Anti-TNF agents are widely used in rheumatoid arthritis (RA). Their effect on inflammation results from the neutralization of soluble TNF (sTNF), but also supposedly from the induction of reverse signaling through their binding to membrane TNF (tmTNF). Despite possible clinical relevance, reverse signaling has been described only in vitro but has not been proven in vivo.
Objectives
In this study we aim to demonstrate for the first time the existence of tmTNF reverse signaling in vivo and its importance in anti-TNF response during arthritis.
Methods
Triple transgenic mouse model (3TG), KO for TNFR1/TNFR2 and KI for tmTNF, thus secreting no sTNF was developed. To analyze reverse signaling, mice were injected either with etanercept (ETA, 10 mg/kg), an anti-mouse TNF antibody (MP6-XT22, rat IgG1, 10 mg/kg) or an anti-human IL17 antibody (secukinumab, SEC, 10 mg/kg) as a control. Daily clinical evaluation of K/BxN serum induced-arthritis was performed in 3TG as well as WT mice. Polarization of bone marrow-derived macrophages (BMDM) and cytokine production from non-arthritic WT and 3TG mice under the action of anti-TNF in vitro was evaluated by RT-qPCR, CBA and ELISA.
Results
In vivo, the administration of anti-TNF (ETA or MP6-XT22) decreased arthritic scores in WT mice (p=0.005) as well as in 3TG mice (p<0.001), unlike SEC which had no effect, proving that anti-TNF binding of tmTNF decreased arthritis. In vitro effect of anti-TNF on BMDM from WT as well as 3TG mice induced a decrease in the expression of genes specific of inflammatory macrophages (CD38, GpR18 and FpR2), and an increase in the expression of genes specific of alternative macrophages (Arg1, EgR2, c-Myc). We also observed an inhibition of the secretion of pro-inflammatory cytokines (IL12p70 and IL-6) and an early peak of IL-10 secretion demonstrating an effect of reverse signaling on macrophage polarization and activation. This suggested a switch in macrophage polarization as a probable mechanism for modulation of inflammation during K/BxN serum-induced arthritis.
Conclusions
Our work provides in vivo evidence for the involvement of reverse signaling in the anti-TNF-mediated modulation of arthritis. Reverse signaling is expected to result in the modulation of macrophage polarization from an inflammatory to an alternative functional phenotype in arthritic mice. Our data prompt us to consider new interpretation of the effects of anti-TNF in the treatment of RA.
Monocytes play a key role in both the systemic and local progression of rheumatoid arthritis (RA) by producing molecules that participate to the inflammatory and catabolic events of disease pathogenesis (1). Recently, the spleen has been shown to contribute to the regulation of inflammation through monocytes that are able to exit and rapidly deploy to inflammatory sites (2).
These observations uncover a role for splenic monocytes as a resource exploited by the body to regulate inflammation. Thus, the engineering of vectors tailored to selectively target both tissue resident and circulating monocytes is a promising research track for addressing the role of specific genes in RA pathogenicity. Several lines of evidence imply cytosolic phospholipase A2α (cPLA2α) as a critical enzyme in inflammatory disorders including RA.
Venous haemangioma of the synovium of one knee also involved adjacent, skin and muscle of the leg in a 4-yr-old girl. Phleboliths were seen in the knee on X-ray. Histologically, the synovium contained large venous channels and was laden with haemosiderin. On electron microscopy, the synovial cells were morpholgically most like synthetic (B) cells. Erythrocytes were ingested and broken down, stepwise, to siderosomes. The synovial cell cytoplasm also contained electron-dense punctate iron deposits. The synovial response appeared to represent chronic haemarthrosis.
(1) Background: TNF inhibitors (TNFi) have revolutionized the treatment of rheumatoid arthritis (RA). However, 30-40 % of RA patients do not respond adequately to those biologics. In addition to neutralizing soluble TNF, TNFi have the ability to bind the transmembrane form of TNF, tmTNF. Importantly, tmTNF can act itself as a receptor that induces a “Reverse Signaling” (RS) in cells. We previously showed that certolizumab, a Fab’ TNFi, activates RS in human primary monocytes, at least in part through the transcription factor NRF2 that is known to regulate the expression of genes involved in anti-inflammatory response and oxidative stress. (2) Methods: Here, we have developed an assay for the prediction of clinical response of RA patients to TNF inhibitors. This assay is based on mRNA quantitation of CD36 activation and of 6 genes induced by NRF2 following tmTNF RS in fresh monocytes. (3) Results: We could predict the response to anti-TNF monoclonal antibodies (mAbs) with 93.3 % accuracy. However, our method was not suitable for the prediction of the response to TNF soluble receptor etanercept. (4) Conclusions: We have developed a rather simple, short-term, test that can be standardized. Predicting the response to TNF mAbs will help physicians offer the best available treatment and provide patients with personalized medicine.
