bluuebunny/crossref_metadata_2025 huggingface dataset:
NLRP3 inflammasome in immune cells regulates metastatic potential of cancer
Authors: Lee Joo Y , Lee Hye E , Yang Gabsik , Kang Han C , Cho Yong-Yeon , Lee Hye S
Published: January 2023

One of the main features of tumor microenvironment is inflammation accompanied with the activation of immune cells and the production of pro-inflammatory cytokines. IL-1b, one of the critical cytokines released by immune cells to promote tumor growth and metastasis by acting on its receptor, IL-1R, and to culminate in the secondary activation of immune cells and the expression of immune cytokines. Inflammasome plays a critical role in production of mature IL-1b through activation of capspase-1. Therefore, we investigated whether regulation of NLRP3 inflammasome in macrophages affected the metastatic potential of tumor cells. Activation of NLRP3 inflammasome was induced by ATP in bone marrow-derived primary mouse macrophages. The metastatic potential of tumor cells was determined by transwell migration and invasion assays. Wild-type macrophages stimulated with ATP increased the migration and invasion of melanoma cells (B16F10). However, NLRP3- or caspase-1-knockout macrophages showed greatly decreased ability to promote the migration and invasion of melanoma cells. Treatment with celastrol, an inhibitor of NLRP3 inflammasome, blocked the ability of macrophages to stimulate migration and invasion of melanoma cells. The results show that inhibition of the NLRP3 inflammasome in macrophages by genetic deficiency or a pharmacological inhibitor leads to decrease of the metastatic potential of tumor cells. The results would provide a novel anti-cancer immuntherapy to regulate the activity of NLRP3 inflammasome in tumor microenvironment. [Supported by grants from BK21 PLUS program, NRF-2019R1A2C2085739, and NRF-2017R1A4A1015036].
Nur77 controls tolerance induction, terminal differentiation, and effector functions in NKT cells
Authors: Kumar Amrendra , Hill Timothy M , Gordy Laura E. , Suryadevara Naveenchandra , Wu Lan , Flyak Andrew I. , Bezbradica Jelena S. , Van Kaer Luc , Joyce Sebastian
Published: January 2023

Natural killer T (NKT) cells are self-reactive lymphocytes, yet how this lineage attains self-tolerance remains unknown. NKT cells constitutively express high levels of Nr4a1-encoded Nur77, a transcription factor that integrates signal strength downstream of the T cell receptor (TCR) within activated thymocytes and peripheral T cells. Nur77 is associated with tolerance induction in conventional T cells but is of hitherto unknown function in NKT cells. Here we report that sustained Nur77 overexpression (Nur77tg) in mouse thymocytes abrogates NKT cell development. Introgression of a rearranged Vα14-Jα18 a-chain gene into the Nur77tg (Nur77tg;Vα14tg) mouse rescued development up to an early precursor stage 0. NKT cells in bone marrow chimeras that reconstituted thymic cellularity developed beyond stage 0 precursors, yielding interleukin-4-producing NKT2 but not interferon-g-producing NKT1 cell subsets. Nonetheless, NKT cells that emerged in these chimeras expressed the exhaustion marker PD1 and responded poorly to glycolipid agonists. Hence, Nur77 integrates signals emanating from the TCR to control NKT cell tolerance, terminal differentiation, and effector functions.
TNF deficiency induces breach of B cell tolerance by altering the homeostasis of the regulatory signals in activated B cells of lupus prone mice
Authors: Quach Tam D , Diadhiou Catherine M. M. , Raparia Chirag , Martinez Shani , Tao Haiou , Tükel Çağla , Jacob Chaim O. , Davidson Anne
Published: January 2023

The induction of autoantibodies and autoimmunity in patients treated with TNF inhibitors (TNFi) is well-known, but the mechanism by which TNFi induce breach of B cell tolerance is not known. TNF deficiency induces TFH and TH17 cell expansions and disrupts germinal centers (GC), that regulate high affinity antigen specific antibody production and autoreactive B cell selection. We found that TNF deficiency in Sle1 mice (Sle1.TNF−/−) induced high titers of germline encoded, T-dependent IgG anti-cardiolipin. Upon treated with TLR9 ligand (DNA/curli amyloid protein), Sle1.TNF−/− mice developed high IgG anti-dsDNA; although CD95+PNA+/GL7lowGC phenotype B cells were induced, they did not form GC nor acquire dark zone phenotype, and their Ig heavy chains accumulated low numbers of mutations. Similarly, without proper GC, GC phenotype B cells were found in the TNFR1/2−/−NZM2328 mice, who developed accelerated lupus, suggesting disturbed GC by TNF deficiency dysregulated GC phenotype B cell selection. Using an antibody to Neu5Gc (N-Glycolylneuraminic acid, a CD22 high affinity ligand which is the hydrolyzed product of GL7 ligand, N-acetylneuraminic acid, Neu5Ac), we found that GC phenotype B cells from TNF deficient mice failed to stop hydrolyzing Neu5Ac to Neu5Gc, suggesting a failure in recruitment of CD22 to the BCR complex upon activation. Furthermore, activated B cells from Sle1.TNF−/− mice constitutively expressed high level of pS6 and SHIP-1; whereas, additional stimulants, anti-Ig and/or anti-CD40, were required to induce pS6 expression in those of the Sle1 mice. Here, we report a possible mechanism by which TNF deficiency alters the homeostasis of regulatory molecules such as CD22 and FcgRIIB, and thus activated B cell selection.
AMPKα1 in B cells promotes memory B cell longevity and mitochondrial homeostasis while dampening antibody synthesis
Authors: McLetchie Shawna , Cho Sung Hoon , Basso Paulo , Boothby Mark Robin
Published: January 2023

