bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycyrrhizin improves the antimicrobial peptide production by human epidermal keratinocytes (NHEK) transwell-cultured with peripheral blood linage- CD34+ cells, a suppressor cell on the antimicrobial peptide production by NHEK (134.46)
Authors: Yoshida Shohei , Jones Vickie C , Jeschke Marc G , Kobayashi Makiko , Herndon David N , Suzuki Fujio
Published: January 2023

Previously, linage- CD34+ cells isolated form burn patient peripheral blood cells were shown to be inhibitory on the antimicrobial peptide production by NHEK. In the present study, the effect of glycyrrhizin (GL) on the CD34+ suppressor cell activity for the production of β-defensin 1 (BD-1) by NHEK was determined. Linage- CD34+ cells were isolated from peripheral blood of healthy donors after 1-hr of programmed exercise, as previously described. CD34+ cells (1 x 105 cells/ml, upper chamber) were transwell-cultured with NHEK (1 x 105 cells/ml, lower chamber) in the presence or absence of GL (Minophagen Pharmaceutical Co., Ltd., Tokyo, Japan). Forty-eight hrs after cultivation, cells in the lower chamber were assayed for human BD-1 and cathelicidin (CAP-18) mRNAs by RT-PCR. BD-1 and CAP-18 mRNAs were consistently expressed by NHEK. However, NHEK transwell-cultured with CD34+ cells did not express these peptide mRNAs. When transwell-cultures between these 2 cells were performed in the presence of 100 μg/ml of GL, mRNAs for BD-1 and CAP-18 were expressed by NHEK. The mRNA expression for BD-1 and CAP-18 by NHEK was not directly influenced by GL. These results indicate that the suppressor cell activity of linage- CD34+ cells on the antimicrobial peptide production by NHEK is modulated by GL. This study was supported by SHC NA #8610.
Improved survival in experimental sepsis with conjugated cholic acids preventing activation of dendritic cells by LPS (134.48)
Authors: Kim Youn-Hee , Seong Seung-Yong
Published: January 2023

The incidence and mortality of sepsis have steadily increased during the last few decades. The development of targeted therapy has been hampered due to multiple targets of LPS. Here, we tested bioemulsifiers inhibiting interaction of LPS with dendritic cells (DCs) in vitro and in an experimental mouse model. We found that glycine-conjugated cholic acids (CCA) and taurine-CCA reduced LPS-mediated NF-κB activation by inhibiting LPS binding with target cells. CCAs were also able to inhibit expression of costimulatory molecules, production of inflammatory cytokines and NO of DCs by LPS. In addition, CCAs inhibit T cell proliferation by DCs activated with LPS. In vivo, CCA administration reduced blood proinflammatory cytokines and improved mice survival following LPS challenge. Considering functional impairment of DCs in sepsis patients, protecting DCs from hyperactivation by LPS might be crucial in the physiological outcome of sepsis. Taken together, CCA might mitigate the development of severe sepsis.
The role of IL-1β in the vaccine adjuvant Monophosphoryl Lipid A’s TRIF-biased TLR4 signaling and low toxicity (134.49)
Authors: Eaves Chelsea Anne , Cekic Caglar , Mitchell Thomas C.
Published: January 2023

Vaccines often require adjuvants to elicit responses to otherwise weakly immunogenic proteins. The TLR4 agonist, monophosphoryl Lipid A (MLA) retains the immunogenicity, but not the toxic inflammatory response, of its parent molecule S. minnesota LPS. We previously reported that MLA is a TRIF-biased agonist of TLR4, and have now determined that a single phosphate group difference is sufficient for this signaling difference, as well as for a loss of IL-1β production. These two characteristics of MLA contribute greatly to its reduced toxicity. In order to determine the relationship between TRIF-biased signaling and the loss of IL-1β, we compared IL-1β production by RT-PCR and ELISA in primary bone marrow-derived dendritic cells. Preliminary studies show that MyD88 signaling is required for post-transcriptional IL-1β processing, indicating that TRIF-bias may cause MLA to produce low levels of IL-1β protein. Discovering the mechanisms responsible for these low-toxicity adjuvant properties of MLA will aid in the generation of safer and more effective vaccines. This project was supported by funding from the NIH/NIAID.
Rational design of enhanced affinity at the MICA-NKG2D interface (134.51)
Authors: Stoufer Lisa K. , Davies Luke D. , Culpepper David , McFarland Benjamin J.
Published: January 2023

MICA is an MHC-like protein expressed on stressed cells. It binds the NKG2D immunoreceptor and activates natural killer cells. Our research seeks to enhance binding affinity at contacts between MICA and NKG2D. Previous research has focused on mutating the area beneath the binding site, an indirect design strategy that has increased the association and affinity between MICA and NKG2D by more than an order of magnitude. We currently aim to increase binding affinity directly by designing point mutations at MICA-NKG2D protein-protein contacts. RosettaDesign was used to predict which contact mutations would be most beneficial at the MICA-NKG2D interface. We have produced multiple point mutants of MICA in an undergraduate research environment and measured NKG2D binding kinetics and thermodynamics using surface plasmon resonance. In the first round of design, 4 out of 6 mutations enhanced NKG2D affinity by 0.5 kcal/mol or more, up to 1.2 kcal/mol. This success rate is similar to published accounts of antibody-antigen design and other protein design strategies. Once a panel of point mutations is complete, we will investigate the kinetics and thermodynamics of combining mutations, from direct and indirect design strategies.
Meprin metalloproteases are involved in monocyte trans-extracellular matrix migration and meprin deficiency aggravates LPS-induced septic shock (134.56)
Authors: Jin Hong-Jian , Banerjee Sanjita , Yura Renee , Bao Lei , Bond Judith , Sun Qi
Published: January 2023

