bluuebunny/crossref_metadata_2025 huggingface dataset:
Natural antibodies recognize cytoplasmic myosin that is cleaved and transferred to the cell membrane during apoptosis (89.4)
Authors: Cui Xiaoxuan , Zhang Lu , Magli Amanda , Catera Rosa , Kolitz Jonathan , Allen Steven , Rai Kanti , Chiorazzi Nicholas , Chu Charles
Published: January 2023

Natural antibodies (Abs) and Abs from patients with chronic lymphocytic leukemia (CLL) bind a subset of apoptotic cells that expose intracellular myosin on their surface. To test the mechanisms that produce myosin exposed apoptotic cells (MEACs), Jurkat T cells were induced to undergo intrinsic (spontaneous or camptothecin-induced) or extrinsic (Fas ligand- or anti-Fas-induced) apoptosis. After prolonged incubation by all methods, a high percentage of apoptotic cells became MEACs. Thus, both intrinsic and extrinsic apoptotic pathways lead to MEAC formation, suggesting that a common downstream mediator is involved. Consistent with this, a caspase-3 inhibitor reduced both apoptosis and MEAC formation, whereas a caspase-1 inhibitor had no effect. To test if caspase-3 cleavage resulted in membrane exposure of myosin, cytoplasmic and membrane protein extracts were examined for caspase-3 cleaved myosin fragments. MEAC extracts predominantly exhibited the expected 149 and 94 kDa myosin fragments in the membrane fraction. In contrast, live cell extracts exhibited predominantly the full-length myosin protein in the cytoplasmic fraction. These results indicate that MEAC formation occurs during intrinsic and extrinsic apoptosis when caspase-3 is activated. Caspase-3 cleavage of myosin produces fragments that reside in the cell membrane, where they become available to react with CLL and natural Abs.
ANCA antigen proteinase 3 acts as a phagocytotic receptor of bacteria via PAR2 pathway in human neutrophils. (89.40)
Authors: Kim Yongchul , Choi Youngnim , Shin Ji Eun , Kim Young suk
Published: January 2023

The molecular mechanisms underlying the phagocytosis of bacteria by neutrophils are pooly understood. We previously reported the efficient uptake of Streptococcus sangunis by human neutrophils in the absence of opsonins. To characterize the phagocytosis receptor, protein lysates of neutrophils and HL-60 cells were subjected to affinity chromatography using epoxy beads immobilized with S. sangunis. Denaturing electrophoresis of the eluted proteins revealed several neutrophil-specific bands. One of these proteins was identified as proteinase 3 (PR3) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which was confirmed by western blotting. PR3 is a serine proteinase of the azurophil granules and also co-expressed with NB1, a glycosylphosphatidylinositol (GPI)-linked receptor, on the surface of neutrophils. Enzymatic cleavage of GPI anchors or blocking of PR3 significantly reduced the ability of neutrophils to phagocytose S. sangunis. Protease activity was required for phagocytosis, and S. sanguinis and protease-activated receptor 2 (PAR2) agonist induced calcium mobilization in a homologous desensitization manner, indicating the involvement of PAR 2 in phagocytosis. Finally, exposure to S. sanguinis preferentially activated RhoA to Rac1 and Cdc42, which was blocked by an intracellular calcium chelator. Collectively, our data show that PR3 acts as a phagocytotic receptor via PAR2 and the Rho family GTPases in neutrophils.
Modulation of human immune responses by ginseng extracts (89.56)
Authors: Madrenas Joaquin , Toth Judit , Hewson Gillian , Frodermann Vanessa , Chau Luan
Published: January 2023

Ginseng (GS) has been used as an herbal remedy for centuries but its immunomodulatory effects remain unclear. Here we report the effects of standardized North-American ginseng (Panax quinquefollius) extracts (ethanol, aqueous and polysaccharide extracts) on innate and adaptive immune responses of human peripheral blood mononuclear cells (PBMCs) of healthy volunteers. We found that endotoxin (LPS)-free GS by itself induced the production of pro-inflammatory cytokines (IL-1β, IL-6, TNFα) and of IL-10 by PBMCs in a dose-dependent manner. Of the three extracts of GS tested, the aqueous extract was the most potent. GS extracts did not inhibit but rather enhanced the pro-inflammatory response induced by LPS. However, the T cell IL-2 response to bacterial superantigens was down-regulated in the presence of GS. Next, we examined the signalling pathways involved in the immune response to GS. We found that the GS aqueous extract activated the MAPK (ERK1/2, p38), the PI3K/Akt, and the NFκB pathways. Inhibition of src kinases and PKC decreased GS-induced cytokine production whereas inhibition of PI3K selectively blocked IL-10 production. Based on these results, we conclude that GS, and in particular its aqueous extract, has modulatory properties on innate and adaptive immunity. This work should help to focus the search for compounds in these extracts with specific immunomodulatory activities.
Gamma delta T cell subpopulations defined by expression of distinct sets of the co-receptor WC1 genes. (89.6)
Authors: Baldwin Cynthia , Chen Chuang
Published: January 2023

