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Platelets are mediators of inflammation with immune effector cell properties, and have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). This study investigated the role of platelet associated lectin galactoside-binding soluble 3 binding protein (LGALS3BP) as a mediator of inflammation in SLE, and a potential biomarker associated with clinical phenotypes.We performed RNA sequencing on platelets of patients with SLE (n=54) and age, sex, and race-matched controls (n=18) and measured LGALS3BP in platelet releasate and in circulating serum. We investigated the association between levels of LGALS3BP with the prevalence, disease severity, and clinical phenotpyes of SLE, and studied platelet-mediated effects on myeloid inflammation.Platelets from patients with SLE exhibit increased expression of LGALS3BP (fold change = 4.0, adjusted p value = 6.02 x 10These data support that platelets act as potent effector cells contributing to the pathogenesis of SLE by secreting proinflammatory LGALS3BP, which also represents a novel biomarker of SLE clinical activity.

Little is known regarding T cell translational regulation. We demonstrate that T follicular helper (TFH) cells use a previously unknown mechanism of selective messenger RNA (mRNA) translation for their differentiation, role in B cell maturation, and in autoimmune pathogenesis. We show that TFH cells have much higher levels of translation factor eIF4E than non-TFH CD4 + T cells, which is essential for translation of TFH cell fate-specification mRNAs. Genome-wide translation studies indicate that modest down-regulation of eIF4E activity by a small-molecule inhibitor or short hairpin RN impairs TFH cell development and function. In mice, down-regulation of eIF4E activity specifically reduces TFH cells among T helper subtypes, germinal centers, B cell recruitment, and antibody production. In experimental autoimmune encephalomyelitis, eIF4E activity down-regulation blocks TFH cell participation in disease pathogenesis while promoting rapid remission and spinal cord remyelination. TFH cell development and its role in autoimmune pathogenesis involve selective mRNA translation that is highly druggable.

Myocardial injury after non-cardiac surgery (MINS) is common. We investigated the incidence and outcomes of MINS, and mechanistic underpinnings using pre-operative whole blood gene expression profiling in a prospective cohort study of individuals undergoing lower extremity revascularization (LER) for peripheral artery disease (PAD). Major adverse cardiovascular and limb events (MACLE) were defined as a composite of death, myocardial infarction, stroke, major lower extremity amputation or reoperation. Among 226 participants undergoing LER, MINS occurred in 53 (23.5%). Patients with MINS had a greater incidence of major adverse cardiovascular events (49.1% vs. 22.0%, adjusted HR 1.87, 95% CI 1.07–3.26) and MACLE (67.9% vs. 44.5%; adjusted HR 1.66, 95% CI 1.08–2.55) at median 20-month follow-up. Pre-operative whole blood transcriptome profiling of a nested matched MINS case–control cohort (n = 41) identified upregulation of pathways related to platelet alpha granules and coagulation in patients who subsequently developed MINS. Thrombospondin 1 (THBS1) mRNA expression was 60% higher at baseline in patients who later developed MINS, and was independently associated with long-term cardiovascular events in the Duke Catheterization Genetics biorepository cohort. In conclusion, pre-operative THBS1 mRNA expression is higher in patients who subsequently develop MINS and is associated with incident cardiovascular events. Pathways related to platelet activity and coagulation associated with MINS provide novel insights into mechanisms of myocardial injury.

Brain metastasis is a significant cause of morbidity and mortality in multiple cancer types and represents an unmet clinical need. The mechanisms that mediate metastatic cancer growth in the brain parenchyma are largely unknown. Melanoma, which has the highest rate of brain metastasis among common cancer types, is an ideal model to study how cancer cells adapt to the brain parenchyma. Our unbiased proteomics analysis of melanoma short-term cultures revealed that proteins implicated in neurodegenerative pathologies are differentially expressed in melanoma cells explanted from brain metastases compared to those derived from extracranial metastases. We showed that melanoma cells require amyloid beta (Aβ) for growth and survival in the brain parenchyma. Melanoma-secreted Aβ activates surrounding astrocytes to a prometastatic, anti-inflammatory phenotype and prevents phagocytosis of melanoma by microglia. Finally, we demonstrate that pharmacological inhibition of Aβ decreases brain metastatic burden. Our results reveal a novel mechanistic connection between brain metastasis and Alzheimer’s disease - two previously unrelated pathologies, establish Aβ as a promising therapeutic target for brain metastasis, and demonstrate suppression of neuroinflammation as a critical feature of metastatic adaptation to the brain parenchyma. Citation Format: Kevin Kleffman, Grace Levinson, Indigo V. Rose, Lili Blumenberg, Sorin A. Shadaloey, Avantika Dhabaria, Eitan Wong, Francisco Galán-Echevarría, Alcida Karz, Diana Argibay, Richard Von-Itter, Alfredo Floristán, Gillian Baptiste, Nicole Eskow, James Tranos, Jenny Chen, Eleazar C. Vega Saenz de Miera, Melissa Call, Robert Rogers, George Jour, Youssef Zaim Wadghiri, Iman Osman, Yue Ming Li, Paul Mathews, Ronald Demattos, Beatrix Ueberheide, Kelly Ruggles, Shane A. Liddelow, Robert J. Schneider, Eva Hernando. Melanoma-secreted amyloid beta suppresses neuroinflammation and promotes brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB052.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19), a pandemic that has claimed over 700,000 human lives. The only SARS-CoV-2 antiviral, for emergency use, is remdesivir, targeting the viral polymerase complex. PF-00835231 is a pre-clinical lead compound with an alternate target, the main SARS-CoV-2 protease 3CLpro (Mpro). Here, we perform a comparative analysis of PF-00835231 and remdesivir in A549+ACE2 cells, using isolates of two major SARS-CoV-2 clades. PF-00835231 is antiviral for both clades, and, in this assay, statistically more potent than remdesivir. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps and validates PF-00835231’s time of action. Both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 in human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective antiviral for SARS-CoV-2, addresses concerns from non-human in vitro models, and supports further studies with this compound

