SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract.

The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting enzyme 2 (ACE2) expression in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings highlight the nasal susceptibility to SARS-CoV-2 with likely subsequent aspiration-mediated virus seeding to the lung in SARS-CoV-2 pathogenesis. These reagents provide a foundation for investigations into virus-host interactions in protective immunity, host susceptibility, and virus pathogenesis.

Rapid Inflammasome Activation following Mucosal SIV Infection of Rhesus Monkeys.

The earliest events following mucosal HIV-1 infection, prior to measurable viremia, remain poorly understood. Here, by detailed necropsy studies, we show that the virus can rapidly disseminate following mucosal SIV infection of rhesus monkeys and trigger components of the inflammasome, both at the site of inoculation and at early sites of distal virus spread. By 24 hr following inoculation, a proinflammatory signature that lacked antiviral restriction factors was observed in viral RNA-positive tissues. The early innate response included expression of NLRX1, which inhibits antiviral responses, and activation of the TGF-ß pathway, which negatively regulates adaptive immune responses. These data suggest a model in which the virus triggers specific host mechanisms that suppress the generation of antiviral innate and adaptive immune responses in the first few days of infection, thus facilitating its own replication. These findings have important implications for the development of vaccines and other strategies to prevent infection.

Anti-inflammatory Roles of Glucocorticoids Are Mediated by Foxp3+ Regulatory T Cells via a miR-342-Dependent Mechanism.

Glucocorticoids (GC) are the mainstay treatment option for inflammatory conditions. Despite the broad usage of GC, the mechanisms by which GC exerts its effects remain elusive. Here, utilizing murine autoimmune and allergic inflammation models, we report that Foxp3+ regulatory T (Treg) cells are irreplaceable GC target cells in vivo. Dexamethasone (Dex) administered in the absence of Treg cells completely lost its ability to control inflammation, and the lack of glucocorticoid receptor in Treg cells alone resulted in the loss of therapeutic ability of Dex. Mechanistically, Dex induced miR-342-3p specifically in Treg cells and miR-342-3p directly targeted the mTORC2 component, Rictor. Altering miRNA-342-3p or Rictor expression in Treg cells dysregulated metabolic programming in Treg cells, controlling their regulatory functions in vivo. Our results uncover a previously unknown contribution of Treg cells during glucocorticoid-mediated treatment of inflammation and the underlying mechanisms operated via the Dex-miR-342-Rictor axis.

Transcript Profiles of Microglia/Macrophage Cells at the Borders of Chronic Active and Subpial Gray Matter Lesions in Multiple Sclerosis.

OBJECTIVE: Microglia/macrophages line the border of demyelinated lesions in both cerebral white matter and the cortex in the brains of multiple sclerosis patients. Microglia/macrophages associated with chronic white matter lesions are thought to be responsible for slow lesion expansion and disability progression in progressive multiple sclerosis, whereas those lining gray matter lesions are less studied. Profiling these microglia/macrophages could help to focus therapies on genes or pathways specific to lesion expansion and disease progression. METHODS: We compared the morphology and transcript profiles of microglia/macrophages associated with borders of white matter (WM line) and subpial gray matter lesions (GM line) using laser capture microscopy. We performed RNA sequencing on isolated cells followed by immunocytochemistry to determine the distribution of translational products of transcripts increased in WM line microglia. RESULTS: Cells in the WM line appear activated, with shorter processes and larger cell bodies, whereas those in the GM line appear more homeostatic, with smaller cell bodies and multiple thin processes. Transcript profiling revealed 176 genes in WM lines and 111 genes in GM lines as differentially expressed. Transcripts associated with immune activation and iron homeostasis were increased in WM line microglia, whereas genes belonging to the canonical Wnt signaling pathway were increased in GM line microglia. INTERPRETATION: We propose that the mechanisms of demyelination and dynamics of lesion expansion are responsible for differential transcript expression in WM lines and GM lines, and posit that increased expression of the Fc epsilon receptor, spleen tyrosine kinase, and Bruton's tyrosine kinase, play a key role in regulating microglia/macrophage function at the border of chronic active white matter lesions. ANN NEUROL 2024;95:907-916.

