bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin-17A is dispensable for myocarditis but essential for the progression to dilated cardiomyopathy (135.25)
Authors: Baldeviano Geral , Barin Jobert , Talor Monica , Srinivasan Sachin , Chen Ping , Bedja Djahida , Zheng Donfeng , Gabrielson Kathleen , Iwakura Yoichiro , Rose Noel , Cihakova Daniela
Published: January 2023

A third of myocarditis cases progress to dilated cardiomyopathy (DCM); however, the mechanisms controlling this process are largely unknown. Immunization of BALB/c mice with myocarditogenic peptide in CFA adjuvant induced the infiltration of pathogenic IL-17A-secreting T helper type-17 cells into the inflamed heart during the acute stage; and mild-to-severe DCM in the chronic stage of the disease. However, IL-17A deficiency did not abrogate the incidence or severity of myosin-induced myocarditis, nor did it ameliorate the severe disease of IFNγ-deficient mice, suggesting that IL-17A plays a minimal role during acute myocarditis. In contrast, during the chronic stage, IL-17A-deficient mice but not WT littermates were fully protected from the progression to DCM. Flow cytometry and cytokine analysis revealed an important role for IL-17A in the upregulation of IL-6, TNFα, and IL-1β in the heart tissue, and the recruitment of CD11b+ monocytes and Gr1+ granulocytes into the heart. These differences were associated with a significant decrease in myocardial fibrosis and dysregulation of matrix metalloproteinases. Furthermore, neutralization of endogenous IL-17A after the onset of myocarditis decreased myocardial fibrosis and improved systolic function. Our findings reveal a critical role for IL-17A in adverse cardiac remodeling leading to end-stage DCM, and suggest that targeting IL-17A may be an attractive therapeutic strategy for patients with inflammatory DCM.
Exaggerated IL-23-induced psoriasis-like inflammation in mice lacking CCR4 (135.32)
Authors: Hedrick Michael , Zhang Hongwei , Farber Joshua
Published: January 2023

Recent evidence strongly implicates the IL-23/Th17/Th22 axis in psoriasis, and intradermal injection of IL-23 produces local psoriasis-like inflammation, which depends on IL-22 and other IL-10-family cytokines. CCR4 is a chemokine receptor thought to be important for T cell trafficking to skin, and is expressed on all Th2 cells, as well as many Th17 cells and Treg. We tested CCR4 deficient mice in the IL-23 injection model and found, surprisingly, that they had an exaggerated inflammatory response, with increased swelling of the injected skin, greater acanthosis, parakeratosis, and microabscess formation, higher numbers of infiltrating neutrophils, T cells and dendritic cells, and higher numbers of pro-inflammatory cytokines, including: IL-17a, IL-17f, IL-19, IL-22 and TNFα. Remarkably, despite the greater inflammation, Foxp3+ cells were not increased in the IL-23-injected skin of the CCR4 deficient mice, producing a higher effector T cell/Treg ratio as compared with wild-type mice. Our results suggest that CCR4 is important, not for effector functions, but for recruiting cells to down-modulate Th17/Th22-mediated inflammation in skin. These findings may help in understanding how inflammation is regulated in psoriasis and related disorders. Supported by the Intramural Research Program, NIH, NIAID.
The structural mechanism of Serum Amyloid A -mediated Secondary amyloid formation (135.34)
Authors: Lu Jinghua , Du Clos Terry , Mold Carolyn , Sun Peter
Published: January 2023

Secondary amyloidosis is a severe complication of many chronic inflammatory diseases, such as rheumatoid arthritis and atherosclerosis, when the C-terminally cleaved fragments of acute phase protein Serum Amyloid A (SAA) are deposited in tissues as amyloid fibrils. The pathologic process of secondary amyloidosis involves a conversion of the structure of native SAA into a predominantly antiparallel β-sheet secondary structure. We have now determined the X-ray structure of SAA in a monomeric and a hexameric form at 2.2Å and 2.7Å resolution, respectively. The hexameric SAA structure revealed a dimeric architecture arranged as two trimers. Strong interactions between the C-terminal loop region and four-helix-bundle core of the SAA monomers create a stable structure to prevent secondary structure refolding during amyloidogenesis. The presence of multimeric forms in human serum was confirmed by size-exclusion experiments. Recombinant monomeric, trimeric and hexameric SAA protein were purified from Escherichia coli and characterized. Binding studies show that the monomeric and trimeric but not the hexameric SAA bind to Toll-like receptor 2 and 4. In line with these experiments, SAA may undergo a transition between hexamers and monomers, which would activate Toll-like receptors to further complicate the chronic inflammation in disease situation.
Prolonged CD154 expression in pediatric lupus CD4 T cells correlates with increased CD154 transcription, increased NFAT activity, and glomerulonephritis (135.4)
Authors: Cron Randy , Genin Anna , Brunner Michael , Scalzi Lisabeth , Mishra Nilamadhab , Beukelman Timothy , Mehta Jay
Published: January 2023

To explore a mechanism explaining dysregulated CD154 expression in lupus, cell surface CD154 expression was examined, pre- and post-activation, on peripheral blood CD4 T cells from 29 children with lupus and matched controls by flow cytometry. CD154 mRNA levels and transcription rates were studied by real-time RT-PCR and nuclear run-on assays, respectively. NFAT transcriptional activity and NFAT mRNA levels in lupus CD4 T cells were explored by reporter gene analysis and real-time RT-PCR, respectively. We found CD154 surface protein levels were increased 1.44-fold on lupus CD4 T cells compared to controls at one day post-activation ex vivo and this correlated with the presence of nephritis. Increased CD154 protein also correlated with increased CD154 mRNA levels and rates of CD154 transcription, particularly at later time-points post-T cell activation. Reporter gene analyses revealed a trend for increased NFAT, but decreased AP-1 and similar NFkB, activity in lupus CD4 T cell compared to controls. NFAT1 and, in particular, NFAT2 mRNA levels were notably increased in lupus CD4 T cells compared to controls. In summary, following activation, cell surface CD154 is increased on pediatric lupus CD4 T cells compared to controls, and this correlates with the presence of nephritis, increased CD154 transcription rates, and NFAT activity. These results suggest that NFAT/calcineurin inhibitors, such as tacrolimus and cyclosporine, will be beneficial treating lupus nephritis.
Expression of chitinase-like proteins in inflamed tissues of Sjögren’s Syndrome (SS) patients (135.6)
Authors: Greenwell-Wild Teresa , Moutsopoulos N. , Gliozzi M. , Kapsogeorgou E. , Rangel Z. , Munson P. , Moutsopoulos H. , Wahl S.
Published: January 2023

