bluuebunny/crossref_metadata_2025 huggingface dataset:
High dose antigen stimulation leads to binding defects caused by persistent spatial segregation of TCR and CD8 in cytotoxic T cells (178.9)
Authors: Perica Karlo , Bieler Joan , Schneck Jonathan
Published: January 2023

The lateral organization of surface receptors controls ligand binding and activation. Our group has shown that activated T Cells exhibit persistent clustering of T Cell Receptor (TCR) several days after stimulation, leading to enhanced binding of soluble Major Histocompatibility Complex (MHC) dimers at low concentrations. In contrast, other groups have shown a complete loss of binding in activated T Cells during the same time period. Here, we resolve this contradiction by demonstrating that persistent binding behavior is controlled by the dose of antigen during primary activation. T Cells from TCR transgenic mice stimulated with high but not low dose of peptide are unable to specifically bind soluble Kb-SIY or Db-GP100 MHC dimer four days after stimulation. The behavior is not due to differences in the surface expression of TCR, CD8alpha, or CD8beta. Cytokine production mirrors the loss of binding. However, both high and low dose stimulated cells are able to bind the so-called ‘CD8 independent’ Ld-QL9 dimer, implicating the coreceptor in the binding defect. Using biophysical binding assays, k-space Image Correlation Spectroscopy, and Forster Resonance Energy Transfer between TCR and CD8, we demonstrate that TCR and CD8 are segregated, and TCR unclustered, on high dose activated T Cells, impairing CD8-dependent binding. Thus, membrane organization derived binding defects represent a mechanism for controlling T Cell responses to supraoptimal antigen.
TOLL-LIKE RECEPTOR 4 PLAYS AN ESSENTIAL ROLE IN EARLY SKIN WOUND HEALING (180.1)
Authors: Chen Lin , Guo Shujuan , Ranzer Matthew , DiPietro Luisa
Published: January 2023

Toll-like receptor 4 (TLR4) plays a key role in the initiation of innate immunity and in the regulation of adaptive immune responses. Using microarray analysis and PCR, TLR4 expression was observed to increase in experimental murine skin wounds at the early stages, suggesting a role in wound inflammation. Closure of excisional wounds was significantly delayed in TLR4 deficient (C3H/HeJ) as compared to wild type mice, and both IL-1β and IL-6 production were significantly lower in the wounds of TLR4 deficient mice. EGF also markedly decreased in the wound edge of epidermis in TLR4 deficient mice. In vitro studies confirmed that a wound stimulus induces TLR4 mRNA expression in primary normal human epidermal keratinocytes (NHEK). In vitro injury also induced the phosphorylation of p38 and JNK MAPK, as well as the expression of IL-1β and TNF-α by NHEK. Blockade of TLR4 abolished the phosphorylation of p38 and JNK MAPK, and blockade of TLR4 and/or p38/JNK abolished IL-1β production. The results strongly suggest that inflammatory cytokine production by injured NHEK is stimulated via the TLR4-p38 and JNK MAPK signaling pathway. Together, the current results provide evidence for a role of TLR4 at sites of injury, and suggest that TLR4 is an important regulator of wound inflammation.
The Asp299Gly polymorphism alters TLR4 signaling by interfering with recruitment of adapter proteins MyD88 and TRIF (180.5)
Authors: Medvedev Andrei , Figueroa Leandra , Xiong Yanbao , Song Chang , Piao Wenji
Published: January 2023

The Asp299Gly (D299G) and, to a lesser extent, Thr399Ile (T399I) TLR4 polymorphisms have been associated with Gram negative sepsis and infectious diseases, but the mechanisms by which they affect TLR4 signaling are unclear. In this study, we determined the impact of these polymorphisms on TLR4 expression, TLR4- MD2 interactions, LPS binding, and activation of MyD88- and TRIF-dependent pathways. Complementation of HEK293/CD14/MD2 transfectants with WT or mutant YFP-TLR4s revealed similar TLR4 expression levels, TLR4-MD2 interactions, and LPS binding. The presence of the D299G polymorphism led to impaired LPS-induced phosphorylation of p38 and TBK-1, activation of NF-κB and IRF-3, and induction of IL-8 and IFN-β mRNA. The T399I TLR4 species elicited responses that showed no statistical difference with those caused by WT receptor. In contrast to WT TLR4, expression of the D299G mutant in TLR4-/- mouse macrophages failed to elicit LPS-mediated induction of TNF-α and IFN-β genes. Co-immunoprecipitation revealed deficient LPS-elicited interaction of MyD88 and TRIF with D299G TLR4, in contrast to robust adapter recruitment to WT TLR4. Our data indicate the D299G polymorphism compromises TLR4 signaling by impairing recruitment of MyD88 and TRIF, suggesting that its interference with TLR4 dimerization and assembly of docking platforms in the TIR domains that enable adapter recruitment
Partial organization of bovine variable-heavy chain gene locus and influence of recombination signal sequences (RSS) on various variable region gene expression (42.6)
Authors: Pasman Yfke , Kaushik Azad
Published: January 2023

An attempt has been made to determine the organization of variable-heavy chain gene locus in cattle, based on 91% assembled Hereford cow genome sequence, where both functional VH and pseudogenes are identified on chromosome 7 and 21. Of a total of 13 VH genes identified unambiguously, 6 VH genes are located on chromosome 21 and 7 VH genes are present on chromosome 7. Interestingly, the functional VH genes are distributed on both chromosome 7 and 21 raising questions about VDJ recombination occurring on two different chromosomes (Hosseini, A. et al. 2004. Int. Immunol.16:843) or via as yet unidentified mechanism that possibly permitted allelic exclusion. The presence of 10 DH-genes in the Hereford genome is confirmed as earlier reported by our laboratory (Koti, M. et al. 2010. Mol Immunol. 47:2119). Come cryptic recombination signal sequences (RSS) seem to exist within the VH encoding region requiring elucidation of their role in VDJ recombination. Nevertheless, V-gene expression in bovine B cells is influenced by the composition of RSSs. These findings, especially the presence of variable heavy chain locus on two chromosomes, raise fundamental questions about the phenomenon and mechanisms of allelic exclusion in bovine B cells.
A Dual-regulatory and Compartmental-specific Role for FCRL5 in Innate-like B Cell Receptor Signaling (42.8)
Authors: Davis Randall , Li Ran , Zhou Tong , Zhu Zilu
Published: January 2023

