bluuebunny/crossref_metadata_2025_split huggingface dataset:
Host mRNA deadenylation machinery selectively targets interferon mRNAs, regulating antiviral immunity
Authors: Braun Rachel M , Ferretti Max , Miller Jesse , Castellana Lauren , Lee Jae Seung , Lynch Kristen W , Cherry Sara 
Published: August 2023

Abstract
Elucidating host-mediated regulation of viral infection is critical in the development of antivirals and in understanding viral pathogenesis. Here, through high-throughput RNA interference screening, we determine that CNOT2 promotes SARS-CoV-2 infection in human respiratory epithelial cells. CNOT2 is a non-enzymatic subunit of the CCR4-NOT complex, which plays conserved roles in the control of gene expression by deadenylating the poly(A) tails of host mRNAs, thus targeting them for decay. CCR4-NOT can selectively degrade cohorts of mRNAs that are delivered by RNA Binding Proteins. Tristetraprolin (TTP) is among the best characterized of such cargo adapters and is known to recruit mRNAs containing AU-rich elements in their 3′ UTR to CCR4-NOT for deadenylation and decay. We found that in addition to CNOT2, another non-catalytic subunit of CCR4-NOT, CNOT1, in addition to the selectivity factor, TTP, also promote SARS-CoV-2 infection. Global transcriptomics revealed that CNOT2, CNOT1, and TTP specifically regulate interferons (IFNs) and interferon stimulated genes (ISGs) and that depletion of these mRNA decay factors leads to increased levels of IFNs and ISGs. Moreover, the pro-viral activity of CCR4-NOT components and TTP is dependent on IFN signaling: the CCR4-NOT complex has no effect on SARS-CoV-2 infection when IFN signaling is blocked. This led us to discover that CNOT2 promotes the selective deadenylation of IFN mRNAs, and that depletion of CNOT2 leads to increased poly(A) tail lengths of IFN mRNAs. Therefore, we found that the basal levels of IFNs, and thus IFN signaling, are post-transcriptionally regulated by deadenylation machinery, which in turn impacts susceptibility to viral infections including SARS-CoV-2.
Supported by grants from NIH (T32-AI-007324)
NF-κB signaling promotes SARS-CoV2 infection in human lung epithelial cells
Authors: Etwebi Zienab , Ayyanathan Kasirajan , Miller Jesse , Kamalia Brinda , Li Minghua , Lee Jae Seung , Ferretti Max , Braun Rachel M , Srivastava Tamanna , Dohnalova Lenka , Cox Timothy , Thaiss Christoph , Schultz David , Cherry Sara 
Published: August 2023

Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is responsible for over 660 million infections, and more than 6 million deaths worldwide. There is an increasing need for more treatment options that addresses SARS-CoV-2 infection and its replication. Innate immune pathways including interferon signaling and inflammatory NF-κB signaling are important players in regulating immune responses, these pathways are often targeted by viral pathogens to enhance viral replication, host immune evasion, and host cell survival. The respiratory tract is the major target for SARS-CoV-2; thus, we are exploring the role of innate immune signaling in regulating infection in these cells. We found that both interferon and NF-κB signaling are induced by SARS-CoV-2 infection as measured by increases in transcription factor localization and gene expression. Moreover, pharmacological inhibition of NF-κB signaling but not interferon signaling blocks SARS CoV-2 infection of these respiratory cells. We are exploring how NF-κB signaling impacts viral infection. Indeed, we found that the activity of the NF-κB pathway is independent of interferon signaling. Further mechanistic studies are underway to define the role of NF-κB in coronavirus infection and suggest that inhibitors of the NF-κB signaling pathway may be a novel pharmacological target for the treatment of SARS-CoV-2.
Investigating links between persistent symptoms after COVID-19 and longitudinal changes in memory T cell responses to EBV, CMV, HSV-1, and SARS-CoV-2.
Authors: Zhong Jiabao , Smith Erika L , McConney Christina S , Bean David J , Belkina Anna C , Snyder-Cappione Jennifer E 
Published: August 2023