SS represents a chronic autoimmune disease of salivary and lacrimal glands that may be accompanied by multi-organ systemic manifestations. Among the locally involved innate immune defense populations are macrophages, which have been linked to IL-12 and IL-23 that moderate T lymphocyte lineage commitment. To further an understanding of the immunopathologic sequelae associated with SS and to define therapeutic targets, we performed microarray analysis of target tissues from SS patients with severe histopathologic lesions compared to those with sicca symptomatology but negative minor salivary gland biopsy. We identified the expression of multiple genes, including members of the mammalian chitinase family, not previously associated with exocrinopathies, among the most highly expressed SS genes as compared to diseased, but non-SS tissues. Both chitinase-3-like-1 (CHI3L1/YKL-40) and chitinase 1 (CHIT1), highly conserved members of the mammalian chitinase-like glycoprotein family, one with and one lacking enzymatic activity, were evident at the transcriptome levels, as well as detected as glycoproteins within inflamed salivary glands. Monocyte to macrophage differentiation is accompanied by an increase in chitinase expression, which can be augmented by cytokine exposure. Since the elevated expression of these molecules corresponded with more advanced disease in SS patients, these observations suggested a potential immunopathologic involvement.
Positioning of the HA Globular Head within the TLR5 Agonist, Flagellin, Modulates the Immunogenicity and Reactogenicity of a Novel-H1 Pandemic Influenza Vaccine (136.13)
Authors: Umlauf Scott , Gathuru John , Bell Rodney , Hou Fu , Reiserova Lucia , Song Langzhou , Weaver Bruce , Liu Ge , Tussey Lynda
Published: January 2023

The recent appearance of a novel H1N1 (nH1N1) virus has highlighted the importance of rapid response to emerging pandemic threats. VaxInnate has developed an influenza vaccine production system that permits rapid response on a global scale. The vaccine comprises the protective globular head subunit of HA fused covalently to the TLR 5 ligand flagellin. Using animal models that have successfully predicted the tolerated dose range in humans we have identified an optimal position for the HA globular head. Three constructs evaluated the globular head fused to the C terminus of flagellin (C-term), in place of domain 3 (R3) and in both positions (R3.2x). The three formats were tested in mouse immunogenicity and rabbit reactogenicity models. HAI assays were performed using CA/07/2009 virus. Rabbits were monitored for body temperature, food consumption and CRP. In mice, HAI titers >1:40 were achieved with 1 μg doses of all vaccines, but R3 and R3.2x vaccines yielded higher titers. In rabbits, the C-term vaccine elicited elevated temperature and CRP levels and reduced food consumption at doses well below R3 and R3.2x. Replacement of domain 3 of flagellin with the HA globular head, either R3 or R3.2x, proves safe and immunogenic in mice and rabbits. Our clinical experience with other flagellin-linked vaccines has correlated well with the rabbit model. The results predict that the R3 or R3.2x nH1 vaccines would be both safe and efficacious in humans.
Alternative TLR signaling restores Francisella tularensis LPS-mediated protection against Francisella tularenis LVS challenge in TLR2-/- mice (136.17)
Authors: Cole Leah , Vogel Stefanie
Published: January 2023

The Category A select agent Francisella tularensis (Ft) is the causative agent of tularemia, a potentially fatal infectious disease. Intraperitoneal infection of mice with ~10 Ft Live Vaccine Strain (Ft LVS) organisms causes a lethal infection that resembles human tularemia. Immunization with 100 ng Ft LVS lipopolysaccharide (Ft-LPS) provokes an antibody response that protects wild-type (WT) C57BL/6J mice against an otherwise lethal Ft LVS challenge, but offers limited protection to TLR2-/- mice (WT, 85% survival vs. TLR2-/-, <25% survival; p < 0.0001, N = 4 separate experiments). Since macrophage recognition of Ft LVS is overwhelmingly TLR2-dependent (Cole, L. E., et al. 2007. Infect. Immun. 75:4127-37), we hypothesized that treatment of TLR2-/- mice with an alternative MyD88-dependent, TLR agonist would compensate for the failure of Ft LVS to signal in TLR2-/- mice and thereby lead to increased Ft-LPS-mediated protection. Concurrent administration of the TLR4 agonists, E. coli LPS (5μg) or monophosphoryl lipid A (MPL; 100μg), a synthetic non-toxic derivative of E. coli lipid A, with either the Ft-LPS immunization or the Ft LVS challenge, increased the survival of Ft-LPS-pretreated TLR2-/- to 100% (N=4 separate experiments). This suggests that effective Ft-LPS-mediated protection against Ft LVS challenge requires both Ft-LPS recognition and MyD88-dependent, TLR signaling. Supported by NIH AI157168.
In vitro examination of the role of TLR5 in enhancement of antigen presentation. (136.19)
Authors: Atif Shaikh , Letran Shirdi , McSorley Stephen
Published: January 2023

