bluuebunny/crossref_metadata_2025 huggingface dataset:
SARS-CoV-2 induced oxidative stress promotes HMGB1 secretion to induce inflammation
Authors: Hosakote Yashoda Madaiah , Rayavara Kempaiah , Corri Levine B , McLellan Susan , Weaver Scott C , Chopra Ashok , Tseng Chein-Te K
Published: January 2023

The coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a major ongoing pandemic, which results in life-threatening pneumonia and multiple-organ failure. Although the molecular mechanisms by which SARS-CoV-2 lead to various disease sequalae remain elusive, exaggerated inflammatory and immune responses can lead to the debilitating and lethal outcomes of this disease. Understanding how immune mechanisms contribute to the development of severe symptoms is essential for designing effective countermeasures against SARS-CoV-2. Our studies show that SARS-CoV-2 induces increased extracellular secretion of high-mobility group box 1 (HMGB1), a prominent damage-associated molecular pattern family member that alerts the immune system to potential damage and triggers immediate response. The goal of this study is to dissect the molecular mechanisms that promotes the release of HMGB1 and its associated cellular signaling in response to SARS-CoV-2 infection. Studies were conducted in human lung epithelial cells including A549-ACE2 and Calu-3/2B4 cells, mice, and COVID-19 patient samples. HMGB1 levels and oxidative stress markers were measured by an immune assay, Western blot, and qRT-PCR. SARS-CoV-2 infection induced severe oxidative stress, leading to enhanced extracellular release of HMGB1 and subsequent activation of signaling cascades to promote inflammation. Therefore, modulation of HMGB1’s proinflammatory signaling may reduce SARS-CoV-2-induced inflammation by controlling oxidative and inflammatory stress in COVID-19 disease.

This study was supported by a Young Clinical Scientist Award from the Flight Attendant Medical Research Institute (FAMRI) to YMH (grant I.D. number 123385), NIH/NIAID R21 AI35619 to YMH, and Rapid Response Pilot grant from NIEHS P30 Center Grant ES030285 to YMH. Additional support was from the Institute for Translational Science at UTMB, supported by a Clinical and Translational Science Award Mentored Career Development (KL2 Award; KL2TR001441), and Pilot grant (85393) from Institute for Human Infections & Immunity at UTMB, supported by Sealy & Smith Foundation to YMH.
Molecular mechanisms of dexamethasone-mediated modulation of inflammatory and interferon responses in severe COVID-19 patients.
Authors: Chimote Ameet A , Alshwimi Abdulaziz , Chirra Martina , Gawali Vaibhavkumar S , Hudock Kristin M , Powers-Fletcher Margaret V , Conforti Laura
Published: January 2023

Severe COVID-19 is characterized by cytokine storm, an excessive production of pro-inflammatory cytokines, which results in acute lung damage and death. Dexamethasone (Dex), a corticosteroid administered as a standard of care for severe COVID-19, has been shown to reduce the onset of severe complications in these patients. However, the mechanisms underlying the beneficial effects of Dex are poorly understood. We conducted a transcriptomic analysis (NanoString) of peripheral blood mononuclear cells (PBMCs) from COVID-19 patients with mild (no hospitalization) and severe (on ventilator in the ICU) disease. The latter group included patients ± Dex. Treatment of severe COVID-19 patients with Dex inhibited pro-inflammatory pathways, circulating cytotoxic and Th1 cells, interferon (IFN) signaling and the genes involved in cytokine storm (IL-1, IL-6 and IFN-γ), which were altered in severe vs mild disease. Pro-inflammatory immune cell functions are regulated by calcium signaling, which in turn is under the control of Kv1.3 potassium channels, but their role in the complications associated with COVID-19 remains elusive. RT-qPCR experiments in PBMCs from COVID-19 patient cohorts showed an increase in Kv1.3 expression in severe patients that was significantly reduced by Dex treatment. In agreement with these findings, in vitro treatment with Dex reduced Kv1.3 protein abundance in CD3+, CD4+, CD8+ T cell and CD56dim NK cells in healthy donor (HD) PBMC, while NanoString showed inhibition of pro-inflammatory genes and cytokines. Functional studies showed that Dex significantly reduced Kv1.3 activity and IFN-γ in HD T cells. Thus, our findings suggest that Kv1.3 inhibition contributes to the immunosuppression by Dex.

This work was supported by Senior Pilot Grant, University of Cincinnati, Department of Internal Medicine to LC, NIH (R38HL155775 to MC) and Biospecimens and associated data were provided by the Cincinnati COVID-19 Repository at the University of Cincinnati and Cincinnati Children’s Hospital Medical Center.
A surprising pathogenic role of B cells in mice with Alzheimer’s disease
Authors: Biragyn Arya , Kim Ki , Wang Xin , Ragonnaud Emeline , Bodogai Monica , McDevitt Ross A , Okun Eitan
Published: January 2023

The role of adaptive immune cells in Alzheimer’s disease (AD), a progressive neurodegenerative disease that associates with impaired clearance of toxic amyloid-b (Ab) peptide aggregates from the brain parenchyma, is rarely explored and mostly assumed to be beneficial. Here, using three, widely utilized transgenic AD mouse models (3xTgAD, APP/PS1, and 5xFAD mice), we provide counterintuitive evidence that the manifestation of AD requires B cells. Despite expression of the AD-fostering transgenes, the genetic loss of B cells or antibody-mediated depletion of circulating B cells at the onset of the disease is sufficient to reverse Aβ plaque burden and disease-associated microglia in the brains. The B-cell loss also ameliorates behavioral and memory deficits in AD mice. Mechanistically, AD we linked to infiltration of B cells into the brain parenchyma and production of immunoglobulins and pro-inflammatory factors. These results warrant a new look to B cells as important, pathogenic players of AD. As such, it underscores therapeutic value of B-cell inactivation in control AD progression 1.

