bluuebunny/crossref_metadata_2025 huggingface dataset:
5-Hydroxyndole-3-acetic acid suppresses activation of glutamate oxaloacetate transaminase 1 linked to Th1/17 cell differentiation and ameliorates rheumatoid arthritis
Authors: Lee Jong Yeong , Lim Hyo Jung , Choi Jin kyeong
Published: January 2023

It has been established that cell proliferation and survival activities can be controlled according to metabolic resources in inflammation. However, it is not clear how T cell-mediated inflammatory immune responses in rheumatoid arthritis (RA) affect the activity and regulation of inflammatory cells by meeting biosynthesis and bioenergy requirements. This study evaluated the effect of serotonin-derived metabolite 5-Hydroxyndole-3-acetic acid (5-HIAA) on the increase in glutamate oxaloacetate transaminase 1 (GOT1) by the differentiation of Th1, Th17, or Th1/17 cells. The differentiation of Th1, Th17, or Th1/17 cells increased GOT1 and showed that GOT1 promotes the differentiation of Th1, Th17, or Th1/17 cells. Conversely, selective inhibition of GOT1 using 5-HIAA blocked the differentiation of Th1, Th17, or Th1/17 cells. Furthermore, inhibition of GOT1 using 5-HIAA ameliorated RA through up-regulation of regulatory T cells (Foxp3+ CD4+), suppression of pro-inflammatory cytokines, and inhibition of Th1/17 responses in mouse joints. Therefore, targeting glutamate-dependent metabolic pathways would be an effective therapeutic strategy for rheumatoid arthritis and a wide spectrum of autoimmune diseases.

Supported by 21st Century Medical Science Creative Human Resource Development Center
CXCR6 is required for tissue resident memory T cell formation across diverse peripheral non-lymphoid tissues
Authors: Heim Taylor , Steele Maria M , Mudianto Tenny , Lund Amanda W
Published: January 2023

CD8 T cell control of viral infections in peripheral tissues is largely dependent upon their spatial positioning. Clearance of pathogens requires direct contact between CD8 T cells and infected cells. Chemokine receptors play an essential role in this process by facilitating T cell entry into tissues, directing T cell positioning within tissues and by influencing egress out of tissues. Tissue resident memory T cells (TRM), which fail to egress after pathogen clearance and scan cells for signs of infection, are potent sources of immunity to secondary encounters with pathogens. Here we show that expression of the chemokine receptor CXCR6 is a conserved feature of TRM after viral infection in many non-lymphoid tissues. Using CXCR6KO TCR-tg T cells, we found that CXCR6 is necessary for proper formation of TRM in all non-lymphoid tissues examined. CXCR6 was necessary for early accumulation of effector T cells in several tissues such as the kidney and salivary gland but CXCR6 was dispensable for early accumulation in the skin. After vaccinia infection in the skin, CXCR6 expression is reinforced upon antigen re-encounter and promotes CD8 T cell accumulation in a migration-independent manner. Using single cell transcriptional analysis, we show that CXCR6KO TRM appear to exhibit a survival disadvantage and are outcompeted by wildtype T cells. This work reveals a critical requirement for generating TRM in diverse anatomical locations and may benefit strategies for vaccine design, anti-tumor immunity and reducing T-cell mediated autoimmunity.

Supported by R01CA238163 T32Al100853-10
The E3 Ubiquitin ligase Cul4b is required for CD8+ T cell-mediated antiviral immunity
Authors: Dar Asif Amin , Gordon Scott , Klinzing Kathleen , Dale Kim , Porter Nadia , Guha Ipsita , Behrens Edward M , Oliver Paula M
Published: January 2023

CD8+ T cells play crucial roles in cellular immune mechanisms that clear invading viruses and prevent reinfection. Virus specific CD8+ T cells expand approximately 10,000 fold, undergoing up to 20 cell divisions in the first week of infection. While this rapid expansion is needed to reduce viral loads, replication can lead to DNA damage and telomere shortening, resulting in growth arrest. T cells must be equipped with mechanisms that promptly promote DNA repair to protect cells from premature senescence and death. We recently determined that the E3 Ubiquitin ligase Cullin-4b (Cul4b) regulates genome integrity in CD4+ T cells by helping to resolve DNA damage. In keeping with this, aberrant expression of Cul4b and its ortholog Cul4a has been reported in many cancers. However, whether Cul4b is important in controlling CD8+ T cell numbers or viral clearance is not known. To address this, we infected control and Cul4bcKO mice with LCMV. Following infection, virus-specific CD8+ T cells lacking Cul4b failed to expand or acquire effector functions. As a consequence, Cul4bcKO mice were unable to clear LCMV. Furthermore, Cul4b-deficient CD8+ T cells were unable to differentiate into memory T cells. Using IP and tandem mass spectrometry, we found Cul4b associated with the chromatin remodeling BAF complex (SWI/SNF). Specifically, our data supports that Brg1, a component of SWI/SNF complex, is a client of the Cul4b ubiquitin ligase. Loss of Cul4b increased Brg1 binding to chromatin following CD8+ T cell activation, impacting gene expression and/or cell cycle arrest. Together, these data support that Cul4b limits the amount of Brg1 associated with chromatin, and allows the expansion and differentiation of CD8+ T cells thus promoting antiviral immunity.
Hepcidin deficiency increases susceptibility to disseminating candidiasis and renal failure
Authors: Scindia Yogesh , Kasem Sadat , Mansouri Samira , Desai Dhruv , Agarwal Annanya , Khodayari Nazli , Lionakis Michail , Mehrad Borna
Published: January 2023

Background

Candida albicans is the single most common human fungal pathogen, and invasive candida infections carry a mortality rate of about 25%. C. albicans possesses a range of iron acquisition mechanisms that contribute to its persistence, and virulence. Patients with fungal infection have substantially increased transferrin saturation and serum iron concentrations independent of underlying hematological disorder, suggesting that host iron handling is critical to the outcome of the infection. Using a murine model of systemic iron overload, we investigated whether C.albicans-induced pathology is influenced by iron availability.

