Differential lipid metabolism in monocytes and macrophages: influence of cholesterol loading.

The influence of the hypercholesterolemia associated with atherosclerosis on monocytes is poorly understood. Monocytes are exposed to high concentrations of lipids, particularly cholesterol and lysophosphatidylcholine (lyso-PC). Indeed, in line with recent reports, we found that monocytes accumulate cholesteryl esters (CEs) in hypercholesterolemic mice, demonstrating the need for studies that analyze the effects of lipid accumulation on monocytes. Here we analyze the effects of cholesterol and lyso-PC loading in human monocytes and macrophages. We found that cholesterol acyltransferase and CE hydrolase activities are lower in monocytes. Monocytes also showed a different expression profile of cholesterol influx and efflux genes in response to lipid loading and a different pattern of lyso-PC metabolism. In monocytes, increased levels of CE slowed the conversion of lyso-PC into PC. Interestingly, although macrophages accumulated glycerophosphocholine, phosphocholine was the main water-soluble choline metabolite being generated in monocytes, suggesting a role for mono- and diacylglycerol in the chemoattractability of these cells. In summary, monocytes and macrophages show significant differences in lipid metabolism and gene expression profiles in response to lipid loading. These findings provide new insights into the mechanisms of atherosclerosis and suggest potentials for targeting monocyte chemotactic properties not only in atherosclerosis but also in other diseases.

NFκB2/p100 is a key factor for endotoxin tolerance in human monocytes: a demonstration using primary human monocytes from patients with sepsis.

Endotoxin tolerance (ET) is a state of reduced responsiveness to endotoxin stimulation after a primary bacterial insult. This phenomenon has been described in several pathologies, including sepsis, in which an endotoxin challenge results in reduced cytokine production. In this study, we show that the NFκ L chain enhancer of activated B cells 2 (NFκB2)/p100 was overexpressed and accumulated in a well-established in vitro human monocyte model of ET. The p100 accumulation in these cells inversely correlated with the inflammatory response after LPS stimulation. Knocking down NFκB2/p100 using small interfering RNA in human monocytes further indicated that p100 expression is a crucial factor in the progression of ET. The monocytes derived from patients with sepsis had high levels of p100, and a downregulation of NFκB2/p100 in these septic monocytes reversed their ET status.

CD16 regulates TRIF-dependent TLR4 response in human monocytes and their subsets.

Blood monocytes recognize Gram-negative bacteria through the TLR4, which signal via MyD88- and TRIF-dependent pathway to trigger an immune-inflammatory response. However, a dysregulated inflammatory response by these cells often leads to severe pathologies such as sepsis. We investigated the role of CD16 in the regulation of human monocyte response to Gram-negative endotoxin and sepsis. Blood monocytes from sepsis patients demonstrated an upregulation of several TRIF-dependent genes as well as a selective expansion of CD16-expressing (CD16(+)) monocytes. Gene expression and biochemical studies revealed CD16 to regulate the TRIF-dependent TLR4 pathway in monocytes by activating Syk, IFN regulatory factor 3, and STAT1, which resulted in enhanced expression of IFNB, CCL5, and CXCL10. CD16 also upregulated the expression of IL-1R-associated kinase M and IL-1 receptor antagonist, which are negative regulators of the MyD88-dependent pathway. CD16 overexpression or small interfering RNA knockdown in monocytes confirmed the above findings. Interestingly, these results were mirrored in the CD16(+) monocyte subset isolated from sepsis patients, providing an in vivo confirmation to our findings. Collectively, the results from the current study demonstrate CD16 as a key regulator of the TRIF-dependent TLR4 pathway in human monocytes and their CD16-expressing subset, with implications in sepsis.

Impaired antigen presentation and potent phagocytic activity identifying tumor-tolerant human monocytes.

Monocyte exposure to tumor cells induces a transient state in which these cells are refractory to further exposure to cancer. This phenomenon, termed "tumor tolerance", is characterized by a decreased production of proinflammatory cytokines in response to tumors. In the past, we found that this effect comprises IRAK-M up regulation and TLR4 and CD44 activation. Herein we have established a human model of tumor tolerance and have observed a marked down-regulation of MHCII molecules as well as the MHCII master regulator, CIITA, in monocytes/macrophages. These cells combine an impaired capability for antigen presentation with potent phagocytic activity and exhibit an M2-like phenotype. In addition circulating monocytes isolated from Chronic Lymphocytic Leukemia patients exhibited the same profile as tumor tolerant cells after tumor ex vivo exposition.

Potent phagocytic activity with impaired antigen presentation identifying lipopolysaccharide-tolerant human monocytes: demonstration in isolated monocytes from cystic fibrosis patients.

Monocyte exposure to LPS induces a transient state in which these cells are refractory to further endotoxin stimulation. This phenomenon, termed endotoxin tolerance (ET), is characterized by a decreased production of cytokines in response to the proinflammatory stimulus. We have established a robust model of ET and have determined the time frame and features of LPS unresponsiveness in cultured human monocytes. A large number of genes transcribed in tolerant monocytes were classified as either "tolerizable" or "nontolerizable" depending on their expression levels during the ET phase. Tolerant monocytes exhibit rapid IL-1R-associated kinase-M (IRAK-M) overexpression, high levels of triggering receptor expressed on myeloid cells-1 (TREM-1) and CD64, and a marked down-regulation of MHC molecules and NF-kappaB2. These cells combine potent phagocytic activity with impaired capability for Ag presentation. We also show that circulating monocytes isolated from cystic fibrosis patients share all the determinants that characterize cells locked in an ET state. These findings identify a new mechanism that contributes to impaired inflammation in cystic fibrosis patients despite a high frequency of infections. Our results indicate that a tolerant phenotype interferes with timing, efficiency, and outcome of the innate immune responses against bacterial infections.