bluuebunny/crossref_metadata_2025 huggingface dataset:
Development and characterization of new swine immune reagents to understand immune correlates for vaccines, infection, and biomedical research outcomes
Authors: Gourapura Renukaradhya J , Loving Crystal L , Kenney Scott , LaBresh Joanna , Patil Veerupaxagouda , Byrne Kristen , Walker Kristen , Dai Chaohui , Hailstock Taylor , Lunney Joan K
Published: February 2023

The USDA-NIFA Swine Immune Toolkit Initiative has a goal to generate priority immune reagents, based on inputs from veterinary immunology researchers worldwide, and pipeline them for marketing. Our efforts are aimed at expression of soluble proteins and production of panels of monoclonal antibodies (mAbs) using collaborations with commercial partners for protein expression and mAb production. Generation of new panels of mAbs reactive with porcine IL-5 and IL-21 have been initiated. Panels of mAbs to IL-6, IL-13, IL-17A, IL-28B, CXCL10, and BAFF are being screened for their reactivity in multiple immune assays. Reactivity tests of labeled anti-IL-6, -IL-17A, -IL-13 and -CXCL10 mAbs for intracellular staining of porcine myeloid and T cells using flow cytometry-based assays are in progress. Sensitive sandwich ELISA assays are now available for IL-17A, IL-13 and CXCL10; other targets are being screened for best mAb pairs for such assays. Planning for the generation of SLA-I & -II tetramers to identify swine CD4 and CD8 T cells specific for influenza virus peptides has been initiated. For each target, our goal is to provide the veterinary community with new commercial reagents and standardized techniques using these reagents for their research efforts. Tools and reagents generated by this project will undoubtedly advance swine immune, disease, vaccine, and biomedical research efforts.
Avian-specific immune reagents and immunoassays to analyze poultry immune response
Authors: Lillehoj Hyun S , Kim Woohyun , Lu Mingmin , Panebra Alfredo , Lee Yungsub , Hong Yeongho , Panebra Alfredo
Published: February 2023

NIFA-funded grant to identify chicken immune molecules, particularly cytokines and chemokines and to characterize their function resulted in new sets of avian-specific immune tools useful in the studies host immune responses after infections and vaccinations. Total 65 new monoclonal antibodies (mAbs) have been validated for their specificity against avian molecules using several assays including ELISA, immunohistochemistry, Western blot, flow cytometry, qPCR, and cell proliferation. All the target molecules that we selected have shown to have critical functions in host defense against pathogens and include 13 cytokines (interleukin-4, 7, 10, 12, 13, 17F, 21, 22, 23, 26, 34, IFN-α, IFN-κ, TNF-α, CSF-1, and TGF-β) and 3 chemokines (CXCLi2, CCL4 and 5). Functional similarity and dissimilarity were discovered between immune molecules of avians and mammals. These immune reagents will be useful for studies in host-pathogen immunobiology of infections and vaccinations in poultry.

(This research was supported by USDA NIFA award number: 2017-67015-26793)
Development of antigen sandwich ELISA to detect interferon-alpha (IFN-α) using monoclonal antibodies in chicken
Authors: Lee Youngsub , Kim Woo hyun , Lillehoj Hyun S
Published: February 2023

Interferon alpha (IFN-α) belongs to the type I interferon family which mediates an early innate immune response to viral infections. In the present study, we developed a sandwich ELISA using specific mouse monoclonal antibodies (mAbs) to measure IFN-α production in chickens. Recombinant chicken IFN-α (chIFN-α) expressed in yeast was used to immunize the mice. Five mAbs which specifically recognize chIFN-α antigen were selected and characterized. For sandwich ELISA development, mAbs were labeled with biotin, followed by a pairing assay to identify the best capture and detection antibodies. Two sets of mouse anti-chIFN-α mAb pairs were identified as optimum pairs of antibodies for detection of chIFN-α. The sandwich ELISA effectively detected native chicken IFN-α in chicken macrophage cells stimulated by polyinosinic:polycytidylic acid and its minimum detectable level was about 25 pg/mL. The anti-viral activity of chIFN-α against vesicular stomatitis virus (VSV) was evaluated in chicken embryonic fibroblast and the mouse anti-chIFN-α mAbs which neutralize the activity of chicken IFN-α against VSV were identified. The newly developed antigen capture sandwich ELISA for the detection of chIFN-α will be a useful tool to monitor IFN-α production in chickens during viral infections.
Host responses to feline coronavirus are significantly different in primary macrophages compared to CRFK cells.
Authors: Drechsler Yvonne , Vasconcelos Elton J.R. , Griggs Lisa M , Diniz Pedro P.P.V.
Published: February 2023

Feline coronavirus (FCoV) is a highly contagious virus that exists as two pathotypes, with feline infectious peritonitis (FIP) caused by the pathogenic FIPV. The pathogenesis of FIPV remains poorly understood, and several different theories are still being discussed. A strong argument can be made for a role of the immune status and genetics of affected cats in pathogenesis. Most studies have focused on viral entry or differential replication instead of host responses. Considering the importance of genetics and host immune responses in viral infections, transcriptome studies to elucidate host pathogen interactions in primary macrophages and Crandell-Rees Feline Kidney cells (CRFK) were performed by next-generation sequencing of mRNA. CRFK and macrophages from healthy male cats infected with FIPV 79-1146 ex vivo displayed differential gene expression in response. While CRFK gene expression was enriched for many classical virally induced genes and pathway networks, gene expression in macrophages was quite different. Interestingly, some of the commonly downregulated genes in macrophages involved innate anti-viral factors, while upregulated genes fell within unexpected pathways, such as autophagy, cellular senescence and other cellular processes not directly linked to infection. Our results highlight how individual host responses play a strong role when exposed to the virus, consistent with the fact that cats developing feline infectious peritonitis are very young or old, immune suppressed or of a particular genetic background. This study will give more insight into interactions of FCoV with feline macrophages and contrast it with the responses of a cell line that readily replicates the virus.
Butyrate alters lipopolysaccharide-induced inflammatory response by porcine monocytes
Authors: Becker Sage R , Byrne Kristen , Loving Crystal L
Published: February 2023

