bluuebunny/crossref_metadata_2025 huggingface dataset:
TRAF6-TAK1 signaling drives Th9 differentiation
Authors: Park Sang A. , Nakatsukasa Hiroko , Chen WanJun
Published: January 2023

Naive CD4+ T cells differentiate into different subsets depending upon milieu of cytokine exposure. Interleukin-9 (IL-9)-producing CD4+ helper T cells (Th9 cells) are induced from naive CD4+ T cells by transforming growth factor-β (TGF-β) together with interleukin-4 (IL-4) during stimulation of the T cell antigen receptor (TCR) in vitro. The master regulator of Th9 cells as well as the molecular mechanisms by which TGF-β signals and IL-4 signals co-operatively drive the differentiation of Th9 cells are not fully understood.

Previously, our group has reported that inhibitor of DNA-binding 3 (Id3), a transcription factor which inhibits DNA-binding of basic helix-loop-helix protein, such as E2A, controls the TGF-β-mediated differentiation of Th9 cells. We found that this process required activation of the kinase transforming growth factor beta-activated kinase 1 (TAK1). In addition, RNA-seq revealed that tumor necrosis factor receptor-associated factor 6 (TRAF6) was highly up-regulated by TGF-β together with IL-4 in CD4+ T cells. Since TRAF6 is one of the TAK-1 activator, we hypothesized TRAF6 is also involved in the differentiation of Th9 cells. We found that the expression and ubiquitination of TRAF6 was upregulated under the Th9 differentiation condition and it resulted in activation of TAK1. These regulations were attenuated both in TGF-β and Smad3 deficient mice, suggesting TRAF6-TAK1 signaling is involved in TGF-β-mediated Th9 differentiation.
Bcl6 interacting corepressor contributes to CD4+ T helper 17 cell formation by repressing CD27 and Foxo1 signaling
Authors: Kotov Jessica Amy , Gearhart Micah D. , Bardwell Vivian J. , Jenkins Marc K.
Published: January 2023

CD4+ T helper 17 (Th17) cells protect against pathogens at mucosal barriers by secreting cytokines that drive clearance of the pathogen by neutrophils. We found that Bcl6 interacting corepressor (BCOR) drives Th17 cell formation after Streptococcus pyogenes infection and was required for optimal expression of IL-17A and CCR6. RNA and chromatin immunoprecipitation sequencing revealed that BCOR enhances Th17 differentiation by directly repressing genes that negatively regulate Th17 cells, including genes related to the CD27 and Foxo1 signaling pathways. We have also found that Streptococcus infection generates a novel BCOR-driven Tfh-Th17 hybrid population, which may provide unique B cell help during mucosal infections. This work provides insight into a novel mechanism that enhances Th17 differentiation and function, which may be informative for the design of T cell based vaccines against extracellular pathogens at mucosal barriers.
STAYING ALIVE! SIGNAL PROCESSING BY NF-κB PROTECTS NKT CELLS FROM TNFR1-TRIGGERED DEATH
Authors: Kumar Amrendra , Gordy Laura E. , Bezbradica Jelena S. , Stanic Aleksandar K. , Hill Timothy M. , Boothby Mark R. , Van Kaer Luc , Joyce Sebastian
Published: January 2023

A unique ontogenetic program controls the thymic development of type I nature killer T (NKT) cells. For example, signal integration by NF-κB plays a critical role in NKT cell development such that RelA and RelB play cell intrinsic and cell extrinsic roles, respectively. The key cell extrinsic role played by NF-κB seems to be the induction of IL-15 production that is required for the development and homeostasis of NKT cells. On the other hand, cell intrinsic NF-κB activity is essential for the development of both NK1.1neg and NK1.1pos NKT cells. We have demonstrated that NKT cells in mice expressing a dominant negative mutant IκBDNtg that prevents NF-κB activation were arrested at a very early stage of their development. Importantly, introgression of Bcl-xL-coding Bcl2l1 transgene into IκBDNtg mice lead to the development of NK1.1pos NKT cells. Further investigations revealed that NKT cell intrinsic NF-κB activation was required to protect developing NKT cells from tumor necrosis factor (TNF) receptor 1-induced cell death and not from Fas/FasL- or NKT T cell receptor-induced apoptosis. Consistent with this finding, we found that TNF-α induces the activation of caspase 8- and 9-mediated apoptosis in NKT cells in which NF-κB activation was blocked. Thus, akin to hepatocytes and some cancer cells, TNF-α induces both cell intrinsic (caspase 8-mediated) and cell extrinsic (caspase 9-mediated) apototic pathways in developing NKT cells that are offset by the activation of NF-κB and the expression of one of its target gene, Bcl2l1.
LSD1 regulates PD-1 expression through interactions with Blimp-1 during acute viral infection
Authors: Neeld Dennis Kyle , Bally Alexander , Majumder Parimal , Barwick Benjamin , Boss Jeremy M.
Published: January 2023