By virtue of their evolutionarily conserved antibody repertoires, sensitivity to T cell-independent stimuli, and rapid responsiveness, splenic marginal zone (MZ) and peritoneal cavity (PEC) B1 B lymphocytes share critical responsibility in primary humoral responses. However, despite their similar functionality they have different B cell receptor (BCR) signaling characteristics. A discrete marker of these innate-like subsets possessing tyrosine-based dual regulatory potential termed FCRL5 was investigated to explore these discrepancies. Although FCRL5 dominantly repressed BCR activation in MZ cells, it had no influence in B1 B cells. The molecular basis for its inhibition derived from SHP-1 phosphatase recruitment to its cytoplasmic ITIM. Remarkably, mutagenesis of this docking site unearthed co-activating properties for FCRL5 that were orchestrated via independent association of the Lyn kinase with its ITAM-like sequence. SHP-1 and Lyn-deficient mice validated the requirement of these elements in balancing the receptor’s unique dualistic function. These studies found that the requisite and opposing roles of SHP-1 and Lyn in modulating FCRL5’s unique binary properties directly correlated with the cellular pools of these signaling elements and consequently the disparate antigen receptor-mediated biology evident between innate-like MZ and B1 B cells.
Activated B-cells in the Granulomas of non-human primates infected with Mycobacterium tuberculosis (43.5)
Authors: Phuah Jia Yao , Mattila Joshua , Lin Philana Ling , Flynn JoAnne
Published: January 2023

To date, the contribution of humoral immunity, including antibodies and specific functions of B cells, on Mycobacterium tuberculosis (Mtb) infection in humans remains largely unknown. However, recent studies in mice have revealed that humoral immunity can alter Mtb infection outcomes. The cynomolgus macaque (M. fascicularis) model for Mtb infection recapitulates many aspects of human TB such as the spectrum of granulomas, variable infection outcomes, including latency and active disease, and response to treatment. As a first step to understanding the importance of humoral immunity to control of Mtb infection in primates, we characterized the B-cell and plasma cell populations in infected animals and found that B-cells are primarily present within the granuloma along with T-cells in the lymphocytic cuff. The B cells tend to be present in clusters within the granulomas are in close proximity to PNAd+ cells, have elevated HLA-DR and contain proliferating cells. Singular B-cells were also noted. Tissues containing M. tuberculosis bacilli had higher levels of Mtb specific IgG, compared to uninvolved tissue samples from the same monkeys. Plasma cells were detected within the granuloma and are functional active. This study demonstrates that B cells are present and actively secreting antibodies specific for Mtb antigens at the site of infection, in lung granulomas and thoracic lymph nodes. These antibodies likely have the capacity to modulate the host’s ability to control infection.
HMGB1 mediates Splenomegaly, Anemia and Leukocytosis in Mice Surviving Sepsis (44.7)
Authors: Valdes-Ferrer Sergio , Rosas-Ballina Mauricio , Lu Ben , Olofsson Peder , Chavan Sangeeta , Tracey Kevin
Published: January 2023

Severe sepsis, a syndrome complicating infection or injury, is a leading cause of death. It also has long-term deleterious effects on survivors, including high mortality and diminished quality of life. High-mobility group box 1 (HMGB1) is a critical late mediator of acute sepsis that remains elevated after other inflammatory cytokines have normalized. Here we studied the kinetics of circulating HMGB1 in mice surviving severe abdominal sepsis, and the effects of administering neutralizing anti HMGB1 mAb. In a standardized model of murine sepsis, we observed that mice surviving sepsis developed splenomegaly for at least four weeks. Splenocytes from sepsis survivors produced significantly more cytokines following exposure to TLR ligands. Circulating HMGB1 levels are significantly increased for at least 4 weeks after sepsis. Treatment with anti-HMGB1 neutralizing monoclonal antibodies effectively prevented the development of splenomegaly, leukocytosis and anemia. Chronic administration of recombinant HMGB1 to healthy mice induced significant splenomegaly and leukocytosis. Recombinant HMGB1 also sensitized splenocytes and significantly increased the release of TNF and IL-6. In conclusion, serum HMGB1 levels are persistently elevated for at least 4 weeks in mice that survive lethal sepsis. Anti-HMGB1 mAb administration reverses the development of splenomegaly, leukocytosis and anemia, which may have implications for understanding the pathogenesis of post-sepsis complications.
Generation of NK cell memory requires IL-12 and STAT4 signaling (45.3)
Authors: Madera Sharline , Sun Joseph
Published: January 2023

Recent studies have demonstrated that natural killer (NK) cells can become long-lived and can contribute to secondary immune responses like T and B cells; however, the precise signals that promote the generation of these long-lived memory NK cells remain unknown. Using cytokine receptor deficient mice we investigated the role of cytokine IL-12 in the activation, expansion, and generation of the virus-specific NK cell response to mouse cytomegalovirus (MCMV) infection. We demonstrate that IL-12 receptor-deficient NK cells exhibit a profound expansion defect following MCMV infection. This defect extended to the formation of long-lived memory cells, as IL-12 receptor-deficient NK cells were undetectable 2 weeks after infection whereas wildtype NK cells proliferated robustly and generated long-lived cells detectable months after MCMV infection. These findings were corroborated through the use of mice deficient in STAT4, which exhibited a defect in the expansion and formation of long-lived memory cells compared to wildtype NK cells in a competitive setting. This work demonstrates a key role for IL-12 and STAT4 during NK cell priming, and describes the requirements for generating robust virus-specific NK cell memory. Understanding the full contribution of inflammatory cytokines and the signals necessary for a long-lived NK cell response against viral infection will be of interest in the development of vaccines and therapeutics.
NKT cells enhance C5a generation by CD11b+Gr-1+ peritoneal cells via IL-10 and IFN-gamma mediated cross-talk, thereby promoting sepsis in mice (45.9)
Authors: Oh Sae Jin , Kim Ji Hyung , Chung Doo Hyun
Published: January 2023

