ObjectivesMethotrexate (MTX) is the anchor drug for treatment of rheumatoid arthritis (RA), but the mechanism of its anti-inflammatory action is not fully understood. In RA, macrophages display a proinflammatory polarisation profile that resembles granulocyte-macrophage colony-stimulating factor (GM-CSF)-differentiated macrophages and the response to MTX is only observed in thymidylate synthase+ GM-CSF-dependent macrophages. To determine the molecular basis for the MTX anti-inflammatory action, we explored toll-like receptor (TLR), RA synovial fluid (RASF) and tumour necrosis factor receptor (TNFR)-initiated signalling in MTX-exposed GM-CSF-primed macrophages.MethodsIntracellular responses to TLR ligands, TNFα or RASF stimulation in long-term low-dose MTX-exposed human macrophages were determined through quantitative real-time PCR, western blot, ELISA and siRNA-mediated knockdown approaches. The role of MTX in vivo was assessed in patients with arthritis under MTX monotherapy and in a murine sepsis model.ResultsMTX conditioned macrophages towards a tolerant state, diminishing interleukin (IL)-6 and IL-1β production in LPS, LTA, TNFα or RASF-challenged macrophages. MTX attenuated LPS-induced MAPK and NF-κB activation, and toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF1)-dependent signalling. Conversely, MTX increased the expression of the NF-κB suppressor A20 (TNFAIP3), itself a RA-susceptibility gene. Mechanistically, MTX-induced macrophage tolerance was dependent on A20, as siRNA-mediated knockdown of A20 reversed the MTX-induced reduction of IL-6 expression. In vivo, TNFAIP3 expression was significantly higher in peripheral blood cells of MTX-responsive individuals from a cohort of patients with arthritis under MTX monotherapy, whereas MTX-treated mice exhibited reduced inflammatory responses to LPS.ConclusionsMTX impairs macrophage proinflammatory responses through upregulation of A20 expression. The A20-mediated MTX-induced innate tolerance might limit inflammation in the RA synovial context, and positions A20 as a potential MTX-response biomarker.

Background and AimsGM-CSF-dependent macrophage polarization has been demonstrated in rheumatoid arthritis (RA). Our aim was to seek diagnostic/prognostic biomarkers for undifferentiated arthritis (UA) by analyzing GM-CSF expression and source, macrophage polarization and density in joints of patients with UA evolving to RA or PsA compared with established RA or PsA, respectively.MethodsSynovial tissue (ST) from patients with UA evolving to RA (UA>RA, n=8), PsA (UA>PsA, n=9), persistent UA (UA, n=16), established RA (n=12) and PsA (n=10), and healthy controls (n=6), were analyzed. Cell source and quantitative expression of GM-CSF and proteins associated with pro-inflammatory (GM-CSF-driven) and anti-inflammatory (M-CSF-driven) macrophage polarization (activin A, TNFα, MMP12, and CD209, respectively) were assessed in ST CD163+ macrophages by multicolor immunofluorescence. GM-CSF and activin A levels were also quantified in paired synovial fluid samples. CD163+ macrophage density was determined in all groups by immunofluorescence.ResultsSynovial stromal cells (FAP+ CD90+ fibroblast, CD90+ endothelial cells) and CD163+ sublining macrophages were the sources of GM-CSF. ST CD163+ macrophages from all groups expressed pro-inflammatory polarization markers (activin A, TNFα, and MMP12). Expression of the M-CSF-dependent anti-inflammatory marker CD209 identified two macrophage subsets (CD163+ CD209high and CD163+ CD209low/-). CD209+ macrophages were more abundant in ST from healthy controls and PsA patients, although both macrophage subtypes showed similar levels of pro-inflammatory markers in all groups. In paired synovial fluid samples, activin A was detected in all patients, with higher levels in UA>RA and RA, while GM-CSF was infrequently detected. ST CD163+ macrophage density was comparable between UA>RA and UA>PsA patients, but significantly higher than in persistent UA.ConclusionsGM-CSF is highly expressed by sublining CD90+ FAP+ synovial fibroblasts, CD90+ activated endothelium and CD163+ macrophages in different types of arthritis. The polarization state of ST macrophages was similar in all UA and established arthritis groups, with a predominance of pro-inflammatory GM-CSF-associated markers. CD163+ macrophage density was significantly higher in the UA phases of RA and PsA compared with persistent UA. Taken together, our findings support the idea that GM-CSF is a strong driver of macrophage polarization and a potential therapeutic target not only in RA but also in PsA and all types of UA.