Continuous, low-level inflammation can have a negative impact on intestinal health. Butyrate is a short-chain fatty acid produced in the lower intestinal tract by microbial fermentation of dietary fibers and modulates immune cell responses through various mechanisms. Blood monocytes migrate into the intestine and respond to signals, including potential modulation by butyrate or in response to microbial components, such as lipopolysaccharide (LPS). To investigate the impact of butyrate on inflammatory response to LPS, porcine monocytes were either cultured with a combination of LPS and varying butyrate concentrations or pre-treated with butyrate before LPS exposure. Independent of butyrate, LPS stimulation of monocytes induced IL-1β and TNF production. Monocytes cultured just three hours (h) prior to LPS stimulation had reduced overall cytokine production, suggesting rapid phenotypic changes in monocytes. Butyrate had varying effects on the amount of cytokine produced in response to LPS. Specifically, low butyrate concentrations (0.25 mM) limited IL-1β production, but did not impact TNF production. Higher butyrate concentrations (> 4 mM) limited TNF production to LPS stimulation. Pre-treatment of monocytes with butyrate for 3 h prior to LPS stimulation altered IL-1β production. Pre-treatment with 0.25 mM butyrate did not limit IL-1β production, but concentrations > 4 mM did. In conclusion, reduction of LPS-induced cytokine production by porcine monocytes was dependent on butyrate concentration and exposure time relative to LPS exposure. Further work is ongoing to understand the mechanism of butyrate modulation of pig monocytes and inflammatory response to LPS for potential application in vivo.
Purification, biotinylation, and testing of a monoclonal antibody to identify B cells in Trachemys scripta, the red-eared slider turtle
Authors: Paton Hanna D , Mool Allison , Green Whitney , Bowden Rachel , Vogel Laura A
Published: February 2023

There is a shortage of research in reptile immunity which is further hampered by lack of reptile-specific reagents. Evidence suggests there are important differences between reptile and mammalian immune strategies and our laboratory is interested in reptile B cell development and function. Our undergraduate research project involved the preparation of a previously developed monoclonal antibody (HL673) that recognizes turtle light chain protein. To begin, culture supernatant from the HL673 mAb murine cell line was received and was applied to a protein A affinity column. Unbound proteins were then washed away, and the bound proteins were removed from the column using low pH glycine-HCl buffer. The purified antibody proteins were collected in fractions, OD at 280nm measured, then positive fractions were pooled and dialyzed. The concentration of the purified antibodies was determined, and reactivity tested using an ELISA plate coated with dilute turtle serum. Dilutions of the purified HL673 were detected by anti-mouse IgG-horseradish peroxidase (HRP). Furthermore, some of the antibody preparation was conjugated to biotin. After dialysis, the HL673-biotin was tested by ELISA with dilute turtle serum and detected by streptavidin-HRP. The newly biotinylated antibody was incubated with blood and spleen samples from both adult and hatchling red-eared slider turtles. Bound antibodies were detected using streptavidin-fluorochrome and B cell populations identified using flow cytometry. Our results showed successful detection of turtle B cells using the labeled mAb in both hatchling and adult turtle cell samples. Future studies will use this reagent to investigate the distribution and function of B cells in reptile gut immunity.
Characterization of circulating porcine immune cells using single-cell RNA-sequencing and comparison to human datasets
Authors: Loving Crystal L , Wiarda Jayne E , Sivasankaran Sathesh K , Daharsh Lance , Liu Haibo , Byrne Kristen A , Tuggle Christopher K
Published: February 2023

Pigs are part of global food security and serve as a valuable human biomedical model. Immunoreagent unavailability limits resolution of pig leukocyte populations by protein markers; thus, single-cell RNA-sequencing (scRNA) was used to characterize pig peripheral blood mononuclear cell (PBMC) populations and infer function based on transcriptional profiles and comparison to human datasets. Across seven PBMC samples partitioned and sequenced, 28,810 cells distributed across 36 clusters were classified into 13 general cell types including plasmacytoid dendritic cells (DC), conventional DCs, monocytes, more than ten B cell clusters including plasmablasts, conventional CD4 and CD8 αβ T cells, NK cells, and four clusters of γδ T cells. Signature gene sets for a publicly available human bulk RNA-seq dataset were assessed for relative enrichment in genes expressed in pig cells and substantial overlap in gene expression between specific pig and human PBMC populations was detected. Integration of pig scRNA with a public human scRNA dataset provided further validation for similarity between human and pig. Of four pig γδ T cell clusters, two had high prediction and mapping scores with human γδ T cells. The other two pig γδ T cell clusters had gene expression profiles predictive of human γδ T cells, but also human CD4 T central memory (Tcm) and innate lymphoid cells, suggesting innate and adaptive function of some pig γδ T cells. Pigs have a population of circulating double-positive (DP) CD4+CD8αα+ αβ T cells, and putative DP clusters had high prediction scores to human CD4 Tcm cells, supporting their classification in literature as memory cells. Overall, the data provided the first single-cell atlas for porcine PBMCs and comparison to human datasets.
Comparative annotation of porcine bone marrow cell types using scRNAseq
Authors: Daharsh Lance N , Sivasankaran Sathesh K , Byrne Kristen A , Herrera-Uribe Juber , Loving Crystal L , Tuggle Christopher K
Published: February 2023