This work was supported by the Intramural Research Program, NIA/NIH.
Inhibition of LRRK2 Kinase Activity in the Treatment of Crohn’s Disease
Authors: Deloer Sharmina , Fuss Ivan , Suebsuwong Chalada , Kumar Kunal , Devita Robert J , Peter Inga , Strober Warren
Published: January 2023

The leucine-rich repeat kinase 2 (LRRK2) gene is a genetic hotspot for gain-of-function mutations associated with various diseases such as Crohn’s and Parkinson’s disease. These mutations enhance LRRK2 kinase activity and, consequently, various inhibitors of this activity are currently being tested as treatment modalities. Here, we report studies of two novel kinase inhibitors (Termed CS-190 and CS-82) with LRRK2 inhibition activity. In vitro studies disclosed that the two inhibitors suppress LRRK2 phosphorylation as well as its ability to phosphorylate the LRRK2 targets, Rab 10 and 12; in addition, they suppress human dendritic cell production of TNFa stimulated by several known LRRK2 activators including the Dectin-1-ligand, zymosan-depleted S. cerevisiae extract. Finally, the inhibitors exhibited powerful suppression of the ability of LRRK2 to mediate NLRC4 inflammasome production of IL-1beta. These in vitro finding correlated with in vivo studies showing that IP administration of one of the kinase inhibitors (CS-82) ameliorates colitis in the DSS-colitis model as evaluated by weight loss and inflammation scores. These findings suggest that these newly developed inhibitors are potential agents for treatment of Crohn’s disease.
Contrasting roles for neutrophils and macrophages during experimental pyelonephritis
Authors: de Dios Ruiz Rosado Juan , Cortado Hanna , Robledo-Avila Frank , Spencer John David , Becknell Brian , Partida-Sanchez Santiago
Published: January 2023

Background

Urinary tract infections (UTIs) account for 7 million annual office visits and over $1.6 billion in health care spending in the United States. Uropathogenic Escherichia coli (UPEC) initiates over 80% of UTIs. When UPEC ascends from the bladder to the kidneys, the resulting pyelonephritis can lead to renal scarring and end-stage renal disease.

Objective

We determined the specific roles of monocytes and neutrophils in bacterial clearance and the development of kidney fibrosis during pyelonephritis.

Methods

Female C3H/HeOuJ mice were treated with neutrophil depleting or monocyte depleting antibodies (monoclonal). After cell depletion, the animals were transurethrally infected with UPEC (CFT073 strain). The progression of UTI was assessed via biophotonic imaging and quantification of bacterial burden in the urinary tract. Transcriptomic analyses were performed using customized TaqMan Arrays. Histopathological scores were performed by H&E and Sirius-Red stainings. The renal function was assessed by measuring blood levels of waste products and electrolytes.

Results

Monocytes and neutrophils function in a synchronized manner, while neutrophils engulf and eliminate UPEC in the urinary tract, monocytes differentiate into macrophages and promote inflammation in the bladder and kidney to boost the recruitment of neutrophils. However, the absence of neutrophils results in increased macrophage infiltration and exaggerated inflammatory responses in the urinary tract. Macrophage-dependent inflammation leads to renal scarring and renal dysfunction during UTI.

Conclusions

Our findings uncover a detrimental role for the neutrophil-macrophage imbalance in promoting renal scarring during pyelonephritis.

Supported by grants from NIH (K01 DK128379-01)
The role of phosphatidylserine receptor-mediated phagocytosis of amyloid-β by microglia in Alzheimer’s Disease.
Authors: Bauchle Casey Jordan , Owlett Laura , Karaahmet Berke , Olschowka John , O’Banion Kerry , Elliott Michael Rusty
Published: January 2023

The phagocytosis of apoptotic cells and cellular debris by microglia critically regulates immune homeostasis in the brain. In Alzheimer’s Disease (AD), microglial phagocytosis of amyloid-β (Aβ) aggregates is thought to play a protective role in AD pathogenesis, but the mechanisms by which this occurs are not well understood. Recent work has suggested that phosphatidylserine (PtdSer)-sensing receptors may induce Aβ plaque engulfment, and data from our lab and others’ supports this idea. To better understand the role of PtdSer-sensing receptors in Aβ clearance, we utilized immortalized cell lines and isolated primary microglia to examine PtdSer-mediated phagocytosis of Aβ aggregate targets in vitro and compared this to phagocytosis of apoptotic cells, as well as to phagocytosis by Fcγ receptors and complement receptors. Phagocytosis was measured by live-cell imaging and flow cytometry, and revealed distinct kinetics between the different forms of phagocytosis. We further investigated the role of specific PtdSer receptors using genetic and pharmacologic blocking approaches. In vivo results using the APP/PS1 AD mouse model support a role for the PtdSer binding protein Gas6 in mediating Aβ plaque clearance. Our work supports a model in which the effective clearance of Aβ by microglia occurs via a combination of phagocytic mechanisms.
mTOR-associated Mitochondrial Energy Metabolism Limits Mycobacterium ESX-1-induced Cytotoxicity
Authors: Pagán Antonio J , Lee Lauren J , Edwards-Hicks Joy , Moens Cecilia B , Tobin David M , Pearce Erika L , Busch-Nentwich Elisabeth , Ramakrishnan Lalita
Published: January 2023

Necrosis of macrophages in the tuberculous granuloma represents a major pathogenic event in tuberculosis. Through a zebrafish forward genetic screen, we identified the mTOR kinase, a master regulator of metabolism, as an early host resistance factor in tuberculosis. We found that mTOR protects macrophages from mycobacterium-induced death by enabling infection-induced increases in mitochondrial energy metabolism fueled by glycolysis. These metabolic adaptations were required to prevent mitochondrial damage and death caused specifically by the mycobacterial ESX-1 secretion system. Our finding that the host can effectively counter this early critical mycobacterial virulence mechanism simply by regulating energy metabolism may help explain why Mycobacterium tuberculosis, albeit humanity’s most lethal pathogen, only causes disease in a minority of infected individuals.

