bluuebunny/crossref_metadata_2025 huggingface dataset:
A Multi-armed Approach for Identifying Circulating Bacterial Proteins in Melioidosis.
Authors: Hau Derrick , Pflughoeft Kathryn J , Green Heather R , Hannah Emily E , Thorkildson Peter N , Pandit Sujata G , Demers Haley , Magee D Mitchell , Song Lusheng , LaBaer Joshua , Woosley Rebekah , Quilici David R , Mayo Mark , Currie Bart J , Sahl Jason W , Keim Paul S , AuCoin David P
Published: January 2023

The detection of pathogen-associated macromolecules in clinical samples is a powerful alternative to more traditional gold standards of diagnosing infectious diseases. Burkholderia pseudomallei, the causative agents of melioidosis, is a facultative, intracellular bacteria categorized as a Tier 1 Select Agent by the United States Federal Government for its capacity for large-scale dissemination, elevated rates of mortality and morbidity, and minimal medical countermeasures in place. B. pseudomallei is prevalent in tropical regions and is intrinsically resistant to many first-line antibiotics. The current gold standard for diagnosing melioidosis is blood culture, however this method is inadequate and timely. The multifaceted display of clinical presentations of melioidosis further emphasize the need for specific, yet rapid diagnostics. To further characterize viable bacterial targets present during an infection, a multi-armed approach was used to analyze clinical melioidosis samples through direct and indirect platforms. First, the In vivo Microbial Antigen Discovery (InMAD) platform utilized syngeneic CD1 mice to indirectly detected bacterial proteins in clinical samples in conjunction with a high-density nucleic acid protein array (HD-NAPPA). Secondly, patient serology was evaluated on the HD-NAPPA to determine generated antibody response in the host as these proteins may be shed targets from these intracellular pathogens. Third, protein profiling by liquid chromatography with tandem mass spectrometry (LC-MS/MS) would depict proteins in an unbiased proteomic methodology. Converging data from each approach resulted in multiple targets of interest to be evaluated as biomarkers of melioidosis.
NSAIDs-induced oxidative stress sensitizes tumor cells to T cell cytotoxicity through the TRAIL-DR5 axis
Authors: Aboelella Nada S , Zhou Gang
Published: January 2023

Adoptive T cell therapy (ACT) has shown promising therapeutic outcomes in patients with hematological malignancies. However, the success of ACT is limited to a small fraction of treated patients. Hence, developing new strategies to enhance the potency of ACT are urgently needed for better therapeutic outcomes. Our previous study demonstrated that inducing oxidative stress in tumor cells is critically involved in tumor rejection by ACT. In the current study, we report that indomethacin, a prototypical non-steroidal anti-inflammatory drug (NSAID), can intensify ROS in tumor cells and sensitize them to ACT. Mechanistically, our data indicate that indomethacin treatment leads to enhanced expression of death receptor 5 (DR5) in tumor cells, rendering them more susceptible to TNF-related apoptosis-inducing ligand (TRAIL)-induced cell death. We found that tumor-specific T cells upregulated TRAIL expression upon adoptive transfer. Using genetic-based approaches, we showed that DR5-deficient tumor cells were unresponsive to the beneficial effect of indomethacin, while overexpressing TRAIL in donor T cells led to further improvement in mouse survival. Collectively, our study demonstrates that the pro-oxidative feature of selected NSAIDs can be exploited to augment the efficacy of T cell therapy. Our findings provide a strong rationale for targeting tumor redox to achieve more effective tumor destruction through the combination of pro-oxidants and ACT.
Combination therapy for treating drug-resistant multiple myeloma
Authors: Kim Byung-Gyu , Choi Sung Hee , Luo George , Kim Seong-Jin , Malek Ehsan , Driscoll James , Letterio John
Published: January 2023

Proteasome inhibitors (PIs) have remarkably improved the survival of multiple myeloma (MM) patients but dose-limiting toxicities and the development of drug resistance limit their long-term utility. Elevated TGF-β levels in MM patient sera correlate with chemoresistance, disease progression, metastasis and poor prognosis. Therefore, we evaluated the anti-MM therapeutic potential of a TGF-β type I receptor kinase inhibitor, Vactosertib. In vitro treatment of Vactosertib synergistically inhibited the growth of PI-resistant MM cells in combination with the ixazomib by suppressing TGF-β activation of Smad2/3 and expression of PSMB5, which encodes the proteasome 5 catalytic subunit targeted by PIs. Oral administration of Vactosertib as a single agent inhibited MM progression and induced a decrease in mortality and an increase in body weight of the mice bearing MM. Vactosertib alone also attenuated TGF-β activation of Smad2/3, reduced the expansion of CD11b+ Gr-1+ myeloid derived suppressor cells (MDSCs) in bone marrow tumor microenvironment, and diminished the population of Foxp3+ regulatory T cells (Treg) in the spleen. Combination therapy of Vactosertib plus ixazomib was exhibited a synergistic anti-tumor effect when compared to either Vactosertib or ixazomib alone by greatly prolonged survival and significant a reduction in both MDSCs and Tregs. Taken together, our data provide the rationale for clinical evaluation of Vactosertib in MM and demonstrate proof-of-concept that combination of Vactosertib and PIs may overcome chemoresistance and enhance durable patient responses.