Abstract Introduction Our objective was to assess the capacity of dendrimer aza-bis-phosphonate (ABP) to modulate phenotype of monocytes (Mo) and monocytes derived dendritic cells (MoDC) activated in response to toll-like receptor 4 (TLR4) and interferon γ (IFN- γ) stimulation. Methods Mo ( n = 12) and MoDC ( n = 11) from peripheral blood of healthy donors were prepared. Cells were preincubated or not for 1 hour with dendrimer ABP, then incubated with lipopolysaccharide (LPS; as a TLR4 ligand) and (IFN-γ) for 38 hours. Secretion of tumor necrosis factor α (TNFα), interleukin (IL) -1, IL-6, IL-12, IL-10 and IL-23 in the culture medium was measured by enzyme-linked immunosorbent assay (ELISA) and Cytokine Bead Array. Differentiation and subsequent maturation of MoDC from nine donors in the presence of LPS were analyzed by flow cytometry using CD80, CD86, CD83 and CD1a surface expression as markers. Results Mo and MoDC were orientated to a pro-inflammatory state. In activated Mo, TNFα, IL-1β and IL-23 levels were significantly lower after prior incubation with dendrimer ABP. In activated MoDC, dendrimer ABP promoted IL-10 secretion while decreasing dramatically the level of IL-12. TNFα and IL-6 secretion were significantly lower in the presence of dendrimer ABP. LPS driven maturation of MoDC was impaired by dendrimer ABP treatment, as attested by the significantly lower expression of CD80 and CD86. Conclusion Our data indicate that dendrimer ABP possesses immunomodulatory properties on human Mo and MoDC, in TLR4 + IFN-γ stimulation model, by inducing M2 alternative activation of Mo and promoting tolerogenic MoDC.
The authors have previously shown that phosphorus-based dendrimer ABP targets monocytes to inhibit inflammation and osteoclastogenesis in two models of experimental arthritis. The authors have now further explored this model and tested the frequency and route of administration of dendrimer ABP.
Materials and methods
The authors used two models of mouse arthritis: IL-1 ra-/- which develops a spontaneous arthritis at the age of 6 weeks. The KxB/N serum transfer arthritis, which is more severe, and develops upon the injection of auto-antibodies from KxB/N mice. Inflammation and paw-swelling were scored, cytokines were measured by Elisa, and histology was performed.
Results
In the IL-1 ra-/- model of spontaneous arthritis, the i.v. injection of dendrimer was found to be optimal when administered every 3 weeks. To test for other route of administration, gavage was also performed once a week after solubilisation of dendrimer in an aqueous buffer. This suggests that dendrimer ABP is not degraded in the gastrointestinal tract and is absorbed efficiently using this route of administration. In the KxB/N serum transfer model, which is more severe, several i.p. injections of serum interspaced with dendrimer ABP (every 3 days over the course of the experiment) were performed. The authors showed that injections of KxB/N serum (days 0 and 2) induced arthritis which was well controlled by dendrimer. Subsequent injection of serum (day 11) reactivated arthritis, thus mimicking flares of arthritis that are observed in RA. Again, arthritis was controlled by dendrimer. On day 20, 3 days after the last injection of serum performed on day 17, mice were euthanised and blood drawn for analysis. Quantification of serum pro-inflammatory cytokines (IL-1β, TNFα, IL-6, IL-17) showed that i.v. dendrimer completely abrogated the inflammation seen in untreated arthritic mice. Bone destruction and osteoclast (OC) formation were also controlled in mice treated with dendrimer.
Conclusion
Our data further argue in favor of strong anti-inflammatory and anti-OC properties of dendrimer ABP. Our data are also in favor of a possible oral administration of the compound.
In order to ascertain the significance of transmembrane tumor necrosis factor (tmTNF) reverse signaling in vivo, we generated a triple transgenic mouse model (3TG, TNFR1−/−, TNFR2−/−, and tmTNFKI/KI) in which all canonical tumor necrosis factor (TNF) signaling was abolished. In bone-marrow-derived macrophages harvested from these mice, various anti-TNF biologics induced the expression of genes characteristic of alternative macrophages and also inhibited the expression of pro-inflammatory cytokines mainly through the upregulation of arginase-1. Injections of TNF inhibitors during arthritis increased pro-resolutive markers in bone marrow precursors and joint cells leading to a decrease in arthritis score. These results demonstrate that the binding of anti-TNF biologics to tmTNF results in decreased arthritis severity. Collectively, our data provide evidence for the significance of tmTNF reverse signaling in the modulation of arthritis. They suggest a complementary interpretation of anti-TNF biologics effects in the treatment of inflammatory diseases and pave the way to studies focused on new arginase-1-dependent therapeutic targets.
