bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential gene expression profile analysis of RIG-I- versus type I IFN-stimulated primary human nasal epithelial cells and protection from influenza virus replication
Authors: Hartmann Evelyn , von Lilien Waldau Vanessa , Stümpel Jan Phillip , Ludwig Janos , Schlee Martin , Coch Christoph , Hartmann Gunther
Published: January 2023

Nasal mucosa is a key barrier against upper airway infection including influenza. Trained innate antiviral immunity has the potential to broadly protect the host from a variety of respiratory viruses. Although exposure of cells to type I IFN confers some degree of protection, direct stimulation of innate immune sensors may induce a more effective and longer lasting antiviral immunity.

In the present study we analysed the gene expression profile and functional consequence of direct activation of RIG-I by its specific ligand 5′-triphosphate RNA (3pRNA) in primary human nasal epithelial cells, and compared these effects to stimulation of other innate receptors and stimulation by type I IFN. Gene expression analysis was carried out by Lexogen QuantSeq 3′mRNA sequencing. For functional analysis, epithelial cells were exposed to H1N1 influenza virus strain A/PR/8/34. Viral replication was analysed by qPCR.

Gene set enrichment analysis revealed upregulation of pathways related to antiviral immune response and type I Interferon signaling. Activation of RIG-I in epithelial cells induced a broad panel of antiviral defense mechanisms which extends beyond those stimulated by recombinant type I IFN. RIG-I-stimulated cells were completely protected from influenza viral replication, while viability of the cells was not reduced. Recombinant IFN-b resulted in lower protection against viral replication compared to 3pRNA, demonstrating that RIG-I induces additional functional antiviral activities not induced by exogenous type I IFN acting alone.

Thus, our findings support the concept that stimulation of RIG in epithelial cells is a promising strategy to protect cells from viruses including emerging viral infections of the respiratory tract.
Unveiling the functional role of hsa-miR-4634 in the innate immunity against respiratory pneumovirus infection
Authors: Uche Ifeanyi Kingsley , Guerrero-Plata Maria Antonieta
Published: January 2023

Human metapneumovirus (HMPV) is a clinically important single stranded RNA virus that belongs to the genus Metapneumovirus in the new virus family, Pneumoviridae. HMPV is among the major causes several respiratory infections such as bronchiolitis and pneumonia worldwide. The lack of vaccine for this respiratory pathogen warrants further research toward understanding the host immune response induced by this pneumovirus. In fact, the molecular mechanisms regulating the antiviral response against this virus is still unclear, specifically miRNAs in HMPV infection are poorly understood. We have recently demonstrated that HMPV induces predominant expression of hsa-miR-4634 in infected cells. However, the functional role of this miRNA during HMPV infection is unknown. To determine the role of hsa-miR-4634 in HMPV infection, we use an in vitro model of human epithelial cells. Our results show that inhibition of hsa-miR-4634 significantly increased the induction of type I and III IFN responses. In addition, overexpression of hsa-miR-4634 led to an increased viral gene expression with concurrent lower expression of IRF7 and Mx1. Furthermore, based on predicted target for hsa-miR-4634, we explored the effect of hsa-miR-4634 on TLR8 activation and the role of TLR8 in HMPV infection. Using an engineered TLR8 knock out cells, we observed that TLR8 is necessary for an adequate IFN response in HMPV-infected cells. Overall, our data strongly suggest that hsa-miR-4634 impairs the innate immune response to HMPV infection. These studies contribute to our understanding of host-pneumovirus interaction, which is critical to design future therapeutic approaches against HMPV and other highly relevant viral infections.
Pro-autoimmune genetic risk variant, PTPN22 1858C>T accelerates formation of tertiary lymphoid structures in a mouse model of Type 1 Diabetes
Authors: Schmiel Shirdi E , Marquardt Kristi , Sherman Linda A
Published: January 2023

Protein tyrosine phosphatase non-receptor type 22 (PTPN22) encodes a lymphoid tyrosine phosphatase adaptor molecule involved in many biological pathways. Studies have shown an association with the minor allelic variant of PTPN22, R620W, and increased incidence of numerous autoimmune disorders, including type 1 diabetes (T1D). The exact mechanisms by which this pro-autoimmune risk allele enhances the incidence of T1D is unknown. To examine the immunological pathways altered by this risk variant, our lab introduced the murine ortholog of the human R620W allele, R619W, into the endogenous locus of Non-obese diabetic (NOD) mice that develop spontaneous T1D. NOD mice harboring this mutation (NOD 619W/W) demonstrated a similar phenotype seen in affected humans with accelerated onset of T1D and enhanced anti-islet autoantibodies (IAA). Preliminary data generated in our lab show enhanced islet infiltration in the pancreata of NOD 619 W/W compared to wild type NOD mice starting at 4–5 weeks of age. Studies have shown that infiltrating lymphocytes can organize into tertiary lymphoid structures (TLS) at the insulitis lesions of pre-diabetic mice. In addition to enhanced infiltration, NOD 619W/W pancreata also show characteristics of TLO formation at the sites of insulitis. Studies have shown that TLOs contribute to disease manifestation and prevention of TLO formation in the pancreas can prevent pathogenesis. Therefore, we are actively examining the immune cell types and pathways altered by the PTPN22 risk allele that may enhance early islet infiltration and promote the formation of TLOs in the pancreata of NOD 619 W/W hosts.
A hypomorphic Ephb2 gene variant contributes to autoimmune diabetes in NOD mice associated with altered Mapk3 activities in B-lymphocytes
Authors: Wang Qiming , Chapman Harold D , Racine Jeremy J , Serreze David
Published: January 2023

