bluuebunny/crossref_metadata_2025 huggingface dataset:
Defining a role for PARP1 in driving RSV induced pulmonary pathology
Authors: Mire Mohamed , Elesela Srikanth , Rasky Andrew , Morris Susan , Lukacs Nicholas
Published: August 2023

The mechanisms underlying how Respiratory Syncytial Virus (RSV) subverts dendritic cell (DC) immune function to enhance pathology are not well defined, but we have recently shown RSV alters DC metabolism. Analysis of Reverse Phase Protein Array (RPPA) data, which identifies phosphorylated cytoplasmic proteins (>400 in the analysis), revealed one of the most differentially activated signaling molecules, PARP1, had increased activation (phosphorylation) in RSV infected DC. PARP1 is a DNA damage enzyme that, in an NAD+ depleting manner, polymerizes deposition of poly-ADP-ribosylation (PAR) units in cellular proteins, possibly altering their physiological properties. Our data shows genomic ablation of PARP1 negates the capacity of RSV to manipulate DC metabolism at the early stage of infection. Enzymatic inhibition or genomic ablation of PARP1 resulted in increased key innate immune cytokine expression including type I interferon and IL-12, which together promote a more appropriate anti-viral environment. PARP1−/− mice and PARP1 inhibitor treated mice were protected against RSV induced immunopathology including airway inflammation and mucus hypersecretion as indicated by PAS staining of lung tissue. Assessment of isolated airway epithelial cells demonstrated genomic depletion of PARP1 did not alter RSV induced innate cytokines, suggesting predominately an immune cell effect upon RSV infection. Together, these data highlight PARP1 may be a logical target for therapeutic intervention during RSV infections to diminish the immunopathology and reduce disease severity.

Supported by grants from NIH (R35 HL150682, R01 AI138348)
Fibrinogen is increased in the spleen over the course of murine endotoxemia
Authors: Nunez Kathryn Elizabeth , Hunat Junela Cecile , Falguni Mehnaz M , Harberts Erin M
Published: August 2023

The fibrinogen protein is composed of three subunits encoded for by the genes Fga, Fgb, and Fgg. This trimer is a main component of blood clots. Irregular blood clotting contributes to low blood pressure during septic shock, which eventually leads to death from total system organ failure. A previously published data set finds Fga, Fgb, and Fgg to be dysregulated during sepsis in the serum of both wildtype (WT) and toll-like receptor 4 (TLR4) deficient mice. Deficiency in the innate immune receptor TLR4 models immune ataxia that occurs in patients who recover from septic shock, surviving patients have an increased risk of mortality due to secondary infection. We hypothesize that fibrinogen contributes to symptoms of septic shock independent of the TLR4 pathway. In this study we confirm that fibrinogen is transcriptionally upregulated in the liver of both WT and TLR4−/− mice over the course of LPS induced endotoxemia. Western blots were completed using organ lysates from liver, spleen, kidney, and brain of WT and TLR4−/− mice from 0, 6, 12, and 18 hours after induction of endotoxemia with intraperitoneal injection of 30 mg/kg lipopolysaccharide (LPS). Western blots show that WT and TLR4−/− control mice express similar amounts of fibrinogen in organs, and that there is more fibrinogen in the liver than the spleen, which is consistent with current literature. Western blots conducted with endotoxemic samples show that over the course of disease in WT mice, fibrinogen levels are consistent in the liver but increase in the spleen. Continued experiments in TLR4−/− mice will determine the level of TLR4-independent fibrinogen activation during sepsis and provide evidence for use of serum clotting modulators in patients who have recovered from septic shock.

This study is supported by a Towson University FDRC grant awarded to EMH, a Fisher College of Science and Mathematics undergraduate research award to KEN, and the 2022 Linda Sweeting Summer research fellowship awarded to KEN by the Towson University Department of Chemistry.
Repurposing FDA-approved drugs to inhibit IL-33 mediated mast cell function
Authors: Long Dionna L , Burchett Jason R , Ryan John J
Published: August 2023

IL-33 is an IL-1-family cytokine that is released from fibroblasts, epithelial, endothelial, and some immune cells in response to tissue damage or inflammatory signals. IL-33 activates mast cells, innate leukocytes that play a key role in asthma. IL-33 induces production of inflammatory cytokines that worsen allergic diseases like asthma. It is essential to better understand IL-33 function and how to suppress it. Computer modeling studies using the SYBYL and GOLD algorithms to screen 3035 FDA-approved drugs for interactions with the IL-33 receptor, ST2, at the IL-33 binding site revealed several potential ST2 antagonists. In this study, we show that mouse bone marrow derived mast cells (BMMCs) treated with the antiviral drug lopinavir had diminished IL-33 responsiveness. Specifically, lopinavir (15mM) significantly reduced IL-33-mediated IL-6 and IL-13 secretion. Lopinavir effects noted when the drug was given simultaneously with IL-33 and were maximal when given 1 hour prior to IL-33 and suppression. These rapid effects suggest a direct drug-receptor interaction. We treated BMMCs with drug for up to 24 hours to assess for alternative mechanisms such as ST2 down-regulation, which was not noted. Other candidate drugs included clindamycin, which had no effect on IL-33-mediated cytokine production, and ritonavir and cobcistat, which increased cytokine secretion. Our data demonstrate that lopinavir can inhibit IL-33 mediated mast cell function, likely as an off-target effect of the drug. These data support further investigation of lopinavir and suggest the utility of repurposing FDA-approved drugs in allergy and asthma treatment.
Linking CD36-dependent lipid processing to the alveolar macrophage transcriptional program via GATA2
Authors: Jackson-Strong Morgan , Shoger Karsen E , Michalides Brandon Alan , Kruszelnicki Sonia M , Kohan Alison B , Gottschalk Rachel A
Published: August 2023