This work was supported by a Wellcome Trust Principal Research Fellowship (103950/Z/14) and an NIH MERIT award (R37 AI054503) (L.R.).
The role of ERK2 in dendritic cell function
Authors: Abdul-Baki Nawal Samer , Negron Austin , Forsthuber Thomas
Published: January 2023

Dendritic cells (DCs) are an important link between the innate and adaptive immune systems, specifically by serving as key regulators of T cell-dependent (TD) immune responses. In DCs, several processes critical for initiating TD immune responses, such as migration, provision of costimulatory signals, and antigen uptake and presentation, utilize the mitogen-activated protein kinase (MAPK) signaling pathway. The dysregulation of these processes can result in the aberrant activation of autoreactive CD4+ T cells, resulting in autoimmune disease. Extracellular signal-regulated kinase 1 and 2 (ERK1/2) are key effectors of the MAPK pathway, and while ERK1 has been shown to be a critical regulator of DC migration and TLR-induced IL-10 secretion, the function of ERK2 remains unknown. Preliminary data has shown that Erk2Δ bone marrow-derived DCs (BMDCs), as well as Erk2Δ splenic DCs, exhibit altered expression of surface markers associated with T cell costimulation, antigen presentation, and migration compared with DCs from littermate control. Taken together, this suggests that ERK2 plays a unique role in the regulation of several DC functions, potentially opening a new approach to treating CD4+ T cell-mediated autoimmune diseases.

Supported by grant R21AI44731 from the NIH/NIAID (TGF)
Effector memory CD4 T cells engage myeloid cells through TNFR and CD40 to induce innate inflammation and autoimmune pathology independent of pattern recognition receptors
Authors: Meibers Hannah , McDaniel Margaret M , Jain Aakanksha , Chawla Amanpreet Singh , Pasare Chandrashekhar
Published: January 2023

T cell mediated autoimmune diseases and associated tissue pathology is commonly attributed to inflammatory cytokines produced by the innate immune system. Similarly, CAR-T cell therapy and checkpoint blockades lead to innate cytokine storms and inflammation. While blocking individual cytokines can alleviate some of this pathology, the upstream mechanisms permitting self-reactive T cells to facilitate an innate inflammatory response remain unknown. We have discovered critical players that are engaged by effector memory CD4 (Tem) T cells to induce a broad transcriptional program in cells of the innate immune system. The pro-inflammatory response induced in innate immune cells is independent of pattern recognition receptors (PRRs), but mirrors genes induced by microbial recognition. We find that the innate inflammation is driven by combined engagement of TNFR and CD40 on myeloid cells presenting cognate antigen to Tem cells. Pro-inflammatory cytokine production and autoimmune pathology can be completely rescued by ablating the CD40 and TNFR pathways in several models of T cell driven inflammation. Thus, we have discovered a previously unknown trigger of innate inflammation and autoimmune pathology and propose that targeting this signaling node could be a more effective therapeutic approach than previously characterized cytokine-specific remedies.

Supported by grant from NIH (R01 AI123176)
IRF5 regulation of CD4+ T cell metabolism controls CD40L expression
Authors: Brune Zarina , Matta Bharati , Barnes Betsy
Published: January 2023

Systemic Lupus Erythematosus (SLE) is a devastating autoimmune disease that results from failure of the immune system to distinguish “self” from “non-self”. Studies in our lab and others demonstrated that human SLE CD4+ T cells have elevated levels of IRF5 and increased metabolism, while Irf5 knockout murine CD4+ T cells show diminished utilization of oxidative phosphorylation and glycolysis, respectively. However, how IRF5 contributes to CD4+ T cell support of B cell function and dysfunction has not been fully elucidated. Here, using IRF5 KO C57BL/6J mice, we show that loss of IRF5 in CD4+ T cells directly contributes to defects in plasmablast generation. Examination of the CD40-CD40L interaction shows significant decreases in CD40L expression in Irf5 KO CD4+ T cells. Examination of Cd40lg transcript expression shows no difference in mRNA expression in Irf5 KO murine CD4+ T cells when compared to WT. This finding indicated a novel post-transcriptional regulatory role for IRF5. Examination of the canonical mTOR pathway, a key translational regulator, shows decreased phosphorylation of the S6 ribosomal protein upon loss of Irf5, indicating diminished activity of the mTOR pathway. mTOR inhibition with rapamycin in turn results in decreased CD40L expression, supporting a role for mTOR in CD40L regulation. As mTOR signaling is known to be regulated by cellular metabolism, we examined if loss of IRF5 results in altered T cell metabolism. We found that Irf5 KO T cells have decreased glucose metabolism and increased glutamine metabolism. This data has increased our understanding of how metabolism influences CD4+ T cell function and provided insight into novel post-translational regulatory roles for IRF5 via T cell metabolic regulation.
Enhancing lysosomal lipid metabolism prevents the loss of Kupffer cells in non-alcoholic steatohepatitis and attenuates liver pathology
Authors: Chan Mandy M , Daemen Sabine , He Li , Gainullina Anastasiia , Artyomov Maxim N , Razani Babak , Schilling Joel D
Published: January 2023

During the development of non-alcoholic steatohepatitis (NASH), liver resident Kupffer cells (KCs) are progressively lost and replaced by monocyte-derived macrophages (MdMs). The impact of KC loss on NASH pathology is not currently known. Given the important role of KCs in maintaining tissue homeostasis and clearing blood-borne antigens, we hypothesized that KC depletion contributes to NASH pathogenesis. Thus, we aimed to elucidate the mechanism(s) of KC loss and to devise a strategy to enhance KC survival during NASH.