Meprins are metalloproteases first discovered in kidney and intestinal epithelial cells, and have been implicated in autoimmune diseases. We have shown that homeostasis of monocytes is compromised in meprin alpha/beta null (alpha-/-/beta-/-) mice. However, the mechanisms responsible for the involvement of meprins in the immune and hematological system remain uncharacterized. We report here that human monocytes expressed meprins, and the expression of meprins was down-regulated in mature macrophages and dendritic cells but not their immature counterparts. We further show that bone marrow monocytes and peritoneal macrophages from meprin-deficient mice had decreased efficiency in migration through extracellular matrix (ECM) compared to those from the wild-type mice in Boyden assays without marked defects in cell mobility in the absence of ECM. In a sepsis shock model, meprin-deficient mice exhibited more severe hypothermia than the wild-type mice in response to peritoneally injected lipopolysaccharide (LPS), and the prevalence of blood monocytes in the merpin-deficient mice were only 25% of that in the wild-type mice, with compensatory increase of lymphocytes. While meprin-deficient mice in the steady state had moderately lower blood levels of IL-12, MCP-1 and IL-6 than the wild-type mice, LPS-challenged meprin-deficient mice exhibited a drastic elevation of the above inflammatory cytokines in comparison to the wild-type mice, with comparable blood concentrations of IFN-gamma and IL-10 between the two mice. These results indicate that meprin metalloproteases are required for efficient trans-ECM migration of monocytes, and meprin deficiency is associated with defects in monocyte functions which have pathological consequences.
Heat shock protein 90 protects degradation of retinoic acid-inducible gene-I (134.75)
Authors: Matsumiya Tomoh , Imaizumi Tadaatsu , Yoshida Hidemi , Topham Matthew K. , Stafforini Diana M. , Satoh Kei
Published: January 2023

Retinoic acid-inducible gene I (RIG-I) plays a pivotal role in innate immune responses as an intracellular sensor of viral RNAs. Recognition of dsRNA by RIG-I activates downstream IFN-inducible molecules including IRF3, resulting in the induction of type I IFN. However, the mechanisms that participate in downstream events following activation of RIG-I are incompletely understood. The objective of this study was to identify proteins that associated with RIG-I. Studies combining immunoprecipitation and mass spectrometry showed that RIG-I formed part of a protein complex that included heat shock protein 90 (HSP90), a molecular chaperone. Treatment with geldanamycin, a HSP90 inhibitor, dissociated the RIG-I: HSP90 complex and also resulted in the degradation of RIG-I protein. In contrast, the levels of RIG-I mRNA were not affected. In addition, the proteasomal inhibitor MG-132 significantly abolished geldanamycin-mediated RIG-I degradation, suggesting that HSP90 protected RIG-I from proteasomal degradation. Our results also showed that pretreatment with geldanamycin significantly reduced RIG-I mediated IFN-β promoter activity induced by polyinosinic: polycytidylic acid. Taken together, these data suggest that HSP90 plays a role in innate immune responses by shielding RIG-I from proteasomal degradation.
STAT6 Independent Alternative Activation of the Pulmonary Environment in Response to Helminth Infection. (134.78)
Authors: Siracusa Mark  C. , Craig John M. , Urban Joseph F , Artis David , Pearce Edward J
Published: January 2023

Macrophages adopt an alternatively activated phenotype in the context of a polarized Th2 response as a result of STAT6 (Signal transducer and activation of transcription 6)-dependent IL-4 and IL-13 signaling. Given the strong connection between IL-4/IL-13/STAT6 signaling and the induction of the alternatively activated macrophage phenotype, it was unexpected to observe that infection with Nippostrongylus brasiliensis (Nb), a strong inducer of IL-4 and IL-13, in Stat6-deficient mice resulted in an apparent alternate activation of lung macrophages with the induction of arg1, ym1 and fizz1. The goal of the work presented here was to test the hypothesis that there is a STAT6-independent pathway for alternative activation of lung macrophages in response to the pulmonary inflammation induced by the transient passage of Nb larvae through the parenchyma of the lungs. To test this hypothesis, we isolated CD11c+ lung macrophages from STAT6-/- mice on days 0, 2, 4, 8, and 12 post-Nb to test for the alternative activation. We demonstrate the macrophage-specific induction of Arg1 and to a lesser extent YM1 protein in an IL-4 and IL-13 independent manner. It was also determined that epithelial cells, but not macrophages, produce Fizz1 in a Stat6-independent manner. These results demonstrated the partial alternative activation of macrophages in the absence of IL-4 and IL-13 signaling and underscore the need to study the innate immune mechanisms capable of altering macrophage phenotypes.
Select procyanidins prime the γδ T cell by modulating transcriptome degradation (134.88)
Authors: Holderness Jeff S , Daughenbaugh Katie , Kimmel Emily , Freedman Brett , Hedges Jodi , Jackiw Larissa , Jutila Mark A
Published: January 2023

Tannins from many plant-derived traditional medicines demonstrate immune regulatory activity. Recently, we reported that condensed tannins (procyanidins) isolated from a select number of traditional medicines act as γδ T cell agonists. γδ T cells play an important role in mucosal immunity by regulating the mucosal immune response and maintaining epithelial health. Procyanidins induce a priming state on the γδ T cell, thus improving its ability to quickly respond to secondary stimuli. This priming state is characterized, in part, by an increased half-life of select transcripts in the γδ T cell relevant to innate immunity. This increase in transcript half-life could enable the cell to more quickly produce appropriate immune responses to secondary stimuli. Previously we reported that oligomeric procyanidin fractions, but not monomeric procyanidins, act as γδ T cell agonists. We have further defined the activity of different sizes of procyanidins. The tetrameric and pentameric procyanidins from apple peel have greater specificity for γδ T cells, whereas larger oligomers demonstrate less specificity. Our results suggest procyanidins with specific oligomeric structures prime γδ T cells by stabilizing transcripts with critical functions in innate immunity. Funded by: NIH/ NIAID Contract HHSN266200400009/N01-AI40009 and NCCAM P01 AT004986-01
Divergent macrophage programs for phagocytosis and antimicrobial responses regulate innate immunity to microbial pathogens (134.90)
Authors: Montoya Dennis , Cruz Daniel , Zhang Xiaoran , Krutzik Stephan , Ferguson Benjamin G , Modlin Robert L
Published: January 2023