Alveolar macrophages (AMs) catabolize lipid-rich pulmonary surfactant to support gas exchange and have steady-state anti-inflammatory functions to limit lung tissue damage in response to minor challenge. While it is well appreciated that the lung microenvironment shapes tissue-specific AM function and that this linked with lipid metabolism, little is known about the persistent signaling events that support these functional and metabolic programs. We recently demonstrated that the absence of CISH, a suppressor of cytokine signaling (SOCS) family member, led to a lipid-laden, foamy AM phenotype and the accumulation of pulmonary surfactant. These changes were associated with increased AM expression of the transcription factor GATA2 and RNA-seq analysis of Cish−/− AMs revealed dysregulated expression of lipid-processing genes and increased expression of genes enriched for GATA2-binding motifs. While GATA TFs have been implicated in anti-inflammatory macrophage function and in dysregulation in of lipid-laden athlerosclerotic plaque macrophages, their function in AMs is largely unknown. Our preliminary data suggest that lipid uptake through the scavenger receptor CD36 enhances GATA2 activity and limits AM pro-inflammatory function. We are currently investigating mechanisms by which pulmonary surfactant lipids regulate GATA2 and AM programming, using mice with myeloid specific deletion of CD36; our data suggest that AMs from these mice are impaired in their ability to uptake DPPC, the primary lipid species in surfactant. Defining signaling processes that link CD36 with GATA2 and AM transcriptional and metabolic programs will provide insight into lung homeostasis and support our ability to manipulate AMs therapeutically.

R01 HL162658
Innate immune responses to enteric Escherichia coli dictate neonatal sepsis outcomes
Authors: Muske Josey , Greenfield Kara G , Knoop Kathryn A
Published: August 2023

Late-onset neonatal sepsis (LOS), a leading cause of neonatal death, can result from pathogenic bacteria disseminating to the bloodstream from the intestinal microbiota. E. coli is the second most commonly isolated pathogen causing LOS. During sepsis, there are significant immune discrepancies between infants and adults, including elevated IL-6 levels in neonates, demonstrating the need for age-specific research. We have developed a murine model of LOS that uses clinical isolates of gut-resident E. coli, which results in rapid pro-inflammatory cytokine production within 6 hours and neonatal mortality within 2–3 days post-infection. Not all enteric E. coli results in inflammation and mortality, and we have classified isolates as “commensal” or “pathogenic”. TLR4−/− mice infected with pathogenic E. coli have a delayed mortality to sepsis, suggesting innate bacterial recognition is required for acute inflammation. To further determine the source of this delay, we have begun investigating signaling pathways during bacterial infection. We found the immune signaling pathway NFκB/AP1 is consistently activated in murine macrophages by commensal and pathogenic bacterial strains. However, only pathogenic bacteria induce elevated IL-6 production by murine macrophages. Interestingly, this response required live bacteria, suggesting lipopolysaccharide alone from E. coli is not sufficient to recapitulate LOS inflammation. Our future directions will explore alternative signaling pathways that are activated uniquely during pathogenic infection and induce a robust inflammatory response. Through this work, we will determine the consequence of specific pro-inflammatory cytokines in neonatal mice during pathogenic infection.

Research supported by R03 DK122187.
The nature of the stimulant differentially impacts the innate memory response of monocytes to Mycobacterium tuberculosis
Authors: Subbian Selvakumar , Kumar Ranjeet , Kolloli Afsal , Singh Pooja
Published: August 2023

Innate immune cells, such as monocytes, can generate a non-specific ‘memory’ response (a.k.a. trained immunity) upon stimulation with an antigen/bacterium (e.g., LPS or BCG), which confers heterologous protection against another infectious agent. However, the association between the nature of stimulants and the corresponding immune correlate of trained immunity is not fully understood. Similarly, the response of macrophages “trained” by various stimulants to subsequent infection by different clinical Mycobacterium tuberculosis (Mtb) strains remains unknown. Here, we report the trained immunity profile of the blood-derived monocytes (BDM) of BCG-vaccinated adults and monocytic THP-1 cells after stimulation with BCG, LPS, Purified Protein Derivative (PPD), heat-killed MtbHN878 or MtbCDC1551 strains. The response of monocytes trained by these agents to subsequent infection with clinical Mtb isolates HN878 and CDC1551, which differ in their virulence potential, was evaluated using standard molecular and cellular immunologic assays. The BCG- and LPS-trained macrophages produced higher proinflammatory, antimicrobial responses, and autophagy induction than unstimulated, PPD, HK-HN878, or CDC1551-stimulated cells. The HN878-infected trained THP-1 cells elicited a more robust inflammatory response than the CDC1551 infection. A significantly reduced intracellular Mtb growth was noted in the BCG and LPS-trained THP-1 cells, which was augmented by adding rapamycin, an autophagy inducer. A similar immunological response was noted in the BDM of BCG-vaccinated adults infected with these Mtb strains. Thus, the nature of the stimulant affects the macrophages’ trained immunity profile and its response to Mtb infection.