β-cell destruction in type 1 diabetes (T1D) is considered a T-cell mediated autoimmune disease, but based on studies in the NOD mouse model, B-lymphocytes also play a critical pathogenic role. They do so by serving as a preferential subset of pathogenic APC that expand diabetogenic T cells. Immunological tolerance induction mechanisms that normally eliminate or inactivate autoreactive B-lymphocytes are defective in NOD mice and also in T1D patients. We previously mapped a faulty NOD B-lymphocyte tolerance phenotype to a 6.22Mb region on Chromosome 4. With a combination of differential gene expression and ingenuity pathway analyses (IPA) we identified a hypomorphic Ephb2 allelic variant within this region on Chromosome 4 as a potential candidate gene interacting with human T1D susceptibility genes in a common pathway modulating Mapk3 activities. Ephrin-B1 ligand engagement of Ephb2 attenuated B-cell receptor (BCR) induced Mapk3 phosphorylation in vitro. We tested the candidacy of Ephb2 by introducing into NOD mice a transgenic higher expression variant of Ephb2 derived from the B6 strain (NOD-Ephb2B6). Elevated expression of Ephb2 afforded significant T1D protection due to changes in hematopoietic cells. Despite having enhanced proliferative capacity, B-lymphocytes from NOD-Ephb2B6 mice had diminished diabetogenic activity associated with attenuated Mek-Erk signaling. Introduction of the Ephb2 transgene into NOD BCR transgenic models expressing autoreactive peripherin (Per) specific Ig molecules also inhibited the accelerated T1D development characterizing these stocks. Thus, signaling via Ephb2 in B-lymphocytes can fine-tune BCR signaling that ultimately determine their diabetogenic activities.
Monocyte activation in cardiovascular disease through inflammatory signaling associated with CD11c function
Authors: Hernandez Alfredo Alexander , Foster Greg A , Dai Yao , Rivara Kathryn Rose , Bailey Keith , Singh Gagan D. , Simon Scott I
Published: January 2023

Circulating monocytes are critical in the development and progression of atherosclerotic disease. During disease, prolonged metabolic dysregulation and low-grade inflammation promote monocytosis and subsequent skewing of the circulating monocyte repertoire to favor an expanded frequency of intermediate monocytes (CD14+CD16+CCR2, iMo). Intermediate monocytes display a hallmark upregulation of β2-integrin CD11c/CD18 which works in cooperation with β1-integrin VLA-4 to mediate monocyte capture and firm arrest to endothelial VCAM-1 at sites of disturbed flow at sites of nascent plaque. Notably, the expanded frequency, quantity of iMo CD11c receptors, and CD11c/VLA-4 dependent arrest of iMo to VCAM-1 gauge the severity of cardiovascular disease and are predictive of poor clinical outcome. Using these observations, we sought to examine the functional activity of arrested iMo on a substrate of VCAM-1 under arterial shear stress through the assessment of CD11c outside-in signaling using microfluidic platforms. We find that iMo undergo rapid transmigration (~90sec) at near 100% efficiency following by characteristic ADAM17 dependent shedding of CD16 that coincided with nuclear translocation of NFκβ, and elaboration of intracellular MMP-9 and IL-1β inflammatory molecules. Phenotypic conversion towards activated M1-like macrophages was governed by an allosteric shift in CD11c from high to low affinity on a majority of adherent iMo from CAD and NSTEMI patients, but not those from age-matched healthy subjects. We conclude that CD11c is a molecular biomarker and functionally relevant inflammatory signaling molecule on iMo. Its expression and function provides the most sensitive measure for diagnosing progression of CAD to MI.
CNS HIV virus persistence irrespective of CD4 T cell trafficking
Authors: Kohlmeier Alison , Lipscomb Jonathan , Li Jin-fen , Tino Ariana Santos , Anderson Albert M , Johnson Jeffrey A
Published: January 2023

Incomplete HIV virologic suppression in the central nervous system (CNS) can cause persisting neurologic disorders even with suppressed plasma viremia. To better understand immune properties that support CNS virus persistence we characterized T cells and virus genotypes from cerebrospinal fluid (CSF) of HIV-infected participants to understand local cell trafficking behavior and the influence of combination antiretroviral therapy (ART). Compared to blood, CSF T cells expressed higher frequencies of CCR5****, CD69****, CX3CR1****, CD39**, and HLADR**(P values:*≤0.05, **≤0.01, ***≤0.001,****≤0.0001). Among CSF T cells, CD4 T cells were more likely to express CCR7****, CX3CR1****, and CD39**, and less likely to express CCR5*. CX3CR1 expression on CSF CD4 T cells correlated with CSF viral load (VL)*, whereas, CD39 expression mirrored CSF** and plasma VL*. In the absence of ART, CSF HIV (VL>900 copies/mL) shared majority sequence similarity to plasma virus demonstrating a migratory influence of susceptible cells into the CNS. However, CSF CD4 T cells from persons on ART expressed decreased CCR5**, CX3CR1****, HLADR****, and CD39***, regardless of viral suppression, while matched-PBMC CD4 T cells did not exhibit these trends. CSF CD4 T cells expressed features consistent with immune-restricted localization but with a migratory profile indicative of immune surveillance function and lymphoid trafficking. ART use associated with reduced migratory properties regardless of CNS viremia, suggesting ongoing CNS HIV replication under therapy involves local susceptible cells distinct from circulation. This CSF cellular profile could serve as a reference to target biomedical interventions that improve HIV suppression in the CNS.
Human interleukin-2 receptor beta mutations associated with defects in immunity and peripheral tolerance
Authors: Zhang Zinan , Gothe Florian , Pennamen Perrine , James John , McDonald David , Mata Carlos , Modis Yorgo , Alazami Anas , Acres Megan , Haller Wolfram , Bowen Claire , Doffinger Rainer , Sinclair Jan , Brothers Shannon , Zhang Yu , Matthews Helen , Naudion Sophie , Pelluard Fanny , Alajlan Huda , Yamazaki Yasuhiro , Notarangelo Luigi , Thaventhiran James , Engelhardt Karin , Al-Mousa Hamoud , Hambleton Sophie , Rooryck Caroline , Smith Kenneth G.C. , Lenardo Michael J
Published: January 2023