The mechanisms by which monocytes differentiate into macrophages (Mø) contribute to the outcome of the host response to microbial infection. Previously, we found that IL-10 derived Mø (CD209+ M2) had enhanced phagocytic receptor expression and function versus IL-15 derived Mø (CD209+ M1). Here we show that CD209+ M2 Mø unlike CD209+ M1 Mø, were unresponsive to vitamin D for the induction of the antimicrobial peptide, cathelicidin, known to kill intracellular mycobacteria, demonstrating a divergence of phagocytosis and host defense function.. The divergent Mø programs for phagocytosis and antimicrobial responses regulate the effector arm of innate immunity relative to pathogenesis in microbial infection.
Receptor Like Tyrosine Phosphatases CD45 and CD148 Regulate Integrin- and GPCR-Mediated Pathways in Murine Neutrophils (135.18)
Authors: Zhu jing W , Doan Kristen , Zhang Hong , Lowell Cliff , Weiss Arthur
Published: January 2023

Reciprocal regulation of tyrosine phosphorylation by protein tyrosine kinases and phosphatases is central to normal immune cell function. CD45 is a very abundant and important receptor like tyrosine phosphatase in the immune system that regulates key proximal signaling molecules, Src family kinases. Interestingly, in contrast to its requirement in T cell development, the phenotype of CD45 deficient B cells and myeloid cells is rather subtle. Using a doubly deficient CD45 and CD148 mouse model, we discovered the overlapping function of these structurally distinct phosphatases in regulating immunoreceptor signaling via dephosphorylation of the inhibitory tyrosine of Src-family kinases in B cells and macrophages.

Since SFKs are also critical regulators of many other signaling pathways, including integrin and GPCR signaling, we studied the functions of CD148 and CD45 in these pathways. We focused on integrin- and GPCR-mediated pathways in neutrophils. We found that the superoxide release in response to integrin stimulation was impaired in both CD45 and CD148 deficient neutrophils and even more so in doubly deficient cells. Similar defects were observed when cells were stimulated with the GPCR ligand fMLP. Interestingly, we also observed impairment of calcium flux in CD148 single deficient neutrophils following MIP-2 stimulation, indicating unique function of CD148 in response to a chemokine signal. More detailed investigations on the biochemical and functional basis of these defects are under way.

This work is supported by NIH grant A1066120
Conditional targeting of C-terminal Src kinase (Csk) in NK cells (135.2)
Authors: Hampshire Daniel , Saudemont Aurore , Tarakhovsky Alexander , Colucci Francesco
Published: January 2023

Csk is a key negative regulator of Src-family kinases and has been shown to be a strong inhibitor of immunoreceptor signalling in T cells and granulocytes. Our hypothesis is that an increase in Src kinase activity might take place in NK cells following the elimination of Csk, resulting in an enhanced cellular response even at low levels of stimulation. We aim to establish the role of Csk in NK cells using conditional knockout mice. A transgenic mouse has been developed where the Csk gene is flanked by LoxP (floxed) sites. Crossing Csk floxed transgenic mice with transgenic mice expressing Cre recombinase under the control of a human CD2 gene (hCD2-iCre) will enable the deletion of the Csk gene in a tissue-specific manner. The ROSA26 reporter gene system suggests that Cre deletes the floxed gene in approximately 30% of splenic NK cells and, as reported before, in the vast majority of T cells and B cells. Intracellular staining for Csk demonstrates a correlation between Cre targeting of the ROSA26 locus and the target locus. There is a reduction in the number of lymphocytes in the Csk knockout (KO) mouse. More specifically, there is a decrease in B cells, suggesting a role for Csk in B cell development. However, no obvious phenotype is detected in NK cells. Ongoing studies of NK cell functions will be presented.
Fc-receptor crosslinking stimulates cell cycle progression of macrophages via the ERK pathway (135.25)
Authors: Casadevall Arturo , Pollard Jeffrey W. , Luo Yong
Published: January 2023

Our previous study showed that phagocytosis and Fc receptors (FcR) crosslinking of macrophages could enhance their cell proliferation. In the present study, we further explored the activation of the signaling pathways responsible for this phenomenon. Our investigation revealed an increase in cyclin D1 and cyclin E protein expression in murine macrophages after FcR crosslinking. Specifically, we further identified the extracellular signal-regulated kinase (ERK) signaling pathway as a potential mediator of signals from FcR activation to cyclin D1 expression, since cyclin D1 expression associated with FcR crosslinking was attenuated by specific inhibitors of the ERK1/2 signaling pathway, PD98059 and U0126 and the spleen tyrosine kinase (Syk) inhibitor, Piceatannol. Our findings establish a link between the ERK and cyclin signaling pathways thus providing a mechanism by which FcR activation produces a mitogenic effect that stimulate macrophage proliferation. The results suggest the possibility of new treatment options for certain infectious diseases, chronic inflammatory diseases and leukemias based on interference with FcR-mediated cell cycle progression.