During prolonged exposure to antigens, such as during chronic viral infections, sustained T cell receptor (TCR) signaling via cognate peptide: MHC interactions results in T cell exhaustion mediated in part by the expression of Programmed cell death protein 1 (PD-1). PD-1 blockade has been implemented in the clinical setting as a promising treatment for several different forms of cancer and therefore obtaining a complete understanding of the epigenetic mechanisms that regulate PD-1 is of great value to improving existing therapies. Here, we demonstrate that CD8 positive T cells lacking lysine-specific histone demethylase 1A (LSD1) expressed approximately 2-fold higher levels of PD-1 mRNA following TCR stimulation. Antigen specific CD8 T cells lacking LSD1 also showed increased surface levels of PD-1 during acute infection with LCMV but not during chronic infection. Molecular analysis revealed that Blimp-1, a known repressor of PD-1, recruits LSD1 for silencing. Although Blimp-1 is able to bind the PD-1 locus during both acute and chronic LCMV infection, it fails to recruit LSD1 in the chronic setting. LSD1 is responsible for removing activating histone modifications H3K4me1 and H3K4me2 and our results show that these marks are removed from the PD-1 locus only during acute infection. During viral infection, loss of DNA methylation is associated with with PD-1 expression. Examination of the PD-1 locus during acute infection revealed that LSD1 null CD8 T cells had less methylation compared to cells expressing LSD1 further demonstrating the role of LSD1 in regulating PD-1 expression. Taken together, these results demonstrate LSD1 downregulates PD-1 by interactions with Blimp-1.
A transient Malt1 aggresome sustains T cell receptor signaling to NF-κB
Authors: Traver Maria K. , Campanello Leonard , Huaman Celeste , Paul Suman , Shroff Hari , Losert Wolfgang , Schaefer Brian C.
Published: January 2023

The T cell receptor (TCR) to NF-κB signaling pathway is crucial for T cell activation and differentiation. Upon engagement of the TCR with cognate antigen, a series of events leads to the formation of the Carma1-Bcl10-Malt1 (CBM) complex, increasingly recognized as vital to a properly functioning immune response. We have demonstrated that in effector T cells, the CBM complex gives rise to a polymeric, filamentous signalosome called POLKADOTS that directs terminal activation of NF-κB. POLKADOTS consist of the core protein Bcl10, its constitutive binding partner Malt1, and their recruited signaling partners. We have previously shown that Bcl10 is degraded via selective autophagy following T cell activation. Here, we show that Malt1, which serves both as an adaptor transmitting signals to NF-κB and as a protease which cleaves a variety of substrates, is not concurrently degraded. Instead, Malt1-containing POLKADOTS coalesce via microtubule transport to a canonical aggresome. Aggresomes are thought to be depots of misfolded protein destined for degradation via macroautophagy; however, the Malt1 aggresome promotes late stage NF-κB activation through prolonging pIKK-Malt1 interactions. Additionally, this aggresome enhances the proteolysis of a subset of Malt1 targets. These results establish a mechanism for sustaining cytoplasmic signals from the TCR. More broadly, our findings demonstrate that aggresomes can serve as a stable platform for signal transduction.
The utilization of scientific language communication techniques to enhance Immunology Education for Postgraduate Clinical Health Science students
Authors: Sika-Paotonu Dianne , Tait Carolyn
Published: January 2023

Classroom discourse describes the spoken face-to-face interactions which happen in the classroom, with language being the dominant communication medium. It has been reported that teachers can easily talk for more than two thirds of a classroom lesson. To facilitate and support effective learning for students within the postgraduate setting where in-class time can be restricted, it is important that teachers carefully consider the language utilized particularly when introducing scientific material to students for the first time. The purpose of this work was to analyse the scientific language communication occurring within the Immunological content delivery for postgraduate Clinical Health Science students during in-class time. Verbal feedback from postgraduate Clinical Health Science nursing, midwifery and health students was obtained after Immunology based in-class learning and teaching sessions. Formal teaching evaluation of the Immunology teaching sessions was also undertaken. The in-class scientific language utilization was also considered within the literature context. Positive student feedback and favorable evaluation responses within specific teaching categories indicate the scientific language communication being utilized in the postgraduate setting for clinical health science students are providing support and promoting scientific language learning for students. The in-class scientific language utilization within this Immunology teaching content and was considered consistent when compared with the literature context. These results reinforce the importance of appropriate scientific language communication for effective of Immunological course content.
The utilization of a clinically relevant Immunological research case example within undergraduate biomedical sciences education
Authors: Sika-Paotonu Dianne
Published: January 2023

Mechanistic and cellular Immunology related teaching and learning content can be difficult to assimilate when being introduced for the first time even when appropriate scientific language communication is being utilized. To support the application of scientific Immunological content information and facilitate the in-depth understanding within the clinical context, integration of clinically relevant Immunological course elements and related research teaching examples into undergraduate biomedical sciences education are needed. Acute rheumatic fever (ARF) is an autoimmune condition caused by untreated Group A Streptococcal (GAS) infection of the upper respiratory tract (and possibly skin). Multiple or severe attacks of ARF can cause permanent cardiac damage known as rheumatic heart disease (RHD). Rates of ARF in New Zealand, Australia and the Pacific are among the highest in the world. Management for ARF requires painful monthly injections for at least 10 years of a type of Penicillin known as Benzathine Penicilin G (BPG). A less painful and longer acting BPG formulation is urgently needed would help prevent recurrence of ARF. Work being undertaken in New Zealand seeks to make a more appropriate form of Peniclilin for ARF and RHD. The purpose of this work was to successfully incorporate ARF as a clinically relevant case example within undergraduate Immunological course teaching. Students demonstrated improved improved in-class engagement and ability to answer in-class questions in addition to better understanding and recall of Immunological concepts. Student knowledge and understanding of Immunology was achieved using integration of a clinically relevant Immunological condition for undergraduate biomedical education.
The successful use of Immunology based Peer Teaching strategies within postgraduate clinical health sciences blended learning education frameworks
Authors: Sika-Paotonu Dianne
Published: January 2023