Interleukin-2, which conveys essential signals for effective immunity and immunological tolerance, operates through a heterotrimeric receptor. Genetic deficiency of the alpha or gamma chain causes debilitating disease. Here we identify human interleukin-2 receptor (IL-2R) beta chain (IL2RB) gene defects as a cause of life-threatening immune dysregulation. We report three homozygous mutations in the human IL2RB gene of eight individuals from four consanguineous families that cause disease by distinct mechanisms. Nearly all patients presented with autoantibodies, hypergammaglobulinemia, bowel inflammation, dermatological abnormalities, and cytomegalovirus disease. Patient T lymphocytes lacked surface expression of IL-2Rbeta and were unable to respond normally to high-dose IL-2 stimulation. By contrast, patient natural killer (NK) cells retained partial IL-2Rbeta expression and cytotoxic function. IL-2Rbeta loss of function was recapitulated in a recombinant system, in which endoplasmic reticulum sequestration was revealed as the mechanism by which certain missense mutations cause disease. Hematopoietic stem cell transplant resulted in resolution of clinical symptoms in one patient. The hypomorphic nature of this disease highlights the significance of variable IL-2Rbeta expression between different lymphocyte subsets and during their maturation as a means of modulating immune function. Insights from these patients can inform the development of IL-2-based therapeutics for immunological diseases and cancer.
Development of a high throughput, bead-based flow cytometry panel for T-helper cell cytokine quantitation
Authors: O’Rourke John , Weldon Caroline , Keenan Lisa , Carter mark B
Published: January 2023

Upon T-cell activation, naïve CD4+ cells differentiate into various types of T-helper (Th) cells, which is influenced by the cytokine milieu present during antigen engagement and is further driven by autocrine mechanisms. Th subsets orchestrate adaptive immune responses to specific pathogens and dysregulation of Th cells are major contributors to autoimmune and allergic diseases. Th subsets can be identified, in part, by their unique cytokine secretion profile.

To measure cytokine secretion from Th cells, Intellicyt has developed multiplex bead-based panels (QPanels) to quantitate Th1 and Th2 (4-, 6- or 9-plex) and Th1, Th2, & Th17 (7-plex) associated cytokines in cell supernatants. These miniaturized assays require sample volumes of only 10 μl. Data acquisition is performed using 96 or 384 well plates on the iQue Screener platforms. Pre-mixed reagents and cytokine standards, and templated data analysis, including event gates, gating strategy, 4 parameter standard curves and sample quantitation are auto-generated to allow for ease of use.

For proof of concept studies, peripheral blood mononuclear cells (PBMCs) from 3 donors were cultured in media containing human sera and IL-2. Cells were stimulated with anti- CD3/CD28 beads and cytokine secretion was measured 1, 2, and 3 days post-stimulation. Donor to donor variation and temporal differences in secreted cytokine levels were observed. All donor PBMCs secreted IFNγ, GM-CSF and TNFα by 3 days post-stimulation. Notably, one donor showed a more robust Th2 (IL-4, IL-6, and IL-10) and Th17 (IL-17A) response compared to other donors. These data highlight the use of QPanels in T cell screening, which allows easy assessment of effector function and the identification of Th subsets.
Function of Protein Kinase CK2 in Innate Immune Cells in Neuroinflammation
Authors: Qin Hongwei , Yang Wei , Yan Zhaoqi , Benveniste Etty
Published: January 2023

Immune dysregulation is critical to the pathogenesis of autoimmune diseases, including multiple sclerosis (MS). Studies in MS patients and an animal model of MS, experimental autoimmune encephalomyelitis (EAE), have highlighted the importance of both innate and adaptive immune responses in MS/EAE pathology. Antigen-presenting cells of the innate immune system, especially dendritic cells (DCs), not only activate naive CD4+ T cells by antigen presentation, but also control T cell differentiation by expressing co-stimulatory molecules and cytokines. Our current studies demonstrate that Tamoxifen-induced global deletion of CK2a in CK2αfl/flERT2Cre mice results in delayed onset and significantly slower progression of EAE, which is not observed in CK2α T cell-specific deleted CK2αfl/fldLckCre mice, which exhibit lower EAE clinical scores during the peak and resolution phases of disease. EAE mice with global CK2α deletion express reduced pathogenic Th1 and Th17 cells, and enhanced anti-inflammatory Treg cells. We speculate that CK2 kinase activity in DCs is indispensable for the initiation and progression of EAE. Specifically, our data indicate that deletion of CK2α reduces the activation of bone marrow-derived DCs, but not macrophages, by suppressing the expression of CD40, TNF-α, iNOS and IL-12 and activation of AKT and p38. Furthermore, we found that activation of DCs is prevented in EAE mice with global CK2α deletion, which correlates with differential regulation of Th1, Th17 and Treg cells. Collectively, our findings establish a novel regulatory role for CK2 kinase in DCs during EAE initiation and progression, which may provide evidence to establish CK2 as a potential novel therapeutic target for MS treatment.
Perturbation of Developmental Hematopoiesis Shapes Lung ILC2 Immune Memory
Authors: Lopez Diego A , Beaudin Anna E.
Published: January 2023