Supported by 1R03CA259901-01A1
Lactobacillus rhamnosus GG re-shapes gut microbiota and triggers STING-type I IFN-dependent antitumor immunity
Authors: Wang Liangliang , Liang Hua Laura , Weichselbaum Ralph
Published: January 2023

Lactobacillus rhamnosus GG (LGG) is one of the most well characterized and widely used probiotics. The beneficial roles of LGG in modulating immune response and inflammatory state during cancer development have been well demonstrated. However the true therapeutic importance of its role in current radiotherapies and immunotherapies is still unclear. Using murine models of colorectal cancer and melanoma, we demonstrate that oral administration of live LGG augmented the antitumor activity of antiPD-1 immunotherapy and/or radiotherapy in either germ-free mice or specific pathogen-free mice. Whole genome shotgun metagenome sequencing analyses revealed that the combination treatment shifted the gut microbial community towards enrichment in Bacteroidetes, Actinobacteria, Proteobacteria, and Cyanobacteria. Notably, among the enriched species, Lactobacillus murinus and Bacteroides uniformis are known to increase DC activation and CD8+ T cell tumor recruitment. Immunologically, the synergistic antitumor effect of LGG and anti-PD1 relied on the increased tumor-infiltrating dendritic cells (DCs) and cytotoxic CD8+ T cells. Mechanistically, treatment with live LGG triggered cGAS/STING-dependent type I interferon (IFN) production in DCs, which resulted in enhanced cross-priming of tumor-specific CD8+ T cells. In DCs, LGG induced IFN-β production depended on cGAS/STING/TBK1/IRF7 axis, as further evidenced by the transcriptome sequencing. Overall, our results demonstrate that oral administration of LGG mediates antitumor immunity through activating DCs in a cGAS/STING-type I IFN-dependent manner. The study provides an alternative and effective strategy to advance cancer immunotherapies and radiotherapies.
High glucose drinks improve the anti-tumor efficacy of brain tumor immunity by gut microbiome modulation
Authors: Kim Jaeho , Lee Heung Kyu
Published: January 2023

Over the past decades, consumption of high sugar drinks has been linked to diverse diseases such as obesity, high blood pressure and diabetes. It has been found that most of these high sugar drinks have negative effect on the host including progression of colorectal malignancies. However, there were little literatures about the effect of intake of these high sugar drinks on distant tumors such as glioblastoma (GBM). We observed the GBM survival rate by supplying 20% glucose water in the GL261 mouse syngeneic GBM model and found consumption of high glucose water increased the survival outcomes. Administration of such high glucose water enhances the anti-tumor efficacy of CD8+ T cells in GBM tumor microenvironment. This change induced a synergistic effect with anti PD-1 immune checkpoint inhibitor in GBM mouse syngeneic model. We found that the gut microbiota induced the anti-tumor effect of high glucose water with negative result of germ-free mice experiment. Through 16S rRNA sequencing analysis of fecal samples of mice, administration of high glucose water induced more abundance of Desulfovibrio genus and proved that this strain played a decisive role in improving the survival rate of the GBM mouse model. As a result, we identified that the intake of high sugar drinks could affect the survival rate of GBM through changes in gut microbiome.
Role of RIG-I in tumor endothelium.
Authors: Baris Adrian , Anand Sudarshan , Khou Sokchea
Published: January 2023

The immune microenvironment plays a critical role in the efficacy of cancer treatment. The manipulation of cytosolic nucleic acid sensors has become a strategy to boost the immune response in cancer therapy, but it is critical to understand how these nucleic acid sensors work in the tumor microenvironment. One aspect of the immune microenvironment is the nucleic acid sensor, which recognizes pathogenic nucleic acids and activates an immune response. Retinoic acid-inducible gene I (RIG-I) is an RNA sensor that responds to dsRNA by activating a type I interferon response. It has been found that RIG-I activation can induce an anti-tumor immune response, but its role in the tumor microenvironment is unknown. Prior studies show regulation of the innate immune response in endothelium can increase the anti-tumor response. Understanding the role of RIG-I in endothelial cells, and how this can be used to potentially reprogram the tumor microenvironment, is critical for developing more effective cancer treatments. In addition, my research on the effects of RIG-I in endothelium has wide-reaching implications beyond cancer treatment. I hypothesize RIG-I activation in endothelial cells will produce an anti-tumor immune response. My findings show that endothelial RIG-I activates a robust type I interferon response and inhibits EC function including, inducing apoptosis and inhibiting migration, which is indicative that RIG-I plays a role in the endothelial immune response. RIG-I also plays a role in the expression of the several angiogenic regulators in a tissue-specific manner. This research will provide critical knowledge about the role of RIG-I in tumor angiogenesis, and provide novel treatment avenues for solid tumors.

Supported by grants from NIH (T32) and Knight (CVP-002)
Investigating the role of interleukin-4 in anti-opioid vaccine efficacy
Authors: Crouse Bethany , Hicks Dustin , Pravetoni Marco
Published: January 2023

Vaccination against opioids is a novel therapeutic strategy to combat opioid use disorder (OUD) which can be used alone or in combination with currently available pharmacotherapies. Vaccination with opioid-carrier conjugates stimulates the production of opioid-specific antibodies that sequester the target drug in the serum and prevent distribution into the brain, attenuating the opioid’s pharmacological effects including analgesia, respiratory depression, and it’s reinforcing effects. An anti-oxycodone vaccine has shown promising preclinical efficacy, selectivity, and safety, and is now being tested in a Phase I clinical trial. Clinical trials for conjugate vaccines against cocaine and nicotine have shown that clinical efficacy is met in vaccinated subjects displaying high concentration of high affinity IgG titers. To increase anti-opioid vaccine clinical efficacy, we sought to elucidate their underlying immunobiology and identify predictive biomarkers of vaccine efficacy. Previously, our lab has found that the depletion of the cytokine interleukin-4 (IL-4) during anti-oxycodone immunization increases antibody titers, enhances class-switching from IgG1 to IgG2a, and leads to improved vaccine efficacy after oxycodone challenge in mice. Here, we investigated the association between increased IgG2a and improved vaccine efficacy by directly testing the role of this subclass during immune response after vaccination and during drug challenge. Furthermore, pre-immunization levels of IL-4 are explored as a predictive biomarker of vaccine efficacy in mice. Results from these studies will inform next generation vaccine design and biomarker identification in human clinical trial participants.