Gamma delta T cells can be 60% of peripheral blood mononuclear cells in young ruminants with the majority expressing the WC1 co-receptor. WC1 co-receptors are composed of up to eleven extracellular scavenger receptor (SRCR) domains with the interdomain pattern organized as a-[b-c-d-e-d’]-[b-c-d-e-d’] and the greatest differences occurring in the most distal domain 1 (‘a’ in the pattern). Two main subpopulations are defined by the presence of either the WC1.1 or the WC1.2-antigenic epitopes and these are functionally distinct since only a small subpopulation of WC1.1+ cells proliferate to antigens of Leptospira and produce IFN-γ in response to leptospira or IL-12. Prior to the onset of the studies described here the copy number of WC1 genes and the WC1 forms that account for the serologically-defined WC1.1 and WC1.2 were unknown. By applying real-time QPCR and relative quantification analysis, 13 WC1 domain 1 genes were found to be present in cattle from two breeds. In addition, we found 6 subpopulations of WC1+ γδ T cells defined by anti-WC1 subpopulation-specific mAbs and showed that this correlated with expression of different sets of WC1 genes. The studies described here demonstrate that more than one WC1 gene is expressed on a single WC1+ γδ T cells, that specific WC1 genes are associated with γδ T cell subpopulations defined by a panel of anti-WC1 subpopulation-specific mAbs and that these differences are likely to influence their response to various stimuli.
Intestinal ischemia/reperfusion-induced lipid alterations do not correlated with damage or PGE2 production (90.12)
Authors: Archer Slone Emily , Sparkes Byron , Roth Mary , Welti Ruth , Fleming Sherry
Published: January 2023

Ischemia/reperfusion (IR)-induced injury results in severe tissue damage and causes up to 30,000 human deaths per year in the US. In a mouse model of intestinal IR, C57Bl/6 mice experience significant intestinal damage; however Rag-1-/- mice, which are antibody deficient, do not sustain significant damage. Complement activation is also critical as indicated by the lack of damage in complement receptor 2 deficient (CR2-/-) mice. Additionally, when Rag-1-/- or CR2-/- mice are pretreated with C57Bl/6 antibodies or specific anti-phospholipid monoclonal antibodies, damage is restored to C57Bl/6 levels. We hypothesized that antibodies recognize a specific lipid antigen produced during IR. After subjecting C57Bl/6, CR2-/- and Rag-1-/- mice to either Sham or intestinal ischemia followed by reperfusion, we determined jejunal prostaglandin E2 (PGE2) production and lipid profile. Mass spectrometry analysis of extracted lipids indicated that lysolipids and arachidonic acid (AA) levels increased by 15 min post-ischemia in C57Bl/6 mice. Differences in lysolipids or AA will be detected between C57Bl/6, CR2-/- and Rag-1-/- mice. C57Bl/6 mice produced significantly more Cox-2 mRNA and PGE2 than either CR2-/- or Rag-1-/- mice. Pretreating Rag-1-/- mice with antibodies purified from C57Bl/6 mice restored IR-induced PGE2 production to C57Bl/6 levels. We conclude that antibodies do not influence lipid metabolism, but are required for activation of Cox-2 and subsequent production of PGE2.
Induction of Th17 responses during vaccination: influence of route of immunization. (90.16)
Authors: Norton Elizabeth , Louise Lawson , Clements John , Cardenas-Freytag Lucia
Published: January 2023

The role of Th17 cells in vaccine-induced immune responses is poorly understood. A growing body of evidence suggests that some adjuvants, such as cholera toxin from Vibrio cholerae and heat-labile toxin (LT) from Escherichia coli, can promote development of Th17 responses. We investigated the presence of Th17 cells after immunization with tetanus toxoid (TT) with and without the inclusion of a detoxified form of LT designated LT(R192G/L211A), or dmLT. Groups of BALB/c mice were immunized twice (day 0, 30) with TT alone or in conjunction with dmLT either mucosally (oral, intranasal, intrarectal) or parentally (subcutaneous). Animals were sacrificed 30 days after the last immunization and splenocytes were harvested for in vitro restimulation assays. Recipients of TT + dmLT by all mucosal routes had elevated levels of TT-specific Th17 cells compared to groups immunized parenterally or with TT alone by any route. Intranasal immunization with TT + dmLT induced particularly strong Th17 responses, with significant levels of IL-17 detected in antigen-restimulated splenocyte supernatants. Collectively, these results suggest that mucosal immunization in the presence of an appropriate immunostimulant, such as the adjuvant dmLT, can direct the development of Th17 responses. The observation that antien-specific Th17 cells were only detected following mucosal immunization suggests that Th17 responses may play a critical role in mucosal immune responses and mucosal vaccine-based protection.
Essential role of membrane trafficking factor AP-1B in gut immune homeostasis (90.4)
Authors: Takahashi Daisuke , Hase Koji , Kimura Shunsuke , Ohno Hiroshi
Published: January 2023

Intestinal epithelium covering the mucosal surface play an important role to maintain homeostatic balance between immunity and tolerance in the gut; however, the molecular mechanisms that control this function of intestinal epithelium remains to be elucidated. Epithelium-specific adaptor protein (AP) complex AP-1B, contributes to basolateral sorting of certain membrane proteins, which is essential for polarity formation and maintenance. We found that mice deficient in AP-1B caused severe chronic inflammation in the colon, characterized by crypt elongation, decrease in goblet cells, massive infiltration of CD11b+ dendritic cells (DCs) and TH17 cells. CD11b+ DCs prepared from the colon of AP-1B-deficient mice produced high amounts of IL-1β and TNF-α no matter if stimulated with LPS or not, whereas those from control mice did not release detectable amount of these cytokines even upon stimulation with LPS, suggesting a defect in immunological tolerance in AP-1B-deficient mice. This could be due to abrogated epithelial innate immune function, because reduced expression of antimicrobial proteins by colonic epithelium and bacterial penetration into the lamina propria were evident in AP-1B-deficient mice. Our data clearly suggest that appropriate protein sorting by AP-1B is necessary for formation and/or maintenance of epithelial integrity that is important for containment of excessive inflammatory response in the colon.
Type I interferon reverses human Th2 commitment and stability by repressing GATA3 (91.11)
Authors: Huber Jonathan , Ramos Hilario , Gill Michelle , Farrar J.
Published: January 2023

