bluuebunny/crossref_metadata_2025 huggingface dataset:
Cross-talk between NF-kB and Neuronal Nuclei (NeuN) in HT 22 mouse hippocampus neuron: a mechanism for neuronal survival versus damage reaction (54.19)
Authors: Dasgupta Subhajit , Bandyopadhyay Mausumi
Published: January 2023

The Neuronal Nuclei or NeuN is well known as a marker for post mitotic neuron cell body in central nervous system of fetal as well as adult brain. However, functional role of NeuN has not been well elucidated yet. The present study demonstrated a co-association between NF-kB and NeuN in HT22 mouse hippocampus neuron cell. We found that, intracellular TLR7/8 ligand imidazoquinoline compound induced NF-kB activation in HT22 cells. The nuclear extracts of HT22 cells were found to demonstrate NeuN as well as NF-kB. Further, co-immunoprecipitation of NeuN from nuclear extracts of imidazoquinoline treated HT 22 hippocampus cells demonstrated an association with NF-kB. The observations suggested an interaction between NeuN and NF-kB in HT22 hippocampus neuron. A study has been initiated to determine a regulatory role of NeuN in management of neuronal dysfunction during dementia and epileptic seizures. Acknowledgement. The HT22 cells were provided by Dr Mark S Kindy (MUSC). We are thankful to Eileen McFadden for her support.
Anti-inflammatory activity of Tribulus terrestris in RAW264.7 Cells (54.2)
Authors: Oh Joa Sub , Baik Seung Ho , Ahn Eun-Kyung , Jeong Wonsik , Hong Seong Su
Published: January 2023

Inflammation is involved in various diseases, including chronic inflammatory disease and arthritis. The inflammation induced by the production of cyclooxygenease (COX)-2 and inducible nitric oxide synthase (iNOS), which are important mediators of inflammation. In this study, we investigated the ethanol extract of Tribulus terrestris (ETT) inhibited expression of COX-2 and iNOS in lipopolysaccharide(LPS)- stimulated RAW264.7 cells. We observed that ETT significantly decreased release of NO production in a dose-dependent manner in LPS-stimulated RAW264.7 cells. ETT also suppressed expression of proinflammtory cytokines, such as TNF-α and IL-4 in macrophage cell line. Our findings suggest that ETT inhibits expression of mediators related to inflammation and expression of inflammatory cytokines, which has a beneficial effect on various inflammatory diseases.
Effects of high-fat diet on B1 B cells in obesity-associated inflammatory diseases (54.28)
Authors: Hall Matthew , Zhu Andrew , Lovell John , Aprahamian Tamar , Rifkin Ian , Zhong Xuemei
Published: January 2023

Objective: High-fat diet (HFD)-induced abdominal obesity is a major risk factor for diabetes and atherosclerosis, yet the immunological mechanisms underlying these associations are still unclear. B1 B lymphocytes reside in the peritoneal cavity and omentum in direct contact with abdominal visceral adipose tissue. Their physiological location and functions make them an ideal intermediary for obesity-induced inflammatory disease. This study aimed to dissect the B1 B cell response to HFD-induced obesity in diabetes and atherosclerosis. Methods: gld/ApoE-/- mice fed a high-fat Western diet (WD) were used to model diet-accelerated atherosclerosis and autoimmunity. Wild type C57BL/6 mice fed a HFD were used to model diet-induced obesity and diabetes. Control mice from each group were fed with a low-fat control diet (CD). Cells from the peritoneal cavity and omentum were studied using flow cytometry. Hematoxylin/Eosin and immunohistochemistry stainings were performed on paraffin-embedded tissues. Results: Compared to their CD-fed counterparts, WD-fed gld/ApoE-/- mice exhibited enlarged omental milky spots and decreased peritoneal B1 cells. HFD-fed C57BL/6 mice demonstrated enlarged omental milky spots and reduced peritoneal B1 B cells compared to CD-fed counterparts. Conclusion: The similar B1 B cell response pattern to diet in both gld/ApoE-/- and WT mice indicates that HFD has a profound influence on the B1 B cell immune response regardless of genetic susceptibility.
Determination of the Proinflammatory Potential of Spores from Different Basidiomycetes Species with a Human Whole Blood Assay. (55.13)
Authors: Rivera-Mariani Félix , Vysyaraju Kranthi , Levetin Estelle , Hartung Thomas , Breysse Patrick
Published: January 2023

Airborne fungal spores could pose a proinflammatory risk when inhaled by susceptible individuals. Most studies have examined fungal spores from mitosporic fungi (e.g. Aspergillus sp, Penicillium sp., Alternaria sp.). Less is known about the proinflammatory potential of spores from basidiomycetes (meiotic fungi). In this study, we evaluated the proinflammatory potential of spores (basidiospores) from three species of basidiomycetes (Chlorophyllum molybdites, Pluteus cervinus, and Coprinus micaceus) in a human whole blood (HWB) system. This system provides the flexibility to evaluate the proinflammatory potential of a given sample based on the release of proinflammatory cytokines (e.g. IL-1β) by leukocytes. Different concentrations (100 ug, 10 ug, 1.0 ug) of spore deposits from each basidiomycete were incubated (18 hrs) with human whole blood from healthy subjects, and the release of IL-1β determined with ELISA. HWB was also incubated with endotoxin as a positive control. Similar to endotoxin, the basidiospores induced the release of IL-1β in a dose-dependent manner. In most cases, there were no significant differences between the response of endotoxin and that of the basidiospores. To our knowledge, this is the first study to evaluate the proinflammatory potential of basidiospores in a HWB system. Our results suggest that basidiospores could pose a proinflammatory inhalation risk comparable to that of endotoxin in subjects with respiratory diseases.
Brucella melitensis induces the Unfolded Protein Response in macrophages (55.14)
Authors: Khan Mike , Magnani Diogo , Harms Jerome , Liu Yi-Ping , Splitter Gary , Smith Judith
Published: January 2023

Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. As no current vaccine is available, and current therapy suboptimal, a better understanding of how Brucella establishes an intracellular replicative niche is necessary. Brucella fuses with the endoplasmic reticulum (ER) in order to replicate, and thus may provoke an ER stress response called the Unfolded Protein Response (UPR). In this study, we found upregulation of the UPR target genes BiP and CHOP, as well as XBP1 mRNA splicing in both RAW 264.7 murine macrophages and bone marrow macrophages infected with B. melitensis. These data indicate activation of all 3 major axes of the UPR. BiP and CHOP upregulation was MyD88 independent. Furthermore, upregulation of BiP, CHOP and the XBP1 target ERdj4 was ablated in the heat killed and VirB (ER fusion) Brucella mutant. Finally, taurodeoxycholic acid, a UPR inhibitor, significantly impaired Brucella replication. The JNK inhibitor SP600125 also suppressed replication, consistent with previous reports implicating the IRE-1-dependent UPR pathway. Together, these results suggest Brucella induces the UPR to aid in its intracellular replication, and that the UPR could be a novel therapeutic target for treatment of brucellosis.
Epithelial STAT3-induced antimicrobial protein, Reg3γ is required for host defense against MRSA pneumonia in mice (55.15)
Authors: Choi Sun-Mi , McAleer Jeremy , DeLeo Frank , Hooper Lora , Qin Shulin , Reinhart Todd , Ouyang Wenjun , Kolls Jay
Published: January 2023

Incidence of community-associated Methicillin-resistant Staphylococcus aureus (CA-MRSA) pneumonia has increased and is associated with high mortality rates. Patients with Hyper IgE Syndrome (HIES), resulting from mutations in signal transducer and activator of transcription 3 (STAT3), are susceptible to recurrent SA pneumonia. USA300 (2x107 CFU), the most prevalent pneumonia-causing strain, was oropharyngeally administered to C57BL/6 (WT) and various knockout mice. Bacterial burden, gene expression, and protein levels were determined in the lung 20 hours post-infection. Although, Rag2γc-/- mice clear SA as well as WT mice, GP130 or STAT3 inhibited mice had increased bacterial burden. USA300 infection leads to IL-6 induction. IL-6 binds to GP130 and activates STAT3 in lung epithelium during infection as seen by IHC. IL-6 induction also occurred in Rag2γc-/- mice indicating that B, T, NK, and NK T cells are dispensable for this response. IL-6 induces STAT3 signaling and Reg3γ production by mouse lung epithelial (MLE12) cells. Furthermore, STAT3-dependent Reg3γ limits USA300 growth in vitro. Therefore, STAT3 signaling is necessary for host defense against MRSA pneumonia. Although, HIES patients have decreased Th17 cells, recurrent SA pneumonias are likely due to impairment of STAT3 signaling in innate immune and epithelial cells. Reg3γ, with antimicrobial activity against USA300, may be the basis for new therapies for CA-MRSA pneumonia.
Production of an NKT cell stimulatory glycosphingolipid by a prominent member of the human gut microbiota (55.22)
Authors: Penaranda Cristina , Wieland Brown Laura , Kashyap Purna , Clardy Jon , Kronenberg Mitchell , Sonnenburg Justin , Comstock Laurie , Bluestone Jeffrey , Fischbach Michael
Published: January 2023

While the human gut microbiota are suspected to produce diffusible small molecules that modulate host signaling pathways, few of these molecules have been identified. Species of Bacteroides and their relatives, which often comprise >50% of the gut community, are unusual among bacteria in that their membrane is rich in sphingolipids, a class of signaling molecules that play a key role in modulating the host immune response. Although known for more than three decades, the full repertoire of Bacteroides sphingolipids has not been defined or linked to immune modulatory activity. We report that Bacteroides fragilis produces the glycosphingolipid α-galactosylceramide (α-GalCerBf), which is structurally related to a sponge-derived glycosphingolipid (α-GalCer, KRN7000) that is the prototypical agonist of CD1d-restricted natural killer T (NKT) cells. We demonstrate that α-GalCerBf binds to CD1d and stimulates the production of cytokines by both mouse and human NKT cells. In vivo, we show that α-GalCerBf stimulates NKT cells to induce the activation markers CD25 and CD69, secrete IFN-γ in a CD1d-dependent manner and can prevent cyclophosphamide-induced diabetes. These data suggest that NKT cells directly sense an endogenous antigen produced by a prominent human gut symbiont, uncovering a new mechanism by which the gut microbiota modulate a subset of immune cells relevant to pathogen response and autoimmune disease.
The Macrophage galactose-specific lectin (MGL) recognizes Taenia crassiceps antigens and plays a role in resistance to the infection (55.3)
Authors: Terrazas Luis I , Montero Daniel , Vera-Arias Laura , Terrazas Cesar A , Gomez-Garcia Lorena , Ledesma-Soto Yadira
Published: January 2023

