bluuebunny/crossref_metadata_2025 huggingface dataset:
Migratory properties of skin-resident γδ T cell populations (49.20)
Authors: Jiang Xiaodong , Yoo Min Jae , Kupper Thomas
Published: January 2023

γδ T cells preferentially reside in epithelial tissues like skin. Dendritic epidermal T cells (DETC) in epidermis have been studied extensively. Recently, a novel IL-17-producing γδ T cell population in dermis was identified and demonstrated to have pivotal role in skin inflammation. Nevertheless, their migration patterns are largely undefined. In this study, we found that almost 30% γδ T cells in dermis, lymph nodes (LNs) and spleen are Vγ2+. This proportional distribution led us to hypothesize that γδ T cells may re-circulate between dermis and secondary lymphoid tissues. To test this hypothesis, we created GFP+:GFP- parabiotic mice, and demonstrated that dermal γδ T cells are constantly circulating, whereas DETC are stationary and non-recirculating. We next focused on skin homing of dermal γδ T cells and found that these γδ T cells were not diminished in lymphotoxin α-/- mice (lacking LNs), suggesting that dermal γδ T cells can be stably maintained after seeding from perinatal thymus. Further study revealed that dermal γδ T cells express high levels of CD69, CD103, and E- and P-selectin ligands, all of which are required for mature T cell migration to skin. However, in E- and P-selectin ligand-deficient (Fuc T IV/VII-/-) mice, dermal γδ T cells are paradoxically increased, while DETC are remarkably diminished. Taken together, these findings may help better define the role of γδ T cells in peripheral epithelial tissues.
The recognition and internalization of apoptotic cells and Gram negative bacteria share the BAI1-ELMO1 pathway in the gastrointestinal tract (49.4)
Authors: Ernst Peter , Sarkar Arup , Owen Katherine , Ravichandran Kodi , Casanova James , Das Soumita
Published: January 2023

Clearance of apoptotic cells, debris and pathogens by phagocytes is essential role for the homeostasis of host responses. It has been shown previously that the BAI1-ELMO1-Dock180 pathway stimulates Rac1 activity which has a pivotal role in enabling phagocytosis. ELMO1 was first identified as a protein required for the phagocytosis of apoptotic cells bound by BAI1. Recently we found that BAI1 recognizes LPS on Gram-negative bacteria and helps in bacterial attachment. The objective of these studies was to determine if the BAI1-ELMO1 pathway contributes to the internalization of targets relevant to the digestive tract. Both BAI1 and ELMO1 were expressed by macrophages in the murine small intestine. Inhibition of BAI1 function by siRNA decreased the binding of cellular and microbial targets to macrophages from culture or freshly isolated from the gut. Inhibition of ELMO1 reduced internalization of both apoptotic epithelial cells and Salmonella in association with decreased Rac1 activity. This loss of ELMO1 function had no effect on binding suggesting these events are downstream of the surface interaction mediated by BAI1. Interestingly, inhibition of ELMO1/Rac function impaired the pro-inflammatory response to infection. These results suggest a novel role for the BAI1-ELMO1 pathway in Rac1 activation and the scavenging of apoptotic cells and bacteria by macrophages in the digestive tract.
Colitis-associated glycome for local memory CD4+ T cell expansion (49.5)
Authors: Nishida Atsushi , Lau Cindy , Kobayashi Taku , Hisamatsu Tadakazu , Hibi Toshifumi , Mizoguchi Emiko , Fukuda Minoru , Andoh Akira , Blumberg Richard , Mizoguchi Atsushi
Published: January 2023

Immune responses are modified by a diverse and abundant repertoire of carbohydrate structures on cell surface, which is known as the “glycome”. We herein report a colitis-associated glycomethat is created on the colonic, but not systemic, memory CD4+T cells under intestinal inflammatory conditions. Through gene screening approaches, we found that CAG represents an immature core-1 O-glycan that is created through the decreased expression of an enzyme, core-2 β1,6-N-acetylglucosaminyltransferase1 that is responsible for the production of core-2 O-glycan branch through an addition of GlcNAc to a core-1 O-glycan. Indeed, restoration of C2GnT1 expressions using T-cell-specific C2GnT transgenic mice abolished the inflammation-induced development of CAG on colonic CD4+T cells. Functionally, the CAG promoted the proliferation of memory CD4+T cells, resulting in the exacerbation of colitis. Mechanistically, CAG induced the “super rafts” formation on memory CD4+T cells, which provide a place for initiating the signaling machinery for T cell activation. Indeed, the CAG contributed for sustaining the activation of protein kinase C θ, a key signaling molecule for T cell activation. These findings suggest that a specific glycome, which is created on memory CD4+T cells under intestinal inflammatory conditions, leads to further exacerbation of diseases by promoting memory T cell expansion.
Re-utilization of germinal centers in multiple PP results in highly synchronized, oligoclonal and affinity matured gut IgA responses (49.6)
Authors: Lycke Nils , Bergqvist Peter , Stensson Anneli , Mehr Ramit , Bemark Mats
Published: January 2023

Whereas recent studies have pointed to multi-centered and diverse gut IgA responses to the microbiota, little information is available on IgA responses following oral immunization with T cell-dependent antigens. Here we have addressed how gut IgA responses develop using a novel approach where NP-hapten was conjugated to cholera toxin (CT), which allowed us to follow, at the molecular level, the site of initiation, expansion, differentiation and distribution of a specific IgA B cell response. Clonal relationships and affinity maturation of specific IgA cells at gut inductive and effector sites were investigated. Unexpectedly, we found gut IgA B cell responses to be oligoclonal and dominated by high affinity maturation. Extensive lineage trees of gut NP-specific IgA cells were generated, revealing strong clonal relationships throughout the entire gut mucosal immune system. Thus, clonally related IgA cells were found in Peyer’s patches, mesenteric lymph nodes and the small and large intestine, suggesting an effective expansion and selection process. This was achieved through synchronization of multiple PP hosting the same high affinity B cell clones. Adoptive transfer experiments showed that this was possible through re-utilization of already existing germinal centers (GC) in multiple PP by previously activated GC GL7+ B cells. The re-utilization required that oral antigen was given prior to cell transfer.
Reversal of normal ovarian function in experimental autoimmune ovarian failure mouse model post-dexamethasone treatment (51.10)
Authors: Mande Purvi , Khole Vrinda
Published: January 2023

