bluuebunny/crossref_metadata_2025_split huggingface dataset:
MyD88-mediated signaling in myo-/fibroblasts is required for control of macrophage maturation under mucosal tolerance in the gut
Authors: Chulkina Marina , Uribe Gabriela , He Bing , McAninch Steven , Khanipov Kamil , Golovko George , Markov Nikolay S , Powell Don W , Beswick Ellen , Pinchuk Iryna V 
Published: January 2023

Abstract

Introduction
Colonic myo-/fibroblasts (MFs) are cells of mesenchymal origin in a healthy gut are among the key players in the mucosal tolerance that involve MF-restricted MyD88 signaling. MFs and macrophages interact in colonic tissue. However, the mechanisms of MF mediated regulation of macrophages under intestinal mucosal tolerance has emerging evidence. We hypothesized that MF-restricted MyD88 signaling regulates macrophage maturation under colonic mucosal tolerance.

Methods
RNAseq, qRT-PCR, H&E, and flow cytometry was used for analysis of colonic tissue/cell derived from B6ACTA2CreMyD88fl/fl, B6Col1a2CreMyD88fl/fl and B6RagCol1a2CreMyD88fl/fl MF-specific conditional KO mice.

Results
MF-specific deletion of MyD88 in vivo resulted in the development of microcolitis and was associated with dysbiotic decrease in Firmicutes to Bacteroidetes bacterial ratio, a hallmark of intestinal inflammation. This process was associated with the increase in colonic expression of genes linked to the inflammatory activity of myeloid cells and accumulation of immature monocyte-derived macrophages. In vitro deletion of MyD88 from MFs (isolated from MF-specific MyD88 conditional KO mice) resulted in changes of basal and TLR4 mediated pathways involved in myeloid cell migration/maturation and inflammation. Using MF-macrophage co-cultures we demonstrated the MF-restricted MyD88 signaling suppresses myeloid cell inflammatory activity.

Conclusion
Intrinsic MyD88 signaling within colonic mesenchymal cells is critical to control myeloid cell differentiation and inflammatory activity under mucosal tolerance in the gut.
Supported by CTSA/TL1 5TL1TR001440-04 UTMB Institute for Translational Science and R01 DK103150 National Institute of Diabetes and Digestive and Kidney Diseases.
The role of progesterone in barrier responses to intravaginal viral infection.
Authors: Dulson Sarah J , Lopez Cesar A , Lazear Helen M 
Published: January 2023

Abstract
The female reproductive tract (FRT) is a robust physical and immunological barrier tasked with balancing tolerogenic immunity to non-self antigens such as commensal bacteria, sperm, and fetal tissues, with protection against sexually transmitted pathogens such as herpes simplex virus (HSV) and Zika virus (ZIKV). The FRT is dynamically regulated by hormones, but the mechanisms by which hormones affect antiviral responses in immune and non-immune cells are incompletely understood. We found that a high progesterone state, such as pregnancy or treatment with Depo-medroxyprogesterone acetate (DMPA, Depo-provera), was necessary to render mice susceptible to vaginal infection with HSV-1 or ZIKV, and that this was true for both wild-type and immunocompromised Ifnar1−/− mice. The effect of progesterone was specific to vaginal infection as treatment with DMPA did not enhance susceptibility to HSV or ZIKV infection by dermal or subcutaneous inoculation, respectively. Importantly, we found that only DMPA-treated mice were protected against secondary vaginal infection, suggesting that productive viral replication is necessary for the establishment of protective memory responses in the vagina. Although we found that DMPA increased dye permeability of the vagina, mechanical abrasion without DMPA treatment was not sufficient to induce susceptibility, implicating other roles of progesterone in modulating epithelial antiviral responses. Together, these data underscore a crucial endocrine-immune axis involved in early antiviral responses in the vagina. Ongoing studies seek to identify progesterone-responsive cells in the vagina and to define the mechanism by which progesterone renders these cells permissive to viral infection.
Supported by grants from NIH K12-GM000678 (SJD), NIH F31 AI143237 (CAL), NIH R01 AI139512 (HML), and NIH R21 AI44631 (HML).
Enzymatic/mechanical digestion outperforms explant culture for isolation of tissue resident memory t cells from human lungs
Authors: Geber Adam B , Sportiello Michael John , Hyuck Heidie , Pryhuber Gloria , Topham David J 
Published: January 2023

