bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-2 producing T Follicular Helper cells self restricts entry of T follicular helper cells in the germinal centers
Authors: Dave Shivangi
Published: August 2023

Upon infection or immunization, dendritic cells (DCs) present antigens (Ags) to naïve CD4+ T cells. As a result, a fraction of the responding CD4+ T cells upregulate Bcl6 and differentiate into T follicular helper (Tfh) cells, which migrate into the B cell follicles. Once in the B cell follicles, Tfh cells help promote the development and maintenance of the germinal centers (GC). Importantly, limiting the number of Tfh cells is critical for maintaining the selective pressure in the GC, thereby allowing the Darwinian selection that leads to affinity maturation. However, the mechanisms that regulate Tfh cell numbers and prevent excessive Tfh help remains unknown. Using an influenza virus infection model, we show here that Tfh cell differentiation is limited to the first 72h of the infection. After this time, whereas DCs continue to present viral antigens and efficiently prime naïve T cells, responding CD4+ T cells fail to upregulate Bcl6 and preferentially differentiate into effector Th1 cells. Mechanistically, we demonstrate that the lack of Tfh cell differentiation after day three was due to the presence of the early-primed Tfh cells. Our results indicate that early-primed Tfh cells produce large amounts of IL-2 in the proximity of the B cell follicles, thereby limiting the differentiation of new Tfh cells after day three. Collectively, our data demonstrate that early-primed Tfh cells act as a self-regulating factor that shields the B cell follicles early after infection, thereby preventing the continuous differentiation of Tfh cells regardless of the presence of viral antigens. These data demonstrate a new self-competitive mechanism that critically contributes to limiting Tfh cell expansion after infection.

This work was supported by The University of Alabama at Birmingham (UAB) and the National Institutes of Health grant 1R01AI162698-01A1 to A.B.-T
Opposing functions of circadian protein DBP and atypical E2F family E2F8 in anti-tumor Th9 cell differentiation
Authors: Lim Yun-Ji , Park Sang-A , Ku Wai Lim , Zhang Dunfang , Cui Kairong , Tang Liu-Ya , Zanvit Peter , Chen Zuojia , Jin Wenwen , Xu Junji , Wu Chuan , Zhang Ying E , Zhao Keji , Chen WanJun
Published: August 2023

Interleukin-9 (IL-9)-producing CD4+ T helper cells (Th9) have been implicated in allergy/asthma and anti-tumor immunity, yet molecular insights on their differentiation from activated T cells, driven by IL-4 and transforming growth factor-beta (TGF-β), is still lacking. Here we show opposing functions of two transcription factors, D-binding protein (DBP) and E2F8, in controlling Th9 differentiation. Specifically, TGF-β and IL-4 signaling induces phosphorylation of the serine 213 site in the linker region of the Smad3 (pSmad3L-Ser213) via phosphorylated p38, which is necessary and sufficient for Il9 gene transcription. We identify DBP and E2F8 as an activator and repressor, respectively, for Il9 transcription by pSmad3L-Ser213. Notably, Th9 cells with siRNA-mediated knockdown for Dbp or E2f8 promote and suppress tumor growth, respectively, in mouse tumor models. Importantly, DBP and E2F8 also exhibit opposing functions in regulating human TH9 differentiation in vitro. Thus, our data uncover a molecular mechanism of Smad3 linker region-mediated, opposing functions of DBP and E2F8 in Th9 differentiation.

Open Access funding provided by the National Institutes of Health (NIH).
A method for in vitro polarization of pathogenic ST2-expressing memory Th2 cells
Authors: Liu Caini , Xiao Jianxin , Qian Wen
Published: August 2023

IL-33 and its receptor subunit ST2 (IL1RL1) have been linked to type 2 eosinophilic asthma in multiple human and mouse studies. In particular, recent clinical trials demonstrate that monoclonal antibodies targeting IL-33/ST2 are beneficial to subsets of asthma patients. ST2+ memory Th2 cells (mTh2) are important effector cells in IL-33-mediated type 2 eosinophilic asthma. Herein we describe a unique method for polarizing a subset of pathogenic ST2+ mTh2 (ST2+ mTh2p) cells from naïve mouse CD4 T cells in vitro, which does not require multi-round activation/resting or co-culturing with antigen-presenting cells as reported in prior studies. The ST2+ mTh2p cells proliferate and produce large amounts of type 2 cytokines upon IL-33 treatment [without involving T-cell receptor (TCR) ligation]. IL-33 stimulation induces metabolic reprogramming of ST2+ mTh2p, as indicated by the increased mitochondrial respiration and glycolytic rate in Seahorse assays as well as upregulated key metabolites and metabolic genes involved in these two bioenergetic pathways. Interestingly, IL-33 stimulation also increases the metabolites and the expression of key enzymes for the production of polyamines (the important regulators of cell proliferation). Moreover, ST2+ mTh2p cells are highly pathogenic, as they are capable of propagating and producing type 2 cytokines in the lung, resulting in strong eosinophilic inflammation induced by intranasal IL-33 administration alone after adoptive transfer to Rag2−/−IL2r−/− mice (lacking both innate and adaptive lymphocytes). In summary, the current study provides useful tools to investigate IL-33/ST2-regulated molecular mechanisms and immunological functions of ST2+ mTh2p cells both in vitro and in vivo.