Longitudinal analysis of nursing home residents' T cell responses after SARS-CoV-2 mRNA vaccinations shows influence of biological sex and SARS-CoV-2 infection history.

BACKGROUND: Vaccines and vaccine boosting have blunted excess morbidity and mortality from SARS-CoV-2 infection suffered by older nursing home residents (NHR). However, the impact of repeated vaccination on the T cell response based on biological sex and prior infection of NHR remain understudied. METHODS: We examined T cell responses to mRNA vaccines to SARS-CoV-2 in a cohort of NHR and healthcare workers (HCW) over 2 years. We used IFN-γ ELIspot and flow cytometry to assess T cell response before, two weeks and 6 months after the initial series and each of two booster vaccines. We analyzed these data longitudinally with mixed-effect modeling and also examined subsets of our cohorts for additional changes in T cell effector function. RESULTS: We show that prior SARS-CoV-2 infection and female sex contribute to higher T cell response in NHR but not HCW. When looking across time points, NHR but not HCW with prior infection had significantly higher T cell responses than infection-naive subjects. These patterns of response were maintained across multiple booster vaccinations and suggest that the age, multimorbidity, and/or frailty of the NHR cohort may accentuate sex and infection status differences in T cell response to mRNA vaccination.

Loss of function of ribosomal protein L13a blocks blastocyst formation and reveals a potential nuclear role in gene expression.

Ribosomal proteins play diverse roles in development and disease. Most ribosomal proteins have canonical roles in protein synthesis, while some exhibit extra-ribosomal functions. Previous studies in our laboratory revealed that ribosomal protein L13a (RPL13a) is involved in the translational silencing of a cohort of inflammatory proteins in myeloid cells. This prompted us to investigate the role of RPL13a in embryonic development. Here we report that RPL13a is required for early development in mice. Crosses between Rpl13a+/- mice resulted in no Rpl13a-/- offspring. Closer examination revealed that Rpl13a-/- embryos were arrested at the morula stage during preimplantation development. RNA sequencing analysis of Rpl13a-/- morulae revealed widespread alterations in gene expression, including but not limited to several genes encoding proteins involved in the inflammatory response, embryogenesis, oocyte maturation, stemness, and pluripotency. Ex vivo analysis revealed that RPL13a was localized to the cytoplasm and nucleus between the two-cell and morula stages. RNAi-mediated depletion of RPL13a phenocopied Rpl13a-/- embryos and knockdown embryos exhibited increased expression of IL-7 and IL-17 and decreased expression of the lineage specifier genes Sox2, Pou5f1, and Cdx2. Lastly, a protein-protein interaction assay revealed that RPL13a is associated with chromatin, suggesting an extra ribosomal function in transcription. In summary, our data demonstrate that RPL13a is essential for the completion of preimplantation embryo development. The mechanistic basis of the absence of RPL13a-mediated embryonic lethality will be addressed in the future through follow-up studies on ribosome biogenesis, global protein synthesis, and identification of RPL13a target genes using chromatin immunoprecipitation and RNA-immunoprecipitation-based sequencing.

Upregulation of PTPRC and Interferon Response Pathways in HIV-1 Seroconverters Prior to Infection.

Human immunodeficiency virus 1 (HIV-1) exposed seronegative (HESN) individuals may have unique characteristics that alter susceptibility to HIV-1 infection. However, identifying truly exposed HESN is challenging. We utilized stored data and biospecimens from HIV-1 serodifferent couple cohorts, in which couples' HIV-1 exposures were quantified based on unprotected sex frequency and viral load of the partner with HIV-1. We compared peripheral blood gene expression between 15 HESN and 18 seroconverters prior to infection. We found PTPRC (encoding CD45 antigen) and interferon-response pathways had significantly higher expression among individuals who went on to become seropositive and thus may be a signature for increased acquisition risk.

Host transcriptomic signatures of tuberculosis can predict immune reconstitution inflammatory syndrome in HIV patients.