C-type lectins are multifunctional sugar-binders on dendritic cells (DCs) and macrophages that internalize antigens for processing and presentation. MGL recognizes galactose and N-acetylgalactosamine and it is expressed on immature DCs and alternatively activated macrophages (AAM). Helminth parasites contain large number of glycosilated components which appears to play a role in the immuneregulation induced by such infections. To address whether MGL may influence the immune response and resistance to T. crassiceps we infected i.p. MGL deficient (MGL-/-, C57BL/6 background) and wildtype mice (MGL+/+) with 15 non-budding T. crassiceps metacestodes. Parasite burden, cellular and humoral responses were measured during 8 wks of infection. mMGL-/- DCs displayed lower ability to bind T. crassiceps antigens. Compared with wild-type mice, mMGL-/- mice failed to produce significant levels of inflammatory cytokines during the first two weeks of Taenia infection. MGL-/- mice developed a Th2-dominant whereas wild-type mice developed a Th1-dominant immune response after Taenia infection. Associated with the insufficient production of early inflammatory cytokines, mMGL-/- mice harbored significantly higher numbers of parasites whereas WT mice were resistant. Finally, FACS analysis showed overexpression of IL-4Rα, PDL1 and PDL2 on macrophage surface in MGL-/- group unrevealing AAM phenotypes. Thus, MGL may be involved in T. crassiceps recognition and subsequent innate immune activation.
Signaling via an adapter molecule CARD9 is essential for the host defense to infection with Cryptococcus neoformans (55.5)
Authors: Yamamoto Hideki , Nakamura Yuri , Takahashi Yurie , Sato Ko , Yamamoto Natsuo , Ishii Keiko , Hara Hiromitsu , Kawakami Kazuyoshi
Published: January 2023

CARD9 is a critical adaptor molecule signaling for NF-κB activation triggered via C-type lectin receptors (CLRs), which recognize carbohydrate structures of microbes. Cryptococcus neoformans, a yeast-like fungus is recognized by some CLRs, such as mannose receptor or DC-SIGN, whereas the role of CLRs or CARD9 in the host defense to this infection remains to be elucidated. In this study, we analyzed the role of CARD9 in the protective immune response to C. neoformans infection. C57BL/6 and CARD9KO mice were infected intratracheally with C. neoformans (B3501). The number of live microbes, histopathological findings and cytokine synthesis in lungs and the cytokine response of paratracheal lymph node (LN) cells upon antigenic re-stimulation were evaluated. CARD9KO mice were highly susceptible to infection with C. neoformans, as shown by increased number of live microbes in lungs compared to control, which was associated with less marked granulomatous response and remarkable multiplication of yeast cells in the alveolar spaces. Expression of RORγt and IL-17A mRNA and intracellular expression of IL-17A by T, NKT and γδ T cells were strongly attenuated in the lungs of CARD9KO mice. IL-17A production by the re-stimulated LN cells was mostly abrogated in these mice. By contrast, Th1-cytokine response was not much affected. These results suggest that CARD9 is a key molecule in the Th17 response to C. neoformans, which is associated with the host defense against this infection.
GM-CSF-Induced Regulatory T cells Selectively Inhibit Anti-Acetylcholine Receptor-Specific Immune Responses in Experimental Myasthenia Gravis (56.1)
Authors: Sheng Jianrong , Prabahakar Bellur , Meriggioli Matthew
Published: January 2023

We and others have demonstrated the ability of granulocyte-macrophage colony-stimulating factor (GM-CSF) to suppress autoimmunity by increasing the number of CD4+CD25+ regulatory T cells (Tregs). In the current study, we have explored the critical role of induced antigen specific Tregs in the therapeutic effects of GM-CSF in murine experimental autoimmune myasthenia gravis (EAMG). Specifically, we show that Tregs from GM-CSF treated EAMG mice (GM-CSF/AChR-induced-Tregs) adoptively transferred into animals with EAMG suppressed clinical disease more potently than equal numbers of Tregs from either GM-CSF untreated EAMG mice or healthy mice treated with GM-CSF. In addition, GM-CSF/AChR-induced-Tregs selectively suppressed antigen specific T cell proliferation induced by AChR relative to that induced by an irrelevant self antigen, (thyroglobulin) and failed to significantly alter T cell proliferation in response to an exogenous antigen (ovalbumin). These results are consistent with the hypothesized mechanism of action of GM-CSF involving the mobilization of tolerogenic dendritic cell precursors which, upon antigen (AChR) capture, suppress the anti-AChR immune response through the induction/expansion of AChR-specific Tregs.
Use of Fungal-like Particles to probe Dectin-1 Responses in Innate Immunity (58.12)
Authors: Khan Nida , Tam Jenny , Mansour Michael , Vyas Jatin
Published: January 2023

The presence of multiple types of carbohydrate moieties on fungal cell walls prohibits elucidation of the exact immunological response to pathogen-derived carbohydrate epitopes. To dissect the immunological mechanisms used to recognize pathogens, a system of “fungal like particles” was developed that consisted of polymer beads, which mimicked the three dimensional shape of the fungus, coated covalently with purified b-1,3-glucan (BG) derived from S. cerevisiae. The morphology of the BG layer was examined by immunofluorescence, flow cytometery, and immuno-transmission electron microscopy. The covalent linkages of the BG to the polystyrene surface were stable after subjecting the beads to detergents. By pre-treating BG beads with laminarinase the reactivity of the anti-BG antibody was abrogated in comparison to treatment with proteinase K indicating that the coating of these beads was predominantly BG. TNF-a was also measured by stimulating bone-marrow derived macrophages with the BG beads and showed a dose dependent response. Finally, BG beads were incubated with GFP-Dectin-1 expressing macrophages and imaged using confocal microscopy. BG beads were taken up within minutes and retained Dectin-1, the major receptor for BG, to the phagosome as compared to uncoated beads. This data describes a unique fungal-like particle system that will permit immunologists to probe the critical steps in early recognition of pathogen-derived fungal carbohydrate antigens by innate immune cells.
Enhancement of MICA affinity for NKG2D by combining multiple rational design strategies (58.17)
Authors: McFarland Benjamin , Henager Samuel , Hale Melissa
Published: January 2023