Immunology education remains a vital component for in-depth learning within clinical health sciences. The inclusion of undergraduate Immunology teaching content within nursing, midwifery and health related courses however remains inconsistent. The blended learning context provides avenues where active learning in-class activities that are learner centered can support and form synergies with more traditional learning forms. To support effective teaching and learning of Immunological related content at postgraduate level for classes with students with mixed experiences of Immunology education, integration of active learning and teaching strategies into postgraduate clinical sciences education are needed. The purpose of this work was to explore and assess the impact of Immunological based peer-teaching within a blended learning environment for postgraduate clinical health sciences students. Face-to-face Immunology teaching sessions for the postgraduate Pathophysiology course were delivered with the incorporation of short peer teaching sections allocated throughout the in-class session time. Instructions were carefully presented to ensure clarity and that communication was culturally appropriate for all students. Students demonstrated marked improvements in their understanding, memory recall of immunological concepts in addition to improved in-class engagement and when responding to in-class questions. In addition to enjoying the session time in class, improved confidence regarding immunological content covered in class was also noted. The integration of clinically active learning and teaching strategies such as Immunologically based peer teaching within postgraduate clinical sciences education.
Play your way to the finals: An immunology board game project to teach Immunology to undergraduate students.
Authors: Kucknoor Ashwini S.
Published: January 2023

It is a normal practice to use the last class day of class to review the course materials before the finals. Most of the time, all the students are seldom engaged and are not participating in these review sessions. It is important to engage the students and make the session more student-centered than the common instructor-centered ones. Here I describe the use of a semester long Immunology board game group project, as a tool to review the course material on the last day of the class. The board game project serves as a perfect learning tool to review the course material learnt during the semester in several ways. First of all, it helps all the students in the group to actively participate and be engaged in learning the concept assigned to each of the group. Secondly, since the students have to transform the board game of their choice to incorporate one specific concept of Immunology, they have to understand clearly and think deeply about the concept to come up with the details of the game, for example the board strategy, clues, game piece etc. Third and most importantly, to encourages creativity, teamwork and active participation of all students. I use this Board game group project for an upper division Immunology class with class size ranging from 24-28 students. On the day of the game, each group will play their game with their peers. Each group will design their board game to include a specific concept in detail. When all the groups (usually 4-5 groups for about 25 students) have played their games, most important concepts of the course material would be reviewed. More than 90% agreed that the Board game project helped them understand a given concept way better and that playing the board games before finals helped them tie up all the concepts well before the final exams.
Too Many B cells, When Food Fights Back, and Hacking T cells: The effective use of case studies to teach methods in an undergraduate Immunology course.
Authors: Dinkel Brittney A. , Walser-Kuntz Debby R.
Published: January 2023

The rapidly changing nature of immunology makes it a challenging course for both undergraduate students and faculty. A teaching approach that incorporates essential immunological techniques and an introduction to the primary literature helps undergraduates navigate the discipline and remain current. Case studies are an effective active learning approach that provide real world examples to engage undergraduates and an opportunity for hands on problem solving, data analysis, and critical thinking as students must consider outcomes and consequences. Although case studies that explore clinical connections already exist for undergraduates, they do not include key techniques or provide experience working with the primary literature. We have developed several case studies to help undergraduate immunology students understand and analyze data involving: flow cytometry, CRISPR/Cas, CAR T cells, and ELISAs. The case studies 1) introduce students to techniques and methods they may not have access to in their laboratory courses due to the lack of appropriate instruments or the cost of reagents, 2) build on difficult concepts covered in the course, and 3) incorporate figures from papers that challenge students to apply what they have learned. Assessment of student learning includes in-class quizzes and small group responses to data analysis questions, the use of “clickers”/personal response system, and related exam questions. Students self-report that the case studies are a highly effective teaching method that allow them to both reinforce their understanding of the course material and apply it to practical problems.
Role of Acinetobacter baumannii thioredoxin in pulmonary bacterial dissemination following LPS induced lung injury
Authors: May Holly , Yu Jieh-Juen , Guentzel M. Neal , Arulananadam Bernard P.
Published: January 2023

Despite medical advances, sepsis remains an increasing and major cause of death in hospitalized patients in the United States. Multiple-drug resistant Acinetobacter baumannii has emerged as an important hospital-acquired pathogen in both pulmonary infections and sepsis. Previously, our lab has shown the importance of bacterial thioredoxin during infection. A TrxA-null mutant (ΔTrxA) generated from A. baumannii Clinical isolate 79 (Ci79) showed a 100-fold decrease in virulence during systemic challenge. To further define the role of thioredoxin, our lab has created a pulmonary model of infection that leads to sepsis and death in C57BL/6 mice. This model is dependent on the induction of acute lung injury through E. coli lipopolysaccharide (LPS) injection. Lungs were analyzed for increased permeability using FITC-labeled dextran. Mice receiving the LPS showed significantly higher FITC-dextran particles in their serum compared to those that did not. Additionally, histopathology and cytokine production indicated limited immune response to the dose of LPS used. Using this model, mice showed significantly increased WT organ burdens in the lungs, liver, kidneys, cervical lymph nodes, and spleen compared to the mutant. Concurrently, mortality was significantly greater in the WT compared to ΔTrxA (~85%). Histopathological assessment showed increased cellular infiltrates and pulmonary inflammation during WT vs ΔTrxA infection. Finally, when the WT was treated with a thioredoxin blocker (PX-12) before challenge, mice survival improved from ~15% to ~75% with treatment. Together, this data demonstrates that thioredoxin plays a vital role in dissemination and virulence and could be a potential target to attenuate sepsis.
ALCOHOL ENHANCES TYPE 1 INTERFERON-α AND MORTALITY OF YOUNG MICE INFECTED WITH Mycobacterium tuberculosis
Authors: Tripathi Deepak , Welch Elwyn , Cheekatla Satyanarayana Swamy , Radhakrishnan Rajesh Kumar , Venkatasubramanian Sambasivan , Paidipally Padmaja , Van Abhinav , Tvinnereim Amy R. , Samten Buka , Devalraju Kamakshi P. , Neela Venkata Sanjeev Kumar , Valluri Vijaya Lakshmi , Mason Carol , Nelson Steve , Vankayalapati Ramakrishna
Published: January 2023