R0 1HL144497
Gut microbiome and metabolites relate diabetes risk among Native Hawaiians and Pacific Islanders
Authors: Peres-David Rafael Aniu , Kunihiro Braden P , Wells Riley K , Phankitnirundorn Krit Aniu , Umeda Lesley P , Lee Rosa Aniu , Juarez Ruben , Maunakea Alika K
Published: August 2023

Native Hawaiians and Pacific Islanders (NHPIs) suffer higher rates of type 2 diabetes mellitus (T2DM) prevalence and mortality compared to other racial-ethnic groups in Hawaii. Gut microbiome is recognized as a partial determinant of T2DM progression, the role of its associated metabolome is not well understood in the context of immunometabolic health disparity. We employed community-based research strategies among Oahu, Hawaii residents to form a cohort enriched with NHPIs (N=98). Gut microbiome profiling was achieved via 16s rDNA metagenomic sequencing from stool. Plasma inflammation factors were measured using magnetic bead-based multiplex assays. Stool metabolites were identified using mass spectrometry. T2D individuals had reduced relative abundance of gut bacteria belonging to Bacteroidetes (P<0.05). Similar reduction was observed for known butyrate-producers, including Roseburia sp. (P<0.05), Faecalibacterium sp.(P=0.01), and Eubacterium sp (P=0.03). Metabolomic data revealed that phenylbutyric acid (PBA), a short-chain fatty acid (SCFA) derivative of butyrate, was differentially present between diabetics and control (P=0.001). Furthermore, PBA concentrations correlated negatively with A1c (%; R=−0.34, P<0.01), CRP(ng/mL; R=−0.32, P<0.01), and positively with Adiponectin (ng/mL; R=0.38, P<0.01) and IL-10 (ng/mL; R=0.47, P<0.01). Higher stool levels of PBA were associated with lower levels of IL-1 (P=0.03), MCP-1 (P=0.01), and IFN-g (P=0.02). Further elucidation of biological mechanisms underlying gut bacterial modulation of systemic inflammation through SCFA metabolites may allow for future development of mitigative strategies for T2DM risk in such health-disparate communities as NHPIs.

This project was supported in part by the National Institutes of Health (NIH), National Institute on Minority Health and Health Disparities (NIMHD) under grant numbers R56MD014630, U54MD007601-35, and R01MD016593 and by the National Institute of General Medical Sciences (NIGMS) under the IDeA Networks of Biomedical Research Excellence (INBRE) program (P20GM103466).
IL21 Receptor-Deficient Regulatory T Cells Promote Resolution of Intestinal Inflammation by Resisting Metabolic Disturbance
Authors: Bamidele Adebowale , Klatt Emily , Hirsova Petra , Mishra Shravan , Fehrenbach Patrick , Sagstetter Mary , Valenzuela-Perez Lucia , Lee Hyun Se Kim , Faubion William
Published: August 2023

Background

Inflammatory regulatory T cells (Tregs) are one of the hallmarks of therapy resistance. Yet mechanisms leading to inflammatory Treg development and its contribution to inflammatory bowel disease (IBD) are unexplored.

Methods

Tregs derived from healthy PBMCS were characterized by immune and mitochondrial phenotyping. Transcripts were validated in dataset from IBD intestinal lesions. Wildtype (WT) and IL21 receptor-deleted (Il21r−/−) Tregs were examined in murine models of CD4+ T cell-induced colitis.

Results

Through in vitro cytokine challenge of Tregs and subsequent analysis, we found that IL21 induces Tregs co-expressing TNF, IFNγ and IL17. Transcriptomic and metabolomic integration revealed upregulation of mRNA and metabolites associated with glycolysis, and amino acid, nucleotide and lipid metabolism. Moreover, IL21-induced metabolic transcripts were more enriched in Treg cluster within scRNA-seq dataset from intestinal lesions of anti-TNF non-responders vs. non-responders. Mechanistically, cell imaging and real-time bioenergetic analysis revealed disruption to mitochondria-ER ultrastructure and function, coinciding with glycogen synthase kinase 3β (GSK3β) activation and glycolytic switch rather than pyruvate-mediated oxidative phosphorylation. GSK3β inhibition or methyl pyruvate supplementation rescued IL21-induced metabolic and inflammatory phenotypes. In contrast to WT Tregs, murine Il21r−/− Treg transfer efficiently prevented and rescued CD4+CD45Rbhigh T cell-induced colitis.

Conclusions

IL21-induced metabolic dysfunction can incapacitate Treg immunosuppressive function. Desensitizing Tregs to IL21 through various therapeutic approaches can alleviate IBD.

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) K01DK124358, the Pilot & Feasibility Award by the Center for Cell Signaling in Gastroenterology (P30DK084567), and the Mayo Clinic Center for Biomedical Discovery Career Development Award, and David F. and Margaret T. Grohne Cancer Immunology and Immunotherapy (to AOB).
Targeting Multiple Costimulatory Molecules Prevents Persistence of Lung Tissue-resident Memory T cells and Leads to Airway Tolerance
Authors: Sethi Gurupreet Singh , Croft Michael
Published: August 2023

Pools of tissue-resident memory T (Trm) cells are thought to maintain and contribute to exacerbations of inflammatory or autoimmune disease. We hypothesized that several costimulatory molecules would control the expansion and maintenance of these populations when responding to antigen, and tested the potential of targeting OX40L, CD30L, and ICOSL, using a house dust mite (HDM) allergen-based mouse model of asthmatic lung inflammation. We found that allergen induced persisting CD4+CD44hiCD62Llo CD69+ and CD69− lung-localized Trm cells and RNA-seq revealed the heterogeneity of these memory pools, containing Th2, Tfh, Th17, and Th1 clusters. Showing their reliance on costimulation for recall activity to allergen, blocking CD30L and OX40L together, or blocking ICOSL alone, resulted in ~50% reduction in the expansion of both CD69+ and CD69− tissue-resident effector T cells during a secondary allergen exposure or a tertiary allergen exposure. Blocking all three molecules at once further inhibited T cell accumulation by ~90%, corresponding to a similar reduction in lung tissue inflammation, including eosinophilia. Moreover, triple costimulation blockade resulted in a diminished persisting lung tissue-resident memory T cell pool, which failed to expand to normal levels and drive further exacerbations of lung inflammation upon subsequent exposure to allergen, resulting in a state of airway tolerance. Overall, our data suggest that the concurrent inhibition of OX40, CD30, and ICOS signaling, can impair the expansion and persistence of lung-localized Trm cells and could engender long-lasting tolerance in asthma and similar autoimmune or inflammatory diseases that are maintained by tissue-resident memory T cells.
Deletion of protein kinase D1 in myeloid lineage cells suppresses development of IFNγ+IL-17A+ CD4 T cells and ameliorates hypersensitivity pneumonitis
Authors: Yi Ae-Kyung , Snyder John D , Yoon Tae Won , Fitzpatrick Elizabeth A
Published: August 2023