This work was supported by NIH grants AI033142, AI033774, AI052733, HL059842, 5T32CA009173, 5R01HD030280-09, 5RO1CA089617-03 and the Northeastern Biodefense Center grant AI057158-05.
Ku protein regulates interferon regulatory factor 1 expression in response to CpG-ODN (135.39)
Authors: Riolo Matthew , Chu Wen-Ming
Published: January 2023

Interferon regulatory factor 1 (IRF1) expression is induced in response to toll-like receptor 9 (TLR9) stimulation by bacterial and viral CpG-DNA. Recent studies have demonstrated that IRF1's activity is specific to promoting the expression of interleukin-12p70, but not p40, in a novel signaling pathway that is myeloid differentiation protein-88 (MyD88)-dependent yet nuclear factor kappa B (NF-κB)-independent. Further evidence suggests that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), is upstream of IRF1 activation and required for its expression and subsequent production of interferon in response to CpG- oligodeoxynucleotides (ODNs) stimulation. Here we have demonstrated that regulatory protein Ku is critical for IRF1 expression in response to CpG-ODN stimulation and the subsequent production of IL12p70. In Ku70 and Ku80 knock out mice bone marrow-derived dendritic cells (BMDCs) IRF1 expression was nearly abolished in response to CpG-ODN stimulation, and IL12p70 production was reduced to one fourth that of their wild type counterparts, while IL12p40 levels showed no differences. Our results suggest a novel pathway where Ku proteins regulate DNA-PKcs activation in response to CpG stimulation critical for the expression of IRF1 and the subsequent antiviral response.

This research is supported by the GAANN Training Grant.
A Role for RIP1/RIP3-Dependent Programmed Necrosis in Innate Immune Responses Against Vaccinia Virus Infections (135.44)
Authors: Chan Francis K , Cho YoungSik , Challa Sreerupa
Published: January 2023

Cell death by programmed necrosis (also known as caspase-independent cell death or necroptosis) is characterized by rapid loss of plasma membrane integrity prior to the exposure of phagocytic signal. The release of endogenous "danger signals" from necrotic cells trigger inflammation and may have important physiological and pathological consequences in immune responses, inflammatory diseases and cancer growth. However, the role of programmed necrosis in immune responses and inflammation has not been adequately tested, in part because the molecular pathway that regulates programmed necrosis was ill-defined. The serine/threonine kinase RIP1 plays crucial role in programmed necrosis induced by TNF and other stimuli. We performed a RNA interference (RNAi) screen to identify downstream regulators of RIP1-dependent programmed and identified RIP3 to be another kinase critically required for TNF-induced programmed necrosis. In order to test the role of programmed necrosis in inflammation and anti-viral immune responses, we infected RIP3-deficient mice with vaccinia virus and found that the RIP3-deficient mice were highly sensitive to vaccinia virus infections. The details of these studies and the physiological implications of programmed necrosis will be discussed.
Polymorphisms associated with human Dectin-1 result in disrupted protein folding and loss of function (135.47)
Authors: Wolf Andrea J , Simmons Randi M , Goodridge Helen S , Underhill David M
Published: January 2023

Recognition of fungal pathogens is primarily mediated by a combination of TLRs and Dectin-1, the C-type lectin receptor for fungal β-glucans. While fungal infections in U.S. hospitals primarily arise among immune compromised patients, there has been an increased incidence in fungal infections that has been complicated by the emergence of drug resistance among fungal pathogens. Even though Dectin-1 functions as one of the primary phagocytic receptors in macrophages for fungal pathogens, the importance of Dectin-1 for controlling fungal pathogens is still unclear and may be pathogen dependent. Therefore we set out to analyze the functional consequences of single nucleotide polymophisms (SNPs) in human Dectin-1. When we mutated the full length form of the A isoform of human Dectin-1, two SNPs in the coding region resulted in a loss of phagocytic function. While both mutant forms of the protein were detectable they no longer supported phagocytosis of yeast zymosan particles in non-phagocytic cells. Analysis of alternate amino acid substitutions demonstrated an essential structural role for amino acid 223. Consequently, individuals possessing either of these mutations are potentially at a higher risk for certain fungal pathogens.
TLR7-induced protective CD8+ T cell immunity is mediated by a Type I IFN and IL-12 co-dependent pathway (135.48)
Authors: Oh Jason Zee Seug , Kedl Ross M.
Published: January 2023

Covalent conjugation of a TLR7 agonist to a protein antigen enhances the immunogenicity of the antigen and elicits protective CD8+ T cell responses. This study identifies divergent effects of two major cytokines induced by the TLR7 agonist-protein conjugate (conjugate), IFNα and IL-12, in leading to efficient priming of CD8+ T cells. In examining dendritic cell responses to the conjugate, we found that signaling through the Type I IFN receptor was required for the timely recruitment and accumulation of activated dendritic cells in the draining lymph nodes. Furthermore, Type I IFN receptor-deficient dendritic cells failed to cross-present appreciable levels of the antigen even though the capacity for antigen uptake was not impaired. Consequently, conjugate-induced CD8+ T cell responses were substantially reduced in Type I IFN receptor-deficient mice. In contrast, IL-12 deficiency had no effect on the capacity of dendritic cells to respond to the conjugate. Yet, IL-12 was required for the induction of CD8+ T cells. Collectively, the data suggest that there is a co-dependency of both IFNα and IL-12 during the primary response to immunization with the conjugate possibly due to distinct roles on dendritic cells and CD8+ T cells, respectively. These results point to the possibility of targeting the induction of both IFNα and IL-12 as a novel strategy in vaccine development for optimal induction of cell-mediated immunity.

