Measuring the T-cell down-regulation of TCR-zeta, ZAP-70 and CD28 in arthritis patients: An old tool for new biomarkers.

Low T-cell receptor (TCR)/CD28 signaling lymphocytes are expanded in arthritis. We asked whether the down-expression of TCR-related molecules correlates with specific arthritis characteristics and if it has clinical implications. TCR-ZETA, ZAP-70 and CD28 expression was measured by flow cytometry in synovial fluid (SF) and peripheral blood (PB)-derived T cells. In PB, ZETA-downregulation in CD4+ CD28+ and consequent CD4+ CD28lowZETAlow cell expansion correlate with CRP elevation, leukocyte recruitment into SF and, primarily, disease activity (DAS). In some patients, ZETA-downregulation extends to CD8+ CD28null and/or CD8+ CD28+ cells, and this correlates with enhanced leukocyte recruitment, multiple joint involvement, and disability index (HAQ). ZETA-downregulation in CD4+ CD28+ may also lead to CD4+ CD28+ ZETAnull cell expansion, which strongly correlates with HAQ. In SF, ZETA-downregulation in CD8+ CD28null and consequent CD8+ CD28nullZETAlow/null cell expansion correlate with CRP elevation and neutrophilic influx into SF, whereas ZAP-downregulation in CD8+ CD28+ and consequent CD8+ CD28lowZAPlow cell expansion strongly correlate with HAQ and DAS. ZETA-downregulation is preponderant in SF of seronegative arthritides, with seronegative rheumatoid arthritis showing significant down-regulation in CD8+ CD28null, and non-rheumatoid arthritides showing significant down-regulation in CD4+ CD28+ . Altogether, we identified new molecular and cellular biomarkers of arthritis-related T-cell inflammation, useful for assessing arthritis activity, predicting polyarticular progression and functional impairment, characterizing seronegative arthritides, and possibly tailoring immunotherapies.

IL-17 stimulates differentiation of human anti-inflammatory macrophages and phagocytosis of apoptotic neutrophils in response to IL-10 and glucocorticoids.

Exposure of human monocytes/macrophages to anti-inflammatory agents, such as IL-10 or glucocorticoids, can lead to two separate fates: either Fas/CD95-mediated apoptosis or differentiation into regulatory and efferocytic M2c (CD14(bright)CD16(+)CD163(+)Mer tyrosine kinase(+)) macrophages. We found that the prevalent effect depends on the type of Th cytokine environment and on the stage of monocyte-to-macrophage differentiation. In particular, the presence of IFN-γ (Th1 inflammation) or the prolonged exposure to IL-4 (chronic Th2 inflammation) promotes apoptosis of monocytes/macrophages and causes resistance to M2c differentiation, thus provoking impaired clearance of apoptotic neutrophils, uncontrolled accumulation of apoptotic cells, and persistent inflammation. In contrast, the presence of IL-17 (Th17 environment) prevents monocyte/macrophage apoptosis and elicits intense M2c differentiation, thus ensuring efficient clearance of apoptotic neutrophils and restoration of anti-inflammatory conditions. Additionally, the Th environment affects the expression of two distinct Mer tyrosine kinase isoforms: IL-4 downregulates the membrane isoform but induces an intracellular and Gas6-dependent isoform, whereas IFN-γ downregulates both and IL-17 upregulates both. Our data support an unexpected role for IL-17 in orchestrating resolution of innate inflammation, whereas IFN-γ and IL-4 emerge as major determinants of IL-10 and glucocorticoid resistance.

Efficient clearance of early apoptotic cells by human macrophages requires M2c polarization and MerTK induction.