Abstract
Tissue resident memory T cells (TRM) comprise a distinct population which persists in the tissue after infection. Lung TRM help to coordinate early immune responses to infections of the respiratory tract, but sampling of the relevant anatomical sites in humans is logistically difficult and not easily standardized. We used fresh human tissues to compare methods for organ dissociation and lymphocyte enrichment with regard to T cell phenotype and viability. The two main approaches to tissue processing were explant culture and combined enzymatic/mechanical digestion. While cytokine supplementation was able to induce T cell egress from intact respiratory samples, the majority of TRM remained in situ and the viability of all cells was compromised after three days of ex vivo culture. More T cells were recovered after IL-2 supplementation than with IL-7, but it remains unclear whether IL-2 is inducing proliferation in vitro. Enzymatic and mechanical digestion did not appear to alter the antigenic phenotype of the lymphocyte population and yielded enriched and purified cellular samples that were suitable for downstream flow cytometric or transcriptomic assays. Results suggest that minimizing processing time and using a targeted enzymatic cocktail are important considerations for working with human lung TRM.
Supported by NIH grant GR531572.
Bystander CD8 T cell memory responses partially protect mice against lethal vaginal HSV-2 challenge
Authors: Arkatkar Tanvi , Dave Veronica , Talavera Irene , Prlic Martin , Lund Jennifer M 
Published: January 2023

Abstract
Herpes Simplex Virus type-2 (HSV-2) infection is one of the most prevalent sexually transmitted infections, yet no vaccine is currently available. Detailed analysis of HSV-infected human tissue revealed tissue-resident CD8 T cells (TRM) limit the duration and severity of HSV-2 episodes. Interestingly, recent studies indicate that the sensing and alarming function of CD8 TRMs is not restricted to cognate antigen interaction, but CD8 TRM can mediate protection against antigenically unrelated pathogens, termed “bystander activation.” Here, we investigated if antigen non-specific CD8 T cells could provide some degree of protection in a bystander fashion in the context of HSV infection. To test this, we created antigen non-specific memory compartments through immunization of mice with Listeria expressing ovalbumin (OVA) (LM-OVA). Mice were then challenged with wild-type HSV-2 to assess the degree of bystander-mediated protection. Immunization with LM-OVA-delayed disease progression from lethal viral challenge, suggesting that bystander CD8 T cells may mediate protection despite the lack of antigen-specificity. Furthermore, we found early infiltration of antigen-non-specific CD8 T cells (OVA-specific) in the vaginal tract in the infection setting. Additionally, assessment of the memory CD8 compartment in human vaginal tissue upon in-vitro treatment with cytokines caused bystander activation of memory CD8 T cells. Finally, we found that in-vitro treatment of CD8 T cells with cytokines delayed disease progression and reduced viral burden upon adoptive transfer. Altogether, our findings suggest that local bystander-activation of CD8 memory T cells can participate in protection from HSV-2.
Intranasal, not parenteral, vaccination induces the formation of tissue-resident memory CD8 T cells in nasal mucosa that rapidly clear influenza virus infection
Authors: Matysiak Colette , Kazer Samuel , Ordovas-Montanes Jose , von Andrian Ulrich H 
Published: January 2023

Abstract
Respiratory tract infections are among the leading causes of death. While current vaccines reduce severe disease, they provide suboptimal mucosal protection (e.g. against influenza virus and SARS-CoV-2). Nasal vaccines offer an advantage by quickly limiting viral shedding, in part by generating tissue-resident memory (Trm) CD8 T cells in the nasal mucosa. But little is known about the molecular requirements for this Trm formation. The common paradigm is that lymphocytes home to tissues after having been imprinted within draining lymph nodes to express a tissue-specific combination of trafficking molecules, and Trm cells form rapidly from this pool. To probe the paradigm of tissue homing to the nasal mucosa, we compared CD8 T cell trafficking following intranasal and intramuscular vaccination. To investigate the trafficking molecules expressed by CD8 T cells and nasal cells we generated a single-cell RNA sequencing profile of the nasal mucosa over the course of an acute influenza infection. We found significantly more CD8 Trm cells in the nasal mucosa following intranasal vaccination that rapidly cleared influenza infection. Notably, CD8 T cells could be “pulled” into the nasal mucosa with an inflammatory stimulus following intramuscular vaccination, suggesting a new strategy for generating nasal CD8 Trm cells. Unlike canonical T cell homing to the gut and skin, nasal CD8 T cell trafficking depended on a4/VCAM1 (not a4b7/MADCAM1 or P and E-Selectin), and CD8 T cells expressed high levels of CXCR3 and CXCR6. This combination of trafficking molecules suggests a distinct multi-step adhesion cascade for CD8 T cell recruitment to the nasal mucosa and provides critical insights to rationally design vaccines for respiratory tract protection.
Supported by grants from NIH (R01 AR068383-01, P01 AI 112521) and HMS-AbbVie Alliance
Defining the transcriptional signature and heterogeneity of lung resident memory B cells
Authors: Padilla-Quirarte Herbey Oswaldo , Williams Elliot , Scharer Christopher 
Published: January 2023