Hypersensitivity pneumonitis (HP) is an extrinsic allergic alveolitis characterized by inflammation of the interstitium, bronchioles, and alveoli of the lung that leads to granuloma formation and ultimately irreversible fibrosis. Previous studies found that exposure to HP inciting antigen S. rectivirgula (SR) leads to the activation of protein kinase D1 (PKD1) in the mouse lung in vivo and that PKD1 contributes to the acute pulmonary inflammation, IL-17A expression in the lungs, and development of HP caused by repeated exposure to SR. In the present study, we investigated whether PKD1 in myeloid cells affects the pathogenic course of the SR-induced HP. Compared to control PKD1-sufficient mice, mice with deficiency of PKD1 in myeloid cells (PKD1mKO) showed significantly suppressed expression of IL-6, TNFα, IFNγ, CCL2, CCL3, CCL4, CXCL1, CXCL2, and CXCL10 and neutrophilic alveolitis after single intranasal exposure to SR. Substantially reduced levels of alveolitis and granuloma formation were observed in the PKD1mKO mice repeatedly exposed to SR. Surface expression of MHC-II and CD86 on myeloid cells, and expression of IFNγ, IL-17A, CCL20, CXCL9, and CXCL10 in lungs were significantly reduced in the PKD1mKO mice repeatedly exposed to SR. In addition, surface expression of CD69, CXCR3, and CCR6 on CD4 T cells and frequency of IFNγ+IL-17A+ CD4 T cells in the lungs were significantly reduced in PKD1mKO mice repeatedly exposed to SR. Our results demonstrate PKD1 in myeloid lineage cells plays an essential role in the initial proinflammatory responses and neutrophil influx in the lung after exposed to SR and contributes to the development of IFNγ+IL-17A+ CD4 T cells and HP caused by repeated exposure to SR.

Supported by grants from NIH (R01 AR064723, R01 AR069010) and UTHSC.
The Effects of Fever on Macrophage Metabolism and Function in Young and Aged Cells
Authors: Taylor Kenneth H , Hassan Intisar , Durdik Jeannine M
Published: August 2023

Fever is an adaptive immune response to infections. With aging comes a rise in background inflammation that does not protect against infection. Here, we investigate the metabolism and function of macrophages at fever temperatures in a mouse cell line, in young and aged peritoneal resident macrophages, and bone marrow derived macrophages. Fever temperatures generally enhance macrophage functions although the effects are complex and dynamic. We show in the RAW264.7 murine cell line, that a raise in the baseline body temperature of 37°C to a fever temperature of 39°C increased the speed in which macrophages shift from mitochondrial oxidative phosphorylation to glycolysis. Driving this metabolic reprogramming, is the production of nitric oxide. There is also an increase in global protein synthesis and an increase in the mRNA for iNOS, the enzyme that produces NO and inflammatory cytokine production. The metabolic shift was measured via the oxygen consumption and the proton efflux rate over time. Cells were stimulated with lipopolysaccharide and/or interferon-gamma (IFNg).

Our data also shows that tissue resident PRMs from aged mice exhibit a much stronger response to fever than those of young mice, while bone marrow derived microphages (BMDM) appeared to show a similar response to that of RAW264.7 cell line. However, with the addition of a cyclooxygenase2 inhibitor to block the production of prostaglandin E2, BMDMs of aged mice show an exaggerated increase in their response to LPS+IFNg activation. This work uncovers differences in fever responses between young and aged individuals that may explain the overall outcomes of the aged to infection.

Arkansas Immunology Foundation
Role of TLR In The Cell Fate Decision of Fibroblastic Reticular Cells
Authors: Jayakumar Preethi , Jiang Ting , Hu Jiannan , Huang Hai , Deng Meihong
Published: August 2023

During sepsis, massive PAMPs and DAMPs are released and initiate the TLR signaling to mediate immune response and cell fate decision in cells. We have shown that Fibroblastic Reticular Cells (FRC)-based therapy improves outcome in a mouse polymicrobial sepsis model. Modulating TLR4 and TLR9 signaling in FRC regulates the efficacy of FRC-based therapy in sepsis. However, whether TLR signaling regulates FRC cell fate decision and thus affects the efficacy of FRC therapy is unknown. Here we aim to understand the role of TLR9 and TLR4 in FRC cell fate decision. FRCs were treated with or without LPS/ODN1585 for 6, 12, and 24h. To test the role of TLR4/9 in FRC proliferation, Ki67 expression was assessed using quantitative PCR and flow cytometry. To test the role of TLR4/9 in FRC cell death, the expression levels of protein related to apoptosis and necroptosis were assessed using flow cytometry and western blot. Treatment of ODN1585 decreased the expression levels of Ki67, indicating that TLR9 signaling suppressed FRC proliferation. While the expression levels of Ki67 remain at similar levels in the LPS-treated FRC as the control. Furthermore, treatment of ODN1585 drove FRC towards apoptosis cell death, evidenced by increased levels of activated caspase-3 and caspase-8. Interestingly, treatment of LPS increased the expression levels of p-RIPK1 and MLKL, suggesting that TLR4 signaling drives FRC towards necroptotic cell death. Our result indicated that TLR4 and TLR9 signaling are important for the cell fate decision of FRC. Understanding the mechanism of TLR4/9 in controlling FRC cell fate decision may help with designing new therapeutic strategies to improve the efficacy of FRC-based therapy for sepsis.