Emerging evidence indicates that metabolic programs regulate B cell activation and antibody responses. However, the metabolic mediators that support the durability of the memory B cell and long-lived plasma cell populations are not fully elucidated. Adenosine monophosphate-activated protein kinase (AMPK) is an evolutionary conserved serine/threonine kinase that integrates cellular energy status and nutrient availability to intracellular signaling and metabolic pathways. Here, we use genetic mouse models to show that B cell restricted loss of the catalytic subunit, AMPKα1, leads to a defect in the long-term survival of the memory B cell population. AMPKα1-deficient memory B lymphocytes exhibited aberrant mitochondrial activity including impaired mitophagy and decreased mitochondrial spare respiratory capacity. In contrast, AMPKα1 was dispensable for the stability of the bone marrow-resident long-lived plasma cell population yet its absence led to increased rates of Ig synthesis and elevated serum antibody concentrations elicited by immunization. Collectively, our findings fit a model in which AMPKα1 supports longevity in the memory B cell compartment by promoting mitochondrial homeostasis but restrains rates of immunoglobulin production by plasma cells.
Lipin-1 transcriptional co-regulatory activity enhances anti-inflammatory responses in lipid-loaded macrophages and induces atheroprotection
Authors: Blackburn Cassidy , Schilke Robert M , Vozenilek Aimee E , Finck Brian N. , Woolard Matthew D
Published: January 2023

Metabolic disorders, like atherosclerosis, increase the lipid burden in macrophages leading to chronic inflammation. How macrophages sense and respond to excess lipid is not well understood. Lipin-1 is a phosphatidic acid phosphatase that also functions as a transcriptional co-regulator and is critical to regulating lipid burden in cells. We have shown that lipin-1 enzymatic activity promotes macrophage pro-inflammatory responses and accelerates atherosclerosis. Lipin-1 transcriptional co-regulatory activity binds to and augments transcription factors such as PPARγ. PPARγ increases macrophage anti-inflammatory responses. The objective of this study was to determine if after increased lipid burden in macrophages, lipin-1 transcriptional co-regulatory activity tailors PPARγ to promote anti-inflammatory responses by macrophages and reduce atherosclerosis. Upon RNA analysis in lipid stimulated macrophages, we demonstrated lipin-1 tailors PPAR-associated gene expression and promotes anti-inflammatory responses. Using mice that lack just lipin-1 enzymatic activity or all lipin-1 in myeloid cells, we examined the contribution of lipin-1 transcriptional co-regulatory activity to atherosclerosis progression. Our mice lacking both myeloid-associated lipin-1 activities had larger, more necrotic plaques. Using Nanostring analysis, we observed increased expression of pro-inflammatory genes such as IL-23 in the plaque and increased IL-23 in serum suggesting lipin-1 transcriptional co-regulatory activity in macrophages promotes anti-inflammatory responses to reduce atherosclerosis. Our work highlights the dynamic nature of lipid metabolism on macrophage function and contribution to disease progression.
Cationic Cyclodextrins Regulate Nucleic Acid-induced Proinflammatory Responses
Authors: Sharma Rahul , Venkatadri Rajkumar , Dogan Murat , Palakurthi Sushesh , Mohammad Saleh , Hudspeth Andrew , Palakurthi Srinath
Published: January 2023

Cyclodextrins (CDs) were discovered over a century ago for their ability to complex with several macromolecules and have found use in pharmaceutical industry to enhance solubility of drugs. Cationic CDs can bind nucleic acids (NA) and are being tested for delivery of NA to cells. Extracellular NAs can act as pro-inflammatory triggers and their inefficient clearance may activate Toll-like receptors (TLR) and contribute to autoimmune diseases such as lupus. We hypothesized that NA-binding CD (NABCD) may be used for scavenging extra-cellular NA to prevent pro-inflammatory responses. Here we investigated whether NABCD can modulate immune activation triggered by DNA or NA-TLR ligands. Indeed, in vitro assays using bone marrow derived macrophages (BMM) showed that NABCD induced a statistically significant inhibition of the expression of innate immune activation genes including Mx1, Prkr, Il1b and Tnfa when exposed to DNA or poly I:C. Treatment of C57BL/6 mice with TLR ligand polyI:C induced an acute pro-inflammatory response indicated by upregulation of interferon response genes and cytokine production. Treatment with NABCD not-only attenuated the innate-immune activation, but also modulated the adaptive immune response by significantly preventing a Th1 skewing of the immune response. The NABCD-treated mice also had higher numbers of IL-10 producing CD4 T-cells and more Foxp3+T-regulatory cells in the spleens as compared to mice treated only with polyI:C. Our data indicate a feasibility for the use of NABCD for modulating NA-induced proinflammatory responses and may find applications for treatment of autoimmune diseases such as lupus, where extra-cellular DNA plays a pathogenic role.
Mononuclear phagocytes drive Mertk-dependent T cell regulation at autoimmune and tumor sites
Authors: Whitesell Jennifer C , Lindsay Robin S , Dew Kristen , Rodriguez Erika , Tracy Dayna , Sandor Adam M. , Yannacone Seth , Jacobelli Jordan , Friedman Rachel S.
Published: January 2023