Both NKT cells and complement protein C5a have been implicated in the pathogenesis of sepsis, but the issue of whether NKT cells regulate C5a generation during sepsis has been unclear. To investigate this issue, we examined immune responses in WT and NKT cell-deficient mice during cecal ligation and puncture (CLP)-induced sepsis. The levels of C5a, IFN-gamma, and IL-10 during sepsis were higher in the serum and peritoneal fluid of WT mice than in those of CD1d-/- mice. Furthermore, the mortality rate was lower in CD1d-/- mice than in WT mice. Adoptive transfer of liver mononuclear cells (LMCs) of WT mice increased mortality and C5a generation during sepsis in CD1d-/- mice, whereas transfer of IL-10- or IFN-gamma-deficient LMCs did not. In co-culture experiments, IL-10 and IFN-gamma produced by NKT cells enhanced C5a generation by CD11b+Gr-1+ peritoneal cells. Furthermore, treatment with anti-C5a antibody during sepsis decreased CD4+ T-cell apoptosis, serum D-dimer levels, and numbers of bacterial colony-forming units, but increased responsiveness against lipopolysaccharide in WT and WT-LMC-treated CD1d-/- mice, increasing survival rates. Taken together, these results suggest that CD1d-restricted NKT cells enhance C5a generation through IL-10- and IFN-gamma- mediated cross-talk with CD11b+Gr-1+ peritoneal cells, thereby aggravating CLP-induced sepsis.
Immunization with “self” peptide induces a novel phenotype of splenic dendritic cells contributing to peripheral tolerance (48.10)
Authors: Farkas Adam , Finn Olivera
Published: January 2023

A 100mer peptide (MUC1p) derived from the tumor associated antigen MUC1 is seen as “self” by the immune system of MUC1 Tg mice. TCR.Tg CD4+ T cells specific for MUC1 proliferate less in MUC1 Tg mice after vaccination with DC loaded with MUC1p (DC/MUC1p) relative to WT. We performed gene array on splenic RNA from WT and Tg mice vaccinated with DC/MUC1p to globally query mechanisms of peripheral tolerance to MUC1p. Expression of well-characterized pancreatic proteases (Trypsin, Elastase, CPB1) was dramatically down regulated in DC from immunized MUC1 Tg mice relative to WT, providing a signature of ensuing tolerance to self-peptide. Additionally, there was a decrease in the number of splenic DC in MUC1 Tg mice concurrent with an increase in the number of NK cells, in an IL-10 dependent fashion. IL-10 is also required to mediate down-regulation of proteases in DC linking enzyme expression with immunogenicity. Preliminary data suggest that this IL-10 is derived from increased numbers of MDSC in the spleens of immunized MUC1 Tg mice. Splenic DC from DC/MUC1p immunized MUC1 Tg mice migrate shorter distances ex vivo compared to DC recovered from WT, suggesting impaired ability to home in vivo. These DC produce less IL-12p40 and more Aldh1 compared to WT DC and express less CD40, CD86 and MHCII. Finally, DC from DC/MUC1p vaccinated MUC1 Tg mice differentiate naïve CD4+ T cells into Foxp3+ Treg in an antigen-specific manner ex vivo more effectively than DC recovered from WT mice.
Death Receptor 5 (DR5) marks the highly pathogenic interacting GM-CSF+ T helper cells and IL-23+ macrophages rendering it as an attractive therapeutic target of autoimmunity (51.4)
Authors: Li Jun , Hsu Hui-Chen , Yang PingAr , Wu Qi , Li Hao , Spalding David , Chatham W. , Kimberly Robert , Bridges S. , Mountz John
Published: January 2023

The highly pathogenic granulocyte-macrophage colony-stimulating factor (GM-CSF)+ IL-17+ CD4 T cells and IL-23+IRF5+M1 macrophages (MØ) exhibit a bidirectional interaction in autoimmunity. DR5 is a pro-apoptotic molecule upregulated by IRF5. DR5 deficient mice develop increased inflammation. We analyzed DR5 expression and apoptosis function of TRA-8, an anti-human DR5 antibody, in T cells and MØ in autoimmune conditions. Expression of DR5 was highly correlated with that of GM-CSF in CD4 T cells and that of IL-23 and IRF5 in MØ from synovia fluid or PBMC of rheumatoid arthritis and lupus subjects (p<0.01). In vitro treatment of these samples with TRA-8 for 48 hrs resulted in 23.4% and 35.9% depletion of GM-CSF+ CD4+ T cells and IL-23+ MØ, respectively. Similar DR5 expression pattern and TRA-8 depletion effects were observed in a 3kb mouse promoter/humanized DR5 Tg mouse crossed with the SHP-1- deficient viable motheaten mice which exhibit a profound activation of inflammatory MØ and Th17 cells. Three doses of TRA-8 (0.1 mg IP, weekly) lead to a 35.7% reduction of GM-CSF+ CD4+ T cells and 45.1% reduction of IL-23+IRF5+ MØ in the draining LN, 74.4% and 94.7% decrease of Csf-2 and p19 in joints, resulting in amelioration of pneumonitis and arthritis, reduction of autoantibodies, and increase of the lifespan. Our study indicated that DR5 is highly correlated with GM-CSF in CD4+ T cells and IL-23 in MØ, and demonstrated the high therapeutic efficacy of an anti-human DR5 antibody.
Th17 cell LFA-1 engagement-mediated IL-17A and GM-CSF mRNA stabilization is HuR-dependent (54.14)
Authors: Huntenburg Sarah , Ramgolam Vinod , Yarovinsky Timur , Bender Jeffrey
Published: January 2023