We had previously shown that lipids can cause lysosomal cell death in macrophages. Using a mouse model of NASH, we discovered that KCs also develop phagolysosomal pathology. Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and lipid metabolism that has been shown to rescue lysosomal dysfunction in other metabolic diseases. To test whether TFEB activation could protect against KC death in NASH, we generated a mouse model in which a KC-specific Cre induces expression of a TFEB overexpression construct (KCTFEB). Despite having similar body and liver weight as WT mice fed a NASH-inducing diet, KCTFEB mice had a striking preservation of KC number. Moreover, KCTFEB mice had reduced recruitment of pro-inflammatory MdMs and this was associated with lower liver triglyceride and injury. RNA sequencing of KCs from transgenic and WT mice revealed that TFEB induced the expression of several lysosomal and lipid metabolic genes. Together these findings provide important proof-of-concept evidence that KC loss contributes to disease pathology in NASH and suggest that targeting macrophage lysosomal/lipid metabolic function could be a useful strategy to enhance resident macrophage survival.

Supported by grants from ADA-118IBS280, DDRCC-P30DK052574, NIH T32-AI007163, and NSF GRFP-DGE-1745038
Transcription factor profiling reveals activation of Type II Natural Killer T cell effector functions in response to Francisella tularensis infection.
Authors: Torres Cameron M
Published: January 2023

The bacterium Francisella tularensis (Ft) is the causative agent of human tularemia. When untreated, tularemia can reach a mortality rate as high as 30% in healthy adults with inhalation of as few as 10 colony forming units (CFU) inducing respiratory tularemia. While Ft is spread from zoonotic sources in nature, the ease of aerosol spread and high mortality in respiratory form led to its weaponization and concern for bioterrorism. Exposure to Ft elicits the cytokine storm, in which the immune system over-responds to the presence of the bacterium releasing massive amounts of proinflammatory and regulatory cytokines. Natural Killer T (NKT) cells are important immune cells that have both activating and inhibitory effector mechanisms. NKT cells are activated very early following Ft infection suggesting a critical role in the innate immune response to the bacterium. NKT cells are categorized into two distinct subsets based on their antigen recognition of CD1d-tetramers loaded with α-galactosylceramide (αGalCer-tetramer), though their individual characteristics in regulating inflammation are ill-defined. Type I NKT cells, similar to CD4+ αβ T helper cells, have been shown to express varied transcription factor (TF) profiles, e.g., T-bet, GATA3, and RORγT, contributing to either Th1, Th2 or Th17 phenotypes. It is not currently known whether this same TF profile classification exists within type II NKTs. The purpose of this research project is to investigate the activation of the type II NKT cell subset and characterize their TF profiles in response to Ft infection. This data will lead to a better understanding of the role of type II NKT cells in Francisella-mediated immunity.

Supported by National Institute of General Medical Sciences (NIGMS) Research Initiative for Scientific Enhancement (RISE) grant R25 GM 06921-18
Key contribution of NK cells to inflammation after muscle injury
Authors: Cevik Hilal , Gangadin Isabelle , Boyer Justin G , Millay Douglas , Waggoner Stephen N
Published: January 2023

Immune activation after tissue injury is required for removal of dead cells and debris to permit subsequent regenerative healing. Natural killer (NK) cells are innate lymphocytes that are essential for immune defense and for regulation of inflammation. NK cells limit fibrosis after cardiac muscle damage, yet the role of these cells during skeletal muscle inflammation is less clear. We hypothesize that NK cells promote acute inflammation after muscle damage but that NK cell responses must be resolved to permit regenerative healing. Following bilateral injection of cardiotoxin into tibialis anterior and gastrocnemius muscles of C57BL/6 mice, NK cell accumulation in injured muscle was detectable within 18 hours and peaked four days post-injury. Selective depletion of NK cells using a titrated dose of anti-NK1.1 antibody prior to cardiotoxin injection resulted in a substantial reduction in the overall infiltration of numerous immune cell types, including T and B cells, into the injured muscle. Thus, NK cells are an important mediator of cellular inflammation following muscle damage. Future studies will determine the mechanism by which NK cells contribute to this inflammatory response and how ablation of NK-cell mediated inflammation impacts healing of damaged muscles.

