bluuebunny/crossref_metadata_2025 huggingface dataset:
Where will our next antibiotic come from? Investigating the effects of plant extracts on the growth of E. coli (50.2)
Authors: Herrington Lori , Teyton Luc
Published: January 2023

In April 2011, the World Health Organization adopted “Combating Drug Resistance and its Global Spread” for its World Health Day theme. With this recent urgent call for global action and after working at an immunology research laboratory through the American Association of Immunologists High School Teachers Summer Research Program in Immunology, I developed science curriculum that develops students’ understanding of basic human immune responses, how antimicrobial resistance (AMR) develops and how the discovery of new therapies can alleviate problems associated with AMR. Students will perform internet-based research on antimicrobial resistance public health issues and a hands-on experiment to discover if certain plant extracts have antimicrobial properties. Upon completion of the curriculum, students will not only understand the human immune system response and how antimicrobial resistance presents the world with a looming public health issue, but students will also demonstrate, first hand, the process of drug discovery and research. The ultimate goal of this curriculum is to help students understand how science touches their everyday lives and to provide students with the opportunity to learn about the exciting field immunology.
The immune system and the mechanisms of cell proliferation: an introduction to cancer and the IDO pathway (50.3)
Authors: Pascal Lyndonia
Published: January 2023

Cancer is a disease that arises when abnormal cells divide exponentially in any part of the body resulting in tumorigenesis. My lab experience at The University of Miami School of Medicine exposed me to the IDO pathway and its relationship with cancer cells. This introduction to 4T1 IDO knockdown cell lines inspired me to design an Immunology Unit aligned with Florida Biology Next Generation Sunshine State Standards (NGSSS). Students will be introduced to the immune system and the mechanisms of cancer. Students will learn the organization of the immune system. They will also gain an understanding that cancer has many faces. Virtual inquiry and laboratory activities will provide students with an opportunity to practice aseptic technique, cell culture, and cell counting. A research component will require students to construct an explanation of how cancer develops. Students will also be required to identify some of the agents that cause cancer and to differentiate between normal cells and cancer cells.
Treatment of type 1 diabetes by α7nAChR pathway activation. (51.11)
Authors: Hanes William , Chavan Sangeeta , Pavlov Valentin , Tracey Kevin
Published: January 2023

Type 1 diabetes is characterized by autoimmune destruction of insulin-producing pancreatic β cells. The α7 nicotinic acetylcholine receptor (α7nAChR) acts as part of the “cholinergic anti-inflammatory pathway” to inhibit the production of cytokines by monocytes, macrophages, dendritic cells, and other cytokine producing cells. Administration of highly selective α7nAChR agonists in vivo have significantly reversed or prevented the inflammatory damage associated with several preclinical disease models, including collagen induced arthritis, ileus, colitis, ischemia reperfusion injury, sepsis, and autoimmune diabetes. To determine the protective effects of the cholinergic pathway in type 1 diabetes, NOD mice were treated with cholinergic pathway stimulators beginning at an early age (3-4 weeks). Blood glucose was monitored weekly to determine the onset of hyperglycemia, as determined by blood glucose >200 mg/dl. Intervention with cholinergic pathway stimulators at an early age reduced the age of onset of hyperglycemia in NOD mice and reduced the total prevalence of type 1 diabetes. This demonstrates that activation of the α7nAChR may be useful in the early prevention of type 1 diabetes.
TGF-β inhibits central memory cell differentiation in CD8 T-cells (52.14)
Authors: Takai Shinji , Schlom Jeffrey , Greiner John
Published: January 2023

Generation of a robust long-term T-cell memory would be expected to fortify vaccines and enhance host protection against infectious diseases and cancer immunotherapy. TGF-β is a well-known immune suppressive cytokine and in initial studies its ability to interfere with the generation of CD8+ central memory (CM) T-cells was confirmed. Exogenous TGF-β addition to isolated splenocytes from C57BL/6 or T-cells from F5 transgenic mice inhibited the differentiation of CD44+/CD62L+ CM T-cells in a dose-dependent manner. The addition of 0.5 ng/ml of TGF-β reduced the number of CD44+/CD62L+ CM by 50%, independent of any changes in CD8+ T-cell proliferation or apoptosis. Inhibition CM T-cell generation by TGF-β was reversed with the addition of SD208, a TGF-β RI kinase inhibitor. Signal transduction studies revealed that TGF-β selectively activates SMAD2, not SMAD independent pathways such as p38 MAPK in CD8+ T cells. SD208 interfered with SMAD2 activation and reversed the TGF-β-mediated suppression of CM T-cells. Interestingly, the TGF-β RI kinase inhibitor also suppressed S6, a down-stream mTORC1substrate. This study suggests that inhibiting TGF-β with the addition of TGF-β RI kinase inhibitor facilitates the generation of a more robust CM T-cell response, suggesting that combining TGF-β inhibition with vaccination could generate long-lasting CD8+ central memory immunity.
Combination immunotherapy integrating HSP90 inhibitor 17-DMAG reconditions the tumor microenvironment for improved T cell recruitment and anti-tumor efficacy (52.19)
Authors: Rao Aparna , Kawabe Mayumi , Taylor Jennifer , Storkus Walter
Published: January 2023