Recent studies demonstrate that the β2 integrin LFA-1 is required for entry of encephalitogenic Th17 cells into the brain parenchyma during murine experimental autoimmune encephalomyelitis (EAE). We previously showed that LFA-1 engagement by its ligand, intracellular adhesion molecule-1 (ICAM-1), in human T cells results in translocation of the RNA-binding protein HuR and stabilization of TNFα and IFNγ mRNAs. The role of HuR in the regulation of Th17 cell-mediated inflammation is unknown. We hypothesize that LFA-1 engagement in Th17 cells leads to increased neuropathology-relevant cytokine expression at inflammatory sites through HuR-mediated mRNA stabilization. LFA-1 engagement led to HuR nuclear-to-cytoplasmic translocation and stabilization of both IL-17A and GM-CSF transcripts in mouse EL4 T cells, in vitro differentiated mouse Th17 cells and human primary T cells. EL4 and human primary T cell adhesion to ICAM-1 led to increased levels of IL-17A and GM-CSF mRNA in HuR immunoprecipitates. LFA-1 engagement-stimulated IL-17A and GM-CSF mRNA stabilization was lost in HuR-deficient Th17 cells generated from RORcγt-Cre/HuRflox/flox mice. We conclude that LFA-1 engagement induces HuR-dependent stabilization of the otherwise labile GM-CSF and IL-17A transcripts in Th17 cells, and that this may be highly relevant in Th17 cell-induced CNS pathology. Future experiments will focus on the role of Th17 cell HuR in the EAE model.
Rheumatoid arthritis synovial tissue fibroblast Mucin 3 expression and function (54.27)
Authors: Volin Michael , Zanotti Brian , Klosowska Karolina , Smith Kelley , Birly Jennifer , Rodriguez Manuel , Woods James
Published: January 2023

Mucin 3 (MUC3) is elevated in rheumatoid arthritis (RA) synovial tissues (STs). We hypothesized that MUC3 is expressed by RA synovial fibroblasts, that it is cytokine inducible and chemotactic for RA synovial fibroblasts. To test this, RA synovial fibroblast MUC3 expression was determined by immunohistochemistry, flow cytometry and Western blot analysis. Levels of MUC3 from arthritic synovial fluids (SFs) were measured by a novel MUC3 ELISA. RA synovial fibroblast chemotaxis to RA SFs was assessed using a modified Boyden chamber assay. RA ST cells stained for both MUC3 and vimentin indicating that RA synovial fibroblasts express MUC3. Flow cytometry experiments showed that most of the RA synovial fibroblast MUC3 is cytoplasmic. Cytokine stimulation of these cells significantly increased MUC3 production (P<0.05). RA SFs contain on average 8.7μg/ml (n=8) of MUC3. Immunodepletion of MUC3 from RA SFs resulted in significantly reduced RA synovial fibroblast chemotaxis (P<0.05, n=5 RA SFs and n=8 RA synovial fibroblasts). These results show that MUC3 is produced by synovial fibroblasts in RA STs, that MUC3 is inducible by cytokine stimulation and that RA SF induced synovial fibroblast chemotaxis can be inhibited by depleting MUC3. Thus, MUC3 synovial fibroblast production may be induced by the inflammatory milieu of the RA joint and may drive synovial fibroblast chemotaxis within the RA ST potentially aiding pannus formation and cartilage destruction.
Modulation Of IL-17A And TL1A Largely Abrogates House Dust Mite-Induced Lung Inflammation In A Murine Model Of Allergic Airway Disease (54.5)
Authors: Hubeau Cedric , Kubera John , Hammerman Katherine , Wright Jill , Denz Michelle , Juang Yuang-Taung , Williams Cara
Published: January 2023

Recent studies suggest a role for Th17 responses in the increased airway neutrophilia associated with severe asthma. House dust mite (HDM) is a natural allergen to which asthmatics are often sensitized. Mice repeatedly challenged with HDM extract developed robust airway neutrophilia rapidly evolving into asthma-like disease with increased numbers of eosinophils and lymphocytes in bronchoalveolar lavages (BAL) as well as inflammatory infiltrates, vascular/muscular hypertrophy, interstitial fibrosis, epithelial hyperplasia and mucus accumulation in lung tissues. RNA and protein screening revealed a robust Th17 component post-HDM exposure. We thus evaluated whether IL-17A deficiency could modulate HDM-induced allergic airway disease. Airway neutrophilia was indeed abrogated in IL-17A deficient mice weekly challenged with HDM (acute model), however total BAL cellularity and lung mechanics remained comparable to those of HDM-challenged WT mice. In contrast, IL-17A deficient mice daily exposed to HDM (chronic model) had decreased BAL cellularity associated with reduced numbers of BAL macrophages, neutrophils, eosinophils and lymphocytes. Interestingly antibody neutralization of TL1A, a member of the TNF superfamily known to promote Th2 and Th17 responses, reduced BAL cellularity to baseline levels in HDM-challenged WT mice. Our results thus indicate that targeting Th17 responses can alleviate HDM-induced airway neutrophilia, and can also broadly modulate allergic airway disease.
IRAK4 kinase-deficient mice exhibit increased susceptibility to S. pneumoniae that is overcome by poly IC:LC treatment (55.23)
Authors: Pennini Meghan , Salazar Andres , Vogel Stefanie
Published: January 2023