Mer tyrosine kinase (MerTK) is a major macrophage apoptotic cell (AC) receptor. Its functional impairment promotes autoimmunity and atherosclerosis, whereas overexpression correlates with poor prognosis in cancer. However, little is known about mechanisms regulating MerTK expression in humans. We found that MerTK expression is heterogenous among macrophage subsets, being mostly restricted to anti-inflammatory M2c (CD14(+)CD16(+)CD163(+)CD204(+)CD206(+)CD209(-)) cells, differentiated by M-CSF or glucocorticoids. Small numbers of MerTK(+) "M2c-like" cells are also detectable among circulating CD14(bright)CD16(+) monocytes. MerTK expression levels adapt to changing immunologic environment, being suppressed in M1 and M2a macrophages and in dendritic cells. Remarkably, although glucocorticoid-induced differentiation is IL-10 independent, M-CSF-driven M2c polarization and related MerTK upregulation require IL-10. However, neither IL-10 alone nor TGF-ß are sufficient to fully differentiate M2c (CD16(+)CD163(+)MerTK(+)) macrophages. M-CSF and IL-10, both released by T lymphocytes, may thus be required together to promote regulatory T cell-mediated induction of anti-inflammatory monocytes-macrophages. MerTK enables M2c macrophages to clear early ACs more efficiently than other macrophage subsets, and it mediates AC clearance by CD14(bright)CD16(+) monocytes. Moreover, M2c cells release Gas6, which in turn amplifies IL-10 secretion via MerTK. IL-10-dependent induction of the Gas6/MerTK pathway may, therefore, constitute a positive loop for M2c macrophage homeostasis and a critical checkpoint for maintenance of anti-inflammatory conditions. Our findings give new insight into human macrophage polarization and favor a central role for MerTK in regulation of macrophage functions. Eliciting M2c polarization can have therapeutic utility for diseases such as lupus, in which a defective AC clearance contributes to initiate and perpetuate the pathological process.

Assessing SARS-CoV-2-specific T-cell reactivity in late convalescents and vaccinees: Comparison and combination of QuantiFERON and activation-induced marker assays, and relation with antibody status.

OBJECTIVES: Monitoring of SARS-CoV-2 spread and vaccination strategies have relied on antibody (Ab) status as a correlate of protection. We used QuantiFERON™ (QFN) and Activation-Induced Marker (AIM) assays to measure memory T-cell reactivity in unvaccinated individuals with prior documented symptomatic infection (late convalescents) and fully vaccinated asymptomatic donors (vaccinees). METHODS: Twenty-two convalescents and 13 vaccinees were enrolled. Serum anti-SARS-CoV-2 S1 and N Abs were measured using chemiluminescent immunoassays. QFN was performed following instructions and interferon-gamma (IFN-γ) measured by ELISA. AIM was performed on aliquots of antigen-stimulated samples from QFN tubes. SARS-CoV-2-specific memory CD4+CD25+CD134+, CD4+CD69+CD137+ and CD8+CD69+CD137+ T-cell frequencies were measured by flow cytometry. RESULTS: In convalescents, substantial agreement was observed between QFN and AIM assays. IFN-γ concentrations and AIM+ (CD69+CD137+) CD4+ T-cell frequencies correlated with each other, with Ab levels and AIM+ CD8+ T-cell frequencies, whereas AIM+ (CD25+CD134+) CD4+ T-cell frequencies correlated with age. AIM+ CD4+ T-cell frequencies increased with time since infection, whereas AIM+ CD8+ T-cell expansion was greater after recent reinfection. QFN-reactivity and anti-S1 titers were lower, whereas anti-N titers were higher, and no statistical difference in AIM-reactivity and Ab positivity emerged compared to vaccinees. CONCLUSIONS: Albeit on a limited sample size, we confirm that coordinated, cellular and humoral responses are detectable in convalescents up to 2 years after prior infection. Combining QFN with AIM may enhance detection of naturally acquired memory responses and help stratify virus-exposed individuals in T helper 1-type (TH1)-reactive (QFNpos AIMpos Abshigh), non-TH1-reactive (QFNneg AIMpos Abshigh/low), and pauci-reactive (QFNneg AIMneg Abslow).

Effectiveness and safety of secukinumab in ankylosing spondylitis and psoriatic arthritis: a 52-week real-life study in an Italian cohort.