Pigs are important as a biomedical model and for worldwide meat production. Therefore, a better understanding of porcine hematopoiesis and immune system development could have a wide impact on human disease research and food production. The bone marrow represents a unique environment that is the major site of differentiation of stem cells and progenitors into mature blood cells. We performed scRNA-seq on bone marrow from healthy adult pigs and sequenced a total of 3,576 cells. After QC, 2,707 cells and 16,432 genes were used for downstream analysis. The porcine bone marrow cells were grouped into 15 distinct clusters and these relationships were mapped using non-linear dimensional reduction. To find markers that defined each cluster, we found a total of 3,335 differentially expressed genes using the non-parameteric Wilcoxon rank sum test. Further, pairwise differential expression testing and random forest models were used to evaluate the similarity between clusters and further define the unique features of each cluster. Using these defined markers, we were able to annotate the cell types for each cluster. The clusters represented a diverse set of cell types including myeloid and lymphoid cells, myeloid and lymphoid cell progenitors, and platelet and red blood cell progenitors. We validated these annotations using publicly available datasets including the CITE-seq database of human bone marrow mononuclear cells (Stuart et al., 2019). We also compared the expression profiles between similarly annotated cell types between human and pig. Our novel annotation of porcine bone marrow cells will be a resource for the study of porcine hematopoiesis and immune system development and help inform human translational biomedical research.
Genetic Variation in the RSV fusion protein controls strain-dependent inflammasome activation and the development of a pathogenic Th17 response
Authors: Waldstein Kody , Weiss Kayla , Hartwig Stacey , Varga Steven M
Published: February 2023

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in young children worldwide. RSV-induced disease ranges from mild to severe, with severe disease correlating with increased levels of pro-inflammatory cytokines, including the Th17-associated cytokine, IL-17. To understand the viral factors that influence the induction of a pathogenic Th17 response, we compared the inflammatory responses elicited by different RSV strains. In contrast to the commonly used RSV A2 strain, RSV 2–20 infection resulted in increased protein levels of IL-6 and IL-1β, which were required to induce a pathogenic Th17 response. RSV 2–20 infection increased mucus production and airway hyperreactivity indicating strain-specific disease induction. RSV 2–20 and A2 induced similar expression of caspase-1, however, 2–20 infection resulted in increased levels of pro-IL-1β suggesting differential activation of inflammasome signal 1. Increased production of IL-1β following RSV 2–20 infection was dependent on TLR4 expression and signaling, a canonical inflammasome signal 1 activator. A recombinant A2 virus expressing the 2–20 fusion (F) protein induced IL-1β to similar levels as wild-type 2–20 indicating the 2–20 F protein is sufficient to increase pro-inflammatory cytokine production. Reversion of three-point mutations in a region likely to interact with host cells in the RSV A2/2–20 F recombinant virus to the A2 sequence significantly reduced IL-6 and IL-1β protein production. Our results demonstrate that genetic variation in the RSV F protein plays a crucial role in the induction of pro-inflammatory cytokines and modulating disease severity in the animal model and may contribute to differential RSV pathogenesis.
Molecular insights into host-virus interaction of human and rhesus cytomegalovirus UL144 mediated by immune checkpoint BTLA and CD160
Authors: Nemcovicova Ivana , Lenhartová Simona , Škrabana Rostislav , Benko Mário , Nemčovič Marek
Published: February 2023

Cell-cell communication is a crucial mode for multi-cellular organisms to accomplish complex biological functions and various signaling molecules have evolved to meet ever complicated demands to communicate extracellular stimuli with intracellular components. Thus, signaling molecules, whose expression is responsive to local environments; balance the communication between host innate cells and viral pathogens. This intimate interaction between virus and the host immune system covers a wide range of contacts. HVEM, a member of the TNF receptor superfamily, is commonly recognized as a co-stimulatory receptor for LIGHT and LTα. Interestingly, HVEM was also found to interact with BTLA and CD160, and thus deliver a suppressive signal to T cells, therefore the complex molecular network between HVEM and its cellular partners makes bidirectional signaling feasible. Here, we study the viral glycoprotein UL144 of human and rhesus cytomegalovirus that is orthologous to cellular HVEM, thus resembling some of its promiscuity on the cell surface. This molecular network is quite well described, however, the engagement of CD160 by UL144 has not yet been satisfactorily studied. We measured both human and rhesus CMV UL144 binding to BTLA or CD160 using purified HVEM or UL144 proteins and their mutants. We confirmed that human CMV UL144 only bound BTLA and not CD160, whereas HVEM bound both BTLA and CD160. Surprisingly, we have observed binding between UL144-glycan free mutant not only to BTLA but also CD160. Moreover, considering that the ectodomain of UL144 is highly polymorphic across primate CMV we examined both human and rhesus CMV UL144 selectivity for its cellular receptors. We found rhesus CMV UL144 bound CD160 with lower KD when compared to BTLA.
NK cells exhibit memory and stem cell like features after Zika virus infection
Authors: Kujur Weshely , Gomez Oscar M , Adduri Raju S.R. , Vankayalapati Ramakrishna , Konduru Nagarjun V , Mulik Sachin
Published: February 2023