In the current study, we determined the effects of chronic alcohol consumption on the mortality of young and old mice and immune responses during Mycobacterium tuberculosis (Mtb) infection. Eighty percent of Mtb H37Rv infected alcohol-fed young mice died in five months compared to twenty-five percent in Mtb infected alcohol-fed old mice. There is no significant difference in lung bacterial burden of control and alcohol diet fed young and old mice. IFN-α levels were significantly higher in the lungs of Mtb infected alcohol-fed young mice and treatment with anti-IFNAR-1 antibody enhanced their survival. There are significantly higher numbers of CD11b+Ly6G+ neutrophils in the lungs of Mtb infected alcoholic young mice compared to Mtb infected alcohol-fed old mice and Mtb infected control diet-fed young mice. CD11b+Ly6G+ neutrophils are the major source of IFN-α in Mtb-infected alcohol-fed young mice and IFN-α enhanced the expression of RIP-1 and RIP-3 molecules, which are known to be involved in necroptosis. Alcohol-fed old Mtb infected mice and Mtb infected control diet-fed old and young mice expressed low level of IFN-α and RIP-1 and RIP-3 in their lungs. In response to Mtb stimulation, peripheral blood mononuclear cells (PBMC) from young healthy alcoholic individuals with latent tuberculosis infection (LTBI) produced significantly higher amounts of IFN-α compared to non-alcoholic young, alcoholic and non-alcoholic old healthy LTBI+ individuals. Our findings demonstrate that in Mtb infected young mice, alcohol enhances CD11b+Ly6G+ neutrophil infiltration in lungs and excess IFN-α production by neutrophils causes lung macrophage necroptosis and enhanced mortality.
Tissue Specific Overexpression of Human Heat Shock Protein 70 in Mouse Oviduct Epithelium Reduces Chlamydia Induced Immunopathology
Authors: Murthy Ashlesh K. , Keeler Kelly Rae , Do Jennifer , Seetharaman Aravind , Ramsey Kyle H. , Ciancio Mae , Li weidang
Published: January 2023

Chlamydia trachomatis genital infections lead to severe immunopathological consequences in the upper genital tract (UGT), including pelvic inflammatory disease and infertility, in a small subset of infected women. Heat shock protein 70 (Hsp70) is an evolutionarily conserved stress-induced protein and has been shown to exhibit significant cytoprotective and immunoregulatory activities. However, the role of Hsp70 in genital chlamydial pathologies had not been examined. Hsp70 transgenic (Hsp70 Tg) mice were created by injecting a villin promoter-driven human Hsp70 targeting vector into C57BL/6 oocytes. The villin promoter is active only in the epithelium of the mouse oviduct but not in other parts of the female mouse genital tract. Female, 6-8 week old, Hsp70 Tg and non-transgenic (NTG) littermates were infected intravaginally with 5×104 IFU of C. muridarum. Vaginal chlamydial shedding, bacterial burden in the upper genital tract including oviducts and uterine horns, Chlamydia-specific IFN-γ, TNF-α, and IL-17 production, and serum anti-Chlamydia antibody levels were comparable between NTG and Hsp70 Tg mice. However, at day 80 after inoculation, the incidence and severity of pathology in oviducts, not neighboring uterine horns, was significantly reduced in Hsp70 Tg mice compared to NTG mice. Given the highly polymorphic nature of hsp-70 gene and associated susceptibility to various inflammatory conditions, and the occurrence of immunopathology in only a subset of Chlamydia-infected women, these results also underscore the need to further evaluate the role of hsp-70 in chlamydial immunopathogenesis in the female upper reproductive tract.
Novel Pycard gene polymorphism impairs Nlpr3 inflammasome-induced IL-1β production in mice selected for low inflammatory response.
Authors: Ibañez Olga M. , Starobinas Nancy , Monteleone Leticia Figueiredo , Borrego Andrea , Icimoto Marcelo Yudi , Cabrera Wafa Koury , Ribeiro Orlando Garcia , De Franco Marcelo , Jensen José Ricardo , Dragani Tommaso Antonio
Published: January 2023

A SNP based linkage study in mouse lines phenotypically selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory response (AIR), mapped a major locus, Inflammatory response modulator 1 (Irm1) at 128Mb (3.5Mb interval) in chr 7 controlling AIR, measured by leukocyte and IL-6 levels in exudates and IL-1β production by circulating leukocytes after Nlpr3 inflammasome activation. Sequencing of this region in mice with extreme high or low IL-1β levels revealed 14 SNPs between the two groups, narrowing the locus interval to 420Kb. Candidate genes at Irm1 include Pycard with an undescribed exon 3 (C/T) mutation leading to E19K substitution at the pyrin domain, and Itgam, Rgs10 and BC017158 with intronic SNPs. In Pycard, the C allele was fixed in high responder AIRmax mice whereas the C and T alleles frequencies were 39% and 61%, respectively in AIRmin. We then investigated the effect of this novel Pycard SNP in inflammation phenotypes.