Immune reconstitution inflammatory syndrome (IRIS) can be a complication of antiretroviral therapy (ART) in patients with advanced HIV, but its pathogenesis is uncertain. In tuberculosis (TB) endemic countries, IRIS is often associated with mycobacterial infections or Bacille-Calmette-Guerin (BCG) vaccination in children. With no predictive or confirmatory tests at present, IRIS remains a diagnosis of exclusion. We tested whether RISK6 and Sweeney3, validated immune-based blood transcriptomic signatures for TB, could predict or diagnose IRIS in HIV+ children and adults. Transcripts were measured by RT-qPCR in BCG-vaccinated children and by microarray in HIV+ adults with TB including TB meningitis (TBM). Signature scores before ART initiation and up to IRIS diagnosis were compared between participants who did or did not develop IRIS. In children, RISK6 and Sweeney3 discriminated IRIS cases from non-IRIS controls before ART, and at diagnosis. In adults with TB, RISK6 discriminated IRIS cases from controls after half-week on ART and at TB-IRIS onset. In adults with TBM, only Sweeney3 discriminated IRIS cases from controls before ART, while both signatures distinguished cases from controls at TB-IRIS onset. Parsimonious whole blood transcriptomic signatures for TB showed potential to predict and diagnose IRIS in HIV+ children and adults.

Germinal Center T follicular helper (GC-Tfh) cell impairment in chronic HIV infection involves c-Maf signaling.

We have recently demonstrated that the function of T follicular helper (Tfh) cells from lymph nodes (LN) of HIV-infected individuals is impaired. We found that these cells were unable to provide proper help to germinal center (GC)-B cells, as observed by altered and inefficient anti-HIV antibody response and premature death of memory B cells. The underlying molecular mechanisms of this dysfunction remain poorly defined. Herein, we have used a unique transcriptional approach to identify these molecular defects. We consequently determined the transcriptional profiles of LN GC-Tfh cells following their interactions with LN GC-B cells from HIV-infected and HIV-uninfected individuals, rather than analyzing resting ex-vivo GC-Tfh cells. We observed that proliferating GC-Tfh cells from HIV-infected subjects were transcriptionally different than their HIV-uninfected counterparts, and displayed a significant downregulation of immune- and GC-Tfh-associated pathways and genes. Our results strongly demonstrated that MAF (coding for the transcription factor c-Maf) and its upstream signaling pathway mediators (IL6R and STAT3) were significantly downregulated in HIV-infected subjects, which could contribute to the impaired GC-Tfh and GC-B cell functions reported during infection. We further showed that c-Maf function was associated with the adenosine pathway and that the signaling upstream c-Maf could be partially restored by adenosine deaminase -1 (ADA-1) supplementation. Overall, we identified a novel mechanism that contributes to GC-Tfh cell impairment during HIV infection. Understanding how GC-Tfh cell function is altered in HIV is crucial and could provide critical information about the mechanisms leading to the development and maintenance of effective anti-HIV antibodies.

Differential RNA Transcriptome in Ustekinumab Responders vs Non-Responders.

Despite numerous available psoriasis treatments, no "one size fits all" regimen provides complete disease control without side effects, logistical obstacles, and/or expense. Despite increasingly efficacious drugs, only 20-25% of patients treated with biologic therapies achieve completely clear skin (PASI 100) and even fewer achieve this if they have experienced failures of multiple biologics.

Impaired estrogen signaling underlies regulatory T cell loss-of-function in the chronically inflamed intestine.