Experiment

Iron overloaded hepcidin knockout (Hamp−/−) and wild type (WT) litter mates were infected with C. albicans SC5314 and outcomes of infection were evaluated after 3 days.

Results

Compared to WT mice, Hamp−/− mice displayed increased renal fungal burden, had more renal injury, inflammation and mortality. C. albicans was in yeast form in the kidneys of WT mice but had transformed into hyphae in the iron rich renal tubular segments of Hamp−/− mice. Despite the greater pathogen burden, kidneys of Hamp−/− mice had reduced numbers of neutrophils with increased expression of p21.

Conclusion

Our data identify systemic iron overload as a susceptibility factor to C. albicans induced renal failure. Hepcidin deficiency was associated with increased renal iron burden and transformation of yeast into hyphae, suggesting increased virulence. Increased p21 expression in neutrophils suggests impaired NETosis and may possibly explain increased fungal burden. Our data highlight importance of dysregulated iron metabolism in disseminating candidiasis.
Effective clearance of Chlamydia from the murine female reproductive requires IL-12p40, but not T-bet driven Th1 responses
Authors: Rixon Jordan , McSorley Stephen J
Published: January 2023

Protective immune responses to Chlamydia infection within the female reproductive tract (FRT) are incompletely understood. MHC class II-restricted IFN-γ+ Th1 responses are thought to be vital for bacterial clearance, but the Th1 master transcription factor T-bet had not yet been fully interrogated in primary infection. Here, we investigated T-bet and IFN-γ involvement in clearance of primary infection from the FRT. Surprisingly, IFN-γ producing CD4+ T cells from the FRT expressed low levels of T-bet, indicating that Chlamydia-specific CD4 T cells are not classical T-bet+ Th1 cells. Additionally, mice deficient in T-bet competently cleared infection with wild-type kinetics and showed significant skewing towards Th17 responses, indicating compensation pathways through alternate Th fates. On the other hand, IFN-γ-, and IFN-γR-deficient mice resolved much of FRT infection, but displayed systemic dissemination and 100% mortality. Thus, IFN-γ protects mice from fatal systemic disease, but classical T-bet+ Th1 cells are not required for primary clearance. In investigating alternative Th fates, RORγt mutant mice deficient for Th17 responses exhibited a short delay in clearance of FRT infection, demonstrating an unanticipated role for Th17 cells in Chlamydia clearance. However, mice deficient in the cytokine subunit IL-12p40 that promotes both Th1 and Th17 cells displayed a prolonged delay in FRT clearance despite CD4 T cells retaining the capacity for IFN-γ production. In summary, the type 1 cytokine IFN-γ plays an essential role in systemic control of Chlamydia, while clearance of primary infection within the FRT is largely dependent on an IL-12p40 driven mechanism that does not require T-bet or classical IFN-γ producing Th1 cells.

Supported by grants from NIH (RO1 A1103422, T32 AI060555)
Using Dictyostelium discoideum as a model for phagocytosis resistance in Escherichia coli
Authors: Riddick Terra L B , Perez-Vazquez Marta , Snyder Michelle
Published: January 2023

Escherichia coli related illnesses continue to be a problem around the world, especially in developing countries that lack clean water and proper sanitation. Dictyostelium discoideum is a soil dwelling amoeba that phagocytizes bacteria for nutrients. D. discoideum is a great model to study host pathogen interactions, and it has been used to identify virulence factors in bacteria. Our objective is to use Dictyostelium discoideum as a model to identify bacterial genes involved in resistance to phagocytosis. We hypothesize that D. discoideum can be used as a model organism to identify virulence genes associated with E. coli. The aims of this study are to identify E. coli genes predicted to grant resistance to D. discoideum grazing and phagocytosis and further characterize genes identified as potential virulence factors and observe the genes’ effects on D. discoideum grazing and phagocytic killing. Intestinal pathogenic, extraintestinal pathogenic, and laboratory E. coli strains were classified according to grazing phenotype. Previous comparative genomic analysis by large-scale BLAST score ratio (LS-BSR) identified genes prevalent amongst strains resistant to D. discoideum grazing. The gene gipA (growth in Peyer’s patches) was one gene found in various E. coli strains resistant to D. discoideum grazing. We are currently working to characterize the role of E. coli genes associated with resistance to D. discoideum grazing by manipulating E. coli strains for expression of these genes. We will observe the interactions between these strains and D. discoideum grazing and phagocytosis. This experiment will give insight into bacterial genes and virulence factors responsible for evading phagocytosis and the immune system.