NIH Grant R21: AI70038
Distinct regulatory roles of NF-kB in CpGA and CpGB-induced IFN-alpha production by plasmacytoid dendritic cells (135.50)
Authors: cui chongwei , Singh Sukhwinder , George Thaddeus , Fitzgerald-Bocarsly Patricia
Published: January 2023

Plasmacytoid dendritic cells (PDC) are a major source of IFN-α production in response to the TLR9 ligand CpGA, but not CpGB. TLR9, MyD88, and IRF7 have been established to be in the major pathway of PDC IFN-α production. We found that the inhibition of NF-κB activation by SN50 or NBD conferred on CpGB the ability to induce IFN-α in PDC, while CpGA-induced IFN-α was decreased. Using the ImageStream imaging flow cytometer, we found that although CpGA-induced IRF-7 translocation was inhibited by SN50, SN50 conferred the ability of CpGB to induce IRF7 nuclear translocation. The early endosomal localization of CpG has been shown to be important in IFN-α production. We found that both CpGA and CpGB moved from early to late endosomes from 30 min to 2 hr. However, neither CpGA nor CpGB was found in either early or late endosomes in large quantities upon NF-κB inhibition. Unlike in cell lines, in PDC, TLR9 was found to be constitutively localized in the early endosomes and also recruited from ER upon stimulation with CpGA. These results suggest that NF-κB plays an inverse role in the regulation of IFN-α production by PDC in response to CpGA or CpGB stimulation. Supported by AI26806.
GSK3 β Regulates the mTOR -mediated inflammatory response in TLR-stimulated innate immune cells (135.55)
Authors: wang huizhi , Garcia Carlos A. , Martin Michael
Published: January 2023

The tuberous sclerosis complex-mammalian target of rapamycin (TSC-mTOR) pathway has been shown to play a critical role in regulating the inflammatory response. Here we identity the downstream signaling molecules within this pathway that are responsible for differentially controlling pro- and anti- inflammatory cytokines production by TLR-stimulated innate immune cells. Utilizing a kinase screen, we demonstrate that inhibition of mTOR by rapamycin or siRNA-mediated gene silencing results in the loss of GSK3-β (Ser 9) phosphorylation and subsequently increases its kinase activity. The ability of rapamycin to inhibit the TSC-mTOR pathway and thus augment pro-inflammatory cytokines while concurrently suppressing the anti-inflammatory cytokine IL-10 was shown to be dependent on its regulation of GSK3-beta activity. Analysis of downstream kinases of mTOR identified that p70S6K was responsible for the ability of the TSC-mTOR pathway to control GSK3 activity. mTOR inhibition in vivo induced a potent adaptive and innate inflammatory response that was abrogated by preventing rapamycin from increasing GSK3 activity. These findings identify the molecular mechanism by which the TSC-mTOR pathway regulates the host inflammatory response.
Cathepsin B activity is required for NALP3-induced caspase-1 activation, lysosomal dysfunction and cell death in macrophages. (135.81)
Authors: Bruni Antonio , Ha Soon-Duck , Franchi Luigi , Nunez Gabriel , Kim Sung O
Published: January 2023

The nucleotide-binding and leucine-rich repeats (NLRs) are a family of innate immune receptors sensing various pathogen- and danger-associated molecules within the cytosol. Activation of NLRs such as NALP3 induces the secretion of inflammatory cytokines such as interleukin (IL)-1ß and rapid cell death, through caspase-1 activation. To date, the mechanisms associated with NALP3 activation and cell death remain poorly defined. This study examined whether lysosomal membrane dysfunction or the lysosomal protease, cathepsin B (CTSB), are involved in cell death induced by NALP3. Wild-type (WT), NALP3-, ASC-, caspase-1- and CTSB-deficient immortalized bone marrow-derived macrophages were exposed to the NALP3 activator ATP. ATP treatment caused lysosomal dysfunction measured by loss of acridine orange, caspase-1 activation and rapid cell death in WT but not in NALP3-, ASC-, caspase-1- or CTSB-deficient macrophages. Mutant Pseudomonas aeruginosa (exotoxin U deficient) induces IPAF-dependent IL-1ß release and cell death. The mutant bacteria induced cell death and IL-1ß release in WT and CTSB-deficient macrophages but not in IPAF-, ASC- or caspase-1-deficient macrophages. Altogether, these results suggest that cathepsin B is required for NALP3-induced but not IPAF-induced caspase-1 activation and lysosomal dysfunction.
Aberrant DNA demethylation of PD-1 during chronic viral infection (136.16)
Authors: Youngblood Ben , Oestreich Kenneth J , Ha Sang-Jun J , Duraiswamy Jaikumar , Akondy Rama , Boss Jeremy M , Ahmed Rafi
Published: January 2023

Chronic infection results in CD8 T cell exhaustion characterized by reduced cytokine expression, cell proliferation and an inability to clear the source of antigen. Central to the development of exhausted CD8 T cells is the retained upregulation of the inhibitory receptor programmed death (PD)-1. It is now known that PD-1 upregulation in exhausted CD8 T cells is coincident with the progression of many chronic human diseases including human immunodeficiency virus (HIV), hepatitis C virus, (HCV), and Epstein Barr virus (EBV). To date, relatively little is known regarding PD-1 regulation. Using Lymphocytic Choriomeningitis virus (LCMV) infection to study anti-viral CD8 T cells in mice, we observe that DNA methylation of the PD-1 promoter is present in naïve (PD-1lo) and memory (PD-1lo) CD8 T cells, while effector cells (PD-1hi) are partially demethylated. Consistent with these observations, exhausted CD8 T cells from chronically infected mice have near complete demethylation of the PD-1 promoter. Resolution of the persistent LCMV infection results in a moderate reduction of PD-1 expression and a lack of promoter methylation. These findings provide insight into antigen induced expression of PD-1 associated with many human diseases. Moreover prolonged signals for demethylation in the antigen specific cells may result in heritable transcriptional dysregulation accounting for the deviant gene profile of functionally exhausted CD8 T cells.
A novel non-Rel subunit of NF-kB confers its regulatory specificity (136.2)
Authors: Wan Fengyi , Lenardo Michael J.
Published: January 2023