CD4+ T helper-type 2 (Th2) cells regulate inflammatory responses to helminth infections while also mediating pathological processes of asthma and allergy. IL-4 drives Th2 development by inducing the expression of the GATA3 transcription factor. GATA3 positively reinforces its own expression thereby promoting IL-4-independent stability in Th2 cells. In this study, we found that type I interferon (IFN-α/β) potently blocked human Th2 development and inhibited cytokine secretion from pre-committed Th2 cells. Further, IFN-α/β inhibited Th2 cytokine secretion from CRTH2+ cells isolated from peripheral blood. This negative regulatory pathway was operative in human, but not mouse CD4+ T cells and was specific to type I interferon as neither IL-12 nor IFN-γ mediated such inhibition. IFN-α/β blocked Th2 cytokine secretion through the inhibition of GATA3 expression during both Th2 development and in fully committed Th2 cells. Thus, we demonstrate a novel role for IFN-α/β in blocking Th2 commitment, highlighting its potential as a therapy for atopy and asthma.
Airway epithelium response to IFN-γ regulates allergic airway inflammation (91.7)
Authors: Mitchell Charlotte , Provost Karin , Niu Naiqian , Homer Robert , Cohn Lauren
Published: January 2023

IFN-γ produced by Th1 cells inhibits Th2-induced allergic airway disease by decreasing airway eosinophilia, mucus production and chitinase activity. To define pathways by which IFN-γ inhibits allergic airway inflammation, we generated bone marrow chimeras with IFN-γ receptor (IFN-γR) deficient mice, transferred OVA-specific Th1, Th2 or Th1+Th2 cells and exposed mice to inhaled OVA. In mice receiving Th1+Th2 cells, IFN-γR was required on both hematopoietic and non-hematopoietic cells for maximal inhibition of mucus and chitinase activity, while IFN-γR expression on hematopoietic cells was sufficient for inhibition of airway eosinophilia. We hypothesized that airway epithelial cells are essential non-hematopoietic cells required to mediate the inhibitory effects of IFN-γ during airway inflammation. We generated epi-IFN-γR mice that express IFN-γR only on airway epithelial cells. Epi-IFN-γR mice that received Th1+Th2 cells showed near total inhibition of mucus, suggesting that IFN-γ directly inhibits mucus in epithelial cells. In epi-IFN-γR mice that received Th1+Th2 cells there was partial inhibition of chitinase activity, likely due to direct effects of IFN-γ on the epithelium. In epi-IFN-γR mice, airway eosinophilia was moderately inhibited through effects on eosinophil development in the bone marrow. These studies show the importance of airway epithelial cells in the regulation of allergic airway disease and highlight the epithelium as a target for therapy in airway disease.
Glutamate signaling through the kainate receptor is critical for human B cell IgE production (91.9)
Authors: Sturgill Jamie , Conrad Daniel
Published: January 2023

Glutamate, the major excitatory neurotransmitter, has recently been implicated in a variety of diseases. Patients with HIV, certain cancers, and autoimmune illnesses have elevated levels of glutamate in the periphery. While glutamate receptor signaling has been examined in T cells, there are currently no published observations on the effects of glutamatergic stimuli on B cells. Human B cells were first examined for the presence of glutamate receptors via RT-PCR and Western Blot. The kainate receptor (KAR), a specific subtype of ionotropic glutamate receptor was found on human B cells. We found that KAR activation increased ADAM10 mRNA which is in agreement with receptor activity in the CNS. A significant corresponding increase in CD23 released into the supernatant was seen as ADAM10 has been identified as the protease responsible for CD23’s ectodomain shedding. Remarkably, we also detect a strong increase in cellular proliferation and IgE synthesis. Thus, we report for the first time the unique presence of a neurotransmitter receptor and that activation of this receptor could serve as a novel mechanism for enhancing B cell activation. This enhancement has implications for allergic and autoimmune diseases.
Targeted Deletion of Cannabinoid Receptors 1 and 2 Results in Enhanced Inflammation and Exacerbated Immune Response Phenotype to Influenza Challenge (92.10)
Authors: Karmaus Peer , Chen Weimin , Kaplan Barbara , Kaminski Norbert
Published: January 2023

In previous studies, mice lacking cannabinoid receptors 1 and 2 (CB1-/-CB2-/-) showed reduced lung viral burden after a single instillation of mouse-adapted A/PR8/34 influenza compared to C57Bl/6 (WT) mice. In this study we investigated potential mechanisms involved in the differences in viral burden. In a kinetic study, CB1-/-CB2-/- and WT mice were challenged with A/PR8/34 and 1, 3, 5 and 7 days post infection (dpi) several immune parameters were assessed. In the bronchoalveolar lavage fluid, increases in neutrophils and macrophages were observed in response to viral challenge, and both populations were increased in CB1-/-CB2-/- mice as compared to WT at 3 dpi. Furthermore, increases in T cell activation as assessed by CD69 and IFNγ production were observed in CD4+ and CD8+ T cells from CB1-/-CB2-/- compared to WT as early as 3 dpi. To determine potential factors involved in modulating the outcome of the T cell response we performed a low density mouse immune panel gene array on lung RNA 1 and 3 dpi. Differences in chemokine and inflammatory cytokine levels, with enhancement in CB1-/-CB2-/- compared to WT were observed. In a repeat study we observed CD4+NK1.1+CD44- and NK1.1+CD4-CD8- producing significant amounts of IL-17 in CB1-/-CB2-/- over WT at 3 dpi. Overall, these studies suggest an enhanced inflammatory response in CB1-/-CB2-/- leading to faster T cell kinetics and a more robust immune response, thereby limiting viral replication.
Characterization of the monocyte response in the influenza-infected lungs. (92.12)
Authors: Gorski Stacey , Kim Taeg , Sun Jie , Braciale Thomas
Published: January 2023

