Recently our group described that Nattectin, a C-type lectin of the venom of Thalassophryne nattereri shows a potent pro-inflammatory capacity. Here, we demonstrated that Nattectin is able to induce M1 macrophage marker iNOS, and up-regulate the expression of MHC class II, CD80, CD86 and CD40 molecules. The increase in MHC class II and CD49a integrin expression with MMP-9 production and endocytic capacity depend on lectin function of Nattectin. Moreover, the polarization of peritoneal and bone marrow-derived macrophages induced by Nattectin to M1 profile is dependent on Th1 cytokines (IL-12 and IFN-γ), and negatively regulated by Th2 cytokines (IL-4, IL-10 and IL-13). Also we reveal that IL-4 play a dual role in this polarization: a regular action of IL-4 was seen in the negative regulation of the CD40 expression, but an unexpected positive regulation was seen in the expression of CCR7 and MHC class II. Finally, our in vivo studies showed that the influx of neutrophils and small peritoneal macrophage--F4/80(low)MHCII(hi) induced by Nattectin is totally dependent on IL-4 and IFN-γ cytokines. Furthermore, the induction of IL-6 release is negatively regulated by IL-4 and positively regulated by IL-12 and IFN-γ. Together, the results allowed us to expand the knowledge about the regulation of macrophage activation, as well as confirmed the ability of Nattectin, a fish C-type lectin, as an important immunomodulatory agent.

Platelet-activating factor receptor (PAFR) is a G protein-coupled receptor (GPCR) implicated in many diseases. Toll-like receptors (TLRs) play a critical role in shaping innate and adaptive immune responses. In this study, we investigated whether PAFR signaling changes the macrophages responsiveness to agonists of TLR2 (Pam3Cys), TLR4 (LPS), and TLR3 agonist Poly(I:C). Exogenous PAF inhibited the production of pro-inflammatory cytokines (IL-12p40, IL-6, and TNF-α) and increased anti-inflammatory IL-10 in macrophages challenged with Pam3Cys and LPS, but not with Poly (I:C). PAF did not affect mRNA expression of MyD88, suggesting that PAF acts downstream the adaptor. PAF inhibited LPS-induced phosphorylation of NF-κB p65 and increased NF-κB p105 phosphorylation, which is processed in the proteasome to generate p50 subunit. The PAF potentiation of IL-10 production was dependent on proteasome processing but independent of NF-κB transactivation domain. Inhibition of p50 abolished the PAF-induced IL-10 production. These findings indicate that the impaired transcriptional activity of the p65 subunit and the enhanced p105 phosphorylation induced by PAF are responsible for down regulation of pro-inflammatory cytokines and up regulation of IL-10, respectively, in LPS-challenged macrophages. Together, our data unveil a heretofore unrecognized role for PAFR in modulating activation of NF-κB in macrophages.

PAFR in adipose tissue macrophages is associated with anti-inflammatory phenotype and metabolic homoeostasis. Clinical Science, 130(8), pp. 601-612. (doi:10.1042/cs20150538) This is the author's final accepted version.There may be differences between this version and the published version. You are advised to consult the publisher's version if you wish to cite from it.http://eprints.gla.ac.uk/120744/ cells. Blood monocytes of PAFRKO mice also exhibited a pro-inflammatory phenotype (increased frequency of Lys6C+.cells) and PAFR-ligands were detected in the serum of both PAFRKO and WT. Regarding metabolic parameters, compared to WT, PAFRKO mice had: i) higher weight gain andserum glucose concentration levels; ii) decreased insulin-stimulated glucose disappearance; iii) insulin resistance in the liver; iv) increased expression of Ldlr in the liver. In mice fed HFD, some of these changes were potentiated, particularly in the liver. Thus, it seems that endogenous ligands of PAFR are responsible for maintaining the anti-inflammatory profile of blood monocytes and adipose tissue macrophages under physiological conditions. In the absence of PAFR signaling, monocytes and macrophages acquire pro-inflammatory phenotype, resulting in adipose tissue inflammation and metabolic dysfunction. SummaryWe found an essential role of PAFR in adipose tissue macrophages. The PAFRdeficiency leads to infiltration of pro-inflammatory macrophages in the adipose tissue resulting in weight gain, reduced glucose tolerance, hepatic insulin resistance, followed by hepatic steatosis.Short Title: PAFR is associated with anti-inflammatory macrophages and glucose metabolism

Chronic granulomatous disease (CGD) is an innate immune deficiency of phagocytic cells caused by mutations that affect components of the NADPH oxidase system, with resulting impairment in reactive oxygen species production. Patients with CGD are susceptible to recurrent infections and hyperinflammatory responses. Mutations in CYBB lead to the X-linked form of CGD and are responsible for ~ 70% of cases. In this study, we report the case of a 2.5-year-old male patient with recurrent pneumonia and Bacillus Calmette-Guérin infection (BCGitis). As his first clinical manifestation, he presented with bullous impetigo at 18 days of age, which was followed by recurrent pneumonia and regional BCGitis. Genetic analysis revealed a de novo mutation in exon 5 of the CYBB gene: a single-nucleotide substitution, c.376T > C, leading to a C126R change.