We increased the affinity of MICA for the dominant immunoreceptor NKG2D by 50-fold using seven rationally designed mutations found through a stepwise process similar to affinity maturation. First we evaluated the stability of amino acid mutations in MICA located beneath the interface with NKG2D using RosettaDesign. These mutations were near a flexible region that folds when NKG2D binds. Sets of mutations predicted to mildly destabilize this region of MICA also increased its affinity for NKG2D as measured by surface plasmon resonance, with the best results from three mutations that increased affinity by 20-fold. The second design strategy optimized MICA residues that contact NKG2D. Ten single mutations proposed by RosettaDesign increased MICA-NKG2D affinity by 2-fold to 8-fold by SPR. Most combined surface mutants tested had subadditive effects on binding, but some enhanced binding through enthalpic stabilization. A combination of four of the five best single mutants increased affinity by 19-fold from wild-type. These sets of three and four mutations were combined in a MICA variant that bound NKG2D with 50 nM affinity. This variant in complex with NKG2D persisted through a size-exclusion column under conditions where the wild-type complex fell apart. These mutants were purified and tested in the context of undergraduate biochemistry courses in large quantities, so samples of purified recombinant high-affinity MICA are readily available for more tests.
Altered Th17 and Th1 cells responses in patients with primary Sjogren syndrome (59.10)
Authors: Kang Gi Soo , Kim Sang Hyun , Bunin Viviane , Shin Min Sun , Lee Naeun , Parke Ann , Kang Insoo
Published: January 2023

Primary Sjogren syndrome (pSS) is a systemic autoimmune disease characterized by dry eyes, dry mouth and extraglandular abnormalities, of unknown etiology. IL-17 produced largely by CD4+ Th17 cell is a major proinflammatory cytokine involved in host defense and development of inflammatory disease. We analyzed CD4+ Th17 and Th1 cells in peripheral blood from patients with pSS (n=16) and healthy subjects (n=14). We also measured cytokines, including IL-1β, IL-6, IL-7, IL-10, IL-12 and IL-23, in supernatants of lipopolysaccharide- and zymosan- stimulated PBMCs. The frequency of CD4+ T cells producing IL-17 (Th17) and IFN-γ (Th1) was significantly higher in patients with pSS than in healthy subjects. In measuring Th17-polarizing cytokines, levels of IL-1β, IL-6 and IL-23 were not different between the two groups. However, patients with pSS had higher production levels of IL-7, which could also promote Th17 response, compared to healthy subjects. In addition, the Th1-polarizing cytokine IL-12 was produced at higher levels in patients with pSS. Of interest, production levels of the anti-inflammatory cytokine IL-10 were lower in patients with pSS than in healthy subjects. In conclusion patients with pSS had an increased frequency of Th17 and Th1 cells in peripheral blood, which could be related to increased IL-7 and IL-12 production as well as to decreased IL-10 production from innate immune cells.
IL-22 attenuates IL-25 production by lung epithelial cells and inhibits antigen-induced eosinophilic airway inflammation (59.8)
Authors: Takahashi Kentaro , Hirose Koichi , Kawashima Saki , Niwa Yusuke , Wakashin Hidefumi , Iwata Arifumi , Tokoyoda Koji , Nakayama Toshinori , Nakajima Hiroshi
Published: January 2023

BACKGROUND: IL-22 functions as both a proinflammatory and an anti-inflammatory cytokine in various inflammations. However, the roles of IL-22 in the allergic airway inflammation are still largely unknown. OBJECTIVE: We sought to determine whether IL-22 is involved in the regulation of allergic airway inflammation. METHODS: We examined IL-22 production and its cellular source at the site of antigen-induced airway inflammation in mice. We also examined the effect of IL-22 neutralization, as well as IL-22 administration. We finally examined the effect of IL-22 on IL-25 production from a lung epithelial cell line (MLE-15 cells). RESULTS: Antigen inhalation induced IL-22 production in CD4(+) T cells. Only one third of IL-22-producing CD4(+) T cells also produced IL-17A. Anti-IL-22 antibody administration enhanced antigen-induced IL-13 production, eosinophil recruitment, and goblet cell hyperplasia in the airways. On the other hand, intranasal administration of rIL-22 attenuated eosinophil recruitment. Moreover, anti-IL-22 antibody enhanced IL-25 production in the airways, and anti-IL-25 antibody reversed the enhancing effect of anti-IL-22 antibody on eosinophil recruitment into the airways. Finally, IL-22 inhibited IL-13-mediated enhancement of IL-25 expression in IL-1β- or LPS-stimulated MLE-15 cells. CONCLUSION: IL-22 attenuates antigen-induced airway inflammation, possibly by inhibiting IL-25 production by lung epithelial cells.
Development of spontaneous vitiligo in human tyrosinase reactive co-receptor independent TCR transgenic mice (60.11)
Authors: Chatterjee Shilpak , Al-Khami Amir , Nishimura Michael , Le Poole Caroline , Mehrotra Shikhar
Published: January 2023

Vitiligo is an autoimmune disease of the skin presenting with progressive depigmentation in approximately 0.5% of the population worldwide. Traditionally, vitiligo has been ascribed to a combination of genetic, neurologic and immune deficiencies. However, limited animal models for the disease have hampered its understanding. Clinical trial targeting melanoma has shown that destruction of melanocytes in the skin could be correlated to the presence of auto reactive T cells against melanoma-associated antigen. To gain insight into the crucial involvement of immune system in the development of autoimmune vitiligo, we generated TCR transgenic mice (h3TA2) using the human tyrosinase epitope reactive, CD8 independent, high affinity TCR isolated from MHC class-I restricted CD4+ T cells obtained from tumor infiltrating lymphocytes of a metastatic melanoma patient. These h3TA2 transgenic mice develop spontaneous hair depigmentation that progressed with age. Strikingly, the transgenic TCR was primarily expressed by CD3+CD4-CD8- double-negative (DN) T cells in h3TA2 mice. Further our data shows that development of vitiligo in these transgenic mice strongly depends on IFN-gamma as genetic ablation of IFN-gamma controls the disease, whereas CXCR3, TNF-alpha and perforin are dispensable. Our data suggest that IFN-gamma ablation controls development of T cell mediated spontaneous vitiligo and provides a model for testing other therapeutic modalities.
Investigating the novel protein, Ttc7, and its role in transitional B cells (60.2)
Authors: Walker Jennifer , Hill Beth , Harris Kimberly , Hakker Lauren , Agris Paul , Pelsue Stephen
Published: January 2023