BACKGROUND: Secukinumab has shown high efficacy in randomized controlled trials in both ankylosing spondylitis (AS) and psoriatic arthritis (PsA). Here, we investigated its real-life effectiveness and tolerability in a cohort of AS and PsA patients. METHODS: We retrospectively analyzed medical records of outpatients with AS or PsA treated with secukinumab between December 2017 and December 2019. ASDAS-CRP and DAS28-CRP scores were used to measure axial and peripheral disease activity in AS and PsA, respectively. Data were collected at baseline and after 8, 24, and 52 weeks of treatment. RESULTS: Eighty-five adult patients with active disease (29 with AS and 56 with PsA; 23 males and 62 females) were treated. Overall, mean disease duration was 6.7 years and biologic-naïve patients were 85%. Significant reductions in ASDAS-CRP and DAS28-CRP were observed at all time-points. Body weight (in AS) and disease activity status at baseline (particularly in PsA) significantly affected disease activity changes. ASDAS-defined inactive disease and DAS28-defined remission were achieved in comparable proportions between AS and PsA patients, at both 24 weeks (45% and 46%) and 52 weeks (65.5% and 68%, respectively); male sex was found an independent predictor of positive response (OR 5.16, P = 0.027). After 52 weeks, achievement of at least low disease activity and drug retention were observed in 75% of patients. Secukinumab was well-tolerated and only mild injection-site reactions were recorded in 4 patients. CONCLUSION: In a real-world setting, secukinumab confirmed great effectiveness and safety in both AS and PsA patients. The influence of gender on treatment response deserves further attention.

A vascular endothelial growth factor deficiency characterises scleroderma lung disease.

OBJECTIVES: Vascular endothelial growth factor (VEGF) is thought to play an important role in systemic sclerosis (SSc) pathogenesis. It was found to be upregulated in the serum and in the affected skin of scleroderma patients. However, its involvement in scleroderma lung disease is not clear. This study aimed to evaluate VEGF concentration in the bronchoalveolar lavage fluid (BALF) of scleroderma patients with interstitial lung disease, to correlate the cytokine levels in plasma and in the lung with pulmonary functional, radiological and cellular parameters, and with the progression of lung disease. METHODS: BALF and plasma VEGF concentrations were analysed by ELISA in 55 SSc patients with lung disease and 17 controls. Cytokine real-time PCR messenger RNA expression in alveolar macrophages was assessed. Lung involvement progression was evaluated after a 1-year follow-up. RESULTS: VEGF was found to be significantly lower in the BALF of scleroderma patients compared with controls. The lowest concentrations were observed in SSc patients with alveolitis. A decreased VEGF expression in alveolar macrophages was found in SSc patients with alveolitis. VEGF concentration in BALF correlated inversely with the ground glass score on high-resolution CT and with BALF neutrophil cell count. Moreover, SSc patients with a lower VEGF concentration showed a worsening in the interstitial score at follow-up. CONCLUSIONS: Scleroderma interstitial lung disease is characterised by a VEGF deficiency. Lower concentrations were found in patients with progression of lung disease.

Interstitial Lung Disease in Rheumatoid Arthritis: A Practical Review.

Rheumatoid arthritis (RA) is a systemic inflammatory disease, which primarily causes symmetric polyarthritis. An extrarticolar involvement is common, and the commonly involved organ is lungs. Although cardiac disease is responsible for most RA-related deaths, pulmonary disease is also a major contributor, accounting for ~10-20% of all mortality. Pulmonary disease is a common (60-80% of patients with RA) extra-articular complication of RA. Optimal screening, diagnostic, and treatment strategies of pulmonary disease remain uncertain, which have been the focus of an ongoing investigation. Clinicians should regularly assess patients with RA for the signs and symptoms of pulmonary disease and, reciprocally, consider RA and other connective tissue diseases when evaluating a patient with pulmonary disease of an unknown etiology. RA directly affects all anatomic compartments of the thorax, including the lung parenchyma, large and small airways, pleura, and less commonly vessels. In addition, pulmonary infection and drug-induced lung disease associated with immunosuppressive agents used for the treatment of RA may occur.

Immunotherapy of COVID-19: Inside and Beyond IL-6 Signalling.