Influenza virus infection of the respiratory tract triggers a rapid and vigorous innate immune response. Monocyte/macrophages make up a large fraction of this inflammatory infiltrate and are the major contributors to lung inflammation associated with infection. The monocyte population can be defined as CD11b+Ly6G-Dx5-SiglecF-, and subsets can be further discriminated based on Ly6C expression levels. Here we show rapid accumulation of Ly6Chi monocytes in the infected lungs during the inductive phase of the host immune response, resulting in the predominant if not exclusive accumulation of the Ly6Chi monocyte population at the peak of the inflammatory response. The Ly6Chi monocytes are potent producers of TNF alpha and their presence during early infection strongly correlates with overall lung inflammation. Ly6Chi monocytes slowly decline during the recovery phase following virus clearance, resulting in an accumulation of a Ly6Clo monocyte subset. The presence of this Ly6Clo subset, and the subsequent down-regulation of TNF alpha production in the Ly6Chi subset, strongly correlates with recovery (e.g. weight gain and reduced inflammation) in this mouse model of influenza infection.
The role of different virus-specific effector memory T-cell subsets in response to influenza A/H3N2 and pandemic A/H1N1 strains in older compared to young adults (92.2)
Authors: Zhou Xin , McElhaney Janet
Published: January 2023

Age-dependent changes in the cellular immune response have been mainly described in CD8+ T cells, with relative sparing of CD4+ T cells. However, a potential compensatory role for CD4+ T cells in the presence of severe effector functional defects in CD8+ T cells in older adults has not been described. We show that effector memory (CD45RA-CCR7-) and effector (CD45RA+CCR7-) CD8+ T-cell subsets in older compared to young adult PBMC responding to influenza A/H3N2 challenge have limited cytolytic activity against influenza-infected autologous targets. In contrast, effector CD4+ T-cell subsets in older adults demonstrate cytolytic activity in response to influenza challenge and share similar phenotypic and functional characteristics with virus-specific T cell subsets from young adults. Furthermore, we identified influenza virus-specific CD45RA+CCR7-CD25+CD127+ T-cell subsets in both CD4+ and CD8+ T cells , observed the limited effector T cell reponse in healthy young adults who received seasonal influenza split-virus vaccine. We conclude that CD4+ T cells may provide a compensatory response to influenza infection when CD8+ T cells become compromised during the aging process.
Preventing lung immunopathology during a heterologous infection by functional inactivation of cross-reactive influenza A memory T cells (92.20)
Authors: Wlodarczyk Myriam , Kraft Anke , Chen Hong , Kenny Laurie , Selin Liisa
Published: January 2023

Heterologous immunity may help explain why any respiratory infection can cause a wide range of disease from asymptomatic to extremely severe, even resulting in death. Prior immunity to influenza A led to significantly increased viral load and lung pathology upon LCMV infection, the severity of which varied between genetically identical mice, but did not correlate with viral load. Flu-specific memory CD8 T cells mediated this increased pathology as evidenced by CD8 depletion studies and the observation that disease severity directly correlated with the expansion of two cross-reactive Flu-epitope specific memory CD8 T cell responses during LCMV infection. H2-Kb-restricted IAV-PB1703 and H2-Db-restricted IAV-PA224 responses cross-reacted with LCMV-GP34, and -GP276, respectively. Functionally inactivating these memory populations by peptide tolerization or blocking IFNγ activity greatly decreased lung pathology demonstrating novel therapeutic interventions. Flu-specific memory CD4 T cells mediated the enhanced viral load during LCMV infection as demonstrated by CD4 depletion studies and depletion of the early enhanced Th2 cytokine IL-4. These findings have important implications for understanding and potentially controlling variability in disease outcome during respiratory infections.
Altering hemagglutinin binding to B cells modulates original antigenic sin responses to influenza viruses (92.23)
Authors: Powers Ryan , Kim Jin , Steinhauer David , Jacob Joshy
Published: January 2023

Original antigenic sin is a phenomenon in which exposure to a novel influenza virus strain leads to production of antibodies against a related viral strain encountered in the past. These cross-reactive antibodies are produced at the expense of antibodies against novel epitopes in the current strain. The existence of original antigenic sin has been demonstrated in humans as well as mice, rabbits, guinea pigs and ferrets. Despite the fact that this phenomenon was first described in 1953, the mechanisms involved are poorly understood. We hypothesize that original antigenic sin results from selective activation of B cells, particularly memory B cells specific for cross-reactive antigenic determinants. The hemagglutinin of influenza viruses can bind to all B cells, irrespective of their B cell receptor specificity, via sialic acid binding. Such binding may activate memory B cells better because they have lower activation thresholds than naïve B cells. To test whether original antigenic sin resulted from targeting of the virus to B cells and activation of pre-existing memory B cells specific for influenza strains encountered in the past, we generated influenza viruses whose ability to bind to sialic acid receptors on B cell surfaces is severely diminished. Our results suggest such diminished B cell targeting modulates the induction of original antigenic sin.
IL-15 is required for the migration of influenza-specific effector CD8+ T cells to the lung airways (92.24)
Authors: Verbist Katherine , Cole Charles , Field Mary , Klonowski Kimberly
Published: January 2023

