Siglecs Modulate Activities of Immune Cells Through Positive and Negative Regulation of ROS Generation.

Reactive oxygen species (ROS) are a group of oxygen-containing highly-reactive molecules produced from oxidative metabolic processes or in response to intracellular signals like cytokines and external stimuli like pathogen attack. They regulate a range of physiological processes and are involved in innate immune responses against infectious agents. Deregulation of ROS contributes to a plethora of disease conditions. Sialic acids are carbohydrates, present on cell surfaces or soluble proteins. Sialic acid-binding immunoglobulin-like lectins (Siglecs) recognize and bind to sialic acids. These are widely expressed on various types of immune cells. Siglecs modulate immune activation and can promote or inhibit ROS generation under different contexts. Siglecs promote ROS-dependent cell death in neutrophils and eosinophils while limiting oxidative stress associated with chronic obstructive pulmonary disease (COPD), sickle cell disease (SCD), coronavirus disease-2019 (COVID-19), etc. This review distinguishes itself in summarizing the current understanding of the role of Siglecs in moderating ROS production and their distinct effect on different immune cells; that ultimately determine the cellular response and the disease outcome. This is an important field of investigation having scope for both expansion and medical importance.

Interplay Between Sialic Acids, Siglec-E, and Neu1 Regulates MyD88- and TRIF-Dependent Pathways for TLR4-Activation During Leishmania donovani Infection.

TLR4 activates two distinct signaling pathways involving adaptors MyD88 and TRIF to produce proinflammatory cytokines and type-I interferon respectively. How Leishmania donovani suppresses these pathways is not well studied. We earlier reported, TLR4 is hypersialylated due to reduced membrane-bound neuraminidase (Neu1) on infected-macrophages. We hypothesized that such enhanced sialoglycoconjugates on host cells may modulate the interactions with siglecs- which are the inhibitory receptors. Here, we examined the impact of such sialylation on overall TLR4 activation both in murine cell line J774A.1 and primary bone marrow derived macrophages (BMDM). Supporting this hypothesis, we demonstrated siglec-E engages hypersialylated TLR4 during infection. Such sialic acids-siglec-E interaction enhanced siglec-E phosphorylation that mediated its strong association with SHP1/SHP2 and also upregulated their phosphorylation in both types of macrophages. Pre-treatment of parasites and host cells with neuraminidase reduced SHP1/SHP2 phosphorylation and triggered TLR4 activation respectively through enhanced nuclear translocation of p-65. Moreover, a reciprocal interplay between Neu1 and siglec-E differentially regulates MyD88- and TRIF-pathways through sialic acids on TLR4 as their common substrate during infection. Correspondingly, Neu1 overexpression enhanced MyD88-signaling while still suppressing TRIF-activation. However, silencing siglec-E specifically activated TRIF-signaling. Pro-inflammatory cytokines corresponding to MyD88 and TRIF pathways were also upregulated respectively. Additionally, Neu1 overexpression or siglec-E silencing prevented TLR4 ubiquitination and subsequent degradation by Triad3A. Neu1-overexpression and siglec-E-silencing together followed by infection activated both MyD88 and TRIF-signaling through their enhanced TLR4-association. This elevated the MyD88-specific cytokines and TRIF-mediated IRF3 and IFN-ß genes, thus upregulating the pro-inflammatory cytokines and nitric oxide levels and reduced anti-inflammatory cytokines. All these significantly inhibited parasite survival in macrophages thus demonstrating a previously unidentified dualistic regulation of TLR4signaling pathways activation through sialic acids by interplay of Neu1 and siglec-E during Leishmania infection.

Status of IL-4 and IL-10 driven markers in experimental models of Visceral Leishmaniasis.