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a critical component of Toll-like receptor signaling, and patients with IRAK4 mutations are extremely susceptible to recurrent bacterial infections. In these studies, we used mice homozygous for a mutant IRAK4 that lacks kinase activity (IRAK4KDKI) to address the role of IRAK4 in response to TLR agonists and infection with whole bacteria. IRAK4KDKI macrophages have a diminished response to the TLR4 agonist LPS, little to no response to the TLR2 agonist Pam3Cys, but normal response to the TLR3 agonist poly I:C when compared to wild-type (WT) macrophages as measured by cytokine mRNA expression and MAPK activation. Thus, diminished responses of IRAK4KDKI cells are specific to TLR pathways that are MyD88-dependent. Importantly, we have identified two proteins downstream of MAPK signaling, MNK1 and MSK1, whose phosphorylation is deficient in IRAK4KDKI macrophages when compared to WT. These data suggest a role for IRAK4 in propagating a TLR signal beyond initial phosphorylation of p38 and ERK. Additionally, IRAK4KDKI mice were more susceptible to infection with S. pneumoniae, demonstrating the usefulness of this murine model for the exploration of strategies to overcome IRAK4 deficiencies. Co-treatment of infected mice with Poly IC:LC, a TLR3 agonist, significantly improved the survival rate of both WT and IRAK4KDKI mice providing a potential treatment strategy in both normal and immunocompromised patients.
Role of IL-17A in airway tolerance in a murine model of asthma (57.14)
Authors: Graser Anna , Zhivkova Nataliya , Finotto Susetta
Published: January 2023

Allergic asthma is a world-wide increasing disease. The mechanism controlling this disease is not yet completely understood. In the attempt to design vaccination strategies against it, we previously established a model of airway tolerance. Airway tolerance is characterized by decreased pro-inflammatory immune responses. In this model we described in wild type as well as in T-bet deficient mice a down-regulation of both Th2 and Th17 cytokines indicating a T-bet independent mechanism in this model. In this study we found that this feature is accompanied by induction of the suppressive cytokine IL-10 in the airway tolerance model only in the T-bet deficient mice. Because IL-17A is a T cell-derived pro-inflammatory cytokine playing a critical role in immune and inflammatory responses we asked about the role of IL-17A in the airway tolerance model by using IL-17A deficient and wild type mice. In our experiments we found that IL-17A deficient mice had decreased airway hyper-responsiveness after methacholin challenge in the airway tolerance model as compared to allergic asthma model. Moreover we found an increase of IL-10 protein release in the airways of IL-17A deficient mice as compared to the wild type littermates in this model. These results indicate a suppressive function of IL-17A on IL-10 in the presence of T-bet in the lung that should be further investigated.
In utero exposure to EPFR-containing PM inhibits pulmonary Th1 maturation and enhances adult asthma development in mice (57.15)
Authors: Cormier Stephania , Wang Pingli , You Dahui
Published: January 2023

Environmentally persistent free radicals (EPFRs), with half-lives on the order of weeks have been detected in association with airborne particulate matter (PM) and early exposure to environmental hazards, such as PM, has been associated with an increased incidence of asthma in childhood. We hypothesized that in utero exposure to EPFR-containing PM disrupts immune development resulting in aberrant immune responses and enhancing susceptibility to asthma. To test this hypothesis, we exposed pregnant mice to ultrafine particles containing persistent free radicals (EPFR-UFP) and determined postnatal immune development and response to allergen sensitization and challenge. Intrauterine exposure to EPFR-UFP inhibited pulmonary T helper (Th1/Th2/Th17) and T regulatory (Treg) responses in early life (<1 week of age). Th2 and Treg responses recovered to normal levels; whereas Th1 responses remained attenuated at 6 weeks of age. In the asthma model, intrauterine EPFR-UFP-exposed mice exhibited higher airway hyperresponsiveness and pulmonary eosinophilia. In addition, levels of IL-4 and IL-5 (Th2 cytokines) in the bronchoalveolar lavage fluid of these mice were significantly higher than that of non-exposed mice that were sensitized and challenged with ovalbumin. Our data suggest that in utero exposure to EPFR-UFP enhances adult asthma development in mice, which might be related to the inhibition of pulmonary Th1 maturation.
Gas6/TAM signaling differentially modulates chronic fungal asthma with the expansion of myeloid regulatory cell subsets in mice (57.7)
Authors: Shibata Takehiko , Ismailoglu Ugur , Coelho Ana , Lukacs Nicholas , Kunkel Steven , Moreira Ana , Hogaboam Cory
Published: January 2023

Gas6, a Tyro3, Axl, Mertk (TAM) receptor ligand, is detected in a variety of diseases and has various roles. Herein, we show that Gas6 differentially modulates experimental fungal asthmatic response via the expansion and modulation of myeloid-derived regulatory cells (MDRCs). Aspergillus fumigatus-sensitized mice were challenged with live Aspergillus conidia and received approximately 2 μg (low) or 7 μg (high) of recombinant Gas6 via intranasal installation from days 14 to 28 after conidia challenge. In the low dose Gas6 group, significant airway hyperresponsiveness (AHR), airway remodeling, and whole lung IL-13 were observed compared with the control group. Although high dose Gas6 treatment significantly suppressed AHR and the whole lung levels of inflammatory cytokines compared with control, this treatment exacerbated airway remodeling. MDRCs have both disease enhancing and suppressing cells in asthma. Indeed, low dose Gas6 treatment increased the accumulation of CD11b+F4/80+Ly6C-Ly6G+ MDRC with pro-inflammatory properties into asthmatic lung whereas high dose Gas6 promoted the accumulation of immunosuppressive CD11b+F4/80+Ly6C+Ly6G- MDRC during chronic asthma. Anti-Axl Ab, but not anti-Mer Ab, treatment significantly suppressed not only AHR but also airway remodeling in asthmatic mice compared with IgG control asthmatic groups. Together, these results demonstrate that Gas6-TAM receptor interactions modulate fungal asthma, in part through effects on MDRCs.
Evaluation of three next generation sequencing platforms for immune repertoire sequencing (58.10)
Authors: Wang Chunlin , Levy Shawn , Yang Qunying , Byrne-Steele Miranda , Han Jian
Published: January 2023