Natural Killer (NK) cells are the components of innate immune system known for their role in virus infection and tumor immunity. Their importance is appreciated from the case studies where lack of NK cells leads to life threatening herpes virus infection and increased host susceptibility to certain cancers. Recent development in the NK field have opened a new frontier bringing to light that innate immune cells can demonstrate memory features too. In line with the above-mentioned development, we report NK memory cell generation post Zika virus infection in mice. Our detailed transcriptomics, epigenetic and functional analysis revealed that CD27+ memory like NK cell that developed post Zika virus infection exhibited stem cell like properties and shared gene signatures with memory CD8 T cells, stem cells and stem like T cells from chronic infection and cancer. We termed these cells as “NK memory stem cells” which also possessed greater antiviral function when adoptively transferred into Zika infected mice. We next performed single cell RNA-seq on NK memory stem cells and identified multiple subpopulations which differed in gene expression profiles. Our work revealed NK cells with memory like and stemness features that are helpful in fighting viral infection. We also detected subpopulations in NK memory stem cells and efforts are ongoing to evaluate the role of these NK subpopulations in antiviral immunity.
Expression of Nutrient Transporters on NK Cells during Murine Cytomegalovirus Infection is MyD88-Dependent
Authors: Khan Abrar Ul Haq , Almutairi Saeedah Musaed , Ali Alaa Kassim , Salcedo Rosalba , Stewart C. Andrew , Lee Seung-Hwan
Published: February 2023

Natural killer (NK) cells are the predominant innate lymphocytes that provide the first line of defence without prior sensitization. In the inflammatory milieu, NK cells modify their metabolism to cope with the high energy demand required to support their proliferation, activation, and functional plasticity. This metabolic reprogramming is usually accompanied by the upregulated expression of nutrient transporters on the cell surface, leading to increased nutrient uptake needed for robust proliferation. The members of interleukin-1 family of inflammatory cytokines are critical in activating NK cells during infection; however, their function in NK cell metabolism is not fully elucidated. Previously, IL-18 has been shown to upregulate the expression of solute carrier transmembrane proteins and thereby induces a robust metabolic boost in NK cells. However, we demonstrated that IL-18 signaling is dispensable during viral infection in vivo whereas the expression of nutrient transporters on NK cells is primarily regulated by MyD88-pathway since NK cells from Myd88−/− mice displayed significantly reduced expression of nutrient receptors. Moreover, we identified that IL-33, another cytokine employing MyD88 signaling, can induce the expression of nutrient receptor but requires a sequential exposure to IL-12. Furthermore, signaling through NK cells activating receptor, Ly49H, can also promote the expression of nutrient transporters. In summary, our findings revealed multiple pathways that induce the expression of nutrient transporters on NK cells while highlighting the imperative role of MyD88 in NK cell metabolism during infection.
Murine Roseolovirus Induces Autoimmune Gastritis and Disrupts Thymic Antigen Presenting Cells.
Authors: Bigley Tarin , Yang Li-ping , Kang Liang-I , Saenz Jose B , Mills Jason C , Yokoyama Wayne M
Published: February 2023

Murine roseolovirus (MRV) is a beta-herpesvirus that is a natural murine pathogen and is genetically highly related to the human roseoloviruses, HHV6 and HHV7. The human roseoloviruses have been associated with autoimmune disease but demonstrating causality has been difficult due to the ubiquitous and chronic nature of roseolovirus infections. Neonatal MRV infection resulted in transient thymic and peripheral CD4+ T cell depletion, including CD4+Foxp3+ regulatory T cells (Tregs). We found that neonatal MRV infection induced autoimmune gastritis in adult mice. Our studies showed that autoimmune gastritis occurs in the absence of detectable virus in gastric mucosa. Ganciclovir (GCV) inhibited MRV replication as well as MRV-mediated CD4+ T cell depletion and development of autoimmune gastritis. This suggests that MRV replication is necessary for altering immunologic tolerance early in life. Interestingly, neonatal MRV infection resulted in the production of a wide array of autoantibodies in adult mice. The inflammatory infiltrate into the gastric mucosa was characterized by an increase in T cells, neutrophils and eosinophils. Furthermore, the CD4+ T cells in the gastric mucosa exhibited Th2 and Th17 differentiation. The development of autoimmune gastritis was CD4+ T cell and IL-17 dependent, while B cells were only necessary early in life. Neonatal MRV infection resulted in a transient decrease in medullary thymic epithelial and thymic dendritic cells numbers, as well as reduced expression of AIRE and tissue restricted self-antigens in the thymus. These findings strongly suggest that infection with a roseolovirus early in life results in disruption of central tolerance and development of autoimmune disease.
Novel Mechanisms of Innate Immune System Regulation Following SARS-CoV-2 Exposure
Authors: Allen Irving C , Ivester Hannah , Morrison Holly , Finkielstein Carla , Auguste Jonathan , Duggal Nisha , Smyth Jamie , Meng Xiang-Jin , Kehn-Hall Kylene , Weger James
Published: February 2023