NF-kB regulates many important immunity, inflammation and anti-apoptosis genes. It still remains largely unknown how NF-kB activates selected gene expression. We hypothesized that other protein components could form an integral part of the NF-kB and control its action on target genes. To understand the selective and high affinity recruitment of NF-kB, we used p65 in a proteomic screen and identified ribosomal protein S3 (RPS3) as a novel non-Rel component of p65 homodimer and p65-p50 heterodimer DNA binding complexes that enhances the p65-DNA interaction. Reduction of RPS3 by RNAi impaired NF-kB function, but did not affect its nucleocytoplasmic shuttling. NF-kB activating stimuli dissociated RPS3 from its cytoplasmic berth by phosphorylation and caused a parallel translocation of RPS3 to the nucleus where it promoted NF-kB binding to gene regulatory sites. RPS3 was essential for normal expression of specific NF-kB target genes requiring p65, including key physiological events, such as IL-2 and IkBa, but not CD25 and CD69 in T cells and the induction of immunoglobulin k light chain gene expression in B cells. Thus, RPS3 is a novel non-Rel subunit of NF-kB that confers specific binding to certain kB DNAs. Our discovery sheds new light on the long-standing question of how NF-kB regulates distinct sets of genes in response to specific inducers. This research was supported by the Intramural Research Program of the NIH, NIAID.
High resolution mapping in Experimental Autoimmune Neuritis identifies Ncf-1 as a candidate gene (136.26)
Authors: Beyeen Amennai Daniel , Huberle Alexander , Öckinger Johan , Jagodic Maja , Olsson Tomas , Weissert Robert
Published: January 2023

We here dissect the polygenetic regulation of Experimental Autoimmune Neuritis (EAN), an animal model of Guillain-Barré Syndrome (GBS). GBS is a multifocal demyelinating disorder of the peripheral nervous system. In our work with a rat EAN model, we have crossed the EAN-susceptible DA strain with the EAN-resistant PVG for 12 generations, allowing us to investigate the relative effects of small genetic regions from the two different strains. We identified one such region, Ean6, linking to clinical disease severity. Ean6 contains the gene Ncf-1, a disease protective gene in animal models of Multiple Sclerosis and Rheumatoid Arthritis. Ncf-1 is a member of the NADPH oxidase complex and regulates T cell activation in these two models. As T lymphocytes have an essential role in EAN disease progression we sought to investigate the role of Ncf-1 in our model. We stimulated Ncf-1 through the NADPH complex with the compound phytol, resulting in a milder disease in DA rat females compared to controls. Although other mechanisms of action of phytol in inflammation can not be excluded, we suggest Ncf-1 to be a candidate gene in EAN and Guillain Barré Syndrome.
Antigen-Specific Tolerance Induction Using Genetically Modified Marginal Zone B Cells (136.35)
Authors: Zhang Ai-Hong Allan , Su Yan , Li Xin , Skupsky Jonathan , Scott David W.
Published: January 2023

Antigen-specific tolerance induction can be induced in mouse models of autoimmune diseases by adoptive transfer of genetically modified spleen B cells. We hypothesized that genetically modified marginal zone (MZ) B cells are more efficient in mediating tolerance induction. B cell-depletion using an IgG1 mouse anti-mouse CD20 monoclonal antibody (aCD20), which spares MZ B cells, was used to test our hypothesis. Mice were injected i.v. with IgG1 type aCD20 or isotype control IgG1. Two weeks later, spleens from control mice contained 75% follicular B cells (FO; CD19+CD23hiCD21low) and 8% MZ B cells (CD19+CD23intCD21hi). IgG1 aCD20 treatment reduced FO B cells by 98% but MZ B cells were relatively enriched (51% of the residual B cells). To test whether MZ B cells are enriched to function as tolerogenic antigen-presenting cells (APC), we used B cells from mOVA B cell transgenic mice (mOVA mice), which express membrane-bound OVA. C57Bl/6 recipient mice were adoptively transferred with 5 ×10E6 LPS-activated MZ B cells or whole spleen B cells from mOVA or B6 mice, as control. One week after B-cell transfer, the mice were challenged with OVA323-339/CFA, and T-cell proliferation response to OVA323-339 analyzed two weeks later. Adoptive transfer of LPS activated mOVA spleen B cells, but not B6 B cells, were tolerogenic and significantly decreased the T-cell response to OVA323-339 (p<0.001). Importantly, transfer of LPS-activated mOVA MZ B cells further dampened the T-cell response to OVA323-339 (p<0.05). Taken together, we conclude that genetically modified MZ B cells are more efficient APC in mediating tolerance induction than whole spleen B cells. (Supported by NIH RO1 AI035622 and an Innovative Grant from the Juvenile Diabetes Research Foundation)
A novel zinc finger protein represses the MHC-II transactivator (CIITA) during B cell differentiation and interacts with the polycomb complex factor EZH2 (136.36)
Authors: YOON HYESUK , Boss Jeremy M.
Published: January 2023