Supported by National Institute of Heath (NIH) (R01-AI148080)
The Effect of Malnutrition on T-cell Circadian Rhythms
Authors: Foster Takesha R , Dadzie Kwesi A , Adams Olivia , Gubbels-Bupp Melanie R
Published: January 2023

In mammals, T-cell migration is under circadian control, likely to anticipate daily rhythms in infection risk. Glucocorticoids are a major controller of circadian processes and malnutrition is associated with increased glucocorticoid secretion. Previous studies suggest malnutrition may impart a “super-quiescent” phenotype to T-cells, enabling a greater number of naïve T-cells to survive short-term malnutrition albeit with diminished function. Thus, we hypothesize that malnourished T-cells may conserve energy by disengaging from rhythmic migration under circadian control and/or foregoing migration to reside in the bone marrow instead. To test this hypothesis, the total number of nucleated cells and naïve CD4+ and CD8+ T-cells in the blood, spleen, bone marrow, and brachial and mesenteric lymph nodes were enumerated by flow cytometry every four hours over the course of one day from control and malnourished mice. Additionally, expression levels of CD127 and CXCR4 in both T-cell populations and the concentration of glucocorticoids in the blood were assessed. A better understanding of how malnutrition affects the circadian rhythm of T-cell migration will not only help identify the mechanisms of how circadian rhythms work, but also how organisms’ circadian rhythms change in response to malnutrition. This knowledge of how malnutrition disrupts the circadian rhythm of T-cells may help improve vaccination strategies in malnourished children.

Supported by NSF-MRI [DBI- 1920116] NSF -RUI [IOS-1951881]
Characterization of the cross-reactive T cell repertoire in the context of heterosubtypic influenza A virus infection
Authors: Gaevert Jessica A , Duan Susu , Thomas Paul G
Published: January 2023

The T cell receptor (TCR) repertoire is extremely diverse and plays an instrumental role in fighting off pathogens individuals encounter in a lifetime. A key aspect of the immune system combatting the diverse array of pathogens an individual sees may be TCR cross-reactivity, where a TCR can recognize more than one peptide displayed on an MHC-I molecule. The number of possible peptides presented to the immune system exceeds 1015, and this number is greater than the number of possible T cells the immune system can generate and sustain. Thus, a certain portion of the T cells in an immune system must be cross-reactive. Specifically, cross-reactive TCRs may play a key role in allowing the immune system to adapt to rapidly evolving pathogens, such as influenza A virus (IAV). Utilizing 8 variant influenza viruses for priming and secondary challenge, our preliminary data has shown that cross-reactive TCRs seen in primary infection with one IAV can mount a robust and effective memory response to a different IAV, across a range of mutations. However, selection for cross-reactive receptors may create a narrowed repertoire consisting of a few clonotypes over the course of many repeated heterosubtypic IAV infections, covering a narrow range of antigenic space representing the mutant epitopes encountered. Paradoxically, this selection for cross-reactivity may provide a substantial escape opportunity with future antigenic variations. Ongoing work focuses on how repeated IAV challenges shape the repertoire, and how previous infections inform future responses by selected cross-reactive T cell pools.

Supported by grants from NIH (R01 112404040) and the St. Jude Children’s Research Hospital Graduate School
A fluorescent reporter model to study the role of TDC during infection and cancer
Authors: Kuka Mirela , Fiore Alessandra , Sala Eleonora , Oberrauch Federico , Provero Paolo , Riba Michela , Cristofani Claudia , Iannacone Matteo
Published: January 2023

TDC are hematopoietic cells that combine dendritic cell (DC) and conventional T cell markers and functional properties. They were identified in secondary lymphoid organs (SLO) of naïve mice as cells expressing CD11c, MHC-II, and TCRβ chain. TDC expand in response to TCR-mediated stimulation, but they also express the DC lineage marker Zbtb46. Moreover, TDC are characterized by a cytotoxic gene signature, including high expression of IFNγ and granzymes. Unfortunately, using CD11c as a marker for TDC has the caveat of its upregulation on different cells (including T cells) upon activation. Therefore, a more specific marker could be useful to further investigate TDC functions in peripheral organs or during inflammation. We exploited the unique genetic profile of TDC to obtain a reporter mouse model in which TDC are marked with two different fluorescent proteins (Zbtb46-GFP and Gzmb-Tomato). This model allowed us to use Zbtb46 instead of CD11c as a marker for TDC, in order to investigate the frequency and localization of TDC in peripheral tissues such as liver, small intestine, and lung. RNA sequencing analysis confirmed that TDC identified with this reporter model have an overlapping gene signature with the one already reported for TDC. In addition, frequency and total numbers of Zbtb46+ TDC in the SLO recapitulated those previously found using CD11c, thus confirming the validity of our model. Interestingly, we found that TDC numbers in the SLO increased upon LCMV infection, indicating that TDC might play a role during viral infections. Further studies aimed at identifying the localization of TDC in peripheral organs, and their role in several pathological settings are ongoing.

M.K. is supported by the Italian Ministry of Education, University and Research grants SIR-RBSI14BAO5 and PRIN-2017ZXT5WR
Tissue- and age-specific adaptations of human γδT cells in early life
Authors: Gray Joshua I , Chin Athena , Connors Thomas J , Guyer Rebecca S , Rybkina Ksenia , Bradley Marissa C , Matsumoto Rei , Farber Donna L
Published: January 2023

