CTLA4-Ig treatment induces M1–M2 shift in cultured monocyte-derived macrophages from healthy subjects and rheumatoid arthritis patients

Background In rheumatoid arthritis (RA), macrophages play an important role in modulating the immunoinflammatory response through their polarisation into “classically” (M1) or “alternatively activated” (M2) phenotypes. In RA, CTLA4-Ig (abatacept) reduces the inflammatory activity of macrophages by interacting with the costimulatory molecule CD86. The study aimed to investigate the efficacy of CTLA4-Ig treatment to induce an M2 phenotype both in M1-polarised monocyte-derived macrophages (MDMs) obtained from healthy subjects (HS) and in cultured MDMs obtained from active RA patients. Methods Cultured MDMs were obtained from peripheral blood mononuclear cells of 7 active RA patients and from 10 HS after stimulation with phorbol myristate acetate (5 ng/mL) for 24 h. HS-MDMs were then stimulated with lipopolysaccharide (LPS, 1 mg/mL) for 4 h to induce M1-MDMs. M1-MDMs and RA-MDMs were treated with CTLA4-Ig (100 μM and 500 μM) for 3, 12, 24, and 48 h. The gene expression of CD80, CD86, and TLR4 (M1 markers); CD163, CD204, and CD206 (surface M2 markers); and MerTK (functional M2 marker) was evaluated by qRT-PCR. The protein synthesis of surface M2 markers was investigated by Western blotting. The statistical analysis was performed by the Wilcoxon t-test. Results In LPS-induced HS-M1-MDMs, CTLA4-Ig 100 μM and 500 μM significantly downregulated the gene expression of M1 markers (3 h p<0.01 for all molecules; 12 h p<0.05 for TLR4 and CD86) and significantly upregulated that of M2 markers, primarily after 12 h of treatment (CD163: p < 0.01 and p < 0.05; CD206: p < 0.05 and p < 0.01; CD204: p < 0.05 by 100 mg/mL). Moreover, in these cells, CTLA4-Ig 500 μM increased the protein synthesis of surface M2 markers (p < 0.05). Similarly, in RA-MDMs, the CTLA4-Ig treatment significantly downregulated the gene expression of M1 markers at both concentrations primarily after 12 h (p < 0.05). Furthermore, both concentrations of CTLA4-Ig significantly upregulated the gene expression of CD206 (after 3 h of treatment; p < 0.05), CD163, and MerTK (after 12 h of treatment, p < 0.05), whereas CD204 gene expression was significantly upregulated by the high concentration of CTLA4-Ig (p < 0.05). The protein synthesis of all surface markers was increased primarily by CTLA4-Ig 500 μM, significantly for CD204 and CD206 after 24 h of treatment (p < 0.05). Conclusions CTLA4-Ig treatment seems to induce the in vitro shift from M1 to M2 macrophages, of both HS-M1-MDMs and RA-MDMs, as observed by the significant downregulation exerted on selected M1 markers and the upregulation of selected M2 markers suggesting an additional mechanism for its modulation of the RA inflammatory process.

Role of Macrophage Polarization in Acute Respiratory Distress Syndrome

Acute Respiratory Distress Syndrome is a familiar and destructive clinical condition characterized by progressive, swift and impaired pulmonary state. It leads to mortality if not managed in a timely manner. Recently the role of imbalanced macrophage polarization has been reported in ARDS. Macrophages are known for their heterogeneity and plasticity. Under different microenvironmental stimuli, they (M0) can switch between classically activated macrophage (M1) and alternatively activated (M2) states. This switch is regulated by several signaling pathways and epigenetic changes. In this review, the importance of macrophage M1 and M2 has been discussed in the arena of ARDS citing the phase-wise impact of macrophage polarization. This will provide a further understanding of the molecular mechanism involved in ARDS and will help in developing novel therapeutic targets. Various biomarkers that are currently used concerning this pathophysiological feature have also been summarized.

β2-Adrenergic Receptor Enhances the Alternatively Activated Macrophages and Promotes Biliary Injuries Caused by Helminth Infection

The autonomic nervous system has been studied for its involvement in the control of macrophages; however, the mechanisms underlying the interaction between the adrenergic receptors and alternatively activated macrophages (M2) remain obscure. Using FVB wild-type and beta 2 adrenergic receptors knockout, we found that β2-AR deficiency alleviates hepatobiliary damage in mice infected with C. sinensis. Moreover, β2-AR-deficient mice decrease the activation and infiltration of M2 macrophages and decrease the production of type 2 cytokines, which are associated with a significant decrease in liver fibrosis in infected mice. Our in vitro results on bone marrow–derived macrophages revealed that macrophages from Adrb2−/− mice significantly decrease M2 markers and the phosphorylation of ERK/mTORC1 induced by IL-4 compared to that observed in M2 macrophages from Adrb2+/+. This study provides a better understanding of the mechanisms by which the β2-AR enhances type 2 immune response through the ERK/mTORC1 signaling pathway in macrophages and their role in liver fibrosis.