Macrophages are major effector cells in inflammatory rheumatisms such as Rheumatoid arthritis (RA) and Psoriatic arthritis (PsA). Depending on their microenvironment, especially cytokines content, they can display various phenotypes or "polarizations" described as inflammatory (in the presence of IFNγ or GM-CSF) or as alternative (in the presence of IL-4 or IL-10). These cytokines involve JAK/STAT signaling pathway, and their action is expected to be inhibited by JAK inhibitors (JAKi) developed in RA and PsA. How JAKi modulate polarization of macrophage, and whether this phenomenon explains the clinical benefit in RA and PsA is not fully understood.
Objectives
To evaluate whether JAKi modulate in vitro polarization of monocytes derived macrophages.
Methods
[Cell culture] CD14+ monocytes were isolated from healthy donors and differentiated as macrophages in the presence of M-CSF. Macrophages were then polarized as inflammatory macrophages (by LPS + IFNγ, or GM-CSF), or as alternative macrophages (by IL-4, or IL-10), in the presence or not of JAKi (500 nM): Baricitinib (JAK1/JAK2 specific), Tofacitinib (JAK1/JAK2/JAK3/Tyk2 specific). [Membrane markers] Surface polarization markers were evaluated by flow cytometry. [Functional assays] Cytokines production in supernatants of macrophages were quantitated by bead-based immunoassay and by ELISA. Reactive oxygen species (ROS) production triggered by LPS and/or IFNγ was assessed by flow cytometry. Phagocytosis of E.coli bioparticles was assessed by flow cytometry.
Results
We analyzed 12 donors. Membrane polarization markers were: CD40/CD80/CD206 for inflammatory macrophages, CD163/CD16/CD206/CD200R for alternative macrophages. Both JAKi modulated these markers. Regarding the inflammatory polarizations, Baricitinib and Tofacitinib respectively reduced CD40 and CD206 expression. Regarding the alternative polarizations, both drugs inhibited the expression of CD163, CD206 and CD200R. At thecytokines level, both drugs did not significantly modulate the pro-inflammatory/anti-inflammatory balance in supernatants. We observed a slight increase in TNF and IL-6 resulting from LPS/NFkB pathway [1]. However, Baricitinib decreased the inflammatory IP-10, and increased IL-10 production. Both JAKi did not affect ROS production in the presence of LPS. Consistently with the absence of modulation of CD16 surface expression, Baricitinib and Tofacitinib did not affect CD16-dependent phagocytosis.
Conclusion
Our study shows that despite distinct JAK specificity, Tofacitinib and Baricitinib maintained surface phenotypes close to those of non-activated macrophages This finding was observed whatever was the polarizing condition, thus supporting the potential benefit of JAK inhibitors in different immune diseases.
Macrophages contribute to the pathogenesis of rheumatoid arthritis (RA). They can display different states of activation or “polarization”, notably the so-called inflammatory “M1” and the various alternative “M2” polarizations, characterized by distinct functions. Data regarding the effects of RA anti-cytokine biological disease-modifying anti-rheumatic drugs (bDMARDs) on macrophage polarization are scarce. We aimed to assess in vitro modulation of macrophage polarization by bDMARDs targeting pro-inflammatory cytokines in RA. We generated monocyte derived macrophages using blood samples from 20 RA patients with active RA and 30 healthy controls. We evaluated in vitro the impact on M1 inflammatory macrophages of: etanercept (ETA), adalimumab (ADA), certolizumab (CZP), tocilizumab (TCZ), and rituximab (RTX). We assessed the impact on macrophage polarization using flow cytometry and RTqPCR to study the expression of surface markers and perform functional studies of cytokine production, phagocytosis, and negative feedback control of inflammation. Among evaluated bDMARDs, anti-TNF agents modulated the polarization of inflammatory macrophages by decreasing inflammatory surface markers (CD40, CD80) and favoring alternative markers (CD16, CD163, MerTK). Anti-TNF agents also induced alternative functions in macrophages activated in inflammatory condition with (i) the inhibition of inflammatory cytokines (TNF, IL-6, IL-12), (ii) an increase in phagocytosis. These findings were mechanistically related to an increase in early IL-10 production, responsible for higher negative feedback control of inflammation involving SOCS3 and Gas6. This IL-10 effect was STAT3-dependent. Anti-TNF agents not only inhibit in vitro inflammatory functions of macrophages, but also favor resolution of inflammation through polarization towards alternative features specifically involving the IL-10/STAT3 axis.