Understanding mechanisms of immune regulation is key to developing effective immunotherapies for autoimmunity and cancer. We examined the role of mononuclear phagocytes (MNPs) in regulating effector T cells in type 1 diabetes and melanoma. MNPs in the islets impaired T cell responsiveness to antigen by preventing antigen-mediated T cell arrest. MNPs in the autoimmune lesion express the TAM family receptor tyrosine kinase Mertk which functions in efferocytosis. Inhibition or deficiency of Mertk led to a release from T cell regulation characterized by enhanced T cell arrest in pre-diabetic islets and at the tumor site. T cell arrest was accompanied by increased T cell-antigen presenting cell interactions as well as increased antigen experience and effector function by T cells in the islets. Single cell RNA-seq analysis of islet MNPs identified multiple myeloid subsets. Inhibition of Mertk induced the most significant changes in gene expression among the islet resident macrophages including upregulation of inflammatory cytokine and adhesion genes. In the NOD mouse model, inhibition of Mertk-dependent T cell regulation culminated in the rapid acceleration of autoimmune pathology and disease. In human islets, the number of Mertk-expressing cells were increased in remaining insulin-containing islets from type 1 diabetic patients, suggesting that they might have a protective role in human disease. These data indicate that Mertk signaling in MNPs drives T cell regulation that functions specifically at the disease site in peripheral tissues through a mechanism that prevents T cell arrest and response to antigen.
E3 Ubiquitin Ligase Cbl-b Restrains Priming of Pathogenic Th17 Cells via Inhibiting IL-6 Production by Macrophages
Authors: Tang Na , Zeng Qiuming , Guo Hui , Zhang Jian
Published: January 2023

E3 ubiquitin ligase Cbl-b is involved in the maintenance of a balance between immunity and tolerance. Mice lacking Cbl-b are highly susceptible to experimental autoimmune encephalomyelitis (EAE), a Th17-mediated autoimmune disease. However, how Cbl-b regulates Th17 cell response remains unclear. In this study, utilizing adoptive transfer and cell type-specific Cblb knockout strains we show that Cbl-b expression in macrophages but not T cells or dendritic cells (DCs) restrains the generation of pathogenic Th17 cells and the development of EAE. Cbl-b inhibits the production of IL-6 by macrophages induced by the signaling derived from C-type lectin receptors, which directs T cells to generate pathogenic Th17 cells. Therefore, our data are the first to unveil a previously-unappreciated function for Cbl-b in the regulation of pathogenic Th17 response.
Investigating the role of mast cell activation by nitrogen mustard in lung toxicity
Authors: Cruz-Hernandez Angela Alexandra , Mendoza Ryan , Brown Jared M
Published: January 2023

Chemical warfare agents (CWA) were widely used during the Gulf War (1990–1991) and as a result left ~30% of veterans suffering from Gulf War Illness (GWI). The vesicating agent, sulfur mustard (SM) is one of the most noteworthy CWA used during this time. Prior research has demonstrated that SM targets the bone marrow and has the potential to influence immune cells. In the skin, SM is reported to induce mast cell degranulation. Therefore, the aim of this study was to determine if nitrogen mustard (NM) (a surrogate for SM) promotes activation of mast cells. Using murine bone marrow derived mast cells (BMMCs) we assessed activation through degranulation, eicosanoid expression, and pro-inflammatory cytokine production. While NM exposure (1μM–50μM) did not result in mast cell degranulation, we observed a 30-fold increase of cyclooxygenase-2 (COX-2) mRNA production at 10μM at 6h. In addition, IL-6 production by BMMCs post 24h exposure to NM demonstrated a dose dependent increase indicative of late phase activation. To further demonstrate a pivotal role for mast cells in NM exposure, we compared the effects of NM exposure on lung pathology between C57BL/6 and B6.Cg-KitW-sh/ HNihrJaeBsmJ (mast cell deficient) mice. Significant lung injury was observed in C57BL/6J following NM exposure at 72 hrs as indicated by H&E staining. In contrast, significantly less injury was observed in mast cell deficient mice. Similarly, increases in total protein and IL-6 was observed in C57BL/6 mice but not mast cell deficient mice. These results suggest that mast cells play a prominent role in the injury induced by NM and may contribute to symptoms observed in GWI.
Genetic regulation of nitric oxide-dependent mitochondrial metabolism and immune function in dendritic cells
Authors: Snyder Julia Priscilla , Lahue Karolyn , Krementsov Dimitry N , Amiel Eyal
Published: January 2023

Dendritic cell (DC) activation is characterized by a rapid “metabolic switch” towards increased glycolysis that supports maturation. Sustained glycolysis post-activation is a requirement for survival in DC subsets that express inducible nitric oxide synthase (iNOS), due to the production of nitric oxide (NO) which acts as an inhibitor of mitochondrial respiration. Long-term metabolic reprogramming in activated DCs has almost exclusively been studied in the strain C57BL/6, which experience loss of mitochondrial function due to NO accumulation. To assess the conservation of NO-driven metabolic regulation in DCs, we generated and compared cells from C57BL/6 mice to the wild-derived genetically diverse PWD/PhJ (PWD) strain. We show attenuated iNOS induction and significantly lower NO production in PWD DCs. Decreased NO production allows PWD DCs to maintain mitochondrial respiration following activation, and significantly alters their glucose-dependent carbon flux through the TCA cycle compared to C57BL/6 DCs. We utilize a consomic strain which includes the PWD chromosome segment containing the iNOS locus on an otherwise C57BL/6 genetic background to demonstrate that intrinsic differences in the iNOS locus drive differential iNOS regulation. We demonstrate that differential iNOS regulation amongst these strains impacts DC post-activation survival and immune effector functions. These studies provide a more comprehensive understanding of NO-driven metabolic reprogramming in the murine myeloid lineage and establish a natural genetic model for investigating the contribution of NO in regulating the interplay between DC metabolism and immune function.
Maternal hypothyroxinemia alters the immune function of the adipose tissue in the adult the progeny
Authors: Gutierrez Barbara Cecilia , Alba Arturo , De luca Natalia , Vecchiola Andrea , Osses Maricarmen , Gomez Felipe , Santos Johana , Mendez Luis , Aguayo Daniel , Opazo Maria Cecilia
Published: January 2023

Obesity and thyroid hormone deficiency are global problems. Chile has 31,2% of obesity. Obesity is characterized by adipose tissue, that’s able to influence the immune system. Gestational environment can increase the offspring susceptibility to have obesity. It’s been suggested that maternal hypothyroxinemia (HTX) could be a factor for obesity development in the offspring imprinting the adipose tissue contributing to the altered immune response seen in HTX progeny.

