participated in the study conception and design. This engagement includes collaboration with key stakeholders, such as members of the National Rheumatoid Arthritis Society and Patient Research Partners (PRPs), who provided continuous feedback and guidance throughout the research process. Specifically, the qualitative element was coproduced with two PRPs, who were involved in co-leading the focus groups and data analysis.

Human in vitro-induced IL-17A+ CD8+ T-cells exert pro-inflammatory effects on synovial fibroblasts.

IL-17A+ CD8+ T-cells, termed Tc17 cells, have been identified at sites of inflammation in several immune-mediated inflammatory diseases. However, the biological function of human IL-17A+ CD8+ T-cells is not well characterized, likely due in part to the relative scarcity of these cells. Here, we expanded IL-17A+ CD8+ T-cells from healthy donor PBMC or bulk CD8+ T-cell populations using an in vitro polarization protocol. We show that T-cell activation in the presence of IL-1ß and IL-23 significantly increased the frequencies of IL-17A+ CD8+ T-cells, which was not further enhanced by IL-6, IL-2, or anti-IFNγ mAb addition. In vitro-generated IL-17A+ CD8+ T-cells displayed a distinct type-17 profile compared with IL-17A- CD8+ T-cells, as defined by transcriptional signature (IL17A, IL17F, RORC, RORA, MAF, IL23R, CCR6), high surface expression of CCR6 and CD161, and polyfunctional production of IL-17A, IL-17F, IL-22, IFNγ, TNFα, and GM-CSF. A significant proportion of in vitro-induced IL-17A+ CD8+ T-cells expressed TCRVα7.2 and bound MR1 tetramers indicative of MAIT cells, indicating that our protocol expanded both conventional and unconventional IL-17A+ CD8+ T-cells. Using an IL-17A secretion assay, we sorted the in vitro-generated IL-17A+ CD8+ T-cells for functional analysis. Both conventional and unconventional IL-17A+ CD8+ T-cells were able to induce pro-inflammatory IL-6 and IL-8 production by synovial fibroblasts from patients with psoriatic arthritis, which was reduced upon addition of anti-TNFα and anti-IL-17A neutralizing antibodies. Collectively, these data demonstrate that human in vitro-generated IL-17A+ CD8+ T-cells are biologically functional and that their pro-inflammatory function can be targeted, at least in vitro, using existing immunotherapy.

Efficacy and safety of ciclosporin versus methotrexate in the treatment of severe atopic dermatitis in children and young people (TREAT): a multicentre parallel group assessor-blinded clinical trial.

BACKGROUND: Conventional systemic drugs are used to treat children and young people (CYP) with severe atopic dermatitis (AD) worldwide, but no robust randomized controlled trial (RCT) evidence exists regarding their efficacy and safety in this population. While novel therapies have expanded therapeutic options, their high cost means traditional agents remain important, especially in lower-resource settings. OBJECTIVES: To compare the safety and efficacy of ciclosporin (CyA) with methotrexate (MTX) in CYP with severe AD in the TREatment of severe Atopic Eczema Trial (TREAT) trial. METHODS: We conducted a parallel group assessor-blinded RCT in 13 UK and Irish centres. Eligible participants aged 2-16 years and unresponsive to potent topical treatment were randomized to either oral CyA (4 mg kg-1 daily) or MTX (0.4 mg kg-1 weekly) for 36 weeks and followed-up for 24 weeks. Co-primary outcomes were change from baseline to 12 weeks in Objective Severity Scoring of Atopic Dermatitis (o-SCORAD) and time to first significant flare (relapse) after treatment cessation. Secondary outcomes included change in quality of life (QoL) from baseline to 60 weeks; number of participant-reported flares following treatment cessation; proportion of participants achieving ≥ 50% improvement in Eczema Area and Severity Index (EASI 50) and ≥ 75% improvement in EASI (EASI 75); and stratification of outcomes by filaggrin status. RESULTS: In total, 103 participants were randomized (May 2016-February 2019): 52 to CyA and 51 to MTX. CyA showed greater improvement in disease severity by 12 weeks [mean difference in o-SCORAD -5.69, 97.5% confidence interval (CI) -10.81 to -0.57 (P = 0.01)]. More participants achieved ≥ 50% improvement in o-SCORAD (o-SCORAD 50) at 12 weeks in the CyA arm vs. the MTX arm [odds ratio (OR) 2.60, 95% CI 1.23-5.49; P = 0.01]. By 60 weeks MTX was superior (OR 0.33, 95% CI 0.13-0.85; P = 0.02), a trend also seen for ≥ 75% improvement in o-SCORAD (o-SCORAD 75), EASI 50 and EASI 75. Participant-reported flares post-treatment were higher in the CyA arm (OR 3.22, 95% CI 0.42-6.01; P = 0.02). QoL improved with both treatments and was sustained after treatment cessation. Filaggrin status did not affect outcomes. The frequency of adverse events (AEs) was comparable between both treatments. Five (10%) participants on CyA and seven (14%) on MTX experienced a serious AE. CONCLUSIONS: Both CyA and MTX proved effective in CYP with severe AD over 36 weeks. Participants who received CyA showed a more rapid response to treatment, while MTX induced more sustained disease control after discontinuation.