Ttc7 is a novel protein of unknown function. Ttc7fsn mutant mice develop an autoimmune phenotype that includes an increase in immature B lymphocytes and marginal zone cells. Here we show that the transitional T1 population is increased two-fold in Ttc7fsn mice and that these cells proliferate upon BCR ligation. Preliminary data also indicate that T1 cells survive BCR stimulation rather than undergo apoptosis. Thus far we've identified two signaling abnormalities in transitional B cells: elevated Erk phosphorylation and decreased CD22 expression. Together, these data suggest that autoreactive transitional B cells are inappropriately activated and allowed to survive rather than undergo clonal deletion or anergy. Results from in silico analyses reveal a number of physical and chemical changes to the Ttc7fsn protein including decreased hydrophobicity, increased polarity and net charge, and the addition of helix-breaking residues. Computational models comparing the TPR domains of Ttc7 and Ttc7fsn show that the mutation disrupts the structure of both the TPR-1 domain and the linking sequence connecting TPR-1 and TPR-2. Further evidence of structural change is provided by preliminary CD spectroscopy, which indicates that the α-helical nature of Ttc7 is altered by the fsn mutation. Based on these findings, we hypothesize that structural changes in the Ttc7fsn protein alter its function and result in faulty molecular and functional regulation of B cell development and function.
A novel murine α4 integrin splicing variant is a specific receptor for VCAM-1 (61.2)
Authors: Kon Shigeyuki , Kouro Hitomi , Matsuda Tadashi
Published: January 2023

Integrins affect multiple cell motility in control of cell survival, growth, or differentiation mediated by cell-cell and cell-extracellular matrix interaction. We previously reported that α9 integrin splicing variant, SFα9, promotes wild type α9 integrin-dependent adhesion. In this report, we introduce a new murine α4 integrin splicing variant mSFα4, which have new short cytoplasmic tail. In inflamed tissues including rheumatoid joint, the expression of mSFa4, as well as wild type α4 integrin, is upregulated. Interestingly, cells expressing mSFα4 bind to VCAM-1 but not other α4 integrin ligand such as fibronectin CS1 or osteopontin. Binding of cells coexpressing mSFα4 and wild type α4 integrin to α4 integrin ligands is promoted than cells expressing wild type α4 integrin. Thus, mSFα4 functions modulation of α4 integrin. Now, we pursue the metastatic function of mSFα4 by using mSFα4-specific knockdown tumor cells.
Migratory properties of pulmonary dendritic cells are developmentally programmed (61.4)
Authors: Cook Donald , Burgents Joseph , Nakano Hideki
Published: January 2023

Pulmonary dendritic cells (DCs) take up inhaled allergens and migrate to draining lymph nodes (LN) to present antigens to naïve T cells. However, pulmonary DCs are also required to elicit inflammation after allergen challenge, suggesting the presence of both migratory and non-migratory DCs. There are two phenotypically distinct major lung DC subsets, defined by their expression of CD103 and CD11b, respectively. We used a novel Ccr7-gfp knock-in reporter mouse to study Ccr7 expression in lung DCs. Ccr7-gfp was expressed in many CD103 DCs, but in few CD11bhi DCs. Using the dye PKH26 to track lung DCs, we found that many CD103+ DCs migrate to draining LNs, whereas relatively few CD11bhi DCs do so. Thus, CD11bhi lung DCs are comprised of both migratory and non-migratory cells. Further analysis revealed that monocyte-derived (mo)CD14hi CD11bhi DCs, including LPS-elicited Ly-6Chi inflammatory DCs, do not express Ccr7 or migrate to LNs. Flt3l-/- mice, which lack DCs derived from the common DC precursor (CDP) but retain moDCs, possessed almost no migratory Ccr7-expressing DCs in the lung. Conversely, mice lacking both Ccr2 and Cx3cr1 lacked moDCs, but retained migratory, CDP-derived DCs. These findings show that the migratory activity of CD11bhi DCs is developmentally programmed; CDP-derived CD103+ and CD11bhi pulmonary DCs are migratory, whereas monocyte-derived CD11bhi DCs do not express Ccr7 and therefore remain in the lung, even during acute pulmonary inflammation.
Differentiation and detection of mouse CD4+ Th cells by measuring intracellular cytokine production (62.6)
Authors: Santos Mark , Williams Yuko , Weldon Don , Olcott Angelica , Hsu Matthew
Published: January 2023

Naïve CD4+ T cells can give rise to a variety of subsets of Th cells depending on the nature of the immune response, and subsequently release a distinct subset of cytokines. Conventionally these cytokines can be measured in a secreted format (e.g. Luminex or ELISAs), but here we describe a novel method to reliably differentiate mouse CD4+ T cells, and further characterize the differentiated lineages by measuring intracellular cytokines using flow cytometry. In addition, we also describe the use of a proprietary fixable viability dye in order to eliminate the false positives associated with non-specific staining. Based on an optimized protocol for differentiating CD4+ T cells, we have developed differentiation tools designed to obtain the desired Th cell lineages. Following naïve T cell differentiation toward specific Th lineages, we further developed a flow cytometry assay to measure intracellular cytokine production by blocking the secretion of the cytokine with Brefeldin A. We further eliminate non-specific staining due to dead or dying cells using a proprietary fixable viability dye and accurately determine the percentage of Th lineages using guava benchtop flow cytometer and InCyte analysis software.
Thymic Selection of T cells expressing CD8 independent high affinity human TCR αβ in TCR transgenic mice (63.7)
Authors: Thyagarajan Krishnamurthy , Al-Khami Amir , Naga Osama , Fang Quan , Nishimura Michael , Mehrotra Shikhar
Published: January 2023