Data from our laboratory suggest that TLR5 can enhance the presentation of flagellin eptiopes to CD4 T cells in vivo. In this present study we have examined this process in vitro using antigen-presenting cells harvested from wild-type and TLR5-deficient mice and examining early activation of flagellin-specific SM1 CD4 T cells. Our in vivo data suggest that TLR5 expression in the spleen is absolutely required for activation of SM1 T cells. Recapitulation of this response in vitro will allow us to identify the TLR5+ antigen presenting cells in this anatomical site. As previous studies have failed to identify any TLR5+ population in the spleen, these experiments have the potential to enhance our understanding of flagellin antigen presentation and splenic dendritic cell subsets.
TLR reporter cell lines for screening TLR agonists and antagonists (136.20)
Authors: Rosenberg Jonathan , Lee Hyun-Ku , Singh Gita , Andrew Kody , Singh Sujay
Published: January 2023

We have developed novel TLR/NF-κB SEAPorter™ HEK 293 cell lines each expressing an intact Toll-like Receptor (TLR) as well as a secreted alkaline phosphatase (SEAP) reporter whose expression is under control of an NF-κB response element. These cell lines are suitable for TLR-dependent functional assays and are an ideal fit for high-throughput screening of TLR agonists or antagonists. TLR expression of each cell line has been validated by Flow Cytometry. Using a 96-well plate format, functional activity has been evaluated by criteria including cell number titration, ligand dose response, cell stimulation time and DMSO tolerance. Validation results yield an average signal-to-background (S/B) ratio of 16.9±0.7 as well as Z' value of 0.75±0.05 for ligand dose-response tests. Assay validation is based on the 96-well plate format screening assay, but are also suitable for 384- or 1536-well plate format screening assays. For TLR agonist screening, each compound in a library can be directly applied to cells and induced SEAP in culture media can be measured without additional steps. TLR antagonist screening can be performed as agonist screening except the addition of TLR ligands is done before or after treatment with library compounds. Consequently, the TLR-specific cell-based high-throughput feature of our novel TLR/NF-κB SEAPorter™ HEK 293 cell lines will provide a fast, flexible and convenient means for lead compound screening in the pharmaceutical drug development fields.
Evidence that gC1qR and DC-SIGN associate on the surface of immature dendritic cells (136.24)
Authors: Hosszu Kinga , Vinayagasundaram Uma , Vinayagasundaram Rama , Santiago-Schwarz Frances , Peerschke Ellinor , Ghebrehiwet Berhane
Published: January 2023

Complement protein C1q is involved in many immunoregulatory functions, such as modulating T-cell proliferation and regulating dendritic cell (DC) differentiation and maturation. C1q has at least two multi-functional cell surface receptors, cC1qR and gC1qR. While these receptors ostensibly lack a direct conduit into intracellular elements, they are known to form docking/signaling complexes with transmembrane partners. DC-SIGN (dendritic cell-specific-ICAM-grabbing-non integrin) is a C-type lectin expressed on the surface of DCs and involved in T-cell activation and endocytosis. Because SIGN-R1, a C-type lectin related to DC-SIGN has been shown to bind directly to C1q, we investigated whether DC-SIGN also binds to C1q and if DC-SIGN may act as a transmembrane partner for gC1qR and/or cC1qR. Using flow cytometry, we found that DC-SIGN, gC1qR and cC1qR are all present on the surface of human immature DCs (iDC) grown in culture. While antigen-capture ELISA experiments using iDC lysates showed that C1q, gC1qR and cC1qR directly or indirectly associate with DC-SIGN, immunofluorescence microscopy revealed that C1q and gC1qR, but not cC1qR co-localize with DC-SIGN on the surface of iDCs. The data suggest that DC-SIGN and gC1qR may directly associate on the surface of iDCs and regulate DC differentiation in a stage specific manner. Since both gC1qR and DC-SIGN have been shown to bind HIV, it is possible that the two receptors may play a role in the pathogenesis of HIV-AIDS.
TLR5 functions as a scavenger receptor to enhance flagellin-specific CD4 T cell Responses (136.29)
Authors: Letran Shirdi , Lee Seung-Joo , Atif Shaikh , McSorley Stephen
Published: January 2023

Innate immune activation via Toll-like Receptors (TLRs) causes dendritic cell maturation and the production of inflammatory mediators. Our data also demonstrate that TLR5 functions as a scavenger receptor for flagellin, enhancing the activation of flagellin-specific CD4 T cells. Flagellin-specific CD4 T cells were found to expand poorly in TLR5-deficient mice immunized with flagellin and this deficiency persisted even when additional TLR agonists were provided. In marked contrast, TLR5-deficient mice generated a robust flagellin-specific T cell response to processed flagellin peptide. The deficiency in flagellin-specific T cell activation was also detected in TLR5-deficient mice that were orally infected with a flagellated pathogen, demonstrating that TLR5 is responsible for directing a dominant CD4 response to flagellin in the context of infectious disease. Together, these data demonstrate that in addition to its function as an initiator of innate inflammation, TLR5 functions as a scavenger receptor allowing optimal presentation of flagellin epitopes to CD4 T cells.
Novel signaling interactions between proteinase-activated receptor 2 and Toll-like receptors in vitro and in vivo (136.36)
Authors: Nhu Quan , Rallabhandi Prasad , Shirey Kari Ann , Teijaro John , Farber Donna , Netzel-Arnett Sarah , Antalis Toni , Fasano Alessio , Vogel Stefanie
Published: January 2023