Brain injury in COVID-19 is associated with dysregulated innate and adaptive immune responses.

COVID-19 is associated with neurological complications including stroke, delirium and encephalitis. Furthermore, a post-viral syndrome dominated by neuropsychiatric symptoms is common, and is seemingly unrelated to COVID-19 severity. The true frequency and underlying mechanisms of neurological injury are unknown, but exaggerated host inflammatory responses appear to be a key driver of COVID-19 severity. We investigated the dynamics of, and relationship between, serum markers of brain injury [neurofilament light (NfL), glial fibrillary acidic protein (GFAP) and total tau] and markers of dysregulated host response (autoantibody production and cytokine profiles) in 175 patients admitted with COVID-19 and 45 patients with influenza. During hospitalization, sera from patients with COVID-19 demonstrated elevations of NfL and GFAP in a severity-dependent manner, with evidence of ongoing active brain injury at follow-up 4 months later. These biomarkers were associated with elevations of pro-inflammatory cytokines and the presence of autoantibodies to a large number of different antigens. Autoantibodies were commonly seen against lung surfactant proteins but also brain proteins such as myelin associated glycoprotein. Commensurate findings were seen in the influenza cohort. A distinct process characterized by elevation of serum total tau was seen in patients at follow-up, which appeared to be independent of initial disease severity and was not associated with dysregulated immune responses unlike NfL and GFAP. These results demonstrate that brain injury is a common consequence of both COVID-19 and influenza, and is therefore likely to be a feature of severe viral infection more broadly. The brain injury occurs in the context of dysregulation of both innate and adaptive immune responses, with no single pathogenic mechanism clearly responsible.

miR-155-overexpressing monocytes resemble HLAhighISG15+ synovial tissue macrophages from patients with rheumatoid arthritis and induce polyfunctional CD4+ T-cell activation.

MicroRNAs (miRs) are known to regulate pro-inflammatory effector functions of myeloid cells, and miR dysregulation is implicated in rheumatoid arthritis (RA), a condition characterized by inflammation and destruction of the joints. We showed previously that miR-155 is increased in myeloid cells in RA and induces pro-inflammatory activation of monocytes and macrophages; however, its role at the interface between innate and adaptive immunity was not defined. Here, RNA-sequencing revealed that overexpression of miR-155 in healthy donor monocytes conferred a specific gene profile which bears similarities to that of RA synovial fluid-derived CD14+ cells and HLAhighISG15+ synovial tissue macrophages, both of which are characterized by antigen-presenting pathways. In line with this, monocytes in which miR-155 was overexpressed, displayed increased expression of HLA-DR and both co-stimulatory and co-inhibitory molecules, and induced activation of polyfunctional T cells. Together, these data underpin the notion that miR-155-driven myeloid cell activation in the synovium contributes not only to inflammation but may also influence the adaptive immune response.