The SARS-CoV-2 pandemic has wreaked havoc on our public health system and economy. The outcome of SARS-CoV-2 infection is determined by host-virus interactions and anti-viral immune responses. A delicate balance must be struck between mounting a vigorous immune response to eradicate the virus and quelling overactive inflammation to protect the host. Indeed, overzealous inflammation is associated with the “cytokine storm” reported in an alarming number of COVID-19 patients and underlies the Multisystem Inflammatory Syndrome reported in pediatric cases. Initial studies have suggested that SARS-CoV-2 is capable of inhibiting type-I interferon signaling, hijacking cell death processes, and inducing robust pro-inflammatory cytokine production in patients with poor outcomes. Here, we confirm many of these previous findings using whole genome transcriptomics approaches in human patients and complementary mechanistic studies using SARS-CoV-2 infection in K18-hACE2 mice. Using these approaches, we further identify 12 critical cytokines that appear to be associated with the overzealous immune response in COVID-19 patients. Further studies and pathway analysis identified members of the NBD-LRR (NLR) family of pattern recognition receptors that regulate distinct mechanisms underlying overzealous inflammation. Together, these data provide insight into novel mechanisms of immune system regulation that appear responsible for modulating several aspects of SARS-CoV-2 pathobiology.
Platelet-rich neutrophil-platelet micro-emboli contribute to cigarette smoke induced flu severity.
Authors: Kaminski Tomasz W. , Brzoska Tomasz , Li Xiuyang , Vats Ravi , Tutuncouglu Egemen , Robinson Keven , Nyuonya Toru , Sundd Prithu
Published: February 2023

Epidemiological evidence suggests that prior exposure to cigarette smoke (CS) or habitual smoking increases the risk of influenza A virus (IAV)-triggered respiratory failure. Although emerging evidence supports the role of thrombo-inflammation in the development of CS and IAV-triggered lung injury, the innate immune mechanism remains poorly understood. We have developed a two-hit model of CS-induced severe flu in mice. Mice were exposed to four weeks of room air (air) or CS followed by intra-nasal administration of A/PR/8/34 IAV. The body weight was measured every day for two weeks after IAV administration followed by assessment of lung injury at day 7. Lungs were harvested for histological assessment and viral titration by qPCR. Quantitative fluorescence intravital lung microscopy (qFILM) was conducted at 2-, 3- and 4-days post IAV-infection to visualize dynamics of neutrophil and platelet recruitment in the lung of mice IV administered with fluorescent dextran, anti-Ly6G Ab and anti-CD49Abs. Mice exposed to CS+IAV manifested significantly more weight loss, lung injury, lung congestion, hemorrhage and hypoxemia compared to mice administered IAV only. QFILM revealed that severity of lung injury was associated with significantly larger area with impaired blood flow and more vascular leakage secondary to vascular occlusion by platelet-rich neutrophil-platelet aggregates in the lung of CS+IAV than IAV administered mice. These initial results suggest that CS primes innate immune signaling in neutrophils and platelets to promote their recruitment in the lung following flu, leading to severe acute lung injury. Currently, studies are underway to identify innate immune pathways that drive hyper thrombo-inflammatory response.
In vitro infection of human lung tissue with SARS-CoV-2: Heterogeneity in host defense and therapeutic response
Authors: Schaller Matthew , Sharma Yamini , Dupee Zadia , Nguyen Duy , Uruena Juan , Smolchek Ryan , Machuca Tiago , Sawyer W. Gregory , Mehrad Borna
Published: February 2023

Cell lines are the mainstay in understanding the biology of COVID-19 infection, but do not recapitulate many of the complexities of human infection. The use of human lung tissue is one solution for the study of such novel respiratory pathogens. We hypothesized that a cryopreserved bank of human lung tissue allows for the in vitro study of the inter-individual heterogeneity of host response to SARS-CoV-2 infection, thus providing a bridge between studies with cell lines and studies in animal models. We generated a cryobank of tissues from 16 donors, most of whom had risk factors for severe illness from COVID-19. Cryopreserved tissues preserved 90% of cell viability and contained heterogeneous populations of metabolically active epithelial, endothelial, and immune cell subsets of the human lung. Samples were readily infectible with HCoV-OC43 and SARS-CoV-2 coronavirus strains, and demonstrated comparable susceptibility to infection. In contrast, we observed a marked donor-dependent heterogeneity in the expression of IL-6, CXCL8 and IFNβ in response to SARS-CoV-2 infection. Treatment of tissues with dexamethasone and the experimental drug, N-hydroxycytidine, suppressed viral growth in all samples, whereas chloroquine and remdesivir had no detectable effect. Metformin and sirolimus, molecules with predicted antiviral activity, suppressed viral replication in tissues from a subset of donors. In summary, we developed a novel system for the in vitro study of human SARS-CoV-2 infection using primary human lung tissue from a library of donor tissues. This model may be useful for drug screening and for understanding basic mechanisms of COVID-19 pathogenesis.
SARS-CoV-2 infection drives endothelial dysfunction indirectly through hypoxia and increased pro-inflammatory cytokine production
Authors: Won Taejoon , Wood Megan K , Hughes David M , Talor Monica V , Johns Roger A , Gilotra Nisha A , Pekosz Andrew S , Hooper Jody E , Cihakova Daniela
Published: February 2023