Glucocorticoids (GCs) are in widespread clinical use for treatment of autoimmune diseases including Premature Ovarian Failure and In Vitro fertilization-Embryo Transfer failures. Immunosuppression by GCs in these patients inhibits potential adverse effects of Anti-Ovarian Antibodies (AOAs) on oocyte quality, gamete interaction, implantation and embryo development. The present study highlights the restoration of normal follicular development and fertility in experimental autoimmune ovarian failure mouse model after dexamethasone treatment, thus establishing the significance of corticosteroids in human autoimmune ovarian failures. However, their mode of action in these cases remains unknown. Female mice were immunized with immunodominant epitopes of three dominant human ovarian autoantigens and monitored for various ovarian parameters. Immunized female mice showed high AOA titers and specific reactivity to different ovarian cell types. Adverse effects on cyclicity, pregnancy, pre-implantation embryos and fertility were noticed. Ovaries from these mice exhibited fewer healthy corpora lutea (CL) and growing follicles and increased atretic follicles. However, the number of healthy follicles, CL, pre-implantation embryos and fertility was restored to normal in dexamethasone treated mice in a dose dependent manner, thereby establishing that the three putative autoimmune ovarian antigens can be used for developing targeted therapies in human autoimmune ovarian pathologies.
Vitamin D mediated inhibition of Helicobacter species protects mice from colitis (51.7)
Authors: Ooi Jot Hui , Cantorna Margherita
Published: January 2023

Vitamin D regulates experimental inflammatory bowel disease (IBD). Mice that are deficient in the ability to convert vitamin D to the active form (Cyp27B1KO) were highly susceptible to dextran sodium sulphate (DSS)-induced colitis. Depletion of gut bacterial flora using broad-spectrum antibiotics protected Cyp27B1KO mice from DSS-induced colitis, suggesting that the susceptibility of Cyp27B1KO mice to DSS-induced colitis was due to the alterations in gut bacterial flora. 454 pyrosequencing showed that Cyp27B1KO mice had increased numbers of Proteobacteria and Actinobacteria and decreased numbers of Lachnospiraceae. In particular, the Helicobacteraceae family members from the Proteobacteria phylum were significantly elevated in Cyp27B1KO mice. Treating Cyp27B1KO mice with the active form of vitamin D (1,25(OH)2D3) reversed the phenotype of the Cyp27B1KO mice, reduced Helicobacteraceae expression and protected mice from DSS-induced colitis. Full vitamin D-deficiency in WT mice also resulted in increased Helicobacteraceae expression and more severe DSS-induced colitis compared to vitamin D-sufficient WT mice. The presence of elevated Helicobacteraceae results in the increased susceptibility to DSS colitis in vitamin D- or 1,25(OH)2D3-deficient mice. The data show that vitamin D through 1,25(OH)2D3 plays a role in regulating the composition of the intestinal microflora to suppress Helicobacter species and symptoms of experimental IBD.
Fyn kinase is required for B cell IL-4Ralpha-mediated activation of Stat3, Stat5 and Stat6, germinal center formation and antibody production (52.10)
Authors: Falanga Yves , Natalia Chaimowitch , Conrad Daniel , Ryan John
Published: January 2023

The generation of antigen-specific antibodies and the development of immunological memory require collaboration between B and T cells. T cell-secreted IL-4 is important for B cell survival, isotype switching, affinity maturation and the development of germinal centers (GC). Fyn-deficient (KO) T cells have reduced IL-4 production after α-CD3/α-CD28 stimulation. Here we demonstrate that Fyn-deficient mice fail to develop robust humoral immunity following a T-dependent antigen immunization. In fact, Fyn KO mice display low Ag-specific IgG1, IgG2a but not IgM, correlating with low numbers of Follicular Helper T cells (TFH) and spleen plasma cells. More importantly, we demonstrate that Fyn kinase is required for IL-4-induced Stat3, Stat5 and Stat6 phosphorylation, describing a previously unreported role for Fyn in B cell IL-4R signaling. Taken together, our data demonstrate that Fyn kinase plays a critical role in the development of humoral immunity, affecting both B cells and T cells.
Innate memory of human Vγ2Vδ2 T cells: Distinct proliferative, migratory and tumorcidal capabilities of γδ memory subsets (52.17)
Authors: Morita Craig , Ness-Schwickerath Kristin , Nam Ki-Hoan , Siddiqui Akbar , Leon Annette , Brandes Marlene , Moser Bernhard , Oldfield Eric , Wang Hong , Puan Kia-Joo , Jin Chenggang
Published: January 2023