Signaling of 17ß-estradiol (estrogen) through its two nuclear receptors, α and ß (ERα, ERß), is an important mechanism of transcriptional regulation. Although ERs are broadly expressed by cells of the immune system, the mechanisms by which they modulate immune responses remain poorly understood. ERß-specific signaling is reduced in patients with chronic inflammatory diseases, including systemic lupus erythematosus and inflammatory bowel disease, and our previous work suggests that dysregulation of ERß-specific signaling contributes to enhanced intestinal inflammation in female SAMP/YitFC mice, a spontaneous model of Crohn's disease-like ileitis. The present study builds on these prior observations to identify a nonredundant, immunoprotective role for ERß-specific signaling in TGF-ß-dependent regulatory T cell (Treg) differentiation. Using a strain of congenic SAMP mice engineered to lack global expression of ERß, we observed dramatic, female-specific exacerbation of intestinal inflammation accompanied by significant reductions in intestinal Treg frequency and function. Impaired Treg suppression in the absence of ERß was associated with aberrant overexpression of Tsc22d3 (GILZ), a glucocorticoid-responsive transcription factor not normally expressed in mature Tregs, and ex vivo data reveal that forced overexpression of GILZ in mature Tregs inhibits their suppressive function. Collectively, our findings identify a pathway of estrogen-mediated immune regulation in the intestine, whereby homeostatic expression of ERß normally functions to limit Treg-specific expression of GILZ, thereby maintaining effective immune suppression. Our data suggest that transcriptional cross-talk between glucocorticoid and steroid sex hormone signaling represents an important and understudied regulatory node in chronic inflammatory disease.

Significant Reduction in Vaccine-Induced Antibody Levels and Neutralization Activity Among Healthcare Workers and Nursing Home Residents 6 Months Following Coronavirus Disease 2019 BNT162b2 mRNA Vaccination.

Antibody decline occurred from 2 weeks to 6 months post-BNT162b2 mRNA vaccination in nursing home (NH) residents and healthcare workers. Antispike, receptor-binding domain, and neutralization levels dropped >81% irrespective of prior infection. Notably, 69% of infection-naive NH residents had neutralizing antibodies at or below the assay's limit of detection.

Macrophage maturation from blood monocytes is altered in people with HIV, and is linked to serum lipid profiles and activation indices: A model for studying atherogenic mechanisms.

People with HIV (PWH) are at increased risk for atherosclerotic cardiovascular disease (ASCVD). Proportions of vascular homing monocytes are enriched in PWH; however, little is known regarding monocyte-derived macrophages (MDMs) that may drive atherosclerosis in this population. We isolated PBMCs from people with and without HIV, and cultured these cells for 5 days in medium containing autologous serum to generate MDMs. Differential gene expression (DGE) analysis of MDMs from PWH identified broad alterations in innate immune signaling (IL-1ß, TLR expression, PPAR ßδ) and lipid processing (LXR/RXR, ACPP, SREBP1). Transcriptional changes aligned with the functional capabilities of these cells. Expression of activation markers and innate immune receptors (CD163, TLR4, and CD300e) was altered on MDMs from PWH, and these cells produced more TNFα, reactive oxygen species (ROS), and matrix metalloproteinases (MMPs) than did cells from people without HIV. MDMs from PWH also had greater lipid accumulation and uptake of oxidized LDL. PWH had increased serum levels of free fatty acids (FFAs) and ceramides, with enrichment of saturated FAs and a reduction in polyunsaturated FAs. Levels of lipid classes and species that are associated with CVD correlated with unique DGE signatures and altered metabolic pathway activation in MDMs from PWH. Here, we show that MDMs from PWH display a pro-atherogenic phenotype; they readily form foam cells, have altered transcriptional profiles, and produce mediators that likely contribute to accelerated ASCVD.

CX3CL1 and IL-15 Promote CD8 T cell chemoattraction in HIV and in atherosclerosis.