Acting on the cytokine cascade is key to preventing disease progression and death in hospitalised patients with COVID-19. Among anti-cytokine therapies, interleukin (IL)-6 inhibitors have been the most used and studied since the beginning of the pandemic. Going through previous observational studies, subsequent randomised controlled trials, and meta-analyses, we focused on the baseline characteristics of the patients recruited, identifying the most favourable features in the light of positive or negative study outcomes; taking into account the biological significance and predictivity of IL-6 and other biomarkers according to specific thresholds, we ultimately attempted to delineate precise windows for therapeutic intervention. By stimulating scavenger macrophages and T-cell responsivity, IL-6 seems protective against viral replication during asymptomatic infection; still protective on early tissue damage by modulating the release of granzymes and lymphokines in mild-moderate disease; importantly pathogenic in severe disease by inducing the proinflammatory activation of immune and endothelial cells (through trans-signalling and trans-presentation); and again protective in critical disease by exerting homeostatic roles for tissue repair (through cis-signalling), while IL-1 still drives hyperinflammation. IL-6 inhibitors, particularly anti-IL-6R monoclonal antibodies (e.g., tocilizumab, sarilumab), are effective in severe disease, characterised by baseline IL-6 concentrations ranging from 35 to 90 ng/mL (reached in the circulation within 6 days of hospital admission), a ratio of partial pressure arterial oxygen (PaO2) and fraction of inspired oxygen (FiO2) between 100 and 200 mmHg, requirement of high-flow oxygen or non-invasive ventilation, C-reactive protein levels between 120 and 160 mg/L, ferritin levels between 800 and 1600 ng/mL, D-dimer levels between 750 and 3000 ng/mL, and lactate dehydrogenase levels between 350 and 500 U/L. Granulocyte-macrophage colony-stimulating factor inhibitors might have similar windows of opportunity but different age preferences compared to IL-6 inhibitors (over or under 70 years old, respectively). Janus kinase inhibitors (e.g., baricitinib) may also be effective in moderate disease, whereas IL-1 inhibitors (e.g., anakinra) may also be effective in critical disease. Correct use of biologics based on therapeutic windows is essential for successful outcomes and could inform future new trials with more appropriate recruiting criteria.

Synovial fluid-derived T helper 17 cells correlate with inflammatory activity in arthritis, irrespectively of diagnosis.

We analyzed peripheral blood (PB) and synovial fluid (SF) mononuclear cells from 16 rheumatoid arthritis (RA), 9 spondyloarthritis (SpA), 3 microcrystal arthritis patients, to define the presence of Th17 and Th1 and their relationship with inflammatory activity, and TCR-zeta chain and ZAP-70 levels. Th17 were significantly higher in SF than in PB and more abundant in microcrystal arthritis patients compared to the other groups. Irrespectively of the diagnosis, SF Th17 percentages correlated with joint (SF total leukocyte count, neutrophil percentage) and systemic (C reactive protein [CRP], fibrinogen, erythrocyte sedimentation rate) inflammation markers. SF Th1 percentages directly correlated with inflammation and disease activity (CRP, swollen joint count [SJC]) indices in SpA, but not in RA patients. These observations support the role of Th17 in the pathogenesis of inflammatory arthritides. The TCR-zeta(dim) lymphocytes in SF were found to produce the highest amounts of cytokines including IL-17, whereas no ZAP-70 impairment was associated to Th17.

ß-Thymosins and interstitial lung disease: study of a scleroderma cohort with a one-year follow-up.

BACKGROUND: ß-thymosins play roles in cytoskeleton rearrangement, angiogenesis, fibrosis and reparative process, thus suggesting a possible involvement in the pathogenesis of systemic sclerosis. The aim of the study was to investigate the presence of thymosins ß4, ß4 sulfoxide, and ß10 in bronchoalveolar lavage fluid of scleroderma patients with interstitial lung disease and the relation of these factors with pulmonary functional and radiological parameters. METHODS: ß-thymosins concentrations were determined by reverse phase-high performance liquid chromatography-electrospray-mass spectrometry in the bronchoalveolar lavage fluid of 46 scleroderma patients with lung involvement and of 15 controls. RESULTS: Thymosin ß4, ß4 sulfoxide, and ß10 were detectable in bronchoalveolar lavage fluid of patients and controls. Thymosin ß4 levels were significantly higher in scleroderma patients than in controls. In addition, analyzing the progression of scleroderma lung disease at one-year follow-up, we have found that higher thymosin ß4 levels seem to have a protective role against lung tissue damage. Thymosin ß4 sulfoxide levels were higher in the smokers and in the scleroderma patients with alveolitis. CONCLUSIONS: We describe for the first time ß-thymosins in bronchoalveolar lavage fluid and their possible involvement in the pathogenesis of scleroderma lung disease. Thymosin ß4 seems to have a protective role against lung tissue damage, while its oxidation product mirrors an alveolar inflammatory status.