Supported by grants from NIH (R01DA041730, T32DA007097, F31DA054760, HHSN272201800048C, U01DA051658)
Polyanhydride based nanovaccine promotes long term humoral and cellular response to PDAC associated protein MUC4β
Authors: Christiansen John C , Liu Luman , Kshirsagar Prakash , Wafa Emad , Gautam Shailendra , Aithal Abhijit , Gulati Mansi , Kumar Sushil , Solheim Joyce , Batra Surinder , Salem Aliasger , Jain Maneesh , Wannemuehler Michael , Narasimhan Balaji
Published: January 2023

Patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) have an unrelentingly poor prognosis, in part because of the lack of neoantigen targets associated with PDAC tumors. However, another approach would be to take advantage of differentially expressed antigens on PDAC tumor cells. It has been demonstrated that mucin 4 (MUC4) is overexpressed on the tumor cells of many PDAC patients and is considered a promising antigenic target for PC immunotherapy. Previous work by our lab has demonstrated that poly-anhydride (PA) nanovaccines based on 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 1,6-bis(p-carboxyphenoxy)hexane (CPH) nanoparticles (NPs) both induced a humoral immune response to the beta subunit of MUC4 (MUC4β) and an anti-tumor response against OVA in an EG-7 tumor model. In these studies, we show that both a PA-based nanovaccine formulation and a PLGA-based nanovaccine induced MUC4β-specific T cell response. Results demonstrated that restimulation of LN and splenic lymphocytes 40 days after initial vaccination induced both CD4+ and CD8+ MUC4β-specific T cells. The MUC4ß-specific T cells could be detected for at least 180 days post-vaccination. Cytokine secretions from the antigen specific T cells included IL-2, INFγ, and TNF-α; in addition, tetramer analysis detected MUC4ß-specific CD8+ T cells using the MUCβ peptide FTPVFYSQL. Collectively, these results demonstrate that a PA nanovaccine can function as an effective vaccine platform for induction of MUC4ß-specific cellular immunity, and lays the ground work for forthcoming studies in MUC4β-expressing tumor model systems.

Supported by the National Institute of Health (U01 CA213862) and the Iowa State University Nanovaccine Institute. B. N. is grateful to the Vlasta Klima Balloun Faculty Chair.
Characterization of cellular subpopulations and their gene expression by single-cell RNA sequencing in canine atopic dermatitis
Authors: Drechsler Yvonne , Sparling Brandi Ann Terrance Elize , Moss Nicholas Grimm , Kaur Gagandeep , Hawkins R David
Published: January 2023

Atopic Dermatitis (AD) is the most common, chronic, and inflammatory allergic skin disease worldwide, with a high prevalence both in children and adults, particularly in developed countries. There are limited effective treatments for severe allergic skin diseases available, thus, there is an unmet need of better understanding the molecular mechanisms and cellular populations involved in AD. Since AD in canines closely resembles the disease in humans, they are an attractive animal model for further studies.

Leveraging single-cell RNA sequencing, we identified the cellular composition and shifts in cell populations in canine epidermis of atopic dogs compared to the epidermis from healthy controls. Fibroblasts and keratinocytes represented the largest cell populations, with fibroblasts decreasing in the atopic epidermis. Other populations such as endothelial vascular cells showed differences as well. Shifts in immune cell distribution included changes in subpopulations of dendritic cells and monocytes in the atopic skin. NK and T cells, particularly CD8 T cells were increased. Subclustering revealed specific cellular changes in AD dogs. We found signifiant changes in gene expression in most cell populations, with some genes differentially expressed across cells, while others were expressed in a cell specific manner. We also observed subtle differences between samples from animals with different disease progression, identifying gene expression markers changing with AD severity.

Here we show that single-cell sequencing is a useful tool to define the cellular composition in canine atopic dermatitis, resolving changes in gene expression at the cellular level to advance our understanding of disease development and progression.

Supported by intramural funds from Western University of Health Sciences
Changes of small non-coding RNAs by severe acute respiratory syndrome coronavirus 2 infection
Authors: Wu Wenzhe , Zhang Ke , Choi Eunjin , Buck Darby , Wang Binbin , Adam Awadalkareem , Huang Gengming , Lee Inhan , Dong Jianli , Wang Tian , Bao Xiaoyong
Published: January 2023

The ongoing pandemic of coronavirus disease 2019 (COVID-19), which results from the rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a significant global public health threat. In the context of viral infections, sncRNAs have been shown to regulate the host responses, viral replication, and host-virus interaction. Compared with other subfamilies of sncRNAs, including microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), tRNA-derived RNA fragments (tRFs) are relatively new and emerge as a significant regulator of host-virus interactions. Using T4 PNK-RNA-seq, a modified next-generation sequencing (NGS), we found that nasopharyngeal swabs (NPS) samples from SARS-CoV-2 positive and negative subjects show a significant difference in sncRNA profiles. There are about 166 impacted sncRNAs. Among them, tRFs are the most significantly affected and almost all impacted tRFs are derived from the 5′-end of tRNAs (tRF5). Using qRT-PCR, we validated that tRF5s derived from tRNA GluCTC (tRF5-GluCTC), LysCTT (tRF5-LysCTT), ValCAC (tRF5-ValCAC), CysGCA (tRF5-CysGCA) and GlnCTG (tRF5-GlnCTG) are enhanced in NPS samples of SARS-CoV2 patients and SARS-CoV2-infected airway epithelial cells. In addition to host-derived ncRNAs, we also identified several sncRNAs derived from the virus (svRNAs), among which a svRNA derived from CoV2 genomic site 346 to 382 (sv-CoV2-346) has the highest expression. The induction of both tRFs and sv-CoV2-346 has not been reported previously, as the lack of the 3′-OH ends of these sncRNAs prevents them to be detected by routine NGS. In summary, our studies demonstrated the involvement of tRFs in COVID-19 and revealed new CoV2 svRNAs
Maternal and Neonatal Antibody And T Cell Responses To SARS-CoV-2 Following Maternal Infection And/Or Vaccination
Authors: Trinh Ivy , Eyoh Enwono , Murrell Amelie , Desai Srushti , Lauritsen Cody , Rambaran Saraswatie , Stone Addison , Agbodji Ayitevi , Chandra Sruti , Elliott Debra , Smira Ashley , Dugas Courtney , Satou Ryosuke , Pridjian Gabriella , Longo Sherri , Robinson James , Piedimonte Giovanni , Gunn Bronwyn M , Norton Elizabeth B
Published: January 2023

