Functional evaluation of immunoregulatory molecules HLA-G, galectin-1, and IL-10 in people living with HIV.

OBJECTIVES: Investigate polymorphisms and expressions of human leukocyte antigen-G (HLA-G), galectin-1 (Gal-1), and interleukin-10 (IL-10) in people living with HIV (PLHIV) with and without comorbidities to help understanding the mechanisms involved in triggering these disorders in PLHIV and in their prognosis. DESIGN: Here we evaluated the potential correlation between the genetic polymorphism and/or protein levels of HLA-G, Gal-1, and IL-10 with and without comorbidities of PLHIV. METHODS: Two hundred HIV patients under antiretroviral treatment (83 with comorbidities and 117 without comorbidities) and 200 healthy individuals (controls) were genotyped, using PCR, for HLA-G 14-base pair polymorphism located at the 3' untranslated region in exon 8 insertion/insertion (Ins/Ins: low HLA-G expression) or deletion/deletion (Del/Del: high HLA-G expression). Soluble levels of HLA-G (sHLA-G), Gal-1, and IL-10 were quantified by enzyme-linked immunosorbet assay. RESULTS: HIV patients without comorbidities exhibited higher frequency of 14-base pair Del/Del genotype than HIV patients with comorbidities. As expected, HIV patients Ins/Ins with and without comorbidities produced less sHLA-G than controls. However, HIV patients Del/Del with comorbidities expressed sHLA-G more than controls and HIV patients Del/Del without comorbidities. Interestingly, patients that showed low levels sHLA-G, and presence of comorbidities, exhibited high Gal-1 serum levels. However, an increase in soluble levels of IL-10 in PLHIV was observed when compared to controls, especially in the PLHIV group without comorbidities suggesting, a protective role of IL-10 in the development of comorbidities. CONCLUSIONS: These data suggested that the high expression of sHLA-G and IL-10 or Gal-1 could be associated and could be associated with the development or not of comorbidities in PLHIV.

Examination of galectin localization using confocal microscopy.

Confocal microscopy provides a unique modality to examine the expression and localization of biomolecules in a variety of settings. Using this technique, an image is acquired from the focal plane of the objective using focused laser light, making it possible to work within the resolution limit of the optical system. In addition, by acquiring multiple images from a variety of focal planes, stacked series of images can provide clear spatial localization of a probed structure or protein. We describe herein the immunofluorescence methods for galectin staining in frozen sections of tissue for galectin localization using confocal microscopy.

Galatrox is a C-type lectin in Bothrops atrox snake venom that selectively binds LacNAc-terminated glycans and can induce acute inflammation.

Previous studies indicate that snake venom contains glycan-binding proteins (GBPs), although the binding specificity and biological activities of many of these GBPs is unclear. Here we report our studies on the glycan binding specificity and activities of galatrox, a Bothrops atrox snake venom-derived GBP. Glycan microarray analysis indicates that galatrox binds most strongly to glycans expressing N-acetyllactosamine (LacNAc), with a significant preference for Galß1-4GlcNAcß over Galß1-3GlcNAcß compounds. Galatrox also bound immobilized laminin, a LacNAc-dense extracellular matrix component, suggesting that this GBP can bind LacNAc-bearing glycoproteins. As several endogenous mammalian GBPs utilize a similar binding LacNAc binding preference to regulate neutrophil and monocyte activity, we hypothesized that galatrox may mediate B. atrox toxicity through regulation of leukocyte activity. Indeed, galatrox bound neutrophils and promoted leukocyte chemotaxis in a carbohydrate-dependent manner. Similarly, galatrox administration into the mouse peritoneal cavity induced significant neutrophil migration and the release of pro-inflammatory cytokines IL-1α and IL-6. Exposure of bone marrow-derived macrophages to galatrox induced generation of pro-inflammatory mediators IL-6, TNF-α, and keratinocyte-derived chemokine. This signaling by galatrox was mediated via its carbohydrate recognition domain by activation of the TLR4-mediated MyD88-dependent signaling pathway. These results indicate that galatrox has pro-inflammatory activity through its interaction with LacNAc-bearing glycans on neutrophils, macrophages and extracellular matrix proteins and induce the release of pro-inflammatory mediators.

Expanding the universe of cytokines and pattern recognition receptors: galectins and glycans in innate immunity.

Effective immunity relies on the recognition of pathogens and tumors by innate immune cells through diverse pattern recognition receptors (PRRs) that lead to initiation of signaling processes and secretion of pro- and anti-inflammatory cytokines. Galectins, a family of endogenous lectins widely expressed in infected and neoplastic tissues have emerged as part of the portfolio of soluble mediators and pattern recognition receptors responsible for eliciting and controlling innate immunity. These highly conserved glycan-binding proteins can control immune cell processes through binding to specific glycan structures on pathogens and tumors or by acting intracellularly via modulation of selective signaling pathways. Recent findings demonstrate that various galectin family members influence the fate and physiology of different innate immune cells including polymorphonuclear neutrophils, mast cells, macrophages, and dendritic cells. Moreover, several pathogens may actually utilize galectins as a mechanism of host invasion. In this review, we aim to highlight and integrate recent discoveries that have led to our current understanding of the role of galectins in host-pathogen interactions and innate immunity. Challenges for the future will embrace the rational manipulation of galectin-glycan interactions to instruct and shape innate immunity during microbial infections, inflammation, and cancer.