Amiodarone-induced organizing pneumonia mimicking COVID-19: a case report.

BACKGROUND: Differential diagnosis of interstitial lung diseases (ILDs) during the COVID-19 pandemic is difficult, due to similarities in clinical and radiological presentation between COVID-19 and other ILDs on the one hand, and frequent false-negative swab results on the other. We describe a rare form of interstitial and organizing pneumonia resembling COVID-19, emphasizing some key aspects to focus on to get the right diagnosis and treat the patient properly. CASE PRESENTATION: A 76-year-old man presented with short breath and dry cough in the midst of the COVID-19 outbreak. He showed bilateral crackles and interstitial-alveolar opacities on X-ray, corresponding on computed tomography (CT) to extensive consolidations with air bronchograms, surrounded by ground glass opacities (GGO). Although his throat-and-nasopharyngeal swab tested negative, the picture was overall compatible with COVID-19. On the other hand, he showed subacute, rather than hyperacute, clinical onset; few and stable parenchymal consolidations, rather than patchy and rapidly evolving GGO; pleural and pericardial thickening, pleural effusion, and lymph node enlargement, usually absent in COVID-19; and peripheral eosinophilia, rather than lymphopenia, suggestive of hypersensitivity. In the past year, he had been taking amiodarone for a history of ventricular ectopic beats. CT scans, in fact, highlighted hyperattenuation areas suggestive of amiodarone pulmonary accumulation and toxicity. Bronchoalveolar lavage fluid (BALF) investigation confirmed the absence of coronavirus genome in the lower respiratory tract; conversely, high numbers of foamy macrophages, eosinophils, and cytotoxic T lymphocytes with low CD4/CD8 T-cell ratio were detected, confirming the hypothesis of amiodarone-induced cryptogenic organizing pneumonia. Timely discontinuation of amiodarone and initiation of steroid therapy led to resolution of respiratory symptoms, systemic inflammation, and radiographic opacities. CONCLUSIONS: A comprehensive analysis of medical and pharmacological history, clinical onset, radiologic details, and peripheral and BALF cellularity, is required for a correct differential diagnosis and management of ILDs in the COVID-19 era.

Imperfect storm: is interleukin-33 the Achilles heel of COVID-19?

The unique cytokine signature of COVID-19 might provide clues to disease mechanisms and possible future therapies. Here, we propose a pathogenic model in which the alarmin cytokine, interleukin (IL)-33, is a key player in driving all stages of COVID-19 disease (ie, asymptomatic, mild-moderate, severe-critical, and chronic-fibrotic). In susceptible individuals, IL-33 release by damaged lower respiratory cells might induce dysregulated GATA-binding factor 3-expressing regulatory T cells, thereby breaking immune tolerance and eliciting severe acute respiratory syndrome coronavirus 2-induced autoinflammatory lung disease. Such disease might be initially sustained by IL-33-differentiated type-2 innate lymphoid cells and locally expanded γδ T cells. In severe COVID-19 cases, the IL-33-ST2 axis might act to expand the number of pathogenic granulocyte-macrophage colony-stimulating factor-expressing T cells, dampen antiviral interferon responses, elicit hyperinflammation, and favour thromboses. In patients who survive severe COVID-19, IL-33 might drive pulmonary fibrosis by inducing myofibroblasts and epithelial-mesenchymal transition. We discuss the therapeutic implications of these hypothetical pathways, including use of therapies that target IL-33 (eg, anti-ST2), T helper 17-like γδ T cells, immune cell homing, and cytokine balance.