This project was funded in part by federal funds from the National Institute of Allergy and Infectious Diseases, National Institute of Health, and Department of Health and Human Services under grant number U19 AI110820. Dr. M. Snyder was funded by an NIH NIGMS grant F33 GM116618, a Franklin Research Grant from the American Philosophical Society and a grant from the TU FDRC committee. This research was also supported by an NIH NIGMS R25 GM19970.
TNF-α signaling is required for fungal clearance during brain infection with Cryptococcus neoformans via promoting the recruitment of CD4+ T cells and inflammatory monocytes
Authors: Chen Yanli , Strickland Ashley , Shi Meiqing
Published: January 2023

Cryptococcus neoformans is a pathogenic fungus accounting for 180,000 deaths worldwide per year. The infection starts in the lung; however, the fungus can migrate to the brain, causing fatal meningoencephalitis primarily in immunocompromised individuals. TNF-α has been shown to mediate protective immunity in the lung during pulmonary infection with C. neoformans. However, the functions of TNF-α signaling during brain infection with C. neoformans remain poorly understood. Recent studies have shown that CD4+ T cells and inflammatory monocytes (IMs) mediate cryptococcal clearance in the brain at the cost of inducing immunopathology. Here, we study the role of TNF-α in the context of CD4+ T cells and IMs during brain infection with C. neoformans. We found that TNF-α levels in the brain were significantly increased during infection. The increase of TNF-α was associated with enhanced mRNA expression of various chemokines and accumulation of CD4+ T cells and IMs. Genetic ablation of TNF-α receptor (TNF-αR) in mice led to significantly higher brain fungal burdens and early mortality during C. neoformans infection. Infected TNF-αR−/− mice displayed significantly reduced recruitment of CD4+ T cells and IMs in the brain compared to infected wild-type mice. This was accompanied with reduced mRNA levels of KC, MIP-2, CCL2 and CCL7. Interestingly, deficiency of TNF-αR also resulted in a significant drop in the mRNA levels of ICAM-1 and VCAM-1 as well as the expression of CD11a and VLA4 in CD4+ T cells and IMs. Collectively, these results suggest TNF-α signaling is essential for the recruitment of CD4+ T cells and IMs, mediating fungal clearance during brain infection with C. neoformans.

Supported by AI131219 and AI131905
Dynamics of the Immune Response to MRSA Subcutaneous Skin Infection
Authors: Bose Jeffrey L , Gilchrist Aubrey K , Ridder Miranda J , Dai Hongyan , Pritchard Michele T , Markiewicz Mary A
Published: January 2023

Staphylococcus aureus is a leading cause of skin and soft tissue infections (SSTIs). While studies have examined how the immune system responds to S. aureus SSTIs, most are limited in scope to a single time point or outcome. To set a foundation for future studies, we conducted a S. aureus skin infection experiment using female C57BL/6J mice and USA300 S. aureus to examine the host-pathogen interface up to 15 days post-infection (d.p.i.). We measured lesion size, bacterial titers, local cytokine and chemokine levels, phenotyped responding leukocytes, and collected skin for histopathologic analysis and Gram staining. Lesions were largest at 1 d.p.i. with peak necrotic tissue area at 3 d.p.i. and were largely resolved by 15 d.p.i. IL-6, IL-4, CCL2, and IL-1β were highest during early infection, while IL-17A/F peaked 7 days post-infection. Neutrophils were abundant in the skin throughout the infection and were the primary immune cell present until 9 d.p.i. Histopathologic analysis demonstrated swift and extensive keratinocyte death and robust and persistent neutrophil infiltration. Gram staining revealed subdermal S. aureus colonization and, later, limited migration into upper skin layers. Most leukocyte subsets decreased within 8h after infection and returned during wound resolution coincident with declining bacterial titers. These (re)emerging leukocytes included CD4+ T cells, innate lymphoid cells, inflammatory monocytes, macrophages, and dendritic cells. Collectively, these data illustrate a dynamic immune response to S. aureus skin infections and provide a foundation for further studies characterizing the host-pathogen interface.

This research was supported by funding from the National Institute of Allergy and Infectious Diseases (NIAID) award R01AI121073 as well as pilot project grants from K-INBRE (P20GM130418) and University of Kansas Chemical Biology of Infectious Disease COBRE grant P20GM113117 to JLB. We acknowledge the Kansas Intellectual and Developmental Disabilities Research Center Histology Core which is sponsored in part by the NIH/NICHD (U54 HD090216) and the Flow Cytometry Core Laboratory, which is sponsored, in part, by the NIH/NIGMS COBRE grant P30 GM103326 and the NIH/NCI Cancer Center grant P30 CA168524.
Airway Prevotella promote TLR2-dependent neutrophil activation and rapid clearance of Streptococcus pneumoniae from the lung
Authors: Clark Sarah E , Horn Kadi J , Schopper Melissa A
Published: January 2023

This study investigates how the lung microbiome influences the early immune response to infection. Prevotella are a major component of the endogenous airway flora, and Prevotella melaninogenica is one of the most commonly isolated species in the human lung. Increased abundance of P. melaninogenica correlates with reduced infection with the bacterial pathogen Streptococcus pneumoniae, indicating a potentially beneficial role. Using a mouse co-infection model, we found that P. melaninogenica enhanced protection against S. pneumoniae, resulting in rapid clearance from the lung and improved survival. This response required recognition of P. melaninogenica lipoproteins by toll-like receptor (TLR)2, the induction of TNFa, and neutrophils, as the loss of any of these factors abrogated Prevotella-induced protection. Improved clearance of S. pneumoniae was associated with increased serine protease-mediated killing by lung neutrophils. IL-10 was also important for restraining P. melaninogenica-induced inflammation in co-infected mice. Together, these findings highlight innate immune priming by the airway microbiome as an important protective feature in the respiratory tract.

