Siglec-9 Restrains Antibody-Dependent Natural Killer Cell Cytotoxicity against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection alters the immunological profiles of natural killer (NK) cells. However, whether NK antiviral functions are impaired during severe coronavirus disease 2019 (COVID-19) and what host factors modulate these functions remain unclear. We found that NK cells from hospitalized COVID-19 patients degranulate less against SARS-CoV-2 antigen-expressing cells (in direct cytolytic and antibody-dependent cell cytotoxicity [ADCC] assays) than NK cells from mild COVID-19 patients or negative controls. The lower NK degranulation was associated with higher plasma levels of SARS-CoV-2 nucleocapsid antigen. Phenotypic and functional analyses showed that NK cells expressing the glyco-immune checkpoint Siglec-9 elicited higher ADCC than Siglec-9- NK cells. Consistently, Siglec-9+ NK cells exhibit an activated and mature phenotype with higher expression of CD16 (FcγRIII; mediator of ADCC), CD57 (maturation marker), and NKG2C (activating receptor), along with lower expression of the inhibitory receptor NKG2A, than Siglec-9- CD56dim NK cells. These data are consistent with the concept that the NK cell subpopulation expressing Siglec-9 is highly activated and cytotoxic. However, the Siglec-9 molecule itself is an inhibitory receptor that restrains NK cytotoxicity during cancer and other viral infections. Indeed, blocking Siglec-9 significantly enhanced the ADCC-mediated NK degranulation and lysis of SARS-CoV-2-antigen-positive target cells. These data support a model in which the Siglec-9+ CD56dim NK subpopulation is cytotoxic even while it is restrained by the inhibitory effects of Siglec-9. Alleviating the Siglec-9-mediated restriction on NK cytotoxicity may further improve NK immune surveillance and presents an opportunity to develop novel immunotherapeutic tools against SARS-CoV-2 infected cells. IMPORTANCE One mechanism that cancer cells use to evade natural killer cell immune surveillance is by expressing high levels of sialoglycans, which bind to Siglec-9, a glyco-immune checkpoint molecule on NK cells. This binding inhibits NK cell cytotoxicity. Several viruses, such as hepatitis B virus (HBV) and HIV, also use a similar mechanism to evade NK surveillance. We found that NK cells from SARS-CoV-2-hospitalized patients are less able to function against cells expressing SARS-CoV-2 Spike protein than NK cells from SARS-CoV-2 mild patients or uninfected controls. We also found that the cytotoxicity of the Siglec-9+ NK subpopulation is indeed restrained by the inhibitory nature of the Siglec-9 molecule and that blocking Siglec-9 can enhance the ability of NK cells to target cells expressing SARS-CoV-2 antigens. Our results suggest that a targetable glyco-immune checkpoint mechanism, Siglec-9/sialoglycan interaction, may contribute to the ability of SARS-CoV-2 to evade NK immune surveillance.

Human galectin-9 promotes the expansion of HIV reservoirs in vivo in humanized mice.

OBJECTIVE: The human endogenous protein galectin-9 (Gal-9) reactivates latently HIV-infected cells in vitro and ex vivo , which may allow for immune-mediated clearance of these cells. However, Gal-9 also activates several immune cells, which could negatively affect HIV persistence by promoting chronic activation/exhaustion. This potential 'double-edged sword' effect of Gal-9 raises the question of the overall impact of Gal-9 on HIV persistence in vivo . DESIGN: We used the BLT (bone marrow, liver, thymus) humanized mouse model to evaluate the impact of Gal-9 on HIV persistence in vivo during antiretroviral therapy (ART). METHODS: Two independent cohorts of ART-suppressed HIV-infected BLT mice were treated with either recombinant Gal-9 or phosphate-buffered saline control. Plasma viral loads and levels of tissue-associated HIV DNA and RNA were measured by qPCR. Immunohistochemistry and HIV RNAscope were used to quantify CD4 + T, myeloid, and HIV RNA+ cells in tissues. T cell activation and exhaustion were measured by flow cytometry, and plasma markers of inflammation were measured by multiplex cytokine arrays. RESULTS: Gal-9 did not induce plasma markers of inflammation or T cell markers of activation/exhaustion in vivo . However, the treatment significantly increased levels of tissue-associated HIV DNA and RNA compared to controls ( P  = 0.0007 and P  = 0.011, respectively, for cohort I and P  = 0.002 and P  = 0.005, respectively, for cohort II). RNAscope validated the Gal-9 mediated induction of HIV RNA in tissue-associated myeloid cells, but not T cells. CONCLUSIONS: Our study highlights the overall adverse effects of Gal-9 on HIV persistence and the potential need to block Gal-9 interactions during ART-suppressed HIV infection.

Markers of fungal translocation are elevated during post-acute sequelae of SARS-CoV-2 and induce NF-κB signaling.

Long COVID, a type of post-acute sequelae of SARS-CoV-2 (PASC), has been associated with sustained elevated levels of immune activation and inflammation. However, the mechanisms that drive this inflammation remain unknown. Inflammation during acute coronavirus disease 2019 could be exacerbated by microbial translocation (from the gut and/or lung) to blood. Whether microbial translocation contributes to inflammation during PASC is unknown. We did not observe a significant elevation in plasma markers of bacterial translocation during PASC. However, we observed higher levels of fungal translocation - measured as ß-glucan, a fungal cell wall polysaccharide - in the plasma of individuals experiencing PASC compared with those without PASC or SARS-CoV-2-negative controls. The higher ß-glucan correlated with higher inflammation and elevated levels of host metabolites involved in activating N-methyl-d-aspartate receptors (such as metabolites within the tryptophan catabolism pathway) with established neurotoxic properties. Mechanistically, ß-glucan can directly induce inflammation by binding to myeloid cells (via Dectin-1) and activating Syk/NF-κB signaling. Using a Dectin-1/NF-κB reporter model, we found that plasma from individuals experiencing PASC induced higher NF-κB signaling compared with plasma from negative controls. This higher NF-κB signaling was abrogated by piceatannol (Syk inhibitor). These data suggest a potential targetable mechanism linking fungal translocation and inflammation during PASC.