R01 AI152044
Mutating Amino Acids to Analyze the Effects on NADase Activity in Uropathogenic Escherichia coli and Acinetobacter baumannii TIR proteins
Authors: Vallejo-Schmidt Tamara E , Obiorah Trinity E , Kudjra Rachida E , Snyder Greg , Snyder Michelle E
Published: August 2023

Toll/interleukin-1 receptor/resistance protein (TIR) domains are crucial components in the innate immune system. In mammalian cells, TIR domains are found in Toll-like receptors (TLRs) which activate innate immune responses upon recognition of pathogen-associated molecular patterns and danger-associated molecular patterns. In contrast, TIR domains in prokaryotic cells may be linked to bacterial evasion of the innate immune system. To evade immune system defenses, Uropathogenic E. coli utilize TIR domains, like TcpC, to inhibit TLR signaling pathways. TcpC also has NADase activity which could have a role in immune evasion; we are interested in identifying amino acids important for TcpC’s NADase activity. Previously, we were able to determine that a conserved amino acid, glutamate, is important for NADase activity and that a conserved aspartic acid was crucial for NADase functionality for the additional bacterial TIR protein found in bacteria Acenitobacter baumanii (AbTIR). We hypothesized that the conserved aspartic acid may play a role in TcpC NADase activity. To support our hypothesis, we created an aspartic acid (D) to alanine (A) mutation using site-directed mutagenesis, tested to see if the mutation in TcpC affected NADase activity, and compared the effect of the TcpC DA mutation with the DA mutation from AbTIR. Results show that the DA mutants lost NADase activity, suggesting that the conserved aspartic acid is important for NADase activity in both TcpC and AbTIR. By determining which amino acids are critical for NADase activity, we can use this information for drug development for diseases that might be associated with TIR activity and innate immune evasion.

Funded by NIH R25Act GM058264
Differential regulation of natural killer cells by tumor necrosis factor receptors 1 and 2
Authors: McCulloch Timothy R , Rossi Gustavo Rodrigues , Lam Allie , Wong Joshua , Kane Lawrence P , Herold Marco , Wells Timothy , Souza-Fonseca-Guimaraes Fernando
Published: August 2023

Tumor necrosis factor (TNF) is a key cytokine across homeostasis, health, and disease. However, the role of TNF is often dichotomous in that it is critical for protective anti-microbial and anti-tumour responses, yet it is also a major player in immune dysfunction and immune-related pathology. Surprisingly, while the effects of TNF on myeloid responses are well studied, how TNF impacts lymphocyte responses is yet to be elucidated. This is particularly the case for natural killer (NK) cells, innate lymphoid cells which play a key role in immune protection against both cancer and infection. To address this knowledge gap, we have generated novel transgenic mouse models which lack the receptors for TNF (TNFR1 and/or TNFR2) specifically on NK cells, allowing us to precisely dissect how TNF directly regulates NK cell function and survival, along with the relative importance of each receptor. Using single cell RNA sequencing among other techniques, we have discovered TNF regulates expression of co-inhibitory receptors on NK cells, including Tim3 and TIGIT, through both TNFR1 and TNFR2. In models of infection and cancer we have found that TNFR1 plays an inhibitory role through depletion of NK cells, leading to increased disease burden. Conversely, TNFR2 plays a protective role through maintenance of optimal NK function and cytokine production, promoting disease control. This ongoing work establishes the importance of TNF in the regulation of NK cell function, which could have significant clinical implications.
Myeloid PTEN inhibits innate host defense during methicillin-resistant Staphylococcus aureus-induced sepsis
Authors: Faneuff Eden , Shili Cedrick , Kim Min Joo , Salina Ana , Penteado Leticia , Blackman Amondrea , Serezani C Henrique
Published: August 2023

Sepsis is defined as life-threatening organ dysfunction and damage caused by an aberrant host immune response to systemic infection. Due to the abundant production of different classes of inflammatory mediators, investigating the role of endogenous proteins that influence multiple signaling pathways might provide important clues in understanding sepsis pathogenesis. Phosphatase and tensin homolog (PTEN) is a protein/lipid phosphatase, tumor suppressor, and a negative regulator of the PI3K/AKT/mTORC pathway. PTEN inhibits FcR-mediated phagocytosis, bacterial killing, and the expression and actions of the Toll-IL1R (TIR) adaptor MyD88. Whether PTEN influences the outcome of Methicillin-resistant Staphylococcus aureus (MRSA) infection remains to be determined. Given the pleiotropic effects of this phosphatase on both antimicrobial response and TLR activation, we hypothesize that PTEN is a key signaling intermediate that perturbs cellular function and increases organ damage in sepsis. Our data showed that MRSA infection increased PTEN expression in the murine kidney and PTEN deletion in myeloid cells (PTENΔmyel) decreases MRSA loads and kidney abscess area compared to PTENfl/fl mice. PTENΔmyel leads to increased neutrophil but not monocyte/macrophage migration to the kidney. Furthermore, infected PTENΔmyel show reduced IL-1β and TNF-α in the kidney. In vitro data demonstrate that PTEN−/− macrophages have increased phagocytosis and reactive oxygen species production. These data indicate that during MRSA-induced sepsis, PTEN expression in myeloid cells increases the inflammatory response while decreasing antimicrobial effector functions in the kidney.