Supported by grants from NIH (R01 AI161822) and the Bill and Melinda Gates Foundation (INV-009099; formerly OPP1157210).
Investigating the role of BHLHE40 in host responses to Mycobacterium tuberculosis infection and myeloid cell development
Authors: Hendrix Skyler V , Mreyoud Yassin , Nehls Michael , Smirnov Asya , Bradstreet Tara , Webber Ashlee , Edelson Brian T , Stallings Christina L
Published: August 2023

We currently lack a complete understanding of the immune responses required to control Mycobacterium tuberculosis (Mtb) infection. Our lab has shown that the transcription factor BHLHE40 is essential during Mtb infection. Deletion of Bhlhe40 in CD11c+ lung macrophages and dendritic cells (DCs) is sufficient to increase mouse susceptibility to Mtb infection, though the role of BHLHE40 in these cells during infection is unknown. Murine bone marrow cultured in the presence of GM-CSF, an abundant cytokine in the lung, develop into populations of CD11c+ macrophages, DCs, and granulocytes, all of which express Bhlhe40. Using this system, we examined cytokine and transcriptional responses before and after exposure to heat-killed Mtb to understand how BHLHE40 impacts cell development and inflammatory responses. We found that in WT mice, GM-CSF-cultured cells develop into 9 distinct populations and expression of Bhlhe40 was required for GM-CSF-mediated pro-inflammatory cytokine and transcriptional programs. Additionally, loss of Bhlhe40 led to decreased DCs accompanied by upregulation of the transcription factor C/EBPβ in DC progenitor populations, which negatively impacts DC development. ChIP-seq analysis revealed BHLHE40 binds directly to C/EBPβ’s gene locus, indicating that BHLHE40 may directly repress Cebpb. In contrast, loss of Bhlhe40 did not affect macrophage development, but led to a transcriptional shift towards an anti-inflammatory state. These findings demonstrate that in the presence of GM-CSF, BHLHE40 is required for DC development and inflammatory macrophage polarization. Defects in either of these aspects could contribute to the essential role for BHLHE40 in lung macrophages and DCs during Mtb infection in vivo.
Determining the fate of pulmonary iILC2s in helminth infection
Authors: Kashyap Amita , Miller Mindy M , Reinhardt R Lee
Published: August 2023

Group 2 Innate Lymphoid Cells (ILC2s) are innate counterparts to Th2 cells and key early responders in helminth infection. ILC2s have two subpopulations: tissue-resident “natural” nILC2s, present in the lungs at homeostasis, and “inflammatory” iILC2s, which appear transiently in the lungs during the response to helminth infection. We aim to determine the fate of pulmonary iILC2s and hypothesize that pulmonary iILC2s acquire an nILC2-like phenotype and make biologically relevant long-term contributions to the tissue-resident ILC2 pool. Early studies demonstrated that iILC2s can convert to an nILC2 phenotype, but the prevailing view in the field is that the nILC2 population is maintained through self-renewal. However, fate-mapping models show an external contribution to the pulmonary ILC2 population which corresponds with an influx of iILC2s. We have further characterized the pulmonary ILC2 population during helminth infection and found an intermediate-phenotype population 8 days post infection. Preliminary flow cytometry and scRNA-seq data from congenic transfer experiments indicate that iILC2s may acquire an nILC2 phenotype in vivo. Preliminary multiple infection experiments indicate that mice lacking a pulmonary iILC2 response have smaller nILC2 compartments compared to wildtype counterparts after 3 infections. We are further testing our hypothesis using fate-mapping models and congenic transfer experiments in conjunction with single-cell sequencing approaches to more rigorously confirm these findings. Understanding the contribution of iILC2s to the long-term tissue-resident ILC2 population could lead to therapies in diseases with ILC2 involvement including helminth infection and steroid-resistant asthma.

Supported by NIH grant R01 AI56901-0
Leukocyte associated immunoglobulin-like receptor 1 modulates immune response to Staphylococcus aureus infection
Authors: Dorando Hannah K , Rich Helen , Warheit-Niemi Helen , Mutic Evan , Tomaszewski Kelly , Tian Ling , Wurtz Mellisa , Quinn Chaz , Veis Deborah , Wardenburg Juliane Bubeck , Moore Bethany B , Payton Jacqueline E
Published: August 2023

Immunity is a balance of maximal pathogen defense with minimal host damage. Inhibitory immune receptors are important for maintaining this balance. We recently identified elevated levels of leukocyte-associated immunoglobulin (Ig)-like receptor protein1 (LAIR1), an inhibitory immune receptor, in cutaneous lymphoma patients, in whom invasive Staphylococcus aureus (S. aureus) infections are frequent. We therefore tested Lair1 knock-out (KO) mice in two models of invasive S. aureus infection: pneumonia and subcutaneous skin infection. In WT mice, skin infections resolve by 14 days post-infection (dpi), while pneumonia infections cause >50% lethality by 2dpi. In both models, Lair1 KO mice showed consistent patterns of myeloid hyper-activation. Lair1 KO had consistently larger skin lesions than WT throughout the 14 days, but no difference in bacterial clearance. Lair1 KO with pneumonia had higher survival (100% KO vs. 33% WT) despite comparable measures of lung injury and immune cell recruitment. In both models, KO mice produced higher levels of proinflammatory cytokines (IL-6, IL-1b) and neutrophil chemokines (CCL2, 3, 4) in lung, bronchoalveolar lavage, and skin by multiplex cytokine array. KO bone marrow-derived macrophages infected with S. aureus in vitro also secreted higher levels of proinflammatory cytokines (IL-6) and neutrophil chemokines (CCL2, 3, 4). Together, our results demonstrate that LAIR1 loss enhances myeloid activation, resulting in excessive tissue damage in skin infection, yet is protective against death in pneumonia. We posit that LAIR1 plays a crucial role in balancing myeloid immune response; LAIR1 activity can be beneficial or detrimental to the host depending on the site and type of infection.
Antibacterial functions of neutrophils against cystic fibrosis isolates of Staphylococcus aureus are compromised by short chain fatty acids
Authors: Rada Balazs , Miller Arthur , Fantone Kayla , Tucker Samantha L , Goldberg Joanna B
Published: August 2023