Antibody Cross-Linking of CD14 Activates MerTK and Promotes Human Macrophage Clearance of Apoptotic Neutrophils: the Dual Role of CD14 at the Crossroads Between M1 and M2c Polarization.

Mer receptor tyrosine kinase (MerTK) is key for efficient phagocytosis of apoptotic neutrophils (ANs) and homeostasis of IL-10 production by human anti-inflammatory M2c monocytes/macrophages. We asked whether stimulation of M2c surface receptors contributes in turn to MerTK activation. For this purpose, human monocytes/macrophages were differentiated under M1, M2a, and M2c polarizing conditions. The effects of antibody-mediated cross-linking of M2c receptors (i.e., CD14, CD16, CD32, CD163, CD204) on MerTK phosphorylation and phagocytosis of ANs were tested. MerTK expression was also studied by flow cytometry and western blot in the presence of LPS and in M2c-derived microvesicles (MVs). Antibody cross-linking of either CD14 or CD32/FcγRII led to Syk activation and MerTK phosphorylation in its two distinct glycoforms (175-205 and 135-155 kDa). Cross-linked CD14 enhanced efferocytosis by M2c macrophages and enabled M1 and M2a cells to clear ANs efficiently. In M1 conditions, LPS abolished surface MerTK expression on CD14bright cell subsets, so disrupting the anti-inflammatory pathway. In M2c cells, instead, MerTK was diffusely and brightly co-expressed with CD14, and was also detected in M2c macrophage-derived MVs; in these conditions, LPS only partially downregulated MerTK on cell surfaces, while the smaller MerTK glycoform contained in MVs remained intact. Altogether, cooperation between CD14 and MerTK may foster the clearance of ANs by human monocytes/macrophages. CD14 stands between M1-related LPS co-receptor activity and M2c-related MerTK-dependent response. MerTK interaction with CD32/FcγRII, its detection in M2c MVs, and the differential localization and LPS susceptibility of MerTK glycoforms add further new elements to the complexity of the MerTK network.

Abatacept in the treatment of psoriatic arthritis: biological and clinical profiles of the responders.

Abatacept (CTLA4Ig), a selective T-cell costimulation modulator, has been approved for the treatment of psoriatic arthritis patients with an inadequate response to conventional synthetic disease-modifying antirheumatic drugs, but not for those with uncontrolled skin lesions, nor with axial involvement. In this review, we will try to interpret such a differential efficacy of abatacept on the psoriatic arthritis clinical domains, on the basis of its differential effectiveness on the diverse T-cell subsets at different sites. Clinical and biological profiles of possible responders to abatacept will be provided.

Effectiveness and safety of secukinumab in ankylosing spondylitis and psoriatic arthritis: a 52-week real-life study in an Italian cohort

Abstract Background Secukinumab has shown high efficacy in randomized controlled trials in both ankylosing spondylitis (AS) and psoriatic arthritis (PsA). Here, we investigated its real-life effectiveness and tolerability in a cohort of AS and PsA patients. Methods We retrospectively analyzed medical records of outpatients with AS or PsA treated with secukinumab between December 2017 and December 2019. ASDAS-CRP and DAS28-CRP scores were used to measure axial and peripheral disease activity in AS and PsA, respectively. Data were collected at baseline and after 8, 24, and 52 weeks of treatment. Results Eighty-five adult patients with active disease (29 with AS and 56 with PsA; 23 males and 62 females) were treated. Overall, mean disease duration was 6.7 years and biologic-naïve patients were 85%. Significant reductions in ASDAS-CRP and DAS28-CRP were observed at all time-points. Body weight (in AS) and disease activity status at baseline (particularly in PsA) significantly affected disease activity changes. ASDAS-defined inactive disease and DAS28-defined remission were achieved in comparable proportions between AS and PsA patients, at both 24 weeks (45% and 46%) and 52 weeks (65.5% and 68%, respectively); male sex was found an independent predictor of positive response (OR 5.16, P = 0.027). After 52 weeks, achievement of at least low disease activity and drug retention were observed in 75% of patients. Secukinumab was well-tolerated and only mild injection-site reactions were recorded in 4 patients. Conclusion In a real-world setting, secukinumab confirmed great effectiveness and safety in both AS and PsA patients. The influence of gender on treatment response deserves further attention.