CD8 T cells targeted to conserved viral proteins can provide protection against multiple strains of seasonal and highly pathogenic influenza virus, mediating heterosubtypic immunity. However, in mice this protection declines over time as the number of influenza-specific CD8 T cells in the lung airways falls below a set numerical threshold, despite influenza-specific splenic memory CD8 T cells continually migrating to the lung airways. Thus, understanding how to enhance both the frequency and longevity of the Tmem population in the lung airways is critical to an effective CD8 T cell-mediated vaccine. Since IL-15 regulates the survival of anti-viral CD8 Tmem, we sought to determine whether local deficiencies in IL-15 were responsible for the attrition of influenza-specific CD8 T cells in the lung airways. Using wild type and IL-15 deficient animals, we found IL-15 to be dispensable for both the generation and maintenance of influenza nucleoprotein (NP)-specific CD8 Tmem in all tissues. However, IL-15-/- mice harbored fewer influenza-specific CD8 Teff cells in the airways. Furthermore, influenza-specific Teff migrated to IL-15 complexes in vitro and in vivo. Overall, our studies describe a unique role for IL-15 in regulating the trafficking of CD8 Teff to the lung airways following influenza infection and suggest that using IL-15 complexes as an adjuvant in the mucosa may represent a methodology to prolong protection by lung airway-resident CD8 T cells.
Expression of the orphan nuclear receptor NR4A2 is required for IL-17 production by Th17 cells and the induction of Th17-mediated autoimmunity (93.14)
Authors: Raveney Ben , Oki Shinji , Yamamura Takashi
Published: January 2023

Previously, we reported that NR4A2, an orphan nuclear receptor, is upregulated by peripheral blood cells during the human autoimmune disease multiple sclerosis (MS), as well as by peripheral blood cells and leukocytes infiltrating the central nervous system (CNS) during experimental autoimmune encecephalomyelitis (EAE). The pathology of MS and EAE is mediated by infiltration of both Th1 and Th17 cells into the CNS, but we reveal that upregulation of NR4A2 observed in CNS-infiltrating leukocytes and peripheral blood cells is most apparent amongst IL-17-secreting CD4+ T cells. Therefore, we examined the role of NR4A2 in the differentiation of these Th17 cells: transfection of naïve T cells with NR4A2 siRNA precludes IL-17 secretion when cultured under Th17 polarising conditions. However, such cells do proliferate and upregulate RORγt to the same extent as fully functional Th17 cells. IL-23 stimulation is critical in licensing IL-17 production by Th17 cells and that this depends on the upregulation of IL-23R by Th17 cells subsequent to autocrine IL-21 signalling. NR4A2 siRNA knock-down in CD4+ T cells prevents IL-21 production under Th17 polarising conditions and consequently such cells do not upregulate IL-23R. Addition of exogenous IL-21 circumvented NR4A2 knock-down, leading to IL-23R upregulation and IL-17 production. Furthermore, NR4A2 appears to be critical in generating Th17-mediated autoimmunity as systemic administration of NR4A2 siRNA prevented EAE induction.
Identification of a critical CYP2E1 epitope in patients with drug-induced liver injury from anesthetics (93.16)
Authors: Njoku Dolores , Li Zhaoxia , Li Haiyan , Ligons Davinna , Rose Noel , Njoku Dolores
Published: January 2023

Susceptible individuals develop immune-mediated drug-induced liver injury (DILI) after receiving halogenated anesthetics, anti-seizure medications, antibiotics or non-steroidal anti-inflammatory drugs. In anesthetic DILI, serum cytochrome P4502E1 (CYP2E1) autoantibodies and diminished CYP2E1 activity assist in confirming the diagnosis, while responsible epitopes are unknown. We hypothesize that anesthetic DILI is initiated by pathogenic CYP2E1 epitopes centered on the Ser129 active site and presented by MHC II molecules in susceptible haplotypes. To test our hypothesis, we identified 15 mer human CYP2E1 candidate epitopes centered on Ser129 that bind to mouse MHC II using competitive inhibition of OVA323 - 329 in I-Ad DO11.10 T cell hybridomas + LS102.9 activation. Next we measured splenocyte T cell proliferation to candidate epitopes using our model of anesthetic DILI in BALB/c mice two weeks following TFA-S100 immunizations. Two CYP2E1 epitopes were detected by competitive inhibition; however, only one epitope Gly113 - Leu135 was recognized by proliferation and human sera ELISA assays. SYFPEITHI prediction of the probability of binding confirmed H2-Ad and H2-Ed haplotypes in BALB/c mice and identified 6 human MHC II haplotypes that could bind this epitope: HLA-DRB1*0101, HLA-DRB1*0301 HLA-DRB1*0401, HLA-DRB1*0701 HLADRB*1101 and HLADRB1*1501. This is the first demonstration of a CYP2E1 epitope and MHC II haplotypes that may increase the risk of developing anesthetic DILI.
Pathogenic Potential of Trypanosoma cruzi-Induced Autoimmunity (93.2)
Authors: Bonney Kevin , Daniels Melvin , Taylor Joann , Epting Conrad , Engman David
Published: January 2023

Chagas heart disease (CHD), caused by the parasite Trypanosoma cruzi, is a potentially fatal myocarditis associated with cardiac autoimmunity. Immunization of A/J mice with heat-killed T. cruzi (HKTC) induces acute cardiac damage and polyantigenic humoral and cellular autoimmunity of a specificity and magnitude similar to that observed in infected mice, yet it does not cause cardiac inflammation. The goal of the present study was to determine the differences between the autoimmune response induced by T. cruzi infection and that initiated by HKTC immunization that may be responsible for the presence or absence of myocarditis. We hypothesized that certain inflammatory conditions or secondary immune stimuli might be required in order for T. cruzi-induced autoimmunity to lead to myocarditis. The antigen specificity, autoantibody titers, and DTH responses to a panel of known cardiac autoantigens were similar in infected and HKTC-immunized mice. However, the HKTC-immunized mice failed to develop the robust Th1/Th17-mediated immune response present in infected mice. This suggested that Th1/Th17 responses may be important for the development of myocarditis associated with live T. cruzi infection. Coadministration of Th1-promoting but not Th2-promoting adjuvants resulted in the development of myocarditis in HKTC-immunized mice, supporting this hypothesis.
HLA alleles not associated with arthritis predispose by forming HLA- DQbeta-DQalpha trans-heterodimer (93.27)
Authors: Taneja Veena , Behrens Marshall , David Chella
Published: January 2023

