Cholesterol and sphingomyelin are critical for Fcγ receptor-mediated phagocytosis of Cryptococcus neoformans by macrophages.

Cryptococcus neoformans is a fungal pathogen that causes life-threatening meningoencephalitis in lymphopenic patients. Pulmonary macrophages comprise the first line of host defense upon inhalation of fungal spores by aiding in clearance but can also potentially serve as a niche for their dissemination. Given that macrophages play a key role in the outcome of a cryptococcal infection, it is crucial to understand factors that mediate phagocytosis of C. neoformans. Since lipid rafts (high-order plasma membrane domains enriched in cholesterol and sphingomyelin [SM]) have been implicated in facilitating phagocytosis, we evaluated whether these ordered domains govern macrophages' ability to phagocytose C. neoformans. We found that cholesterol or SM depletion resulted in significantly deficient immunoglobulin G (IgG)-mediated phagocytosis of fungus. Moreover, repletion of macrophage cells with a raft-promoting sterol (7-dehydrocholesterol) rescued this phagocytic deficiency, whereas a raft-inhibiting sterol (coprostanol) significantly decreased IgG-mediated phagocytosis of C. neoformans. Using a photoswitchable SM (AzoSM), we observed that the raft-promoting conformation (trans-AzoSM) resulted in efficient phagocytosis, whereas the raft-inhibiting conformation (cis-AzoSM) significantly but reversibly blunted phagocytosis. We observed that the effect on phagocytosis may be facilitated by Fcγ receptor (FcγR) function, whereby IgG immune complexes crosslink to FcγRIII, resulting in tyrosine phosphorylation of FcR γ-subunit (FcRγ), an important accessory protein in the FcγR signaling cascade. Correspondingly, cholesterol or SM depletion resulted in decreased FcRγ phosphorylation. Repletion with 7-dehydrocholesterol restored phosphorylation, whereas repletion with coprostanol showed FcRγ phosphorylation comparable to unstimulated cells. Together, these data suggest that lipid rafts are critical for facilitating FcγRIII-mediated phagocytosis of C. neoformans.

Group IIA secreted phospholipase A2 is associated with the pathobiology leading to COVID-19 mortality.

There is an urgent need to identify the cellular and molecular mechanisms responsible for severe COVID-19 that results in death. We initially performed both untargeted and targeted lipidomics as well as focused biochemical analyses of 127 plasma samples and found elevated metabolites associated with secreted phospholipase A2 (sPLA2) activity and mitochondrial dysfunction in patients with severe COVID-19. Deceased COVID-19 patients had higher levels of circulating, catalytically active sPLA2 group IIA (sPLA2-IIA), with a median value that was 9.6-fold higher than that for patients with mild disease and 5.0-fold higher than the median value for survivors of severe COVID-19. Elevated sPLA2-IIA levels paralleled several indices of COVID-19 disease severity (e.g., kidney dysfunction, hypoxia, multiple organ dysfunction). A decision tree generated by machine learning identified sPLA2-IIA levels as a central node in the stratification of patients who died from COVID-19. Random forest analysis and least absolute shrinkage and selection operator-based (LASSO-based) regression analysis additionally identified sPLA2-IIA and blood urea nitrogen (BUN) as the key variables among 80 clinical indices in predicting COVID-19 mortality. The combined PLA-BUN index performed significantly better than did either one alone. An independent cohort (n = 154) confirmed higher plasma sPLA2-IIA levels in deceased patients compared with levels in plasma from patients with severe or mild COVID-19, with the PLA-BUN index-based decision tree satisfactorily stratifying patients with mild, severe, or fatal COVID-19. With clinically tested inhibitors available, this study identifies sPLA2-IIA as a therapeutic target to reduce COVID-19 mortality.

Group IIA Secreted Phospholipase A 2 Plays a Central Role in the Pathobiology of COVID-19.