Thymocytes undergo a number of selection events before egressing the thymus and moving to lymphoid organs as mature T cells. The chronology of these molecular and cellular events is crucial for determining the function of mature T cells. We have established a transgenic mouse model using a CD8 independent human TIL1383I αβTCR that was isolated from MHC I restricted CD4+ T cells obtained from tumor infiltrating lymphocytes of a metastatic melanoma patient and recognizes tyrosinase epitope presented by HLA-A2. We analyzed the cell fate of thymocytes in these mice in the presence of different selecting ligands. Surprisingly, this HLA-A2 restricted TCR was expressed on peripheral CD4+ and CD8+ T cells in HLA-A2- C57BL/6 mice. When bred into MHC I and MHC II deficient mice, CD8+ and CD4+ T cells respectively were absent from the periphery indicating that transgenic T cells are selected by MHC I and MHC II ligands in the mouse. Development of T cells in murine MHC deficient thymus directed majority of the transgenic T cells to CD3+CD4-CD8- phenotype. The presence of HLA-A2 also resulted in >90% of the transgenic T cells to egress the thymus with a CD3+CD4-CD8- double-negative phenotype. While peripheral transgenic T cells in MHC deficient or MHC sufficient HLA-A2+ background were CD4 and CD8 coreceptor deficient, they retained antigen recognition function. The above data suggests that CD8 independent TIL1383I αβTCR does not strictly follow rules of MHC-specific thymic selection.
IL-9 is Required for TSLP-mediated Allergic Airway Inflammation (64.3)
Authors: Yao Weiguo , Zhang Yanlu , Tepper Robert , Kaplan Mark , Zhou Baohua
Published: January 2023

Thymic stromal lymphopoietin (TSLP) is an epithelial cell derived cytokine important for the initiation and development of Th2-mediated allergic inflammation. In this study, we identified a significant association between IL-9 and TSLP concentration in the serum of infants with atopic dermatitis. Th9 cultures increased IL-9 production in a TSLP dose-dependent manner, parallel with an increase in pSTAT5 binding to Il9, Irf4 and Gata3 loci. Lung-specific transgenic expression of TSLP stimulated IL-9 production in the lung, and anti-IL-9 treatment attenuated TSLP-induced airway inflammation. Using an adoptive transfer model, we observed that TSLP promoted Th9-induced allergic inflammation in an IL-9-dependent manner, with affects directly on T cells. Together, our results demonstrate that TSLP promotes Th9 differentiation, and define a requirement for IL-9 in TSLP-induced allergic inflammation.
Transglutaminase 2 expressed in mast cell activation induces IgE production in B cells, airway inflammation and remodeling via up-regulating CD40L and cytokine expression in mouse allergic asthma (64.4)
Authors: Ro Jai , Lee Yun Song , Kim Soo Youl , Hong Gwan
Published: January 2023

TGase 2 is over-expressed in a variety of inflammatory diseases including allergic asthma. This study aimed to investigate the role of TGase 2 in signaling pathways in mast cells related to OVA-induced allergic asthma in vitro and in vivo. BMMCs isolated from C57BL/6 WT or TGase 2-/- mice were activated with Ag/Ab. Mice were sensitized and challenged with OVA to induce asthma. [Ca2+]i level were determined by confocal, mast cells by May-Giemsa, LT, IgE and cytokine levels by ELISA, expression of TGase 2, PLA2, EMBP, Muc5ac, collagen, cytokines, MMPs and TIMPs by Western blotting or RT-PCR, NF-kB activity by EMSA, the recruitment of inflammatory cells into BAL fluid or lung tissues by Diff-Quik and H&E, AHR by Fexivent system. Act-WT-BMMCs expressed TGase 2, and increased the [Ca2+]i level, activities of I-kB, NF-kB and PLA2, LTs level, expression of CD40/CD40L, cytokines and MMPs, collagen deposition, and IgE production in act-B cells, whereas act-KO-BMMCs reversed all of them. TGase 2-/- mice protected against OVA-specific IgE production as well as AHR, expression of signaling molecules and mediator release in BAL cells and lung tissues versus those in WT mice. Our data suggest that TGase 2 expression and Ca2+ influx required by bidirectional events in mast cell activation facilitate IgE production in B cells via regulating mast cell CD40L expression, and induce the expression of numerous molecules associated with airway inflammation and remodeling in allergic asthma.
Antigen-specific B cells are not required for soluble OVA protein induced T-cell tolerance through i.v. route (65.8)
Authors: Zhang Ai-Hong , Scott David
Published: January 2023

Intravenous exposure to soluble proteins can lead to antigen-specific immune tolerance. Moreover, resting B cells have been shown to be tolerogenic antigen-presenting cells (APC) in vivo. However, it is still not clear whether this APC function is fulfilled by the rare antigen-specific B cells or the abundant antigen non-specific B cells. To address this, we used soluble chicken ovalbumin (OVA) as a model antigen and employed B-cell receptor (BCR) transgenic mice (8.18C5 Tg) of C57BL6 (B6) background. In the homozygous 8.18C5 Tg mice, ~97% of peripheral B cells contain recombined immunoglobulin heavy chain specific for non-related myelin oligodendrocyte glycoprotein. As expected, a single i.v. dose of soluble OVA in wt B6 mice induced significant B+T cell tolerance. The 8.18C5 Tg mice immunized with OVA+CFA failed to produce any detectible anti-OVA antibody response, confirming the lack of OVA-specific endogenous B cells in these mice. Importantly, pretreatment of the 8.18C5 Tg mice with 25µg soluble OVA i.v. also resulted in profound T-cell tolerance upon OVA+CFA challenge a week later. Since antigen non-specific B cells can present processed antigen after soluble protein exposure in vivo, our results support the notion that antigen non-specific resting B cells in the steady state may participate in the peripheral tolerance to self proteins.
ROR-alpha is required for natural helper cell development in all tissues from a bone-marrow derived progenitor and the induction of allergic inflammation (67.11)
Authors: Halim Timotheus , MacLaren Aric , Romanish Mark , Gold Matthew , McNagny Kelly , Takei Fumio
Published: January 2023

