Inflammasomes are multiprotein complexes nucleating around an NLR (Nucleotide-binding domain and Leucine-rich Repeat containing protein), which regulate the secretion of the pro-inflammatory interleukin (IL)-1β and IL-18 cytokines. Monocytes and macrophages, the main cells expressing the inflammasome genes, adapt to their surrounding microenvironment by a phenotypic polarization towards a pro-inflammatory M1 phenotype that promotes inflammation or an anti-inflammatory M2 phenotype important for resolution of inflammation. Despite the importance of inflammasomes in health and disease, little is known about inflammasome gene expression in relevant human cells and the impact of monocyte and macrophage polarization in inflammasome gene expression. We examined the expression of several members of the NLR, caspase and cytokine family, and we studied the activation of the well-described NLRP3 inflammasome in an experimental model of polarized human primary monocytes and monocyte-derived macrophages (M1/M2 phenotypes) before and after activation with LPS, a well-characterized microbial pattern used in inflammasome activation studies. Our results show that the differentiation of monocytes to macrophages alters NLR expression. Polarization using IFN-γ (M1 phenotype), induces among the NLRs studied, only the expression of NOD2. One of the key results of our study is that the induction of NLRP3 expression by LPS is inhibited in the presence of IL-4+IL-13 (M2 phenotype) at both mRNA and protein level in monocytes and macrophages. Unlike caspase-3, the expression of inflammasome-related CASP1 (encodes caspase-1) and CASP4 (encodes caspase-4) is up-regulated in M1 but not in M2 cells. Interestingly, the presence of LPS marginally influenced IL18 mRNA expression and secretion, unlike its impact on IL1B. Our data provide the basis for a better understanding of the role of different inflammasomes within a given environment (M1 and M2) in human cells and their impact in the pathophysiology of several important inflammatory disorders.

Circulating serum amyloid A (SAA) is increased in various inflammatory conditions. The human SAA protein family comprises the acute phase SAA1/SAA2, known to activate a large set of innate and adaptive immune cells, and the constitutive SAA4. The liver synthesis of SAA1/SAA2 is well-established but there is still an open debate on extrahepatic SAA expression especially in macrophages. We aimed to investigate the ability of human primary monocytes and monocyte-derived macrophages to express SAA1, SAA2 and SAA4 at both the transcriptional and protein levels, as previous studies almost exclusively dealt with monocytic cell lines. Monocytes and derived macrophages from healthy donors were stimulated under various conditions. In parallel with SAA, pro-inflammatory IL1A, IL1B and IL6 cytokine expression was assessed. While LPS alone was non-effective, a combined LPS/dexamethasone treatment induced SAA1 and to a lesser extent SAA2 transcription in human monocytes and macrophages. In contrast, as expected, pro-inflammatory cytokine expression was strongly induced following stimulation with LPS, an effect which was dampened in the presence of dexamethasone. Furthermore, in monocytes polarized towards a pro-inflammatory M1 phenotype, SAA expression in response to LPS/dexamethasone was potentiated; a result mainly seen for SAA1. However, a major discrepancy was observed between SAA mRNA and intracellular protein levels under the experimental conditions used. Our results demonstrate that human monocytes and macrophages can express SAA genes, mainly SAA1 in response to an inflammatory environment. While SAA is considered as a member of a large cytokine network, its expression in the monocytes-macrophages in response to LPS-dexamethasone is strikingly different from that observed for classic pro-inflammatory cytokines. As monocytes-macrophages are major players in chronic inflammatory diseases, it may be hypothesized that SAA production from macrophages may contribute to the local inflammatory microenvironment, especially when macrophages are compactly organized in granulomas as in sarcoidosis.

Secreted phospholipases A2 (sPLA2s) are present in atherosclerotic plaques and are now considered novel attractive therapeutic targets and potential biomarkers as they contribute to the development of atherosclerosis through lipoprotein‐dependent and independent mechanisms. We have previously shown that hGX‐sPLA2‐phospholipolyzed LDL (LDL‐X) induces proinflammatory responses in human umbilical endothelial cells (HUVECs); here we explore the molecular mechanisms involved. Global transcriptional gene expression profiling of the response of endothelial cells exposed to either LDL or LDL‐X revealed that LDL‐X activates multiple distinct cellular pathways including the unfolded protein response (UPR). Mechanistic insight showed that LDL‐X activates UPR through calcium depletion of intracellular stores, which in turn disturbs cytoskeleton organization. Treatment of HUVECs and aortic endothelial cells (HAECs) with LDL‐X led to activation of all 3 proximal initiators of UPR: eIF‐2α, IRE1α, and ATF6. In parallel, we observed a sustained phosphorylation of the p38 pathway resulting in the phosphorylation of AP‐1 downstream targets. This was accompanied by significant production of the proinflammatory cytokines IL‐6 and IL‐8. Our study demonstrates that phospholipolyzed LDL uses a range of molecular pathways including UPR to initiate endothelial cell perturbation and thus provides an LDL oxidation‐independent mechanism for the initiation of vascular inflammation in atherosclerosis.—Gora, S., Maouche, S., Atout, R., Wanherdrick, K., Lambeau, G., Cambien, F., Ninio, E., Karabina, S.‐A. Phospholipolyzed LDL induces an inflammatory response in endothelial cells through endoplasmic reticulum stress signaling. FASEB J . 24, 3284–3297 (2010). www.fasebj.org