Human Vγ2Vδ2 T cells are unconventional T cells that monitor isoprenoid metabolism by using their antigen receptors to recognize foreign and self prenyl pyrophosphates. Although important for immunity to infections and for tumor immunotherapy, little is known about the development of γδ memory. Elucidation of γδ differentiation pathways will help to develop ways to boost their immunity. Here we report that there are many unique features compared with αβ T cells. Unlike αβ T cells, at birth half of Vγ2Vδ2 cells have memory phenotypes and ~25-40% express inflammatory chemokine receptors. In adults, 98.4% of Vγ2Vδ2 T cells are memory cells that can be separated into CD28+CD27+ TEarly (central); CD28+CD27- TEarly 27-; CD28-CD27+ TIntermediate (effector); and CD28-CD27- CD45RA+ TLate RA (effector) subsets. The memory subsets display distinct functional properties with differential expression of effector molecules, NK receptors, and chemokine receptors. Intermediate and late effector memory cells express higher levels of cytotoxic effector molecules, innate CXCR1, CXCR2, and CX3CR1 chemokine receptors, KIR, CD16, CD56, and CD57 NK receptors, and lower levels of the β7 integrin, and proliferate poorly compared with early memory cells that express CXCR6, CCR1, and CCR2. Thus, different inflammatory chemokines attract each Vγ2Vδ2 memory subset. The rapid conversion of Vγ2Vδ2 cells from naive to memory allows them to mount memory responses to primary bacterial and protozoan infections.
TLR activated-dendritic cells (*) enhance the response of aged naïve CD4 T cells in vivo (52.5)
Authors: Brahmakshatriya Vinayak , Jones Stephen , Rymeski Barbara , Brauner Matthew , Swain Susan
Published: January 2023

Declines in T cell immune function occur as we age and poor responses of naïve T cells contribute to making the elderly population resistant to immunization with new vaccines and vulnerable to rapidly evolving infectious viral diseases such as influenza. In the young, triggering “danger pathways” such as Toll like receptors (TLR) is an effective adjuvant strategy. We found that cognate antigen presentation by bone marrow-derived and splenic DC, directly activated by TLR ligands (DC*), enhanced expansion and IL-2 production of aged naïve CD4 T cells in vitro, due to small increases in proliferation and better survival of responding cells during effector generation. IL-6, produced by the DC*, was key for these effects. Since only the Ag-specific T cells are targeted, this has therapeutic potential as an adjuvant approach that avoids undesirable bystander effects. Here we are investigating if this approach can work in vivo. We find that DC*, used as APC, can enhance the expansion of both aged and young naïve CD4 T cells in vivo, causing increased proliferation and survival and that IL-6 production by DC* plays an important role in TLR-mediated enhanced responses including enhancing the ability of the CD4 T cells to make IL-2, IFNγ and IL-17. This suggests it should be possible to selectively enhance an Ag-specific response of naïve CD4 T cells in vivo in the elderly, while avoiding systemic exposure to an adjuvant.
Substrate rigidity regulates human T cell activation and proliferation (52.9)
Authors: O'Connor Roddy , Hao Xueli , Shen Keyue , Bashour Keenan , Kam Lance , Milone Michael
Published: January 2023

Adoptive immunotherapy using cultured T cells holds promise for the treatment of cancer and infectious disease. Culture platforms based upon hard materials, such as polystyrene plastic, form the basis of many culture systems. The mechanical properties of a culture substrate can influence cellular adhesion, proliferation, and differentiation. We explored the impact of substrate stiffness on ex vivo T cell activation and polyclonal expansion using substrates with variable rigidity manufactured from poly(dimethylsiloxane) (PDMS), a biocompatible silicone elastomer. We show that the IL-2 production and ex vivo proliferation of human CD4+ and CD8+ T cells are increased an average of 4-fold following stimulation on softer (Young’s Modulus [E] < 100 kPa) compared with stiffer (E >2 MPa) substrates. Mixed peripheral blood T cells cultured on the stiffer substrates also demonstrate a trend towards a greater proportion of CD62Lneg, effector-differentiated CD4+ and CD8+ T cells. Culture of naïve CD4+ T cells on softer substrates yields an average 3-fold greater proportion of IFN-γ producing TH1-like cells. These findings reveal that the rigidity of the substrate used to immobilize T cell stimulatory ligands is an important and previously unrecognized parameter for T cell culture systems used for adoptive immunotherapy. These results also have implications for studies of T cell activation and signal transduction that use immobilized TCR/CD3 and CD28 ligands.
RTA-specific monoclonal antibodies neutralize ricin toxin intracellularly (53.13)
Authors: O'Hara Joanne , Klokk Tove Irene , Sandvig Kirsten , Mantis Nicholas
Published: January 2023

According to the current paradigm, antibodies neutralize protein toxins by either preventing toxin binding to cell surfaces or promoting toxin clearance via Fc-receptor mediated uptake. In the case of the biothreat agent ricin, although this toxin uses its B-subunit (RTB) to gain cell entry the majority of antibodies are produced against its enzymatic A-subunit (RTA). We have previously demonstrated using a collection of monoclonal antibodies (mAbs) against RTA that neutralizing activity and protection in a mouse model is associated with epitope specificity, and not mAb affinity or Ig subclass. We now put forth evidence to indicate that at least two RTA-specific mAbs, R70 and IB2, neutralize ricin intracellulary. Neither R70 nor IB2 prevented ricin binding to cell surfaces, as measured by flow cytometry. Moreover, R70 (and likely IB2) retained toxin neutralizing activity in a Vero cell assay when added after ricin had attached to cell surfaces. Using in vitro assays, we also found that R70 and IB2 affected the ability of protein disulfide isomerase to reduce the single disulphide bond linking RTA to RTB and interfered with RTA’s capacity to arrest protein synthesis. We are currently using a combination of techniques to define more precisely the mechanism by which these and other RTA-specific mAbs neutralize ricin.
A lipidomics analysis of influenza infection and PPARγ activation (53.17)
Authors: Monroe Jacob , Bassaganya-Riera Josep , Ray Keith , Helm Richard , Lu Pinyi , Kronsteiner-Dobramysl Barbara , Hontecillas Raquel
Published: January 2023