In the Covid-19 patients, vascular thrombosis is observed in the heart, lung, and other organs, leading to multiorgan morbidity and mortality. We obtained heart and lung tissues from autopsies of the patients who died from Covid-19 or unrelated causes as a control. We found that endothelial cells of the hearts and lungs were in dysfunctional state during Covid-19. Cardiac and pulmonary endothelial cells in the Covid-19 group showed decreased level of thrombomodulin, an anticoagulant, and increased level of von Willebrand factor, a procoagulant, compared to non-Covid-19 control. Endothelial cells in the Covid-19 autopsy hearts and lungs also exhibited reduced ICAM-1, VCAM-1, and E-selectin expression. Most of the hearts from the Covid-19 autopsies tested negative for SARS-CoV-2 genome, while all the lungs tested positive. Human coronary artery endothelial cell (HCAEC) in vitro culture was not infected by SARS-CoV-2, showing no dysfunctional phenotype. In the HCAEC culture and in vivo mouse experiments, hypoxia and pro-inflammatory cytokine treatment caused endothelial cell dysfunction including thrombomodulin downregulation similar to what we observed in Covid-19 autopsy tissues. Collectively, endothelial cell dysfunction associated with thrombus formation in Covid-19 is not caused by direct infection with SARS-CoV-2, but it can be induced by pathogenic microenvironments such as hypoxia and increased pro-inflammatory cytokine milieu.
SARS-CoV-2 plasma viral load polarizes the systemic myeloid cell compartment towards an immunosuppressive/regulatory phenotype in severe COVID-19 patients
Authors: Trombetta Amelia Chiara , de Carvalho Farias Guilherme Borlido , Godinho-Santos Ana , Gomes André MC , Rosmaninho Pedro , Conceição Carolina M. , Laia Joel , Santos Diana F. , Neiva Maria Inês Tostão , Almeida Afonso , Sousa Ana E , Fernandes Susana M.
Published: February 2023

Severe COVID-19 determines high death rates and health resources overwhelming. Aim of the present study was to shed light on the immune-pathogenesis of severe COVID-19, evaluating the effects of detectable plasma viral load on circulating myeloid cell phenotype and activation. Adult COVID-19 patients with pneumonia stratified according to the degree of respiratory insufficiency were longitudinally followed. Flow cytometry data of monocytes (Mo) and dendritic cells (DCs) subsets were analysed by supervised and unsupervised approaches, in parallel with SARS-CoV-2 plasma viral load by digital droplet PCR and serum cytokines and chemokines. All patients featured systemic immune-suppressive myeloid cell responses, characterized by a reduction of CD14lowCD16+ Mo and reduced expression of CD80, CD86, and SLAN. All DC subset were significantly reduced and both Mo and DC showed increased expression of CD163, CD204, CD206 and PD-L1 immune-regulatory markers. These changes correlated with an unbalanced cytokine/chemokine response, in which G-CSF, M-CSF, IL-6 increased similarly in all patient groups, while low levels of IFN-alpha, beta, IL-1a and IL-1b persisted, associated with increased concentrations of immune-regulatory cytokines as IL-1RA and IL-10. A significantly greater expansion of a PD-L1 positive M2-like population was seen in classical monocytes from viremic patients. Our data favour the conclusion that massive replication of SARS-CoV-2, leading to release in blood, polarize the systemic myeloid cell compartment response towards a more differentiated immunosuppressive/regulatory phenotype.
Susceptibility to Low Dose Influenza in Mice is Increased by Administration of Ketamine/Xylazine
Authors: Nash Paul B
Published: February 2023

Ketamine/xylazine is an anesthetic commonly used in both research and practice. This study demonstrates a side effect that may have important ramifications. Mice where ketamine/xylazine was used compared to mice not administered the anesthetic were more susceptible to illness caused by administration of low pathogenic preparations of influenza strain A/Puerto Rico/8/34 (PR8). When using one lot of virus, severity of illness as measured by weight loss was increased dramatically by the use of the anesthetic. A slightly more pathogenic lot of virus in other experiments showed that mice with no anesthetic had very mild if any signs of disease, while those with anesthetic succumbed to the illness. In weight loss experiments, mice were affected by the anesthetic even if they were allowed to wake up before inoculation with virus, showing that it was exposure to the drugs that caused the effect, not simply them being asleep. The asleep mice however did recover more slowly from the infection. These results suggest that anesthesia with ketamine/xylazine for treatments may increase the likelihood of post-treatment illness. Likewise experimental results could be affected by using this drug combination such that the immune responses analyzed could be suppressed or the diseases investigated could be more severe.
Rapid activation and expansion of peripheral gammadelta (γδ) T cells in SARS-CoV-2-infected nonhuman primates following aerosol exposure
Authors: Fears Alyssa , Walker Edith , Chirichella Nicole , Slisarenko Nadia , Beddingfield Brandon , Maness Nicholas J , Roy Chad , Rout Namita
Published: February 2023

