CD5L promotes M2 macrophage polarization through autophagy-mediated upregulation of ID3

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.Peer ReviewedPostprint (published version

Paper de la proteïna scavenger AIM en l’activació de macròfags i desenvolupament de cèl·lules escumoses

[cat] La proteïna secretada Apoptosis inhibitor expressed by macrophages (AIM) és expressada per macròfags tissulars en condicions d’inflamació. És una glicoproteïna que pertany a la família dels Receptors Scavenger Rics en Cisteïnes (SRCR-SF). Mitjançant models murins in vitro i in vivo s’ha observat com l’expressió d’AIM depèn de l’activació de l’heterodímer LXR/RXR i diversos estudis han demostrat com la forma murina AIM (mAIM) evita l’apoptosi de macròfags i altres tipus cel•lulars. En aquest context, estudis amb models murins d’aterosclerosi, deficients en el LDLr, han suggerit que mAIM actua com a factor aterogènic. En aquest sentit s’ha demostrat que AIM juga un paper rellevant en el procés ateroscleròtic murí incrementant l’apoptosi del macròfag, però es desconeix la seva implicació en l’aterosclerosi humana. A més, resultats recents la descriuen com un possible marcador de sèrum de condicions d’inflamació. La present tesi té com a objectiu principal aprofundir en el coneixement del paper de la proteïna humana AIM en els processos moleculars implicats en l’aterosclerosi. En la tesi presentada s’observa com pacients afectats per PAD tenen menors nivells de hAIM en sèrum que pacients sans. Tot i que aquestes diferències són estadísticament significatives, no són suficients per utilitzar els nivells de hAIM en sèrum com a biomarcador de la patologia, a no ser que es combini amb altres factors analítics. Aquests resultats es correlacionen amb els resultats obtinguts amb els macròfags cultivats in vitro, on la presència de la proteïna hAIM disminueix en el sobrenedat dels macròfags incubats amb d‘OxLDL mentre que augmenta en els llisats cel•lulars, suggerint el possible segrest de hAIM pel macròfag en presència d‘OxLDL. Per contra, en sèrum de models murins d’aterosclerosi els nivells de mAIM són majors que en el ratolins control, la qual cosa suggereix diferències en la regulació de la forma murina d’AIM en sèrum respecte la seva homòloga humana (hAIM). A més s’observa que la regulació del RNAm de hAIM augmenta amb l’activació dels receptors nuclears LXR/RXR, al igual que la seva homologa murina, mAIM. Mitjançant estudis funcionals es mostra que hAIM manté les característiques antiapoptòtiques de mAIM en front de les LDL modificades. I per primer cop, es suggereix que hAIM augmenta la formació dels macròfags escumosos, com s’observa per tincions amb Oil Red O o Nile Red O i dels resultats de colesterol total o èsters de colesterol. Aquest augment no és degut a la disminució del transport de colesterol cap a l’espai extracel•lular, ja que hAIM no modifica significativament l’eflux dels macròfags cap als acceptors lipídics plasmàtics. De fet, mitjançant estudis de citometria s’observa que hAIM incrementa la captació de les lipoproteïnes OxLDL i AcLDL marcades amb fluorescència, resultats correlacionats amb l’augment de l’expressió dels principals Receptors Scavenger implicats en la captació de LDL modificades (CD36 i SR-A1). En concordança amb aquests resultats, mitjançant assajos d’ELISA es suggereix que hAIM s’uneix a OxLDL i a AcLDL, i a més a més, mitjançant estudis de microscòpia de fluorescència s’observa que hAIM augmenta la capacitat de les cèl•lules HEK-293 transfectades amb CD36 en captar OxLDL i d’aquesta manera es suggereix que hAIM podria facilitar la captació d’OxLDL via CD36. Per altra banda, durant els estudis de participació de hAIM en l’adhesió del macròfag, s’observa que hAIM augmenta l’expressió de LFA-1 i la conseqüent adhesió a la molècula d’adhesió ICAM-1. Els resultats de la tesi doctoral presentada suggereixen que hAIM està involucrada en la supervivència del macròfag, la formació del macròfag escumós i l’adhesió del macròfag, així doncs hAIM podria contribuir significativament en els mecanismes relacionats en l’evolució de l’aterosclerosi.[eng] Apoptosis inhibitor expressed by macrophages (AIM) is expressed by tissue macrophages under inflammatory conditions. 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 to date. This study aimed to investigate the contribution of human AIM (hAIM) to the macrophage events that lead to atherosclerosis. Our data show that serum levels of hAIM in patients with peripheral arterial disease (n=16) decreased with respect to controls (n=35) (median [interquartile range]): 98 [61] vs. 116 [72] ng/ml (p= 0.017). These findings are consistent with reduced hAIM secretion by monocytic THP1 cells and macrophages differentiated from peripheral blood monocytes upon OxLDL treatment. Functional studies with these cell types indicated that hAIM retains the anti-apoptotic effects against modified LDL (mLDL), 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 total and esterified cholesterol content. This was not due to decreased reverse cholesterol transport, because hAIM did not significantly affect the efflux from 3H-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 OxLDL receptor CD36. Moreover, hAIM bound to OxLDL in ELISA assays 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. In conclusion, our data represent the first evidence that macrophage hAIM is involved in in atherosclerosis: macrophage survival, adhesion and foam cell formation, and suggest a significant contribution to atherosclerosis-related mechanisms in the macrophage