Abstract
Lung resident memory cells have been shown to drive protection against viruses and bacteria, and recently resident memory B cells (BRM) have been also recognized in humans and animal models as critical immune mediators of protection from pathogens. Compared to naïve B cells, circulating influenza-specific memory B cells harbor a unique epigenetic and transcriptional landscape. Although lung-resident memory T cells have distinct molecular programming, it is not known if lung BRM are similarly programmed or distinct. Recent work has defined the emergence and requirement for T cell help in the formation of BRM, suggesting a distinct set of signals are required to promote lung residency compared to circulating memory. Here we investigated the unique programming of lung BRM and plasma cells to determine how their unique transcriptional, metabolic, and epigenetic properties. We used single-cell RNA sequencing (sc-RNA-seq) on sorted lung resident and mediastinal LN (mLN) memory B cells and antibody secreting cells from influenza-infected C57BL/6 mice 35 days post-infection. As a comparison, circulating naïve B cells were also sorted. Here, we investigated BRM heterogeneity and transcriptional programming in the lung tissue. Comparisons to tissue resident memory T cells define metabolic characteristics that promote survival in the lung microenvironment. These data defined the BRM molecular properties compared to circulating B cells and their unique relationship to plasma cells in the lung.
Supported by grant 5R01AI148471-02 from NIH
Human Defensin-5 (HD-5) and HD-6 mRNA Produced by Intestinal Crypt Paneth Cells are Decreased in Fecal Samples of Patients Positive for SARS-CoV2
Authors: Ryan Lisa K , Robinson Tanya O , Figgins Erika H , Do Uyen ‘Emily’ , Wilson Kenneth J , Glover Sarah C , Diamond Gill 
Published: January 2023

Abstract
Alpha-defensins (AD) are antimicrobial peptides (AMP) that are found on mucosal surfaces, providing innate immune defense against pathogens. Human defensin-5 (HD-5) and HD-6 are AD produced only by Paneth cells of small intestinal crypts. SARS-CoV2 virus has been shown to infect the small intestinal cells via the ACE2 receptor and suppress inflammation in the gut. HD-5 and HD-6 bind ACE2 to prevent entry to intestinal cells. Based on the observation that human beta defensin-1 (hBD-1) is downregulated by influenza virus in airway epithelial cells, we hypothesized that HD-5 and HD-6 could be similarly downregulated by SARS-CoV2. To examine this in situ, we sampled patient cohorts with similar co-morbidities, with and without SARS-CoV2 positivity, for HD-5 and HD-6 gene expression via qRT-PCR. Stool samples were collected from all patients, diluted in Qiagen RNA Later and frozen at −80°C prior to total RNA extraction. Results revealed a significant decrease in both HD-5 (p=0.000006) and HD-6 (p=0.002) of SARS-CoV2 positive patients compared with non-Covid19 patients negative for SARS-CoV2. The results were consistent in fecal swabs or fecal streams. Gene expression in other significant markers of inflammation were also blunted: sPLA-2 (p=0.006), alpha-1-anti-trypsin (p=0.0001) and trypsin 2 (p=0.005). Other gut inflammatory markers PSTI, HIP/PAP, trypsin 3 and IL-8 were unchanged. There was a trend in IL-8 upregulation in Covid19 patients. This is the first demonstration of defensin mRNA detection in stool samples. The results suggest that decreasing AMPs may be a mechanism by which the virus increases its chance for survival.
Supported by R21DE028378
Nasopharyngeal inflammatory lymphocytes perturbation in COVID-19 severity prediction
Authors: Ayass Mohamad Ammar , Tripathi Trivendra , Griko Natalya , Zhang Jin , Nair Ramya Ramankutty , Dai Jun , Zhu Kevin , Cao Wanying , Pashkov Victor , Singh Sharda Kalla , Mosleh Lina Abi 
Published: January 2023