Supported by the National Institute of Allergy and Infectious Diseases grant K22AI143922, an American Thoracic Society Research Program Grant, an American Lung Association Innovation Award, and a Boettcher Foundation Webb-Waring Biomedical Research Award.
Optimization of cell culture protocols and immunoassays in a non-model teleost.
Authors: Collias Alexandra , Steinel Natalie C
Published: January 2023

The threespine stickleback fish and its natural helminth parasite Schistocephalus solidus are an emerging model for studying host-parasite immune modulation. However, to serve as an effective immunological and host-pathogen interaction model in vitro protocols must be developed and optimized to assess immune function, including leukocyte viability, apoptosis, production of reactive oxygen species (ROS), phagocytosis, and proliferation. Unique features of stickleback leukocytes make them incompatible with many commercially available reagents and their small size make them undetectable by some instruments, presenting challenges in the development of these assays. Flow cytometry has proven to be a successful tool for performing streamlined cell counts and we have succeeded in identifying live, apoptotic, and dead leukocytes in this species. We have also optimized flow cytometry assays to detect the production of multiple ROS species using DHR-1,2,3 as well as a superoxide-specific detection assay. The small size of stickleback leukocytes makes them incompatible with large phagocytosis particles, such as zymosan. However, using a combination of lipophilic cell membrane dye and fluorescently tagged E. coli particles, in conjunction with confocal microscopy, we successfully quantified the phagocytic activity of stickleback splenocytes and confirmed internalization of the particles. Lastly, identifying proliferating stickleback leukocytes has been difficult; both EdU and Ki67 assays have been unsuccessful. Although challenging, identifying and documenting successful techniques to use with non-model organisms is essential for expanding our immunologic tool box and furthering our knowledge of these organisms.

Supported by grants from NIH (R01 AI146168)
Influenza-induced cytokine response predisposes hosts to secondary Aspergillus fumigatus infections
Authors: Grau Madeleine S , Wang Xi , Obar Joshua J
Published: January 2023

Influenza infected patients admitted to the ICU are at significant risk of developing secondary Aspergillus fumigatus (Af) infections, termed influenza-associated pulmonary aspergillosis (IAPA). IAPA affects both immunocompromised and immunocompetent patients, causing a higher morbidity and mortality −50–72% mortality rate–in comparison to other secondary infections. Influenza-induced secondary bacterial infections have been extensively studied, yet little is known about IAPA pathogenesis. We have developed a murine IAV-Af superinfection model in which immunocompetent C57BL/6J mice infected with influenza A virus (IAV) results in increased susceptibility to secondary Af challenge. IAV infection 6 days, but not 3 days, prior to Af challenge causes IAPA in these mice with significant fungal growth and germination leading to increased mortality. With this murine model of IAPA, we examined the potential role of cytokine dysregulation in driving IAV infection to secondary Af challenge. We observed a significant reduction in IAV-induced interferon gamma (IFNγ), which may be associated with impaired immune response during IAV-Af coinfection. However, IAPA infected C57BL/6J and Ifngr−/− mice developed similar disease. IAV-Af superinfected mice produce elevated TNFα during superinfection in comparison to IAV infection. Since myeloid immune cells stimulated with these cytokines have reduced bactericidal activities in COVID-19 patients this combination has the potential to similarly impair IAPA immune responses. Using our novel IAPA model we will assess the role of IFN dysfunction on innate immune cell populations, thereby increasing susceptibility to secondary Af infection by single cell RNA sequencing.

Supported by grant from NIH (1 R21 AI152019-1)
Sialophorin is an essential host element for immunity against fungal pneumonia
Authors: Mudalagiriyappa Srinivasu , Nanjappa Som Gowda
Published: January 2023

Invasive fungal infections (IFI) are emerging diseases accounting for 1.7 million deaths and 150 million life-threatening diseases worldwide annually. The increased population of immunocompromised individuals and lack of arsenal of antifungal drugs necessitate an understanding of mechanisms of host-pathogen interactions to define new preventive and therapeutic targets. Here, we show that sialophorin (CD43), a transmembrane glycoprotein, is necessary to mediate protective immunity against primary pulmonary fungal infection. Using a mouse model of lethal fungal infection with Blastomyces dermatitidis, we learned that ablation of sialophorin enhanced the severity of disease, pathology, and accelerated death. At the cellular level, alveolar macrophages, one of the first lines of innate cellular barriers, were blunted in the absence of sialophorin. Additionally, sialophorin deficiency truncated the numbers of inflammatory monocytes, CD103+ DC, and interstitial macrophages. Surprisingly, sialophorin was dispensable for neutrophils. Despite sialophorin implication, the defect seen under sialophorin deficiency was independent of the cell trafficking. Finally, we uncovered that sialophorin augmented IL-17A responses from innate lymphoid cells.

Collectively, we show that sialophorin is an essential host element for innate immunity against pulmonary fungal infection and can be an immune target for the control of fungal infections.

Funding support: Innovation Award by the American Lung Association to SGN.

Supported by Innovation Award by the American Lung Association to SGN.
The preterm infant microbiome impairs lung immune responses to respiratory syncytial virus infection
Authors: Brown Julia A , Carrow Hannah , Jin Jenny C , Ananthanarayanan Aparna , Sanidad Katherine Z , Johnson Elizabeth L , Perlman Jeffrey M , Worgall Stefan , Zeng Melody Y
Published: January 2023

The gut microbiome plays a critical role in neonatal immune development; early-life perturbations to the microbiome have been linked to later susceptibility to respiratory conditions, including asthma and chronic obstructive pulmonary disease. Preterm infants are at higher risk for adverse outcomes to respiratory infections such as respiratory syncytial virus (RSV). The microbiome in premature infants is significantly altered, but it remains unclear how this affects lung immune cell development and susceptibility to RSV infection independent of lung immaturity. In this study, we have generated a biorepository of stool specimens from preterm and term infants during the first weeks of life. Metagenomic sequencing studies indicate significant alterations in the gut microbiome between these two groups, including a lack of fiber-fermenting Bacteroides species in preterm infants. Using these stool specimens, we find that the altered gut microbiome in premature infants induces IL-17/neutrophil-biased immune responses but weaker anti-viral interferon responses in the lung, both at steady state and following RSV infection. Mice colonized with preterm infant stool bacteria produced lower levels of neutralizing antibodies against RSV than mice colonized with term infant stool bacteria. Taken together, our findings suggest that the altered gut microbiome in premature infants dysregulates immune cell development in the lung and impairs the infant’s ability to develop long-term immunity against respiratory infections. These findings might be potentially leveraged in the development of gut bacteria-based interventions to improve the development of protective immunity against respiratory viral pathogens in preterm infants.

