Abbreviations: 3-MA, 3-methyladenine; AKT, v-akt murine thymoma viral oncogene homolog; ALB, albumin; ATG7, autophagyrelated 7; CD, cluster of differentiation; CD5L, CD5 molecule-like; FCS, fetal calf serum; FSL1, pam2CGDPKHPKSF; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL, interleukin; MAP1LC3A/B (LC3), microtubule-associated protein 1 light chain 3 a/b; LPS, lipopolysaccharide; LTA, lipoteichoic acid; MK, macrophages; MAPK, mitogen-activated protein kinase; moAb, monoclonal antibody;; NFKB, nuclear factor of kappa light polypeptide gene enhancer in B-cells; oxLDL, oxidized low-density lipoprotein; PB monocytes, peripheral blood monocytes; PBS, phosphate-buffered saline; PI3K, phosphoinositide 3-kinase; PIK3C3, phosphatidylinositol 3-kinase, catalytic subunit type 3; poAb, polyclonal antibody; PMA, phorbol 12-myristate 13-acetate; PtdIns3K, phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol 3-phosphate; r-HsCD5L, recombinant human (Homo sapiens) CD5L; RELA, v-rel avian reticuloendotheliosis viral oncogene homolog A; siRNA, short interference RNA; SRCR, scavenger receptor cysteine-rich; TBS, tris-buffered saline; TLRs, toll-like receptors; TNF, tumor necrosis factor.CD5L (CD5 molecule-like) is a secreted glycoprotein that participates in host response to bacterial infection. CD5L influences the monocyte inflammatory response to the bacterial surface molecules lipopolysaccharide (LPS) and lipoteichoic acid (LTA) by inhibiting TNF secretion. Here we studied the intracellular events that lead to macrophage TNF inhibition by human CD5L. To accomplish this goal, we performed functional analyses with human monocytic THP1 macrophages, as well as with peripheral blood monocytes. Inhibition of phosphatidylinositol 3-kinase (PtdIns3K) reversed the inhibitory effect of CD5L on TNF secretion. Among the various PtdIns3K isoforms, our results indicated that CD5L activates PtdIns3K (whose catalytic subunit is termed PIK3C3), a key modulator involved in autophagy. Further analysis revealed a concomitant enhancement of autophagy markers such as cellular LC3-II content, increased LC3 puncta, as well as LC3-LysoTracker Red colocalization. Moreover, electron microscopy showed an increased presence of cytosolic autophagosomes in THP1 macrophages overexpressing CD5L. Besides preventing TNF secretion, CD5L also inhibited IL1B and enhanced IL10 secretion. This macrophage anti-inflammatory pattern of CD5L was reverted upon silencing of autophagy protein ATG7 by siRNA transfection. Additional siRNA experiments in THP1 macrophages indicated that the induction of autophagy mechanisms by CD5L was achieved through cell-surface scavenger receptor CD36, a multiligand receptor expressed in a wide variety of cell types. Our data represent the first evidence that CD36 is involved in autophagy and point to a significant contribution of the CD5L-CD36 axis to the induction of macrophage autophagy.
CD5L (CD5 molecule-like) is a secreted glycoprotein that controls key mechanisms in inflammatory responses, with involvement in processes such as infection, atherosclerosis, and cancer. In macrophages, CD5L promotes an anti-inflammatory cytokine profile in response to TLR activation. In the present study, we questioned whether CD5L is able to influence human macrophage plasticity, and drive its polarization toward any specific phenotype. We compared CD5L-induced phenotypic and functional changes to those caused by IFN/LPS, IL4, and IL10 in human monocytes. Phenotypic markers were quantified by RT-qPCR and flow cytometry, and a mathematical algorithm was built for their analysis. Moreover, we compared ROS production, phagocytic capacity, and inflammatory responses to LPS. CD5L drove cells toward a polarization similar to that induced by IL10. Furthermore, IL10- and CD5L-treated macrophages showed increased LC3-II content and colocalization with acidic compartments, thereby pointing to the enhancement of autophagy-dependent processes. Accordingly, siRNA targeting ATG7 in THP1 cells blocked CD5L-induced CD163 and Mer tyrosine kinase mRNA and efferocytosis. In these cells, gene expression profiling and validation indicated the upregulation of the transcription factor ID3 by CD5L through ATG7. In agreement, ID3 silencing reversed polarization by CD5L. Our data point to a significant contribution of CD5L-mediated autophagy to the induction of ID3 and provide the first evidence that CD5L drives macrophage polarization.