The advent of next generation sequencing (NGS) techniques, combined with a semi-quantitative multiplex PCR method, enables us to comprehensively profile the lymphocyte receptor diversity (immune repertoire) of the entire collection of B or T cells from a particular sample. Different NGS platforms vary in read length, throughput, sequencing error rate and error pattern, hands-on time, turnaround time and price. Three popular NGS techniques (Illumina HiSeq, 454 FLX and Ion Torrent) were employed to sequence amplicons from T cell receptor clones generated with arm-PCR in order to evaluate the usage of those techniques on immune repertoire sequencing. The error pattern and rate of the three techniques in the context of T cell receptor sequences were profiled. Based on the differences of the three techniques, we discuss the usage of each NGS platform in the context of immune repertoire sequencing.
IL-27 sustains T-bet expression and promotes the development of terminally differentiated CD4 T cells during tuberculosis (59.5)
Authors: Torrado Egidio , Fountain Jeffrey , Pearl John , Cooper Andrea
Published: January 2023

IL-27 is a member of the IL-12 family of cytokines that was originally described to induce the expression of the transcription factors STAT1 and T-bet, thus promoting Th1 differentiation. Despite this apparent Th1 inducing profile, mice deficient in IL-27R signaling are more resistant to Mtb infection, even though CD4 T cells in the lungs of these mice produce lower amounts of IFN-γ on a per cell basis. Here we show that, in the absence of IL-27 signaling, CD4 T cells express lower levels of the transcription factor T-bet during the chronic phase of infection which corresponds with a reduced accumulation of terminally differentiated CD4 T cells in the lungs of these mice. Mechanistic analysis of the impact of IL-27 signaling in Ag-specific T cells suggest improved competitive fitness of CD4 T cells deficient in IL-27 signaling. Accordingly, the transcription profile of CD4 T cells from the lungs of Mtb-infected wild-type mice suggests that these cells are under oxidative stress, unlike CD4 T cells from the lungs of IL-27R-/- mice. These data suggest that IL-27 has an important impact in the maintenance of effector CD4 T cell responses during tuberculosis.
Cytotoxic effect of IL-17A on neural stem cells as a mechanism underlying Th17-cell-induced proinflammatory demyelination (59.7)
Authors: Zhang Guag-Xian , Lee Eric , Li Ke , Zhu Lin , Kumar Priyanka , Xu Hui , Ciric Bogoljub , Rostami Abdolmohamad
Published: January 2023

IL-17A, a Th17 cell-derived proinflammatory molecule, has been found to play an important role in the pathogenesis of autoimmune diseases, including multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). The IL-17A receptor (IL-17RA), through which IL-17A signals, is expressed in many immune-related cells, including central nervous system resident cells such as microglia and astrocytes. However, whether IL-17A exerts a direct effect on neural stem cells (NSCs) and oligodendrocytes, thus inducing inflammatory demyelination in the central nervous system is not known. Here we report that IL-17RA is constitutively expressed on NSCs, oligodendrocytes and their precursors. IL-17A significantly reduced NSC proliferation, which was mainly due to cytotoxicity, but not to induction of apoptosis. IL-17A did not influence the differentiation of NSCs into neural cells. The effects of IL-17A are similar to those of TNF-α, another proinflammatory cytokine that plays a pathogenic role in EAE; however, a synergistic/additive effect of these two molecules has not been observed. We conclude that IL-17RA is expressed in NSCs, through which IL-17A blocks proliferation of these cells, resulting in reduced numbers of myelinating cells such as oligodendrocytes and their precursors. Thus, in addition to its proinflammatory role in the immune system, IL-17A also plays a direct role in inducing demyelination and blocking neuronal repair in EAE/MS.
The lupus-susceptibility Sle2c1 sublocus regulates T cell expansion through several mechanisms (60.1)
Authors: Xu Zhiwei , Morel Laurence
Published: January 2023

The lupus-susceptibility (278-37)rec1 sublocus is derived from the NZB-derived Sle2 locus found in the NZM2410 lupus-prone mouse. Our previous results showed that the candidate gene p18 in the (278-37)rec1 sublocus demonstrated significantly reduced mRNA expression and the (278-37)rec1 sublocus enlarged spleen, more notably peripheral lymph nodes in Fas-deficient mice. Our current research discovered firstly that the mouse with both p18 gene deletion and lpr mutation displayed expanded peripheral lymph organs as the same as the B6.(278-37)rec1.lpr mouse, meaning an association of p18 deficiency and lymphocyte hyperproliferation. Secondly, the B6.(278-37)rec1.lpr mouse showed significantly more Ki67+ T cells in lymph nodes than B6.lpr mouse, but T cells from both B6.(278-37)rec1.lpr and B6.lpr mice have the same proliferation response in vitro with stimulation by anti-CD3 and anti-CD28. Furthermore, both Treg cell percentage and the ration of Treg/T effective cell in B6.(278-37)rec1.lpr mouse showed markedly decreased in spleen and lymph nodes compared with B6.lpr mouse, but both mouse stains have the same Treg function in inhibiting T cell proliferation. Finally, the T cell apoptosis of B6.(278-37)rec1.lpr mouse seems unimpaired in activation-induced cell death assay. In summary, the T cell hyperproliferation controlled by the (278-37)rec1 sublocus involves several mechanisms, but their interrelation needs further to be elucidated in the future.
Rapid replacement by apoptosis in the draining lymph nodes sustains dynamics of skin-migratory dendritic cells in the steady state. (61.3)
Authors: Tomura Michio , Kabashima Kenji , Kanagawa Osami
Published: January 2023