The identification of “trained immunity/tolerance” in myeloid cells has changed our perception of the performance of monocytes and macrophages during inflammatory and immune responses. Pemetrexed (PMX) and methotrexate (MTX) are blockers of the one-carbon metabolism (OCM) and commonly used therapeutic agents in cancer and rheumatoid arthritis (RA). We have previously showed that MTX promotes trained immunity in human macrophages. In the present manuscript, we have assessed the anti-inflammatory effects of PMX and MTX and found that OCM blockers alter the functional and gene expression profile of human macrophages and that OCM blockade reprograms macrophages towards a state of lipopolysaccharide (LPS) tolerance at the signaling and functional levels. Moreover, OCM blockade reduced macrophage LPS responsiveness by impairing the expression of membrane-bound and soluble CD14 (sCD14). The therapeutic relevance of these results was later confirmed in early RA patients, as MTX-responder RA patients exhibit lower sCD14 serum levels, with baseline sCD14 levels predicting MTX response. As a whole, our results demonstrate that OCM is a metabolic circuit that critically mediates the acquisition of innate immune tolerance and positions sCD14 as a valuable tool to predict MTX response in RA patients.

BackgroundUndifferentiated arthritis (UA) is defined as an inflammatory oligo-or polyarthritis that does not fulfil criteria for a definitive diagnosis. Earlier diagnosis would permits a better functional prognosis. Synovial tissue (ST) macrophages have been associated with disease activity, radiographic erosion and response to therapy in RA. Furthermore, it has been reported that M1 polarised macrophages predominate in RA synovitis, whereas M2 predominate in SpA synovitis.1 ObjectivesTo analyse polarised macrophages (M1 proinflammatory and M2 anti-inflammatory) in ST of patients with UA which evolved to RA or PsA, after a long follow-up, to explore their diagnostic value.MethodsTo determine the polarisation state of macrophages in ST obtained by arthroscopy from patients with UA that evolved to RA (UA-RA=8) or PsA (UA-PsA=9), the expression of proteins associated to GM-CSF-driven polarisation M1(INHBA, TNFα and MMP12) and M-CSF-driven polarisation M2(CD209) was assessed in ST CD163+macrophages. Patients with established RA(n=12) or PsA(n=10) were included as control. 4 µm cryosections of ST in OCT were blocked for 10 min with 1% human immunoglobulins and incubated with primary (1–5 µg/ml) and secondary antibodies. Imaging was performed with an inverted confocal microscope (SPE, Leica Microsystems) and glycerol immersion objective (ACS-APO 20x/NA 0.60). Single cell mean fluorescence intensity(MFI) was assessed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). At least three random fields were evaluated for each type of ST, quantifying the expression of INHBA, TNF-α, MMP12 and CD209 in all segmented CD163+macrophages. Macrophages density was normalised based on selected tissue area(mm2). After background subtraction, data were plotted using GraphPad software (GraphPad Software, La Jolla, CA, USA).ResultsCD163+ sublining (SL) macrophages from UA-RA expressed abundantly the INHBA-encoded activin A, whereas TNFα and MMP12 were variably detected. Regarding the M-CSF-associated marker CD209, 2 populations of CD163+ macrophages were found in the SL of UA-RA, CD163+CD209+ and the other CD163+CD209-, with higher than 100 arbitrary units (au) for CD163+CD209+ and lower than 100 au for CD163+CD209-. Similarly, INHBA, MMP12 and TNFα expression and 2 populations of CD209 were detected in CD163+ macrophages from UA-PsA. Macrophage density was also found comparable between UA, with 650/mm2 in UA-RA and 649/mm2 in UA-PsA. Quantification of the above indicated markers in CD163 +ST macrophages from established RA and PsA revealed similar levels of INHBA, TNFα, MMP12 and CD209 than those from UA-RA and UA-PsA.ConclusionsThis study shows for the first time that the polarisation state of ST CD163+ macrophages in UA progressing to RA and PsA is similar to that of established RA and PsA in terms of INHBA, MMP12, TNFα and CD209 expression. Therefore, the inflammatory polarisation state of macrophages is similar in RA and PsA and it is already detected at the earlier steps.Reference[1] Vandooren B, et al. Arthritis...

CD28 expression is generally considered to be T lymphocyte specific. We have previously shown CD28 mRNA expression in M‐CSF‐dependent anti‐inflammatory monocyte‐derived macrophages (M‐MØ), and now demonstrate that CD28 cell surface expression is higher in M‐MØ than in GM‐CSF‐dependent macrophages, and that macrophage CD28 expression is regulated by MAFB and activin A. In vivo, CD28 was found in tumor‐associated macrophages and, to a lower extent, in pro‐inflammatory synovial fluid macrophages from rheumatoid arthritis patients. Analysis of mouse macrophages confirmed Cd28 expression in bone‐marrow derived M‐MØ. Indeed, anti‐CD28 antibodies triggered ERK1/2 phosphorylation in mouse M‐MØ. At the functional level, Cd28KO M‐MØ exhibited a significantly higher capacity to activate the OVA‐specific proliferation of OT‐II CD4+ T cells than WT M‐MØ, as well as enhanced LPS‐induced IL‐6 production. Besides, the Cd28KO M‐MØ transcriptome was significantly different from WT M‐MØ regarding the expression IFN response, inflammatory response, and TGF‐β signaling related gene sets. Therefore, defective CD28 expression in mouse macrophages associates to changes in gene expression profile, what might contribute to the altered functionality displayed by Cd28KO M‐MØ. Thus, CD28 expression appears as a hallmark of anti‐inflammatory macrophages and might be a target for immunotherapy.