Deficiencies in MHC Class I antigen presentation by tumor cells is a significant barrier to effective cancer immunotherapy. Therefore, combinational therapies that enhance tumor-associated antigen (TAA) presentation in tumor MHC Class I molecules while coordinately stimulating TAA-specific immunity would be expected to yield a greater clinical benefit. We have previously showed that in vitro treatment of tumor cells with 17-DMAG results in transient (proteasome-dependent) degradation of the EphA2, a TAA that is over expressed in several cancers, accompanied by increased presentation of EphA2 peptides on MHC class I complexes on tumor cells. To further characterize the therapeutic potential of this approach, we assessed the efficacy of a rationally-designed combinational therapy incorporating 17-DMAG and EphA2-targeted immunotherapy in murine tumor models. In vivo administration of 17-DMAG promoted: i) dramatically slowed tumor growth; ii) Enhanced recognition of tumor cells by anti-EphA2 CD8+ T cells; iii) Reduction in the frequency of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) in the tumor microenvironment, and iv) activation of tumor-associated vascular endothelial cells thereby facilitating Type-1 T cell infiltration. When combined with EphA2-specific active vaccination or the adoptive transfer of EphA2-specific CD8+ T cells, 17-DMAG co-treatment yielded a superior tumor therapeutic regimen that was capable of rendering animals free of disease.
Immunological Characterization of the Local Tissue Response to Atypical Antipsychotics (52.22)
Authors: Paquette Sara , Hickey Magali , Dawit Had , Deaver Daniel
Published: January 2023

It is recognized that the immune response plays a key role in the progression of many neurological diseases. Orally administered atypical antipsychotics (ATAPs) are generally thought to have anti-inflammatory actions; yet few studies investigating this phenomenon have been conducted. In order to increase patient compliance, injectable formulations of ATAPs that release over time have been developed. However, subcutaneous administration of aripiprazole monohydrate (ARP) and olanzapine pamoate monohydrate (OLZ) leads to a significant local tissue response (LTR) in rat models. Consequently, knowing the effect of long residing injections of ATAPs on the immune system and characterizing the LTR is of interest. LTRs begin to form as early as day 1and can persist for at least 21 days. Currently, there is no detailed immunological description of these LTRs in terms of the cellular or cytokine profile reported. We describe here the immunological characteristics of the LTR to ARP and OLZ. Injection sites and sera were isolated from rats and analyzed for the presence of cytokines. Peripheral blood cells were characterized to determine if circulating cell populations were affected by the LTR. Based on these data, a significant local foreign body reaction occurs at the site of injection following administration of ARP and OLZ. This local response is not associated with changes in circulating immune cells or in serum cytokines.
Interplay of alphavirus-based replicons with dendritic cells induces innate immune activation and enhances adaptive immunity (53.1)
Authors: Tonkin Daniel , Barro Mario , Johnston Robert
Published: January 2023

Venezuelan equine encephalitis virus replicon particles (VRP) are infectious, non-propagating particles which function as a safe and robust humoral, mucosal and cellular adjuvant when codelivered with antigen. VRP are known to infect dendritic cells (DCs) in vitro and in vivo, and we have explored the role of DCs in the adjuvant activity of VRP. Upon footpad injection VRP are rapidly transported to the draining lymph node, resulting in cytokine secretion and recruitment of leukocytes. We found that DCs were preferentially infected in the lymph node, although other cell types were also infected. TNF-secreting monocyte-derived inflammatory DCs were the cell type most dramatically recruited to the lymph node and also the most readily infected by VRP. These inflammatory DCs also efficiently took up antigen. By depletion of DCs we demonstrated that both VRP transport to the lymph node and the subsequent inflammatory response were at least partly DC-dependent. By in vitro analysis we found that VRP-infected DCs can amplify CD4 and CD8 T cell responses to antigen, which is mediated at least partly by DC-secreted factors. We also show that VRP-infected DCs are sufficient generate an adjuvant effect comparable to that achieved by injection of VRP alone. Altogether, these data provide strong evidence that in vivo targeting of DCs by VRP matures and alters those DCs in a manner that potentiates the adjuvant activity of VRP.
Modulation of tumor microenvironment by Staphylococcus aureus and the effects of immunotherapy using novel adjuvants (53.16)
Authors: Roy Chowdhury Roshni , Ghosh Swapan
Published: January 2023