Toll-like receptors (TLRs) and proteinase-activated receptors (PARs) function as innate immune biosensors in mucosal epithelial cells (ECs). We previously reported the functional and physical interactions between TLR4 and PAR2, and more recently, between PAR2 and the TLR adapter, TRIF, and established a novel mechanism of “receptor cooperativity” between PAR2 and TLR4. We hypothesized that intracellular signaling pathways utilized by TLRs and PAR2 would converge either cooperatively or non-cooperatively when co-engaged. We demonstrate herein the cooperation between PAR2 and TLR2, TLR3, or TLR4 for activation of NF-κB-dependent signaling in mucosal EC lines. In contrast, activation of PAR2 negatively regulated TLR3-dependent antiviral pathway, blunting the expression of TLR3/IRF-3-driven genes, as well as activation of IRF-3 and STAT1. Similarly, TLR4-TRIF-activated, IRF-3-driven genes were also attenuated by PAR2 co-activation. Consistent with these in vitro observations, PAR2−/− and TLR4−/− mice, which were refractory to footpad edema induced by PAR2 agonist peptide, were protected from mouse-adapted H1N1 influenza A virus-induced lethality when compared to wild-type mice. These data support and extend our recently described, novel model of PAR2-TLR4 “receptor cooperativity” and highlight the complexity of signaling integration between heterologous innate immune biosensors.
Role of PI3K and autophagy in virus-stimulated IFN-α production by human plasmacytoid dendritic cells (136.46)
Authors: Deng Jing , Singh Sukhwinder , Fitzgerald-Bocarsly Patricia
Published: January 2023

Human plasmacytoid dendritic cells (pDC) produce large amount of IFN-α in response to viral stimuli through TLR7 and TLR9. Phosphatidylinositol-3 kinase (PI3K) has been shown to be activated by TLR stimulation in many cell types and Class I and class III PI3K are essential in the regulation of autophagy. We aimed to study the role of autophagy in both the processing of virus and induction of IFN-α in human pDC, which have not been described. We investigated the effects of Class I and class III PI3K inhibitors wortmannin, 3-methyladenine (3MA) and LY294002 on IFN-α production by HSV, Flu, SeV, CpG A and HIV-MN/AT2-stimulated human pDC. pDC were pretreated with the inhibitors then stimulated with virus for 6hr for detection of intracellular IFN-α by intracellular flow cytometry or overnight for stimulation and release of IFN-α, which was detected by ELISA. Inhibition of PI3K with wortmannin, 3MA and LY294002 inhibited IFN-α production by pDC. The PI3K inhibitors did not affect endocytosis or macropinocytosis. In addition, HSV dramatically induced autophagosome double-membrane structures in pDC as visualized by TEM and LC3B aggregation as seen in imaging flow cytometry. All of the inhibitors inhibited HSV-induced LC3B aggregation in pDC. These results support key roles for PI3K and autophagy in mediating IFN-α secretion and virus detection by human pDC. Supported by AI26806.
TIM-4 mediated phagocytosis of apoptotic cells induces regulatory cytokine secretion by mouse peritoneal macrophages (136.47)
Authors: Bu Xia , Zhu Baogong , Umetsu Dale , DeKruyff Rosemarie , Freeman Gordon
Published: January 2023

TIM-4 is a member of the T cell immunoglobubulin mucin (TIM) family and is expressed on macrophage and dendritic cell populations. TIM-4 specifically binds phosphatidylserine (PtdSer) on the surface of apoptotic cells and mediates phagocytosis of apoptotic cells by macrophages. NIH3T3 cells transfected with TIM-4 can phagocytose apoptotic thymocytes and this phagocytosis can be blocked by PtdSer liposomes, indicating that TIM-4 is sufficient to mediate phagocytosis of apoptotic cells in a PtdSer-dependent manner. We examined cytokine secretion by mouse peritoneal macrophage after phagocytosis of apoptotic thymocytes and found that production of a set of cytokines including TGF-β, IL-10, and others was increased dramatically. We performed qRT-PCR assays to detect the mRNA level of these cytokines upon peritoneal macrophage phagocytosis of apoptotic thymocytes. We found that the increased cytokine mRNA levels peaked from 30 minutes to 3 hours and protein production peaked at 24h. The anti-mouse TIM-4 antibody, 21H12, can block TIM-4 recognition of PtdSer and reduce phagocytosis of apoptotic cells by peritoneal macrophages. The 21H12 antibody effectively suppressed the cytokine mRNAs induced by TIM-4-mediated phagocytosis of apoptotic cells. These findings indicate that TIM-4 plays an important role in phagocytosis of apoptotic cells and secretion of regulatory cytokines by mouse peritoneal macrophages and may play an important role in immune regulation through these cytokines.
Immunomodulatory effects of Lactobacillus plantarum and Lactobacillus acidophilus strains on a rat intestinal epithelial cell line (137.21)
Authors: Duersteler Megan , Novak Kimberly , Davis Ellen , Smith Alexandra , Rehberger Thomas
Published: January 2023

Lactobacillus plantarum and Lactobacillus acidophilus have the potential to be immunomodulatory probiotics. An experiment was conducted to determine the effects of three L. plantarum and three L. acidophilus strains on the expression of five inflammatory cytokine genes (TNF-α, MIP-2, IL-1β, IL-6, and IL-18) by the IEC-6 rat intestinal epithelial cell line. The Lactobacillus strains had unique genotypic profiles based on RAPD PCR. IEC-6 cells were exposed to 107 cfu viable bacterial cells for one hour with and without lipopolysaccharide (LPS) stimulation. Expression of inflammatory cytokines was analyzed by quantitative PCR. L. plantarum 7-12 and L. acidophilus La-11 had an inflammatory effect on unstimulated cells as indicated by up-regulation of IL-18 by 7-12, and up-regulation of MIP-2 and IL-6 by La-11. L. plantarum 5-39 and L. acidophilus NCFM had an anti-inflammatory effect as indicated by down-regulation of IL-1β. L. plantarum CI15 and L. acidophilus La1 did not affect cytokine expression. LPS increased the expression of TNF-α and MIP-2 in IEC-6 cells > 60 fold. All strains except L. plantarum 7-12 down-regulated expression of TNF-α in cells stimulated with LPS. L. plantarum CI15 and 5-39, as well as L. acidophilus NCFM and La1 down-regulated MIP-2 expression in LPS-stimulated cells. These results indicate that L. plantarum and L. acidophilus have immunomodulatory properties, and the effects on cytokine expression are strain specific.
A role for regulatory T cells and TGF-β in intestinal IgA to rotavirus (137.22)
Authors: Miller Amber , Blutt Sarah , Conner Margaret
Published: January 2023