Methods

AIRmin mice bearing the 3 genotypes at Pycard: CC, CT, TT were produced by genotype-assisted mating. Inflammatory response was measured in AIRmax and in the 3 AIRmin sublines by the number of infiltrating cells and IL-6 concentration in the 24h exudate induced by sc Biogel P-100 bead injection and ex vivo IL-1β production by circulating leukocytes after E coli LPS (1 ug) and ATP (5mM) activation.

Results

IL-1β levels were similar in AIRmaxCC (4.5-±0.4 ng/ml) and AIRminCC (3.4±2.4 ng/ml) whereas AIRminCT produced 0.3±0.5 and AIRminTT <0.05 ng/ml IL-1β. Leukocyte influx and IL-6 levels in inflammatory exudates were not affected.

Conclusion

The E19K substitution in Pycard causes a negative effect in inflammasome activation for IL-1β production, without interfering in other inflammation phenotypes.
Mitochondrial SLC25 proteins interact with NLRP3 to regulate inflammasome function
Authors: Shuvarikov Andrey A. , Davis Michael A. , Esser-Nobis Katharina , Gale Michael J.
Published: January 2023

The NLRP3 protein is a key initiator of inflammation in humans. NLRP3 becomes activated by a multitude of danger signals, including pathogens, markers of metabolic dysfunction, and internalized particulates. Upon activation, NLRP3 nucleates formation of a multiprotein complex called the inflammasome, in which caspase-1 activity mediates processing of the pro-inflammatory cytokines IL-1β and IL-18 and induces pyroptosis, a pro-inflammatory form of cell death that serves to release the mature cytokines along with other inflammatory mediators, thus initiating the inflammatory response. While multiple regulators of the NLRP3 inflammasome have been described, specific ligand(s) of NLRP3 and its mechanism of activation remain largely unknown. We performed a proteomics screen using co-immunoprecipitation (IP) and mass spectrometry to identify cellular proteins that bind NLRP3. Using this screen, we identified multiple NLRP3 interactors, including a family of solute carrier (SLC) proteins that localize to the inner mitochondrial membrane. Co-IP experiments verified that four of these proteins, namely SLC25A1, a citrate transporter, SLC25A6, an ADP/ATP translocase, SLC25A11, a 2-oxoglutarate/malate carrier, and SLC25A13, a calcium-dependent glutamate transporter, specifically interact with NLRP3. Because of its linkage with known metabolic disease, we further assessed the role of SCL25A13 in inflammasome function. When ectopically expressed, SLC25A13 enhanced IL-1β release from cells, while CRISPR/Cas9 knockout of SLC25A13 from THP-1 macrophages partially abrogated NLRP3-dependent pyroptosis. Our studies reveal a novel role for mitochondrial SLC25 proteins in regulation of the NLRP3 inflammasome.
Identification of age-related changes in gene expression and chromatin accessibility in T cells from thymus to periphery
Authors: achour achouak , Chen Guobing , Sharov Alexei , Nugyen Thomas , Peng Weiqun , Patrick Michael , Wood William lll , De Supriyo , Becker Kevin , Weng Nan-ping
Published: January 2023

Aging of immune system is characterized by progressive decline of physiological and cellular function of T cells. Recent studies of naïve T cells from young and old mice have identified age-related altered gene expressions but the mechanisms underlying age associated changes of naïve T cells remain poorly understood. Here we compared the transcriptome and chromatin accessibility in mature CD4 and CD8 thymocytes and in naïve CD4 and CD8 T cells of spleen between young (6–8 weeks) and old (95–110 weeks) C57Bl/6 mice. We used Agilent microarray method and identified the age-related altering gene expressions 1) only in mature CD4 and CD8 T cell from thymus, 2) shared by both thymus and spleen naïve T cells, and 3) only in naïve T cells from spleen. The shared changes include increased chemokine and chemokine receptor expressions and decreased cell cycle regulator expression. We further analyzed the chromatin basis of altered gene expression using ATAC seq between young and old mice. We have identified age-related changes of chromatin accessibility in T cells from thymus and spleen, and some correlated changes between gene expression and chromatin accessibility. Overall, we observed more age-related alterations of gene expression and chromatin accessibility in thymus than in spleen, suggesting some age-related changes of T cells in thymus did not retain in periphery. Together, our findings have identified tissue specific altered gene expressions and chromatin status in T cells during aging and further study of these altered genes will shed new insights into the mechanisms underlying these age-related changes.

This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging.
Kmt2d regulates naive CD8 T-cell development and activation-related survival
Authors: Kim Jaekwan , Ge Kai , Weng Nan-Ping
Published: January 2023