HLA-DQB1*0302/DQA1*0301 renders humans and transgenic mice susceptible to develop arthritis while HLA-DQB1*0601/DQA1*0103 has been associated with resistance to arthritis in both. However, some patients of rheumatoid arthritis carry the non-associated alleles. Most of the studies do not take in to account the role of DQA1 alleles. DQB1*0604 generally occurs in linkage with DQA1*0102. To understand the role of trans-heterodimer, we generated DQB1*0604/ DQA1* 0103 transgenic mice lacking endogenous class II molecules. Previous studies have shown that DQB1*0601/DQA1*0103 transgenic mice are resistant to develop collagen-induced arthritis. The DQB1*0604/A1*0103 and DQA1*0103 mice developed severe arthritis and generated antigen-specific response in vitro. The DQB1*0604/ DQA1*0103 could present CII-derived peptides that are not presented by RA resistant DQ6 allele suggesting that trans-heterodimer molecules between two DQB1 and DQA1 molecules may result in presentation of unique antigens and susceptibility to develop arthritis. The data provides a possible explanation for the presence of HLA alleles not associated with arthritis in some patients.
Donor CD8 T cell activation is critical for sex based differences in lupus-like disease in DBA-into-F1 mice. (93.31)
Authors: Foster Anthony , Haas Mark , Puliaeva Irina , Soloviova Kateryna , Puliaev Roman , Via Charles
Published: January 2023

Transfer of DBA splenocytes into BDF1 mice (DBA->F1) results in donor CD4 T cell mediated lupus-like renal disease that is more severe in females and preceded at day 14 by greater female CD4 T cell engraftment. Donor CD8 T cells have no reported role. To compare sex based differences in donor CD4 T cell function, we transferred CD8 depleted DBA splenocytes and surprisingly observed that greater female renal disease severity and greater day 14 female CD4 engraftment were lost or significantly reduced. Kinetic analyses demonstrated that mice receiving undepleted donor cells exhibited a stronger day 8-12 graft-vs.-host (GVH) and host vs. graft (HVG) responses in males followed by contraction of donor CD8 T cells. The weaker GVH/HVG in females was followed by persistence/increase in donor CD8 T cells. Importantly, sex based differences in GVH/HVG were lost or attenuated in mice receiving CD8 depleted splenocytes, linking sex based differences in day 8-10 GVH/HVG to differences in day 14 CD4 engraftment. We conclude that: 1) donor CD8 T cells enhance the proliferation of donor CD4 T cells in both sexes; and 2) prolonged survival of donor CD8 T cells in females is causally related to greater day 14 engraftment of donor CD4 T cells in females. These results support the novel conclusion that sex based differences in sub optimal donor CD8 CTL activation are critical for shaping sex based differences in donor CD4 T cell engraftment at two weeks and lupus like disease long term.
Increased levels of IgA and IgA deposition in kidney glomeruli of influenza infected ST6Gal I deficient mice (93.38)
Authors: Onami Thandi , Acharya Suneeta , Zeng Junwei , Sangster Mark
Published: January 2023

ST6Gal I, a glycosyltransferase highly expressed by B cells, catalyzes the addition of alpha-2, 6 sialic acid to galactose (Siaα2-6Gal), a modification found on N-linked glycoproteins. Aberrant glycosylation has been associated with the pathogenesis of IgA nephropathy (IgAN), a disease characterized by immune complex deposits in the glomerular mesangium, which can lead to end-stage renal failure in 20-40% of diagnosed patients. Previously, we have shown that ST6Gal I-/- mice infected with influenza A/HKx31 demonstrated profound impairment in the generation of viral-specific humoral responses, although memory IgG responses were mostly normal. We now show that influenza infected ST6Gal I-/- mice had higher levels of influenza-specific IgA and total IgA in sera at memory compared to WT mice. Additionally, we observed increased deposition of IgA in the glomeruli of kidneys of ST6Gal I-/- mice, although no differences were noted in urinalysis. These data suggest that loss of expression of an alpha-2,6 sialyltransferase, together with influenza viral infection, can contribute to increased deposition of IgA in mouse kidney glomeruli, and may contribute to the pathogenesis of one of the most common forms of glomerulonephritis in humans.
Engraftment of peripheral blood mononuclear cells from patients with type 1 diabetes in NOD-scid HLA-A*0201/γc -/-immunodeficient mice (93.5)
Authors: Larry Fatima , Young Ellen , Fudge Elizabeth , Azam Anita , Patel Shipra , Joseph Largay , Warren Byrd , Buse John , Calikoglu Ali , Frelinger Jeffrey
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease characterized by insulitis followed by CD4+ and CD8+ T cell-mediated destruction of the insulin-producing pancreatic beta cells. Studies have demonstrated the presence of circulating islet-antigen specific CD4+ and CD8+ T cells during the clinical onset of disease. Over the past twenty years non-obese diabetic (NOD) mice have provided a useful tool for understanding human T1D. However, the study of human CD4+ and CD8+ T cell-mediated responses and the development of novel therapeutics for T1D requires an in vivo humanized animal model. In order to develop a model where we could investigate the function of human diabetogenic T cells in vivo we transferred peripheral blood mononuclear cells (PBMCs) from patients with T1D into NOD-scid HLA-A*0201/γc-/- transgenic immunodeficient mice. Following adoptive transfer we examined human T cell survival and homing. We isolated human CD4+ and CD8+ T cells from the pancreatic lymph node, spleen and islets of these mice for up to seven weeks. Our data suggest that this may be a useful humanized mouse model system for studying type 1 diabetes.
IL15-DC activate Th17 and Th1 responses upon encounter with wheat gliadin, the dietary antigen in Celiac disease (93.6)
Authors: Harris Kristina , Fasano Alessio , Mann Dean
Published: January 2023