Pregnant women and neonates are at risk for adverse SARS CoV-2 infection outcomes, but few studies have evaluated this population for adaptive immunity. Here, we utilized samples collected from pregnant women 18 years or older reporting two or more respiratory symptoms. Information about trimester of COVID positive test or history of vaccination was also collected. Maternal blood (MB) samples were collected at prenatal clinic visits, or at delivery together with the fetal cord blood (CB)(n=70 dyads). Groups were selected based on trimester of infection, vaccination only, or no history of vaccination or infection (n=9–24) and compared with banked samples from a non-pregnant, age-matched woman from the same time period (n=7–12). Data shows IgG transfer to baby irrespective of trimester of infection (n=9–24 per group). Highest levels of antibodies were observed with vaccinated, uninfected women. Functional antibodies are transferred from mother to fetus following infection and vaccination. CD4 and CD8 T-cell activation and cytokine secretion were detected in maternal PBMCs, with highly variable expression between subjects. In at least one dyad with a PCR-confirmed infection during second trimester, both maternal and cord blood samples had low level expression of anti-S, N, RBD antibodies at delivery and the cord blood mononuclear cells showed CD4 T cell activation (CD134+) and cytokine secretion (IL-2, IL-6, TNFalpha) to spike antigens. These results indicate the unusual immunity to SARS CoV-2 infection during pregnancy and suggest at least one case of a neonate with anti-viral cellular immunity. Ongoing analyses and surveillance for perinatal outcomes may reveal how these immune responses impact infant respiratory outcomes.
Multi-functional HCV-specific CD4+ T cells associated with viral control during the postpartum period express Th1- and Tfh-biasing transcription factors
Authors: Phelps Christopher , Walker Christopher M , Honegger Jonathan R
Published: January 2023

Chronic hepatitis C virus (HCV) infection is characterized by dysfunctional HCV-specific CD8+ T cells, rare CD4+ T responses, and stable high-level viremia. Recovered CD4+ T cell function and unusual declines in viremia have been observed in some women after childbirth. Whether recovered CD4+ T cells are of lineages to help B cells or CD8 T cells is unknown. Preliminary studies suggest both may be true. In 7 women with postpartum viral suppression (>= 1 log10 by 3 months postpartum, “controllers”), and 7 women with more stable viral levels, “non-controllers”, circulating HCV-tetramer+ (tet+) CD4+ T cells expressed chemokine receptors associated with two predominate populations, Th1-biased Tfh (cTfh1) cells and Th1 cells – with a higher cTfh1/Th1 ratio linked to viral control. Here we assessed HCV-specific cTfh1 and Th1 functional responses by combined surface chemokine receptor and intracellular cytokine staining (ICS) after HCV peptide stimulation, and we also assessed expression of lineage-defining transcription factors on HCV-tet+ cells. HCV-specific cytokine+ cells were predominantly cTfh1 or Th1 populations, and multi-functionality correlated with viral control. HCV-tet+ CD4+ T cell Tbet and BCL6 expression levels did not differ by degree of viral control, but both were enhanced in tet+ cTfh1 cells relative to bulk cTfh1 cells, and tet+ Th1 cells expressed significantly more BCL6 than bulk Th1 cells. Together, these data point to a postpartum CD4+ T cell recovery driven by transcription factors favoring cTfh1/Th1-biased phenotypes. Understanding how these cells interact with CD8+ T cells and B cell will be vital for understanding this unique instance of natural control of a chronic viral infection.

Supported by a grant from the NIH (R01 AI096882).
Evidence for metabolic disturbances in the pathogenesis of immune-mediated disease in SAMP1/YitFcs Mice
Authors: Campillo-Gimenez Laure , Drygiannakis Ioannis , Yazgan Ayca Sayi , Rivera-Nieves Jesus , Vera David , Ernst Peter B
Published: January 2023

Background

Inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), affect ~1% of the people in the United States. SAMP1/YitFcs mice are a model of CD with terminal ileitis which is rare in other animal models of IBD. Switch of cell energy metabolism towards aerobic glycolysis is a hallmark of inflammation and has been implicated in immune-mediated diseases. Thus, we sought to determine how energy metabolism is altered in the SAMP mice.

Methods

Glucose metabolism in vivo was determined by 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET). Tissue taking up the 18F-FDG were collected for histology. Metabolic phenotype of activated B and CD4+ T lymphocytes isolated from mesenteric lymph nodes of control and SAMP mice was evaluated by extracellular flux analysis (SeaHorse). To relate the SAMP model to human disease, normal and inflamed ileum from mice and humans were assayed by RNAseq focusing on metabolism-related genes.