The master transcriptional regulator of MHC-II gene expression, CIITA, is silenced as mature B cells differentiate into plasma cells. The mechanism of CIITA silencing is thought to employ the plasma cell differentiation repressor BLIMP-1. However, some ex vivo differentiation systems that result in CIITA silencing suggest that additional factors may be involved. An ex vivo B cell to plasma cell differentiation system was established to identify additional CIITA silencing factor(s). cDNA microarrays were employed to differentially screen for genes expressed in plasma cells. A zinc finger protein (ZFP), which is induced early during ex vivo differentiation of primary B cells, was identified as a potential repressor of CIITA. Ectopic expression of ZFP resulted in a substantial decrease in CIITA expression. ZFP was found by chromatin IP to bind to promoter III of CIITA in a region that is also bound by BLIMP-1 in plasma cells, suggesting a role in CIITA silencing. Moreover, ZFP co-precipitated with BLIMP-1 from lysates derived from plasma cell lines. ZFP also co-precipitated with the polycomb histone methyltransferase EZH2. Intriguingly, plasma cell lines displayed the histone H3K27me3 modification associated with EZH2 at promoter III. Thus, these results suggest that ZFP may function to coordinate repression of CIITA during B cell differentiation in manner that involves the polycomb complex.
Long-range distal elements within the Ifng locus (136.41)
Authors: Collins Patrick L , Henderson Mel , Chang Shojang , Mortlock Doug , Aune Tom
Published: January 2023

Transgenic mice carrying an 8.6 kb Ifng transgene do not recapitulate Th1/Th2 selectivity, but mice carrying a transgene with the surrounding 190 kb of non-coding sequences do. It is unknown why Ifng requires 190 kb of regulatory sequences and space. To understand distal regulation of Ifng, we constructed transgenic mice carrying ~40 kb deletions within the 190 kb transgene. We identified two large regions which regulate Ifng expression. We then created small, 1 kb, deletions to identify specific regulatory elements. We identify two 1 kb regions at -30 kb and at -77 kb which are necessary for IFN-g transcription and repress IFN-g transcription, respectively. Our results demonstrate that multiple long-range distal regulatory elements cooperate to drive the proper temporal and quantitative expression of a single gene.
Induction of severe thyroid epithelial cell hyperplasia (TEC H/P) and fibrosis by in vitro activated splenocytes from IFN-γ-/- NOD.H-2h4 mice (137.11)
Authors: Yu Shiguang , Sharp Gordon C , Braley-Mullen Helen
Published: January 2023

IFN-γ-/- NOD.H-2h4 mice develop TEC H/P and fibrosis characterized by abnormal proliferation of thyrocytes with infiltration of thyroids by CD4+ and CD8+ T cells, and production of anti-mouse thyroglobulin (MTg) autoantibodies. Thyroid inflammatory T cells, especially CD8+ T cells, are the primary effectors for severe TEC H/P and fibrosis. Splenocytes from donors with severe TEC H/P cultured with MTg alone, or with MTg and IL-23 transferred similar severe TEC H/P to SCID recipients, although cells cultured with MTg and IL-23 produced more IL-17 and IL-22 than those cultured with MTg alone. The objective of this study was to determine if splenocytes from naïve IFN-γ-/- mice cultured under Th17 polarization conditions could also transfer severe TEC H/P to SCID recipients. Splenocytes from naïve IFN-γ-/- mice cultured 72 hr with MTg, TGF- β, IL-6 and IL-21 or with MTg and IL-23 transferred severe TEC H/P while the same number of cells cultured with MTg alone did not. These results indicate that cells able to transfer severe TEC H/P can be activated by culturing naïve IFN-γ -/- lymphocytes under Th17 polarizing conditions. Studies are in progress to determine the mechanisms involved in activation of effector cells that induce severe TEC H/P and fibrosis. These results should be relevant for defining potential therapeutic targets for intervention in autoimmune inflammation (VA Merit Review and NIH R56 AI074857).
Environmental causes of autoimmunity: Clues to the loss of tolerance in primary biliary cirrhosis (137.14)
Authors: Naiyanetr Phornnop , Meng Liping , Butler Jeffrey D. , Pfeiff Janice , Kenny Thomas P. , Lam Kit S. , Kurth Mark J. , Leung Patrick S.C. , Gershwin M. Eric
Published: January 2023

Primary biliary cirrhosis (PBC) is a human autoimmune disease that destroys small bile ducts and is characterized by antimitochondrial antibodies (AMAs) directed to the inner lipoyl domain of pyruvate dehydrogenase (PDC-E2). Our thesis is that environmental factors trigger the onset of PBC by breaking tolerance to PDC-E2. In particular, we submit that chemical xenobiotic modification of the lipoyl moiety of PDC-E2 results in creation of a neoantigen with a subsequent immune response in genetically predisposed individuals. Our previous work has been focused in two areas: the use of microarrays to identify chemical xenobiotics that react to AMAs as well as or better than native autoantigen and, second, the demonstration that a PBC-like disease can be generated in mice following immunization with our optimal xenobiotic coupled to BSA. Herein, we have focused on fine mapping of the optimal chemical compounds that mimic lipoic acid. We modified the S-S linkage ring structure of lipoic acid and conjugated an array of such chemicals on a PDC-E2 peptide backbone and then analyzed such compounds for reactivity using sera from patients with PBC and a variety of controls. Importantly, by such chemical fine mapping, we now identify 6,8-bis(acetylthio)octanoic acid as an optimal chemical structure. Finally, we note that this compound has striking structural similarity with a variety of drug metabolites.
CD25+3G11-CD4+ cells: A highly potent Treg subpopulation (137.16)
Authors: Zhang Guang-Xian , Zhao Zhao , Yu Shuo , Li Hongmei , Ciric Bogoljub , Yang Jingxian , Rostami Abdolmohamad
Published: January 2023