Infection with SARS-CoV-2, the novel coronavirus responsible for the current COVID-19 pandemic, results in a wide range of pathologies from asymptomatic/mild outcome to fulminant pneumonia and progression to acute respiratory distress syndrome (ARDS). The cellular mechanisms underlying this spectrum of disease remain unknown. We investigated the dynamic changes in frequency and activation status of peripheral innate lymphocyte subsets in two different species of nonhuman primates. Cohorts of Rhesus macaques (n=8) and African Green monkeys (n=8) were infected with SARS-CoV-2 either by small particle or mucosal (IN/IT) route. Flow cytometric analysis of PBMCs revealed a rapid and significant increase in circulating γδ T cell frequencies and activation at d1 postexposure across all animals inoculated by aerosol, which was correlative with peak viral loads in lung lavage and Vδ2 T cell frequencies. Further, elevated GranzymeB expression in both Vδ1 and Vδ2 subsets of γδ T cells was suggestive of increased cytotoxic function in early infection. In contrast, mucosally-infected animals showed little change in circulating γδ T cell frequencies from d1 through d7 post-infection, nor were there remarkable differences in early MAIT and NK cell frequencies. Collectively, these data suggest γδ T cells may be early responders to deep lung SARS-CoV-2 infection and the kinetics and quality of immune response are dependent upon route of exposure despite resulting in similar peak viral loads from infection. Further understanding of γδ T cell functions within the pulmonary compartment during SARS-CoV-2-driven acute inflammation may reveal tractable therapeutic approaches to further mitigate ARDS-like responses in COVID-19 patients.
Phosphatidylserine Receptor Enhancement of SARS-CoV-2 Entry: AXL as a Therapeutic Target for COVID-19
Authors: Bohan Dana , Van Ert Hanora , Rogers Kai , Ruggio Natalie , Chávez Roberth Anthony Rojas , Stokowy Tomasz , Haim Hillel , Gao Boning , Minna John , Micklem David , Lorens James , Maury Wendy
Published: February 2023

Phosphatidylserine (PS) receptors enhance infection of a wide range of enveloped RNA viruses through virion-associated PS binding that is termed apoptotic mimicry. Here we show that this broadly shared uptake mechanism is utilized by SARS-CoV-2. Under ACE2lo conditions, expression of members of the TIM and TAM families of PS receptors synergized with ACE2 to enhance SARS-CoV-2 infection; however, PS receptors alone did not mediate infection. PS receptors enhanced SARS-CoV-2 binding to the surface of cells. While PS receptors did not interact directly with purified SARS-CoV-2 spike, addition of PS liposomes reduced entry of VSV-SARS-CoV-2 spike pseudovirions, providing evidence that PS/PS receptor interactions are mediating the effect. AXL being abundant on airway cell lines, we evaluated small molecule inhibitors of AXL signaling such as bemcentinib for their ability to inhibit SARS-CoV-2 infection. Bemcentinib robustly inhibited virus infection of some lung cell lines that expressed AXL. This inhibition correlated well with inhibitors that block cathepsin activity, consistent with AXL-mediated uptake of SARS-CoV-2 into the endosomal compartment. RNAseq studies in Vero E6 and A549-hACE2 cells demonstrated that bemcentinib reduced SARS-CoV-2 transcripts dramatically. The ability of bemcentinib to decrease coronavirus infection in vivo was assessed using the betacoronavirus mouse hepatitis virus (MHV). Liver titers and virus load of MHV were significantly inhibited by bemcentinib at day 5 of infection. In total, our findings provide evidence that PS receptors facilitate infection of the pandemic coronavirus SARS-CoV-2 and that inhibition of signaling of the PS receptor AXL has therapeutic potential against SARS-CoV-2.
Endogenous retroviruses mediate IFN-independent protection against vaginal HSV-2 infection
Authors: Tokuyama Maria , Jayewickreme Radeesha , Mao Tianyang , Philbrick William , Kong Yong , Dong Huiping , Treger Rebecca S. , Rakib Tasfia , Iwasaki Akiko
Published: February 2023

Endogenous retroviruses (ERVs) are genomic sequences that originated from retroviruses and are present in most eukaryotic genomes. Both beneficial and detrimental functions are attributed to ERVs, but whether ERVs contribute to antiviral immunity is not known. Here we used herpes simplex virus type 2 (HSV-2) infection and found that mice deficient in Toll-like receptor 7 (Tlr7−/−) that have high systemic levels of infectious ERVs are resistant to intravaginal HSV-2 infection compared with wildtype C57BL/6 (B6) mice. We deleted the endogenous ecotropic murine leukemia virus (Emv2) locus on the Tlr7−/− background (Emv2−/−Tlr7−/−) and found that Emv2−/−Tlr7−/− mice are no longer protected against HSV-2. Intravaginal application of purified ERVs prior to HSV-2 infection in both B6 and highly susceptible interferon-alpha receptor-deficient (Ifnar1−/−) mice delayed disease course. We did not observe enhanced type I interferon (IFN-I) signaling in the vaginal tissues from Tlr7−/− mice or B6 mice treated with purified ERVs. Instead, we observed enhanced expression of epithelial tight junction protein, E-cadherin, in the vaginal epithelium of ERV-treated B6 mice. Similar increase in tight junction proteins was observed in Tlr7−/− but not in the Emv2−/−Tlr7−/− mice. Together, our results showed that IFN-independent modulation of the vaginal epithelium by ERVs protects mice against vaginal HSV-2 infection and lowers disease burden.
Using standardized dry antibody panels for flow cytometry in the assessment of altered immune profiles in response to SARS-CoV2 infection
Authors: Lacaille Vashti , Bowers Rita
Published: February 2023