There is an urgent need to identify cellular and molecular mechanisms responsible for severe COVID-19 disease accompanied by multiple organ failure and high mortality rates. Here, we performed untargeted/targeted lipidomics and focused biochemistry on 127 patient plasma samples, and showed high levels of circulating, enzymatically active secreted phospholipase A 2 Group IIA (sPLA 2 -IIA) in severe and fatal COVID-19 disease compared with uninfected patients or mild illness. Machine learning demonstrated that sPLA 2 -IIA effectively stratifies severe from fatal COVID-19 disease. We further introduce a PLA-BUN index that combines sPLA 2 -IIA and blood urea nitrogen (BUN) threshold levels as a critical risk factor for mitochondrial dysfunction, sustained inflammatory injury and lethal COVID-19. With the availability of clinically tested inhibitors of sPLA 2 -IIA, our study opens the door to a precision intervention using indices discovered here to reduce COVID-19 mortality.

A Mechanism-Based Targeted Screen To Identify Epstein-Barr Virus-Directed Antiviral Agents.

Epstein-Barr virus (EBV) is one of nine human herpesviruses that persist latently to establish permanent residence in their hosts. Periodic activation into the lytic/replicative phase allows such viruses to propagate and spread, but can also cause disease in the host. This lytic phase is also essential for EBV to cause infectious mononucleosis and cancers, including B lymphocyte-derived Burkitt lymphoma and immunocompromise-associated lymphoproliferative diseases/lymphomas as well as epithelial cell-derived nasopharyngeal cell carcinoma. In the absence of anti-EBV agents, however, therapeutic options for EBV-related diseases are limited. In earlier work, we discovered that through the activities of the viral protein kinase conserved across herpesviruses and two cellular proteins, ATM and KAP1, a lytic cycle amplification loop is established, and disruption of this loop disables the EBV lytic cascade. We therefore devised a high-throughput screening assay, screened a small-molecule-compound library, and identified 17 candidates that impair the release of lytically replicated EBV. The identified compounds will (i) serve as lead compounds or may be modified to inhibit EBV and potentially other herpesviruses, and (ii) be developed into anticancer agents, as functions of KAP1 and ATM are tightly linked to cancer. Importantly, our screening strategy may also be used to screen additional compound libraries for antiherpesviral and anticancer drugs.IMPORTANCE Epstein-Barr virus, which is nearly ubiquitous in humans, is causal to infectious mononucleosis, chronic active EBV infection, and lymphoid and epithelial cancers. However, EBV-specific antiviral agents are not yet available. To aid in the identification of compounds that may be developed as antivirals, we pursued a mechanism-based approach. Since many of these diseases rely on EBV's lytic phase, we developed a high-throughput assay that is able to measure a key step that is essential for successful completion of EBV's lytic cascade. We used this assay to screen a library of small-molecule compounds and identified inhibitors that may be pursued for their anti-EBV and possibly even antiherpesviral potential, as this key mechanism appears to be common to several human herpesviruses. Given the prominent role of this mechanism in both herpesvirus biology and cancer, our screening assay may be used as a platform to identify both antiherpesviral and anticancer drugs.

FTY720 reactivates cryptococcal granulomas in mice through S1P receptor 3 on macrophages.

FTY720 is a treatment for relapsing remitting multiple sclerosis (MS). It is an analog of sphingosine-1-phosphate (S1P) and targets S1P receptors 1, 3, 4, and 5. Recent reports indicate an association between long-term exposure to FTY720 and cases of cryptococcal infection. Here, we studied the effect of FTY720 and its derivative, BAF312, which only target S1P receptors 1 and 5, in a mouse model of cryptococcal infection. We found that treatment with FTY720, but not with BAF312, led to decreased survival and increased organ burden in mouse cryptococcal granulomas. Both FTY720 and BAF312 caused a profound CD4+ and CD8+ T cell depletion in blood and lungs but only treatment with FTY720 led to cryptococcal reactivation. Treatment with FTY720, but not with BAF312, was associated with disorganization of macrophages and with M2 polarization at the granuloma site. In a cell system, FTY720 decreased phagocytosis and production of reactive oxygen species by macrophages, a phenotype recapitulated in the S1pr3-/- knockout macrophages. Our results suggest that FTY720 reactivates cryptococcosis from the granuloma through a S1P receptor 3-mediated mechanism and support the rationale for development of more-specific receptor modulators for therapeutic use of MS.