Classically activated mouse macrophages produce methylglyoxal that induces a TLR4- and RAGE-independent proinflammatory response

Abstract

The highly reactive compound methylglyoxal (MG) can cause direct damage to cells and tissues by reacting with cellular macromolecules. MG has been identified as a biomarker associated with increased sepsis-induced mortality. Patients undergoing septic shock have significantly elevated circulating MG levels compared to postoperative patients and healthy controls. Furthermore, MG has been implicated in the development of type II diabetes mellitus and Alzheimer's disease. Because MG is generated during glycolysis, we hypothesized that MG may be produced by classically activated (M1) macrophages, possibly contributing to the inflammatory response. LPS and IFN-treated macrophages acquired an M1 phenotype (as evidenced by M1 markers and enhanced glycolysis) and formed MG adducts, MG-H1, MG-H2, and MG-H3, which were detected using antibodies specific for MG-modified proteins (methylglyoxal 5hydro-5-methylimidazolones). MG adducts were also increased in the lungs of LPS-treated mice. Macrophages treated with LPS and IFN-also exhibited decreased expression of glyoxalase 1 (Glo1), an enzyme that metabolizes MG. Concentrations of exogenous, purified MG > 0.5 mM were toxic to macrophages; however, a nontoxic dose of 0.3 mM induced TNF-and IL-1 , albeit to a lesser extent than LPS stimulation. Despite prior evidence that MG adducts may signal through "receptor for advanced glycation endproducts" (RAGE), MG-mediated cell death and cytokine induction by exogenous MG was RAGE-independent in primary macrophages. Finally, RAGE-deficient mice did not exhibit a significant survival advantage following lethal LPS injection. Overall, our evidence suggests that MG may be produced by M1 macrophages during sepsis, following IFN-dependent down-regulation of Glo1, contributing to over-exuberant inflammation.

The role of RAGE in host pathology and crosstalk between RAGE and TLR4 in innate immune signal transduction pathways

“…Using a polyclonal antibody specific for RAGE (#ab37647, Abcam, Cambridge, United Kingdom), we detected RAGE in mouse lung tissue (Figure 2A). Conversely, in mouse peritoneal macrophages, the antibody detects a similar band, but slightly lower in molecular weight, that we initially speculated might be soluble RAGE in our earlier publication 52 . However, further investigation determined that this band was equivalently present in lysates from both WT and RAGE −/− peritoneal macrophages (Figure 2B), indicating that this is a nonspecific band.…”

“…Initially, it was reported that RAGE‐deficient mice on a CD‐1 background strain were slightly protected against a lethal dose of LPS 15 . However, a subsequent study originating in our laboratory failed to distinguish a significant difference between WT and RAGE‐deficient mice using a different mouse background (C57BL/6J) and a lower LPS dose (50 vs. 20 mg/kg) 52 . Another variation of this basic model of sepsis is the injection of a low dose of LPS to prime the mice, followed by a challenge with a second higher LPS dose.…”

“…Conversely, in mouse peritoneal macrophages, the antibody detects a similar band, but slightly lower in molecular weight, that we initially speculated might be soluble RAGE in our earlier publication. 52 However, further investigation determined that this band was equivalently present in lysates from both WT and RAGE −/− peritoneal macrophages ( Figure 2B), indicating that this is a nonspecific band. This lack of specificity is not unique to this polyclonal antibody either, as two monoclonal antibodies (A9 and A11, Santa Cruz Biotechnology, Dallas, TX) detected only non-specific bands (~37 kDa or ~50 kDa) that were equivalent in lysates from WT versus RAGE −/− peritoneal macrophages (Figure 2C,D).…”

“…AGE‐dependent induction of IL‐1β, TNF‐α, and IL‐6 86 or NF‐κB activation 70 in BMDMs is partially inhibited by an anti‐RAGE antagonist antibody 86 . However, there is a definite need to verify these findings in primary cells more rigorously, especially in light of a recent study that our laboratory has published this past year that showed that MG‐AGE induced pro‐inflammatory cytokines independently of both TLR4 and RAGE in peritoneal macrophages 52 . Given that alveolar macrophages express less RAGE than other cells in the lung tissue, 23 more experiments will be required to measure RAGE expression on multiple cell types to confirm this finding.…”

Role of Peripheral Immune Cells-Mediated Inflammation on the Process of Neurodegenerative Diseases

“…Given the critical role of macrophage-mediated Aβ clearance in both murine models and patients with AD, IL-34 might be relevant to innate immune responses in AD ( 27 ). Besides, in clinical studies, the highly reactive compound, methylglyoxal, has been implicated in the development of AD and methylglyoxal might be produced by macrophages during sepsis and further fasten the pathological process of AD ( 28 ).…”