Our lab has previously identified a developmentally-restricted hematopoietic stem cell (drHSC) that only exists in the perinatal period and specifically gives rise to innate-like lymphocytes. The identification of a developmentally-limited cell of origin for innate-like immune cells that persist across the lifespan defines a “critical window” for immune development, in which the phenotype of the adult immune system can be shaped by extrinsic inputs. To test how developmental perturbation during this critical window drives immune dysfunction, we examined underlying changes to innate-like immune cells in the lung and susceptibility to airway dysfunction following maternal immune stimulation. Lung type-2 innate lymphoid cells (ILC2s) are recognized as potent producers of IL5 and IL13, cytokines important for eosinophil recruitment, activation and goblet cell hyperplasia during allergic airway inflammation. Surprisingly, maternal immune stimulation via a single low-dose injection of poly (i:c) at mid-gestation robustly increased the cellularity of lung ILC2s in offspring at postnatal day 14, concomitant with drHSC and ILC2 progenitor expansion during fetal development. Additionally, lung ILC2s exhibited heightened IL5 and IL13 production upon in-vitro stimulation with PMA and ionomycin in poly (i:c) perturbed offspring. Ongoing experiments examine how altered ILC2 functionality, as a result of developmental perturbation, underlie susceptibility to allergic asthma in response to secondary house-dust mite immune stimulation. Together, our data suggest perinatal immune perturbation may contribute to allergic asthma susceptibility by altering drHSC establishment and training lung ILC2 “innate immune memory”.
m6A RNA modification controls transitions between quiescence and proliferation during B cell development
Authors: Zheng Zhong , Hsu Phillip J , Cui Xiao-Long , Tan Haiyan , Zhang Linda , Sun Hui-lung , Zhang Michelle , Peng Junmin , He Chuan , Huang Haochu
Published: January 2023

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic mRNA. Recent conceptual and technological advances have sparked extensive research interests in the functional roles of m6A. The writers, erasers and readers of m6A have been identified and characterized in a number of biological processes in recent years. However, the physiological roles of m6A and its effectors in lymphogenesis remained unknown. Here, we report that loss of METTL14, a core component of the m6A writer complex, in mouse B lineage cells blocked B cell development. Deletion of METTL14 in developing B cells caused dramatic reductions in the mRNA m6A level, together with an incomplete blockage during the transition from the quiescent pro-B stage to the proliferating large pre-B stage as well as a complete blockage during the proliferation-to-quiescence transition between large pre-B and small pre-B stages. We found that >67% transcripts contain m6A in developing B cells, suggesting that m6A might be widely involved in gene expression regulation. m6A controlled the quiescence-to-proliferation switch partially through its cytoplasmic reader YTHDF2, as deleting YTHDF2 partially mimicked the earlier incomplete blockage between pro-B and large pre-B stages due to METTL14 deficiency, but did not cause the later proliferation-to-quiescence blockage. Altogether, our data identifies m6A, which specifically controls transitions between quiescence and proliferation, as a critical new layer of gene expression regulation in B cell development.
Selective inhibition of gelatinases modulates cellular energetics in CD4+ T-cells
Authors: Onwuha-Ekpete Lillian C. , Tokmina-Roszyk Dorota , Fields Gregg B.
Published: January 2023

The gelatinases, MMP-2 and MMP-9, are proteolytic enzymes with diverse physiological roles. They are linked with various aspects of the immune response, such as leukocyte trafficking and cytokine activation. However, their dysregulation is associated with various pathologies such as metastasis in cancer progression and blood-brain barrier disruption in multiple sclerosis. Much of what is known about the gelatinases is affiliated with their activities in the extracellular milieu. However, recent studies have suggested that they contribute to normal intracellular function of various immune cell types, including T-cells. CD4+ T-cells play a key role in linking the innate and adaptive arms of the immune response, as well as determining the character of the immune response.

Our findings show, for the first time, that intracellular gelatinase activity can modulate T-cell bioenergetics, affecting both oxidative phosphorylation and glycolytic pathways and resulting in a mitigated CD4+ T-cell response to stimulation. Upon stimulation, CD4+ T-cells lacking gelatinase activity demonstrate a reduced capacity to function as determined by the expression of early activation markers, proliferation, and the production of effector cytokines. In addition, RNA-seq analysis of control and gelatinase inhibited CD4+ T-cells highlight key proteins associated with T-cell receptor signaling, cytokine production, and metabolism. These results suggest that gelatinases can be immunomodulatory agents in CD4+ T-cell mediated diseases, such as multiple sclerosis, and thus have therapeutic potential.
T-cell microvilli constitute immunological synaptosomes that are specialized for intercellular communications
Authors: Jun Chang-Duk , Kim Hye-Ran
Published: January 2023