Regulatory T cells (Tregs) play an important role in suppression of effector T cell function and inflammation. Recently Tregs have been shown to play a role in mucosal IgA production through production of TGF-β1, suggesting they also can induce B cell responses. During rotavirus (RV) infection, there is little effector T cell activation but a profound intestinal IgA response. Using flow cytometric based approaches, we observed a significant increase in total number of FoxP3+CD25+CD4+ T cells in both MLNs (5 fold) and PPs (2 fold) 2-4 days post RV infection compared to uninfected mice. We also detected a significant increase in the total number of TGF-β1+ cells and the total number of TGF-β1+CD4+ cells in both tissues isolated from infected mice compared to uninfected mice. The increased number of FOXP3+ Tregs and CD4+/TGF-β1 expression in PP and MLN during RV infection concurrent with previously observed B cell activation and rotavirus-specific IgA production suggests that Tregs might play an important role in the induction of rotavirus specific intestinal IgA in these immune tissues through production of the critical switch factor, TGF-β1.
ANALYSIS OF THE RESPONSE OF HUMAN NEONATAL DENDRITICS CELLS AND MACROPHAGES AGAINST ROTAVIRUS (137.26)
Authors: Rosales Denisse , Gutierrez-Xicotencatl Lourdes , Santana-Calderon Angelica , Ramirez-Pliego Oscar , Cortez-Gomez Rudyard , Esquivel-Guadarrama Fernando
Published: January 2023

Rotavirus (RV) is the most common cause of infectious diarrhea. Dendritic cells (DCs) and macrophages play a crucial role in the immune response against viral infections; therefore it is important to dissect the interaction of RV with these cells. Here we analyzed the interaction of RV with neonatal DCs and macrophages in an in vitro model. Cord blood-derived mononuclear cells were culture in vitro for 6 days in the presence of either hGM-CSF/IL-4 or hM-CSF to generated enriched DCs and macrophages, respectively. These cells were cultured for 24 hrs with rotavirus and the levels of the activation markers CD40, CD86 and MHC class-II, and the intracellular cytokines IL-6, IL-12 and IL-10 were analyzed by flow cytometry. TGF-β was evaluated by ELISA. Finally, the induction of allogenic T-celll proliferation was analyzed. We found that RV could induce the activation of the neonatal DCs and macrophages since all the activation markers analyzed were up regulated, albeit at different levels. As result of this activation, the neonatal DCs and macrophages produced high levels of the regulatory cytokines, but moderate levels of pro-inflammatory cytokines. Finally, the activated DCs and macrophages were able to induce a strong allogenic T-cell proliferation. These results showed that neonatal DCs and macrophages could be activated by RV and that this activation was sufficient to induce the priming of allogenic T-cells. Supported by grant SEP-CONACYT (80149) and DGAPA, UNAM.
The role of TGFβ in the development of Th17 and Treg cells during influenza viral infection in neonatal mice (137.6)
Authors: Hoskins Samantha , Lines Janet , Garvy Beth
Published: January 2023

Influenza virus is responsible for more infant hospitalizations and deaths than any other vaccine preventable disease in America. Our laboratory has established a neonatal murine model to influenza and have reported that T cells in the neonatal lung are less activated than in adults and do not infiltrate the alveolar spaces. It is known that TGFβ is elevated in the lungs of neonatal mice, and since neonatal mice do not respond with a typical Th1 response to influenza virus, the dynamics of T regulatory (Treg) cells and the Th17 response was evaluated. Our current studies show that neonatal mice have higher levels of T regulatory cells (Treg) than adults, and these elevated levels persist during influenza viral infection. However, in the absence of Treg modulation, neonatal T cells were not more activated and did not infiltrate into the alveolar spaces during an influenza viral infection. Larger numbers of neonatal CD4 T cells produced IL-17A than adults during viral clearance. Th17 cells may contribute to the clearance of influenza in the immunosuppressive environment of the developing neonatal lung. Investigation of the importance of TGFβ signaling on T cell activation and migration is currently underway using conditional knockout mice.
Structural basis of DAP12-associated intramembrane immunoreceptor assembly. (138.16)
Authors: Call Matthew , Wucherpfennig Kai , Chou James
Published: January 2023

Many of the molecular sensors that alert lymphocytes to dangerous events such as infection or transformation are modular receptors constructed from ligand-binding subunits and signaling subunits that assemble through specific contacts within the lipid bilayer to form functional complexes. Structure-function studies focusing on the extracellular and cytoplasmic domains of activating immunoreceptors like the T cell receptor (TCR)-CD3 complex have yielded a vast store of knowledge on the mechanisms governing ligand binding and the resulting intracellular biochemical cascades. However, the mechanisms physically linking these events across the membrane to initiate immune activation remain poorly defined, and structural information on the membrane-embedded portions of receptor complexes will be critical for developing more precise models. We previously published the first structure of an immunoreceptor-associated transmembrane (TM) signaling module, the TCR-associated ζζTM dimer. We now present the NMR structure of DAP12, a functional homologue of ζζ that provides the ITAM-based signaling capabilities to more than a dozen activating receptors expressed by NK cells and other hematopoietic lineages. We have also solved a structure of the trimolecular complex DAP12-TM forms with the NK cell receptor NKG2C. These structures reveal features that allow DAP12 to assemble with a multitude of different receptors with disparate TM sequences through focused polar contacts within the membrane.
Modulation of cytokine and nitric oxide production by keratinocytes and mononuclear phagocytes in a model of inflammatory acne. (138.26)
Authors: Watson Michelle , Ping E Ya , Lee Wei Li , Nowakowski Maja
Published: January 2023