Abstract
SARS-CoV-2 causes severe respiratory infections in some patients due to an uncontrolled immune response. The nasal microenvironment plays a crucial role in the transmission, modulation, and clinical progression of COVID-19, however, the immune-responses at the site of viral entry remain poorly understood. We used flow cytometry to decipher nasopharyngeal immune profiling of COVID-19 positive/negative patients; viral detection and load were performed by RT-PCR. Analysis of viral-induced immune-inflammatory responses, lymphocytes population/activation lymphocytes markers, T cell subtypes and associated inflammatory cytokines. Furthermore, we performed in vitro assays using cultured monocytes-derived-macrophages trained with/without SARS-CoV-2 spike protein and co-cultured with/without autologous T cells. Results showed that positive patients had high inflammatory CD45+ and CD8+ T cells including elevated % of Th1/Th17.1, associated with a worse outcome in a subgroup of COVID-19 infected patients. Positive patients showed increased % of cytokines, IFN-γ+, IL-17+, and IL-10+ T cells suggesting a crucial role of these cytokines in recruiting and activating neutrophils that can migrate to target organs (e.g., lungs) and potentially play an important role in the disease severity. Moreover, upregulated Tregs along with monocytic-induced inflammatory T-cell responses and increased TH1/TH17 cytokines in positive patients as compared to negative, potentially having a nefarious effect on disease progression. Our findings indicate a distinct signature of inflammatory lymphocytes/cytokines responses within the nasopharynx of COVID-19 patients that might predict the clinical outcome/progression of the disease.
This work is supported by Dr. Mohamad Ayass, CEO of Ayass Bioscience, LLC
Changes in the structure of lipid A in glycosylated outer membrane vesicles (glycOMVs) suppress the activation of glycan-specific B1 cells in vivo
Authors: Lopez Natalia , Tyagi Srishti , DeLisa Matthew P 
Published: January 2023

Abstract
B1 cells differ from B2 cells in their developmental origin, phenotype, and function. They are anatomically located in various tissues of the immune system, and contribute to around 90% of the circulating antibodies in blood. B1 cells are often activated in a T-cell independent manner promoted by either antigens that contain a B-cell mitogen such as LPS, or by molecules that have highly repetitive structures such as bacterial capsular polysaccharides, with both pathways having the potential to undergo class-switch recombination and somatic hypermutation. Previously, we have shown that immunization with bacterial outer membrane vesicles engineered to display defined glycan structures on their surface (glycOMVs) promotes a strong glycan-specific IgG response for a wide range of bacterial and mammalian glycans. Here, we show that the structure of lipid A in these (glycOMVs) displaying the oncofetal antigen polysialic acid (polySia), influences the activation and class-switching of polySia-specific B1 cells from the spleen and peritoneal cavity in mice. We found that structural remodeling of lipid A from its normal hexa-acylated structure to a penta-acylated variant prevents the activation of polySia-specific B1 cells in the spleen and enhances the activation of polySia-specific B2 cells from the peritoneal cavity. Interestingly, we observed no changes in the glycan-specific IgG titers following immunization of wildtype B6 mice with either construct, even though there was no B cell activation in the spleen of mice receiving penta-acylated glycOMVs. However, these same glycOMVs failed to promote a glycan-specific IgG response in T-deficient mice, suggesting that this response was entirely T-cell dependent.
This work was supported by Bill and Melinda Gates Foundation grant OPP1217652 (to MPD), NSF grants CBET-1159581, CBET-1264701 and CBET-1936823 (to MPD), the Defense Threat Reduction Agency (DTRA) grants HDTRA1-15-10052 and HDTRA1-20-10004 (to MPD), NIH grants 1R01GM137314 and 1R01GM127578 (to MPD) and the Fleming Scholars Fellowship (to NLB).
Heightened levels of circulating cell-free DNA predict aggressive onset of experimental glioblastoma
Authors: Ayasoufi Katayoun , Wolf Delaney , Tritz Zachariah Peter , Jin Fang , Gukbicki Lauren , Hansen Michael , Fain Cori E , Khadka Roman , Johnson Aaron J 
Published: January 2023