Lymphocytes contain abundant flexible projections termed microvilli, the inside of which contain many parallel bundles of actin filaments that extend the cell membrane in the form of a finger. Previous reports demonstrated microvilli on T cells have been proposed to survey surfaces of antigen-presenting cells (APC) or facilitate adhesion under flow. However, in addition to their key role in antigen sensing on APCs, whether they serve essential functions during T cell activation remains largely unclear. In this study, we observed that single T cell contacts with APCs occur through microvillar extensions, which appear to serve as locations for sequestration of immunologically important molecules, including TCR complexes, costimulatory and adhesion molecules, and various cytokines. Strikingly, we found that microvilli were separated from the T cell body by the combined action of two independent mechanisms (trogocytosis and membrane budding) and are deposited at the surface of cognate APCs, thereby potentially acting as the most efficient and effective means of delivering T cell messages to cognate APCs. Consistent with this potential role, these T cell microvilli-derived particles (TMPs) were independently capable of activating cognate dendritic cells (DCs). Therefore, our findings suggest that T cell microvilli might serve as ‘immunological synaptosomes’ with TMPs as a novel class of membrane vesicles serving as conveyors of T cell messages or traits to cognate APCs.
Regulatory T cells perform antigen specific suppression by depleting cognate peptide-MHC class II via trogocytosis
Authors: Akkaya Billur , Oya Yoshihiro , Akkaya Munir , Al Souz Jafar A , Holstein Amanda H , Kabat Juraj , Kamenyeva Olena , Dorward Dave , Glass Deborah , Shevach Ethan M
Published: January 2023

Tregs are professional suppressors of the immune response, yet their mechanism of action in vivo remains unclear. Using electron microscopy and dynamic intravital two-photon microscopy, we revealed that unlike effector T cells, Tregs displayed a distinct morphology with membrane projections that resemble filopodia at the DC binding site increasing the surface area of the contact dramatically. Despite the intense contact with DC, Tregs only abolished the interactions of effector T cells with DC when the effectors shared the same antigen specificity with Tregs. However, this cessation of the effector T cell interactions was not due to a physical hindrance created by the superior binding ability of Treg since the antigen specific inhibition of T cell-DC interaction was still observed after Tregs are removed from the environment. Instead, the antigen-specific inhibition was secondary to the removal of cognate peptide-MHC Class II (pMHCII) from the surface of DC as evidenced by the strictly antigen-specific depletion of the pMHCII from double antigen pulsed DC. pMHCII capture was only performed by antigen specific Treg, not by polyclonal Treg and it was not secondary to the interaction of CTLA-4 with B7 molecules suggesting the functional importance of TCR in Treg suppression. Prominent members of the B7 family, that are not de novo expressed in Tregs, such as CD86, ICOSL and PD-L2 were acquired alongside the TCR, suggestive of a process that includes a membrane transfer event called trogocytosis. Altogether, we propose antigen specific depletion of peptide-MHCII complexes via trogocytosis as a new mechanism for Treg-mediated suppression.
Human natural regulatory T cells recognize peptides of the heavy constant region of immunoglobulins presented by IgG+ B cells
Authors: Hsieh Li-En , Sidney John , Behnamfar Negar , Burns Jane C. , Boyle David , Firestein Gary S. , Sette Alessandro , Franco Alessandra
Published: January 2023

Human natural regulatory T cells (nTreg) recognize conserved peptides derived from the heavy constant region of IgG (Fc). The fine specificity of nTreg and the immunodominance of peptides was determined in healthy donors and rheumatoid arthritis (RA) patients. Eight Fc peptides were identified as highly immunogenic in all subjects with similar ranking and four of eight bound multiple class II HLA alleles. These Fc peptides are presented by IgG+ B cells by internalization and processing of surface IgG and are recognized by Fc-specific nTreg from both healthy donors and RA patients. However, exogenous IgG processed by antigen presenting cells (APC) led to the expansion of this nTreg population only in healthy donors. While nTreg from patients with RA responded to Fc peptides, the response to exogenous IgG presented by APC was compromised. These results demonstrate a hole in the central adaptive immune regulation in RA.
HIF-1α is essential for T cell suppression by murine LP-BM5 retrovirus infection-augmented Monocytic Myeloid Derived Suppressor Cells
Authors: Green Kathy A , Cramer Robert A , Green William R
Published: January 2023

Compared to naïve mice, monocytic myeloid-derived suppressor cells (M-MDSCs) increase in number, and suppressive efficiency, after infection with the immunodeficiency causing mouse retrovirus, LP-BM5. In ex vivo suppression assays, M-MDSCs, enriched from spleens of LP-BM5 infected mice inhibited both T- and as a novel finding, B-cell responsiveness. For M-MDSC suppression of T-cell proliferation and IFN-gamma production, we previously reported the mechanism to be almost completely iNOS/NO dependent. In contrast, the suppression of B-cell proliferation and IL-10 secretion by predominantly T2-B cells was only ~50% iNOS/NO dependent, with V-domain Ig suppressor of T cell activation (VISTA), superoxide and peroxynitrite also playing substantial effector roles. In light of recent reports on the Hypoxia-inducible transcription factor 1, α subunit (HIF-1α) regulating the function and differentiation of MDSCs, and reports of its role as a principal regulator of NO production, we describe here the characterization of M-MDSCs from myeloid-specific HIF-1α deficient, vs. w.t., B6 mice. As early as 5 weeks post infection, enriched M-MDSC preparations from HIF-1α deficient mice were significantly impaired functionally, in some cases with a total inability, to suppress in vitro T cell responsiveness. However, these same M-MDSC preparations suppressed B-cell responsiveness at a level very similar to that of control w.t. M-MDSCs. Experiments are in progress to further understand the role HIF-1α plays in the impairment of M-MDSC ex vivo suppression of T cell proliferation and function, including a possibility that HIF-1α regulates iNOS/NO production in M-MDSCs only when in the process of suppressing T- but not B-cell targets.
Magnolol attenuates the cisplatin-induced muscle wasting via M2c macrophage activation
Authors: Lee Chanju , Park Seon-young , Min Daeun , Bae Hyunsu
Published: January 2023