Short chain fatty acids (SCFA) are generated by anaerobic bacteria as byproducts of their anaerobic fermentation at mucosal sites. The most common SCFAs are acetate, propionate and butyrate. SCFAs have been implicated in several inflammatory diseases including cystic fibrosis (CF). In CF airways SCFAs are present at millimolar concentrations. CF is characterized by lung inflammation dominated by neutrophils and polymicrobial bacterial infections made up of facultative anaerobes, including Staphylococcus aureus. In mammalian organisms, neutrophils represent the main arm of the immune system to fight S. aureus. The reason why neutrophils are unable to clear respiratory pathogens including S. aureus, in CF remains though unclear. Based on published data and our prior results, we hypothesized that SCFAs compromise the ability of human neutrophils to kill S. aureus in vitro. SCFAs do not affect the viability of neutrophils or in vitro S. aureus growth. Our data show that SCFAs do not stimulate release of neutrophil extracellular traps in human neutrophils either. On the other hand, superoxide production, another important effector function of activated neutrophils against S. aureus, was significantly inhibited by SCFAs. SCFAs also compromised the ability of neutrophils to kill some CF isolates of S. aureus in vitro. Overall, our results provide new knowledge on the interactions between SCFAs and the innate immune system, and suggest that SCFAs produced by anaerobic bacteria in the CF lung could interfere with the ability of neutrophils to kill S. aureus, a prominent respiratory pathogen in this disease.

This work was supported by grants provided by the National Institutes of Health (R01HL136707 to B.R. and R21AI154343 to B.R. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Administration of Δ8-tetrahydrocannabinol enhances lung function and attenuates neutrophiles infiltration in a preclinical model of acute respiratory distress syndrome
Authors: Aladhami Ahmed Khaleel , Wilson Kiesha Maria , Carter Tayler , Nagarkatti Prakash , Nagarkatti Mitzi
Published: August 2023

Acute respiratory distress syndrome ARDS, characterized by severe hypoxemia, is an inflammatory response leads to high mortality rate. Patients with ARDS, such as some COVID-19 patients, suffer from pulmonary alveolar damage and major oedema due to diffuse infiltration of immune cells. Neutrophils are predominantly elevated in the bronchoalveolar lavage fluids of ARDS patients. Our laboratory already reported that Δ9-tetrahydrocannabinol (THC) administration effectively attenuated lung inflammation, cytokine storm, and prevents mortality in ARDS. In current study, we tested the effect of Δ8-THC, a cannabinoid which is less psychoactive than Δ9-THC, in lipopolysaccharide-endotoxin (LPS)-induced model of ARDS. Our data showed that Δ8-THC exhibited improvement of pulmonary function tests and decreased bronchoconstriction index in freely conscious-tested animals accompanied with significant decreasing in immune cells infiltration, specifically neutrophiles, and decreasing of serum tumor necrosis factor alpha (TNFα) level. Interestingly, Single cell RNA sequencing analysis of pulmonary cell tSNE clusters showed that Δ8-THC dramatically reduced neutrophils clusters and dysregulates certain genes involved in movement of neutrophils and chemokines production such as CCl6, CCl9, Cxcl3, Cxcl10, and CCl5. Together, these findings suggest that Δ8-THC mitigates ARDS by preventing neutrophiles trafficking into the lungs, and thus, may serve as a therapeutic agent to treat ARDS.

This work was supported in parts by NIH grants R01ES030144, P01AT003961, P20GM103641, and R01AI123947, R01AI160896
Immunoregulatory Roles of TGF-β and IL-4 during Mouse-adapted SARS-CoV-2 Infection
Authors: Sima Solomon Taye , Puebla-Clark Lucinda , Gonzalez-Orozco Maria , Shelite Thomas R , Federman Hannah G , Huante Matthew B , Martinez-Martinez Yazmin Berenice , Dann Sara M , Endsley Mark A , Siracusa Mark C , Menachery Vineet D , Endsley Janice J , Rajsbaum Ricardo , Stephens Robin
Published: August 2023

Severe COVID-19 is due to a dysregulated immune response; however, the mechanisms preventing recovery of airway function are unclear. Cytokines regulate the quality of inflammation, and drive both anti-viral and disease tolerance mechanisms. There was a mostly monocytic infiltration in the lungs with alveolar thickening and consolidation 3 days post-infection with mouse-adapted SARS-CoV-2 (CMA3p20) at the peak of weight loss. Based on the cytokine profiles observed in human and animal studies, we targeted IL-4 and TGF-β as potentially regulatory cytokines, and quantified changes using flow cytometry. Neutralizing TGF-β, which regulates inflammatory cytokine production, significantly increased cellular infiltration of the lung. Neutralization of TGF-β particularly affected inflammatory monocyte (Ly6C+) recruitment, with a slight decrease in alveolar macrophages (CD11c-int). Neutralizing IL-4 prolonged weight loss to 4 dpi but visibly improved resolution of alveolar thickening on 7 dpi, suggesting that IL-4 is protecting from prolonged peak pathology, but delaying resolution. Neutralizing IL-4 also increased the fraction of polymorphonuclear cells and Tregs, as well as improving the ratio of type II to type I alveolar epithelial cells in the lungs compared to the isotype group. To begin to understand mechanisms connecting IL-4 to epithelial repair, we are studying the quality of the monocyte response and found alternatively activated macrophages with Ym1 staining in activated macrophages in the alveoli of infected mice in an IL-4 dependent manner. In order to improve therapeutics for severe COVID-19, it will be important to determine the cellular mechanisms linking regulatory cytokines to epithelial repair.