Cytokines (TNFα, IL-1α, IL-6, IL-8, and IL-10) mediate the inflammatory process in various skin conditions, including acne. An in vitro co-culture model system involving different cell types was used to study the effect of UVB and other treatments on the inflammatory process, with nitric oxide (NO) and tumor necrosis factor (TNFα) as markers of inflammation. Cultured macrophages were stimulated with bacterial lipopolysaccharide (LPS) and exposed to UVB radiation (50 - 200mJ/cm2). The expression of nitric oxide (NO) was measured by modified Griess assay and the expression of TNFα was measured by quantitative ELISA. For co-culture systems, SC monocytes were combined with irradiated human epidermal keratinocytes (NHEK) and cultured macrophages were combined with peripheral blood mononuclear cells (PBMC). Samples of culture supernatants were collected at 1-6 days. When NHEK were irradiated with UVB (290-320 nm), a dose-dependent stimulation of TNFα production was observed (33-200% increase), but NO production was unchanged. In contrast, when macrophages were irradiated with UVB, significant inhibition of NO production (40% suppression, p<0.001) was seen. The macrophage-PBMC co-cultures showed no alteration in TNFα production, whereas NO production showed a dose- and time-dependent increase. These data indicate that keratinocytes and mononuclear phagocytes have distinct products and responses to UVB radiation. This co-culture system is an improved model of cutaneous inflammation.
Analysis of cross-species IgG binding to human and mouse Fcgamma receptors (FcγRs) (138.29)
Authors: Shashidharamurthy Rangaiah , Bozeman Erica , Patel Jaina , Kaur Ramneet , Meganathan Jeyandra , Selvaraj Periasamy
Published: January 2023

Monoclonal antibodies from various species are in therapeutic and diagnostic use and are used extensively in cell depletion studies in rodents. Therefore, in the present investigation the cross-species IgG binding to FcγRs were performed using recombinant dimeric soluble form of human and mouse FcγRs. We observed that human CD16A was able to bind to rabbit IgG better than human CD32AH and CD32AR. The binding specificity is hCD16>hCD32AH>>hCD32R. Both hCD32A alleles were able to bind mouse and rat IgG subtypes, whereas hCD16 binds weakly. hCD32AR binds mouse IgG1, IgG2a and IgG2b but not IgG3 (IgG1>>IgG2a≥IgG2b) whereas CD32AH binds IgG2a and IgG2b (IgG2a≥IgG2b) only not IgG1 and IgG3. With rat IgG subtypes, hCD32AR binds IgG1>IgG2a>>IgG2b and hCD32AH binds IgG1>IgG2b>IgG2a whereas neither hCD32A alleles bound to IgG2c. We observed that hCD32AR allele binds stronger than hCD32AH to rat IgG. With human IgG subtypes, both hCD16A and hCD32AR binds IgG3>IgG1>>>IgG2≥IgG4, whereas hCD32AH binds IgG3>IgG1>IgG2>>>IgG4. We have also characterized binding specificity of mouse FcγRs (mCD16A, mCD32B and mFcRIV) to rabbit and mouse IgGs. All three mFcγRs binds to rabbit IgG. For mouse IgG subtypes, mCD16A binds IgG2b>IgG2a≥IgG1, mCD32B binds IgG1≥IgG2b>IgG2a, whereas FcRIV binds IgG2a≥IgG2b only, none of the mFcγR bound to mIgG3. In conclusion, cross-species FcγR binding to different IgGs must be taken into consideration when IgG based therapeutics and diagnostics are evaluated in rodents.
HIGH DOSES OF TCR STIMULI PREVENT CONVERSION OF NAÏVE CD4+ T CELLS INTO iTREGS IN AN NF-kB-DEPENDENT MANNER (139.10)
Authors: Molinero Luciana , Alegre Maria-Luisa
Published: January 2023

Conversion of naïve CD4+ T cells into FoxP3-expressing regulatory T cells (iTregs) can be achieved in the presence of TGF-b and IL-2 upon TCR triggering by intermediate concentrations of antigen or anti-CD3 mAb, but is prevented by high concentration of antigen. The mechanism for this high dose inhibition of FoxP3 induction has not been elucidated. As TCR stimulation promotes NF-kB signaling, we hypothesized that TCR-induced NF-kB may be responsible for inhibition of FoxP3 expression at high TCR occupancy. Whereas increasing doses of anti-CD3 mAb or antigenic peptide resulted in inhibition of FoxP3 induction in wildtype T cells under iTreg conditions, CD4 transgenic cells expressing an NF-kB superrepressor retained the ability to express FoxP3. Conversely, overactivation of the NF-kB pathway via a constitutively active IKKb reduced Foxp3 expression in wildtype T cells at lower TCR occupancy. Furthermore, CARMA1, a key molecule for NF-kB signaling downstream of the TCR, was critical for Foxp3 inhibition at high TCR occupancy. This was not due to increased TGF-b- or IL-2-mediated signals, or diminished Akt activation in CARMA1-KO T cells, as phosphorylation of Smad2, STAT5 and the Akt target S6 was similar to that in wildtype cells. Rather, the accessibility of the Foxp3 locus was impaired in wildtype but not CARMA1-deficient T cells. Our results suggest that higher levels of NF-kB can inhibit FoxP3 chromatin remodeling, thereby preventing its induction.
Extracellular ATP Acts as a Danger Signal and May Contribute to Tissue Repair by Rapidly Stimulating P2RX7-dependent VEGF Release from Primary Human Monocytes (140.17)
Authors: Hill Lindsay , Gavala Monica , Lenertz Lisa , Bertics Paul
Published: January 2023