Lysine specific methyl-transferase 2D (Kmt2d) catalyzes the mono-methylation of histone 3 lysine 4 (H3K4me1) and play a critical role in enhancer-mediated gene expression regulation. However, its role in CD8 T cell development and differentiation has not been addressed. Here, we analyzed a T-cell specific KMT2D knockout mouse (KMT2Dfl/flCD4cre+) and found that the number of CD8 T cells in spleen and blood was greatly decreased compared to wild type (KMT2Dfl/flCD4cre-, WT) mice. The decrease in CD8 T cells is caused by defective production of T cells in the thymus based on the following observations. First, numbers of recent thymic emigrant (RTE) CD8 cells in blood and spleen of KMT2Dfl/flCD4cre+ mice were significantly less in KMT2Dfl/flCD4cre+RAG-GFP mice. Second, thymic development of T cells was compromised (CD4+/CD8+ population was less and immature CD8SP cells are increased in KMT2Dfl/flCD4cre+ mice). Third, naïve CD8 T cells (CD62Lhi/CD44lo) were more significantly reduced (WT:KO=7:1) than memory (CD62Llo/CD44hi, WT:KO=2:1) CD8 T cells. Furthermore, naïve CD8 T cells isolated from KMT2Dfl/flCD4cre+ mice with anti-CD3 and anti-CD28 in vitro showed increased apoptosis compared to that from WT mice. RNA-seq analysis of naïve CD8 T cells revealed increased expressions of genes related to apoptosis prior stimulation, suggesting Kmt2d regulates activation-induced apoptosis. Together, our findings reveal a role of Kmt2d in naïve CD8 T cell production and survival after activation.

This research was supported entirely by the Intramural Research Programs of the NIH, National Institute on Aging and National Institute of Diabetes, Digestive and Kidney Diseases.
Local and long-range transcriptional regulation of the Bcl6 locus by Ikaros zinc finger factors
Authors: Powell Michael Duane , Read Kaitlin A. , Sreekumar Bharath , Carrillo Gabriela L. , Ringel-Scaia Veronica , Bachus Holly , Allen Irving C. , Ballesteros-Tato Andre , Oestreich Kenneth J.
Published: January 2023

T follicular helper (TFH) cells play a pivotal role in the humoral immune response by providing help to B cells, resulting in germinal center formation and the subsequent generation of pathogen-neutralizing antibodies. The development of TFH cells requires the expression of the transcriptional repressor Bcl-6; however, the mechanisms underlying Bcl6 transcriptional regulation are incompletely understood. Here we identify the Ikaros Zinc Finger (IkZF) transcription factors Aiolos and Ikaros as novel regulators of Bcl6 expression. We demonstrate that the expression of Aiolos and Ikaros is upregulated in antigen-specific TFH cells generated in response to both Listeria monocytogenes and influenza infection. Mechanistically, we find that Aiolos and Ikaros directly associate with the Bcl6 promoter. This association corresponds with chromatin remodeling events consistent with gene activation including the recruitment of chromatin remodeling complexes, increased histone acetylation, and RNA polymerase II binding. Additionally, we find that Aiolos and Ikaros are enriched at a conserved noncoding sequence 273 kb (CNS273) upstream of the Bcl6 transcriptional start site. CNS273 exhibits a covalent histone modification profile indicative of an active enhancer and is capable of enhancing Bcl6 promoter activity in an Aiolos-dependent manner. Collectively, these data provide insight into the multifaceted mechanisms by which Aiolos and Ikaros regulate Bcl6 expression.
Bovine colostrum increases the viability and the healing process of IPEC-J2 intestinal porcine epithelial cells while modulating the barrier function and Escherichia coli-mediated inflammatory responses.
Authors: Bouchard Michaël , Blais Mylène , Deschenes Karine , Robitaille Gilles , Arcand Yves , Asselin Claude , Lessard Martin
Published: January 2023

Piglet gut response to weaning results in impaired intestinal barrier function, increased inflammatory response and susceptibility to enteric diseases. As the use of antibiotics as growth promoters will be restricted in the swine industry in Canada, novel feeding strategies to improve gut health are needed. This study aims to determine the potential of defatted bovine colostrum (BC) and its fractions, the serocolostrum (SC) and caseins (CAS), to promote viability and the healing process and to modulate barrier integrity and the inflammatory response induced by enterotoxigenic E. coli (ETEC) in IPEC-J2 intestinal epithelial porcine cells. To evaluate cell viability and wound healing, XTT cell proliferation and migration scratch assays were performed on IPEC-J2 cells incubated with BC, SC, or CAS. Monolayer permeability of ETEC infected IPEC-J2 cells incubated with BC, SC or CAS was monitored by transepithelial electrical resistance measurements. Inflammatory and oxidative responses were assessed by gene expression analysis of several cytokines and chemokines by qPCR. Incubation with BC derivatives led to 8 to 14% increases IPEC-J2 cell viability, in comparison to untreated cells (P ≤ 0.05). IPEC-J2 cell migration was increased by 42% after SC addition, as opposed to non-treated cells (P ≤ 0.05). BC and SC treatments significantly reduced ETEC-mediated increases in IL-8 and CCL20 expression (P ≤ 0.05). BC treatment also significantly reduced IPEC-J2 cell monolayer permeability induction after ETEC infection. These results show that BC and SC fractions improve intestinal epithelial cell integrity and decrease ETEC-mediated inflammatory responses. Therefore, BC should be considered as a potential alternative to antibiotics.
AlgR controls in vivo persistence of Pseudomonas aeruginosa
Authors: Penaranda Cristina , Hung Deborah
Published: January 2023

