Under normal conditions macrophages provide immune surveillance and host defense in tissues to maintain homeostasis. However, upon sensing changes in the microenvironment, macrophages become activated, undergoing a morphological and functional switch. Activation of these cells is not an “all-or-none” process, but rather a continuum characterized by a wide spectrum of molecular and functional phenotypes ranging from the “classical” M1 activated phenotype, with a highly pro-inflammatory profile, to the “alternative” M2 phenotype, associated with a beneficial, less inflammatory, protective profile. The possibility to promote a macrophage protective phenotype has therefore become a therapeutic goal in the treatment of inflammatory conditions, and the identification of factors that control cell activation is currently an area of active research. Most studies in the field so far have been performed using primary mouse macrophages or macrophage cell lines, and a variety of monocyte differentiation protocols and macrophage activation markers are used by different labs. Moreover, the pharmacological control of human macrophage polarized activation has not been extensively explored. A number of natural and synthetic compounds, including chalcones and curcumin, the major active component isolated from the turmeric plant Curcuma longa, have been shown to induce effects on macrophage function (including antioxidant, anti-microbial, anti-carcinogenic and anti-inflammatory action) through multiple pharmacological mechanisms, including interference with TLR4 signaling. On these grounds, the specific aims of the present thesis were: a) to test cell models and differentiation protocols other than spontaneous blood-derived macrophage differentiation, namely the THP-1 cell line and CSF-1-driven differentiation, respectively; b) to profile the cytokine pattern into the culture medium, and c) to determine the modulation of phenotypic markers by pharmacological agents, namely curcumin derivatives known to suppress microglial activation through reduced production and release of pro-inflammatory mediators, as well as the underlying mechanisms of action.
Macrophages were differentiated from human PBMCs isolated by density gradient centrifugation, and cultured in RPMI 1640 medium with 10% FBS with CSF-1 for 6 days to obtain resting macrophages (M0). Classical (M1) and alternative (M2) phenotypes were generated using specific cytokines (0.1-1 μg/ml LPS or 20 ng/ml IL-4 plus 5 ng/ml IL-13, respectively) in the presence or absence of curcumin analogues or dexamethasone used a reference compound. Macrophage phenotypes were determined by flow cytometry using fluorocrome-labeled antibodies. Gene expression was analysed using qRT-PCR. The composition of macrophage conditioned media (MCM) was assessed with the Luminex technology. Curcumin analogues were kindly provided by Dr. Federica Belluti (University of Bologna).
When M2 polarization was induced with IL-4/IL-13 for 24h, we observed increased expression of M2 markers compared with M0. In terms of gene expression analysis of the CSF-1 driven macrophages, as expected, M1-polarized macrophages after 6 or 48 h showed higher mRNA levels of TNF-α and IL-1β compared with M0. The increase in mRNA was more marked after 48 h for all genes except TNF-α, which rose more sharply after 6 h. The anti-inflammatory cytokine IL-10 mRNA was unexpectedly more abundant in M1- than in M2-polarized macrophages, and peaked after 6 h. Compared with M0, M2 MCM showed higher levels of anti-inflammatory cytokines including CCL22 and IL-4. In contrast, M1 MCM was associated with higher levels of IL-1α, IL-1β, IL-6, IL-8, MCP-1, VEGF and TNF-α. Treatment with the curcumin analogue GG9 as well as CLI095, an inhibitor of TLR4 intracellular domain, reversed the LPS-induced up-regulation of CD80+ (M1) cells. A similar effect was maintained with the double positive CD80+/CCR2+ population. Unlike dexamethasone, which increased the percentage of CD163+ (M2) cells, the curcumin analogue GG9 did not affect M2 markers. Treatment with GG9 significantly blocked IL-1β cytokine production at the cell-bound level, in the protein lysate and in the medium. By contrast, curcumin and GG6 did not affect the levels of intra- and extracellular IL-1β. To investigate intracellular signaling pathways involved in cell activation, we performed Western blot analysis of factors involved in the NF-κB pathway. Activation with LPS significantly decreased the relative expression of IκB-α. By contrast, curcumin and GG6 were able to restore IκB-α amounts as did CLI095, whereas no effect was induced by GG9 treatment.
Therefore, M1 and M2 macrophages showed specific profiles of gene expression and surface markers, which were modulated by pharmacological treatment with dexamethasone or a curcumin analogue. Overall, these data suggest that polarized activation protocols may have an impact on the functional status of macrophages and are critical to further investigate pharmacological macrophage targeting