γδT cells are unconventional T lymphocytes which seed peripheral tissues in early life. They provide frontline anti-pathogen, anti-cancer and homeostatic responses, rapidly initiated in response to signals of cellular stress independent of MHC-recognition. However, the role of γδT cells in humans has been difficult to assess given their purported tissue residency. Using tissues obtained from organ donors, we conducted flow cytometry, TCR-sequencing, and stimulation assays on γδT cells isolated from blood, spleen, lung, intestines and lymph nodes (LN) to create an ‘atlas’ across the human body throughout life. γδT cells were enriched in the blood, spleen, lung, and jejunum in early life, whereas LN proportions were maintained at low levels. Vδ1 and Vδ2 γδT cell subsets also exhibited tissue- and age-specific phenotypes: Vδ1 cells predominated in all organs in infancy except the lung. They were ‘naïve-like’, acquiring a mature phenotype with age, whereas Vδ2 possessed an effector memory signature, even in donors a few days old. Functionally, Vδ2 cells produced IFNγ, TNFα, GzmB and perforin at high levels in childhood. By contrast, Vδ1 cells favored Areg production during infancy but acquired a cytotoxic profile into adulthood. TCR analysis revealed clonal expansion in mucosal organs, whereas lymphoid sites had increased diversity in early life. Despite this site-specific clonal expansion, V-chain usage was stably distributed across organs and did not show the tissue-specific biases seen in mice. Our analyses reveal that γδT cells may play an outsized role in pediatric tissue immune responses and identify potentially divergent roles of specific subsets that contribute to tissue immunity during the most vulnerable years of life.
The functional and molecular determinants of HCV-specific CD8 T cell responses during early infection
Authors: Cai Curtis , Samir Jerome , Adikari Thiruni , Peters Tim , Luciani Fabio
Published: January 2023

T-cell exhaustion is a hallmark of hepatitis C virus (HCV) infection and limits protective immunity in chronic viral infections and cancer. However, limited knowledge exists of the initial dynamics that establish exhaustion in humans. We studied longitudinal blood samples from a unique cohort of primary HCV infection subjects using single cell multi-omics and viral dynamics to identify the functions and phenotypes of HCV-specific CD8+ T cells.

The magnitude of IFN-γ production against the transmitted virus was associated with the rate of immune escape, larger clonal expansion, and a molecular profile consistent with a progenitor of early-exhaustion. Lineage tracing within clones revealed fate plasticity through the course of the infection and differentiation fates of progenitor exhausted subsets. This study provides new insights on the contribution of CD8+ T cells to HCV disease outcome and exhaustion, and supports future studies on early T-cell differentiation in human infections.

Supported by grants from the NHMCR and scholarships from the Australian Government Research Training Program.
Homeostatic cytokines regulate the emergence of human prenatal effector T cells.
Authors: Locher Veronica , Park Sara , Bunis Daniel , Makredes Stephanie , Fragiadakis Gabriela , Halkias Joanna
Published: January 2023

In a healthy pregnancy, prenatal immunity promotes tolerance in utero and transitions to provide protective immunity after birth. In pathologic pregnancies, in utero immune activation and the ensuing fetal inflammatory response are predictive of severe neonatal morbidity and mortality. We previously demonstrated that the majority of prenatal intestinal memory CD4 T cells express the transcription factor PLZF and possess a heightened Th1 effector potential. These PLZF+ CD4+ T cells specifically accumulate in the intestine, are absent from the adult, and are enriched in the cord blood of infants exposed to in utero inflammatory pathology. However, the signals that drive their intestinal accumulation and maturation are not well understood. We show that PLZF+ CD4+ T cells are MHC Class II restricted and display tissue specific functional heterogeneity, including Th2-, Th22- and Th17-like capacities. We provide evidence that differences in homeostatic cytokines between the small intestine and mesenteric lymph node support the preferential accumulation and functional compartmentalization of prenatal PLZF+ CD4+ T cells. Specifically, we show that IL-7 and TGFβ reciprocally modulate the preferential expansion and functional maturation of PLZF+ CD4+ T cells. We provide evidence for the generation of both virtual memory T cells as well as classic antigen driven differentiation of Th1- and Th17-like effector PLZF+ CD4+ T cells. This work identifies critical mechanisms of human prenatal immune maturation and regulation and could inform the development of novel therapeutic strategies to address perinatal inflammatory pathologies.

Supported by K08 Mentored Clinical Scientist Development Award (AI128007) to Joanna Halkias and the Burroughs Wellcome Fund Preterm Birth Initiative (1019828)
Impact of TCR-peptide/MHC class II affinity on the pathogenicity and regulation of prostate-specific CD4+ T cells
Authors: Rodriguez Donald M , Duncombe Ryan , Lee Victoria , Zeng Sharon , Klawon David E J , Adams Erin J , Savage Peter Aidan
Published: January 2023

In both humans and mice, self-reactive CD4+ T cells are implicated in a range of autoinflammatory processes. To promote disease, such cells must evade clonal deletion in the thymus, as well as intrinsic and extrinsic regulation mechanisms in the periphery, including anergy and regulatory T cell-mediated suppression. However, the impact of T cell receptor (TCR) - peptide/MHC class II (pMHCII) binding properties on the autoimmune potential of self-specific cells and their susceptibility to immune regulation remains incompletely defined. Much previous work on this topic has relied on the study of T cells reactive to foreign model antigens. However, immune responses towards such antigens may not recapitulate those targeting bona fide self-antigens. Through the in vivo analysis of CD4+ T cell clones reactive to a natural I-Ab-restricted self-peptide derived from the prostate-specific protein Tcaf3 (termed “C4” peptide), we aim to define the role of TCR-pMHCII binding affinity on the pathogenicity and regulation of self-reactive CD4+ T cell clones. Our findings thus far in both tumor-free and prostate-tumor-bearing mice reveal notable differences in the developmental trajectories and peripheral functions of clones with varying TCR affinity for C4/I-Ab. Ongoing work will continue to leverage our novel and physiologically relevant TCR-ligand system to better understand the fundamental mechanisms by which immune tolerance is established and enforced.