Macrophage Innate Training Induced by IL-4 Activation Enhances OXPHOS Driven Anti-Mycobacterial Responses

Macrophages are key innate immune cells for determining the outcome of Mycobacterium tuberculosis infection. Polarization with IFNγ and LPS into the “classically activated” M1 macrophage enhances pro-inflammatory and microbicidal responses, important for eradicating the bacterium. By contrast, “alternatively activated” M2 macrophages, polarized with IL-4, oppose bactericidal mechanisms and allow mycobacterial growth. These activation states are accompanied by distinct metabolic profiles, where M1 macrophages favor near exclusive use of glycolysis, whereas M2 macrophages up-regulate oxidative phosphorylation (OXPHOS). Here we demonstrate that activation with IL-4 counterintuitively induces protective innate memory against mycobacterial challenge. This was associated with enhanced pro-inflammatory cytokine responses and killing capacity. Moreover, despite this switch towards a phenotype that is more akin to classical activation, IL-4 trained macrophages do not demonstrate M1-typical metabolism, instead retaining heightened use of OXPHOS. Moreover, inhibition of OXPHOS with oligomycin, 2-deoxy glucose or BPTES all impeded heightened pro-inflammatory cytokine responses from IL-4 trained macrophages. Lastly, this work identifies that IL-10 negatively regulates protective IL-4 training, impeding pro-inflammatory and bactericidal mechanisms. In summary, this work provides new and unexpected insight into alternative macrophage activation states in the context of mycobacterial infection.

Macrophages in the uterus are functionally specialised and continually replenished from the circulation

ABSTRACTMacrophages are innate immune cells that fight infection but also regulate tissue regeneration and remodelling. In the uterus, although tissue remodelling is essential for establishment and maintenance of pregnancy, the specialisation of macrophages is not well characterised compared to other mucosal tissues. Here we show that uterine macrophages are functionally specialised, expressing multiple markers of alternative activation associated with tissue remodelling and repair, and responding more highly to the type 2 cytokine IL-4 than other mucosal tissue macrophages. Uterine macrophages were continuously replenished from circulating bone marrow-derived CCR2+ monocytes that fluctuated dramatically in number throughout the reproductive cycle, and had properties distinct from the macrophages that they became, including differential responses to microbial stimulation. Importantly, many of these properties of uterine monocytes and macrophages were conserved between mice and humans. These findings further our understanding of immune regulation of uterine tissue integrity and have important implications for differences in immune responses to infections at different phases of the reproductive cycle.SUMMARYUterine macrophages are specialised, alternatively activated cells that are replenished from circulating bone marrow-derived monocytes. Monocyte and macrophage properties fluctuate markedly throughout the reproductive cycle, with many features conserved between mice and humans, and exhibiting differential responses to microbial stimulation.

Loss of protein tyrosine phosphatase non-receptor type 2 reduces IL-4-driven alternative macrophage activation

Macrophages are a heterogeneous population of innate immune cells that are often divided into two major subsets: classically activated, typically pro-inflammatory (M1) macrophages that mediate host defense, and alternatively activated, tolerance-inducing (M2) macrophages that exert homeostatic and tissue-regenerative functions. Disturbed macrophage function/differentiation results either in inadequate, excessive immune activation or in a failure to induce efficient protective immune responses against pathogens. Loss-of-function variants in protein tyrosine phosphatase non-receptor type 2 (PTPN2) are associated with chronic inflammatory disorders, but the effect of macrophage-intrinsic PTPN2 loss is still poorly understood. Here we report that PTPN2-deficient macrophages fail to acquire an alternatively activated/M2 phenotype. This was the consequence of reduced IL-6 receptor expression and a failure to induce IL-4 receptor in response to IL-6, resulting in an inability to respond to the key M2-inducing cytokine IL-4. Ultimately, failure to adequately respond to IL-6 and IL-4 resulted in increased levels of M1 macrophage marker expression in vitro and exacerbated lung inflammation upon infection with Nippostrongylus brasiliensis in vivo. These results demonstrate that PTPN2 loss interferes with the ability of macrophages to adequately respond to inflammatory stimuli and might explain the increased susceptibility of PTPN2 loss-of-function carriers to developing inflammatory diseases.

SIRPα sequesters SHP-2 to promote IL-4/13 signaling and alternative activation of macrophages

The Th2 cytokines IL-4 and IL-13 through activation of their shared receptor IL-4Rα direct macrophage alternative activation to promote immunosuppression and wound healing. However, the mechanisms that control macrophage responses to IL-4/13 are not fully understood. Apart from driving JAK-STAT and PI3K-Akt pathways to polarize macrophages toward the alternative phenotype, the activated IL-4/13 receptors recruit negative regulators SHP-1 and SHP-2, which dephosphorylate IL-4Rα and decrease its signaling. Here we report that SIRPα spatially restricts SHP-2 and, by such, promotes IL-4/13 signaling and macrophage alternative activation. SIRPα executes this regulation via its cytoplasmic ITIMs/ITSMs that undergo phosphorylation by IL-4/13-induced, Src kinase-activated Brutons tyrosine kinase (Btk), resulting in recruitment of SHP-2 and preclusion of SHP-2 from binding to and inhibiting IL-4/13 receptors. Despite that this regulation occurs independent of CD47, extracellular CD47 ligation of SIRPα facilitates its cytoplasmic phosphorylation and SHP-2 sequestration, leading to stronger IL-4/13 signaling and enhanced macrophage expression of IL-10, TGFβ, CD206, arginase-1, etc. Conversely, deficiency of SIRPα allows SHP-2 to freely bind to γC or IL-13Rα1 and through which dephosphorylate IL-4Rα, dampening its signaling. Consistent with these findings, impaired wound healing in Sirpα-/- mice under experimental colitis correlated with a deficit of immunosuppressive macrophages in the colon, a condition that was corrected by transfusion of ex vivo-produced SIRPαhigh alternatively activated macrophages.