The PPAR-γ antagonist GW9662 elicits differentiation of M2c-like cells and upregulation of the MerTK/Gas6 axis: a key role for PPAR-γ in human macrophage polarization.

BACKGROUND: The nuclear receptors PPAR-γ and LXRs regulate macrophage lipid metabolism and macrophage mediated inflammation. We examined the influence of these molecules on macrophage alternative activation, with particular focus on differentiation of "M2c" anti-inflammatory cells. METHODS: We cultured human monocytes in M0, M1, M2a or M2c macrophage differentiating conditions, in the presence or absence of PPAR-γ and LXR ligands. Flow cytometry was used to analyze membrane expression of phenotypic markers. Basal and LPS-stimulated production of soluble mediators was measured by ELISA. Efferocytosis assays were performed by coincubating monocytes/macrophages with apoptotic neutrophils. RESULTS: We found that PPAR-γ inhibition, using the PPAR-γ antagonist GW9662, elicits differentiation of M2c-like (CD206(+) CD163(+) CD16(+)) cells and upregulation of the MerTK/Gas6 axis. Exposure of differentiating macrophages to IFN-γ, GM-CSF or LPS (M1 conditions), however, hampers GW9662 induction of MerTK and Gas6. When macrophages are differentiated with IL-4 (M2a conditions), addition of GW9662 results into an M2a (CD206(+) CD209(+) CD163(-) MerTK(-)) to M2c (CD206(high) CD209(-) CD163(+) MerTK(+)) polarization shift. Conversely, in the presence of dexamethasone (M2c conditions), the PPAR-γ agonist rosiglitazone attenuates CD163 and MerTK upregulation. The LXR agonist T0901317 induces MerTK independently of M2c polarization; indeed, CD206, CD163 and CD16 are downregulated. GW9662-differentiated M2c-like cells secrete high levels of Gas6 and low amounts of TNF-α and IL-10, mimicking dexamethasone effects in vitro. However, unlike conventional M2c cells, GW9662-differentiated cells do not show enhanced efferocytic ability. CONCLUSIONS: Our results provide new insights into the role of PPAR-γ and LXR receptors in human macrophage activation and reveal the existence of different patterns regulating MerTK expression. Unexpectedly, PPAR-γ appears to negatively control the expansion of a discrete subset of M2c-like anti-inflammatory macrophages.

Increased expression of Mer tyrosine kinase in circulating dendritic cells and monocytes of lupus patients: correlations with plasma interferon activity and steroid therapy.

INTRODUCTION: The requirement for the immunoregulatory Mer tyrosine kinase (Mer) for optimal removal of apoptotic cells prompted us to look at its expression in systemic lupus erythematosus (SLE), in which apoptotic cell clearance is abnormal. We compared the levels of expression of Mer in normal human subjects and in patients with SLE. METHODS: We used flow cytometry of isolated peripheral blood mononuclear cells to compare the levels of Mer on leukocyte subsets. We used a Mer-specific enzyme-linked immunosorbent assay (ELISA) to quantify soluble Mer (sMer) in plasmas. RESULTS: Monocytes, CD1c⁺ myeloid dendritic cells (mDCs), and plasmacytoid dendritic cells (pDCs) from both normal individuals and from SLE patients expressed Mer. In both normal and SLE patients, the CD14⁺⁺CD16⁺ subpopulation of monocytes expressed the highest levels of Mer, with somewhat lower levels on the CD14(int)CD16⁺ population. Mer levels on CD1c⁺ mDCs and pDCs, and sMer levels in blood were increased in SLE patients compared with controls. In patients, Mer levels on CD14(int)CD16⁺, CD14⁺⁺CD16⁻ monocytes, and CD1c⁺ dendritic cells correlated positively with type I interferon (IFN-I) activity detected in blood. In SLE patients treated with corticosteroids, Mer expression on monocytes correlated with prednisone dose, CD1c⁺ myeloid dendritic cells in patients treated with prednisone had higher levels of Mer expression than those in patients not receiving prednisone. CONCLUSIONS: We found no global defect in Mer expression in lupus blood. In contrast, we observed increased levels of Mer expression in DC populations, which could represent a response to increased IFN-I in SLE patients. Enhanced Mer expression induced by corticosteroids may contribute to its beneficial effects in SLE.