Although, understanding of skin-DC homeostasis is important issue, its analysis has been delayed, because of the difficulty of labeling skin-DCs. Here we used newly established photoconvertible fluorescent protein, KikGR-KI mice to label DCs in skin and lymph nodes (LN) by exposure with violet light. This system allows us to directory measure replenishment kinetics and migration of skin-migratory DCs in the steady state. CD103-dermal DC (DDC) in draining (d) LN increased rapidly with peak at d 2 after labeling DCs in skin. On the other hand, skin-derived CD103+DDC gradually increased with peak at d 3. LC increased at similar rate to that of CD103+DDC and reached plateau at d 4. In photoconverted skin at d 5, 40% of DDC, 60% of CD103+DDC and none of LC were non-labeled, indicating presence of labeled-DC in the dLN was closely correlated with decrease of corresponding labeled-DCs in skin. Half replenishment time of DDC, CD103+DDC and LC were 0.6, 1.5 and 2 d measured by following decrease of labeled DC after labeling DCs in LN. Only a few emigrated DCs were detected in other LNs, and skin-migratory DCs had high caspase activity cells. Altogether, DC pool in skin is relatively larger than that of LN, and LN is always filled with newly influx DCs. Skin migratory DCs replaced at rapid turnover rate via apoptosis in LN without emigration, which would limit duration of T cell response and enable to transport carried peripheral antigens from skin-migratory DC to LN-resident DCs.
Omalizumab inhibits human mast cell responses by sequestering pre-bound IgE (64.6)
Authors: Kulka Marianna , Catalli Adriana
Published: January 2023

Mast cells are key effectors of allergic inflammation. IgE-mediated mast cell activation induces degranulation and inflammatory mediator release. Omalizumab (Xolair; Genentech Inc.) is a recombinant humanized monoclonal anti-IgE antibody that prevents IgE binding to its receptor, FcϵRI. Objective: We investigated the effects of omalizumab on IgE pre-sensitized human LAD2 mast cells. Methods: LAD2 degranulation was determined by β-hexosaminidase assay. Chemokine expression and prostaglandin synthesis was measured by quantitative PCR analysis and ELISA, respectively. IgE binding and FcϵRI expression was determined by flow cytometry. Results: Omalizumab pretreatment inhibited IgE binding to LAD2 cells and entirely prevented IgE-dependent upregulation of FcϵRI expression. In addition, omalizumab removed FcϵRI-prebound IgE as early as 24 hrs after treatment. After 5 days, bound IgE was reduced by 92%. Furthermore, omalizumab concomitantly reversed IgE-dependent FcϵRI upregulation by 49% 48 hrs post treatment and by 93% 5 days post treatment. Consequently, omalizumab attenuated ongoing IgE-mediated responses, reducing degranulation by 34%, chemokine expression up to 79% and prostaglandin synthesis by 34% after 7 days of omalizumab treatment. Conclusions: Omalizumab is able to remove pre-bound IgE from sensitized mast cells thereby reducing ongoing response to FcϵRI-dependent signals. This data suggests that omalizumab is an effective inhibitor of sensitized human mast cells.
Glucocorticoids repress the type-I-IFN system and accelerate osteoporosis during Pneumocystis lung infection (70.8)
Authors: Searles Stephen , Gauss Katherine , Mozer Michelle , Meissner Nicole
Published: January 2023

Pneumocystis (PC) infection can range from asymptomatic colonization to fatal pneumonia in severely immunocompromised individuals. Type-I-IFNs are critical regulators of immune responses to PC. In mice lacking type-I-IFN signaling (IFNAR-/-), PC clearance is delayed and transient bone marrow (BM) depression and accelerated osteoclastogenesis are observed. Lack of type-I-IFN-signaling in lymphocyte-deficient mice (IFrag-/-) augments systemic symptoms causing progressive BM failure and significant loss of bone mass due to accelerated osteoclastogenesis. These data highlight the importance of the type-I-IFN system in regulating local and systemic responses to PC infection. Glucocorticoids (GCs) are used to treat chronic inflammatory lung diseases and are immunosuppressive in part by inhibiting type-I-IFN signaling. Interestingly, osteoporosis is a systemic manifestation of chronic inflammatory lung disease and a side-effect of GC treatment. Here we show that GCs inhibit the PC-induced systemic type-I-IFN response in a mouse model. Mice infected with PC and treated with GC have significantly reduce trabecular bone mass compared to uninfected, GC treated mice, mimicking the IFrag-/- phenotype. BM numbers were also reduced and the osteoclastogenic potential increased in PC infected, GC treated mice. These data suggest that PC could act as a co-morbidity factor during GC treatment, ultimately augmenting side effects such as osteoporosis.
Pulmonary endotoxin tolerance increases costimulatory molecule expression on dendritic cells and antigen migration to the lung draining lymph nodes in a mouse model (71.2)
Authors: Natarajan Sudha , Beal Dominic , Kim Jiyoun , Remick Daniel
Published: January 2023

It has been shown that lung-specific endotoxin tolerance (ET) attenuates allergic responses in an environmentally relevant model of cockroach-allergen (CRA) induced asthma. The mechanisms of this response have not yet been elucidated. We hypothesized that ET would reduce antigen migration to lung draining lymph nodes, thereby suppressing development of the adaptive immune response to allergen. ET was induced in outbred (CD-1) mice by direct intratracheal installation of 100ng (low dose) or 1μg (high dose) endotoxin for 4 consecutive days. Controls received PBS. The following day, mice received 2μg of FITC-conjugated CRA and were sacrificed 24h later. Lungs, mediastinal and cervical lymph nodes were collected for FACS analysis. Results showed a dose-dependent increase in the number of antigen-carrying CD11c+FITC+ dendritic cells in the lymph nodes (PBS vs. 100ng LPS vs. 1ug LPS; 1.4*106 cells vs. 2.3 vs. 5.3). An increase was seen in the number of dendritic cells expressing costimulatory molecules CD80 (2.4*104 cells vs. 5.5 vs. 13.0), CD86 (5.1*104 cells vs. 12.7 vs. 30.1) and MHC class II (10.5*104 cells vs. 14.0 vs. 45.7). An increase in the MFI for CD86 was observed for both ET groups compared to control. MFI for MHC class II expression was also elevated in the high-dose ET group compared to controls. These data suggest that dendritic cells in ET mice have an increased capacity for upregulation of costimulatory molecules and antigen transport to the draining lymph nodes.
Aerosolized Influenza Infection During Late Gestation Pregnancy Results in Severe Clinical Outcomes in the Ferret Model (76.7)
Authors: Gage Emily , Reed Doug , Trichel Anita , Dunsmore Tammy , Beigi Richard , Stefano Cole Kelly
Published: January 2023