Pseudomonas aeruginosa (PsA) is one of the most virulent opportunistic human pathogens and is often highly resistant to antibiotic therapy. Although it is traditionally considered an extracellular pathogen, PsA has been shown to invade and persist inside mammalian cells. We hypothesize that this ability to persist intracellularly allows bacteria to become antibiotic insensitive, leading to the formation of reservoirs for recurrent infections. We have developed an in vitro infection model where we identified that the two-component response regulator AlgR is required for infection of human bladder epithelial cells. We demonstrate that AlgR controls invasion, via pilus expression, and intracellular persistence, via an unknown mechanism. Furthermore, we show that although its cognate histidine kinase, AlgZ, is also required, AlgR phosphorylation is not required for persistence. RNA profiling of host cells infected with WT PsA shows induction of hallmark TNFα signaling via NFKβ, which is absent in cells infected with ΔalgR PsA, suggesting that recognition of infecting bacteria may be necessary for their intracellular persistence. Finally, we show that AlgR is also required in an in vivo model of murine urinary tract infection, demonstrating that the in vitro model correlates with chronic infections in a whole animal model. Our results advance our understanding of the requirements for in vivo bacterial persistence and identify a potential target for the development of novel antibiotics.
Chlamydia-Specific CD8+ T cells Enhance Neutrophil Infiltration into Chlamydia-Infected Female Mouse Genital Tract
Authors: Li weidang , Ramsey Kyle H. , Murthy Ashlesh K.
Published: January 2023

We recently demonstrated that Chlamydia-specific TNF-α producing and TNF receptor 2 bearing CD8+ T cells mediate chlamydial pathogenesis in the mouse female upper genital tract (UGT). Additionally, TNF-α production from CD8+ T lymphocytes is necessary for pathogenesis; however, CD8+ T lymphocytes are not a sufficient source of TNF-α production for pathogenesis. Myeloid cells including neutrophils are important sources of TNF-α production and have been implicated in chlamydial pathogenesis. Herein, we evaluated frequency of neutrophils in Chlamydia muridarum-infected female genital tracts of C57BL/6J (WT) mice, mice deficient in Chlamydia-specific CD8+ T cells (OT-1 mice) and TNFR2 knockout (KO) mice. Frequencies of Gr-1+ cells (neutrophils) in UGT were evaluated on days 4, 9, 15, and 24 following genital Chlamydia muridarum infection. OT-1 and TNFR2 KO displayed significant reduction in neutrophil frequency compared to WT animals on day 9 following chlamydial inoculation. Furthermore, we found that OT-1 mice replete with WT CD8+ T cells at the time of chlamydial inoculation display restoration of neutrophil (Ly-6G) frequency to WT levels. These results suggest that Chlamydia-specific CD8+ T cells engage other inflammatory cells such as neutrophils in infected genital tracts in order to cause chronic pathologies in the upper reproductive tract.
Role of Interferon-γ in promoting disease severity in neonatal Bordetella pertussis infection
Authors: Mitchell Ashley E. , Scanlon Karen , Carbonetti Nicholas
Published: January 2023

Pertussis is an infectious disease caused by the bacterial pathogen Bordetella pertussis. In recent years, there has been a re-emergence of pertussis disease. Pertussis morbidity is extensive and spans across all age groups, but a higher incidence of severe disease and mortality is exhibited in young infants. Mortality is associated with severe respiratory infection and other systemic secondary co-morbidities. The infant immune system has reduced capacity to generate proinflammatory/T-helper (Th) 1 cell polarizing responses. Secretion of Interferon gamma (IFN-γ), a Th1 polarizing cytokine, by immune cells plays a critical role in macrophage activation and microbicidal activity in the lung. Furthermore, B. pertussis respiratory challenge of adult IFN-γ receptor deficient mice results in systemic dissemination and lethality. We hypothesize that the dissemination and lethality observed in our neonatal mouse model is a result of inadequate IFN-γ signaling or production, which contribute to insufficient control of the infection. We found that B. pertussis infection results in a significantly lower level of transcription of IFN-γ in the lungs and increased bacterial dissemination in neonatal mice when compared to adult mice. Additionally, infected neonatal mice deficient in IL-10, a potent regulator of IFN-γ, showed reduced dissemination and significantly increased IFN-γ and other pro-inflammatory cytokines as compared to age-matched wild type mice. Future studies include characterization of IFN-γ secreting immune cells during infection. Results generated by this work will contribute to a better understanding of severe disease in infants, addressing the limitations of infant immunity and treatment options for pertussis.
Dynamics and function of group 1 CD1-restricted T cells during disseminated Staphylococcus aureus infection
Authors: Visvabharathy Lavanya , Wang Chyung-Ru
Published: January 2023

Staphylococcus aureus (SA) is frequently associated with nosocomial and community-acquired infections. Some promising vaccine candidates act by priming conventional T cell responses, but less is known about the contribution of non-conventional T cells to SA infection. Group 1 CD1-restricted T cells are a unique subset of T cells that respond to lipid antigens presented by group 1 CD1 (CD1a, -b, –c) molecules. These molecules have low polymorphism in their antigen-binding grooves, making them attractive vaccine targets. Previous studies have shown that group 1 CD1-restricted T cells respond to Mycobacterium tuberculosis (Mtb) lipids. As group 1 CD1 molecules can accommodate a variety of lipid species, we questioned whether and how group 1 CD1-restricted T cells can be activated by SA lipids.