Results

PET scans showed a varying tissue distribution of 18F-FDG uptake in SAMP mice, including ileum, salivary glands and kidneys which all had histological evidence of inflammation. Compared to control, both SAMP B and T cells displayed an increase in glycolysis and oxidative phosphorylation (OXPHOS) rates which is supported by an overall increase in genes related to glucose metabolism, TCA cycle, OXPHOS and fatty acid oxidation in the ileum.

Conclusion

SAMP mice have a multi-organ immune-mediated disease relying on an increase in cell energy metabolism. Therefore, targeting aerobic glucose metabolism to limit inflammation emerges as a new strategy for CD treatment.
Correlation between acute and chronic inflammatory states, a case control study
Authors: Mahesh Seema , Mallappa Mahesh , Vacaras Vitalie , Shah Viraj , Belagaje Amritha , Dhamodar Pooja , Serzhantova Elena , Kubasheva Nadezhda , Chabanov Dmitriy , Tsintzas Dionysios , Jaggi Latika , Jaggi Atul , Vithoulkas George
Published: January 2023

Objective

Suppression of efficient acute inflammation may be one of the mechanisms behind the onset of chronic low-grade inflammation. Efficient acute inflammatory response to pathogenic stimulus may not be possible in the presence of chronic inflammation. We investigated if a correlation exists between chronic and efficient acute inflammation.

Methods

Design

Case control study.

Setting

Homeopathic medical practices in 4 countries Patients with definite improvement in chronic inflammatory conditions with at least 6 months of follow up were selected as cases. Age matched controls involved those who did not improve.

Event of interest

Occurrence of common acute infectious diseases with fever during the follow up period.

Statistical analysis

Odds Ratio of improvement in the chronic condition, with development of acute infectious diseases with fever was calculated. Graphs were plotted to study this correlation in individual cases.

Results

20 cases and 20 age matched controls were selected. Average age was 28.4 and 27.9 years respectively. 18/20 cases and 4/20 controls developed common infectious diseases with fever during the follow up period. Odds Ratio of the chronic condition improving, with development of acute infectious diseases with fever was 36 (95 %, CI: 5.7973 to 223.5513), z statistic: 3.846 and significance level was p = 0.0001. Graphs of individual cases showed distinct patterns confirming the same.

Conclusions

In this case control study, appearance of common acute infectious diseases with fever during treatment was strongly associated with improvement in the chronic inflammatory conditions. Larger studies are needed to further establish the correlation.

Supported by Nil
COVID-19 patients are characterized by increased levels of immune cell membrane-bound and soluble CD48
Authors: Pahima Hadas Tamar , Zaffran Ilan , Jarjoui Amir , Orenbuch-Harroch Efrat , Gaur Pratibha , Puxeddu Ilaria , Zinner Carl , Ben-Chetrit Eli , Tzankov Alexandar , Levi-Schaffer Francesca
Published: January 2023

COVID-19 is a respiratory-centered systemic disorder caused by the severe acute respiratory syndrome (SARS-CoV-2) virus. The disease can progress into a severe form causing acute lung injury (ALI), mainly diffuse alveolar damage (DAD) with thromboinflammation, immunopathology, and cytokine storm. CD48 is an activating/co-activating receptor expressed on most hematopoietic cells, existing as both membrane-bound (mCD48) and soluble (sCD48) forms. Its high-affinity ligand is 2B4 (CD244). We previously found the mCD48 and sCD48 levels are dysregulated in asthma patients regardless of their atopic status. Therefore, we reasoned that CD48 could be dysregulated in COVID-19 ALI too.

CD48 expression was evaluated in tissue sections collected at autopsies of lethal COVID-19 at the first wave (patients not being exposed to high dose dexamethasone), and on peripheral blood leukocytes and in sera of COVID-19 patients by gene expression profiling (autoimmune panel pf HTG), IHC, flow-cytometry and ELISA. Lung tissue of COVID-19 patients showed significantly increased CD48 mRNA expression and infiltration of CD48+ lymphocytes in comparison to other inflammatory conditions (influenza-virus and pneumococcal pneumonia, and non-COVID-19 DAD). In the peripheral blood mCD48 was significantly increased on all the evaluated cells and, additionally, sCD48 levels were significantly higher in COVID-19 patients independent of disease severity. Positive correlation was found between mCD48 levels on monocytes and sCD48 release.

Since both mCD48 and sCD48 are significantly increased in COVID-19, a specific a role for CD48 in COVID-19 can be assumed, suggesting it as a potential target for therapy.

Supported by Israel Science Foundation Grant no. 3933/19 the Aimwell Charitable Trust (UK), Emalie Gutterman Memorial Endowed Fund for COPD related research (USA)
Circulating Monocytes Co-expressing Surface ACE2 and TMPRSS2 upon TLR4/7/8 Activation Are Susceptible to SARS-CoV-2 Infection
Authors: Yao Yi , Subedi Kalpana , Sexton Jonathan Z , Liu Tingting , Khalasawi Namir , Pretto Carla , Wotring Jesse W , Wang Jie , Yin Congcong , Jiang Aimin , Li Jia , Zhou Li , McKinnon John , Mi Qing-Sheng
Published: January 2023