NIH R01 HL124159-09 NIH T32AI138932-03
Spleen tyrosine kinase inhibition restores neutrophil homeostasis in COVID-19
Authors: Wigerblad Gustaf , Warner Seth , Ramos-Benitez Marcos , Kardava Lela , Tian Xin , Mao Rui , Reger Robert , Chakraborty Mala , Wong Susan , Kanthi Yoghandra , Suffredini Anthony , Dell’Orso Stefania , Brooks Stephen , King Christopher , Shlobin Oksana , Nathan Steven , Cohen Jonathan , Moir Susan , Childs Richard , Kaplan Mariana J , Chertow Daniel , Strich Jeff
Published: August 2023

Spleen tyrosine kinase (SYK) is a novel therapeutic target that inhibits neutrophil and macrophage activation in coronavirus disease 2019 (COVID-19). Fostamatinib, a SYK inhibitor, was studied in a phase 2 placebo-controlled randomized clinical trial and was associated with improvements in many secondary end points related to efficacy. Here, we used a multiomic approach to evaluate cellular and soluble immune mediator responses of patients enrolled in this trial. We demonstrated that SYK inhibition was associated with reduced neutrophil activation, increased circulation of mature neutrophils (CD10+CD33−), and decreased circulation of low-density granulocytes (LDG) and polymorphonuclear myeloid-derived suppressor cells (HLA-DR–CD33+CD11b−). Additionally, LDG transcriptional profiles were distinct in COVID-19 compared to during autoimmune conditions. Together, these data suggest that SYK inhibition may mitigate proinflammatory neutrophil cellular and soluble mediator responses thought to contribute to immunopathogenesis of severe COVID-19.

This work has been funded in part by the Intramural Research Program of the NIH Clinical Center (NIHCC) including a Research Award for Staff Clinicians (FY2020/FY2021), NHLBI, National Institute of Allergy and Infectious Diseases (NIAID), and National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAIMS)
IL-4 RECEPTOR SIGNALING REGULATES LUNG MACROPHAGES DURING HELMINTH COINFECTION RESULTING IN ENHANCED GAMMAHERPESVIRUS PERMISSIVENESS
Authors: Petrellis Gerogios , Rolot Marion , Preure Amira , Wathieu Caroline , Dougal Annette , Chetty Alisha , Chariot Alain , Horsnell William , Dewals Benjamin G
Published: August 2023

Helminth infection conditions lung macrophages in the long term, but little is known about how helminths affect lung macrophage responses to respiratory viral coinfection. Experimental helminth infection of BALB/c and C57BL/6 mice revealed an increased type 2 airway inflammation in C57BL/6 mice that was associated with pronounced phenotypic changes in lung macrophages. These changes consist of a disappearance reaction of SiglecF+ alveolar macrophages (AlvMs) and a concomitant recruitment of monocyte-derived macrophages. Monocyte-derived macrophages replenish macrophages in the airways and lung, as shown with Ms4a3TdT reporter mice, and have a distinct profile to tissue resident macrophages. Competent IL-4Rα responsiveness or intra-tracheal instillation of recombinant IL-4 or IL-13 reproduced the contraction of AlvMs and recruitment of monocyte-derived macrophages, while anti-IL13 antibody treatment impaired the phenotypic changes post helminth infection. Helminth infection of C57BL/6 mice resulted in enhanced permissiveness to subsequent infection with murid gammaherpesvirus 4 (MuHV-4) during the acute stage of the infection in vivo with viral early tropism mainly restricted to AlvMs. AlvMs isolated from helminth-infected C57BL/6 mice appear more permissive ex vivo to MuHV-4. This enhanced permissiveness of AlvMs after helminth infection was dependent on direct IL-4Rα signalling. Thus macrophages-specific IL-4Ra signalling is required for the observed macrophage phenotypic changes and renders AlvMs more permissive to gammaherpesvirus infection.

Supported by FNRS-ASP
Vδ1+ and Vδ2+ gamma delta T cell subsets exert distinct functional profiles with aviremic HIV infection and aging
Authors: Smith Erika L , Belkina Anna C , Snyder-Cappione Jennifer
Published: August 2023

Populations of gamma delta T cells exert different effector function profiles (inflammatory (Th1/Th17) vs. immunoregulatory (Th2/Treg)) in response to non-antigen specific signals in a microenvironment. The nature of gamma delta T cell lineage commitment versus functional capacity at the single cell level is unclear. Also, gamma delta T cells are implicated as drivers of the chronic inflammation found with normal aging and ART-suppressed HIV infection; however, the precise subsets involved in this phenomenon are currently unknown. To address these questions, we optimized a 31-color Intracellular Cytokine Staining (ICS) panel enabling measurement of 10 cytokines, as well as Ki67, granzyme B, and CD107a for use with PBMC from participants of a HIV and Aging cohort. Multi-variate analysis (opt-SNE) of the collected dataset revealed striking differences in the overall functional potential of the V delta 1 (Vδ1) and V delta 2 (Vδ2) subsets, with many Vδ1+ cells not producing any of the cytokines measured. Distinct differences of both the Vδ1+ and Vδ2+ subsets were found between the HIV infected and age-stratified subject groups. Individual gamma delta T cells with a ‘Th0’ phenotype, producing both Th1 and Th2 lineage cytokines, were present in both Vδ1+ and Vδ2+ subsets. Also, HIV+ subjects lacked Vδ2+ cells expressing CD107a, indicating a specific defect in the cytotoxic function of this subset with HIV infection. Further investigation of gamma delta T cell functions and phenotypes could provide new insight into the biology of this unique T cell population and reveal novel therapeutic targets.