The aim of this work is to evaluate the effect of HTX in adipose tissue in the progeny and its influence on the adipocyte immune function.

For this purpose, weight and body fat content of the offspring gestated or not under HTX using In vivo MRI was asess. Area and number of adipocytes from offspring conditions was evaluated using a deep convolutional neural network approach. Cell infiltration was also analyzed. Expression of cytokines as PPARs and leptin were evaluated. Adipose tissue infiltrating cells were analyzed by FACS. In vitro, the differentiation capacity of control and HTX pre-adipocytes using Oil red staining and quantification and measure adipokine an cytokine production by these cells was evaluated.

We show that the HTX offspring present an increased body fat content. Image analysis showed an increase in the number and area of the HTX adipocytes. An increased capacity of HTX pre-adipocytes to differentiate to adipocytes was seen. Alteration in the expression of cytokines, PPARs and leptin and an increase in cell infiltration with more predominant activated CD4+ T cells and monocytes was observed.

This work proves that maternal HTX induces alterations of the adipose tissue of the progeny that could contribute to the susceptibility of the HTX to autoimmune diseases.
Transcriptional profiling predicts behavioral performance in experimental mouse model of traumatic brain injury (TBI)
Authors: Jiao Yun , Faden Alan , Sabirzhanov Boris , Stoica Bogdan , Szeto Gregory L. , Leach Jennie
Published: January 2023

Traumatic brain injury (TBI) is a prevailing cause of disability and death. There are no curative therapies or robust predictors of long-term neurodegenerative disease. Treatment is challenging due to poor understanding of the cellular responses and mechanisms underlying TBI. We linked transcriptional profiling, cytokine profiling, and behavioral scoring in experimental TBI to discover new insights into immune-mediated mechanisms of TBI. Mice received ipsilateral moderate controlled cortical impaction. Luminex analyses showed upregulation of inflammatory cytokines in injured cortex, hippocampus, and spinal cord. Neurobehavioral evaluations were performed, then hippocampi isolated at 28d post-TBI for RNAseq. Pathway analyses of upregulated genes were functionally enriched for glial activation, proliferation, and neuronal development. Genes involved in synaptic transmission, glutamate receptor signaling, and neuronal migration were downregulated. Partial least squares (PLS) regression successfully modeled the relationship between differentially expressed genes and behavioral scores (R2=0.94). A core 10-gene signature predicted TBI severity and behavioral score. PLS discriminant analysis (PLSDA) classified TBI severity. VIP scores were used to define a 20-gene signature in TBI based on high VIP score, high fold change, and contribution to biological function. A PLSDA model using only these 20 genes was cross-validated by bootstrap method (AUROC=0.93), indicating that these genes may identify the critical TBI-altered processes. Altogether, our analyses revealed TBI-related biological pathways, potential therapeutic targets, and gene signatures as biomarkers for TBI severity and behavioral outcomes.
A functional interaction between plasmacytoid dendritic cells (pDCs) and CD169+ macrophages in the bone marrow regulates pDCs production of type I interferon during severe malaria
Authors: Moore Jamie M , Fooksman David R , Lauvau Gregoire
Published: January 2023

While type I interferon (IFN-I) is central to antiviral immunity, it can also promote pathology in bacterial and parasitic infections, and autoimmune diseases. Plasmacytoid dendritic cells (pDCs) are able to produce 1,000× more IFN-I than any other cell upon sensing of nucleic acid ligands by TLR7 and TLR9. pDCs can obtain their ligands via direct cell-cell interactions, yet what regulates these interactions and whether pDCs need to receive additional signals to secrete IFN-I is unknown. Using a mouse model of severe malaria, we showed that pDCs initiate IFN-I production in the bone marrow (BM) of infected mice, and that this required CD169+ macrophages (MPs). IFN-I production by pDCs was also dependent on TLR7 and STING sensing in pDCs and CD169+ MPs, respectively. Through intravital microscopy (IVM), we further revealed that pDCs arrest and form clusters with CD169+MPs in the BM of malaria infected mice when peak IFN-I production occurs. Here, we hypothesize that pDC-MP interactions are required to license pDCs to produce IFN-I. We found that pDCs fail to arrest and form clusters with MPs in Tlr7−/− mice, and that blocking chemotaxis prevents pDC-MP interactions and subsequent IFN-I production, consistent with our hypothesis. Interestingly, while pDCs do not make IFN-I in STING-deficient mice, they still arrest and cluster around MPs. These clusters are much larger than in WT mice, suggesting that pDCs fail to receive another STING-dependent signal. Collectively, these results support a model in which pDC production of IFN-I is tightly regulated through a functional interaction with CD169+ MPs providing activating ligands and stimulatory signals. Targeting this interaction may represent a novel way to tune levels of IFN-I produced by pDCs.
Inhaled tigecycline induces change in immune cell modulation that synergizes with antimicrobial activity in chronic pulmonary Mycobacterium abscessus disease
Authors: Pearce Camron M , Walz Amanda , Gonzalez-Juarrero Mercedes , Ali Zohaib , Munoz-Gutierrez Juan , van Ingen Jakko
Published: January 2023