IKZF3/Aiolos Is Associated with but Not Sufficient for the Expression of IL-10 by CD4+ T Cells.

The expression of anti-inflammatory IL-10 by CD4+ T cells is indispensable for immune homeostasis, as it allows T cells to moderate their effector function. We previously showed that TNF-α blockade during T cell stimulation in CD4+ T cell/monocyte cocultures resulted in maintenance of IL-10-producing T cells and identified IKZF3 as a putative regulator of IL-10. In this study, we tested the hypothesis that IKZF3 is a transcriptional regulator of IL-10 using a human CD4+ T cell-only culture system. IL-10+ CD4+ T cells expressed the highest levels of IKZF3 both ex vivo and after activation compared with IL-10-CD4+ T cells. Pharmacological targeting of IKZF3 with the drug lenalidomide showed that IKZF3 is required for anti-CD3/CD28 mAb-mediated induction of IL-10 but is dispensable for ex vivo IL-10 expression. However, overexpression of IKZF3 was unable to upregulate IL-10 at the mRNA or protein level in CD4+ T cells and did not drive the transcription of the IL10 promoter or putative local enhancer constructs. Collectively, these data indicate that IKZF3 is associated with but not sufficient for IL-10 expression in CD4+ T cells.

Presence, function, and regulation of IL-17F-expressing human CD4+ T cells.

The pro-inflammatory cytokine IL-17A has been implicated in the immunopathology of inflammatory arthritis. IL-17F bears 50% homology to IL-17A and has recently been suggested to play a role in inflammation. We investigated the induction and cytokine profile of IL-17F+ CD4+ T cells, and how IL-17F may contribute to inflammation. Upon culture of healthy donor CD4+ T cells with IL-1ß, IL-23, anti-CD3, and anti-CD28 mAb, both IL-17A and IL-17F-expressing cells were detected. In comparison to IL-17A+ IL-17F- CD4+ T cells, IL-17F+ IL-17A- and IL-17A+ IL-17F+ CD4+ T cells contained lower proportions of IL-10-expressing and GM-CSF-expressing cells and higher proportions of IFN-γ-expressing cells. Titration of anti-CD28 mAb revealed that strong co-stimulation increased IL-17F+ IL-17A- and IL-17A+ IL-17F+ CD4+ T cell frequencies, whereas IL-17A+ IL-17F- CD4+ T cell frequencies decreased. This was partly mediated via an IL-2-dependent mechanism. Addition of IL-17A, IL-17F, and TNF-α to synovial fibroblasts from patients with inflammatory arthritis resulted in significant production of IL-6 and IL-8, which was reduced to a larger extent by combined blockade of IL-17A and IL-17F than blockade of IL-17A alone. Our data indicate that IL-17A and IL-17F are differentially regulated upon T cell co-stimulation, and that dual blockade of IL-17A and IL-17F reduces inflammation more effectively than IL-17A blockade alone.

Anti-TNF treatment negatively regulates human CD4+ T-cell activation and maturation in vitro, but does not confer an anergic or suppressive phenotype.

TNF-blockade has shown clear therapeutic value in rheumatoid arthritis and other immune-mediated inflammatory diseases, however its mechanism of action is not fully elucidated. We investigated the effects of TNF-blockade on CD4+ T cell activation, maturation, and proliferation, and assessed whether TNF-inhibitors confer regulatory potential to CD4+ T cells. CyTOF and flow cytometry analysis revealed that in vitro treatment of human CD4+ T cells with the anti-TNF monoclonal antibody adalimumab promoted IL-10 expression in CD4+ T cells, whilst decreasing cellular activation. In line with this, analysis of gene expression profiling datasets of anti-TNF-treated IL-17 or IFN-γ-producing CD4+ T cells revealed changes in multiple pathways associated with cell cycle and proliferation. Kinetics experiments showed that anti-TNF treatment led to delayed, rather than impaired T-cell activation and maturation. Whilst anti-TNF-treated CD4+ T cells displayed some hyporesponsiveness upon restimulation, they did not acquire enhanced capacity to suppress T-cell responses or modulate monocyte phenotype. These cells however displayed a reduced ability to induce IL-6 and IL-8 production by synovial fibroblasts. Together, these data indicate that anti-TNF treatment delays human CD4+ T-cell activation, maturation, and proliferation, and this reduced activation state may impair their ability to activate stromal cells.