AIM: Leishmania donovani, the causative agent for visceral leishmaniasis (VL), modulates host monocytes/macrophages to ensure its survival. However, knowledge regarding the host-parasite interactions underpinning the disease remains limited. As disease progression is associated with polarization of monocytes/macrophages towards M2, which is regulated by cytokines IL-4/IL-13 and IL-10, this study evaluated the status of key IL-4- and IL-10 driven markers in experimental models of VL, as also evaluated their correlation, if any, with parasite load. METHODS: In liver and splenic tissues from L donovani-infected hamsters and BALB/c mice, the parasite burden was determined along with mRNA expression of IL-4-driven markers, that is CD206, Arginase-I, CCL17, CCL22, PPAR-γ, STAT6, KLF4, FIZZ1 and YM1 along with IL-10-driven markers, CXCL13, IL-10, TGF-ß, VDR, CCR2 and CYP27A1. RESULTS: The mRNA expression of IL-4- and IL-10-driven markers was enhanced in both models, but only in the hamster model, the splenic tissues demonstrated a positive correlation between all the IL-10-driven markers and parasite load. CONCLUSIONS: Contrary to human VL, both models demonstrated an increased expression of IL-4- and IL-10-driven markers.

Modulation of TLR4 Sialylation Mediated by a Sialidase Neu1 and Impairment of Its Signaling in Leishmania donovani Infected Macrophages.

Altered sialylation is generally maintained by a fine balance between sialidases and sialyltransferases, which plays an essential role during disease pathogenesis. TLR4 is a membrane-bound highly sialylated glycoprotein predominantly having α2,3-linked sialic acids. It is one of the most important client molecules in the anti-leishmanial innate immune arm. Here, we initiated a comprehensive study on the modulation of TLR4 sialylation in Leishmania donovani (L. d)-infected macrophages by a mammalian sialidase/neuraminidase-1 (Neu1) having substrate specificity toward α2,3-linked sialic acids. We observed reduced membrane-associated Neu1 with its decreased enzyme activity in infected macrophages. Moreover, we demonstrated reduced association of Neu1 with TLR4 leading to enhanced sialylation of TLR4 in these infected cells. Conversely, Neu1 over expression exhibited enhanced association of TLR4 with Neu1 leading to reduced sialylation which possibly linked to increased association of TLR4 with its downstream adaptor protein, MyD88. This, in turn, activated downstream MAP kinase signaling pathway, with enhanced nuclear translocation of NFκB that resulted in increased genetic and protein levels expression of Th1 cytokines and effector molecule nitric oxide secretion which ultimately leads to reduced parasite burden in macrophages. This was further validated by Neu1 silencing in infected macrophages which reversed such a situation. Such events strongly confirm the importance of Neu1 in modulation of TLR4 sialylation during parasite infection resulting in impairment of innate immune response. Furthermore, decreased membrane-bound Neu1 in infected macrophages could be attributed to its reduced tyrosine-phosphorylation as well as diminished association with cathepsin A. Both these phenomenon possibly play significant roles in inhibiting translocation of the sialidase from cytosol to membrane. Taken together, our study first time demonstrated impaired translocation of cytosolic Neu1 to the membrane of L. donovani-infected macrophages due to impaired phosphorylation of this enzyme. This novel finding establishes a link between enhanced α2,3-linked sialic acids on TLR4 and reduced membrane-bound Neu1 which plays a significant role for inhibiting downstream signaling to establish successful infection in the host cells.

Leishmania donovani Internalizes into Host Cells via Caveolin-mediated Endocytosis.

Leishmania donovani is an intracellular protozoan parasite that causes visceral leishmaniasis, a major cause of mortality and morbidity worldwide. The host plasma membrane serves as the portal of entry for Leishmania to gain access to the cellular interior. Although several host cell membrane receptors have been shown to be involved in the entry of Leishmania donovani into host cells, the endocytic pathway involved in the internalization of the parasite is not known. In this work, we explored the endocytic pathway involved in the entry of Leishmania donovani into host macrophages, utilizing specific inhibitors against two major pathways of internalization, i.e., clathrin- and caveolin-mediated endocytosis. We show that pitstop 2, an inhibitor for clathrin-mediated endocytosis, does not affect the entry of Leishmania donovani promastigotes into host macrophages. Interestingly, a significant reduction in internalization was observed upon treatment with genistein, an inhibitor for caveolin-mediated endocytosis. These results are supported by a similar trend in intracellular amastigote load within host macrophages. These results suggest that Leishmania donovani utilizes caveolin-mediated endocytosis to internalize into host cells. Our results provide novel insight into the mechanism of phagocytosis of Leishmania donovani into host cells and assume relevance in the development of novel therapeutics against leishmanial infection.