Pulmonary Mycobacterium abscesssus (pAMB) infection is particularly challenging to treat because this bacillus displays intrinsic antibiotic resistance to most front-line drugs. Tigecycline, a glycyclcycline tetracycline, shows potent bactericidal effects against pMAB, while successfully evading bacterial resistance mechanisms. Tigecycline is FDA approved as a twice daily IV administration but provokes intolerable side effects in patients. More than its potent antimicrobial effects, tigecycline has demonstrated immunomodulatory properties observed in preclinical animal models of bacterial infection. We hypothesized that inhaled therapy of tigecycline reduces side effects and improve efficacy against pMAB. GM-CSF knockout mice with pMAB were treated daily for 28 days with aerosols of tigecycline at 0.25, 1.25 and 2.50 mg/dose. Thereafter, bacterial burden was assessed, and samples were prepared for immunophenotyping of lymphocyte and myeloid cell populations through H&E staining and IHC techniques. The results demonstrated that treatment with aerosols of tigecycline to mice with pAMB was tolerated and highly effective at reducing bacterial burden. Furthermore, this therapy was associated with the influx of macrophages and lymphocytes in both perivascular zones and granulomatous-like formations. There was also an increase in alveoli lined by hypertrophied cells with vesicular nuclei, suggesting induced Type II cell hyperplasia. To summarize, inhaled tigecycline not only demonstrated bactericidal effects against pMAB, but it invokes unique immunomodulatory effects that appear to aid the significant reduction in pulmonary bacteria.
Antigen presentation by CNS-resident microglia and macrophages is required for antiviral CD8 T cell responses in the brain following CNS viral challenge
Authors: Goddery Emma , Lipovsky Chloe , Jin Fang , Hansen Michael , Pavelko Kevin D , Johnson Aaron J
Published: January 2023

CD8 T cell responses are essential for clearance of infections and tumors in the central nervous system (CNS). Under homeostatic conditions, T cell access to the CNS parenchyma is restricted. An intricate process of T cell reactivation by antigen presenting cells (APCs) at CNS barriers is thought to allow activated antigen-specific T cells to infiltrate the parenchyma during neurological disease. The role of CNS-resident APCs during CD8 T cell infiltration of the CNS parenchyma is unclear. We hypothesized that microglia and brain-associated macrophages (BAMs) are APCs critical for antigen specific CD8 T cell reactivation and CNS infiltration. To test this hypothesis, we generated mice with tamoxifen-inducible deletion of MHC class I on microglia and BAMs by crossing CX3CR1creER mice with H-2Db fl/fl mice (Db cKO). We challenged these mice with intracranial infection of Theiler’s murine encephalomyelitis virus (TMEV), which generates a robust CD8 T cell response against a Db restricted antigen (VP2121–130) in the brain. At 7 days post infection (dpi), brain infiltrating Db:VP2-specific CD8 T cells were reduced in Db cKO mice compared to control, while peripheral priming of CD8 T cells was unaffected. Unexpectedly, inflammatory monocytes infiltrating the brain at 7 dpi were also reduced in Db cKO mice, indicating a role for microglia/BAMs and CD8 T cells in recruitment of inflammatory monocytes. Db cKO mice were able to clear virus, indicating functional antiviral responses against TMEV occur despite reduced immune infiltration. These results demonstrate that local antigen presentation by microglia and BAMs facilitates inflammatory monocyte recruitment and antigen-specific CD8 T cell responses in the brain after TMEV infection.
Pathogenic Th17 cell differentiation is negatively regulated by let-7 microRNAs in a mouse model of multiple sclerosis
Authors: Angelou Constance C , Wells Alexandria C , Vijayarhagavan Jyothi , Dougan Carey E , Lawlor Rebecca , Iverson Elizabeth , Lazarevic Vanja , Kimura Motoko Y , Peyton Shelly R , Minter Lisa M , Osborne Barbara A , Pobezinskaya Elena L , Pobezinsky Leonid A
Published: January 2023

Autoimmune disorders such as multiple sclerosis (MS) are caused by proinflammatory events mediated by pathogenic Th17 cells. In MS, these cells arise in response to autoantigen recognition and exposure to the cytokines IL-1β and IL-23, migrate to the central nervous system (CNS) by following gradients of CCR2- and CCR5-cognate chemokines, and secrete GM-CSF. GM-CSF is essential for disease development, as it promotes the activation, differentiation, and recruitment of peripheral inflammatory myeloid cells to the CNS that directly demyelinate neurons and damage axons. Th17 cell pathogenicity in MS has been correlated with microRNA (miRNA) dysregulation, which leads to aberrant post-transcriptional regulation of gene expression and enhanced autoreactive phenotype. We found that the lethal-7 (let-7) miRNA family is abundantly expressed in naive CD4+ T cells, but gets dramatically downregulated over time following antigen encounter, indicating that let-7 may control the differentiation of pathogenic Th17 cells. To investigate a potential regulatory role for let-7 in Th17 cell autoreactivity, we used experimental autoimmune encephalomyelitis (EAE), the animal model of MS. Specifically, we demonstrated that let-7 confers protection from EAE by negatively regulating the proliferation, IL-1β/IL-23-dependent differentiation, and CCR2/CCR5-dependent migration of pathogenic Th17 cells to the CNS. Conversely, absence of let-7 led to enhanced Th17 cell autoreactivity and aggravated disease. Therefore, our results identify a novel regulatory role for let-7 miRNAs in pathogenic Th17 differentiation during EAE development, suggesting a promising therapeutic application for the treatment of MS-related autoimmune diseases.
Eos supports TH1 differentiation through feed-forward propagation of the IL-2/STAT5 signaling pathway
Authors: Read Kaitlin , Sreekumar Bharath , Powell Michael Duane , Jones Devin M , Zafar Shah , Ghoneim Hazem E. , Collins Patrick L. , Oestreich Kenneth J
Published: January 2023