While vaccines are a reliable preventative of influenza infection, useful therapeutics for treatment of the disease would be key to minimizing the impact of a major pandemic. In this study we have focused on the use of the anti-inflammatory compounds Abscisic Acid (ABA) and 61610 in the presence of PPARγ to reduce lung inflammation and damage. The mechanisms by which these therapeutics activate PPARγ have not yet been identified, but a promising avenue is through the modification of known lipid mediators produced during Arachidonic Acid metabolism. In this study, we present a lipidomics analysis of influenza infected C57BL6 Wild Type mice treated with ABA and 61610. Our data demonstrate a rise in lipid mediator levels due to infection as well as in the presence of ABA, and to a lesser extent, 61610. Quantitative Real-Time PCR was performed in order to link the expression of lipid metabolic regulators to observed changes in lipid levels. In addition to this, molecular docking studies of PPARγ were performed to compare predicted binding affinities of previously identified ligands to other eicosanoids. The results of the simulations rank many previously identified PPARγ ligands below other metabolites, indicating that studies analyzing for the presence of previously unexplored eicosanoids may be fruitful in identifying new lipid mediators involved in anti-inflammatory pathways.
Bi-specific TCR anti-CD3 fusions for cancer immunotherapy (53.19)
Authors: Jakobsen Bent , Ashfield Rebecca , Bossi Giovanna , Adams Katherine , Harper Jane , Williams Daniel , McGrath Yvonne , Hassan Namir
Published: January 2023

Although cancer patients frequently mount antigen specific T cell responses, the low affinity of T cell Receptors (TCRs) to self-antigens coupled with low levels of HLA-peptide on cancer cells contribute to the failure of the immune system to clear tumours. Peptide epitopes presented by Class I HLA represent the largest class of tumor associated antigens and are ideal targets for cancer immunotherapy, providing that the issues of low TCR affinity and immune-suppression in the tumor microenvironment can be overcome. The most attractive targets for minimising toxicity are expressed on a restricted number of normal tissue cells, for example cancer testis antigens. We have engineered ImmTAC reagents comprising soluble, high affinity TCRs fused to an anti-CD3 scFv domain that redirects a potent, polyclonal T cell response to kill tumour cells expressing as few as 10-20 epitopes. IMCgp100 targets the HLA-A2 presented epitope gp100 280-288 and kills malignant melanoma cells with pico-Molar potency, leaving other HLA-A2 positive cells untouched. This agent is undergoing clinical testing in a Phase I dose-escalation trial and is well tolerated. A second ImmTAC, IMCmage1, targets MAGE-A3 168-176 in the context of HLA-A1. It specifically kills the myeloma population within CD138+ cells extracted from the marrow of a stage III multiple myeloma patient and can re-direct T cells extracted from the tumor microenvironment. IMCmage1 will enter the clinic in 2012.
Monoclonal antibodies against the ricin toxin B subunit: Evidence for extracellular and intracellular mechanisms of toxin neutralization. (53.2)
Authors: Yermakova Anastasiya , Mantis Nicholas
Published: January 2023

Antibodies are remarkable in their capacity to inactivate even the most potent protein toxins, including anthrax, botulinum, diphtheria, and ricin. However, the underlying mechanisms by which this occurs are poorly understood. In this study, we have produced and characterized a collection of monoclonal antibodies (mAbs) directed against the B subunit (RTB) of ricin toxin. RTB plays two roles in ricin cytotoxicity; it promotes toxin attachment to host cell surfaces, and it mediates the intracellular trafficking of the toxin from the plasma membrane to the endoplasmic reticulum (ER). We screened ~2,000 RTB-specific murine B cell hybridomas, and identified 4 mAbs with toxin neutralizing activity. Two of these mAbs, SylH3 and 24B11, passively protected mice against a 5xLD50 dose of ricin, but only partially prevented ricin binding to cell surfaces, indicating that they must neutralize ricin by a mechanism other than simply interfering with receptor attachment. Using a modified Vero cell cytotoxicity assay we found that both SylH3 and 24B11 were able to completely neutralize ricin in vitro even after the toxin had bound to cell surfaces. These data reveal that SylH3 and 24B11 neutralize ricin downstream of attachment, perhaps by interfering with the ability of the toxin to traffic to the ER. We are currently using biochemical and microscopic techniques to localize where in the cell these mAbs interfere with ricin toxicity.
Tumour eradication and induction of memory against murine mesothelioma by combined immunotherapy (53.22)
Authors: Ireland Demelza , Kissick Haydn , Krishnan Shruti , Madondo Mutsa , Beilharz Manfred
Published: January 2023

Numerous immunotherapy treatments for cancer are undergoing clinical trials or are already approved for use. One particular area of interest is targeting mechanisms of immune tolerance. Using a murine model of mesothelioma, we investigated the roles of regulatory T-cells (Tregs), intra-tumoural transforming growth factor (TGF)-β and the immunosuppressive cytotoxic T lymphocyte-associated (CTLA)-4 molecule in immune tolerance to tumours. It was found that treatments targeting a single immunosuppressive mechanism were not as effective against tumours as targeting multiple mechanisms simultaneously. Combined Treg depletion and intra-tumoural TGF-β neutralisation resulted in the generation of an anti-tumour response however; this treatment regime was limited by the eventual return of the Tregs around 7-10 days following depletion. In this study we report that combining CTLA-4 blockade with our previously published combined Treg depletion/TGF-β neutralisation regime not only permanently cleared established tumours in ~50% of treated mice. Most importantly, it was found that a combined triple treatment of anti-CD25 mAb, anti-CTLA-4 mAb and TGF-β soluble receptor resulted in prolonged depletion of Tregs, long term clearance of tumours and memory against tumour rechallenge. These data suggest that clinical application of immunotherapies against tumours may be improved by targeting multiple immune suppression mechanisms simultaneously.
TLR11-dependent and -independent recognition of Toxoplasma gondii enhances innate immunity to bacterial infection (53.3)
Authors: Neal Lori , Knoll Laura
Published: January 2023