In 2009, the emergence of pandemic A influenza virus (pH1N1) demonstrated greater severity in pregnant women as recognized previously in other influenza pandemics. The Centers for Disease Control and Prevention reported a death rate of 20% in pregnant women in the United States who were hospitalized with influenza-like symptoms. To understand the immunological basis for the enhanced influenza-associated morbidity and mortality observed in pregnant women, pregnant and nonpregnant ferrets were challenged with aerosolized pH1N1 strain A/California/04/09. The pregnant ferrets had poorer gestational outcomes and presented a more rapid and severe clinical disease when compared to the nonpregnant ferret group. Viral load and tissue tropism were similar for both groups as determined by quantitative RT-PCR. Influenza virus was detected in the brains of both groups. Gross pathological analysis and H&E stains identified interstitial pneumonia in the lungs, with no significant pathology in any other tissues. Virus was also detected in placental tissue and kits of the pregnant ferrets suggesting transmission across the placental barrier. This model will be useful in discerning the differences in the host response to infection that results in greater morbidity and mortality during pregnancy.
Characterizing the T cell receptor repertoire of beryllium-responsive CD4+ T cells (P5021)
Authors: Bowerman Natalie , Falta Michael , Mack Douglas , Crawford Frances , Kappler John , Fontenot Andrew
Published: January 2023

Multiple public T cell receptor (TCR) repertoires exist in CD4+ T cells derived from the lung of patients with chronic beryllium disease (CBD). Here, we characterize the TCR Vβ5 repertoire which involves expression of a conserved glutamine (Q)-containing motif that is essential for TCR recognition of HLA-DP2/peptide/Be2+. Using a panel of human TCR Vβ antibodies, we show that beryllium-responsive, Vβ5.1+CD4+ T cell expansions exist in all DP2+ CBD patients tested to date. Sequence analyses revealed oligoclonal expansions consisting of Vβ5+ TCR genes that express a conserved motif composed of identical lengths, conserved joining region expression (Jβ2.5/1.4), and homologous amino acid (Ala/Gly) residues surrounding an essential Q residue. The existence of a public Vβ5+ TCR repertoire suggests selection by an identical antigen in lung, which is likely composed of HLA-DP2, peptide, and Be2+. As expected, these Vβ5+ T cells recognize the same antigen, as revealed by positive staining of CBD T cell lines with a soluble tetrameric HLA-DP2/peptide/Be2+ molecule. Interestingly, this MHCII tetramer was used to identify an additional public Vβ18+ T cell repertoire which expresses the conserved CDR3 motif found in the predominant Vβ5+ T cell population. The presence of beryllium-specific, Vβ5.1+CD4+ T cells in the lung of all DP2+ CBD patients suggests that the MHCII tetramers can be used as an early method of detection of CBD development in beryllium-exposed workers.
Measuring T cell activation in T cell-APC conjugates using the ImageStream imaging flow cytometer (P5043)
Authors: Friend Sherree , Kong Raymond , Hall Brian , Basiji David , Haley Pugsley
Published: January 2023

Adaptive immune responses require antigen presentation to antigen-specific effector cells. Upon interaction between the antigen presenting cell (APC), specific antigen and T cell there is a reorganization of the cytoskeleton and recruitment of adhesive and signaling molecules to the site of intercellular contact forming an immunological synapse (IS). Upon formation of a functional IS, signaling molecules join the clusters and contribute to the signaling cascade leading to translocation of nuclear factors from the cytoplasm to the nucleus culminating in gene activation. Recruitment of these molecules is measured by imaging cell conjugates directly. To analyze a statistically significant number of specific conjugates we used the ImageStream imaging flow cytometer with a 60X objective, which objectively and rapidly collects large numbers of images and provides quantitative image analysis tools to evaluate subcellular localization of molecules. In this study we evaluate the specific location of the adhesion and signaling molecules LFA-1 and Lck within the IS complex in T cells when presented with SEB pulsed APC. In addition we measured the translocation of NFkB in the T cell from the cytoplasm to the nucleus showing the activation of the T cells when conjugated to APC.
Cytosolic flagellin-induced lysosomal pathway regulates inflammasome-dependent and independent macrophage responses (P4141)
Authors: Bortoluci Karina , Lage Silvia , Buzzo Carina , Amaral Eduardo , Matteucci Kely , Massis Liliana , Ferreira Luis Carlos , Rodriques Mauricio , D'Império Lima Regina , Amarante Mendes Gustavo
Published: January 2023

NAIP5/NLRC4 inflammasome activation by cytosolic flagellin results in caspase-1-mediated processing and secretion of IL-1β/IL-18 and pyroptosis, an inflammatory cell death pathway. Here, we found that although NLRC4, ASC and caspase-1 are required for IL-1β secretion in response to cytosolic flagellin, cell death nevertheless occurs in the absence of these molecules. Cytosolic flagellin-induced inflammasome-independent cell death was accompanied by IL-1α secretion and was temporally correlated with the restriction of S. Typhimurium infection. Despite displaying some apoptotic features, this peculiar form of cell death did not require caspase activation but was regulated by a lysosomal pathway, in which cathepsin B and cathepsin D play redundant roles. Moreover, cathepsin B contributed to NAIP5/NLRC4-inflammasome-induced pyroptosis, and IL-1α and IL-1β production in response to cytosolic flagellin. Together, our data describe a novel pathway induced by cytosolic flagellin that induces a peculiar form of cell death and regulates inflammasome-mediated effector mechanisms of macrophages.