Abstract
Post-acute infection syndromes (PAISs) have long been observed following acute infections with bacteria, viruses or other microbes. Overlapping core symptoms across different PAISs suggest underlying mechanism(s) that is putatively independent of the causative agents. During the COVID-19 pandemic, accumulating reports of post-acute sequelae of SARS-CoV-2 infection (PASC), also known as “long Covid”, have provided new clues for the pathogenesis of PAISs, but the mechanistic drivers of persistent symptoms are still unclear. Here, we evaluated temporal changes in the memory T cell repertoire, in both frequency and functional signatures, of twelve individuals initially infected with SARS-CoV-2 in the spring of 2020 (ten outpatient, two hospitalized), with four participants showing evidence of PASC. Longitudinal T cell responses were quantified as the frequencies of cytokine producing cells after overnight (ex vivo) stimulation of PBMCs with peptide pools from cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus type 1 (HSV-1) and SARS-CoV-2; cell readouts were obtained using intracellular cytokine staining (ICS). PBMCs were collected from multiple timepoints from acute illness to 12–25 months post-infection. Our unique dataset serves as an indirect readout of viral replication and provides insights to two possible explanations for PASC: the reactivation of latently infected viruses during and after the acute SARS-CoV-2 infection, and the persistence of SARS-CoV-2 antigens triggering chronic inflammation. For future studies, longitudinal T cell responses after acute infections with other microbes will be assessed in a larger cohort to determine the specificity of the findings to SARS-CoV-2 infection.
Supported by MassCPR PASC Subaward #FP-0034311
SARS-CoV-2 infection and Spike protein exposure alter iPSC-derived human brain organoid homeostasis.
Authors: Cappelletti Gioia , Brambilla Lorenzo , Melzi Valentina , Rizzuti Mafalda , Nizzardo Monica , Trabattoni Daria , Corti Stefania , Clerici Mario , Biasin Mara 
Published: August 2023

Abstract
COVID-19 typically causes respiratory disorders, but several COVID-19 patients (30–60%) manifest also a wide range of neurological conditions, advocating for detrimental effects of SARS-CoV-2 or Spike (S) protein on the central nervous system. However, the molecular mechanisms responsible for these disfunctions as well as the potential neurotropism of SARS-CoV-2, are still under investigation. To assess this issue, by using iPSC-derived-human brain organoids (HBO) we evaluated: the expression of SARS-CoV-2 main receptors: ACE2, CD147, NRP1, Furin, TMPRSS2 (qPCR, IF); their infectability by SARS-CoV-2 over-time (QPCR, TCID50, IF); and the effect of SARS-CoV-2-infection and S exposure on HBO transcriptome (qPCR) and secretome (Multiplex ELISA). All the main SARS-CoV-2 receptors are expressed by HBO with lower NRP1 levels compared to CD147 and ACE2. By analyzing viral N1 and N2 gene sequence over time, infectability of VeroE6 cells by SARS-CoV-2 infected HBO supernatants, and nucleocapsid expression by IF we confirmed that HBO may be productively infected by the virus. Furthermore, SARS-CoV-2 infection was accompanied by the activation of apoptotic and stress pathways (caspase3, caspase8, Bcl2, S100B), inflammatory process (CCL2, NLRP3) Interferon Stimulated Genes (IFITM1, IFITM3, STAT1, NFkB) and antigen presentation pathway (ERAP1, ERAP2, HLA-A, TAP). Notably, the trend of the different targets was comparable following SARS-CoV-2 infection and S-stimulation.
These results confirm that SARS-CoV-2 infect HBO probably by binding CD147 and ACE2 receptors and indicate that the exposure to the S protein can affect their homeostasis and exert neurotoxic effects presumably driving the so called long-COVID symptoms.
Partially supported by grants from Fondazione Alessandro and Vincenzo Negroni Prati Morosini and Fondazione Romeo and Erica Invernizzi.
Potentiation of infiltrating adaptive and innate immune cells and metabolic responses by environmental cadmium exposure following respiratory syncytial virus infection
Authors: Go Young-Mi , Kim Ki-Hye , Kang Sang-Moo 
Published: August 2023

Abstract
Cadmium (Cd) accumulates in humans from regular dietary and environmental exposure including cigarette smoke, and occupational sources, and has a twenty-year biologic half-life. Most individuals have pre-existing immunity from early life exposure to respiratory syncytial virus (RSV) which causes serious lung disease and hospitalizations among the high-risk infants and elderly. Little is known about the effect of low environmental levels of Cd on inflammatory immune cell dynamics causing lung pathology in humans with RSV infection at early life. In this study, we examined the effects of low dose long-term Cd exposure after infant age RSV infection on phenotypes of infiltrating immune cells in a mouse model. C57BL/6J mice at 8 days old were exposed to RSV. A subset of RSV-infected mice was subsequently treated with Cd at a low dose in drinking water (3.0 mg CdCl2/L) for 16 weeks. Flow cytometry and histology data showed that the levels of both T cells (CD4+, CD4+IFNg+, CD4+ IL4+, CD4+ IL13+) and innate immune cells (CD11b+, monocytes, neutrophil, eosinophil, dendritic cells, activated dendritic cells) appear to be linked to inflammatory lung pathology. In particular, the results showed that the levels of T cells and innate immune cells were substantially higher in the group of RSV infection followed by Cd treatment than the other groups (Cd or RSV alone), providing evidence that low dose Cd following early-life RSV infection enhanced lung inflammation. Consistently metabolic disruption was evident in the group of RSV infection followed by Cd treatment. Taken together, our study demonstrates that cumulative Cd burden following RSV infection has a significant impact on inflammatory immune cell infiltration, lung pathology, and metabolism.
NIEHS Grants R01 ES031980 NIAID grans R01 AI54656 R21 AI147042
Metabolic profiling of COVID-19 severity and mortality: A targeted metabolomics approach
Authors: Cyprian Farhan S , Doudin Asmaa , Khatib Malkan , Elrayess Mohamed , Yassine Hadi , Musa Abdallah 
Published: August 2023