Previous studies have shown that mice infected with the obligate intracellular parasite Toxoplasma gondii are resistant to infection with a variety of bacterial, viral, and fungal pathogens, but the factors and mechanisms mediating these phenomena are unknown. This study identifies the TLR11 ligand profilin (TgPRF) as one T. gondii factor sufficient to confer resistance to bacterial infection using a lethal Listeria monocytogenes challenge model. Mice stimulated with a single injection of purified TgPRF had a 3- to 4-log reduction in bacterial burden in the spleen and liver compared to control animals at day 3 post infection, did not develop clinical signs of disease, and survived for over 30 days. TgPRF stimulation was associated with a rapid increase in the number of neutrophils in the spleen and with production of CCL2, TNF-α, IL-12 and IFN-γ. TgPRF stimulation reduced bacterial burden in mice deficient in T cells, NK cells, IL-12 or IFN-γ, but did not provide any benefit in animals treated with neutrophil depleting antibody, suggesting its protective effect was primarily dependent on neutrophil recruitment. T. gondii-infected TLR11-deficient animals showed similar resistance to L. monocytogenes challenge, indicating that other T. gondii factors may function in similar, but TLR11-independent manners. Together these data show that TgPRF and other T. gondii factors may be used as therapeutics to stimulate innate immunity against bacterial infections.
The Role of MyD88 in the Adjuvant Activity of Neisseria meningitidis PorB (53.5)
Authors: Lohsen Mark , Liu Xiuping , Toussi Deana , Massari Paola , Wetzler Lee
Published: January 2023

Adjuvants enhance the response to poorly immunogenic antigens in vaccines. Our laboratory has shown by ELISA that Neisseria meningitidis outer membrane protein PorB acts as an adjuvant by eliciting a greater serum concentration of antigen-specific IgG when compared to immunization with antigen alone, with Ovalbumin as the antigen. PorB is a TLR2 ligand and signals through the adaptor protein MyD88. We compared the adjuvant effect of PorB to other adjuvants (Alum, MPL & CpG) in WT & MyD88 KO mice. PorB adjuvant activity was ablated in MyD88 KO mice and consistent with previous investigations: MPL & CpG adjuvant activity was also MyD88-dependent while Alum acted independently of MyD88. In addition, we previously observed PorB had residual adjuvant activity in TLR2 KO mice. One hypothesis was that PorB could stimulate the Nalp3 inflammasome and induce IL-1β production without a second signal e.g. ATP. Therefore, bone marrow-derived macrophages were stimulated with PorB ± ATP. The IL-1β production was analyzed by ELISA and compared to IL-1β production from stimulation with other TLR ligands. We found that PorB required ATP in order to sufficiently produce IL-1β, disproving our hypothesis. Overall, our results show an importance for MyD88 in both the adjuvant pathway of PorB & other TLR ligand-based adjuvants. One future direction for this work is MyD88 deletion in an antigen-presenting cell-specific manner to examine the role of each cell type in the adjuvant activity of PorB.
Novel hemocyanin from the Fissurella latimarginata exhibits an outstanding immunogenicity and non-specific immunomodulatory effects in a melanoma model (53.6)
Authors: Becker María , Arancibia Sergio , Cecilia Espinoza , Salazar Fabián , Del Campo Miguel , Born Raimundo , Ferreira Jorge , Manubens Augusto , De Ioannes Alfredo
Published: January 2023

The versatile properties of mollusk hemocyanins in biomedical applications, including non-specific immunostimulant during the therapy of bladder cancer and carrier/adjuvant in cutting-edge therapeutic cancer vaccines, has increased the interest in finding new hemocyanins with better immunomodulatory properties. Here, we evaluate the physicochemical and immunological properties of the hemocyanin from Fissurella latimarginata (FLH). This protein shares with keyhole limpet hemocyanin (KLH) and Concholepas hemocyanin (CCH) the typical hollow cylinder structure and it is also made by two subunits (~350 KDa). ConA lectin staining of FLH, demonstrated the presence of mannosylated carbohydrate structures as well as in KLH and CCH. The humoral responses in mice demonstrated that antibody titers induced by FLH itself were significantly higher than KLH and CCH. However, it performance as a carrier protein to induce antibodies to the hapten DNFB, was similar to the other carriers using Freund’s adjuvant as a repository. More interestingly, FLH exhibits an outstanding antitumor activity prolonging mice survival, when was evaluated and compared with KLH and CCH in the B16F10 mouse melanoma model. In addition, flow cytometry analysis showed that FLH induced a significant number of tumor-infiltrating CD4+ cells and also, IFN-δ secretion. Together, these findings introduce a novel hemocyanin, with intrinsically more immunogenic and immunomodulatory capacities for antitumor therapy.
Protective role of R-spondin3 in mesenteric ischemia/reperfusion induced tissue injury (54.11)
Authors: Kannan Lakshmi , Kis-Toth Katalin , Yoshiya Kazuhisa , Thai To-Ha , Tsokos George
Published: January 2023