The Ikaros zinc finger (IkZF) transcription factor Eos is a known regulator of CD4+ regulatory T cell (TREG) populations. However, its role in the differentiation of other CD4+ T cell subsets is unknown. Here, we find that Eos deficiency results in both compromised TH1 cell differentiation and reduced production of the TH1 effector cytokine IFN-γ. Historically, IkZF factors have been associated with changes in gene expression via the alteration of chromatin structure. However, ATAC-seq analysis indicated that there were minimal changes in chromatin accessibility between wild type and Eos-deficient cells cultured under TH1-polarizing conditions. Rather, we find that IL-2 signaling promotes the STAT5-dependent induction of Eos expression, and that Eos both interacts with STAT5 and supports tyrosine phosphorylation-mediated STAT5 activation. Consequently, STAT5 enrichment is reduced at TH1 target genes in the absence of Eos. Thus, modulation of STAT5 signaling represents a novel mechanism by which Eos positively regulates the TH1 gene program. Given the important role for IL-2/STAT5 signaling in the differentiation of TH2, TH9, and TREG cell subsets, it will be of interest to determine whether this mechanism is conserved across additional CD4+ T cell populations.
RNA-binding protein HuR promotes Th17 cell differentiation and targeting HuR reduces autoimmune neuroinflammation
Authors: Chen Jing , Yu Shiguang
Published: January 2023

Dysregulated Th17 cell differentiation is associated with autoimmune diseases such as multiple sclerosis, which has no curative treatment. Understanding the molecular mechanisms of regulating Th17 cell differentiation will help in identifying a novel therapeutic target for treating Th17 cell-mediated diseases. Here, we investigated the cell-intrinsic processes by which RNA-binding protein HuR orchestrates Th17 cell fate decisions by post-transcriptionally regulating transcription factor Irf4 and Runx1 and receptor Il12rb1 expression, in turn promoting Th17 cell and Th1-like Th17 cell differentiation. Knockout of HuR impaired the transcriptome of Th17 cells characterized by reduced levels of RORγt, IRF4, RUNX1, and T-bet, thereby reducing the number of pathogenic IL-17+IFN-γ+CD4+ T cells during experimental autoimmune encephalomyelitis (EAE). In keeping with the fact that HuR increased the expression of adhesion molecule VLA-4 on Th17 cells, knockout of HuR impaired Th17 cell migration to the central nervous system and abolished the disease. Accordingly, targeting HuR by its inhibitor, DHTS, suppressed Th17 cell differentiation and reduced EAE severity. In sum, we uncovered the molecular mechanism underlying HuR’s regulation of Th17 cell differentiation and functions, underscoring the therapeutic value of HuR as a novel treatment for autoimmune neuroinflammation. (Supported by NIH R01 AI119135)
TCR dependent metabolic programming regulates autocrine IL-4 production resulting in self-tuning of the CD8+ T cell activation setpoint
Authors: Crofts Kali F , Holbrook Beth C , Soto-Pantoja David R , Ornelles David A , Alexander-Miller Martha A
Published: January 2023

The ability of T cells to sense and respond to environmental cues by altering their functional capabilities is critical for a safe and optimally protective immune response. One of the important properties that contributes to this goal is the activation setpoint of the T cell. Here we report a new pathway through which CD8+ T cells can self-tune their activation threshold. We find that in the presence of a strong TCR engagement event, there is a shift in the metabolic programming of the cell where both glycolysis and oxidative phosphorylation are significantly increased. This diverges from the switch to a predominantly glycolytic profile that would be predicted following naïve T cell activation. This altered metabolic program results in the production of autocrine IL-4. Both metabolic pathways are required for this cytokine to be made. IL-4 signaling in the activated CD8+ T cell results in modulation of the sensitivity of the cell, establishing a higher activation setpoint that is maintained over time. Together these data demonstrate a novel mechanism for the regulation of IL-4 production in CD8+ T cells. Further, they reveal a new pathway for the self-tuning of peptide sensitivity. Finally, these studies uncover an unexpected role for oxidative phosphorylation in regulating differentiation in CD8+ T cells.
Invariant NKT cells are unusually refractory to glucocorticoid-induced apoptosis but exhibit impaired functional fitness and fail to trigger antitumor immunity following physiological stress
Authors: Rudak Patrick T , Choi Joshua , Haeryfar S.M. Mansour
Published: January 2023

The nervous system serves critical roles in the regulation of immune responses. Consequently, the neuroimmune functional interface can be disrupted by physiological stress, potentially impeding our ability to combat malignancies. Invariant natural killer T (iNKT) cells are innate-like T cells that, upon activation by lipids such as αGC, participate in antitumor immune surveillance. However, whether mediators of stress impact iNKT cell functions in this context remains unexplored. Here, we subjected mice to prolonged psychological stress due to physical restraint or chronic stress due to heterotypic stressors before administering αGC. We report that stress abrogates TH1-type cytokine production by iNKT cells in vivo, an effect that is reversible by glucocorticoid receptor (GR) blockade and that is diminished in mice selectively lacking GRs in their T cell compartment. Instead, iNKT cell responses are skewed towards an abnormal anti-inflammatory and TH17-type cytokine signature. Gene expression analysis revealed that iNKT cells in stressed mice express reduced levels of critical genes supporting their typical differentiation states. Accordingly, iNKT cells in stressed mice fail to protect against pulmonary metastases of B16-F10 melanoma and are also unable to initiate killing activity against MHC class I-deficient splenocytes in vivo or YAC-1 lymphoma cells in vitro. Finally, the suppressive effects of stress on iNKT cells are not due to their apoptotic death. Unlike conventional T cells, human and mouse iNKT cells are remarkably resistant to glucocorticoid-induced apoptosis. Collectively, our findings define a novel mechanism of stress-induced immunosuppression involving innate-like but not conventional T cells.
Anti-CD3 and anti-CD28 antibodies conjugated with lipid microbubbles (MB-TAC) provide a superior expansion of human naive CD4+ and CD8+ T cells in long term culture
Authors: Lustig Ana , Manor Ty’Keemi , Liu Yu-Tsueng , Shi Guixin , Weng Nan-ping
Published: January 2023