R01AI135061 (RS, LPC), R01AI166668 (RR, MGO), R01AI131634 (MCS, HGF)
IL-13 protects epithelial cells from SARS-CoV-2 infection by inhibiting early ACE2-mediated events
Authors: Pivniouk Vadim I , Pivniouk Oksana , Uhrlaub Jennifer , Molzahn Allyson , Kimura Hiroki , Ledford Julie , Kraft Monica , Nikolich-Zugich Janko , Vercelli Donata
Published: August 2023

Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2) infection is mediated by binding of viral spike (S) protein to membrane ACE2 on host epithelial cells, preferentially in the airway. Asthmatics with type 2 (T2) inflammation are reportedly less susceptible to disease, but the underlying mechanisms are unclear.

We recently showed that ACE2 expression is decreased in airway epithelial cells from T2 asthmatics and is consistently suppressed in human primary epithelial cells cultured with IL-13 (PMID: 32422146). To dissect IL-13/SARS-CoV-2 interactions, we assessed the effects of IL-13 on discrete events in SARS-CoV-2 infection. S1 protein binding to green monkey kidney Vero E6, human lung Calu-3 epithelial cells and HEK293T cells stably transfected with human ACE2 (hACE2-HEK293T) was measured by flow cytometry with His-tagged S1 protein. S protein-mediated viral entry into Vero E6 and hACE2-HEK293T cells was assessed using S protein-pseudotyped lentiviral particles carrying a GFP reporter. ACE2 transcription in Vero E6, Calu-3 and primary human airway epithelial cells was measured by RT-qPCR. ACE2 expression on Vero E6, Calu-3 and hACE2-HTK293T cells was evaluated by flow cytometry. USA-WA1/2020 SARS-CoV-2 was used to infect Calu-3 cells.

IL-13 strongly inhibited S protein binding, cell entry, and infection of epithelial cells by SARS-CoV-2. These effects resulted at least in part from IL-13-dependent inhibition of ACE2 expression because IL-13 did not affect S protein binding to, and S protein-pseudotyped lentivirus entry into, hACE2-HEK293T cells that do not downregulate ACE2 in response to IL-13. We suggest that IL-13 protects epithelial cells from SARS-CoV-2 infection by inhibiting early ACE2-mediated events.

Supported by grant from NIAID: U19 AI125357
Understanding of SARS-CoV-2 pathogenicity in COVID-19 cynomolgus macaque model and consideration of its reinfection
Authors: Urano Emiko , Okamura Tomotaka , Yasutomi Yasuhiro
Published: August 2023

Although rapid vaccination has been progressing in the world as a countermeasure against COVID-19, the pandemic of SARS-CoV-2 is still raging worldwide due to the continuous emergence of several variants. We have established the COVID-19 cynomolgus macaque (CM) model using healthy young CMs and advanced-age CMs and the differences observed between the pathologies of young and elderly macaques were similar that those seen in humans.

In the present study, we analyzed viral dynamics and pathology of SARS-CoV-2 variants of concern (VOCs) from Wuhan to Omicrons using the COVID-19 CM model. Delta infection displayed a greater virulence and Omicron infection showed a lesser virulence. Elucidation of how adaptive immunity in patients is induced by SARS-CoV-2 natural infection is critical for establishing a vaccine strategy, serologic therapies, public health control and prediction of infection. Therefore, we conducted the prolonged rechallenge analysis using the COVID-19 CM model reinoculated with either the same or a different strain of SARS-CoV-2 that was used for the initial infection. Re-infection was confirmed between different virus strains and PCR re-positivity was also observed between same VOC strain in some cases. Anti-RBD antibodies against Wuhan, Alpha, and Delta, were well induced by each VOC infection, but not against Omicrons. Antigen-specific cellular responses are also currently being analyzed.

Overall, studies using COVID-19 CM models should contribute to the elucidation of the complicated pathophysiology of COVID-19. In addition, COVID-19 CM model should be useful for understand the virus evolution and to consider vaccine strategy to overcome the COVID-19 pandemic.

AMED JP21fk0108414 AMED JP223fa727002 JSPS 21K19395
Peering into the future of SARS-CoV-2 evolution using a computational model of the SARS-CoV-2 serum antibody repertoire
Authors: Cohen-Lavi Liel , Koren Eilay , Yegorov Yan , Sacharen Sinai , Burkovitz Anat , Hertz Tomer
Published: August 2023

The global emergence of SARS-CoV-2 has provided a wealth of immunological data, including more than 600 solved structures of SARS-CoV-2 Spike antibodies (Abs) available in the Protein Data Bank. We reasoned that this data could be used to generate a computational model of the serum Ab repertoire. We extracted a set of 275 non-redundant Abs, and clustered them using an unsupervised clustering method based on their contact positions with Spike. We identified 9 unique Ab clusters that together summarize the major binding modes of Abs to the Spike protein. To characterize each cluster, we mined 65 published papers extracting detailed information on each Ab. We then demonstrated Spike Ab contacts could be predicted based on the Ab’s CDRs sequence. Next, we developed a computational method for predicting immune escape. We extracted the set of all Spike mutations in SARS-CoV-2 sequences from GISAID. To assess the effects of single mutations on immune escape, we computed the ΔΔG for each Ab-mutation pair and constructed an aggregated escape score for each mutation. Our predicted escape scores were positively correlated with published experimental measurements of Ab escape. We used our escape score to rank novel viral variants and are dynamically ranking strains and novel variants of interest as they appear. To further validate its predictive power, we chose a set of predicted Ab-escaping mutations and are testing their escape from Abs in human sera samples. Taken together, our analysis demonstrates the feasibility of generating a computational model of the serum Ab repertoire, which may be used for the rapid characterization of novel Abs, and to computationally estimate the effects of mutations on immune escape from Ab recognition.