Ischemia reperfusion (I/R) injury represents a leading cause of morbidity and mortality. R-spondins, acknowledged as ligands of Wnt pathways, demonstrate their physiological role as mitogens, morphogens, stem cell factors, and wound healing agents in various development and disease models. We asked whether these proteins could prevent inflammation-associated tissue damage in ischemic disorders. C57BL/6J mice were pretreated with vehicle or R-spondin3 (R-spo3) before inducing mesenteric I/R for 30/180 min. At the end of reperfusion, intestinal tissues were harvested for different experimental analysis. Treatment with R-spo3 significantly decreased local intestinal injury. mRNA and protein analyses revealed that this effect was achieved through the modulation of the expression of pro-inflammatory cytokines (IL-6, TNFα and COX2) and deposition of IgM and complement. The decreased levels of IgM on tissues correlated with the reduced vascular leakage in the intestine of R-spo3 treated mice. Further we determined the effect of R-spo3 on endothelial barrier function in vitro using HUVECs and FITC-dextran (500kDa). R-spo3 pretreated groups showed significant reduction in histamine-enhanced FITC-dextran permeability of endothelial monolayer. Our results demonstrate that R-spo3 mitigates I/R-induced local tissue injury by preventing vascular leakage and decreasing the local inflammatory response. These findings confer the protective effects of R-spondin3 in I/R-related tissue damage.
The role of Syndecan-4 in the development of rheumatoid arthritis (54.23)
Authors: Endo Tsutomu , Morimoto Junko , Ito Koyu , Kanayama Masashi , Iwasaki Norimasa , Minami Akio , Uede Toshimitsu
Published: January 2023

Syndecan-4 (Syn4), a cell-surface heparan sulfate proteoglycan has been implicated as a critical mediator for inflammatory responses. Recent report has demonstrated that dendritic cell-associated heparan sulfate proteoglycan-dependent integrin ligand (DC-HIL) binds to Syn4 on activated T cells, resulting in the inhibition of T cell responses. Here, we have investigated the role of Syn4 in the development of Rheumatoid Arthritis (RA). We have used two RA models; T cell-dependent collagen-induced arthritis (CIA) model and T cell-independent collagen antibodies-induced arthritis (CAIA) model. In CAIA model, Syn4-wild-type and Syn4-deficient (Syn4 KO) mice showed similar levels of clinical severity. In contrast, in CIA model, Syn4 KO mice revealed attenuated RA development. Syn4 expression was detectable on activated CD4+ T cells and B cells in the draining LN (dLN). Syn4 KO mice showed a tendency to generate reduced numbers of Th1 and Th17 cells in the dLN after immunization, and helper T cells generated in Syn4 KO mice revealed different expression patterns of chemokine receptors. Although both groups showed comparable numbers of Tfh and GC (germinal center) B cells, Syn4 KO mice produced lower level of type-II collagen-specific antibody, as compared with WT mice. These results suggest that Syn4 may regulate the development of RA by modulating T and B cell responses. We are currently investigating if Syn4-deficiency affects the functions of Tfh and GC B cells.
Manzamine A (MZA) has differential effects on osteoarthritis (OA) fibroblast expression of MCP-1 and IL-6 (54.26)
Authors: Klosowska Karolina , Mc Dermott Sarah , Zanotti Brian , Volin Michael , Walczak Brian , Woods James
Published: January 2023

MZA is a complex alkaloid compound with anti-inflammatory properties that has recently been synthesized. OA has been characterized to have an inflammatory component involving increased cytokine release by synovial tissue fibroblasts. We hypothesized that MZA treatment of OA fibroblasts could decrease their production of pro-inflammatory cytokines at non-toxic concentrations. OA fibroblasts were isolated with IRB approval, treated with MZA or vehicle, and viability was assessed using multiple methods. OA fibroblast condition media (CM) was collected at various time points after MZA or control treatment, screened by antibody microarrays, and verified by ELISAs. Toxicity studies on OA fibroblasts suggested that concentrations up to 2.5 µM MZA were not toxic using a sensitive flow cytometry kit (results varied in a method-, time-, and concentration-dependent manner). Antibody microarray performed on CM pooled from 5 primary OA fibroblast lines treated with 10 µM MZA for 24 hrs suggested that monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-6 were two of the most prominent proteins detected, and MZA appeared to decrease MCP-1 production. ELISAs suggested that concentrations of 0.5 µM MZA and higher significantly decreased production of MCP-1 (n=5; p<0.05). In contrast, 1.25 µM MZA appeared to increase IL-6 production, while other concentrations inhibited it. Our data suggest that MZA could alter cytokine production at non-toxic concentrations.
Knockdown of Ste20-like proline/alanine-rich kinase (SPAK) attenuates intestinal inflammation in mouse (54.9)
Authors: Yan Yutao
Published: January 2023

Inflammatory bowel diseases (IBDs), principally Crohn’s disease and ulcerative colitis, are characterized by epithelial barrier disruption and alterations in immune regulation. Ste20-like proline/alanine-rich kinase (SPAK) plays a role in intestinal inflammation, but its underlying mechanisms need to be defined. SPAK KO mice exhibited significant increases of intestinal barrier function. Knock-down of SPAK significantly decreased paracellular intestinal permeability to FITC-dextran, and lowered significantly the sodium ion selectivity of tight junctions in intestinal epithelial cells. Further, the expression of junction proteins β-catenin, claudin-2 decreased. In contrast, expression of ZO-1, Occludin, E-cadeherin increased significantly, whereas the change of expressions of Claudin-4 and Claudin-1 was not noticeable. In vivo studies using mouse model of colitis induced by dextran sulfate sodium (DSS) and trinitrobenzene sulfuric acid (TNBS) showed that KO mice were more tolerant to DSS or TNBS treatment than wild-type animals, as demonstrated by clinical and histological characteristics and MPO enzymatic activities. KO mice also demonstrated ameliorated colon inflammation by mouse endoscopy compared to wild type mice. Consistent with this notion, we found that SPAK knockdown attenuated the increases of inflammatory cytokines production in vivo and also ameliorated bacterial translocation under DSS treatment, which together likely reduce intestinal epithelial permeability.
Elevated IL -1β levels are associated with resistance to Yesinia pestis in B10.T(6R) mice (55.11)
Authors: Sarkar Sanghita , Lambert Nathaniel , Nilles Matthew , Bradley David
Published: January 2023