This work was funded by the following sources (to P.A.S.): R01-CA160371, R01-AI110507, U01-AI154560, a Cancer Research Institute Investigator Award, and the University of Chicago Comprehensive Cancer Center. D.R. was supported by an NIH/NCI F30 predoctoral fellowship (F30-CA247264). V.L. was supported by an NIH/NCI F30 predoctoral fellowship (F30-CA217109). D.R. and V.L. were supported by the University of Chicago Medical Scientist Training Program (T32-GM007281).
Chronic Vaccine Boost Prolongs Malaria Vaccine Immunity and CD4 Effector and Memory T cells
Authors: Gbedande Komi , Ibitokou Samad A , Ong Monique L , Brown Michael G , Degli-Esposti Mariapia A , Stephens Robin
Published: January 2023

Vaccines to persistent parasite infections have been challenging to develop, and current iterations lack long-term protection. Persistent infection generates effector and effector memory T cells (Teff, Tem), and both contribute to protection, as we recently showed for Plasmodium infection. Sterilizing immunity from live P. chabaudi vaccination lasts less than 200 days. While specific antibody levels remain stable after the acute phase of infection, the decay of T cells corresponds with loss of protection from re-infection. Chronic vaccination with CMV has been shown to generate Tem and Teff, and so we tested MCMV for the ability to prolong protection to P. chabaudi. We generated MCMV encoding P. chabaudi MSP-1 epitope B5 (MCMV-B5), and used B5 TCR Transgenic CD4 T cells to follow the MCMV-B5 response. We showed that boosting the live malaria vaccine with MCMV-B5: i) prolonged vaccine protection to heterologous infection at day 200, ii) increased parasite-specific T cells and promoted a highly-differentiated Teff phenotype, including iii) IFN-γ+ and TNF+ T cells. Importantly, chronic MCMV infection itself improved protection from P. chabaudi infection and promoted generation of a T cell phenotype similar to P. chabaudi infection. Investigating the mechanisms of MCMV in prolonged vaccine-induced protection, we found an impressive role for IFN-γ, as its neutralization in vivo during MCMV latency is associated with loss of protection to P. chabaudi challenge. Blocking IFN-γ before challenge induced fewer Teff and less plasma IL-12 upon P. chabaudi challenge. Our findings suggest a mechanism to boost CD4 T cell immunity using chronic vaccines, which may be broadly applicable to infections that require T cell based immunity.

Supported by R01AI135061
Age-Dependent Responses of γδ+ T cells During Bloodstream Infection in Early Life
Authors: Yokanovich Lila T , Greenfield Kara , Knoop Kathryn
Published: January 2023

Late-onset neonatal sepsis (LOS) is a bloodstream bacterial infection that occurs during the first two months of life. Despite improved clinical care, LOS remains a significant contributor to neonatal mortality. Clinical observations suggest neonates exhibit unique immune responses and excessive inflammation during sepsis compared to adults and older children, however, factors contributing to these differences remain poorly understood. Two common routes of pathogen entry for neonates are direct introduction of a pathogen into the bloodstream, or the translocation of a gut-resident pathobiont. Our lab has developed a mouse model of LOS that disrupts goblet cell homeostasis to increase gut permeability and facilitate the translocation of virulent bacterial species such as E. coli. During both intraperitoneal and oral infection, we observe several key features of clinical LOS including increased pro-inflammatory serum cytokines, organ failure, and death in 5 day-old pups, but not 15 day-old pups. Strikingly, we have found robust activation of γδ+ T cells in 5 day-old pups, following infection, independent of pathogen entry route. This γδ+ T cell activation occurred in an age-dependent manner and was absent in 15 day-old pups. γδ+ T cell activation in 5 day-old pups was also associated with a pro-inflammatory cytokine profile that was absent in older pups. Finally, 5 day-old TCRδ−/− pups were protected from mortality during LOS, suggesting that γδ+ T cells may also contribute to mortality in this model. This study sheds light on the unique responses of γδ+ T cells to bloodstream infection during two distinct periods of early life.
Exploring cutaneous neuro-immune networks during helminth infection
Authors: Jean E Evonne , Rossi Heather , Herbert De’Broski
Published: January 2023

Sensory nociceptive neurons serve important roles in regulation of cutaneous inflammation directed against microbial and fungal pathogens, but whether neurons serve any role in protective immunity against skin penetrating helminths remains unknown. In this study, we sought to identify the key factors responsible for blocking parasite entry of host skin. Data show that CD4 T cells were essential for restricting parasite entry of the skin during secondary infection, but this resistance was not dependent upon the key TH2 transcription factor Signal Transducer And Activator Of Transcription 6 (STAT6). Interestingly, STAT6 deficient mice generated high levels of TH17 associated cytokines, the latter of which are known to be induced by skin nociceptors. To determine whether skin neurons could prime host protective immunity, we used an optogenetic approach that allowed pre-activation of neurons at the site of skin inoculation that expressed transient receptor potential cation channel subfamily V member 1 (TRPV1). Strikingly, preemptive activation of TRPV1 neurons led to a significant inhibition of parasite entry. Ongoing studies are designed to understand whether depletion of TRPV1 skin neurons impairs immunity and whether skin protection relies upon TH17 dependent immunity.