Cancer chemotherapy induces malnutrition and rapid loss of muscle mass which directly restricts the daily activities leading to poor quality of life and increased mortality. Although the hormone-related therapies have been used to improve appetite and nutritional status, but current treatments are considered palliative. Thus, the maintenance of skeletal muscle mass without side effects is essential in sarcopenia. Magnolol from Magnolia officianlis has been reported to have several pharmacological effects including anti-cancer and anti-inflammatory effects, but the correlations of immune cell infiltrations and muscle protection in muscle atrophy are not well understood. In the present study, we investigated the effects of magnolol on muscle wasting and macrophage subtypes in cisplatin-induced sarcopenia mice model. We showed that magnolol significantly attenuated the body weight loss and muscle loss induced by cisplatin injection. The diameter of tiabialis anterior (TA) muscle was markedly increased by magnolol treatement compared with cisplatin group. Importantly, magnolol increased the percentages of total macrophages without affecting the percentages of T cells. Furthermore, magnolol reduced the CD206−CD163− M1 inflammatory macrophages whereas the CD206+CD163+ M2c tissue reparative macrophages were increased in CD45+CD11b+F4/80+ macrophages. We also showed that cisplatin increased the M1/M2 ratio and magnolol significantly lowered the M1/M2 ratio in TA muscle. Taken together, magnolol may be a promising chemoprotective agent for preventing muscle atrophy by upregulating M2c macrophages.
Knockdown of circular RNA FSCN1 impairs dendritic cell immune function through regulating the NFKB and Foxo3 signaling pathways
Authors: Zheng Xiufen , Zhu Cuilin , Su Yale , Wang Bowen , O’Brien Dana
Published: January 2023

Circular RNAs (circRNAs) that are a new class of endogenously expressed non-coding RNAs produced from back splicing with a covalently closed loop structure have been emerging as an important gene regulator in the physiological and pathological development of cells. It remains unknown about roles of circRNAs in DCs. The objective of the study is to investigate the impact of circular RNA FSCN1 (circFSCN1) on dendritic cell immune function. Bone marrow derived dendritic cells were cultured in vitro and circRNA expression was detected by circRNA microarrays and qRT-PCR. The effect of circFSCN1 on DCs was studied. We found that the expression profiles of circRNAs were significantly different in mature immunogenic DCs v.s immature immunosuppressive DCs. circFSCN1 was the most significantly highly expressed in mature DCs compared with immature DCs. Treatment with immunosuppressive cytokines TGF β and GDF15 reduced circFSCN1 expression in DCs. Knockdown of circFSCN1 using siRNA reduced the phosphorylation of Rel Ap65, but increased phosphorylated Foxo3. Silencing of circFSCN1 impaired DCs to activate T cells, changed inflammatory cytokine production and enhanced Treg generation whereas circFSCN1 siRNA did not affect DC maturation. In conclusion, it is a first report to demonstrate that circFSCN1 is essential for DC immune response.
Student Outcomes in a CURE Course Using Simple Model Organisms to Characterize Host-Pathogen Interactions
Authors: Snyder Michelle , Trauth Amy
Published: January 2023

Course-based undergraduate research experiences (CUREs) can increase the inclusivity of scientific research by breaking down both barriers associated with student awareness and confidence in seeking research experiences, as well as faculty biases in selection of student researchers. We developed a CURE using the social amoeba Dictyostelium discoideum and its bacterial prey Escherichia coli as a model for host-pathogen interactions. D. discoideum phagocytize bacteria and can be used as a simple model to characterize innate immune processes. Our CURE course focuses on characterization of genes potentially associated with resistance to phagocytic killing, which were identified in a screen for mutant E. coli strains with enhanced survival upon D. discoideum phagocytosis. These mutant E. coli strains were sequenced, and students in the course use bioinformatics tools to identify mutated genes. From there, students work with E. coli strains deficient for the mutated genes, performing assays to characterize both bacterial phenotypes as well as D. discoideum phagocytic and signaling responses to these mutant strains. Using various tools, including pre- and post-tests, interviews and the SURE survey to assess student outcomes, we found significant gains in student knowledge and skills, as well as student-reported gains in knowledge of the research process, confidence in working in a research team, and ability to interpret results. Our CURE course was used as a model for implementation of the TU-Research Enhancement Program (TU-REP), which is funded by the HHMI Inclusive Excellence initiative, and seeks to transform the scientific curriculum at TU by offering opportunities for students to engage early and often in authentic research.
Using IF AT strategy in the Dental Immunology curriculum has a beneficial impact on the performance of second year dental students
Authors: Alard Pascale
Published: January 2023