Supported by grants from NIH/NCATS (TL1-TR-002386) and the Biocodex Microbiota Foundation
Understanding the role of maternal antibodies on protection from enteric pathogens
Authors: Wang Bingjie , Torres Stephanie , Koch Meghan
Published: January 2023

A major function of the microbiota is to shield the host from infection with pathogens via a process called colonization resistance. High affinity, T cell-dependent antibodies, produced through interaction between CD4 T follicular helper (Tfh) cells and germinal center (GC) B cells, can modulate gut microbiota diversity and function. We discovered that mice lacking maternal antibodies display increased levels of Tfh and GC B cells in gut draining lymphoid tissues such at the mesenteric lymph nodes and Peyer’s patches. This response is seen in the context of colonization by microbes as germ-free mice lacking maternal antibodies do not show increased Tfh and GC B cell responses.

Given the importance of Tfh cells and TD antibody responses in regulating host-microbe interactions, we tested whether the elevation in Tfh and GC B cells seen in maternal antibody deficient pups alter susceptibility to colonization by the pathogen Salmonella typhimurium. We infected maternal antibody sufficient and deficient pups with the model pathogen Salmonella typhimurium via oral gavage. To block the Tfh and GC B cell response, we treated half of the pups from each group with anti-inducible T cell co-stimulator ligand (aICOSL) while the other half received an isotype control antibody. We found no difference in Salmonella typhimurium burden in the spleen, liver or cecum between maternal antibody sufficient or deficient pups treated aICOSL or isotype control antibody. Taken together, our data suggest that maternal antibodies and the subsequent rise in Tfh and GC B cells do not alter susceptibility to Salmonella typhimurium colonization later in life.

Supported by AAI Careers in Immunology Fellowship
Gamma delta T cells and keratinocytes modulate wound repair via Perforin-2
Authors: Strbo Natasa , Rivas Katelyn , Padula Laura , Fisher Eva , Marjanovic Jelena , Burgess Jamie Lee , Wikramanayake Tongyu , Pastar Irena , Tomic-Canic Marjana
Published: January 2023

Effective and precisely timed immune response is vital for efficient wound closure. Cutaneous gamma delta T (GDT) cells are poised to perform skin-specific roles in inflammation and repair while factors that shape gamma delta T cell activity are still not known. GDT cell deficient mice exhibit significantly delayed wound closure compared to WT mice. We found that GDT cells constitutively express novel antimicrobial protein Perforin-2 (P-2), and we postulated that P-2 contributes to GDT cell activation and recruitment in response to wounding.

We induced full thickness wounds on the dorsal skin of 2- or 10-month-old C57/BL6 WT and P-2 deficient (−/−) mice and assessed healing at day 3 and 6 post wounding. We analyzed re-epithelialization and keratinocyte activation using histomorphometry and Keratin-6 staining.

We found that in 10-month old mice, wound re-epithelialization was significantly delayed at day 6 in P-2 −/− mice compared to WT. This delay was accompanied by significantly decreased Keratin-6 expression at the wound edge. Immunofluorescence staining and flow cytometric analysis revealed significantly fewer GD T cells in the wound bed in aged P-2 −/− mice. The P2 −/− GD T cells also displayed lower expression of activation markers, including significantly lower Skint1 expression in the P-2−/− skin on day 6 post-wounding.

Our data demonstrate impaired keratinocyte activation and GD T cell infiltration in the absence of P-2 that is more pronounced in aged mice, indicating a potential role for this protein in modulating the inflammatory response upon wounding.
CD40 is Required For Protective T Cell Responses Against Cryptosporidium
Authors: O’Dea Keenan , Hunter Christopher A
Published: January 2023

The apicomplexan intestinal parasite Cryptosporidium infects intestinal epithelial cells (IECs) and is a leading cause of life-threatening disease in infants, toddlers, and immunocompromised patients. No vaccine against Cryptosporidium exists, and current treatments are ineffective in immunocompromised individuals. A prominent immunodeficiency associated with susceptibility to Cryptosporidium is hyper-IgM syndrome, which is caused by defects in CD40 signaling. In a murine model of Cryptosporidiosis, mice deficient in CD154 (CD40L) are defective in parasite control and clearance, and complementation of CD40L restores protection. Therefore, characterizing CD40-mediated immune responses against Cryptosporidium may enhance progress toward effective therapy in at-risk individuals. CD40 engagement on dendritic cells (DCs) promotes IL-12 production that induces differentiation and proliferation of T helper type 1 (Th1) cells and their production of interferon-γ (IFN-γ), which is known to be essential for control and clearance of Cryptosporidium infection. Our findings suggest a defect in T cell responses in CD40−/− mice infected with Cryptosporidium. CD40 deletion on Xcr1+ DCs also resulted in increased susceptibility to Cryptosporidium along with a defect in CD8+ T cell expansion. Therefore, we conclude that CD8+ T cell responses against Cryptosporidium require CD40 on cDC1s.