3G11, a sialylated carbohydrate epitope on the disialoganglioside molecule, is expressed predominantly on the surface of mouse CD4+ T cells. Our previous studies suggested that loss of the 3G11 molecule could be a new cell surface marker for regulatory CD4+ T cells. In the present study, we explore the relationship between 3G11- and CD25+ T cells, a well-defined regulatory T cell (Treg) population. We found that large proportion of CD25+CD4+ T cells lack expression of 3G11 and that 3G11-CD4+ T cells express higher levels of Foxp3 compared to the 3G11+CD4+ population. Upon stimulation, 3G11- cells produced a high level of IL-10, but low level of IL-2. Based on 3G11 and CD25 expression we sorted four CD4+ T cell subpopulations and tested their phenotypes. CD25+3G11- T cells expressed the highest levels of Foxp3 and IL-10 and efficiently inhibited mitogenic and antigen-specific immune responses in vitro and clinical EAE in vivo, while CD25-3G11+ T cells exhibited an effector phenotype. Further, upon i.v. antigen, a significant fraction of adoptively transferred 3G11+CD4+ T cells was concerted into 3G11-CD4+ population. Thus, lack of 3G11 expression 3G11-), combined with CD25 expression (CD25+3G11-), defines a cell surface phenotype of a novel, highly potent Treg subpopulation.
Cbl-b-/- T cells are resistant to Treg-expressed TGF-beta: a unifying mechanism for resistance to regulation and autoimmunity (137.18)
Authors: O'Conor Catherine , Housley William J , Clark Robert B
Published: January 2023

Cbl-b is an E3 ubiquitin ligase that negatively regulates T cell activation. Cbl-b deficient (Cbl-b-/-) mice develop spontaneous autoimmunity, and Cbl-b dysregulation has been described in both human lupus and multiple sclerosis. Although the mechanisms underlying the development of autoimmunity in Cbl-b-/- mice are unclear, we have previously reported that Cbl-b-/- CD4+CD25- T cells (Teff) are resistant to regulation mediated by both Tregs and TGF-β in vitro. To characterize the relevance of this resistance in vivo, we now demonstrate that Cbl-b-/- Teff are resistant to Tregs in two models of autoimmunity, colitis and GVHD. To further identify mechanisms underlying this resistance, we asked whether Cbl-b-/- Teff can respond to TGF-β and differentiate into Th17 T cells. In vitro, exogenous TGF-β and IL-6 results in the generation of Th17 cells from Cbl-b-/- Teff; however, unlike WT Teff, Cbl-b-/- Teff are defective in Th17 differentiation when Tregs are the only source of TGF-β. These results suggest that the inability of Cbl-b-/- Teff to respond to TGF-β expressed by Tregs may underlie their resistance to Treg-mediated suppression. Cbl-b-/- T cells may thus offer an important approach for characterizing the functional relationship of Tregs and TGF-β in both suppression and Th17 generation and for identifying related immunoregulatory abnormalities in human autoimmune disease (NIH: 1R56AI072533-01A1).
Hematopoietic TNFR1 signaling mediates protection in a mouse model of arthritis (137.26)
Authors: Scheinman Robert I , Raman Thiagarajan , Williams-Skipp Cheryll , Derry Molly , Helfer Christine
Published: January 2023

TNFα blockade is a common treatment for rheumatoid arthritis (RA). Our hypothesis was that some of the pathological effects of TNFα involved signaling through hematopoietic TNFR1. We tested this using bone marrow transplant. The TNFR1 knockout mouse was backcrossed into the DBA background for 6 generations. DBA mice were irradiated, rescued with bone marrow derived from either DBA mice or from TNFR1-/- mice, and input into the collagen arthritis model. Surprisingly, we found that TNFR1-/-/DBA chimeras developed more intense disease as compared to controls. Increased disease was not caused by an increased immune response to collagen, insufficient backcrossing, or increased TNFα secretion. During intracellular bacterial infections, curiously, TNFα switches function from inflammation to resolution. A central aspect of this switch in function involves inhibition of the secretion of the IL-12/IL-23 cytokine family. We tested this in our system and found that long term culture of DBA/1 macrophages with TNFα resulted in the inhibition of IL-12 p40 subunit secretion and that serum IL-12 p40 subunit levels were elevated in TNFR1-/-/DBA mice at late stages of disease. These data are consistent with the concept that this switch in TNFα function also occurs in chronic inflammatory disease states such as RA. We propose that the genetic strength of this switch may correlate with response to therapy.
Pregnancy-Induced Rheumatoid Arthritis Amelioration in SKG Mice (137.29)
Authors: Dragone Leonard , Shaw Laura A , Rumer Kristen K , Kramer Anita , Sakaguchi Shimon , Winn Virginia D
Published: January 2023

Background: During pregnancy 75% of women with Rheumatoid Arthritis (RA) have amelioration of their disease. The mechanism of pregnancy-induced amelioration of RA is poorly understood. A mouse model of RA has been characterized (SKG). SKG mice have a spontaneous mutation in ZAP-70 and a single dose of zymosan induces arthritis. To determine if the SKG model is valid for studying the effects of the immune system on RA amelioration, we looked at whether remitted arthritis in SKG mice.

Methods: Cohorts of SKG mice were treated with zymosan (Z, n=17), zymosan plus pregnancy (Z+P, n=6), pregnancy (P, n=2) or no treatment (CTL, n=2). A subset of mice was sacrificed on day E14.5 (Z=5, Z+P=3, P=2, CTL=2) to harvest lymphoid and joint tissues.

Results: Pregnancy remitted arthritis in 4 of the 6 Z+P mice with an overall decrease in arthritis progression (Z+P=0.48+0.4 vs Z=1.91+0.2, p=0.01). The Z+P group flared postpartum. Histological analysis showed decreased joint inflammation in the Z+P compared to the Z mice. Flow analysis showed an increased percentage of Th17 cells, known to drive arthritis in SKG mice, in the Z (2.07+0.4) and in the P group (2.72+0.4) vs CTL (0.99+0.005, p<0.04). Interestingly, the Th17 cell increase was additive for the Z+P group (4.37+0.15) despite arthritis improvement.

Conclusions: Pregnancy-induced amelioration of RA can be achieved in SKG mice and is accompanied by an increase in Th17 cells.