Abstract
Glioblastoma (GBM) is an incurable and aggressive form of brain cancer that is associated with severe peripheral immunosuppression. This immunosuppression is a critical barrier to patient survival and the success of immune modulatory therapies. Using the murine GL261 model of GBM, we recapitulated major features of peripheral immunosuppression observed in patients including spleen atrophy, T cell lymphopenia, and T cell sequestration within the bone marrow. We determined that peripheral immunosuppression in GBM is multifaceted and involves release of novel circulating soluble factors that are found in the serum of glioma-bearing mice. This high molecular weight species potently inhibits T cell proliferation in vitro. Cell-free extracellular DNA has been recently identified as an immune suppressive factor in circulation. We, therefore, assessed cell-free DNA levels in the GL261 model. We determined that serum isolated from glioma-bearing mice harbor significantly higher concentrations of double stranded and single stranded cell-free DNA compared to healthy controls. Serial assessment of DNA concentrations in serum of glioma-bearing mice indicated that the most prominent increase was concurrent with end-stage disease. These findings provide a tractable approach to investigate the cellular source and immune suppressive effects of cell-free DNA in the leading murine model of GBM.
Supported by K99NS117799
IFNγ mediated PD-L1 expression mitigate the immunomodulatory effect of irreversible electroporation on pancreatic cancer
Authors: Imran Khan Mohammad , Brock Rebecca , Alinezhadbalalami Nastaran , Aycock Kenneth N , Morrison Holly A , Davalos Rafael V , Allen Irving Coy 
Published: January 2023

Abstract
Pancreatic cancer is one of the deadliest diagnoses leaving patients with few therapeutic options. Irreversible electroporation (IRE), a novel non-thermal ablation system, utilizes very short, high voltage electrical pulses to form micropores in cell membranes and stimulate cell death. IRE has been tested in human clinical trials for pancreatic cancer treatment with a significant improvement in progression-free survival, but not much is understood about the immunomodulatory effects of IRE and the reasons for tumor relapse. We hypothesized that, we could identify a molecular rationale for the tumor recurrence and potential co-therapy targets for IRE treatment. We have utilized Pan02 cells, and a subcutaneous flank model of pancreatic cancer. Here, we show that IRE can significantly delay cancer progression by shifting the tumor microenvironment to a pro-inflammatory state. In doing so, IRE treatment recruits cytotoxic CD8+ T-cells to the tumor sites, reduces contralateral tumor burden, and increases interferon-gamma (IFNγ) levels in the serum. Within two weeks after treatment, immune cells in the tumor site reverts to the pre-treatment condition, tumor relapses, and tumor cells express programmed-cell death-ligand 1 (PD-L1). Our in vitro findings of IFNγ dependent PD-L1 expression and ineffective in vivo blocking of IFNγ hints feedback loop pathway involving IFNγ and PD-L1. Since other findings have shown blocking PD-L1 is not always very effective, our findings indicate another compensatory bypass immune checkpoint mechanism. The effects of IRE can therefore be extended by additional combination therapy methods, including PD-1/PD-L1 antibody immunotherapies, and small molecule inhibitors.
This work was supported, in part, by the Virginia Maryland College of Veterinary Medicine; The Virginia Tech Institute for Critical Technology and Applied Sciences Center for Engineered Health; and The National Institutes of Health (R03AI151494 (ICA); R21EB028429 (ICA). The contents solely the responsibility of the authors and does not necessarily represent the official views of the NIH or any other funding agency.
Triple synergy between vaccine and checkpoint inhibitors in a pre-clinical tumor model
Authors: Becker William J , Olkhanud Purevdorj B , Berzofsky Jay A 
Published: January 2023

Abstract
Despite advances in checkpoint inhibitor (CPI) therapy for cancer treatment, many malignancies remain resistant. Tumors deemed ‘cold’ based on lack of T cell infiltration into the stroma of the tumor show reduced potential for CPI therapy and finding the right combination to balance safety and efficacy is arduous. To study ways to circumvent these limitations, we used the preclinical mouse model of TC1 tumor cells resistant to conventional CPI therapy. The TC1 cells are transfected with oncogenes E6 and E7 of HPV16, and we designed a tumor-vaccine specific for an E7(43–77) peptide. We show the synergy between the tumor-antigen specific vaccine and the combination of two CPIs, anti-TIGIT and anti-PD-L1. The synergistic effect of the triple combination provides more protection against tumor growth than either treatment alone or any pairwise combination and significantly improves survival in a CD8+ T cell dependent manner. Combining the tumor-specific vaccine with CPIs induces tumor-specific CD8+ T cells that infiltrate the tumor in young and aged mice, although aged mice show less protection than their younger counterparts. These data show proof-of-concept for a novel combination of a vaccine designed to elicit de novo anti-tumor T cell responses that can be amplified by synergistic CPIs that lead to greater survival.
Supported by Intramural NCI funding: ZIA-C-004020