M1 macrophages release pro-inflammatory factors during inflammation. They transition to an M2 phenotype and release anti-inflammatory factors to resolve inflammation. An imbalance in the transition from M1 to M2 phenotype in macrophages contributes to the development of persistent inflammation. CD163, a member of the scavenger receptor cysteine-rich family, is an M2 macrophage marker. The functional role of CD163 during the resolution of inflammation is not completely known. We postulate that CD163 contributes to the transition from M1 to M2 phenotype in macrophages. We induced CD163 gene in THP-1 and primary human macrophages using polyethylenimine nanoparticles grafted with a mannose ligand (Man-PEI). This nanoparticle specifically targets cells of monocytic origin via mannose receptors. Cells were challenged with a single or a double stimulation of lipopolysaccharide (LPS). A CD163 or empty plasmid was complexed with Man-PEI nanoparticles for cell transfections. Quantitative RT-PCR, immunocytochemistry, and ELISAs were used for molecular assessments. CD163-overexpressing macrophages displayed reduced levels of tumor necrosis factor-alpha (TNF)-α and monocytes chemoattractant protein (MCP)-1 after a single stimulation with LPS. Following a double stimulation paradigm, CD163-overexpressing macrophages showed an increase of interleukin (IL)-10 and IL-1ra, and a reduction of MCP-1. This anti-inflammatory phenotype was partially blocked by an anti-CD163 antibody (effects on IL-10 and IL-1ra). A decrease in the release of TNF-α, IL-1β, and IL-6 was observed in CD163-overexpressing human primary macrophages. The release of IL-6 was blocked by an anti-CD163 antibody in the CD163-overexpressing group. Our data show that the induction of the CD163 gene in human macrophages under inflammatory conditions produces changes in cytokine secretion in favor of an anti-inflammatory phenotype. Targeting macrophages to induce CD163 using cell-directed nanotechnology is an attractive and practical approach for inflammatory conditions that could lead to persistent pain, i.e. major surgeries, burns, rheumatoid arthritis, etc.

IntroductionPainful diabetic neuropathy is associated with chronic inflammation, in which macrophages are the key effectors. We utilized an in vitro approach to determine the effects of high glucose on macrophage phenotype.Materials and methodsWe exposed human THP-1 macrophages to normal glucose (5 mM) and a clinically relevant high glucose environment (15 mM) and measured the expression and concentration of molecules associated with a diabetic cellular phenotype.ResultsWe found that THP-1 macrophages in high glucose conditions did not influence the basal expression of cyclooxygenase-2, Toll-like receptor-4, or class A scavenger receptor mRNA, or the concentrations of the cytokines interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, and IL-10, but induced a priming effect on tumor necrosis factor (TNF)-α. Then, we stimulated THP-1 macrophages with a strong pro-inflammatory stimulus lipopolysaccharide (LPS; 5 µg/mL). After stimulation with LPS, we observed an exacerbated increase in TNF-α, IL-6, and MCP-1 concentration in the high glucose condition compared to the normal glucose environment. THP-1 macrophages in high glucose conditions developed tolerance to IL-10 anti-inflammatory effects (TNF-α production) when challenged with LPS.ConclusionOur in vitro approach allows the study of macrophages as potential targets for therapeutic purposes since it compares them to primary human macrophages exposed to high glucose and macrophages from patients with diabetes or complications of painful diabetic neuropathy (i.e. ulcers, adipocytes, and pancreas).

IntroductionMonocytes from patients with diabetes mellitus type 2 (DM2) are dysfunctional, persistently primed, and prone to a proinflammatory phenotype. This may alter the phenotype of their differentiation to macrophages and result in diabetic peripheral neuropathy (DPN), nerve damage, nerve sensitization, and chronic pain. We have previously demonstrated that CD163 is a molecule that promotes an anti-inflammatory cellular phenotype in human primary macrophages, but this has not been proven in macrophages from patients with DM2 or DPN. Thus, we hypothesize that macrophages from patients with DM2 or DPN display an altered proinflammatory functional phenotype related to cytokine production and that the induction of CD163 expression will promote a more homeostatic phenotype by reducing their proinflammatory responsiveness.Patients and methodsWe tested these hypotheses in vitro using blood monocyte-derived macrophages from healthy subjects and patients with DM2 with and without DPN. Cells were incubated in the presence or the absence of 5 µg/mL of lipopolysaccharide (LPS). The concentrations of interleukin-10, interleukin-6, tumor necrosis factor-alpha (TNF-α), TGF-β, and monocyte chemoattractant protein-1 (MCP-1) were measured using ELISA assays. Macrophages were transfected with an empty vector plasmid or a plasmid containing the CD163 gene using mannosylated polyethylenimine nanoparticles.ResultsOur results show that nonstimulated DM2 or DPN macrophages have a constitutive primed proinflammatory state and display a deficient production of proinflammatory cytokines upon a proinflammatory challenge when compared to healthy macrophages. CD163 induction produced an anti-inflammatory phenotype in the healthy control group, and this effect was partial in DM2 or DPN macrophages.ConclusionOur results suggest that diabetic macrophages adopt a complex phenotype that is only partially reversed by CD163 induction. Future experiments are focused on elucidating this differential responsiveness between healthy and diabetic macrophages.

Large tissue damage or wounds cause serious comorbidities and represent a major burden for patients, families, and health systems. Due to the pivotal role of immune cells in the proper resolution of inflammation and tissue repair, we focus our current study on the interaction of macrophages with skin cells, and specifically on the effects of CD163 gene induction in macrophages in wound healing. We hypothesize that the over-expression of the scavenger receptor gene CD163 in human macrophages would result in a more efficient wound healing process.

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