Abstract
The analysis of metabolites in response to SARS-CoV-2 infection provides a snapshot of the endogenous host metabolism and its role in shaping the interaction with SARS-CoV-2. In this study, using a targeted metabolomics approach, the metabolic signatures of severity and mortality were studied in COVID-19 patients. Our analysis revealed survival and severity-associated shifts in the metabolome of COVID-19 patients, where amino acids, particularly tryptophan and arginine, and fatty acid metabolism were altered in severe patients as compared to mild and asymptomatic cases. Herein, we also report alterations in the sums and ratios of several essential amino acids as well as kynurenine to tryptophan ratio, which is an indicator of indoleamine-2,3-dioxygenase (IDO) activity. Our findings suggest that metabolites of creatine including symmetric dimethylarginine, asymmetric dimethylarginine, and 1-methylhistidine can be used as biomarkers to identify patients at risk of poor outcomes in COVID-19, and that lower levels of these metabolites are associated with higher survival probabilities. Additionally, elevated levels of short chain acylcarnitines and carnitine palmitoyltransferase 1 and 2 enzymes indicators have been observed in critically ill COVID-19 patients, indicating their potential usefulness as predictors of hospital mortality and poor survival outcome, likely due to increased demand for energy, inflammation, and mitochondrial dysfunction. The metabolome analysis of COVID-19 patients can improve our understanding of disease severity by providing insight into the relationship between metabolites and disease progression, which can lead to the development of potential therapeutics and clinical risk models.
This work was supported by Emergency Response Grant ID: 252
Brain Regional Dependence of Antiviral and Inflammatory Genes Following Simian Immunodeficiency Virus Infection and Antiretroviral Therapy
Authors: Ellison Alysha , Franco Jose Javier Rosado , Alvarez Jade , Williams Dionna 
Published: August 2023

Abstract
Human immunodeficiency virus (HIV) initiates a perpetual cycle of inflammation in the brain that results in neuronal damage and consequent neurologic dysfunction. Since HIV preferentially infects and damages specific brain regions, there is a need for study of antiviral and immune markers in a brain region specific manner. We have evaluated expression of antiviral (IFNa, IFNb, TRAIL, MX1) and immune markers (CCL2, IL-6, IL-1b, IL-10) across nine brain regions in our virally suppressed rhesus macaque model of simian immunodeficiency virus (SIV) infection. Rhesus macaques from four study groups (uninfected, SIV, SIV/ART, SIV/ART cessation) were perfused, brain obtained by region, and snap frozen in liquid nitrogen. RNA was extracted and qRT-PCR performed to quantify the genes of interest in each brain region. Antiviral markers (MX1, TRAIL) were expressed in all brain regions and equally expressed across all groups, with occasional increase of MX1 in SIV infected animals. Alternatively, common immune markers (IL-6, CCL2) were not expressed across the brain regions of any study group, excepting the thalamus. Other antiviral (IFN-a) and immune markers (IL-10) were consistently expressed across brain regions in uninfected animals. However, the effects of SIV and ART on expression of these and other pertinent markers (IFN-b, IL-1b) were brain region dependent. Altogether, these data suggest that antiviral and immune marker expression is dynamic and differs depending on the marker and the brain region under investigation. This knowledge has implications for the way these markers are studied in the brain and implies a need for brain region specific investigation.
Supported by grants from the NIH (5R01DA052859-02) and the NIH funded Johns Hopkins University Center for AIDS Research (P30AI094189-01A1)
Signatures of HIV-infected CD4+ T cell resistance to NK cell-mediated killing
Authors: Grasberger Paula , Kucukural Alper , Zapp Maria , Clayton Kiera 
Published: August 2023

Abstract

BACKGROUND
In vitro experiments in which HIV-infected CD4+ T cells are co-cultured with natural killer (NK) cells yield incomplete elimination of the infected cells. We therefore hypothesize that cell-intrinsic pathways in CD4+ T cells are differentially regulated in HIV-infected cells to mediate resistance to NK cell-mediated killing.