The SARS-CoV-2 virus continues to be a global hazard, further aggravated by the continuous emergence of viral variants. Beside the development of vaccines, the search for immune modulation targets and indicators of disease severity are important objectives of research. A promising approach in order to identify potential crossroads in SARS-CoV2-infection could be to assess proportions, differentiation and functional status of known immune populations. Stimulated by the standardization needs in multi-site research studies, Beckman Coulter has developed several antibody panels for comprehensive profiling of principal human immune populations and commercialized these antibody panels as DURAClone (RUO) kits which are provided off-the-shelf in a dry unitized format, eliminating antibody pipetting and reducing reagent aging. In this technical evaluation, extended versions of these panels with further antibodies of interest added in liquid format have been used to characterize lymphoid and myeloid immune cells in a small number of samples from normal donors and SARS-CoV2-positive donors with either asymptomatic, mild or severe symptoms. Confirming findings in current research of SARS-CoV2, pronounced differences in cellular proportions, differentiation and activation status could be observed in lymphoid and myeloid compartments (not significant in the small number of samples), demonstrating the versatility and usefulness of standardized DURAClone (RUO) antibody panels in hypothesis building and early steps of biomarker discovery.
Malaria infection protects from lupus nephritis at a stage beyond immune complex-induced glomerular inflammation
Authors: Bolland Silvia , Kole Hemanta , Scott Bethany , Qi Chen-Feng , Crompton Peter , Wu Juan , Amo Laura
Published: February 2023

A protective role of malaria infection in SLE was proposed to explain the relatively low prevalence of SLE in West Africa compared to populations of similar genetic background living in the West. Various reports showed an increase of autoantibodies in plasma of West African populations, suggesting that parasite infections were inducing systemic autoreactivity while protecting from tissue pathology. Our own studies from a large longitudinal study in a malaria endemic region confirm the high penetrance of elevated titers of anti-nuclear antibodies in plasma of Plasmodium PCR-positive individuals. In mouse models of malaria, murine Plasmodium infection increases systemic autoreactivity but does not trigger any type of immune-complex induced inflammatory disease. To further investigate the mechanism of kidney protection by Plasmodium, we set up experiments using a malaria parasite in the well characterized SLE mouse model FcγRIIb[KO]. This model system allowed for evaluation of the effect of malaria on autoantibodies, systemic vasculitis and kidney specific pathology, while also assessing the role of parasite-mediated bone marrow alterations, or parasite-produced hemozoin. We found that infection with Plasmodium protects SLE by modifying bone marrow cells and impairing leukocyte infiltration in the kidney, without altering systemic autoimmunity, vasculitis, all independent of the expression of hemozoin. Our results point to a protection of late-stage kidney disease by malaria that targets a step beyond immune complex deposition, hypoxia or innate responses and release of cytokines (IFN, TNF, IL1).
Deficiency of maternal antibodies modulates the development of lupus-like disease in BXSB offspring.
Authors: Zhu Jing , Bowman Nina , Naughton Samantha , Luo Xin M , Leeth Caroline McPhee
Published: February 2023

Maternal factors play an essential role in shaping the immune system of the newborn, yet it is unknown whether maternal factors could modulate the development of systemic lupus erythematosus (SLE) in the offspring. Activation-induced cytidine deaminase (AID) is an enzyme required for somatic hypermutation and class switch recombination. Given that IgG and IgA isotypes account for the vast majority of passive immunity in rodents, our previously established AID-deficient BXSB mice provide a model in which no IgG or IgA antibodies are transferred to the offspring. In this study, we compared genotypically identical mice born to either AID-sufficient dams or AID-deficient dams and evaluated the effects of maternal antibodies in disease progression. Offspring from knockout dams developed disease at a faster rate, as shown by more severe nephritis and elevated pathogenic autoantibodies compared to their counterparts born to wild type dams. In addition, the splenic structure was intensively disrupted with disorganized follicles and fewer discernible germinal centers. A more activated T cell phenotype was also characterized in maternal antibody-deficient mice. When immune competent pups were cross-fostered onto AID deficient dams, these mice exhibited more severe disease phenotypes, indicating the protective antibody effect contributes to the modulation of SLE progression in post-natal period. Overall, these findings highlight the importance of maternal antibodies in programming the immune system and altering the SLE development in the offspring.
Soluble syndecan I (CD138) released by MRL/Lpr T cells enhances APRIL-mediated lupus B cell survival and differentiation
Authors: Liu Lunhua , Akkoyunlu Mustafa
Published: February 2023

High level of soluble serum syndecan-1 (CD138), a heparan sulfate–bearing proteoglycan, in systemic lupus erythematosus (SLE) patients correlates with disease activity and lupus nephritis. Mechanisms responsible for the increase in serum CD138 and the immunopathological function of serum CD138 in lupus development remain poorly understood. In this study, recapitulating the findings in SLE patients, we found a sharp increase in serum CD138 levels parallel to the progression of disease in MRL/Lpr (lupus prone) mice. Although TCRβ+CD138+ T cells expand with age in MRL/Lpr mice, TCRβ+CD138− cells are the likely source of circulating CD138 as the transfer of TCRβ+CD138− cells, but not TCRβ+CD138+ cells, led to an increase in serum CD138 in the recipient mice. We found that CD138 expressed on lupus T cells was resistant to cleavage by MMP9 and collagenase, but it was very sensitive to trypsin. We also found high levels of trypsin production by TCRβ+CD138− cells. Suggesting a role for trypsin expressed by these cells in the elevated serum CD138 in lupus mice, trypsin produced by TCRβ+CD138− cells effectively cleaved CD138 from T cells. Interestingly, soluble CD138 was able to bind APRIL and enhance APRIL-mediated ERK activation and B cell differentiation. The ability of CD138 to potentiate APRIL-induced B cell differentiation was dependent on TACI expression, as the synergistic effect of APRIL and CD138 on plasma cell formation was strongly ablated on TACI deficient B cells. These findings reveal a role for soluble CD138 in B cell differentiation and autoreactive antibody production in MRL/Lpr mice. Understanding the mechanisms by which soluble CD138 is produced and how it functions may reveal novel druggable targets for lupus disease.