Supported by NIH grant U01AI163671
EAE suppression caused by Δ-8-THC treatment is due to downregulation of CNS infiltrating CD4+ T-cells, proinflammatory cytokines and apoptotic genes expression
Authors: Kakar Khadija , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Δ-8-THC a naturally occurring cannabinoid found in trace amounts in Cannabis plants. It possesses potent anti-inflammatory properties along with several other medical benefits. Δ-8-THC has been shown to induce immune system homeostasis particularly in individuals with autoimmune disorders by reducing the activity of T cells and B cells. In current study, we used the experimental model of MS to better understand how Δ-8-THC suppresses neuroinflammation in immune cell-mediated disease called experimental autoimmune encephalomyelitis (EAE). Δ-8-THC administration reduced significantly the clinical severity score with an increase in body weight by suppression of Th17 cells. In the CNS, there was decreased expression of pro-inflammatory cytokine IL-17, an increase in brain infiltrating CD4+ T cells and B cells, while anti-inflammatory phenotype such as FoxP3 was perturbed. Based on preliminary miRNA sequencing data and in silico examination of differential expression of miRNAs, we found that the decrease in neuroinflammation following treatment may be due to up-regulation of miRs, mmu-miR-129–3p, mmu-miR-129-5p, mmu-miR-125-5p and mmu-miR-193-3p as well as down-regulation of mmu-miR-21-3p, mmu-miR-142-5p and mmu-miR147-3p in immune cells in the CNS. Target genes were predicted by TagetScan, miRbase, miRDB. The target genes associated with upregulated miRs included Hrk, CHMP2b, Arid3a and Dcaf7 which are mostly involved in apoptosis, autophagy and migration, while genes associated with down-regulated miRNAs were Abi2, ZFPM2, NDUFA4 that are mostly involved in cell growth, proliferation and differentiation. In summary, Δ-8-THC treatment may offer novel strategies for treatment of neuroinflammation by targeting miRs.

Supported by NIH P01AT003961, P20GM103641, R01AI129788, R01 ES030144, R01 AI160896 and R01AI123947
Non-classical anti-inflammatory drugs (NCAIDs) ameliorates brain inflammation and improves memory in Alzheimer’s diseases mice model
Authors: Islam Md Jahirul , Koh Young-Jae , Chung Chu Young , Seong Seung-Yong
Published: January 2023

Alzheimer’s disease (AD) is the most common cause of dementia due to inflammatory neurodegeneration in the brain. Neuroinflammation caused by damage associated molecular pattern (DAMP: Aβ, ATP) leads to neuronal apoptosis. From our study HY209, a NCAIDs and a TGR5 agonist, inhibits neuroinflammation and neurodegeneration. In vitro, HY209 suppressed NLRP3-ASC oligomerization and production of mature IL-1β/IL-18 in microglia cells by Aβ ± ATP. HY209 inhibited Ca++ influx incurred by ATP or BzATP (P2X7 agonists). Ca++ influx of microglia from TGR5 KO was like that of P2X7 KO compared to wild type mice in response to BzATP. Suggesting that TGR5 is essential in P2X7-mediated Ca++ signaling and HY209 might play a role as an inverse agonist for TGR5 colocalized with P2X7. HY209 activates adenylate cyclase and cAMP. cAMP inhibits priming phase of inflammasomal activation, suppresses pro-inflammatory cytokine (TNF-α) production and oligomerization of NLRP3-ASC complex. Indicating that HY209 suppresses both priming and activation phase of NLRP3 inflammasome. In the 5x Familial Alzheimer’s disease (5xFAD) mice model, HY209 (1 mpk, i.p. or 30 mpk, p.o.) improves memory function, decreases Aβ plaque and neuronal apoptosis in the brain. Structure activity relationships were studied to screen out more potent HY209 derivatives (next generation NCAIDs) with increased bioavailability and higher BBB permeability. Considering efficacies of NSAIDs, PPARγ agonists, specific inhibitors (for TNF-α, ROS, RAGE, HMG-CoA, p38 MAPK, P2X7 and NLRP3) targeting limited number of proinflammatory pathways, NCAIDs could ameliorate broad spectrum of neuroinflammation that is crucial in preventing neurodegeneration and improving cognition and memory in AD.
Novel gene immunotherapy prevents Aquaporin-4 (AQP4) mediated neuroinflammation and demyelination in a mouse model of multiple sclerosis
Authors: Senior Kevin G , Cote Isabelle , Sagadevan Addelynn Sonia , Gaddie Cristina D , Keeler Geoffrey D , Min Daniel , Main Madisyn T , Rechdan Michaela N , Assakawa Stefanie , Hoffman Brad E
Published: January 2023

Neuromyelitis optica (NMO) is an auto-inflammatory demyelinating disease that typically affects optic nerves and spinal cord that is characterized by the presence of serum aquaporin-4 immunoglobulin G antibodies (AQP4-IgG). NMO accounts for >45% of the demyelinating disease in Asians and warrants the development of a suitable therapy other than generalized immunosuppressants. It is thought that autoimmune activated AQP4-specific T cells disrupt the BBB and allow increased entry of AQP4-IgG and other immune effectors into tissues containing astrocytes expressing AQP4 in their membranes. Recently we have established an adeno-associated viral (AAV) gene immunotherapy that effectively prevents and reverses experimental autoimmune encephalomyelitis (EAE) via generation of suppressive antigen-specific regulatory T cells (Tregs). In this report, using a similar antigen-specific approach, we have demonstrated that AAV.AQP4 gene immunotherapy prevented the development of AQP4-mediated neuroinflammation and clinical neurological disability in almost 100% of C57BL/6J mice when AAV.AQP4 vector was administered to mice 2 weeks prior to immunization with an immunogenic epitope of AQP4. Moreover, in contrast to AAV.AQP4 treated mice which remained unremarkable, histological analysis of the spinal cord sections from untreated vehicle only mice showed multiple areas of significant focal inflammation within the spinal cord. Based on our clinical and pathological data, we’ve successfully demonstrated that our novel AAV.AQP4 gene immunotherapy can indeed suppress the induction of AQP4 mediated autoimmune disease. Further evaluation will determine if it can also treat preexisting disease.
MOG variant 45D does not properly activate high-affinity, MOG reactive effector T cells but allows for high-affinity FoxP3+ Treg function
Authors: Faust Michael A , Kolawole Elizabeth M , Evavold Brian D
Published: January 2023