METHODS
Mock-infected and HIV-89.6-infected CD4+ T cells from 6 HIV− donors were co-cultured overnight +/− autologous NK cells. Following co-culture, cells were stained for the surface exposed HIV envelope protein using fluorescently conjugated HIV antibodies. Fluorescence activated cell sorting was then used to isolate infected and uninfected cells, followed by bulk RNA-sequencing. Transcripts from infected and uninfected cells co-cultured overnight with versus without NK cells were compared.

RESULTS
RNA-seq analysis of CD4+ T cells that survived co-culture with NK cells revealed dozens of differentially expressed genes in infected, but not uninfected cells. This included upregulation of interferon-stimulated genes, including PD-L1 (padj=3×10−6), and NF-kB-related genes. Additional upregulated genes in the surviving infected cells included c-Fos (padj=1.9×10−5), c-Jun (padj=4.9×10−8), and cathepsin L (padj=5×10−3).

CONCLUSIONS
NF-kB genes that are downregulated in infected cells, potentially through Vpu activity, are normalized in surviving infected cells. This may be due to NK cell targeting of Vpu-mediated HLA-C downregulation. Deconvolution of the relative contributions of specific genes/proteins to target cell resistance will reveal novel targets for the development of therapeutics to eliminate the HIV reservoir.
Perturbation of Invariant Natural Killer T cells by the Clonal Hematopoiesis-related inflammatory environment
Authors: Luca Francesca , Kuznetsova Valeriya , Franceski Alana , Goel Paran , Costa Amanda , Welner Robert 
Published: August 2023

Abstract
Invariant Natural Killer T (iNKT) cells are T cells characterized by a semi-invariant TCR and NK markers. They respond to glycolipid antigens presented by the major histocompatibility complex (MHC)-like molecule CD1d. Our single-cell transcriptomic profiling of several tissues identified marrow iNKT as a unique subset, possibly modulating marrow homeostasis. However, little is known about their role in hematological malignancies and how the disease-related environment impacts iNKT cells. For example, clonal hematopoiesis of indeterminate potential (CHIP) corresponds to a condition in which mutated hematopoietic cells have an enhanced fitness and expansion, increasing the risk of leukemia and other diseases. Loss of TET2 in CHIP promotes myeloid skewing and inflammation. TET2 is a methycytosine dioxygenase catalyzing DNA demethylation of genes involved in proliferation, differentiation and oncogenesis. However, the impact that TET2 loss has on iNKT lineage output and function is yet to be defined. In our chimeric mouse model of CHIP, only 15% of TET2 mutants differentiate into iNKT. We also observe a decrease of non-transformed iNKT cells in the marrow of our mice, suggesting environmental inhibition. We expect these bystander iNKT cells to be functionally impaired where inflammation leads to their chromatin rewiring potentially contributing to disease. This is addressed by studying CHIP-exposed iNKT transcriptomes and chromatin accessibility. Understanding the molecular basis of the functional reprogramming by the disease-related inflammation will improve clinical outcomes for marrow transplantation and for the development of cellular therapies using iNKT cells.
Targeting lL-38 triggers γδ T cell-dependent anti-tumor immunity
Authors: Silva Priscila , Mora Javier , Ernst Andreas , You Xin , Brüne Bernhard , Weigert Andreas 
Published: August 2023

Abstract
The IL-1 family receptor antagonist IL-38 suppresses auto-inflammatory reactions in murine disease models. Given the connection between auto-immunity and anti-tumor immunity, we wondered if targeting IL-38 would affect the latter. The transgenic PyMT mammary carcinoma model was employed to target IL-38 genetically or with a neutralizing antibody. IL-38-deficient (KO) PyMT mice had a markedly delayed tumor growth compared to WT mice, accompanied by increased NK cells, CD8+, NKT, and γδ T cell infiltrates. Accordingly, in human mammary carcinoma, IL-38 expression negatively correlated with infiltrating T cell subsets. This pattern was largely recapitulated with a IL-38-neutralizing antibody, where antibody-treated PyMT WT mice showed a strong decrease in tumor growth compared to IgG control treated animals, accompanied by an increase in CD8+ and γδ T cells. In a therapeutic model of chemoresistance, IL-38 antibody in combination with doxorubicin prevented tumor relapse, which was accompanied by increased in NK cells, CD8+, CD4+, and γδ T cells. To analyze the involvement of γδ T cells, we used γδ-TCR blocking antibody compared to IgG control in IL-38 KO mice. The delay in growth of IL-38 KO PyMT tumors compared to WT PyMT tumors was abolished upon γδ-TCR blockade. This was accompanied by a strong decrease in CD8+ cells upon effective γδ-TCR blockade, suggesting a cross talking between CD8+ and γδ T cells. Whole transcriptome sequencing analysis of PyMT tumors upon IL-38 neutralization and γδ-TCR blockage suggested NOTCH signaling to mediate the link between CD8+ and γδ T cells. Taken together, these results provide evidence for a therapeutic potential of anti-IL-38 antibodies to activate anti-tumor immunity in mammary carcinoma.
Supported by a grant from Deutsche Krebshilfe (70114051)
Regulation of NK cell chemotaxis by KCa3.1 channels
Authors: Alshwimi Abdulaziz O , Chimote Ameet A , Wise-Draper Trisha , Conforti Laura 
Published: August 2023