Supported by a grant from NIH (R01AG060890-01)
C-type lectin receptor dysregulation in HIV and Mycobacterium tuberculosis co-infection
Authors: Browning Sarah , Paez Reina , Endsley Mark A , Naqvi Kubra , Huante Matthew B , Chauhan Sadhana , Gelman Benjamin , Endsley Janice J
Published: August 2023

Infection with Mycobacterium tuberculosis (Mtb) is a serious and potentially life-threatening condition, especially in people with HIV (PWH). According to the WHO, there were 10.6 million new tuberculosis (TB) cases in 2021, and 700,000 of those cases were among PWH. Macrophages are a host cell for both pathogens and play an important innate immune role in determining the outcome of disease. C-type lectin receptors (CLR) are pattern recognition receptors (PRR) abundantly expressed on the macrophage surface that orchestrate the innate immune response to microbial insults. We previously identified a role for the macrophage galactose-type lectin receptor (MGL) CLR in protection against TB. In subsequent studies, we observed suppression of MGL in tissues of HIV-infected decedents and in human macrophages following in vitro infection with HIV. In human THP-1 cells and a humanized mouse model, we observe that HIV infection markedly impairs activation of MGL by mycobacteria in comparison to other CLRs with defined roles in antimycobacterial immunity including DC-SIGN and Dectin 1. We further determine roles for TLR2/6 heterodimer, TLR4, and TGF-β signaling pathways for transcriptional regulation of MGL. Importantly, these pathways have roles in innate immunity and pathogenesis following Mtb or HIV infections. These findings advance our understanding of MGL regulation as part of the CLR repertoire, as well as its role in the response to Mtb and to Mtb/HIV co-infection.

NIH R31AI138328, R61AI138328
Monocyte-derived IL-6 Programs Microglia to Rebuild Damaged Brain Vasculature
Authors: Choi Bo-Ran
Published: August 2023

Cerebrovascular injury (CVI) is a common pathology caused by infections, injury, stroke, neurodegeneration, and autoimmune disease. Rapid resolution of a CVI requires a coordinated innate immune response. In this study, we sought mechanistic insights into how CNS infiltrating monocytes program resident microglia to mediate angiogenesis and cerebrovascular repair following intracerebral hemorrhage. In the penumbrae of human stroke brain lesions, we identified a subpopulation of microglia that express VEGFA. These cells, termed repair associated microglia (RAM), were also observed in a rodent model of CVI and co-expressed IL-6Ra. Cerebrovascular repair did not occur in IL-6 knockouts or in mice lacking microglial IL-6Ra expression, and single cell transcriptomic analyses revealed faulty RAM programming in the absence of IL-6 signaling. Infiltrating CCR2+ monocytes were the primary source of IL-6 following CVI and were required to endow microglia with proliferative and pro-angiogenic properties. Faulty RAM programming in the absence of IL-6 or inflammatory monocytes resulted in poor cerebrovascular repair, neuronal destruction, and sustained neurological deficits that were all restored via exogenous IL-6 administration. These data provide a molecular and cellular basis for how monocytes instruct microglia to repair damaged brain vasculature and promote functional recovery after injury.
Mycobacterium tuberculosis infection of human neural progenitor cells alters their functions
Authors: Thanthrige Thiunuwan Priyathilaka , Herbath Melinda , Laaker Collin James , Schwartz Michael , Lebakken Connie , Fabry Zsuzsanna , Sandor Matyas
Published: August 2023

Central nervous system tuberculosis (CNS-TB) is the most dangerous form of extrapulmonary tuberculosis. However, the current understanding of infectious mechanisms and anti-mycobacterial immunity within the brain parenchyma is limited due to the absence of an ideal human CNS-TB model. In this study, we developed a novel human in vitro model for the study of CNS-TB by infecting 3D neural organoids and 2D neural progenitor cells (NPC) with Mycobacterium tuberculosis (Mtb). Here we report that the subset of the NPC population can be phagocytized by the apoptotic cell binding receptors. Internalized Mtb bacilli, co-localized within late endosomal and phagolysosomal compartments as well as in the cytoplasm of the NPCs. Mtb infection of NPC induced an elevated cell death and limited proliferation of the infected NPCs Additionally a strong type-1 interferon (IFN) responses were detected in Mtb-infected NPCs by the RNA-sequencing and by immunohistochemistry. We demonstrated that the treatments with anti-IFNα neutralization antibodies could partially rescue Mtb-induced NPC death and proliferation defects. Our findings suggest that these novel neural organoids and NPC-based in vitro platforms provide a new platform for elucidating CNS-TB pathogenesis and can be ultimately used for testing novel anti-mycobacterial treatment strategies for the human CNS-TB.

“Supported by grants from NIH (R56AI162164-01)
LXR Signaling Mediates Protection from Bacterial Toxins
Authors: Lee Minsub S , Sammarco Alessandro , Su Baolong , Williams Kevin , Fong Loren , Tarling Elizabeth , Scumpia Philip O , Bensinger Steven
Published: August 2023

Necrotizing fasciitis (NF) is an aggressive skin and soft tissue infection characterized by massive tissue destruction. Current treatment strategies are limited to aggressive and repeated surgical debridement, coupled with systemic antibiotics. Thus, new therapeutic approaches that preserve tissue integrity would be beneficial. Cholesterol-dependent cytolysins (CDCs) are toxins produced by select gram-positive microbes implicated in NF. CDCs recognize cholesterol in membranes of cells for pore formation and subsequent cellular and tissue damage. Recently, we have shown that interferon signaling is able to protect cells from CDCs via reprogramming cholesterol homeostasis. This observation led to us to ask if pharmacological targeting of cholesterol metabolism provide similar protection. Liver X Receptor (LXRs) are key transcriptional regulators of cholesterol metabolism. We find that pharmacological LXR activation protects mice from Streptolysin O (SLO)-induced tissue damage. LXR stimulation in various cell types, such as macrophages, neutrophils, and endothelial cells induced resistance to CDCs by abrogating the ability of CDCs to directly bind to cholesterol in the plasma membrane. Ongoing mechanistic studies are investigating which metabolic pathways induced by LXRs (e.g., cholesterol efflux, biosynthesis, import, or trafficking) are responsible for the protective effects. Translationally, LXR stimulation may serve as an adjunctive therapeutics for necrotizing fasciitis and other CDC-mediated pathologies (e.g., pneumonia, pharyngitis, and septic cardiomyopathy).