Supposed by the Israel Science Foundation (ISF) grant no. 2683/21, and the NIH\NIAID CEIRR under contract 75N9302100016, St Jude Children’s Research Hospital.
A new mouse model expressing human ACE2, TMPRSS2, and FCGRT for evaluating human antibody responses to SARS-CoV-2
Authors: Ana-Sosa-Batiz Fernanda , Verma Shailendra Kumar , Shafee Norazizah , Miller Robyn , Conner Chris , Hastie Kathryn M , Timis Julia , Maule Erin , Nguyen Michael N , Saphire Erica Ollman , Shresta Sujan
Published: August 2023

Protection against viruses is most often measured by levels of plasma binding and neutralizing antibodies (Abs) in vitro, which do not capture the full picture of Ab immunity, including Fc functions. The continuing evolution of SARS-CoV-2 underscores the need to understand protection mediated by Abs. We generated triple knock-in (TKI) mice expressing human ACE2, TMPRSS2, and FCGRT in place of murine genes. A human anti-SARS-CoV-2 IgG1 with a half-life extending mutation but not wild-type IgG1 exhibited long half-life and protected against BA.2 infection in lungs, thereby validating our TKI mice for evaluating human Ab clearance and function. We next evaluated protective efficacy of plasma samples from vaccinated San Diegans against Delta or BA.5 by performing passive transfer experiments. Treatment with pooled plasma from vaccination plus infection but not triple vaccination individuals cleared infectious Delta in lungs of TKI mice, although Delta-binding and -neutralizing Ab titers in vitro were similar between the two groups of people. In contrast, neither group’s plasma impacted lung viral burden upon BA.5 challenge. Thus, Abs elicited by vaccination plus infection but not triple vaccination reduce Delta infection in this mouse model, and Abs from both groups are ineffective against BA.5. These results are consistent with epidemiological data, and show that in vitro Ab titers do not predict in vivo protection. Our TKI mouse model represents an important tool to test efficacy of human monoclonal Abs against SARS-CoV-2/related CoVs, predict human polyclonal Ab-mediated protection against newly emerging variants/CoVs, and dissect mechanisms of Ab-mediated protection in vivo. Results will aid development of pan CoV vaccines.
Phenotypical and Functional Characterization of Unswitched Memory B cells verses Switched during SARS-CoV-2 Infection
Authors: Castleman Moriah J , Santos Adriana Luna , Lesteberg Kelsey , Maloney James , Janssen William , Mould Kara , Beckham J David , Pelanda Roberta , Torres Raul M
Published: August 2023

Memory B cells are comprised of unswitched (CD27+IgD+) and switched (CD27+IgD-) subsets. The origin and function of unswitched human memory B cells remains controversial, whereas switched memory B cells are primed to respond to recurrent infection. Unswitched memory B cells were described to be reduced in frequency with severe SARS-CoV2 infection and here we report on their activation status, BCR functionality, and contribution to virally-induced cytokine production. Analyses of whole blood from healthy individuals, people immunized against SARS-CoV2, and those with mild or severe SARS-CoV2 infection, confirm a loss of unswitched memory B cells during severe SARS-CoV2 infection and demonstrate this loss is associated with increased levels of systemic TNFα. We further report an increased frequency of ‘IgD+’ only memory B cells that correlate with an increase in IgG autoantibody levels (anti-chromatin, anti-cardiolipin, anti-smith) in severe SARS-CoV2 infection. Unswitched and switched memory B cells from severe SARS-CoV2 infection displayed evidence of heightened activation with concomitant reduction in expression of the inhibitory receptor CD72. Functionally, both populations of memory B cells from severe SARS-COV2 infection harbored a signaling competent BCR that displayed enhanced activity in the unswitched population. Finally, we demonstrate that B cells from mild SARS-CoV2 infection secrete pro-inflammatory cytokines IL-6 and TNFα. Importantly, unswitched memory B cells were a major producer of IL-6 and switched memory B cells were a major producer of TNFα in response to viral TLR ligands. Together these data suggest that B cells contribute to the inflammatory milieu during viral infection.

Supported by grants from NIH R01 AI124474 (RP) and R01 AI136534 (RMT).
Pre-existing SARS-CoV-2-reactive CD4+ T cells are established and highly functional in early childhood
Authors: Olofsson Anna , Humbert Marion , Niessl Julia , Hodcroft Emma , Gao Yu , Sohlberg Ebba , Malmberg Karl-Johan , Lund-Johansen Fritjof , Björkhem-Bergman Linda , Jenmalm Maria C , Ljunggren Hans-Gustaf , Buggert Marcus , Karlsson Annika C
Published: August 2023

The pre-existing SARS-CoV-2-reactive T cells identified in unexposed individuals are likely induced by prior infections with seasonal coronaviruses and may modulate disease outcomes. By assessing humoral and cellular immunity in paired samples from SARS-CoV-2 unexposed children at ages 2 and 6 (n=16), and SARS-CoV-2 seronegative adults (n=94, ages 26–83), this study aims to elucidate how age influences the immune responses against coronaviruses.