Natural Helper (NH) cells are Th2 cytokine-producing innate lymphoid cells (ILCs) in, or associated with the mucosal tissues of the lung and gut. Currently, the lineage relationship between NH cells in different tissues and between NH cells and interleukin-22 (IL-22)-producing retinoid receptor-related orphan receptor (ROR)γt-positive ILCs is unclear. Here, we report that NH cells express RORα but not RORγt. RORα-deficient, but not RORγt-deficient, mice lack NH cells in the lung, small and large intestines while all other lymphocytes including RORγt+ ILCs are unaffected. We have also identified RORα-dependent Lin-Sca-1hic-kit-CD127+CD25+ NH cell progenitors different from common lymphoid progenitors in the bone marrow. The putative NH cell progenitors transplanted into NOD/SCID/Il2rg-/- mice develop into NH cells but not other lymphocytes including RORγt+ ILCs. Thus, all NH cells belong to a distinct cell lineage separate from other lymphoid cell lineages. NH cell-deficient mice generated by RORα-deficient bone marrow transplantation into lymphodeficient Rag2-/-Il2rg-/- mice have normal Th2 cell response but fail to develop acute allergic lung inflammation in response to protease allergens. NH cell-deficient mice further reveal the role of NH cells in allergic lung inflammation.
Efficient capture of infected neutrophils by dendritic cells in the skin inhibits the early anti-leishmanial response (67.13)
Authors: Ribeiro-Gomes Flavia , Peters Nathan , Sacks David
Published: January 2023

Neutrophils and dendritic cells (DC) converge at sites of acute inflammation in the skin following pathogen deposition by the bites of arthropod vectors or by needle injection. How neutrophils might interact with and modulate the function of DCs in these inflammatory settings has not been previously addressed. Neutrophils are the predominant recruited and infected cells during the earliest stage of Leishmania major infection in the skin. The infected neutrophils recovered from the skin expressed elevated levels of apoptotic markers and were preferentially captured by dermal DCs, the majority of which acquired their parasites via uptake of infected neutrophils. Neutrophil depletion augmented the expression of activation markers on infected DCs and their ability to present Leishmania antigen ex vivo, and markedly enhanced CD4+ T cell priming against parasite-derived antigen in vivo. The findings suggest that L. major exploits the apoptotic cell clearance function of DCs to suppress the development of acquired resistance until the acute neutrophilic response is resolved.
The impact of type I interferon on the development of the adaptive immune response (67.14)
Authors: Pinto Amelia , Daffis Stephane , Brien James , Sheehan Kathleen , Schreiber Robert , Diamond Michael
Published: January 2023

Determining the impact of type I interferon on immune responses using mice missing the common IFN-α/β signaling receptor (IFNAR- /-) is limited to studying the functions of type I IFN at all stages of viral infection. To define the temporal functions of type I IFN signaling in the context of infection by West Nile virus (WNV), we treated mice with MAR1-5A3, a neutralizing, non cell-depleting anti-IFNAR antibody at later stages of infection. Inhibition of type I IFN signaling at or before day 2 after infection was associated with markedly enhanced viral burden, whereas treatment at day 4 altered WNV dissemination less dramatically. Blockade of type I IFN signaling starting at day 4 induced dysfunctional CD8+ T cells with depressed cytokine responses and expression of phenotypic markers suggesting exhaustion. The later maturation phase (days 4-8) of anti-WNV CD8+ T cell development requires type I IFN signaling. Collectively, our results suggest that cell non-autonomous type I IFN signaling shapes maturation of antiviral CD8+ T cell response at a stage distinct from the initial priming event.
Genome-wide RNAi screen reveals a novel role of WNT/CTNNB1 signaling pathway in regulation of innate antiviral responses (67.7)
Authors: Lamarre Daniel , Baril Martin , Es-Saad Salwa , Chatel-Chaix Laurent , Karin Fink Karin Fink , Audette Karine , Guenier1 Anne-Sophie , Germain Marie-Anne , Duchaine Jean , Servant Marc , Grandvaux Nathalie
Published: January 2023

To identify new regulators of innate antiviral immunity, we completed the first genome-wide gene silencing screen assessing the transcriptional response at the interferon-β gene (IFNB1) promoter following Sendai virus (SeV) infection. We identified 237 potential modulator genes for which negative or positive actions of gene products were mapped to the different steps of the antiviral responses from virus sensing, signal propagation/amplification up to the feedback regulation. In the present study, we will report on specific proteins that promote IFNB1 expression and innate antiviral response. The functional genomics screen uncovers a novel link between WNT family members and innate antiviral immunity. We show that virus-induced secretion of WNT2B and WNT9B down regulates IFNB1 and ISG56 expression in a β-catenin (CTNNB1)-dependent mechanism. The antiviral response is drastically reduced by GSK3 inhibitors but completely restored in CTNNB1 knockdown cells. The findings confirm a novel regulation of the innate antiviral response by a canonical WNT/GSK3/CTNNB1 pathway in a negative feedback mechanism. The study identifies novel avenues for therapeutically regulating innate immunity for effective treatment of viral infection and prevention of excessive response in autoimmune diseases.
Molecular Mechanisms Responsible for the Selective and Low-grade Induction of Pro-inflammatory Mediators by Lipopolysaccharide (68.10)
Authors: Li Liwu , Maitra Urmila , Deng Hui , Baker Bianca
Published: January 2023