When considering health professions education, providing tools that allow students to become life-long learners is critical. Acquisition of knowledge is more effective in the form of practice activities where learners can think critically and solve problems. Learning Immunology not only requires memorization of new terminology, but also learning concepts. The Immunology program for dental students is very intensive as they learn a tremendous amount of information in a very compressed period of time. In this study, students were divided into groups of 4 – 6 who worked on several questions about the material covered that week using the Immediate Feedback Assessment Technique (IF AT). Students in each group discussed the possible answers before selecting the final answer. If the answer was incorrect, they could choose another answer until they were successful. At the end of each session, the answers and concepts behind the questions and answers were thoroughly discussed. Students appeared to be engaged, and in response to a survey, most found the exercise helpful in achieving a deeper understanding of the concepts as well as good practice for the exam. Their performance on the exam was better than the previous year, with a 50% decrease in students scoring <70, and a 40% increase in students scoring >87. Since the same group of students did not perform better on the following exam on Microbiology, where the instructors did not include this learning strategy, we ruled out the possibility that this group was stronger overall. In addition, the order of the questions and the time spent on the exam had no impact on overall performance. In conclusion, holding IF AT sessions before the exam is highly beneficial and improves the performance of the students.
An Immunological basis for improving interest and participation of underrepresented student groups in High school science classes
Authors: Sika-Paotonu Dianne , Anderson Jane , Cashin Philippa , Cockburn Alana
Published: January 2023

In New Zealand (NZ) children and young people of Māori and Pacific Peoples heritage are underrepresented in high school science and higher education science, medical and health courses. To help support improved engagement and interest in laboratory science learning that was clinically relevant amongst high school students from underrepresented groups that included Māori and Pacific students, a laboratory workshop session was arranged to incorporate a visit from a Pacific Biomedical Scientist teaching laboratory content with an Immunological focus. This visit was organised during the workshop session to support the Immunologically based teaching and also to field questions from the students directly. Amongst other discussion points, the implications of Penicillin as an antibiotic of choice to prevent the autoimmune response to Group A Streptococcal bacteria known as Acute Rheumatic Fever (ARF) was also discussed. It is recognised that Indigenous Māori and Pacific population groups remain disproportionately affected by Acute Rheumatic Fever (ARF) and Rheumatic Heart Disease (RHD) in NZ and in the Pacific Region. Feedback from the high school students indicated that a key source of interest remained the clinical relevance of the scientific discussion emphasised with the Pacific Biomedical Scientist. This work demonstrated the value of Immunology incorporation within the laboratory focus for this wider context and also the role modelling concept for the Pacific students in particular, both as important factors to support heightened interest and engagement levels of underrepresented high school students in science.
Manipulating peripheral blood cell populations to mimic immune disease states: a novel approach to teach flow cytometry to undergraduate immunology students
Authors: Costabile Maurizio , Nguyen Hong , Kenyon Amanda
Published: January 2023

Flow cytometry (FC) allows for the quantification of individual cell populations whose levels are known to change in disease states, such as, the reduction in CD4+ T cells in AIDS. While FC is a powerful technique, it can be difficult to teach its principles and applications to undergraduate students. Assessing changes in cell populations is an excellent approach to teach FC, however obtaining real patient samples is not often possible. In addition, hands on use of a flow cytometer by students is difficult or impossible to achieve when class sizes are large. To address these challenges, we taught the principles of FC, sample preparation (Week 1) and data analysis (Week 2) of mock “control” and “patient” samples. In Week 1, the theory of FC was covered and the students labelled “control” and “patient” blood with anti-CD3, 4, 8, 14, 19 and 66 mAbs. In Wk 2, students were provided with data files where the levels of major cell populations had been manipulated using magnetic beads to mimic immune diseases states. Using written and video instructions for the software and supplied reference ranges, and using a PC, were asked to identify the disease status of their patient sample. Student perceived understanding of FC was found to significantly increase after the practical session, and student feedback was also overwhelmingly positive. The manipulation of cell populations in whole blood to mimic disease states is an excellent way to teach undergraduate students the major principles of FC. These include labelling of cells, learning to use the software, interpreting the data and disease diagnosis. We will continue to expand our list of disease conditions. All data files are freely available to any teaching staff who may wish to use them as part of their teaching.
Novel role of zinc homeostasis in IL-23 regulation and host defense following bacterial infection
Authors: Hall Sannette C , Smith Deandra R. , Katafiasz Dawn M. , Bailey Kristina L. , Knoell Daren L.
Published: January 2023

Zinc homeostasis is crucial for immune cell activation and intracellular zinc is tightly regulated by zinc transporters. Zinc deficiency is associated with increased prevalence of respiratory bacterial infections. Although the association between zinc and immunity is well established, the mechanisms by which zinc protects against bacteria remain to be elucidated. In human monocytes the zinc transporter Zip8 regulates inflammation in a zinc-dependent manner after LPS exposure. Given the critical role of APCs (macrophages and dendritic cells) in guiding the immune response against bacteria, we sought to determine the role of Zip8 in coordinating the immune response after bacterial exposure.