Atherosclerotic cardiovascular disease (ASCVD) remains an important cause of morbidity in the general population and risk for ASCVD is increased approximately 2-fold in persons living with HIV infection (PLWH). This risk is linked to elevated CD8 T cell counts that are abundant in atherosclerotic plaques and have been implicated in disease pathogenesis yet the mechanisms driving T cell recruitment to and activation within plaques are poorly defined. Here we investigated the role of CD8 T cells in atherosclerosis in a non-human primate model of HIV infection and in the HIV-uninfected elderly; we sought to identify factors that promote the activation, function, and recruitment to endothelium of CX3CR1+ CD8 T cells. We measured elevated expression of CX3CL1 and IL-15, and increased CD8 T cell numbers in the aortas of rhesus macaques infected with SIV or SHIV, and demonstrated similar findings in atherosclerotic vessels of HIV-uninfected humans. We found that recombinant TNF enhanced the production and release of CX3CL1 and bioactive IL-15 from aortic endothelial cells, but not from aortic smooth muscle cells. IL-15 in turn promoted CX3CR1 surface expression on and TNF synthesis by CD8 T cells, and IL-15-treated CD8 T cells exhibited enhanced CX3CL1-dependent chemoattraction toward endothelial cells in vitro. Finally, we show that CD8 T cells in human atherosclerotic plaques have an activated, resident phenotype consistent with in vivo IL-15 and CX3CL1 exposure. In this report, we define a novel model of CD8 T cell involvement in atherosclerosis whereby CX3CL1 and IL-15 operate in tandem within the vascular endothelium to promote infiltration by activated CX3CR1+ memory CD8 T cells that drive further endothelial activation via TNF. We propose that these interactions are prevalent in aging and in PLWH, populations where circulating activated CX3CR1+ CD8 T cell numbers are often expanded.

Pediatric screen exposure and school related headache disability.

BACKGROUND AND OBJECTIVES: Prolonged screen exposure is often cited as a trigger for pediatric headache. We present initial findings evaluating the association between adolescent screen use type, duration, and school disability. METHODS: New patients aged 12-17 years presenting to a headache clinic were screened and surveyed regarding headache characteristics, behavioral habits, school attendance, and screen utilization. RESULTS: 99 adolescents (29 M, 70 F) with average age 14.8 years and average headache frequency of 17 days per month completed the survey. Patients missed an average of five full days and three partial days of school due to headaches over the 90 days prior to survey completion.No statistically significant correlation was found between type or duration of screen exposure and monthly headache frequency, school attendance, or school functioning. A small positive association was seen between increasing duration of computer use, total hours screen use, and school absenteeism. While most adolescents reported prolonged screen use (58.6%) and luminosity (64.6%) worsened headaches, no statistical difference was seen in average number of headache days per month. CONCLUSIONS: Average monthly headache frequency in an adolescent population was not significantly correlated with type or duration of screen exposure. Further studies are needed to elucidate how screen utilization impacts school related headache disability.

Impact of Early Antiretroviral Therapy Initiation on HIV-Specific CD4 and CD8 T Cell Function in Perinatally Infected Children.

Early initiation of antiretroviral therapy (ART) in vertically HIV-infected children limits the size of the virus reservoir, but whether the time of treatment initiation (TI) can durably impact host immune responses associated with HIV infection is still unknown. This study was conducted in PBMC of 20 HIV-infected virally suppressed children on ART (mean age 9.4 y), classified as early treated (ET; age at ART initiation ≤0.5 y, n = 14) or late treated (LT; age at ART initiation 1-10 y, n = 6). Frequencies and functions of Ag-specific CD4 (CD40L+) and CD8 (CD69+) T cells were evaluated by intracellular IL-2, IFN-γ, and TNF-α production with IL-21 in CD4 or CD107a, granzyme B and perforin in CD8 T cells following stimulation with HIV gp140 protein (ENV) or GAG peptides by multiparameter flow cytometry. ET showed a higher proportion of cytokine-producing ENV- and GAG-specific CD4 and CD8 T cells compared with LT. In particular, ET were enriched in polyfunctional T cells. RNA sequencing analysis showed upregulation of immune activation pathways in LT compared with ET. Our results suggest that timing of TI in HIV-infected children has a long-term and measurable impact on the quality of the HIV-specific T cell immune responses and transcriptional profiles of PBMC, reinforcing the importance of early TI.

Natural History of Abortive Medication Withdrawal in the Management of Pediatric Medication Overuse Headache.