Supported by grants from NIH (R01 AI095289 and R01 GM083204) and NSF GFRP (Fellow ID:2020297782)
Treponema pallidum specific CD4 T cell epitope discovery
Authors: Reid Tara , Godornes Charmie , Tong Denise , Koelle David M
Published: January 2023

Despite curative therapy and multiple public health campaigns to eliminate syphilis in the US, the incidence of Syphilis is rising in diverse groups: men who have sex with men, women, and children. An effective syphilis vaccine will be a key tool in the eradication of this disease. Prior efforts to create a protective syphilis vaccine focused on raising antibodies against rare outer membrane proteins to facilitate antibody mediated opsonophagocytosis of Treponema pallidum (Tp) in infectious lesions. The inclusion of immunodominant T cell antigens in a subunit vaccine can provide CD4 T cell help to B cells to produce opsonic antibodies. We sought to identify human Tp T cell antigens validated to the epitope level as there are none reported in the literature. Tp specific CD4 T cells were isolated from persons with active or treated syphilis infection by CD137 and CD69 activation–based FACS and polyclonal expansion. These cells were then probed with a panel of 39 recombinantly expressed Tp proteins. Preliminary data show that activation-induced-marker-enriched, expanded T cells can be probed to discover novel CD4 T cell antigens and epitopes using proliferation experiments. The first 4 CD4 T cell antigens identified included antibody targets Tp0435 and Tp0574, a flagellar apparatus protein Tp0870, and a metabolite transporter Tp0684. Further refinement of minimal T cell antigens within these proteins and expansion of the Tp protein panel is ongoing. These are the first human Tp specific T cell epitopes described. Incorporating this information into vaccine design may further increase protection against infection.

Supported by T32AI007140-44
Administration of 3,3-dimethyl-1-butanol is immunomodulatory in collagen induced arthritis murine model independent of TMA lyase activity
Authors: Fechtner Sabrina , Allen Brendan , Chriswell Meagan E , Jubair Widian K , Vroljik M Aaron , Holers V Michael , Kuhn Kristine A
Published: January 2023

Studies of rheumatoid arthritis (RA) and murine collagen-induced arthritis (CIA) link the intestinal microbiome with disease and pathogenic autoantibodies. We have previously identified significant alterations in carnitine metabolism in mice with CIA, providing a potential link between bacterial dysbiosis and autoimmunity. Dietary carnitine is metabolized into trimethylamine (TMA) by bacterial enzyme TMA lyase which is oxidized in the host liver to TMAO. Prior studies determined that 3,3-dimethyl-1-butanol (DMB) and fluoromethylcarnitine (FMC) inhibit TMA lyase. Therefore, we tested if TMA lyase inhibition with DMB or FMC could ameliorate CIA. Male DBA1/j mice at 6 weeks of age were injected with bovine type II collagen in complete Freund’s adjuvant at days 0 and 21. On day 21 drinking water was replaced with vehicle, 1% vol/vol DMB, or 100mg/kg FMC. Mice were monitored for arthritis until day 35. DMB-treated mice had a significant (>50%) reduction in arthritis severity, as well as decreased anti-collagen antibodies, serum IFN-γ, and IL-17 compared to FMC and vehicle-treated mice with CIA. However, only FMC significantly reduced intestinal TMA and circulating TMAO, while DMB did not affect TMA/TMAO, suggesting that the effect of DMB is independent of TMA lyase inhibition. Using gas chromatography, we confirmed that DMB is absorbed and metabolized in mice, thereby identifying a novel metabolite, 3,3-dimethyl-1-butyric acid (DMBut). During CIA, 1% vol/vol DMBut significantly reduced disease, suggesting it is an active metabolite of DMB. Altogether our study suggests that DMB and/or its metabolites are immunomodulatory in CIA. Elucidating its target and mechanism of action may provide directions for RA therapeutic development.

Supported by Grants from NIH (U01HL152405, T32AI074491) and from the State of Colorado Office of Economic Development and International Trade Advanced Industries Accelerator Program
Single-cell mRNA analysis of colon tissue during DSS colitis in CX3CR1cre/+EP4fl/fl mice suggests PGE2 signaling via EP4 promotes survival of protective macrophages
Authors: Murray Page , Emanuel Elizabeth , Kang Byunghyun , Kelsall Brian L
Published: January 2023

Prostaglandin E2 signaling through its receptor EP4 on macrophages (MPs) protects against DSS-induced colitis, however the mechanisms involved are not clear. Here we perform single cell RNA sequencing on the colons of CX3CR1cre/+EP4fl/fl(Cre+) mice and CX3CR1+/+EP4fl/fl(Cre−) littermate controls at days 0, 3 and 7 following DSS treatment. We identified monocyte (MO), MP, DC1, DC2 and pre-DC populations, with MPs clustering into Hes1hi, Mrc1hi  Siglec1(CD169)hi, Retnlahi, and Il1r2hiCd80hi populations. With colitis there was a dramatic increase in the proportions of inflammatory MOs and a loss of all mature MPs, along with an increase in cDC2 but not cDC1 in both mouse strains. However, in Cre+ mice, which develop worse disease with universal GI bleeding, the loss of Mrc1hi  Siglec1hi MPs was accelerated together with an increased proportion of Il1r2hiCd80hi MPs compared to Cre− littermates. Analysis of DEGs showed the Il1r2hiCd80hi MP cluster expresses high levels of leukocyte chemoattractants, suggesting that PGE2 may prevent leukocyte recruitment during inflammation. Cre+ mice also had fewer Retnlahi macrophages than Cre− mice both at days 0 and 7 post DSS. Genes highly expressed in the Retnlahi cluster are associated with wound healing and maintenance of vascular integrity indicating their loss could account for the enhanced GI bleeding in the inflamed Cre+ mice. This is the first detailed scmRNA analysis of immune cell populations involved in DSS colitis. While additional investigation is necessary, the data suggest a role for PGE2 signaling via EP4 on MPs in preventing excessive inflammatory cell infiltration, decreasing vascular permeability, and contributing to tissue repair by maintaining protective MP populations.