Drivers of Inflammation in Psoriatic Arthritis: the Old and the New.

PURPOSE OF REVIEW: The recognition that IL-17 is produced by many lymphoid-like cells other than CD4+ T helper (Th17) cells raises the potential for new pathogenic pathways in IBD/psoriasis/SpA. We review recent knowledge concerning the role of unconventional and conventional lymphocytes expressing IL-17 in human PsA and axSpA. RECENT FINDINGS: Innate-like lymphoid cells, namely gamma delta (γδ) T-cells, invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells, together with innate lymphoid cells (ILCs) are found at sites of disease in PsA/SpA. These cells are often skewed to Type-17 profiles and may significantly contribute to IL-17 production. Non-IL-23 dependent IL-17 production pathways, utilising cytokines such as IL-7 and IL-9, also characterise these cells. Both conventional CD4 and CD8 lymphocytes show pathogenic phenotypes at sites of disease. A variety of innate-like lymphoid cells and conventional lymphocytes contribute towards IL-17-mediated pathology in PsA/SpA. The responses of these cells to non-conventional immune and non-immune stimuli may explain characteristic clinical features of these diseases and potential therapeutic mechanisms of therapies such as Jak inhibitors.

Inflammation and immune resolution.

Inspired by the advances presented at the Inflammation and Immune Resolution Plenary Session at the British Society for Immunology Congress, December 2017, in this issue of Clinical & Experimental Immunology we present a Review Series on Inflammation and Immune Resolution. Our selection ranges from an overview of current genetic understanding of the similarities and differences between immune-mediated inflammatory diseases (IMIDs); discussion of several biological mechanisms underlying the aberrant activation of myeloid cells in RA, and how myeloid cell relevant anti-inflammatory mediators may contribute to immune resolution; presentation of fascinating evidence for the existence of innate immune memory in stromal cells and how this may exacerbate or restrain inflammatory disease; and a review of how the interleukin (IL)-6 family members IL-6 and IL-27 may drive or regulate inflammation. Inflammation and immune resolution are two sides of the same coin: the reviews presented in this series aim to equip readers with greater insight into the delicate balance between the two.

The dynamic co-evolution of memory and regulatory CD4+ T cells in the periphery.

Whereas memory T cells are required to maintain immunity, regulatory T cells have to keep the immune system in check to prevent excessive inflammation and/or autoimmunity. Both cell types must be present during the lifetime of the organism. However, it is not clear whether both subsets are regulated in tandem or independently of each other, especially because thymic involution severely restricts the production of T-cell populations during ageing. In this Opinion article, we discuss recent evidence in both mice and humans that supports the hypothesis that some CD4(+)CD25(+)FOXP3(+)regulatory T cells can differentiate from rapidly proliferating memory T cells in the periphery.

Periodontitis-associated pathogens P. gingivalis and A. actinomycetemcomitans activate human CD14(+) monocytes leading to enhanced Th17/IL-17 responses.