Angiotensin-converting enzyme 2 (ACE2) receptor is required for SARS-CoV-2 entry into human cells. However, emerging evidence shows SARS-CoV-2 infected lung monocytes/macrophages from COVID-19 patients barely express ACE2 mRNA, raising a question how SARS-CoV-2 penetrates macrophages. It’s also under debating whether the peripheral blood cells (HPBCs) can be infected by SARS-CoV-2 that may facilitate viral spread from circulation to other organs besides lung. Herein we demonstrate that resting primary HPBCs harbor abundant cytoplasmic ACE2, regardless of COVID-19 status, and that surface translocation is necessary for viral infection. Upon ex vivo TLR4/7/8 stimulation of HPBCs, ACE2 translocated to the cell surface independent of ACE2 transcription, and this translocation was blocked by an endosomal trafficking inhibitor, suggesting the putative source as ACE2-containing exosomes. However, only stimulated monocytes concurrently expressing ACE2 and cell surface transmembrane serine protease type 2 (TMPRSS2) were efficiently infected by SARS-CoV-2, which was significantly mitigated by remdesivir. Furthermore, ACE2 surface translocation in peripheral myeloid cells from patients with severe COVID-19 correlated with their proinflammatory cytokine production. Collectively, TLR4/7/8-induced ACE2 translocation with TMPRSS2 expression is indispensable for SARS-CoV-2 infection of circulating monocytes. Our work not only provides a new mechanism for the pathogenesis of SARS-CoV-2 and a potential path for its systemic infection, but also unveils a prospective therapeutic strategy by targeting ACE2 trafficking for preventing monocyte/macrophage infection.

This study is partially supported by National Institutes of Health grants R61AR076803, R01AR063611, R01AI119041, and R01AR069681 (Q-S. M.), R01AR072046 (L.Z.), R01DK120623 (J.Z.S.), Henry Ford Immunology Program grants (T71016, Q-S. M.; T71017, L. Z.), and funding for the U-M Center for Drug Repurposing - NCATS CTSA UL1TR002240 (J.Z.S.).
Osteoclast differentiation alters monocyte inflammatory responses to Staphylococcus aureus
Authors: Petronglo Jenna Rose , Eichelberger Kara Rose , Cassat Jim
Published: January 2023

Staphylococcus aureus is the major causative agent of bacterial osteomyelitis, an invasive infection of bone. Osteomyelitis is associated with significant bone destruction, bone-resorbing osteoclasts have been implicated in this process. Osteoclasts differentiate from monocyte precursors in response to the canonical osteoclastogenic cytokine RANKL, or receptor activator of (NF)-κB ligand. The process of osteoclastogenesis involves significant transcriptional reprogramming, thereby altering inflammatory responses of osteoclasts, relative to inflammatory monocytes or macrophages. In this study, we sought to elucidate mechanisms of infection-associated bone loss by defining how RANKL shapes osteoclast responses to S. aureus secreted virulence factors and pathogen-associated molecular patterns. We found that, whereas RANKL-naïve monocytes produce significant amounts of IL-1β in response to priming with S. aureus supernatants, differentiated osteoclasts do not. Osteoclasts instead undergo enhanced caspase-dependent cell death, relative to RANKL-naïve monocytes, without associated IL-1β release. This cell death occurs in response to virulence factors regulated by the S. aureus accessory gene regulator (Agr) quorum-sensing system. These results suggest that RANKL alters monocyte-lineage inflammatory responses to microbial pathogens, that osteoclasts have enhanced susceptibility to S. aureus cytotoxins. In parallel studies, we also found that S. aureus supernatants promote osteoclast differentiation of RANKL-primed monocytes in a TLR-dependent manner. Collectively, these studies contribute to the mechanistic understanding of dysregulated bone homeostasis and bone loss during infection.

Supported by R01AI161022, R01AI145992, R01AI132560, Burroughs Wellcome Fund and T32AR059039.
Tissue resident lung natural killer (NK) cells demonstrate enhanced responses to IL-15 compared to peripheral populations
Authors: Sholevar Cyrus , Judge Sean J , Nielsen Ryan , Farley Lauren E , Dunai Cordelia , Razmara Aryana M , Cooke David T , Brown Erin G , Rebhun Robert B , Murphy William J , Canter Robert J
Published: January 2023

Tissue resident NK cells with unique phenotype and function have been identified in organs such as the lung and liver. We have shown that inhaled (IH) rhIL-15 can lead to durable clinical responses in dogs with naturally occurring osteosarcoma or melanoma lung metastases, suggesting that lung NK populations can be targeted therapeutically. Thus, we set out to evaluate the phenotype of resting and rhIL-15-stimulated lung NK cells isolated from murine and human lung. C57BL/6 and BALB/c mice were exposed to IH or systemic IL-15 at escalating doses, and NK cells were isolated by negative selection from lungs and spleen of wild type mice for co-culture. In human patients undergoing surgery for malignancy, non-tumor lung tissue was isolated for flow cytometry and ex vivo stimulation with IL-15. Using mouse spleen or human blood as controls, we observed a higher proportion of lung NK cells per live CD45+ cells in baseline mouse and human samples. Lung NK also had higher expression of activation markers CD69 and HLA-DR (human), but lower expression of the inhibitory receptor TIGIT. Ex vivo culture with IL-15 also resulted in greater upregulation of Ki67 on lung NK cells. Compared to systemic delivery of IL-15, IH IL-15 resulted in greater upregulation of Ki67 and lower upregulation of TIGIT in lung NK cells. Subset analysis of CD49a+ lung NK cells showed similar results in both mouse and human models for activation marker and Ki67 expression after IL-15. Taken together, our results suggest that lung NK cells possess a more active baseline phenotype and exhibit preferential stimulation in response to rhIL-15 compared to circulating NK cells. Ultimately, targeting of lung resident NK cells may be a viable therapeutic strategy in patients with lung metastases.