Celiac disease (CD) is an autoimmune disease triggered by dietary glutens in genetically susceptible individuals. A direct role for IL-15 has been demonstrated in the disease process; however, the relationship between this cytokine and the adaptive immune response that ensues is not clear. Recent studies show that IL-15 drives monocytes into APC with potent proinflammatory potential, termed IL15-DC. Therefore, we examined the immune response of IL15-DC exposed to a digest of wheat gliadin (PTG), the exogenous factor in CD. Using PBMC from healthy donors, we found that IL15-DC secreted IL-1b, IL-6, IL-23, TNFa and CCL20 and produced surface IL-15 in response to PTG. IL15-DC loaded with PTG subsequently activated Th17 and Th1 responses from autologous CD4+ T cells in a contact-dependent and IL-15-dependent manner, respectively. Importantly, lower concentrations of IL-15 augmented Th1 responses induced by PTG in PBMC from CD patients compared to controls. These findings demonstrate that IL-15 supports Th17 and Th1 responses to a dietary antigen that is normally well-tolerated in healthy individuals by generating IL15-DC. These potentially pathogenic immune responses may occur in CD patients and not healthy individuals as a consequence of being hyper-responsive to IL-15 produced in the intestine upon encounter with PTG. Therefore, genetic and/or environmental factors that control IL-15 expression and responsiveness in the intestine likely participate in the pathogenesis of CD.
G-protein coupled receptor kinase-5 mediates Toll-like receptor-4-induced NFκB activation in primary macrophages and in vivo in mice (94.11)
Authors: Shahi Shipra , Patial Sonika , Saini Yogesh , Appledorn Daniel , Lapres John , Amalfitano Andrea , Parameswaran Narayanan
Published: January 2023

Toll-like receptor-4 (TLR4) activated by lipopolysaccharide (LPS) is involved in a number of inflammatory diseases, in part via activation of NFκB pathway. G-protein coupled receptor kinase-5 (GRK5), a serine-threonine kinase originally discovered for its role in desensitization of G-protein coupled receptors has recently been shown to regulate NFκB pathway stimulated by non-GPCRs. This study was undertaken to determine the potential role of GRK5 in inflammatory signaling in primary macrophages and in vivo using GRK5 knockout (KO) mice. We demonstrate here that TLR4-induced IκBα-NFκB pathway is selectively inhibited in primary macrophage from GRK5 KO mice compared to cells from GRK5 wild type (WT) mice. Consistent with the effects on the NFκB pathway, LPS-induced production of many inflammatory cytokines/chemokines (including Interleukin-2, -3, -4, -5, -12, -17, MCP-1, IFNγ, KC, GMCSF and Eotaxin) are inhibited in GRK5 KO macrophages. Confirming this in vitro data, LPS-induced cytokines/chemokines levels in the plasma are also significantly inhibited in the GRK5 KO mice in vivo. Associated with these effects LPS-mediated liver injury is markedly decreased in the GRK5 KO mice. Taken together, our findings demonstrate that GRK5 acts as a positive regulator of TLR4 signaling in vivo and further suggest that these findings could have potential implications for drug development in inflammatory diseases.
Innate Defence Regulator Peptides as a Novel Anti-infective Strategy (94.2)
Authors: Ma Shuhua , Nijnik Anastasia , Madera Laurence , Elliott Melissa , Easton Donna , Mayer Matthew , Kindrachuk Jason , Jenssen Håvard , Mok Ka Yan (Winnie) , Yang Tom , Hancock R.E.W. (Bob)
Published: January 2023

The therapy of bacterial infections is under great threat as multiple antibiotic resistance increases and there is a paucity of new antibiotic discovery and development. Synthetic Innate Defence Regulator (IDR) peptides, which mimic natural host defence (antimicrobial) peptides, have been designed as a novel anti-infective strategy, working by selectively boosting innate immune protective mechanisms while dampening potentially harmful inflammation. These peptides can resolve serious infections in animal models. The optimal method of use of these peptides was evaluated here using an animal model infection by one of the highly resistant bacteria (Superbugs) afflicting our society, Staphylococcus aureus. A luminescent version of this bacterium was utilized to follow the kinetics of infection non-invasively using IVIS imaging. Protection was achieved by both prophylactic and therapeutic administration. Investigation of the mechanism of protection by examining cells infiltrating the infection site and cytokines/chemokines revealed remarkable parallels between in vitro action of the peptides in primary human cells and these animal models. System biology approaches such as Microarray and InnateDB are being utilized to decipher the complex. This has revealed several receptors, signaling pathways, transcription factors and effector proteins involved in the modulation by peptides of innate immunity.
Critical roles of NKp30 and NKG2D in synapse formation and activation of NK cells (94.8)
Authors: Zheng Xiaodong
Published: January 2023

Natural killer (NK) cell is one of innate immune cells with potential of killing target cell without pre-activation. Activating killer receptors NKp30 and NKG2D play important roles in NK cytotoxicity, however, the critical roles of NKp30 and NKG2D in NK cell immunological synapse remains incompletely understood. In this study, the cytotoxicity of NK92 cell against Hela cell with or without blocking NKp30 or NKG2D was measured. The NK92-Hela conjugates were performed by immunofluorescence and Flow cytometry. Blockade of NKp30 or NKG2D inhibited NK cytotoxicity against several MHC I deficient targets. The cytotoxicity of NK92 cells against Hela was lower with blocking NKp30 than that with blocking NKG2D. The same effect of blocking NKp30 or NKG2D was detected in fresh NK cells from PBMC. However, blockade of NKp30 and NKG2D could not decrease the binding rate of NK92 and Hela cells. These results suggest that NKp30 and NKG2D involve in activation but not complete combining with target cells in NK synapse formation.
Human CD1D gene expression is regulated by LEF-1 through distal promoter regulatory elements (95.4)
Authors: Chen Qiaoyi , Zhang Tao , Pincus Seth , Wu Shixiu , Ricks David , Liu Donald , Sun Zhongsheng , Maclaren Noel , Lan Michael
Published: January 2023