Extracellular ATP has been proposed to act as a danger signal to alert the immune system of cell damage. Release of high local concentrations of ATP activates the nucleotide receptor P2RX7, which triggers the release of pro-inflammatory mediators from monocytic cells. Although the pro-inflammatory actions of P2RX7 are well recognized, little is known regarding the potential function of P2RX7 in repair responses. Because the resolution of inflammation is characterized by monocytic cell-dependent production of pro-angiogenic factors, we evaluated the contribution of P2RX7 to this process. We observed that P2RX7 activation results in a time-dependent increase in VEGF mRNA; furthermore, both transient and long-term P2RX7 activation promote the robust release of VEGF from primary human monocytes. This VEGF release is calcium-dependent and associated with reactive oxygen species production. This previously unrecognized action of P2RX7 suggests that in addition to its established role in inflammation and cell death, P2RX7 signaling may also be important in the control of angiogenesis and wound repair.
Cannabidiol attenuates experimental autoimmune hepatitis by inducing accumulation of myeloid-derived suppressor cells (MDSCs) in liver (140.4)
Authors: Hegde Venkatesh , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Immune-mediated liver diseases are a major health problem worldwide. Cannabidiol (CBD), a non-psychoactive cannabinoid from hemp plant, modulates immune function and has shown therapeutic potential in treating inflammatory diseases. We studied the effects of CBD in a mouse model of autoimmune hepatitis induced by ConA. Administration of CBD after ConA challenge significantly suppressed hepatitis in a dose-dependent manner as shown by decreased aspartate transaminase levels and reduced liver injury. CBD also suppressed crucial inflammatory cytokines. Phenotypic analysis of infiltrating cells revealed that CBD acted by inducing arginase-expressing CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) in liver. Depletion of MDSCs using anti-Gr1 mAb reversed the protective effect of CBD. Furthermore, adoptive transfer of purified MDSCs in to naïve mice conferred partial resistance to ConA-hepatitis. CBD-induced MDSCs could effectively suppress T cell proliferation in vitro. Using vanilloid receptor-deficient Trpv1-/- mice, we observed that CBD acted independently of this receptor in the current model. Interestingly, CBD caused a robust induction of CD11b+Gr1+ MDSCs in the peritoneum which was completely blocked by PPAR-γ inhibitor and was attenuated in mast cell-deficient mice, suggesting that mechanistically, CBD may act through transcription factor PPAR-γ on tissue mast cells thus activating them to release specific mediators resulting in the migration and expansion of MDSCs.
A mouse model of food induced acute allergic angioedema (141.10)
Authors: Gangur Venu
Published: January 2023

Previous studies report that without the use of adjuvant such as cholera toxin, oral exposure to peanut proteins in mice results in oral tolerance. Here we tested the hypothesis that oral administration of peanut butter (instead of soluble peanut proteins) would be sufficient to sensitize C3H/Hej mice for peanut allergy. Groups of C3H/Hej mice were exposed to peanut butter or saline (orally, once per week) and examined for systemic IgE and IgG1 responses. Oral administration of peanut butter, but not saline, resulted in systemic peanut specific IgG1 and IgE antibody responses. Notably, as little as two oral exposures to peanut butter resulted in dramatic IgG1 antibody responses. Peanut butter elicited IgE and IgG1 antibodies were able to bind to peanut protein extract as well as to peanut butter preparation in ELISA system. Furthermore, oral challenge of sensitized mice, but not saline control mice, with peanut butter resulted in clinical symptoms of acute allergic angioedema with swollen lips, nostrils, face and eyelids. Mice also exhibited itching-scratching and rubbing symptoms with abnormal breathing. These data demonstrate for the first time that: oral peanut butter administration to C3H/Hej mice does not result in oral tolerance, but instead results in clinical sensitization for acute allergic angioedema. Funding: US EPA STAR Grant#R833133
TRANSCRIPTOME ANALYSIS OF MOUSE SPLEEN IN RESPONSE TO EGG OVOMUCOID TREATMENT (141.11)
Authors: Husain Mainul , Mine Yoshinori , Karrow Niel , Golovan Serguei
Published: January 2023

Food allergy is a serious health concern among infants and young children. Despite its growing prevalence, our current understanding of the molecular mechanism(s) of food allergy remains incomplete. In this study, transcriptome profile of spleen from BALB/c mice was analyzed using Affymetrix arrays. Female BALB/c mice 6-8 weeks of age were orally exposed twice weekly for 5 wks with 1 mg of egg ovomucoid (OVM) and 10 µg of cholera toxin (CT). Control mice were similarly sensitized with 100 µl of 10 µg/µl amino acid solution and CT. At wk 6, experimental and control mice were challenged by intraperitoneal (i.p.) injection with 1 mg of OVM and amino acid solution respectively. Mice were euthanized 40 min post challenge to collect the spleens. Total RNA extracted from spleen was subjected to microarray hybridization. Analysis of microarray data revealed 87 genes differentially expressed with at least ±1.5 fold (p≤0.05). Gene ontology (GO) analysis revealed several biological processes related to hypersensitivity, inflammation or immune response. Biological network analysis illustrated connections among some differentially expressed genes and several immune or hypersensitivity related processes. Expression of five differentially expressed genes from microarray experiment was validated by real-time RT-PCR. Results from this study revealed a network of genes that may enable us to better understand the underlying molecular mechanism(s) of food allergy.
A non-redundant role for Serpinb3a in the induction of mucus production in asthma (141.17)
Authors: Sivaprasad Umasundari , Askew David , Ericksen Mark , Gibson Aaron , Stier Matthew , Brandt Eric , Bass Stacey , Lindsey Mark , Daines Michael , Chakir Jamila , Stringer Keith , Wert Susan , Whitsett Jeffrey , LeCras Timothy , Aronow Bruce , Wills-Karp Marsha , Silverman Gary , Khurana Hershey Gurjit
Published: January 2023