Although it’s well appreciated that autoreactive T cells can drive autoimmune disease, how T cells precisely distinguish and respond to epitopes is unclear. For example, T cells can proliferate in response to multiple epitopes of myelin oligodendrocyte glycoprotein (MOG), but not all epitopes are encephalitogenic. Here, we investigate why T cells specific for MOG 35–55 are encephalitogenic. We compare the wildtype MOG epitope (wtMOG) to a single amino acid substitute, 45D, which was engineered to expresses the same T cell receptor (TCR) contact residues as wtMOG but is mutated such that its affinity for MHC II is decreased. We employed the micropipette adhesion frequency assay to demonstrate that a monoclonal, MOG-specific, high-affinity TCR binds to 45D with the same affinity as wtMOG. Despite conserved TCR contact residues, the MOG 45D variant is not encephalitogenic and fails to prime high-affinity tetramer-positive TCR clones. We also extended our polyclonal analysis to FoxP3+ regulatory T cells (Tregs) and found that, compared to wtMOG, 45D priming increases the frequency of MOG-specific Tregs. We further show that these Tregs are functional and suppress the proliferation of MOG-specific T cells. Our work begins to dissect the differential T cell receptor kinetic properties that allow for a response, or lack thereof, in Tregs and Teffs to myelin peptides.

Supported by NIH R01 NS071518-09
Targeting proteolytic cleavage of Toll-Like receptors by alpha-1 antitrypsin inhibited dendritic cells activation and function
Authors: Elshikha Ahmed Samir , Abboud Georges , Morel Laurence , Song Sihong
Published: January 2023

Toll-like receptors (TLRs) on dendritic cells (DC) interact with self-antigens and play a critical role in systemic lupus erythematosus (SLE) pathogenesis. TLR3, TLR7/8, or TLR9 are endosomal receptors that require proteolytic processing in the endolysosome to initiate signaling in response to DNA, single-stranded RNA, and double-stranded RNA. Targeting this proteolytic processing may represent a novel strategy for inhibiting TLR mediated pathogenesis. Human alpha 1 antitrypsin (hAAT) is a protease inhibitor with anti-inflammatory and immunoregulatory properties. However, the effect of hAAT on TLRs is not clear. In this study we first tested the effect of hAAT on DCs. We showed that hAAT treatment inhibited TLR7/8 and TLR9 agonists induced DC expressions of CD80, CD86 and MHC-II and significantly reduced the productions of IL-12, IFN-α and CXCL-10. Mechanistically, hAAT lowered the expression of interferon signature genes (MX-I, Isg-15, IRF-7, Il-12p40 and TNF-α). Importantly, western blot analysis showed that hAAT treatment reduced the active form (cleaved) of TLR9 in DCs indicating a novel mechanism of hAAT function in the immune system. Our in vivo studies in mouse models also showed that hAAT attenuated TLR agonist-induced pathogenesis. These data demonstrated that hAAT inhibited TLR7/8 and TLR9 signaling pathways in DCs, which are critical for lupus development. These findings not only advanced the current knowledge of hAAT biology, but also implied an insight for clinical application of hAAT.
Liver metastasis restrains immunotherapy efficacy via macrophage-mediated T cell elimination
Authors: Kryczek Ilona , Liu Jali , Green Michael , Chinnaiyan Arul , Cieslik Marcin , Zou Weiping
Published: January 2023

Metastasis is the primary cause of cancer mortality, and cancer frequently metastasizes to the liver. It is not clear whether liver immune tolerance mechanisms contribute to cancer outcomes. We report that liver metastases diminish immunotherapy efficacy systemically in patients and preclinical models. Patients with liver metastases derive limited benefit from immunotherapy independent of other established biomarkers of response. In multiple mouse models, we show that liver metastases siphon activated CD8+ T cells from systemic circulation. Within the liver, activated antigen-specific Fas+CD8+ T cells undergo apoptosis following their interaction with FasL+CD11b+F4/80+ monocyte-derived macrophages. Consequently, liver metastases create a systemic immune desert in preclinical models. Similarly, patients with liver metastases have reduced peripheral T cell numbers and diminished tumoral T cell diversity and function. In preclinical models, liver-directed radiotherapy eliminates immunosuppressive hepatic macrophages, increases hepatic T cell survival and reduces hepatic siphoning of T cells. Thus, liver metastases co-opt host peripheral tolerance mechanisms to cause acquired immunotherapy resistance through CD8+ T cell deletion, and the combination of liver-directed radiotherapy and immunotherapy could promote systemic antitumor immunity.