Using flow cytometry, the phenotype and function of seasonal coronavirus OC43-specific and pre-existing SARS-CoV-2-reactive memory (m) CD4+ T cells were characterized after a 10h stimulation with viral peptides. The frequency of SARS-CoV-2-reactive mCD4+ T cells were closely linked with the OC43-specific responses in all age groups. However, a positive response to either OC43- or SARS-CoV-2-derived peptides was more common at age 6 than in adults. Similarly, the functional capacity, defined by IFN-γ, IL-2 and TNF expression, of both OC43-specific and SARS-CoV-2-reactive mCD4+ T cells peaked at age 6 and then declined with age. Age-dependent phenotypic markers were also identified, including CD226, TIGIT and CD38. Lastly, in contrast to Rhinovirus, EBV, Influenza and other coronaviruses, OC43 seroprevalence did not increase between ages 2 and 6, or from childhood to adulthood, further establishing OC43 as a childhood infection.

Our results show that memory responses to coronaviruses develop at a young age and exhibit age-dependent qualities, findings which may aid in future vaccine development. In addition, the high prevalence and functional capacity of pre-existing SARS-CoV-2-reactive mCD4+ T cells at age 6 may contribute to the low incidence of severe COVID-19 in this age group.
Autoantibodies produced by SARS-CoV-2 infection trigger damage to the lacrimal and salivary glands and lead to the development of Sjögren’s Disease
Authors: Shen Yiran , Voigt Alexandria , Goranova Laura , Abed Mehdi , Kleiner David , Maldonado Jose , Beach Margaret , Pelayo Eileen , Chiorini John , Craft William , Ostrov David A , Ramiya Vijay , Warner Blake , Nguyen Cuong
Published: August 2023

Sjögren’s Disease (SjD) is a chronic, systemic autoimmune disease characterized by lymphocytic infiltration and the development of dry eyes and mouth as the result of the secretory dysfunction of the lacrimal and salivary glands. In recent years, infectious agents have been shown to be associated with SjD. Several studies indicated that SARS-CoV-2 infection induces autoantibodies production, patients also experienced various autoimmune disorders. This study used SARS-CoV-2 WA1/2020 to infect hACE2 mice to investigate the relationship between SARS-CoV-2 infection and SjD. Infected mice showed dry eyes, decreased salivary flow, lymphocytic infiltration of the lacrimal and salivary glands, elevated antinuclear antibodies (ANA), specifically anti-SSB/La, and apoptotic cells near the acinar cells. Notably, counting of apoptotic cells showed a significantly higher number in the lacrimal glands of mice infected with a low viral load than high viral load. We confirmed with clinical data that we observed the elevation of SjD-specific autoantibodies (ANA, anti-SSB/Ro52, and anti-SSA/La) in sera from COVID-19 patients. Salivary gland biopsies of patients diagnosed with SjD several months after SARS-CoV-2 infection also showed lymphocytic infiltration and fibrosis. Lastly, to identify whether monoclonal antibodies produced by COVID-19 patients are reactive against nuclear antigens, antibodies tested for greater antiviral binding capacity were shown to be more responsive. Overall, by observing SjD-like phenotypes in mouse models and patients, our study confirms a direct pathogenic role of SARS-CoV-2 in SjD. It also provides a different pilot for the study of the mechanisms of autoimmune responses induced by viral infections.

This research was funded by the National Institutes of Health (NIH), National Institute of Dental and Craniofacial Research (NIDCR), Division of Extramural Research (DE028544, PI-Nguyen; DE028544-02S1, PI-Nguyen), and the NIH/NIDCR Division of Intramural Research (Z01-DE000704, PI-Warner; Z01-DE00695, PI-Chiorini).
Prevalence and complement activation of anti-lymphocyte IgM antibodies in hospitalized COVID-19 patients
Authors: Perez-Diez Ainhoa , Liu Xiangdong , Calderon Stephanie , Bennett Ashlynn , Lisco Andrea , Kellog Anela , Galindo Frances , Memoli Matthew , Rocco Joseph M , Laidlaw Elizabeth , Sneller Michael C , Manion Maura , Sereti Irini
Published: August 2023

Recent studies have shown that a proportion of COVID-19 patients develop auto-antibodies against a wide range of secreted and membrane proteins, some of which are expressed by lymphocytes. It is unclear, however, if and what proportion of patients might develop anti-lymphocyte antibodies (ALAb) and whether they might have cytotoxic function. We systematically characterized the presence, isotype and lytic function of ALAb in a cohort of 85 COVID-19 patients during hospital admission from May 2020 to June 2022. As controls we analyzed 20 healthy donors (HD) and seven Flu infected volunteers. Patients were assigned into mild (N=65), or moderate/severe (N=20) groups, based on oxygen requirements, and 37 patients were followed up after recovery. We found that 27% of COVID-19 patients had IgM ALAb (binding CD4, CD8 and/or B cells), compared to 15% of the control populations. IgM ALAb prevalence was independent of disease severity and persisted during early convalescence. Sera from some of the IgM ALAb positive COVID-19 patients, and not from the controls, induced complement dependent cytotoxicity (CDC) of HD PBMCs, mostly targeting B cells. Complement activation was corroborated by detecting complement deposition ex vivo on lymphocytes from 70% of COVID-19 acutely infected patients. The level of C3b deposition on CD4 T cells inversely correlated with CD4 numbers in blood of moderate/severe patients. Our findings indicate that the antibody mediated auto-immune response found in COVID-19 patients includes targeting of lymphocytes with potential contribution to the observed lymphopenia.