Asthma is a major public health burden worldwide. Excessive mucus production and mucus plugging are key pathologic features of asthma, yet the mechanisms responsible for mucus production remain largely unknown and therapies to effectively target mucus hypersecretion are lacking. Using a murine asthma model, we showed that SerpinB3a, the mouse ortholog of the serine protease inhibitors, SERPINB4 and B3, contribute to house dust mite induced airway hyperresponsiveness (AHR), mucus production and goblet cell hyperplasia and expression of SPDEF, a transcription factor that mediates goblet cell differentiation. Microarray analysis revealed attenuated expression of multiple IL-13 regulated genes that contribute to mucus production in the Serpinb3a null mice and IL-13 treated mice showed attenuated AHR and mucus production. Our data have revealed a novel non-redundant role for SERPINB4 and B3 in mediating mucus production through regulation of SPDEF expression.
RGS16 regulation of Th2-mediated inflammatory responses (141.9)
Authors: Shankar Sucharita , Divietro Jeffrey , Xie Zhihui , Wilson Mark , Wynn Thomas , Druey Kirk
Published: January 2023

Chemokine receptors are G-protein coupled receptors (GPCRs) that direct immune/ inflammatory responses. Regulators of G-protein Signaling (RGS) proteins negatively regulate GPCRs by enhancing GTPase activity of Gα, catalyzing its deactivation. RGS16 in T lymphocytes regulates chemokine responses. Transgenic mice overexpressing RGS16 exhibited altered lymphocyte trafficking in an allergic pulmonary inflammation model compared to WT. Rgs16 mRNA increased upon naïve T cell activation with anti-CD3/anti-CD28. Lymph node (LN) T cells polarized towards Th2 in vitro expressed more Rgs16 than naïve cells. To further elucidate the role of RGS16 in Th2 cells, we generated Rgs16-/- (KO) mice. In chemotaxis assays, KO LN Th2 cells migrated more to CCL17 than WT cells, while cells from both groups migrated similarly to CXCL12 and CCL21. Mice were treated either mucosally or intravenously (IV) with Schistosome egg antigen (SEA), causing Th2-mediated eosinophilic pulmonary inflammation or granulomatous lung fibrosis, respectively. In the former model, bronchoalveolar lavage fluid from KO mice exhibited double the lymphocytes and eosinophils versus WT mice after tracheal challenge. In the IV model, granuloma size and liver inflammation markers increased in KO mice versus WT. In both models, KO LN cells secreted more Th2 cytokines (IL-4, 5, 13) versus WT after SEA re-stimulation. In summary, RGS16 in Th2 cells regulates lymphocyte activation and migration to Th2 chemokines.
Glabridin inhibits lipopolysaccharide-induced activation of a microglial cell line, BV-2, by blocking NF-κB and AP-1 (142.3)
Authors: Lee Ki Hoon , Park Sun Hong , Kang Jong Soon , Han Sang-Bae , Lee Kiho , Park Song-Kyu , Kim Hwan Mook
Published: January 2023

Glabridin, a flavonoid present in licorice root, is known to have anti-inflammatory and cardiovascular protective activities. In the present study, we report an inhibitory effect of glabridin on microglial activation. Glabridin dose-dependently attenuated lipopolysaccharide (LPS)-induced production of inflammatory mediators, including nitric oxide, tumor necrosis factor-α and interleukin-1β, in BV-2 cells, a murine microglia cell line. Moreover, mRNA expression of these inflammatory mediators was also suppressed by glabridin in LPS-stimulated BV-2 cells. Further study demonstrated that glabridin inhibited LPS-induced DNA binding activity of NF-κB and AP-1 in BV-2 cells. Collectively, the results presented in this report demonstrate that glabridin inhibits the production of inflammatory mediators in BV-2 cells and this is mediated, at least in part, by blocking NF-κB and AP-1 activation. Our results suggest that glabridin might be a potential therapeutic agent for the treatment of neuroinflammatory and neurodegenerative diseases.
Regulation of the expression of cyclooxygenases and production of PGI2 and PGE2 in human coronary artery endothelial cells by curcumin (142.5)
Authors: Tan Xiaoyu , Poulose Ellen , Zhu Bao-Ting , Stechschulte Daniel , Dileepan Kottarappat
Published: January 2023

Curcumin is a hydrophobic polyphenol present in turmeric (Curcuma longa). PGE2 and PGI2 are produced from arachidonic acid (AA) by the cyclooxygenases (COX1, COX2) and PGE2 synthase (PGE2S) and PGI2S. We evaluated the in vitro effect of CUR on COX1, COX2, PGI2S and PGE2S expression, and PGE2 and PGI2 production in human coronary artery endothelial cells (HCAEC). HCAEC were incubated with CUR (1.25-5.0 μM), and mRNA and protein expression were determined by qRT-PCR and Western blots, respectively. PGI2 and PGE2 levels in the culture supernatants were quantified by EIA. Incubation of HCAEC with CUR led to concentration-dependent increases in COX2 mRNA with a small but significant decrease in COX1 mRNA expression. CUR also induced 2- to 3-fold increase in PGI2S and PGE2S mRNA expression. Although CUR stimulated COX2, PGI2S and PGE2S mRNA, no increase in PGI2 or PGE2 production was noted. Addition of exogenous AA dose-dependently increased prostanoid production by both resting and CUR-treated cells. However, CUR-treated HCAEC produced significantly higher PGE2 when AA concentration was raised to 25µM. 14C-AA uptake by CUR-treated HCAEC was 30% lower than controls suggesting that CUR may inhibit exogenous AA uptake. These results show that CUR acts both as an inducer of COX2, PGI2S and PGE2S genes and a suppressor of COX1 gene. Taken together, these results suggest that CUR regulates AA metabolism and prostanoid homeostasis at both the gene and enzyme levels.