In a novel myeloid-specific Zip8KO mouse model we observed that intranasal instillation of S. pneumoniae resulted in a significant increase of infiltrating neutrophils in the airways of Zip8KO mice when compared to WT counterparts. Analysis of BAL fluid revealed elevated levels of CXCL1 and IL-23 in the Zip8KO mice. To determine whether dendritic cells potentially play a role in the observed response, BMDCs were generated from Zip8KO and WT mice. LPS stimulation produced mature BMDCs with high CD11c and MHC-II expression in both groups. No significant changes were observed in the production of most cytokines examined (TNF-α, CXCL1, IL-6, IL-12), except for IL-23 which was consistently increased in the stimulated Zip8KO cells in comparison to WT control DCs. These results potentially reveal a vital axis that couples zinc homeostasis to IL-23 production in myeloid cells and ultimately host defense against invasive bacterial pathogens. To what extent this alters pathogen clearance and host recovery is the focus of future studies.
Alarmin(g) outcomes of intracellular LPS sensing
Authors: Rathinam Vijay , Russo Ashley , Menoret Antoine , Rabinovich Gabriel , Duduskar Shivalee , Deshmukh Sachin , Vella Anthony T
Published: January 2023

Intracellular sensing of LPS by inflammatory caspases such as caspase-11 has emerged as a key mechanism of innate immune activation during Gram-negative bacterial infections. Cytosolic recognition of LPS by caspase-11 leads to inflammatory cell death or pyroptosis and maturation of IL-1 cytokines. Remarkably, the activation of inflammatory caspases also results in the extracellular release of alarmins and damage associated molecular patterns (DAMPs). These alarmins and DAMPs lack leader signals for classical secretion and are therefore released via a caspase-11 mediated unconventional secretory mechanism. Alarmins and DAMPs—structurally diverse molecules participating in various cellular processes under steady state conditions—are released rapidly upon infections and tissue damage. In the extracellular milieu, these proteins elicit inflammatory reactions by regulating pattern recognition receptor signaling and recruiting innate cells. Despite their potential roles in sepsis, the identity of alarmins and DAMPs released as a result of cytosolic LPS sensing and their contribution to sepsis are poorly understood. Addressing this knowledge gap, we employed an unbiased proteomic approach involving two-dimensional liquid phase fractionation system and mass spectrometry and identified alarmins released in response to caspase-11 activation by cytosolic LPS. In this presentation, the molecular basis of secretion of a candidate alarmin in vitro and in vivo as well as its function in murine models of host defense will be discussed.
Redefining the TIR domain: From Axon Degeneration to Innate Immunity and Beyond
Authors: Essuman Kow , Wan Li , Summers Daniel W. , Anderson Ryan G. , Sasaki Yo , Mao Xianrong , Yim Aldrin Kay Yuen , Monteiro Freddy , Chung Eui-Hwan , Osborne-Nishimura Erin , Dangl Jeffery , Nishimura Marc , DiAntonio Aaron , Milbrandt Jeffrey
Published: January 2023

The Toll/Interleukin-1 Receptor (TIR) domain is an evolutionary ancient protein domain, present in numerous receptors and adaptor proteins, and is the signature-signaling domain of Toll-Like Receptors (TLRs). In animals, these TIR domains generally serve as scaffolds that promote the assembly of signaling complexes to trigger activation of pro-inflammatory cytokines and other defense-related products. In plants, TIR domain proteins are known to mediate disease resistance against pathogens, and trigger hypersensitive cell death. However, their mechanism of action has remained elusive. In bacteria, TIR domains have been associated with virulence and defense against some viruses. Here, we redefine the canonical scaffolding function of the TIR domain by showing that TIR domains can possess intrinsic enzymatic activity, and constitute a family of enzymes that cleave the essential metabolic cofactor Nicotinamide Adenine Dinucleotide (NAD+). We identify the TIR domain of SARM1 as the founding member of the TIR NADase family that triggers axon self-destruction upon axonal injury. We show that the TIR enzymatic activity in conserved in bacteria, and archaea, where in bacteria this NADase activity has recently been linked to defense against viruses. Finally, we extend our findings to plant innate immunity, and show that plant TIR-domain Immune Receptors trigger cell death and transduce recognition of pathogens into an immune response via this conserved enzymatic activity. Altogether, our findings establish TIR domain proteins as a new family of metabolic enzymes, and we posit that the scaffolding function in TLR signaling likely represents a repurposing of this evolutionary ancient enzymatic function.
TRAF3IP3 negatively regulates type I interferon signaling by suppressing TBK1
Authors: Deng Meng , Tam Jason W. , Guo Haitao , Zhang Lu , Davis Beckley K , Brickey June , Sun Shaocong , Ting Jenny P
Published: January 2023

Type I interferon (IFN) signaling is important for antiviral and autoimmune response, and it is subjected to tight control, however molecular mechanisms to tune down these pathways is incompletely understood. Upon sensing cytosolic viral RNA, retinoic acid-induced gene I (RIG-I) and melanoma-differentiation-associated gene 5 (MDA5) interact with mitochondrial antiviral signaling protein (MAVS) and activate TANK binding kinase 1 (TBK1) to induce IFNs. A golgi-associated factor, TRAF3-interacting protein 3 (TRAF3IP3), has been found to be crucial for thymocyte development and T regulatory cell functions. However its role in myeloid cells has not been explored. We find that the overexpression of TRAF3IP3 suppressed cytosolic poly(I:C), 5’ ppp-dsRNA, and vesicular stomatitis virus (VSV) triggered IFN production, whereas deficiency of Traf3ip3 potentiated viral RNA triggered IFN production. In support of the in vitro data, Traf3ip3-deficient mice were infected with VSV and found to exhibit enhanced susceptibility to VSV challenge. Mechanistically, TRAF3IP3 interacted with TBK1 and targeted TBK1 for ubiquitination and degradation. These results uncovered a previously unrecognized role of TRAF3IP3 in the regulation of RNA induced IFN pathway.