Anti-CD3 and anti-CD28 antibodies are a method of choice for general and physiological activation of human primary T cells in vitro. Here we report a comparison of the growth, cell death, and cytokine production of anti-CD3 and anti-CD28 antibodies conjugated with lipid microbubbles (MB-TCA) and the standard Dynal beads (D-TCA). In 56-day cultures with three repeated stimulation cycles (14 days per stimulation), we found that MB-TCA induced a significantly better expansion (~20-fold increase) of naïve CD4 and CD8 T cells than did D-TCA. We found the main difference between MB-TCA and D-TCA was not the speed of activation-induced initial T cell proliferation but rather the survival of activated T cells. Furthermore, we found that MB-TCA activated T cells had a better telomere length maintenance and produced less TNFα and other cytokines than did D-TCA. Finally, blocking TNFα action via antibody against TNFRSF1A significantly improved expansion of T cells activated by D-TCA in vitro. Together, we demonstrated that MB-TCA offers a better expansion of primary human T cells in vitro and will be a useful method for long term culture of primary human T cells for both basic and clinical applications.
Role of tryptophan metabolism on senescent synovial fibroblasts
Authors: Usategui Alicia , López Abigail , Municio Cristina , Del Rey Manuel J , Pablos José L , Criado Gabriel
Published: January 2023

Background

Cellular senescence is connected to inflammation and altered metabolism. Rheumatoid arthritis (RA) patients show premature senescence and increased Trp metabolism along the kynurenine pathway. We aim to analyze the effect of Trp metabolism on cellular senescence and the expression of the Senescence Associated Secretory Phenotype (SASP) in human synovial fibroblasts (SF).

Methods

For inflammation-induced senescence, SF were cultured for 14 days after exposure to IL6/sIL6R (100 ng/ml for 72h on day 2). On day 14, senescence and SASP were analyzed. Senescent SF were quantified in cultures by detection of senescence-associated beta-galactosidase (SA b-gal). IDO1, IDO2, p16, ATG3, ATG5 and SASP components (IL6, IL8, MMP3, MCP1), were quantified by qRT-PCR and IL6 and IL8 proteins by ELISA. IDO1 was silenced using lentiviral vectors. IDO1 protein was detected by WB. Quantitative data were statistically analyzed by Pearson or Spearman correlation coefficient or Mann-Whitney test where appropriate.

Results

IDO1, but not IDO2, was induced by senescence in SF. Conversely, treatment of SF cultures with the IDO1 product kynurenine significantly reduced senescence, as determined by expression of the senescence marker p16 (p=0.018), although the autophagy response and SASP expression were not affected. IDO1 silencing during senescence induction resulted in a significant increase of IL6 expression without affecting other SASP components.

Conclusion

Upregulation of IDO1 by senescence, together with the reduction of senescence by kynurenine and the increased IL6 production by deficient IDO1 activity suggest that IDO1 and the kynurenine pathway play a negative regulatory role on senescence and SASP expression.
CD4+ T cells are more sensitive to inhibition of TCR signaling under Th17 polarizing conditions
Authors: Zinzow-Kramer Wendy M. , Weiss Arthur , Au-Yeung Byron B
Published: January 2023

Following TCR stimulation, in the presence of polarizing cytokines, naïve CD4+ T cells differentiate into effector cells (e.g. Th1, Th2, and Th17). To study how proximal TCR signaling molecules influence T cell differentiation, we used small molecule inhibitors to reduce signaling through the cytoplasmic tyrosine kinases Zap-70 or Lck. We used PP2 to inhibit Lck signaling and a chemical-genetic system to inhibit Zap-70. In this model system, mouse cells expressing an analog-sensitive mutant of Zap-70 are sensitive to inhibitor while wild-type cells are not. Surprisingly, we found that the generation of IL-17 producing Th17 cells was more sensitive to inhibition than the generation of either IFNg producing Th1 cells or IL-4 producing Th2 cells. These results raised the possibility that cells undergoing Th17 differentiation require TCR signaling of a higher intensity. To test this possibility, we analyzed the TCR-induced expression of IRF4 and Nur77-GFP, which are both inducibly expressed in relative proportion to the strength of TCR signaling. For both IRF4 and Nur77-GFP, the magnitude of expression was decreased under Th17 conditions. Additionally, increasing downstream signaling through NFAT rescued the Th17 sensitivity to inhibition. These results suggest that factors in Th17 cultures suppress TCR signal strength, thus cells stimulated under Th17 conditions experience weaker TCR signals and become more sensitive to further inhibition of TCR signaling.
IL-12 derived from CD8α+ dendritic cells drives the differentiation of innate-like T-bethigh memory-phenotype CD4+ T lymphocytes in steady state
Authors: Kawabe Takeshi , Yi Jaeu , Kawajiri Akihisa , Hilligan Kerry , Fang Difeng , Ishii Naoto , Yamane Hidehiro , Zhu Jinfang (Jeff) , Jankovic Dragana , Kim Kwang Soon , Trinchieri Giorgio , Sher Alan
Published: January 2023

CD4+ T lymphocytes consist of naïve, antigen-specific memory, and memory-phenotype (MP) cell compartments in steady state. We recently showed that MP cells exert innate-like effector function in host defense. However, it remained to be defined whether MP cell population comprises multiple subsets, and if so, what factors determine their selective differentiation under homeostatic conditions. Here we characterize MP lymphocytes as consisting of T-bethigh, T-betlow, and T-bet− subsets, with innate Th1-like effector activity exclusively associated with T-bethigh cells. We further show that steady-state differentiation of T-bethigh MP cells is promoted by IL-12-producing CD8α+ dendritic cells (DC1). Finally, we present evidence arguing that this tonic IL-12 production requires TLR-MyD88 signaling triggered independently of foreign agonists and is further enhanced by CD40-CD40L interactions with self-dependent MP CD4+ T lymphocytes. Thus, optimal differentiation of T-bethigh MP lymphocytes in steady state requires IL-12 tonically produced by DC1 in an MyD88 and CD40 signaling-dependent fashion.