Supported by NIH/ NCI 1R03CA252793 NIH/NCI U01 CA224166-01 NIH/NCI T32 CA251007-01
Epigenetic Remodeling by Vitamin C Potentiates the Differentiation of Mouse and Human Plasma Cells
Authors: Lio Chan-Wang Jerry , Chen Heng-Yi , Almonte-Loya Ana , Lay Fang-Yun , Johnson Eric , Gonzalez-Avalos Edahi , Yin Jieyun , Ma Qin , Wozniak Daniel , Harrison Fiona
Published: January 2023

Ascorbate (vitamin C) is an essential micronutrient in humans. The chronic severe deficiency of ascorbate, termed scurvy, has long been associated with increased susceptibility to infections. How ascorbate affects the immune system at the cellular and molecular levels remained unclear. Here, from a micronutrient screen, we identified ascorbate as a potent enhancer for antibody response by facilitating the IL-21/STAT3-dependent plasma cell differentiation in mouse and human B cells. The effect of ascorbate is unique, as other antioxidants failed to promote plasma cell differentiation. Ascorbate is critical during early B cell activation by poising the cells to plasma cell lineage without affecting the proximal IL-21/STAT3 signaling and the overall transcriptome. Consistent with its role as a cofactor for epigenetic enzymes, ascorbate potentiates plasma cell differentiation by remodeling the epigenome via TET (Ten Eleven Translocation), the enzymes responsible for DNA demethylation by oxidizing 5-methylcytosines into 5-hydroxymethylcytosine (5hmC). Genome-wide 5hmC profiling identified ascorbate responsive elements (EAR) at the Prdm1 locus, including a distal element with a STAT3 motif overlapped with a CpG that was methylated and modified by TET in the presence of ascorbate. The results suggest that an adequate level of VC is required for antibody response and highlight how micronutrients regulate the activity of epigenetic enzymes to regulate gene expression. Our findings imply that epigenetic enzymes can function as sensors to gauge the availability of metabolites and influence cell fate decisions.

This research was funded by LJI/KKR young investigator fund; NIH National Cancer Institute K22 (K22CA241290); startup funds from the Department of Microbial Infection and Immunity and from the Pelotonia Institute of Immuno-oncology at the Ohio State University.
Select mucosa-associated intestinal commensal bacteria promote gut barrier repair by inducing IL-1b production
Authors: Wu Wan-Jung , Kim Myunghoo , Chang Lin-Chun , Assie Adrien , Saldana-Morales Fatima B , Ruiz Daniel Fernando Zegarra , Norwood Kendra , Samuel Buck , Diehl Gretchen E
Published: January 2023

Microbiota interactions with host tissue support proper intestinal functions. In diseases such as inflammatory bowel disease, microbial shift along with epithelial dysfunctions are found. Understanding how intestinal epithelial functions are regulated by distinct commensal bacteria is key to improving outcomes in such diseases. Here, we identified a mouse commensal E. coli isolate, GDAR2-2, that promotes intestinal barrier repair in mouse colitis models including Citrobacter rodentium infection and dextran sulfate sodium-induced colitis. In mice, protection after GDAR2-2 colonization depends on CX3CR1+ mononuclear phagocytes (MNPs), which induce expansion of IL-22-secreting type 3 innate lymphoid cells. We detect upregulation of markers of transit amplifying cells, proliferation of which is downstream of IL-22 signaling. Protection is lost if IL-22 is blocked, indicating IL-22 driven epithelial regeneration is downstream of GDAR2-2 colonization. By co-culturing bone marrow derived macrophages with live GDAR2-2, we found GDAR2-2 promotes IL-1b production and the in vivo protection was lost after blockade of IL-1b. We further identified a subset of human commensal E. coli isolates that similarly induce CX3CR1+ MNP expansion and IL-1b mediated protection from C. rodentium infection in mice. This study reveals an unexpected role for IL-1b that promotes intestinal barrier repair and is induced by select commensal bacteria.

Supported by NIH (AI125264, P30DK056338)
Crosstalk between skin homing innate T cells and epithelial cells via cholesterol byproduct messengers is required for tissue immunity
Authors: Frascoli Michela , Ferraj Enxhi , Miu Bing , Esposito Kelsey , Spidale Nicholas , Malin Justin , Cowan Jennifer , Bhandoola Avinash , Kang Joonsoo , Reboldi Andrea
Published: January 2023

Innate T cells and innate lymphoid cells in barrier tissues are essential for both tissue fitness and immunity against pathogens. γδTCR+ lymphocytes that secrete IL-17 (Tγδ17) localize to dermis and are critical regulators of skin immune responses. Despite their functional importance, how Tγδ17 cells mediate their unique function in the skin is largely unknown. Tγδ17 cells express the two prototypic G-protein coupled receptors (GPCRs) CCR6 and GPR183 (EBI2): mice lacking both receptors had significantly diminished dermal Tγδ17 cells and were resistant to psoriasis induction. This was due to a block in Tγδ17 cell maturation in the thymus of Ccr6−/− Gpr183−/− double knock-out mice. EBI2 recognizes oxysterols generated by the cholesterol hydroxylase CH25H and functional oxysterols were detected in both the thymus and skin. Analysis of Ch25h-reporter mice showed that interfollicular epithelial cells (IFEs) and a subset of medullary thymic epithelial cells (mTEC) are the depot for oxysterols in skin and thymus respectively. Ch25h+ TECs were also identified by unbiased single cell transcriptome analyses. These results demonstrate that developing Tγδ17 cells must be conditioned by sensing cholesterol byproducts synthesized by a discrete subset of mTECs to position in specific oxysterol rich niches in the skin. Moreover, in the skin, dietary cholesterol increased tissue oxysterol concentration and modulated homeostatic and pathogenic effector function of Tγδ17 cells in a EBI2 dependent fashion. Given that oxysterols can be regulated by infection and inflammation, this sensory circuit directed by cholesterol is predicted to program skin innate T cells’ ability to rapidly survey for perturbations in the skin.