Low dose endotoxemia is prevalent in humans with adverse health conditions, and correlates with the pathogenesis of chronic inflammatory diseases such as atherosclerosis, diabetes, and neurological inflammation. However, the underlying molecular mechanisms are poorly understood. Here, we demonstrate that subclinical low dose lipopolysaccharide (LPS) skews macrophages into a mild pro-inflammatory state, through cell surface toll-like-receptor 4 (TLR4), interleukin-1 receptor associated kinase-1 (IRAK-1), and the toll-interacting-protein (Tollip). Unlike high dose LPS, low dose LPS does not induce robust activation of nuclear factor kappa-B (NFκB), mitogen-activated protein kinases (MAPK), phosphoinositide 3-kinase (PI3K), or anti-inflammatory mediators. Instead, low dose LPS induces activating transcription factor 2 (ATF2) through Tollip-mediated generation of mitochondrial reactive oxygen species (ROS), allowing mild induction of pro-inflammatory mediators. Our data reveal novel mechanisms responsible for skewed and persistent low grade inflammation, a cardinal feature of chronic inflammatory diseases.
Intrinsic defect in the phosphoinositide 3-kinase - Akt pathway plays a role in cytokine dysregulation in aged mouse macrophages stimulated with TLR ligands or heat killed Streptococcus pneumoniae (68.11)
Authors: Fallah Mosoka , Rogier Eric , Garvy Beth , Bondada Subbarao
Published: January 2023

Age-associated defects in innate immunity have a role in reduced immunity and vaccine efficacy to many Gram positive bacteria like Streptococcus pneumoniae . We have previously shown that splenic macrophages from aged mice exhibited a cytokine dysregulation; impaired production of pro-inflammatory cytokines but enhanced production of IL-10 upon signaling via TLR-2 and TLR-4 or when stimulated with IL-1 or Heat killed S. pneumoniae (HKSP). We showed in this study that aged BMDM demonstrated a cytokine dysregulation phenotype similar to splenic macrophages upon stimulation with TLR2, 4 and 9 ligands, or HKSP. We hypothesized that an intrinsic age-associated increase in the activity of phosphatidyl inositol 3-kinase (PI3K)-Akt- signaling pathway may be playing a causal role in the age-associated cytokine dysregulation. We showed a heightened activity of PI3K in the aged BMDM compared to the young. Inhibition of PI3K in BMDM increased translocation of p65Nf-κB and induction of pro-inflammatory cytokines when stimulated with these ligands. The increase in activity of PI3K-Akt pathway appears to be responsible for cytokine dysregulation in the aged macrophages independent of the splenic microenvironment and could thus be modulated to produce cytokines needed to support B-cell activation and differentiation.
Acidic residues in the juxta-transmembrane region of toll-like receptor 9 are crucial for binding UNC93B1 (68.12)
Authors: Kim Jihee , Brinkmann Melanie , Ploegh Hidde , Kim You-Me
Published: January 2023

The nucleotide-sensing toll-like receptors (TLRs) such as TLR3, 7, 9 traffic from the ER to endolysosomes where they recognize their cognate ligands and initiate signaling. A polytopic membrane protein, UNC93B1 delivers the intracellular TLRs to endolysosomes by binding to the transmembrane region of the receptors. In the cells expressing a mutant UNC93B1 incapable of binding the nucleotide-sensing TLRs, the receptors fail to exit the ER and cannot signal. However, the detailed molecular mechanism underlining the UNC93B1-mediated TLR trafficking is not known. Here we show that acidic residues in the juxta-transmembrane region of TLR9 are critical for the interaction with UNC93B1. Mutation of those acidic residues in TLR9 inhibits its binding to UNC93B1 and prevents it from trafficking to endolysosomes without affecting the localization of UNC93B1. Consequently, the TLR9 mutants are unable to rescue CpG-mediated signlaing in the TLR9 knock-out dendritic cells and macrophages. Based on these observations, we are currently trying to develop a method to pharmacologically disrupt the interaction of endogenous TLR9 and Unc93B1. The specific inhibition of the nucleotide-sensing TLRs by means of preventing the interaction with UNC93B1, without affecting other TLRs, may be beneficial for the treatment of autoimmune diseases.
Toll-like receptor 2 ligands enhance monocyte Fcγ receptor function (68.16)
Authors: Shah Prexy , Justiniano Steven , Dunn Robert , Jarjoura David , Byrd John , Butchar Jon , Tridandapani Susheela
Published: January 2023

Fcγ receptors (FcγR) are major effectors of antibody therapy so it is critical to seek new ways to enhance their effectivness. Toll-like receptors (TLR) recognize a wide range of microbial components, eliciting cellular responses such as cytokine production and generation of antimicrobial factors. Among these receptors, TLR2 recognizes both bacterial and fungal components. Given the strong cellular proinflammatory responses to TLR2 activation, we explored the possibility that TLR2 agonists could strengthen FcγR activity. Human peripheral blood monocytes (PBM) were treated with or without the TLR2 agonist PAM2CSK4 and tested for FcγR expression and function. Results showed that overnight treatment with PAM2CSK4 led to an increase in the expression of both FcγRIIa and the common γ-subunit, while expression of the inhibitory receptor FcγRIIb remained unchanged. Along with this, PAM2CSK4 significantly increased IgG-mediated TNFα production in plate-bound assays. We then tested its effect on phagocytosis, using fluorescently-labeled opsonized sheep red blood cells as targets. PAM2CSK4 significantly enhanced the phagocytic ability of PBM. To test the effect of PAM2CSK4 in vivo, we injected it in combination with anti-CD20 antibody and measured B-cell depletion. Results showed that PAM2CSK4 led to greater depletion of splenic B cells. These findings suggest that TLR2 agonists modulate FcγR expression and function and should be investigated as possible adjuvants for antibody therapy.