P01 HL146358
Viral immunology in the context of COVID-19
Authors: Sika-Paotonu Dianne
Published: August 2023

Early into the COVID-19 pandemic, countries in the Pacific Region worked hard to keep the SARS-CoV-2 virus outside their borders, until COVID-19 vaccines could be developed. Even before made readily available, COVID-19 vaccines became the subject of widespread speculation, misinformation and disinformation, with some of these efforts targeted to the Pacific Region. The demand for appropriate and effective scientific communication efforts suddenly increased. Areas of immunological focus pertaining to the SARS-CoV-2 virus and the COVID-19 vaccine needed clarification for the general public and lay audiences everywhere. Efforts to communicate complex immunological based content pertaining to the SARS-CoV-2 virus in particular while in various degrees of lockdowns became more reliant upon online platforms. Prior to the widespread use of Rapid Antigen Testing for COVID-19, one of the many areas of interest was the PCR testing methodology for COVID-19 which also became a focus of mis/disinformation in the Pacific Region. The purpose of this work was to apply an approach for countering mis/disinformation about the SARS-CoV-2 virus in particular, with a strong focus on the immunological aspects through an online Pacific Fono (gathering) to support appropriate scientific communication. Participants gave positive feedback, indicating this approach was relevant, helpful, with requests for more Pacific Fono. This work showed that an appropriate, contextualised scientific focus upon the immunological aspects of the SARS-CoV-2 virus and COVID-19 vaccines, could help counter mis/disinformation efforts against COVID-19 vaccines.

Wellington School of Medicine & Health Sciences, University of Otago
Localized immune dysregulation is associated with lung injury in severe pediatric COVID-19
Authors: Korenberg Rebecca C , Bell Joseph N , Bradley Marissa C , Connors Thomas J
Published: August 2023

Acute respiratory distress syndrome (ARDS) is a common and devastating consequence of SARS-CoV-2 infection in adults, however severe lung injury is a rare manifestation of COVID-19 in children. Aberrant local immune responses are known to drive lung pathology in adults, but little is known about localized immune responses in children with respiratory failure due to COVID-19. We obtained blood and respiratory tract samples from children (n=35, aged 1 month to 18 years) admitted to the hospital for COVID-19, stratifying by presence of respiratory failure (n=15). We defined the cytokine and chemokine profile of severe disease using a 71 analyte Luminex panel. Children with respiratory failure were found to have increased circulating levels of leukemia inhibitory factor (LIF), a growth factor upregulated during pulmonary inflammation. The local environment revealed significant inflammation with increased levels of pro-inflammatory cytokines including IL-6, IL-8, and IL-33 among others. Application of high dimensional flow cytometry to define immune cell subsets revealed elevated proportions of monocytes in both circulating and local environments. Monocytes demonstrated decreased expression of HLA-DR and CD86, suggesting an immunoparalysis phenotype, previously associated with severe COVID-19 in adults. Further, effector memory T cells in the airway exhibited an exhausted phenotype with high levels of PD-1 expression. Together this analysis reveals a highly inflammatory and dysregulated immune response underlies SARS-CoV-2 induced pediatric lung injury. These findings suggest a shared pathway with adult COVID-19 ARDS and provide new insights into the immune mechanisms associated with severe COVID-19 in children.

Supported by grants from NIH (K23AI141686)
Calming the storm: Immunoregulatory receptor NLRX1 and its role in the immune response to SARS-CoV-2
Authors: Ivester Hannah , Woolls Mackenzie , Morrison Holly A , Tupik Juselyn D , Chuong Christina , Finkielstein Carla , Duggal Nisha , Weger James , Allen Irving C
Published: August 2023

One of the most characteristic clinical disease presentations of severe COVID-19 is the ‘Cytokine Storm,’ stemming from dysregulation of the immune response. This overproduction of proinflammatory cytokines in response to SARS-CoV-2 is responsible for Acute Respiratory Distress Syndrome (ARDS) in adult patients and the Multisystem Inflammatory Syndrome (MIS) of pediatric patients hospitalized with severe COVID-19. Pattern recognition receptor NLRX1 is a member of the NOD-like receptor family with complex involvement in antiviral immunity. It has been reported in the literature in many contexts to be anti-inflammatory by inhibiting NF-kB and interferon signaling-critical pathways in anti-SARS-CoV-2 immunity. Here we show that loss of NLRX1 impacts immune responses to SARS-CoV-2 both in cell culture and in mice. While Nlrx1−/− mice showed similar overall morbidity, there were temporary increases in weight loss at multiple time points post infection. SARS-CoV-2 infection of Nlrx1−/− mice also resulted in an increase in viral titer in the lungs at 2 dpi, despite having higher degrees of airway inflammation. Data in both cell culture and animal challenges report differences in immune activation, showing differences in cytokine production with changes in expression of NLRX1. Together, these experiments uncover another enigmatic function of NLRX1 while simultaneously offering pathways related to NLRX1 as possible therapeutic targets to help mitigate the effects of the ‘cytokine storm’ of severe COVID-19. Finally, these data also add to the growing field regarding SARS-CoV-2 and its pathobiology in an understudied context of immune regulation and continue to explore NLRX1 and its many curious roles in antiviral immunity.
Interferon-β treatment reverses the detrimental effect of B cell depletion therapy on respiratory virus infection
Authors: Allushi Bujana , Chlebicz Magdalena , Turner Sean , Kovats Susan , Axtell Robert C
Published: August 2023

