M2-like polarization of THP-1 monocyte-derived macrophages under chronic iron overload.

Macrophages are characterized by phenotypical and functional heterogeneity. In different microenvironments, macrophages can polarize into two types: classically activated macrophages (M1) or alternatively activated macrophages (M2). M1 macrophages are a well-known bacteriostatic macrophage, and conversely, M2 macrophages may play an important role in tumor growth and tissue remodeling. M1 macrophages have been reported to have high intracellular iron stores, while M2 macrophages contain lower intracellular iron. It has been well-described that disturbances of iron homeostasis are associated with altered immune function. Thus, it is important to investigate if chronic iron overload is capable of polarizing macrophages. Human monocytic leukemia THP-1 cells were maintained in culture medium that contained 100 µM ferrous sulfate heptahydrate (FeSO4) (I-THP-1) and differentiated into THP-1-derived macrophages (I-TDMs) by induction with phorbol 12-myristate 13-acetate (PMA). We characterized that I-TDMs not only enhanced the surface expression of CD163 and CD206 but also increased arginase and decreased iNOS protein expression. I-TDMs enhanced pSTAT6 expression and decreased pSTAT1 and NF-κB expressions. Furthermore, the gene expression profile of I-TDMs was comparable with M2 macrophages by performing human oligonucleotide DNA microarray analysis. Finally, functional assays demonstrated I-TDMs secreted higher levels of IL-10 but not M1 cytokines. Additionally, the conditional medium of I-TDMs had enhanced migration and increased invasion of A375 melanoma cells which was similar to the characteristics of tumor-associated macrophages. Taken together, we demonstrated that THP-1-derived macrophages polarized to a phenotype of M2-like characteristics when subjected to chronic iron overload.

Azithromycin impairs TLR7 signaling in dendritic cells and improves the severity of imiquimod-induced psoriasis-like skin inflammation in mice.

BACKGROUND: The activation of Toll-like receptor 7 (TLR7) in dendritic cells (DCs) plays a crucial role in the pathogenesis of psoriasis. The macrolide antibiotic azithromycin (AZM) had been demonstrated to inhibit the TLR4 agonist-induced maturation and activation of murine bone marrow-derived DCs (BMDCs). OBJECTIVE: To investigate the effects of AZM on the induction of DC maturation and activation by imiquimod (IMQ), a synthetic TLR7 agonist, as well as its potential as a therapeutic agent for psoriasis. METHODS: The effects of AZM on IMQ-induced DC activation were investigated based on the expression of cell surface markers and cytokine secretion. The lysosomal pH, post-translational processing of TLR7, and TLR7 signaling were also examined in DCs. The therapeutic effects of AZM on psoriasis were evaluated in a murine model of IMQ-induced psoriasis-like skin inflammation. RESULTS: AZM significantly inhibited the expression of co-stimulatory molecules (CD40 and CD80) and reduced TNF-α, IL-10, IL-12p40, IL-12p70, IL-23p19 in BMDCs and IFN-α production in plasmacytoid DCs. AZM treatment impaired lysosomal acidification, interrupted TLR7 maturation in the lysosome, and ultimately blocked the IMQ-induced NF-κB and IRF-7 nuclear translocation in DCs. AZM treatment decreased signs of IMQ-induced skin inflammation in BALB/c mice. In addition to decreasing keratinocyte hyper-proliferation and restoring their terminal differentiation, AZM treatment decreased the accumulation of DCs as well as CD4, CD8 T cells and IL-17 producing cells in psoriatic skin lesions. AZM treatment improved splenomegaly, decreased the populations of Th17 and γδ T cells, and reduced the expression of cytokines known to be involved in the pathogenesis of psoriasis, such as IL-17A, IL-17F, IL-22 and IL-23, in the skin and spleen. CONCLUSION: AZM impaired IMQ-induced DC activation by decreasing lysosomal acidification and disrupting TLR7 maturation and signaling. AZM significantly improved the IMQ-induced psoriasis-like inflammation in mice. AZM may be a potential therapeutic candidate for psoriasis treatment.

Chronic Iron Overload Results in Impaired Bacterial Killing of THP-1 Derived Macrophage through the Inhibition of Lysosomal Acidification.

Iron is essential for living organisms and the disturbance of iron homeostasis is associated with altered immune function. Additionally, bacterial infections can cause major complications in instances of chronic iron overload, such as patients with transfusion-dependent thalassemia. Monocytes and macrophages play important roles in maintaining systemic iron homoeostasis and in defense against invading pathogens. However, the effect of iron overload on the function of monocytes and macrophages is unclear. We elucidated the effects of chronic iron overload on human monocytic cell line (THP-1) and THP-1 derived macrophages (TDM) by continuously exposing them to high levels of iron (100 µM) to create I-THP-1 and I-TDM, respectively. Our results show that iron overload did not affect morphology or granularity of I-THP-1, but increased the granularity of I-TDM. Bactericidal assays for non-pathogenic E. coli DH5α, JM109 and pathogenic P. aeruginosa all revealed decreased efficiency with increasing iron concentration in I-TDM. The impaired P. aeruginosa killing ability of human primary monocyte derived macrophages (hMDM) was also found when cells are cultured in iron contained medium. Further studies on the bactericidal activity of I-TDM revealed lysosomal dysfunction associated with the inhibition of lysosomal acidification resulting in increasing lysosomal pH, the impairment of post-translational processing of cathepsins (especially cathepsin D), and decreased autophagic flux. These findings may explain the impaired innate immunity of thalassemic patients with chronic iron overload, suggesting the manipulation of lysosomal function as a novel therapeutic approach.