Toll-like receptors (TLRs) and platelet-activating factor receptor (PAF-R) are highly expressed in macrophages, and are important components of microbicidal and homeostatics processes. There is evidence that PAF-R activation affects the signaling elicited by other receptors. Thus, the aim of this project was investigate the effect of PAF-R activation in the responsiveness of macrophages to TLR agonists MyD88dependent (TLR2 and TLR4) and independent (TLR3). These studies were performed in elicited peritoneal macrophages stimulated concomitantly with cPAF and different TLR agonist. Our results show that PAF addition inhibited the production of proinflammatory cytokines (IL-12p40, IL-6 and TNF-α) and increased anti-inflammatory IL-10 in macrophages stimulated with Pam3Cys and LPS, but not with Poly(I:C). Additionally, cPAF reduced COX-2 expression and PGE 2 production induced by Pam3Cys and LPS, but did not affect the iNOS expression and nitrite formation induced by LPS. Thus, cPAF induced a regulatory phenotype in macrophages activated with TLR2 and TLR4 agonists. This effect of PAF is not due to its action on MyD88 adaptor molecule. When we investigated the PAF effect on signaling program elicited by LPS, we found that PAF inhibited the phosphorylation of NF-κB p65 subunit, and increased phosphorylation of NF-κB p105, the precursor of p50 subunit, which induces IL-10 gene expression. Additionnally, the pretreatment of macrophages with p105/p50 peptide inhibitor reverted the potentiating effect of PAF on IL-10 production. These findings suggest that the decreased transcriptional activity of p65 subunit and enhanced p105 phosphorylation induced by PAF are responsible for the downregulation of pro-inflammatory cytokines and up-regulation of IL-10, respectively, in LPS-stimulated macropages. These results unveil a heretofore unrecognized role for PAF in MyD88-dependent NF-κB in macrophages.

Chronic granulomatous disease (CGD) is an inherited, genetically heterogeneous disease characterized by defective phagocytic cell microbicidal function, leading to increased susceptibility to bacterial and fungal infections. CGD is caused by mutations in components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, which is responsible for reactive oxygen species production during phagocytosis. Mutations in the neutrophil cytosolic factor 2 (NCF2) gene account for <5% of all cases. Here, we report a case of a 2-year-old female with persistent recurrent pneumopathy, even under trimethoprim-sulfamethoxazole (TMP-SMX) and itraconazole prophylaxis combined with IFNγ treatment. Genetic analysis revealed a novel homozygous mutation in NCF2, sequence depletion in a splicing region (c.256_257+2delAAGT NM_000433), leading to a K86Ifs*2 residue change in the p67−phox protein.

Nattectin is a 15 kDa toxin isolated from Thalassophryne nattereri fish venom that has homology with C-type lectin. The aim of this study was to evaluate the Nattectin ability to activate macrophage functions and the influence of Th1/Th2 cytokines in polarized activation of macrophages induced by Nattectin. Our results show that Nattectin was able to induce F4/80 pos CD11b pos CD14 pos Ly6C low macrophage derived from bone marrow activation through CD40, CD80 and CD86 costimulatory molecules and MHC class II upregulation, and also an increase in integrins expression and endocytic capacity. It was found a positive dependence of Nattectin binding to glycosylated structures expressed in macrophages for induction of MHC class II, CD49a integrin expression and an increase in endocytic capacity. The expression of costimulatory molecules in macrophages was dependent the p38 MAPK signaling pathway and primarily PI3K signaling, while endocytic capacity and MMP-9 production was positively regulated through ERK1/2 signaling. We also demonstrated that phenotypic modifications in peritoneal and bone marrow-derived macropahges induced by Nattectin in C57BL/6 mice are consistent with classical (M1) activation of macrophages dependent on IL-12 and IFN-γ cytokines, and negatively regulated by IL-4, IL-10 and IL-13. Finally, it was demonstrated an adjuvant role of IL-4 with IFN-γ in mobilization of SPMs (Small Peritoneal Macrophages) to peritoneal cavity and also in activation (expression of MHC class II and CCR7) of bone marrow-derived macrophages. Thus, Nattectin can be considered an important immunomodulatory agent able to modulate macrophage functions, useful in vaccines development and with therapeutic applications.

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