This work was supported by the Divisions of Intramural Research of NIAID and NIBIB of the NIH
Type I Innate Lymphoid Cells Sense and Proliferate in Response to Distal Viral Infection
Authors: Wise-Dent Olivia , Shannon John P , Reynoso Glennys , Lujan Ramon A , Hickman Heather D
Published: August 2023

Innate lymphoid cells (ILCs) are a diverse group of tissue-resident lymphocytes that lack antigen-specific receptors commonly found on adaptive immune cells. While many different types of ILCs have been described, only group 1 ILCs (ILC1s) are known to exhibit marked antiviral activity. We recently described ILC1-mediated protection of the oral mucosa, a critical barrier tissue and site of continued pathogen exposure. Based on these studies, we hypothesized that these innate antiviral cells might also respond to inflammation induced by a distal viral infection. To address this, we used a common mouse model of vaccinia virus (VACV) infection of the peritoneal cavity, which spreads locally but does not result in infection of the oral mucosa. Confocal microscopy of frozen sections of the lip demonstrated a marked increase in the number and density of oral mucosal ILC1s in response to distal infection. Further, ILC1s remained sustainably elevated for weeks following the initial infection. Ongoing experiments will assess the ability of this enhanced layer of ILC1s to provide heightened antiviral immunity in the oral mucosa and determine the timeframes in which this occurs. Together, our data suggest that the ability of ILC1s to globally sense infection and enhance protection in response to distal infection may represent a previously unappreciated mechanism of trained immunity in the tissue.
Interleukin 27 (IL-27): an immunoregulatory and antiviral cytokine at the maternal-fetal interface during congenital infection
Authors: Merlino Madeline S , Clements Rebecca L , Lopez Alexandra H , Hunter Christopher A , Jurado Kellie Ann
Published: August 2023

Cytokines maintain homeostasis throughout pregnancy and have diverse responses to pathogens at the maternal-fetal interface. Interleukin 27 (IL-27) is a cytokine that is highly expressed in the placenta, however its functional role during pregnancy is unknown. In other contexts, IL-27 is a potent regulator of inflammation and has been shown to mediate antiviral immunity in the skin. Therefore, our objective was to determine how IL-27 contributes to immune responses at the maternal-fetal interface during congenital viral infection. To begin, we utilized an immunocompetent mouse model for Zika virus (ZIKV) infection to assess pregnancy and infection outcomes in the presence and absence of IL-27 signaling. At gestational day 13.5, we observed that the fetuses of IL-27-competent and -deficient dams had similar ZIKV burdens. However, we observed a significantly higher incidence of fetal pathology (resorption) in IL-27-deficient dams. To identify potential effectors of pathology, I evaluated IL-27 receptor (IL27RA) expression in placental tissues using flow cytometry and immunofluorescence and detected IL27RA+ immune cells throughout the maternal-fetal interface. Together these findings suggest that fetal pathology is independent of ZIKV viral burden in IL-27-deficient mice, and that IL-27 signaling may dampen pathologic immune cell responses during pregnancy. Interestingly, the placental tissues of infected, IL-27-deficient dams had significantly higher ZIKV burdens relative to IL-27-competent dams, indicating a potential antiviral role for IL-27 in the placenta. In conclusion, these data support novel antiviral and immunoregulatory roles for IL-27 at the maternal-fetal interface during congenital viral infection.

This work was supported by the University of Pennsylvania Cell and Molecular Biology Training Grant T32 GM-07229. Additional funding provided by the Linda Pechenik Montague Investigator Award and the Pew Charitable Trusts Biomedical Scholars Program.
Mechanism(s) of long-term humoral immunity and immune suppression induced by wildtype Measles virus in rhesus macaques
Authors: Liu Liting , Bartlett Maggie , Rubens Jessica , Ghimire Shristi , Brockhurst Jacqueline , Chan Kwan Pui , Larman H Benjamin , Griffin Diane E
Published: August 2023

Measles virus (MeV) causes a highly contagious systemic viral infection that remains an important cause of morbidity and mortality in children worldwide. While recovery from measles infection induces life-long protective immunity, it also causes immune amnesia which can lead to higher susceptibility to other infections. To better understand the long-term immune responses induced by the MeV vaccine versus wildtype (WT) MeV, first, we studied rhesus macaques for six months after WT MeV or Measles-Mumps-Rubella (MMR) inoculation. We quantified total antibody secreting cells (ASC) and MeV-specific ASCs in peripheral blood mononuclear cells (PBMCs) using well-characterized in house methods for ELISpot; and assessed MeV-specific IgG, avidity, and plaque reduction neutralization titers. Second, we assessed whether the loss in immune memory post WT MeV infection is linked to eviction of B cells from the bone marrow (BM) into circulation. BM and PBMCs from live attenuated MeV (LAMV) vaccinated monkeys were cultured and supernatant were assessed for isotype and antigen specificity by ELISA. PBMC cultures produced detectable amount of MeV-specific antibody and antibody recognition profiles will be compared via Virscan, a phage-display technology for epitope mapping. A novel panel of non-human primate (NHP) pathogen peptides representing Herpesviruses, Retroviruses, Adenoviruses, Pneumoviruses, and Enteroviruses were developed and expressed using the system with plans to use them to characterize changes in antibody production. We hypothesis that the robust immune response to WT MeV is accompanied by eviction of long-lived plasma cells from BM niches resulting in a loss of Ab diversity that does not occur after infection with LAMV.

Supported by grants from NIH (5R01AI153140-03)