The Th17/IL-17 pathway is implicated in the pathogenesis of periodontitis (PD), however the mechanisms are not fully understood. We investigated the mechanism by which the periodontal pathogens Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) promote a Th17/IL-17 response in vitro, and studied IL-17(+) CD4(+) T-cell frequencies in gingival tissue and peripheral blood from patients with PD versus periodontally healthy controls. Addition of Pg or Aa to monocyte/CD4(+) T-cell co-cultures promoted a Th17/IL-17 response in vitro in a dose- and time-dependent manner. Pg or Aa stimulation of monocytes resulted in increased CD40, CD54 and HLA-DR expression, and enhanced TNF-α, IL-1ß, IL-6 and IL-23 production. Mechanistically, IL-17 production in Pg-stimulated co-cultures was partially dependent on IL-1ß, IL-23 and TLR2/TLR4 signalling. Increased frequencies of IL-17(+) cells were observed in gingival tissue from patients with PD compared to healthy subjects. No differences were observed in IL-17(+) CD4(+) T-cell frequencies in peripheral blood. In vitro, Pg induced significantly higher IL-17 production in anti-CD3 mAb-stimulated monocyte/CD4(+) T-cell co-cultures from patients with PD compared to healthy controls. Our data suggest that periodontal pathogens can activate monocytes, resulting in increased IL-17 production by human CD4(+) T cells, a process that appears enhanced in patients with PD.

MicroRNA-155 contributes to enhanced resistance to apoptosis in monocytes from patients with rheumatoid arthritis.

Monocytes and macrophages are key mediators of inflammation in rheumatoid arthritis (RA). Their persistence at the inflammatory site is likely to contribute to immunopathology. We sought to characterise one mechanism by which persistence may be achieved: resistance to apoptosis and the role of mir-155 in this process. CD14+ monocytes from peripheral blood (PBM) and synovial fluid (SFM) of RA patients were found to be resistant to spontaneous apoptosis relative to PBM from healthy control (HC) individuals. RA SFM were also resistant to anti-Fas-mediated apoptosis and displayed a gene expression profile distinct from HC and RA PBM populations. Gene expression profiling analysis revealed that the differentially expressed genes in RA SFM vs. PBM were enriched for apoptosis-related genes and showed increased expression of the mir-155 precursor BIC. Following identification of potential mir-155 target transcripts by bioinformatic methods, we show increased levels of mature mir-155 expression in RA PBM and SFM vs. HC PBM and a corresponding decrease in SFM of two predicted mir-155-target mRNAs, apoptosis mediators CASP10 and APAF1. Using miR mimics, we demonstrate that mir-155 over-expression in healthy CD14+ cells conferred resistance to spontaneous apoptosis, but not Fas-induced death in these cells, and resulted in increased production of cytokines and chemokines. Collectively our data indicate that CD14+ cells from patients with RA show enhanced resistance to apoptosis, and suggest that an increase in mir-155 may partially contribute to this phenotype.

Secretory leukocyte protease inhibitor: a pivotal mediator of anti-inflammatory responses in acetaminophen-induced acute liver failure.

UNLABELLED: Acetaminophen-induced acute liver failure (AALF) is characterized both by activation of innate immune responses and susceptibility to sepsis. Circulating monocytes and hepatic macrophages are central mediators of inflammatory responses and tissue repair processes during human AALF. Secretory leukocyte protease inhibitor (SLPI) modulates monocyte/macrophage function through inhibition of nuclear factor kappa B (NF-κB) signaling. The aims of this study were to establish the role of SLPI in AALF. Circulating levels of SLPI, monocyte cluster of differentiation 163 (CD163), human leukocyte antigen-DR (HLA-DR), and lipopolysaccharide (LPS)-stimulated levels of NF-κBp65, tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6 were determined in patients with AALF, chronic liver disease, and healthy controls. Immunohistochemistry and multispectral imaging of AALF explant tissue determined the cellular sources of SLPI and hepatic macrophage phenotype. The phenotype and function of monocytes and macrophages was determined following culture with recombinant human (rh)-SLPI, liver homogenates, and plasma derived from AALF patients in the presence and absence of antihuman (α)SLPI. Hepatic and circulatory concentrations of SLPI were elevated in AALF and immunohistochemistry revealed SLPI expression in biliary epithelial cells and within hepatic macrophages (h-mψ) in areas of necrosis. H-mψ and circulating monocytes in AALF exhibited an anti-inflammatory phenotype and functional characteristics; typified by reductions in NF-κBp65, TNF-α, and IL-6 and preserved IL-10 secretion following LPS challenge. Culture of healthy monocytes with AALF liver homogenates, plasma, or rhSLPI induced monocytes with strikingly similar anti-inflammatory characteristics which were reversed by inhibiting the activity of SLPI. CONCLUSION: SLPI is a pivotal mediator of anti-inflammatory responses in AALF through modulation of monocyte/macrophage function, which may account for the susceptibility to sepsis in AALF.