There are several disease-modifying therapies (DMTs) used as treatments for multiple sclerosis. Depending on their mechanism, DMTs can increase morbidities of viral infections in patients, including SARS-CoV2 and influenza A virus (IAV). B cell depletion (BCD) with anti-CD20 antibodies and interferon-β (IFN-β) are two popular DMTs for MS. BCD increases the morbidity of acute viral infections in patients, whereas the antiviral properties of IFN-β are postulated to protect against infection. This study determined how BCD and IFN-β alone or in combination modulate IAV infection in mice. For BCD, we treated mice with 3 doses of anti-CD20 antibodies every 5 days beginning 7 days prior to IAV infection. For IFN-β, mice were treated on day 1 and 2 post infection. Mice treated only with BCD lost more weight compared to control mice. This increased morbidity was linked with higher levels of viral RNA and more neutrophils and inflammatory monocytes in the lungs at day 9 post-infection. T cell response to IAV was not altered by BCD. Mice treated only with IFN-β had no alteration in weight loss or myeloid cell infiltration in the lungs compared to controls. When adding IFN-β to BCD therapy, mice had significantly less weight loss and lower numbers of neutrophils and inflammatory monocytes in the lung compared to mice with BCD only. Addition of IFN-β to BCD did not improve IAV clearance. Our findings show that BCD increases virus-induced morbidity in mice, which reflects data from MS patients. Here we provide preclinical data showing that the addition of IFN-β can reverse the detrimental effect of BCD on viral infection. Therefore, the combination of non-interacting biologic therapies may mitigate morbidity during viral infection.
Tlr5 deficiency exacerbates lupus-like disease in the MRL/lpr mouse model
Authors: Alajoleen Razan M , Oakland David N , Estaleen Rana , Shakeri Aida , Lu Ran , Appiah Michael , Sun Sha , Reilly Christopher M , Luo Xin M
Published: August 2023

A leaky gut has been linked to numerous autoimmune disorders, including systemic lupus erythematosus (SLE). We had previously reported significant upregulation of anti-flagellin antibodies in the blood of SLE patients and lupus-prone MRL/lpr mice, which led to our hypothesis that a leaky gut drove SLE through bacterial flagellin-mediated activation of toll-like receptor 5 (TLR5). Here, we investigated the initiation and progression of lupus-like disease in MRL/lpr mice with global Tlr5 deletion generated by CRISPR-Cas9. Contrary to our hypothesis that the deletion of Tlr5 would attenuate lupus, our results showed exacerbation of lupus-like disease with Tlr5 deficiency in MRL/lpr mice. A significantly higher level of proteinuria was observed in Tlr5−/− female MRL/lpr mice compared to Tlr5+/− and Tlr5+/+ female littermates, suggesting aggravated glomerulonephritis with Tlr5 deficiency. In the kidney, the deletion of Tlr5 significantly increased renal infiltration of activated conventional and monocyte-derived dendritic cells. Spleen, mesenteric and axillary/cervical lymph node to body weight ratio were also significantly increased in Tlr5−/− MRL/lpr mice compared to Tlr5+/− and Tlr5+/+ littermates, with the effects more pronounced in female mice, suggesting that the deletion of Tlr5 impacted lymphoproliferation in addition to lupus nephritis. We are currently analyzing the lymphoid tissues to reveal potential cellular mechanisms. Notably, Tlr5 deficiency did not change the existing leaky gut in either female or male MRL/lpr mice. Future studies will elucidate the underlying mechanisms by which the deletion of Tlr5 modulates cellular functions to exacerbate lupus.
Inflammatory bowel disease (IBD)-specific immune cell recruitment and response can be modulated with anti-TNF-α therapies in the human Colon Intestine-Chip
Authors: Kanellias Marianne , Woodham Andrew W , Cespedes Adriana V , Apostolou Athanasia , Kujala Ville J , Ewart Lorna , Carman Christopher V
Published: August 2023

Objective

Inflammatory bowel disease (IBD) is a complex inflammatory disease, for which few effective therapies exist. The goal of our current work was to show: i) that such a complex, immune cell-driven pathogenesis could be captured on Emulate’s human Colon Intestine-Chips; and ii) that this could be used as a novel human-centric system to support IBD drug development including anti-TNF-α antibodies.

Methods

PBMCs were perfused through the vascular (bottom) channel in untreated ‘resting’ or cytokine-‘primed’ Colon Intestine-Chips. Clinically relevant IBD drugs, including anti-TNF-α antibodies (Humira and Cimzia), as well as dexamethasone and AJM300 were administered through the vascular channel during PBMC administration. Total cell recruitment, effluent biomarker secretion (cytokines, calprotectin, C-reactive protein), and barrier function were analyzed for 72 hours.

Results

α4β7+ ‘gut-tropic’ PBMCs (monocytes, CD4+, CD8+, and NK cells) preferentially adhered and transmigrated in a cytokine priming-dependent manner. These PBMCs released clinically relevant IBD cytokines and biomarkers and induced barrier disruption, a hallmark of IBD. Anti-TNF-α antibodies and AJM300 caused reduced PBMC recruitment, inflammatory response and barrier disruption >90%. Dexamethasone had more modest effects, as expected.

Conclusion

The human Colon Intestine-Chip can both model complex immune cell-driven IBD and validate effects of clinically relevant IBD drugs. This system could provide predictive validity for pre-clinical development of new IBD drugs.