This work was in part supported by the Intramural Research Program of the NIAID and NCI, NIH.
Non-typeable Haemophilus influenzae P5 binds C4b-binding protein promoting serum resistance
Authors: Thofte Oskar , Mattsson Emma , Su Yu Ching , Thegerström John , Jalalvand Farshid , Blom Anna Marie , Riesbeck Kristian
Published: January 2023

Haemophilus influenzae is a Gram-negative bacterium considered as a commensal in pre-school children, mainly giving rise to infections locally in the upper and lower respiratory tract. Historically invasive disease has been caused by encapsulated strains, mainly H. influenzae serotype b (Hib). However, since introduction of the Hib vaccine, there has been an increased incidence of invasive disease with unencapsulated non-typeable H. influenzae (NTHi). Since NTHi lacks a capsule they must rely on other mechanisms for serum resistance, including binding of complement regulators. We have previously demonstrated that binding of C4b-binding protein (C4BP) is important for NTHi-dependent complement evasion. Here we identified outer membrane protein 5 (P5) as the C4BP-binding protein in NTHi based on an outer membrane proteomic pull down-assay. NTHi P5 has previously been found to bind factor H, an inhibitor of the alternative complement pathway. We found that NTHi grown in nasopharyngeal-simulated conditions has a lower surface expression of P5. This was also corresponding to decreased serum resistance. Importantly, we found that P5-deficient NTHi mutants showed significantly decreased C4BP binding and serum resistance. Additionally, surface expression of P5 on Escherichia coli conferred increased serum resistance and C4BP binding. Our results further highlights P5 as an important protein for protecting NTHi against complement-mediated killing.
Macrophage replacement in Mycobacterium tuberculosis-induced murine lung granulomas
Authors: Herbath Melinda , Marcus Sarah , Spellman Bailey , Adler Samantha , Gilpin Trey , Thanthrige Thiunuwan , Johnson Lexi , Fabry Zsuzsanna , Sandor Matyas
Published: January 2023

Monocytes/macrophages are the dominant cell type in mycobacterial granulomas. Macrophages both provide a survival niche for the bacteria and play a central role in bacterial control. Most of them are recruited from the blood with a Ly6C+ phenotype and they differentiate into F4/80+ macrophages in the lung lesions. It is well demonstrated that monocytes have access to pre-formed granulomatous lesions, but the rate of cell recruitment and the fate of the recruited cells are not well understood.

Previously, testing BCG-induced liver granulomas using a transplantation model, we detected continuous cell replacement in the preformed lesions. Here, using intravital staining and cytofluorimetric detection of CD45+ blood cells in combination with IHC, we show that CD11b+ cells can access the lung parenchyma and Mtb-induced lesions over the course of 24 hours in Mtb H37Rv infected animals. We measured the level of monocyte/macrophage infiltration in both acute and chronic infection in C57BL/6 and C3HeB/FeJ mice. Although monocytes have a short lifespan in the blood, using intravenous cell transfer of tdTomato-expressing sentinel cells we show that they can live longer than a week in the lung lesions of Mtb-infected animals. Interestingly, a population of adoptively transferred CD11b+tdTomato+ cells can be detected for more than 10 days in the blood vessels in the infected lung. Gene expression profiling of recently or earlier recruited (CD11b+F4/80− or CD11b+F4/80+) cells sorted from the lung shows differential inflammatory gene expression between these populations.

The continuous cell replacement that is required to maintain granulomatous lesions offers a novel possibility to manipulate granuloma pathology for a therapeutic advantage.
Does Dysbiosis Drive CVID Enteropathy by Enhancing Bile Acid Cytotoxicity?
Authors: Mohammed Ahmed Dawood , Chatzistamou Ioulia , Roland Mary , Jolly Amy , Schoettmer Lillian , Nagarkatti Mitzi , Nagarkatti Prakash , Kubinak Jason L.
Published: January 2023

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by defects in the humoral immune response. CVID patients commonly present with a gastrointestinal enteropathy characterized by intestinal malabsorption. CVID is linked with abnormal gut microbiota composition (i.e. dysbiosis), which may be a contributing factor to CVID enteropathy. Recently, we demonstrated that CD19−/− mice represent a spontaneous animal model of CVID enteropathy that develop an associated dysbiosis. We now expand upon this work to identify how microbial dysbiosis may contribute to CVID enteropathy. We first demonstrate a significant association between dysbiosis in CD19−/− mice with defects in bile acid (BA) homeostasis (increased luminal BA titers and decreased abundance of conjugated BA species). Enteropathy in CD19−/− mice is associated with enhanced apoptosis of intestinal epithelial cells (IECs). Adoptive transfer of WT B cells (but not CD19−/− B cells) reduces luminal BA concentrations, protects IECs from apoptosis, and resolves enteropathy, indicating that functional B cells are important regulators of BA homeostasis and gastrointestinal health. Additionally, dietary supplementation with a taurine conjugated BA also protects animals from disease demonstrating that enhanced BA deconjugation enhances disease severity. Finally, direct ablation of the ability of a model commensal microbe to deconjugate BAs also rescues animals from disease. Collectively, these data suggest that enhanced bacterial deconjugation of BAs in the gut contributes to CVID enteropathy by enhancing BA cytotoxicity to IECs. Thus, microbial BA deconjugation may represent a novel therapeutic target for the treat of CVID enteropathy.