Using a transgenic mouse strain expressing human group 1 CD1 molecules (hCD1Tg), we found that group 1 CD1-restricted T cells recognize SA lipid during primary infection. hCD1Tg mice were also protected against SA kidney pathology compared with WT control mice at late times post-infection. Group 1 CD1-restricted T cells produced both IL-17 and IFN-γ in response to SA lipid. Fractionation of SA lipid into 11 discrete parts found that portions enriched in cardiolipins and phosphatidyl glycerol contained immunodominant group 1 CD1-restricted antigens. Finally, group 1 CD1-restricted SA lipid-specific T cells can be specifically detected via FACS to ultimately assay differences in gene expression between these and conventional SA Ag-specific T cells. These findings suggest a role for CD1-restricted, SA lipid-specific T cells in control of systemic SA infection, with strong implications for vaccine design.
Increasing Domain Coverage Improves Neutralizing Potency of C. difficile Toxin-specific Antibodies
Authors: Li Lu , Cole Leah E. , Jetley Utsav , Zhang Jinrong , Pacheco Kristl , Ma Fuqin , Zhang Jianxin , Mundle Sophia , Yan Yanhua , Barone Lucianna , Rogers Christopher , Beltraminelli Nicola , Quemeneur Laurence , Kleanthous Harry , Anderson Stephen F. , Anosova Natalie G.
Published: January 2023

Clostridiumdifficile (C.difficile)is a significant human pathogen. C.difficile infection (CDI) causesclinical symptoms ranging from diarrhea to life-threatening fulminant pseudo membranous colitis. The pathogenesis of C. difficile is mediated by two large exotoxins, toxins A and B. These two toxins are highly homologous, single chain proteins consisting of four functional domains: N-terminal glucosyl transferase domain (GTD), cysteine protease domain (CPD), translocation domain (TLD) and a C-terminal receptor-binding domain (RBD). The important role played by anti-toxin sera and antibodies in the prevention of primary and recurrent CDI has been described and demonstrated in both clinical and pre-clinical studies; however, work focused on the impact of the toxin domain specificity of these antibodies is limited. To address this deficit, sera from a C. difficile vaccine immunized hamsters and toxin-specific human monoclonal antibodies (mAbs) were used to assess the impact of toxin domain specificity on antibody mediated inhibition of cytotoxicity in a Vero cell-based functional assay. Results from toxin domain immunoabsorption assays using hamster anti-toxinsera indicated that no single domain fragment from either toxin A or toxin B could inhibit neutralizing activities. Anti-toxin A activity was prevented with a combination of GTD and CTD fragments while anti-toxin B activity required the GTD, CTD and CPD fragments to block activity. Assays with human mAbs demonstrated that combining mAbs that target multiple toxin domains greatly improves neutralizing potency when compared to equivalent concentrations of either a single mAb or a combination of mAbs against a single domain.
A novel function of ADP-ribosyl cyclases to regulate the length of the small intestine revealed in CD38/CD157 double knockout mice
Authors: ISHIHARA Katsuhiko , YAHAGI Ayano , ISEKI Masanori
Published: January 2023

Mammalian ADP-ribosyl cyclase includes two ectoenzymes; GPI-anchored, Bone marrow stromal cell antigen-1 (BST-1)/CD157 and a type II membrane protein, CD38. Although they are expressed in various tissues including the lung, kidney, intestine, brain, spleen and bone marrow, the enzymatic activity of CD38 is dominant at physiological pH. We generated and analyzed CD157 deficient mice (CD157KO), and reported that CD157 is a positive regulator of humoral immunity in 129 x C57BL/6J (B6) mixed background. Recently, various groups reported novel functions of murine CD157 in intestinal regeneration and depressive or anxious behaviors. Thus, CD157 is now appreciated as an entero-neuro-immune regulator. The phenotypes of CD38KO also revealed the multiple functions of CD38 in immunity, metabolism, social behaviors, as well as in the kidney and lung.

To further clarify the function of ADP-ribosyl cyclase family, we generated CD38/CD157 doubly deficient mice (CD38/157DKO) by sequential targeting at ES cell level, and compared with CD38KO, CD157KO and wild type in B6 background. A novel phenotype unique to B6.CD157KO was enhanced humoral immune responses to TNP-LPS, indicating the suppressive role of CD157 in TLR signal on marginal zone B cell. Although CD38/157DKO developed apparently normal with no histological changes in the major tissues by HE staining, the length of the small intestine increased by approximately 20% at 4 months old. Among the lymphoid organs, the cell numbers in the mesenteric lymph node increased by 1.5 times without changes in the lineage or subset, probably reflecting elongation of the small intestine.

These data suggest that ADP-ribosyl cyclases, CD38 and CD157, have roles in regulating functions of the small intestine.
Differences in psychosocial behavior, salivary IgA, and oral microbiota in student-athletes and sedentary college students.
Authors: Costan Sophia , Appasamy Pierette M. , Edenborn Sherie , Ubinger Elizabeth , Carrillo Andres
Published: January 2023

Identification of behaviors and physiology that positively impact psychosocial and physical well-being in college students is very important. Exercise and social connectedness have been shown to influence psychosocial well-being, immunologic function, and the microbiome. Our previous studies demonstrated reduced anxiety, enhanced oral immunity and an altered oral microbiota in student athletes during the “in-season” period (Lamb et al, 2017). In our present studies, we found that the the composition of the oral microbiota in female NCAA Division III ice hockey players was different in athletes in the “off-season” compared to “in-season.” Specifically, there was a greater proportion of streptococcal strains in the “off-season” athletes, whereas behavioral measures, including anxiety and perceived stress, and salivary cortisol levels, were similar. Salivary IgA levels were different between the two groups. In order to examine whether participation on an athletic team alone is beneficial to students with respect to behavioral and immunologic measures, our most recent study compared female student athletes (NCAA Division III soccer players), “in-season”, with female college students who were not athletes. As expected, student athletes were more “fit”. We found that the degree of perceived stress and depression was higher in the non-athletes, although both groups reported similar degrees of social connectedness. Non-athletes reported a higher rate of illness compared to the athletes. Saliva samples were collected and are being evaluated for microbial composition, IgA and cortisol levels, in order to further define relationships between psychosocial and physical well-being, the microbiome and the immune response.