AIM is expressed by macrophages in response to agonists of the nuclear receptors LXR/RXR. In mice, it acts as an atherogenic factor by protecting macrophages from the apoptotic effects of oxidized lipids. In humans, it is detected in atherosclerotic lesions, but no role related to atherosclerosis has been reported. This study aimed to investigate whether the role of hAIM extends beyond inhibiting oxidized lipid-induced apoptosis. To accomplish this goal, functional analysis with human monocytic THP1 cells and macrophages differentiated from peripheral blood monocytes were performed. It was found that hAIM reduced oxLDL-induced macrophage apoptosis and increased macrophage adhesion to endothelial ICAM-1 by enhancing LFA-1 expression. Furthermore, hAIM increased foam cell formation, as shown by Oil Red O and Nile Red staining, as well as quantification of cholesterol content. This was not a result of decreased reverse cholesterol transport, as hAIM did not affect the efflux significantly from [(3)H] Cholesterol-laden macrophages driven by plasma, apoA-I, or HDL2 acceptors. Rather, flow cytometry studies indicated that hAIM increased macrophage endocytosis of fluorescent oxLDL, which correlated with an increase in the expression of the oxLDLR CD36. Moreover, hAIM bound to oxLDL in ELISA and enhanced the capacity of HEK-293 cells expressing CD36 to endocytose oxLDL, as studied using immunofluorescence microscopy, suggesting that hAIM serves to facilitate CD36-mediated uptake of oxLDL. Our data represent the first evidence that hAIM is involved in macrophage survival, adhesion, and foam cell formation and suggest a significant contribution to atherosclerosis-related mechanisms in the macrophage.
Apoptosis inhibitor of macrophages (AIM), a scavenger protein secreted by tissue macrophages, is transcriptionally regulated by the nuclear receptor Liver X Receptor (LXR) and Retinoid X Receptor (RXR) heterodimer. Given that LXR exerts a protective immune response against M. tuberculosis, here we analyzed whether AIM is involved in this response. In an experimental murine model of tuberculosis, AIM serum levels peaked dramatically early after infection with M. tuberculosis, providing an in vivo biological link to the disease. We therefore studied the participation of AIM in macrophage response to M. tuberculosis in vitro. For this purpose, we used the H37Rv strain to infect THP-1 macrophages transfected to stably express AIM, thereby increasing infected macrophage survival. Furthermore, the expression of this protein enlarged foam cell formation by enhancing intracellular lipid content. Phagocytosis assays with FITC-labeled M. tuberculosis bacilli indicated that this protein was not involved in bacterial uptake; however, AIM expression decreased the number of intracellular cfus by up to 70% in bacterial killing assays, suggesting that AIM enhances macrophage mycobactericidal activity. Accordingly, M. tuberculosis-infected AIM-expressing cells upregulated the production of reactive oxygen species. Moreover, real-time PCR analysis showed increased mRNA levels of the antimicrobial peptides cathelicidin and defensin 4B. These increases were concomitant with greater cellular concentrations of the autophagy-related molecules Beclin 1 and LC3II, as well as enhanced acidification of mycobacterial phagosomes and LC3 co-localization. In summary, our data support the notion that AIM contributes to key macrophage responses to M. tuberculosis.
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