We have previously demonstrated that inbred B10.T(6R) mice are resistant to Yesinia pestis (KIM5 strain) and resistance decreases with age. It has been shown that Y. pestis is cleared by neutrophil engulfment. We evaluate here the potential contribution of IL-1β, a proinflammatory cytokine important for neutrophil recruitment and activation. IL-1β levels were measured from spleens and sera collected 60 and 72 hour post infection from young (resistant) and middle-aged (susceptible) B10.T(6R) mice and control (susceptible) C57BL/6. Splenic IL-1β production was statistically higher in young B10.T(6R), compared with both middle-aged B10.T(6R) and C57BL/6 mice. Serum IL-1β levels were also moderately higher in young B10.T(6R) mice when compared to middle aged B10.T(6R) and C57BL/6 mice. IL-1β produced by Y. pestis-infected peritoneal-derived macrophages taken from uninfected young and middle-aged B10.T(6R) mice and C57BL/6 mice was measured at 2,4,8 and 24 hour. IL-1β levels were significantly elevated in peritoneal macrophages infected in vitro from young B10.T(6R) mice compared to decreased levels in both middle-aged B10.T(6R) and C57BL/6 derived peritoneal macrophages. These studies suggest a direct correlation between elevated IL-1β levels and resistance to Y. pestis infection observed in young B10.T(6R) mice.
Skin wound healing and cytokine response promoted by a quorum-sensing molecule from Pseudomonas aeruginosa (55.16)
Authors: Kanno Emi , Kawakami Kazuyoshi , Miyairi Shinichi , Tanno Hiromasa , Ishii Keiko , Hayashi Denso , Maruyama Ryoko , Tachi Masahiro
Published: January 2023

Quorum-sensing systems regulate virulence factors in Pseudomonas aeruginosa, which produces two autoinducers, N-3-oxododecanoyl or N-butanoyl-L-homoserine lactone (3-oxo-C12- or C4-HSL, respectively). Production of each HSL is regulated by the lasI or rhlI gene. Recently, HSLs are considered to play important roles in modulating immune responses. In this study, we addressed the effect of HSLs on the wound healing in skin. Wounds were created on the back of SD rats. P. aeruginosa PAO1 and its lasI or rhlI-deletion mutant (ΔlasI or ΔrhlI) were inoculated at the wound surface. Rats were injected intraperitoneally with anti-3-oxo-C12-HSL or -C4-HSL antiserum or administered each HSL at the wound surface. The wound tissues were harvested for analysis of re-epithelialization, accumulation of neutrophils and synthesis of TNF-α. Inoculation with PAO1 significantly accelerated the healing process, as evaluated by re-epithelialization, on day 3 post-wounding, which was associated with the local infiltration of neutrophils and synthesis of TNF-α. These responses were not observed when inoculated with ΔlasI or ΔrhlI in place of PAO1 and cancelled when co-treated with these anti HSL antisera, respectively, but not with control rabbit serum. By contrast, administration of either HSL in the absence of PAO1 significantly accelerated the re-epithelialization. These results suggest that 3-oxo-C12-HSL and C4-HSL may be deeply involved in the skin wound healing during P. aeruginosa infection.
Dectin-1 Signaling Recruits Elements of the Autophagic Machinery to Candida albicans-containing Phagosomes (55.2)
Authors: Tam Jenny , Tanne Antoine , Khan Nida , Mansour Michael , Becker Christine , Sokolovska Anna , Stuart Lynda , Xavier Ramnik , Vyas Jatin
Published: January 2023

A classic hallmark in the autophagic process is the reversible conjugation of LC3 to the autophagosomal membrane. Autophagy has been shown to target pathogens that reside in the cytosol or within autophagosomes for lysosomal degradation. Therefore, components of the autophagy pathway can participate in the effective elimination of viruses, bacteria, and parasites, though their role in fungal immunity is poorly understood. We show that activation of Dectin-1 by its ligand β-1,3-glucan leads to recruitment of LC3 to the phagosome and describe multiple intermediate signaling steps. The cytoplasmic tail of Dectin-1 contains an immunoreceptor tyrosine-based activation (ITAM)-like motif which is phosphorylated in response to binding of β-1,3-glucan, leading to relocation from the cell surface to the phagosome. We show that Dectin-1 activation by intact fungal pathogens or by chemically defined ligands leads to LC3 recruitment to the phagosomal membrane. Mutation of the tyrosine phosphorylation site in Dectin-1 abolished LC3 recruitment, suggesting the involvement of spleen tyrosine kinase (Syk) in this subcellular redistribution of LC3. Likewise, impairment of ROS generation by knockout of NADPH oxidase (NCF4) downstream of Syk inhibited LC3 recruitment to the phagosome, whereas knockout of Card9 did not affect LC3 recruitment. These observations suggest a previously unobserved signaling pathway connects Dectin-1 to the autophagic machinery via Syk signaling and ROS generation.
Role of IL-18 in the IFN-γ production by innate lymphcytes in response to contact-dependent interaction with dying bacterial-infected macrophages. (55.20)
Authors: Kubota Koichi
Published: January 2023