Supported by grant from NIH R21AI143225.
The transcription factor LRF promotes Integrin β7 expression by and gut homing of CD8αα intraepithelial lymphocyte precursors
Authors: Nie Jia , Bohrer Andrea , Chopp Laura , Chen Ting , Balmaceno-Criss Mariah , Ciucci Thomas , Xiao Qi , Kelly Michael , McGavern Dorian , Belkaid Yasmine , Bosselut Remy
Published: January 2023

TCRαβ+ CD8α+ CD8β− intraepithelial lymphocytes (CD8αα IEL) differentiate from thymic IEL precursors (IELp) and contribute to gut homeostasis. However, their development remains poorly understood. Here we show that the zinc finger transcription factor LRF, encoded by Zbtb7a, is highly expressed in both CD8αα+ IELs and in IELp and serves in a cell-intrinsic manner to promote IEL differentiation. Mice genetically deficient for LRF (Cd4-Cre Zbtb7afl/fl) lack CD8αα+ IELs, contrasting with largely conserved numbers of conventional CD8αβ+ T cells in the thymus and the secondary lymphoid organs. Unlike their wild-type counterparts, LRF-deficient IELp failed to prevent colitis caused by adoptive transfer of CD4+ T cells in T cell-deficient mice. LRF disruption affects neither the development of thymic IELp, nor their in vitro expansion or differentiation in response to IL-15. However, Cd4-Cre Zbtb7afl/fl mice accumulate CD8αα+ T cells in the spleen, unlike wild-type controls, suggesting impaired migration of IELp to the gut. Single-cell RNA sequencing found LRF necessary for the expression of genes characteristic of the most mature IELp, including Itgb7, encoding the β7 subunit of α4β7. Chromatin immunoprecipitation and gene regulatory network analyses both defined Itgb7 as an LRF target. Our study identifies LRF as an essential transcriptional regulator of IELp maturation in the thymus and subsequent migration to the intestinal epithelium.
Potential Clinical Applicability of The PHENotype SIMulator for In Silico Viral Co-Infection Studies in COVID-19
Authors: Maria Naomi I , Rapicavoli Rosaria Valentina , Alaimo Salvatore , Bischof Evelyne , Pulvirenti Alfredo , Mishra Bud , Duits Ashley John , Ferro Alfredo
Published: January 2023

The PHENotype SIMulator, is a systems biology tool, which leverages available transcriptomic and proteomic databases to model of SARS-CoV-2 host-infection in silico. PHENSIM can determine the viral effects on cellular host-immune responses, which we recently applied to in silico drug repurposing for COVID-19. There is a clear importance of previously imprinted viral infections on the host-immune response against SARS-CoV2. In this study we explore the potential clinical applicability of PHENSIM in addressing co-infection of SARS-CoV2 (SCoV2) and human rhinovirus (HRV), or SCoV2 and Influenza A Virus (IAV).

We leveraged PHENSIM to simulate HRV-infection of A549 lung alveolar cells at 24hours in silico, yielding comparable cellular transcriptomic signatures as previously published for in vitro HRV-infection, including antiviral and inflammatory gene transcriptomics. Next, we simulated viral co-infection of HRV/SCoV2 or IAV/SCoV2 using PHENSIM, and assessed co-infection effects in silico.

Similar to recent in vitro studies, our in silico results indicated HRV-infection prior to SCoV2 exposure induced ISG responses and downregulated SCoV2 transcriptomic activity. Further analysis revealed several key SCoV2 affected pathways, including IFN Type-I & -III and MHC-II pathways, to be negatively affected by HRV-infection. In contrast, co-infection with IAV and SCoV2 resulted in overall increased SCoV2 transcriptomic activity.

Our PHENSIM results confirm HRV-imprinted downmodulation of SARS-CoV2 activity and the importance of timely IFN pathway activation for effective immune response and viral clearance. Identified anti-correlating pathways are of interest for therapeutic targeting and effective drug development.

N.I.M. was funded in part by a fellowship award from the Netherlands-Caribbean Foundation for Clinical Higher Education (NASKHO). S.A., A.F. and A.P. have been partially supported by the MIUR PON research project BILIGeCT “Liquid Biopsies for Cancer Clinical Management”. National Cancer Institute Physical Sciences-Oncology Center Grant U54 CA193313-01 (to B.M.), and US Army grant W911NF1810427 (to B.M.)
Semi and fully automated immunostaining sample preparation platforms improve live leukocyte recovery, reproducibility, and flow cytometry data quality
Authors: Feld Geoffrey K , Lye Melvin , Eberle Christoph , Wang Ann , Ke Chyan Ying
Published: January 2023

Limited innovation in automated cell and organelle sample preparation methodology limits the effectiveness of modern analytical methods, such as single-cell ‘omics, flow and mass cytometry. These techniques traditionally rely on manual centrifugation-based protocols for cell washing and suspension preparation, hampering researchers’ access to the reproducibility and scalability benefits of automation. We have developed a suite of cell suspension preparation systems that enable semi and full automation of cell washing protocols. These Laminar Wash™ technologies robustly, gently, and efficiently remove debris, dead cells, and unbound reagent using laminar flow and liquid handling robotics, rather than turbulent and harsh pelleting-plus-pipetting methods. Adaptation of standard protocols to Laminar Wash automation typically improves repetitive immunostaining processes and workflows, in terms of reduced hands-on time and inter- and intra-operator variability. We demonstrate the superior live cell retention and reproducibility of Laminar Wash over centrifugation in processing murine and humanized mouse peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) for flow cytometry. Furthermore, we show how Laminar Wash improves flow cytometry data quality, in terms of debris removal and separation of immune cell subsets for both PBMCs and TILs. Overall, these results show how Laminar Wash methodology assists in standardizing sample preparation for cytometric analysis, an important and unmet need in cancer immunotherapy discovery and manufacturing workflows.