CD1d expressing cells present lipid antigen to CD1d-restricted natural killer T (NKT) cells, which play an important role in immune regulation and tumor rejection. Lymphoid enhancer-binding factor-1 (LEF-1) is one of the regulators of the Wnt signaling pathway, which is a powerful regulator in cellular growth, differentiation, and transformation. There is little evidence connecting Wnt signaling to CD1d expression. In this study, we have identified LEF-1 as a regulator of the expression of the gene encoding the human CD1d molecule (CD1D). We found that LEF-1 binds specifically to the CD1D promoter. Over-expression of LEF-1 in K562 or Jurkat cells suppresses CD1D promoter activity and down regulates endogenous CD1D transcripts, whereas knockdown of LEF-1 using LEF-1-specific siRNA increases CD1D transcripts in K562 and Jurkat cells but different levels of surface CD1d on these two cell types. Chromatin immunoprecipitation showed that the endogenous LEF-1 factor is situated at the CD1D promoter and interacts with histone deacetylase-1 to facilitate the transcriptional repressor activity. Knockdown of LEF-1 using siRNA potentiates an acetylation state of histone H3/H4, supporting that LEF-1 acts as a transcriptional repressor for CD1D gene. Our finding links LEF-1 to CD1D and suggests a role of Wnt signaling in the regulation of human CD1D gene.
IL-23 receptor deficiency prevents the development of lupus nephritis in B6/lpr mice (96.20)
Authors: Zhang Zheng , Kyttaris Vasilieios , Kuchroo Vijay , Oukka Mohamed , Tsokos George
Published: January 2023

IL-17 producing T cells infiltrate kidneys of patients with lupus nephritis, and IL-23-treated lymphocytes from lupus-prone mice may transfer disease to Rag-1-/- mice. Here we show that IL-23 receptor deficient lupus prone B6/lpr mice display decreased numbers of CD3+CD4-CD8- cells and IL-17 producing cells in the lymph nodes, and produce less total serum IgG and anti-double strand DNA antibodies. In addition, clinical and pathology measures of lupus nephritis are abrogated. The presented experiments imply the importance of IL-23 receptor medicated signaling in the development of lupus nephritis and possible drug target for the treatment of the disease.
APPLICATIONS OF IMMUNOSUPPRESSIVE DNA TO TREAT AUTOIMMUNE AND AUTOINFLAMMATORY DISEASES (96.22)
Authors: Yagci Fuat , Aslan Ozlem , Songur Murat , Karatepe Kutay , Gursel Ihsan
Published: January 2023

Synthetic oligodeoxynucleotides (ODN) expressing suppressive TTAGGG motifs effectively down-regulate the production of proinflammatory and Th1 cytokines elicited either by Toll-Like Receptor (TLR) dependent or independent immune stimuli. Although initially identified by their ability to block CpG-induced immune activation, it was shown that suppressive ODN was able to block multiple forms of immune stimulation and to be effective in the prevention and treatment of pathologic autoimmune and autoinflammatory diseases. Endotoxin-induced uveitis (EIU) is an established animal model of acute ocular inflammation. It is induced by either systemic or intravitreal administration of lipopolysaccharide (LPS). Familial Mediaterranean Fever (FMF) is an autosomal recessive periodic fever disease characterized by recurrent, self-limiting, febrile, inflammatory attacks of the serosal membranes such as peritoneum, pleura, and synovia. Present study aims to demonstrate the downregulatory effect of the suppressive DNA on EIU and FMF as local autoimmune and systemic autoinflammatory diseases respectively. Results indicated that LPS induced EIU in mice, A151 treatment strongly downregulated either expression or protein levels of IL6, IP10, IL1β, MIP1β, MIP3a or iNOS. Moreover, FMF patient PBMCs were more responsive to TLR ligand stimulation compared to healthy subjects and A151 strongly reversed this activation and suppressed several key Th1-cytokine/chemokine levels
Small Modular ImmunoPharmaceutical (SMIP™) Molecules Directed at the TCR Complex (CD3) Block T Cell Activation and Cause Minimal Cytokine Release In Vitro (96.25)
Authors: Wang Camilla , Beckett Travis , Odegard Valerie , Hussell Scott , Mohler Ken , McMahan Catherine
Published: January 2023

SMIP molecules are single chain binding proteins based on a scFv-CH2-CH3 format. We have generated SMIP proteins using binding domains from well characterized mitogenic and non-mitogenic anti-CD3 antibodies, and have found dramatic differences in the biologic consequences on human T cells exposed to the compounds. In particular, compared to antibodies, SMIP proteins cause a unique Ca++ flux, are better able to down modulate the TCR complex from the cell surface, and can inhibit naïve and activated T cells in various in vitro assays. Importantly, SMIP proteins cause a much reduced level of cytokine release, notably, when compared to second generation anti-CD3 antibodies. Most known anti-CD3 antibodies cause activation of T cells, especially when cross linked via FcR on the surface of APCs. Without cross linking, these antibodies have minimal activation of T cells in vitro. In the clinic, second generation anti-CD3 antibodies are still plagued with cytokine release syndrome, despite showing little detectable cytokine release from primary T cells in vitro. We have developed an activated T cell blast assay sensitive enough to detect in vitro cytokine release caused by anti-CD3 antibodies in second generation formats. Results from these assays indicate that features in the SMIP format could generate novel CD3-directed therapeutics with superior efficacy and reduced potential for the serious side effects of Cytokine Storm.