CD161 expression characterizes a subpopulation of human regulatory T cells that produces IL-17 in a STAT3-dependent manner.

Treg cells are critical for the prevention of autoimmune diseases and are thus prime candidates for cell-based clinical therapy. However, human Treg cells are "plastic", and are able to produce IL-17 under inflammatory conditions. Here, we identify and characterize the human Treg subpopulation that can be induced to produce IL-17 and identify its mechanisms. We confirm that a subpopulation of human Treg cells produces IL-17 in vitro when activated in the presence of IL-1ß, but not IL-6. "IL-17 potential" is restricted to population III (CD4(+) CD25(hi) CD127(lo) CD45RA(-) ) Treg cells expressing the natural killer cell marker CD161. We show that these cells are functionally as suppressive and have similar phenotypic/molecular characteristics to other subpopulations of Treg cells and retain their suppressive function following IL-17 induction. Importantly, we find that IL-17 production is STAT3 dependent, with Treg cells from patients with STAT3 mutations unable to make IL-17. Finally, we show that CD161(+) population III Treg cells accumulate in inflamed joints of patients with inflammatory arthritis and are the predominant IL-17-producing Treg-cell population at these sites. As IL-17 production from this Treg-cell subpopulation is not accompanied by a loss of regulatory function, in the context of cell therapy, exclusion of these cells from the cell product may not be necessary.

Interaction with activated monocytes enhances cytokine expression and suppressive activity of human CD4+CD45ro+CD25+CD127(low) regulatory T cells.

OBJECTIVE: Despite the high frequency of CD4+ T cells with a regulatory phenotype (CD25+CD127(low) FoxP3+) in the joints of patients with rheumatoid arthritis (RA), inflammation persists. One possible explanation is that human Treg cells are converted into proinflammatory interleukin-17 (IL-17)-producing cells by inflammatory mediators and thereby lose their suppressive function. The aim of this study was to investigate whether activated monocytes, which are potent producers of inflammatory cytokines and are abundantly present in the rheumatic joint, induce proinflammatory cytokine expression in human Treg cells and impair their regulatory function. METHODS: The presence and phenotype of CD4+CD45RO+CD25+CD127(low) T cells (memory Treg cells) and CD14+ monocytes in the peripheral blood (PB) and synovial fluid (SF) of patients with RA were investigated by flow cytometry. Memory Treg cells obtained from healthy control subjects underwent fluorescence-activated cell sorting and then were cocultured with autologous activated monocytes and stimulated with anti-CD3 monoclonal antibodies. Intracellular cytokine expression, phenotype, and function of cells were determined by flow cytometry, enzyme-linked immunosorbent assay, and proliferation assays. RESULTS: In patients with RA, the frequencies of CD4+CD45RO+CD25+CD127(low) Treg cells and activated CD14+ monocytes were higher in SF compared with PB. In vitro-activated monocytes induced an increase in the percentage of IL-17-positive, interferon-γ (IFNγ)-positive, and tumor necrosis factor α (TNFα)-positive Treg cells as well as IL-10-positive Treg cells. The observed increase in IL-17-positive and IFNγ-positive Treg cells was driven by monocyte-derived IL-1ß, IL-6, and TNFα and was mediated by both CD14+CD16- and CD14+CD16+ monocyte subsets. Despite enhanced cytokine expression, cells maintained their CD25+FoxP3+CD39+ Treg cell phenotype and showed an enhanced capacity to suppress T cell proliferation and IL-17 production. CONCLUSION: Treg cells exposed to a proinflammatory environment show increased cytokine expression as well as enhanced suppressive activity.