CD5L promotes M2 macrophage polarization through autophagy-mediated upregulation of ID3

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.Peer Reviewe

Deletion of Batf3-dependent antigen-presenting cells does not affect atherosclerotic lesion formation in mice

<div><p>Atherosclerosis is the main underlying cause for cardiovascular events such as myocardial infarction and stroke and its development might be influenced by immune cells. Dendritic cells (DCs) bridge innate and adaptive immune responses by presenting antigens to T cells and releasing a variety of cytokines. Several subsets of DCs can be discriminated that engage specific transcriptional pathways for their development. Basic leucine zipper transcription factor ATF-like 3 (Batf3) is required for the development of classical CD8α⁺ and CD103⁺ DCs. By crossing mice deficient in <i>Batf3</i> with atherosclerosis-prone low density lipoprotein receptor (<i>Ldlr</i>^<i>-/-</i><i>)</i>-deficient mice we here aimed to further address the contribution of Batf3-dependent CD8α⁺ and CD103⁺ antigen-presenting cells to atherosclerosis. We demonstrate that deficiency in Batf3 entailed mild effects on the immune response in the spleen but did not alter atherosclerotic lesion formation in the aorta or aortic root, nor affected plaque phenotype in low density lipoprotein receptor-deficient mice fed a high fat diet. We thus provide evidence that Batf3-dependent antigen-presenting cells do not have a prominent role in atherosclerosis.</p></div

CD5L Promotes M2 Macrophage Polarization through Autophagy-Mediated Upregulation of ID3

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

Plaque composition is not altered by <i>Batf3</i> deficiency.

<p>Quantification of the area positive for Mac-2 (a) representative images of immunofluorescence staining are shown; scale bars: 50μm; cell nuclei were counterstained with DAPI (blue), α-smooth muscle actin (b), Sirius-red (c), and of the necrotic core (d) in the aortic root from <i>Ldlr</i>^-/- (n = 12) and <i>Ldlr</i>^-/-<i>Batf3</i>^-/- mice (n = 10) fed with 8 weeks of HFD. Data ara presented as mean ± SEM; ns, non significant.</p

T cell activation is impaired in the spleen of <i>Batf3</i>^<i>-/-</i> mice after 8 weeks of HFD.

<p>Flow cytometric analyses of spleen cells obtained from atherosclerotic <i>Ldlr</i>^-/- (n = 12) and <i>Ldlr</i>^-/-<i>Batf3</i>^-/- mice (n = 10) fed a HFD for 8 weeks. (a) Frequencies of activated CD44^highCD62L^low CD4⁺ and (b) CD8⁺ T cells (representative dot plots are shown; values indicate gated events among CD4⁺ T cells). (c) Frequencies of FoxP3⁺CD25⁺CD4⁺ Tregs, (d) IFNγ⁺CD4⁺ T cells (representative dot plots are shown, values indicate gated events among CD4⁺ T cells), (e) IL-17a⁺CD4⁺ T cells and (f) IFNγ⁺CD8⁺ T cells. Data ara presented as mean ± SEM; *p<0.5;,**p<0.01; ns, non significant.</p

Immune responses are mildy alter in the spleen of <i>Batf3</i>-deficient mice after 8 weeks of HFD.

<p>(a-d) Flow cytometric analyses of Ly6G⁺ neutrophils (a), Ly6C^high and Ly6C^low monocytes (b), and of CD3⁺ T cells (c) among CD45⁺ leukocytes, and of frequencies of CD4⁺ and CD8⁺ T cells among CD3⁺ T cells (d) in spleens from atherosclerotic <i>Ldlr</i>^-/- (n = 12) and <i>Ldlr</i>^-/-<i>Batf3</i>^-/- mice (n = 10) fed a HFD for 8 weeks. Data ara presented as mean ± SEM; **p<0.01; ***p<0.001; ns, non significant.</p

Lipid levels of <i>Ldlr</i>^<i>-/-</i> and <i>Ldlr</i>^<i>-/-</i><i>Batf3</i>^<i>-/-</i> mice fed a high fat diet for 8 weeks.

<p>Lipid levels of <i>Ldlr</i>^<i>-/-</i> and <i>Ldlr</i>^<i>-/-</i><i>Batf3</i>^<i>-/-</i> mice fed a high fat diet for 8 weeks.</p