OBJECTIVE: The objective of this study is to document pain scores during withdrawal of abortive medication in patients diagnosed with medication overuse headache. DESIGN: Cross-sectional study. SETTING: Children's National Hospital's Headache Program. SUBJECTS: Patients 6-18 years of age who presented to the Headache Clinic at Children's National Hospital with presumed medication overuse headache between March 2017 and March 2019 were invited to participate. METHODS: Patients were instructed to abruptly discontinue overused medications and record their headache characteristics daily in a diary for 8 weeks. RESULTS: Fourteen diaries were returned and analyzed at a 4-week follow-up visit. Ninety-three percent of the patients were females, with a median age of 14.9 years (standard deviation [SD] = 2.0). The average headache intensity upon study entry was 4.7 out of 10 (SD = 2.5), and the average headache intensity upon study completion was 3.1 (SD = 2.5). Of the patients, 57% had daily headaches upon study entry, 71% had improved pain intensity from the first diary entry to the last diary entry, and 57% had complete headache resolution at an average of 7.6 days from medication discontinuation (SD = 5.1). Ibuprofen was the most overused medication (71%). CONCLUSIONS: Our findings suggest that medication overuse headache will improve in the majority of pediatric patients who abruptly stop the offending medication(s) in an average of 8 days from withdrawal. Average pain intensity was reduced by more than one point among all patients who stopped taking abortive medications. Further larger-scale studies on medication withdrawal in pediatric patients with medication overuse headache could help us better understand whether this management strategy is effective.

Discovery of a Redox Thiol Switch: Implications for Cellular Energy Metabolism.

The redox-based modifications of cysteine residues in proteins regulate their function in many biological processes. The gas molecule H2S has been shown to persulfidate redox sensitive cysteine residues resulting in an H2S-modified proteome known as the sulfhydrome. Tandem Mass Tags (TMT) multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent in detecting cysteine modifications. Here we developed a TMT-based proteomics approach for selectively trapping and tagging cysteine persulfides in the cellular proteomes. We revealed the natural protein sulfhydrome of two human cell lines, and identified insulin as a novel substrate in pancreatic beta cells. Moreover, we showed that under oxidative stress conditions, increased H2S can target enzymes involved in energy metabolism by switching specific cysteine modifications to persulfides. Specifically, we discovered a Redox Thiol Switch, from protein S-glutathioinylation to S-persulfidation (RTSGS). We propose that the RTSGS from S-glutathioinylation to S-persulfidation is a potential mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress.

Levels of Soluble CD14 and Tumor Necrosis Factor Receptors 1 and 2 May Be Predictive of Death in Severe Coronavirus Disease 2019.

People infected with severe acute respiratory syndrome coronavirus 2 display a wide range of illness, from asymptomatic infection to severe respiratory distress resulting in death. We measured serum biomarkers in uninfected individuals and in individuals with mild, moderate, or critical coronavirus disease 2019 (COVID-19) disease. Levels of monocyte activation (soluble CD14 and fatty acid-binding protein 4) and inflammation (tumor necrosis factor receptors 1 and 2 [TNFR1 and TNFR2]) were increased in COVID-19 individuals, regardless of disease severity. Among patients with critical disease, individuals who recovered from COVID-19 had lower levels of TNFR1 and TNFR2 at hospital admission compared to these levels in patients with critical disease who ultimately died.

Drug perturbation gene set enrichment analysis (dpGSEA): a new transcriptomic drug screening approach.

BACKGROUND: In this study, we demonstrate that our modified Gene Set Enrichment Analysis (GSEA) method, drug perturbation GSEA (dpGSEA), can detect phenotypically relevant drug targets through a unique transcriptomic enrichment that emphasizes biological directionality of drug-derived gene sets. RESULTS: We detail our dpGSEA method and show its effectiveness in detecting specific perturbation of drugs in independent public datasets by confirming fluvastatin, paclitaxel, and rosiglitazone perturbation in gastroenteropancreatic neuroendocrine tumor cells. In drug discovery experiments, we found that dpGSEA was able to detect phenotypically relevant drug targets in previously published differentially expressed genes of CD4+T regulatory cells from immune responders and non-responders to antiviral therapy in HIV-infected individuals, such as those involved with virion replication, cell cycle dysfunction, and mitochondrial dysfunction. dpGSEA is publicly available at https://github.com/sxf296/drug_targeting . CONCLUSIONS: dpGSEA is an approach that uniquely enriches on drug-defined gene sets while considering directionality of gene modulation. We recommend dpGSEA as an exploratory tool to screen for possible drug targeting molecules.