Oxidized LDL induces alternative macrophage phenotype through activation of CD36 and PAFR

OxLDL is recognized by macrophage scavenger receptors, including CD36; we have recently found that Platelet-Activating Factor Receptor (PAFR) is also involved. Since PAFR in macrophages is associated with suppressor function, we examined the effect of oxLDL on macrophage phenotype. It was found that the presence of oxLDL during macrophage differentiation induced high mRNA levels to IL-10, mannose receptor, PPARγ and arginase-1 and low levels of IL-12 and iNOS. When human THP-1 macrophages were pre-treated with oxLDL then stimulated with LPS, the production of IL-10 and TGF-β significantly increased, whereas that of IL-6 and IL-8 decreased. In murine TG-elicited macrophages, this protocol significantly reduced NO, iNOS and COX2 expression. Thus, oxLDL induced macrophage differentiation and activation towards the alternatively activated M2-phenotype. In murine macrophages, oxLDL induced TGF-β, arginase-1 and IL-10 mRNA expression, which were significantly reduced by pre-treatment with PAFR antagonists (WEB and CV) or with antibodies to CD36. The mRNA expression of IL-12, RANTES and CXCL2 were not affected. We showed that this profile of macrophage activation is dependent on the engagement of both CD36 and PAFR. We conclude that oxLDL induces alternative macrophage activation by mechanisms involving CD36 and PAFR

Boosting Adaptive Immunity: A New Role for PAFR Antagonists.

We have previously shown that the Platelet-Activating Factor Receptor (PAFR) engagement in murine macrophages and dendritic cells (DCs) promotes a tolerogenic phenotype reversed by PAFR-antagonists treatment in vitro. Here, we investigated whether a PAFR antagonist would modulate the immune response in vivo. Mice were subcutaneously injected with OVA or OVA with PAFR-antagonist WEB2170 on days 0 and 7. On day 14, OVA-specific IgG2a and IgG1 were measured in the serum. The presence of WEB2170 during immunization significantly increased IgG2a without affecting IgG1 levels. When WEB2170 was added to OVA in complete Freund's adjuvant, enhanced IgG2a but not IgG1 production was also observed, and CD4+ FoxP3+ T cell frequency in the spleen was reduced compared to mice immunized without the antagonist. Similar results were observed in PAFR-deficient mice, along with increased Tbet mRNA expression in the spleen. Additionally, bone marrow-derived DCs loaded with OVA were transferred into naïve mice and their splenocytes were co-cultured with fresh OVA-loaded DCs. CD4(+) T cell proliferation was higher in the group transferred with DCs treated with the PAFR-antagonist. We propose that the activation of PAFR by ligands present in the site of immunization is able to fine-tune the adaptive immune response

Importance of EMT Factor ZEB1 in cDC1 "MutuDC Line" Mediated Induction of Th1 Immune Response.

The role of Epithelial to Mesenchymal Transition (EMT) factor Zeb1 is well defined in metastasis and cancer progression but it's importance in dendritic cells (DCs) is unexplored until now. For the first time we report here that Zeb1 controls immunogenic responses of CD8α <sup>+</sup> conventional Type-I (cDC1) DCs. We found that ZEB1 expression increases significantly after TLR9 stimulation and its depletion impairs activation, co-stimulation and secretion of important cytokines like IL-6, IL-10 and IL-12 in cDC1 MutuDC line. We further confirmed our findings in primary cDC1 DCs derived from bone marrow. Co-culture of these Zeb1 knock down (KD) DCs with OT-II CD4 <sup>+</sup> T helper cells skewed their differentiation toward Th2 subtype. Moreover, adoptive transfer of activated Zeb1 KD DCs cleared intestinal worms in helminth infected mice by increasing Th2 responses in vivo. Integrative genomic analysis showed Zeb1 as an activator of immune response genes in cDC1 MutuDCs as compared to other pathway genes. In addition, differentially regulated genes in Zeb1 KD RNA-seq showed significant enrichment of Th2 activation pathways supporting our in vitro findings. Mechanistically, we showed that decreased IL-12 secreted by Zeb1 KD DCs is the plausible mechanism for increased Th2 differentiation. Collectively our data demonstrate that Zeb1 could be targeted in DCs to modulate T-cell mediated adaptive immune responses

Leukotriene B₄ modulation of murine dendritic cells affects adaptive immunity.

Dendritic cells (DCs) link innate and adaptive immunity. The microenvironment generated during the innate immunity affects DCs and the type of adaptive immunity generated. Lipid mediators are released early in inflammation and could modify the functional state of DCs. Leukotriene B <sub>4</sub> (LTB <sub>4</sub> ) has a wide range of effects on macrophages and in the present study we investigated if it also affects DCs. Murine bone marrow-derived DCs were employed and it was found that stimulation of DCs with LTB4 (10 nM) increased the gene expression of the high affinity receptor BLT-1 but not of BLT-2. It also increased the co-stimulatory molecule CD86 expression but did not affect CD80 and CD40. LTB <sub>4</sub> -stimulated DCs acquired the capacity to present antigen to T lymphocytes, evidenced by antigen-specific proliferation of CD4 <sup>+</sup> lymphocytes in co-cultures of ovalbumin-loaded DCs with DO11.10 splenocytes. LTB <sub>4</sub> -stimulated DCs induced Treg proliferation and increased Th2 cytokine IL-13 in the co-cultures. Expression of transcription factor genes, Gata3 and Foxp3 (Th2 and Treg, respectively) were also found increased. However, the expression of Th1 transcription factor (Tbet) and Th17 (RorγT) were not affected. These results indicate that LTB <sub>4</sub> affects DCs and modulates the type of adaptive immune response

Apolipoprotein A-I and the molecular variant apoA-I Milano : evaluation of the antiatherogenic effects in a knock-in mouse model

No evidence of premature vascular disease is found in apolipoprotein A-I(Milano) (apoA-I(M)) human carriers, despite very low high density lipoprotein (HDL) cholesterol levels. Whether apoA-I(M) may impart a "gain of function" in atherosclerosis protection compared to wild-type apoA-I is hotly debated. To address this question, knock-in mice expressing human apoA-I or apoA-I(M) were crossed with atherosclerosis-susceptible mice expressing the human apoB/A-II transgene (h-B/A-II/A-I(Hu/Hu) and h-B/A-II/A-I(M)(Hu/Hu)). On a chow diet, h-B/A-II/A-I(M)(Hu/Hu) mice were characterized by low HDL cholesterol levels compared to h-B/A-II/A-I(Hu/Hu) mice (35.65+/-8.00 mg/dl versus 58.09+/-13.50mg/dl, respectively; p<0.005). Gender differences in response to high fat diet were observed in both h-B/A-II/A-I(M)(Hu/Hu) and h-B/A-II/A-I(Hu/Hu) lines. h-B/A-II/A-I(M)(Hu/Hu) females had higher total cholesterol levels compared to h-B/A-II/A-I(Hu/Hu) females (895.08+/-183.07 mg/dl versus 544.43+/-116.42 mg/dl; p<0.05) and developed larger atherosclerotic lesions (148,260+/-78,924 microm(2) versus 54,132+/-43,204 microm(2), respectively; p<0.05). On the contrary, no difference in mean lesion area was found between h-B/A-II/A-I(M)(Hu/Hu) and h-B/A-II/A-I(Hu/Hu) males (19,779+/-6,098 microm(2) versus 15,706+/-13,095 microm(2); p=0.685). Our data suggest that, in the atherosclerosis-susceptible human apoB/A-II mouse model, expression of the human apoA-I(M) gene does not have protective advantage over that of the apoA-I gene