The arsenal of SARS-CoV-2 specific antiviral drugs is extremely limited. Only one direct-acting antiviral drug is currently approved, the viral polymerase inhibitor remdesivir, and it has limited efficacy., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease 2019 (COVID-19). There is a dire need for novel effective antivirals to treat COVID-19, as the only approved direct-acting antiviral to date is remdesivir, targeting the viral polymerase complex. A potential alternate target in the viral life cycle is the main SARS-CoV-2 protease 3CLpro (Mpro). The drug candidate PF-00835231 is the active compound of the first anti-3CLpro regimen in clinical trials. Here, we perform a comparative analysis of PF-00835231, the preclinical 3CLpro inhibitor GC-376, and the polymerase inhibitor remdesivir, in alveolar basal epithelial cells modified to express ACE2 (A549+ACE2 cells). We find PF-00835231 with at least similar or higher potency than remdesivir or GC-376. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps in A549+ACE2 cells and validates PF-00835231’s early time of action. In a model of the human polarized airway epithelium, both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 at low micromolar concentrations. Finally, we show that the efflux transporter P-glycoprotein, which was previously suggested to diminish PF-00835231’s efficacy based on experiments in monkey kidney Vero E6 cells, does not negatively impact PF-00835231 efficacy in either A549+ACE2 cells or human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective SARS-CoV-2 antiviral and addresses concerns that emerged based on prior studies in nonhuman in vitro models. IMPORTANCE The arsenal of SARS-CoV-2 specific antiviral drugs is extremely limited. Only one direct-acting antiviral drug is currently approved, the viral polymerase inhibitor remdesivir, and it has limited efficacy. Thus, there is a substantial need to develop additional antiviral compounds with minimal side effects and alternate viral targets. One such alternate target is its main protease, 3CLpro (Mpro), an essential component of the SARS-CoV-2 life cycle processing the viral polyprotein into the components of the viral polymerase complex. In this study, we characterize a novel antiviral drug, PF-00835231, which is the active component of the first-in-class 3CLpro-targeting regimen in clinical trials. Using 3D in vitro models of the human airway epithelium, we demonstrate the antiviral potential of PF-00835231 for inhibition of SARS-CoV-2.

Multiple sclerosis (MS) is a neuroinflammatory disorder resulting from infiltration of T cells into the central nervous system (CNS) and demyelination of neurons. Since T follicular helper (TFH) cells are associated with MS relapse, their selective inhibition could be an ideal therapeutic. TFH cells require transcription factor BCL6 and active mTORC1/2 for development. When mTORC is active, cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) initiates translation of selective mRNAs. However, the requirement of eIF4E for translation of mRNAs necessary for TFH cell development has not been investigated. Disruption of eIF4E binding to the 5′ mRNA cap with drug 4EGI-1 inhibits TFH and germinal center (GC) B cell development while having no effect on differentiation and effector function of TH1, TH2, TH17, or Tregs. Silencing of eIF4E in only CD4 T cells is sufficient to inhibit their formation of TFH cells. We used polysome profiling to determine which mRNAs are selectively translated by eIF4E and identified salient programs regulated by transcription (BCL6, NFAT) and costimulation (CD28, SLAM). eIF4E is required for translation of BCL6 in human lymph node TFH and GC B cells. Administration of 4EGI-1 during experimental autoimmune encephalitis (EAE) results in significantly decreased infiltration of CD4 T cells in the CNS, demyelination, and clinical score. Further, 4EGI-1 treatment following initiation of symptoms results in rapid improvement of symptoms and partial remission earlier than vehicle-treated animals. Thus, eIF4E is required for differentiation of TFH cells and pathogenesis of autoimmune encephalitis, and 4EGI-1 represents a potential therapeutic.