We have previously proposed that macrophage cell death caused by bacteria triggers initial IFN-γ production by innate lymphocytes at an early stage of bacterial infection (Scan. J. Immunol. 71:199-209, 2010; Front. Immun. 2:26, 2011). Here we explore the role of IL-18 in this phenomenon by using functional T cell hybridoma B6HO3, a hybrid between YACUT lymphoma and a relevant innate T cell. B6HO3 cells produced IFN-γ dependently of IL-18, but not IL-12p70, in response to cell-cell contact-dependent interaction with dying Listeria monocytogenes (LM)-infected macrophages. Despite the fact that B6HO3 cells required above 300 pg/ml of IL-18 to produce IFN-γ, the amounts of IL-18 secreted by dying LM-infected macrophages were far below this threshold level. IL-18 binding protein (IL-18BP) inhibited this IFN-γ production, but its ED50 was about 30 nM, far exceeding the physiological level of IL-18BP. LPS-primed macrophages, when stimulated with ATP, underwent cell death and secreted the similar amounts of IL-18, but their co-culture with B6HO3 cells did not result in producing IFN-γ. Inhibitors of caspase-1 and cathepsin B did not inhibit LM-infected macrophage cell death, indicating that neither pyroptosis nor pyronecrosis was involved. These results suggest that when macrophages are killed by bacteria, IL-18 plays a pivotal role in the production of IFN-γ by B6HO3 cells through an as yet undefined cell-cell interaction between dying bacterial-infected macrophages and B6HO3 cells.
Binding of soluble yeast β-1,3/1,6 glucans to complement receptors leads to immunomodulatory effects on both innate and adaptive human immune cells (55.8)
Authors: Grossman William , Chan Anissa , Gorden Keith , Bykowski-Jonas Adria , Walsh Richard , Ertelt Katie , Vasilakos John , Antonysamy Mary , Ottoson Nadine , Bose Nandita
Published: January 2023

β-glucans are considered as fungal pathogen-associated molecular patterns (PAMPs) that are recognized by cells of the human innate immune system. Few studies have focused on recognition and immunostimulatory functions of soluble β-glucan (sβG) on innate and adaptive human immune cells. In this study, we investigated recognition and immunomodulatory functions of an analytically well-characterized form of yeast-derived sβG by human neutrophils, monocytes, and B-cells. Binding studies demonstrated that the sβG bound to human neutrophils and monocytes via complement receptor 3 (CR3) and B-cells via CR2, and that receptor binding was complement-dependent. Opsonization of soluble β-glucan was detected by the presence of complement protein, iC3b on β-glucan-bound cells. Binding of sβG on human neutrophils resulted in enhanced directed migration of neutrophils towards IL-8. In the presence of other PAMPs (eg, LPS), binding of sβG was increased on both neutrophils and monocytes. Interestingly, when sβG was combined with either a TLR7/8 agonist (imidazoquinoline CL097) or a TLR9 agonist (CpG oligonucleotide, CpG 2006), enhanced activation of B-cells was observed as measured by B cell proliferation. These results demonstrate for the first time that complement opsonization of sβG makes it amenable not only for recognition by several complement receptors, but also modulation of immune functions of both innate and adaptive immune cells.
Hydrophobin RodA protein is essential for masking the host immune response and enhancing fungal survival of Aspergillus fumigatus during corneal infection (55.9)
Authors: de Jesus Carrion Steven , Leal Jr. Sixto M. , Paul-Latge Jean , Pearlman Eric
Published: January 2023

Fungal infections of the cornea (i.e keratitis) are caused mainly by filamentous fungi, with Fusarium and Aspergillus species being the most common agents. The surface layer of A.fumigatus spores is composed of hydrophobic RodA protein (RodAp) that coats the conidial surface, making it inert to the immune system. Thus, we proposed that RodA protein (RodAp) masks surface β-glucan immune recognition in the cornea during fungal keratitis. Chemical removal of RodA (using hydrofluoric acid (HFl)) and genetic removal (ΔrodA) lead to increased exposure of surface β-glucan in conidia, and decreased macrophage activation. Dectin-1-/- mice corneas infected with ΔrodA conidia for 6h exhibited decreased cytokine production, and neutrophil infiltration into the cornea. Dectin-1-/- macrophages exposed to ΔrodA and HFl treated conidia showed decreased cytokine production, NFkB activation, and phagocytosis. To study the role of RodAp in innate immune evasion, C57Bl/6 mice corneas infected with ΔrodA conidia for 96h exhibited less corneal opacity and enhanced ability to kill the fungus when compared to the corneas infected with the WT strain. Also, human and murine neutrophils showed increased fungal killing when exposed to ΔrodA hyphae in vitro, compared to the G10 strain. This data shows a role for RodAp in masking the host mediated β-glucan immune response to conidia and establishes a novel role for surface RodAp in enhancing fungal survival in the cornea during fungal keratitis.
Oxidation resistance protein 1 (Oxr1) protects against autoantibody-induced glomerulonephritis by inhibiting inflammation and apoptosis (56.10)
Authors: Li Yajuan , Shao Xinli , Raman Indu , Yan Mei , Du Yong , Mohan Chandra , Li Quanzhen
Published: January 2023

Oxidative stress is a common pathologic feature of immune-mediated nephritis in both human and mouse model. However, the molecular pathways that govern defense against oxidative stress-induced renal injury remain unclear. Using experimental autoantibody-induced nephritis mouse model, we have identified OXR1 may play a protective role against anti-GBM disease and lupus nephritis. Impaired expression of OXR1 was observed in the kidneys of 129/svj and DBA1 mice that suffered from severe anti-GBM-antibody induced nephritis, compared with non-disease controls. The reduced expression of OXR1 was also detected in kidneys of congenic mice with spontaneous lupus nephritis (e.g., B6.Sle1.Sle3 and B6.Mrl/Lpr). Deliberate delivery of human OXR1 via lentiviral vector into mouse kidney attenuated anti-GBM-antibody induced nephritis in 129/svj mice. The overexpression of hOXR1 in recipient mice down-regulated the expression of several inflammatory chemokines, including RANTES, SDF-1, IL1β and IL6, and suppressed the infiltration of macrophage/lymphocyte in the kidneys. The increased OXR1 was also associated with reduced renal cell apoptosis in anti-GBM mice. In vitro experiment further proved that hOXR1-transfected macrophages are more resistant to H2O2-induced apoptosis. These findings suggest that OXR1 may play a protective role in immune-mediated nephritis by modulating inflammation and oxidative stress and therefore may be a potential therapeutic target for lupus nephritis.