Abstract
CD8+ T cells and NK cells are the primary line of defense against tumor cells. However, the tumor microenvironment impairs their anti-tumor capabilities. One of the key functions impaired is the ability of T and NK cells to infiltrate the tumor, essential to allow the direct contact with a target cell and its killing. Kv1.3 and KCa3.1 K+ channels have a multitude of essential functions that have been established in CD8+ T cells: Kv1.3 regulates cytotoxicity and KCa3.1 chemotaxis. However, less is known about K+ channels in NK cells. Herein, we performed electrophysiological and 3D chemotaxis experiments to study the activity and functional role of K+ channels in human NK cells. NK cells were isolated from healthy donors (HD) and head and neck cancer (HNC) patients and activated with IL-2 and IL-15 for 24–48 hr. Activation led to an increase in Kv1.3 currents of 49.6% at 24 hr and 434.1% at 48 hr . KCa3.1 activity increased by 42.8% at 24 hr and 54.2% at 48 hr. Chemotaxis experiment showed that activated NK cells move randomly in the collagen matrix and migrated towards the chemokine gradient in presence of CXCL10. Chemotaxis, but not random migration, was inhibited by the selective KCa3.1 blocker TRAM-34 in NK cells from HD and HNC patients. Shk-Dap22, a Kv1.3 blocker, did not alter either chemotaxis or random migration. Overall, TRAM-34 reduced the distance achieved by NK cells in the chemokine gradient by 157% in HD (4 donors) and 89% in HNC NK cells (1 donor). These data highlight the critical role of KCa3.1 channels in NK cell migration and open the possibility of targeting KCa3.1 to facilitate tumor infiltration by NK cells.
Immune checkpoint blockade unleashes Mucosal Associated Invariant T (MAIT) cells in tumor microenvironment of NSCLC patients
Authors: Birla Pakhi , Zhang Boyang , Zhang Jiajia , Gulati Ananya , Yang Lansaol , Johnson Ramona , Lee Alex , Smith Kellie , Sears Cynthia , Shaikh Fyza , Housseau Franck , Pardoll Drew 
Published: August 2023

Abstract
Mucosal associated invariant T (MAIT) cells are innate like T-cells that recognise non-peptide metabolite antigens presented on monomorphic MHC related-1 (MR1) making them an attractive off the shelf tool for cancer immunotherapy. In this study, we investigate the function of MAITs in the tumor microenvironment of neoadjuvant anti-PD1 treated lung cancer patients (NCT02259621). Paired single cell RNA/TCR sequencing analysis revealed an oligoclonal expansion of MAITs comprising of canonical TRAV1–2+ and non-canonical MAITs TRAV1–2−. TCR capture from canonical MAIT clonotypes confirmed their in vitro recognition of 5-OP-RU, a bacteria derived riboflavin derivative. However, the nature of antigens recognised by non-canonical MAITs remains poorly understood. On mining transcriptional profiles of MAITs, we observed a high clonal amplification of canonical MAITs whereas the non-canonical clonotypes expressed a strong activation/exhaustion gene signature. This suggests that both these MAIT populations recognise different types of antigens, are differentially activated, and respond to immune checkpoint blockade (ICB), the non-canonical MAITs being highly dysfunctional. Using bacterial 16S RNA amplicon and metagenomics sequencing, we seek to identify microbial and metabolomic signatures associated with MAIT activation and response to ICB. Our findings will provide further insights into the nature of ligands recognised by MAITs and the mechanisms associated with MR1 dependent recognition and killing of tumor. Investigating the role of unconventional T-cells in response to ICB has the potential to reveal new tumor-associated antigens which could open up avenues for innovative combination immunotherapies.