This work was supported in part by research grants from the NIH/NCI grants for WZ (CA248430, CA123088, CA099985, CA193136, and CA152470); AC (1UM1HG006508); TSL (U01CA216449); IEN (CA233487); FW (S10OD020053); MAM (CA240515) and the NIH through the University of Michigan Rogel Cancer Center Support Grant (P30CA46592).
The B cell adapter for PI3K (BCAP) promotes M2 macrophage phenotype and safeguards tumors from immune surveillance
Authors: Saha Irene , Pasare Chandrashekhar
Published: January 2023

The tumor microenvironment is a complex milieu consisting of different populations of infiltrating immune cell populations. Macrophages and DCs within the tumor microenvironment are vital in priming naïve T cells within tumors and tumor draining lymph nodes and collectively guide responses to various immunotherapies. Macrophages can exist in both pro-inflammatory (M1) state and produce IL-6, IL-12, TNF-α and ROS to promote anti-tumor responses, and reparative (M2) state that support angiogenesis and secrete IL-10 and TGF-β favoring tumor growth. We have previously identified BCAP as an adapter that links TLRs to PI3K/Akt activation and negatively regulates inflammatory responses. Recent work has shown a critical role of BCAP in promoting the transition of M1 macrophages to M2 phenotype. Since tumor associated macrophages (TAMs) in their M2 state have tumor promoting roles, we wanted to investigate if deficiency of BCAP could drive anti-tumor responses. BCAP null mice with implanted MC38 tumors had significantly reduced tumor burden and higher infiltration of leukocytes. This included higher presence of CD4+ and CD8+ T cells as well as macrophages and inflammatory monocytes in the tumor microenvironment. We are currently investigating macrophage and T cell intrinsic role of BCAP on tumor growth. Finally, to understand the molecular events leading to differential tumor progression in BCAP deficient condition, we will interrogate the transcriptional profile of tumor infiltrating myeloid and lymphoid cells at different stages of tumor growth and examine the potential role of MyD88 and PI3K/Akt signaling in myeloid cells in influencing BCAP dependent tumor growth.

Supported by grant from NIH (R01 AI113125)
Enhanced anti-tumor immunity in ST8Sia6 knockout mice
Authors: Friedman David J , Kizerwetter Monika , Belmonte Paul , Rajcula Matthew , Theodore Keith , Lee Hyun Se Kim , Shapiro Michael J , Dong Haidong , Shapiro Virginia Smith
Published: January 2023

Inhibitory receptors have a critical role in the regulation of immunity. Siglecs are a family of primarily inhibitory receptors expressed by immune cells, which recognize specific sialic acid modifications on cell surface glycans. Sialyltransferases are the enzymes that catalyzing the addition of sialic acids onto glycoproteins and glycolipids. Siglec-E binds to α2,8-linked disialic acids, generated by the enzyme ST8Sia6 on O-linked glycoproteins and functions to inhibit activation of innate immune cells. Many tumors have increased sialic acid incorporation. Overexpression of the sialyltransferase ST8Sia6 on tumors led to altered macrophage polarization and increased tumor growth through engagement of ligands generated by ST8Sia6 with Siglec-E. Here, we examine the role of ST8Sia6 on immune cells in regulating anti-tumor immunity. The loss of ST8Sia6 in tumor bearing mice reduced tumor growth, increased survival, and altered the immune response as compared to ST8Sia6 sufficient tumor bearing mice. Using MC38, B16, and B16-OVA flank tumor mouse models, tumor growth was decreased in ST8Sia6 KO mice as compared to C57Bl/6 mice. Within the tumor microenvironment of tumors present in ST8Sia6 KO mice compared to tumors present in C57Bl/6 mice, macrophages and dendritic cells altered their phenotype towards enhanced immune activation. Tregs from tumors in ST8Sia6 KO mice also displayed an increase in RORγt and a reduction in TNFRII, suggesting a possible shift to a less suppressive Treg. These alterations in the immune response to tumors were due to the loss of ST8Sia6 in macrophages and dendritic cells and not T-cells. Thus, ST8Sia6 generates ligands for Siglecs that dampen anti-tumor immunity.

Supported by grants from NIH (R01CA243545, 2T32AI007425) and Mayo Clinic Center of Biomedical Discovery
Tumor-associated macrophages found at early tumor sites exhibit altered metabolism and contribute to tumor progression
Authors: Kurt Robert A , Chang Musea , Ellmaker Katherine , Esposito Abigail , Pa Sophea , Roberts Meghan
Published: January 2023

Previously, we reported that macrophages at early tumor sites, before the tumors are even palpable, have a mixed phenotype with production of both pro- and anti-inflammatory cytokines, decreased production of reactive oxygen species, and increased dependence on oxidative phosphorylation. Here we show that depleting these tumor associated macrophages (TAM) impacted subsequent growth of the EMT6 tumor and metastasis of the 4T1 tumor supporting the contention that macrophages found at early tumor sites contribute to later stages of tumor progression. Since these TAM could not be clearly defined as M1 or M2 we set out to characterize them further focusing on their metabolism. We found that TAM present at early sites of 168, EMT6 and 4T1 tumors had elevated expression of pkm2, glut1 and ampk, and transwell assays revealed that these tumors could induce expression of pkm2, glut1 and ampk in bone marrow derived macrophages. Since tumor-derived lactate is known to induce alterations in white blood cells we investigated lactate production by these tumors and whether lactate could induce expression of these same genes. The data revealed that the 168, EMT6 and 4T1 tumors produced significant amounts of lactate and that lactate induced expression of pkm2, glut1 and ampk in bone marrow derived macrophages. Finally, we found that targeting lactate dehydrogenase or eIF2 at early tumor sites impacted growth of the EMT6 tumor, and targeting AMPK or GCN2 impacted metastasis of the 4T1 tumor. Collectively, these data support the contention that alterations in macrophages are already present at the early stages of tumor growth, before a tumor is even palpable, and that targeting metabolism at these early tumor sites can impact later stages of tumor progression.