Recognizing and treating myocarditis in recent-onset systemic sclerosis heart disease: potential utility of immunosuppressive therapy in cardiac damage progression.

OBJECTIVES: Scleroderma heart disease is a major risk of death in systemic sclerosis (SSc). Mechanisms underlying myocardial damage are still unclear. We performed an extensive study of SSc patients with recent-onset symptoms for heart disease and examined the efficacy of immunosuppressive therapy. METHODS: A cohort of 181 SSc patients was enrolled. Of these, 7 patients newly developed clinical symptoms of heart disease (heart failure, chest pain, and palpitation); all of them showed mild but persistent increase in cardiac enzymes. These patients underwent Holter ECG, 2D-echocardiography, perfusional scintigraphy, delayed-enhancement-cardiac magnetic resonance (DE-CMR), coronary angiography, and endomyocardial biopsy. Patients were treated for at least 12 months and followed-up for 5 years. RESULTS: Ventricular ectopic beats (VEBs) were found in 4 patients, wall motion abnormalities in 3, pericardial effusion in 6, and DE in CMR in 6 with T2-hyperintensity in 2. In all patients, histology showed upregulation of endothelium adhesion molecules and infiltration of activated T lymphocytes, with (acute/active myocarditis in 6) or without (chronic/borderline myocarditis in 1) myocyte necrosis. Parvovirus B19 genome was detected in 3. None showed occlusion of coronary arteries or microvessels. Compared with SSc controls, these patients more often had early disease, skeletal myositis, c-ANCA/anti-PR3 positivity, VEBs, pericardial effusion, and systolic and/or diastolic dysfunction. Immunosuppressive therapy improved symptoms and led to cardiac enzyme negativization; however, 2 patients died of sudden death during follow-up. CONCLUSIONS: Myocarditis is a common finding in SSc patients with recent-onset cardiac involvement. Its early detection allowed to timely start an immunosuppressive treatment, preventing cardiac damage progression in most cases.

Circulating levels of soluble MER in lupus reflect M2c activation of monocytes/macrophages, autoantibody specificities and disease activity.

INTRODUCTION: Systemic lupus erythematosus (SLE) is characterized by impaired efferocytosis and aberrant activation of innate immunity. We asked if shedding of MER receptor tyrosine kinase (MerTK) and AXL into soluble (s) ectodomains was related to immunological and clinical aspects of SLE. METHODS: Levels of sMER and sAXL in the plasma of 107 SLE patients and 45 matched controls were measured by ELISA. In 40 consecutive SLE patients, we examined potential correlations between either sMER or sAXL and plasma levels of sCD163, a marker of M2 activation. All three soluble receptors were measured in supernatants of monocytes/macrophages cultured in various immunological conditions. Membrane expression of MerTK, AXL and CD163 was assessed by flow cytometry. RESULTS: Both sMER and sAXL were associated with anti-chromatin and anti-phospholipid autoantibodies, and with hematological and renal involvement. However, sMER and sAXL did not significantly correlate with each other; sAXL correlated with growth arrest-specific 6 (Gas6), whereas sMER correlated with reduced free protein S (PROS) levels. Only sMER showed significant associations with lupus-specific anti-dsDNA, anti-Sm, anti-ribonucleoprotein (anti-RNP) and anti-Ro60 autoantibodies. Strong correlations with disease activity indices (Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), complement reduction, titer of circulating anti-dsDNA) were found for sMER, not for sAXL. Patients with active SLEDAI, nephritis, anti-dsDNA and anti-Ro60 positivity showed higher levels of sMER compared to controls. Levels of sMER, not sAXL, correlated with sCD163 levels, and these correlated with SLEDAI. Production of sMER and sCD163 occurred under "M2c" polarizing conditions, whereas sAXL was released upon type-I IFN exposure. CONCLUSIONS: Alterations in homeostasis of anti-inflammatory and efferocytic "M2c" monocytes/macrophages may have a role in immunopathogenesis of SLE.