Chronic inflammation of the tumor microenvironment has been attributed as a hallmark of cancer, for its tumor-promoting effects. Staphylococcus aureus pathogenesis is also known to induce chronic inflammation and it is the second most common Gram-positive bacterium in cancer patients causing lethality. However tumor -S. aureus interaction and its effect on tumor growth and microenvironment remains to be elucidated. The objective of this study was to assess changes in host immune microenvironment at the site of tumor growth with the introduction of S. aureus, followed by adjuvant therapy. Immunotherapy designed to eliminate tumor growth is challenging, because this has to contend not only with tumor progression but also chronic inflammation. Our studies have revealed a number of strategies by which S. aureus can modulate the host’s immune system and create an environment favorable for tumor growth. Cytokine antibody arrays showed that S. aureus induces very high levels of such inflammatory cytokines as IL-1β and IL-17. Quantitative real time PCR revealed that S. aureus down regulates four critical genes, namely, CD40L, Bcl2, Nlrp12 and Tnfsf11; all of which lead to the dysfunctioning of the two most critical players of the immune system: T-cells and dendritic cells. A phytol-based immune adjuvant, phytanol (PHIS-01), developed in our laboratory was found to reverse such effects caused by S. aureus and provide augmented immunity against both bacteria and tumor.
Vaccines Targeting Tumor Blood Vessel Antigens Promote CD8+ T Cell-Dependent Tumor Eradication or Dormancy in HLA-A2 Transgenic Mice (53.21)
Authors: Storkus Walter , Zhao Xi , Bose Anamika , Lowe Devin , Taylor Jennifer
Published: January 2023

Using an HHD model in which HLA-A2neg tumor (MC38 colon carcinoma or B16 melanoma) cells are not recognized by the CD8+ T cell repertoire, we now show that vaccines based on tumor-associated blood vessel antigens (TBVA) elicit protective Tc1-dependent immunity capable of mediating tumor regression or extending overall survival. Vaccine efficacy was not observed if (HLA-A2neg) wild-type C57BL/6 mice were instead used as recipient animals. In the HHD model, effective vaccination resulted in profound infiltration of tumor lesions by CD8+ (but not CD4+) T cells, in a coordinate reduction of CD31+ blood vessels in the tumor microenvironment (TME) and in the “spreading” of CD8+ T cell responses to alternate TBVA that were not intrinsic to the vaccine. Protective Tc1-mediated immunity was durable and directly recognized pericytes and/or VEC flow-sorted from tumor tissue, but not from tumor-uninvolved normal kidneys harvested from these same animals. Strikingly, the depletion of CD8+, but not CD4+, T cells at late time points after effective therapy frequently resulted in the recurrence of disease at the site of the regressed primary lesion. This suggests that the vaccine-induced anti-TBVA T cell repertoire can mediate the clinically-preferred outcomes of either effectively eradicating tumors or policing a state of (occult) tumor dormancy.
A novel iNKT cell-mediated vaccine maneuver induces potent long-lasting anti-tumor immunity for advanced prostate cancer by boosting CD8+T cell memory and function (53.24)
Authors: Varghese Bindu , Lynch Lydia , Vriend Lianne , Kissick Haydn , Dunn Laura , Varghese Sharlin , Dranoff Glenn , Arredouani M. Simo , Balk Steven , Exley Mark
Published: January 2023

iNKT cells have considerable therapeutic promise in cancer, but so far this has not been fully realized. At issue has been delivering iNKT ligand and tumor Ag to the same APC for optimal processing and presentation. Also, although tumor-infiltrating APCs express CD1d in both prostate cancer (PCa) models and patients, tumor induced tolerance and suppression leads to defective iNKT activation. Whole-cell tumor vaccines represent a promising treatment approach and prevent tumor progression in multiple models of cancer. Here, we use an optimized approach consisting of alpha-galactosylceramide (aGalCer) and the GM-CSF-based PCa vaccine, ‘GVax’ combined with IL-12. This approach was capable of breaking tumor-induced tolerance and causing dramatic and substantial tumor regression and survival in large advanced prostate tumors in TRAMP mice. Remarkably, 10/12 vaccinated >9 month old mice rejected tumors, 'breaking tolerance', even at this late stage of tumor growth. The vaccine induced strong immune responses capable of reversing INKT defects in tumor-bearing mice as well as in patient iNKT in vitro. Both IL-12 and aGalCer were crucial for this response. Vaccinated mice had higher levels and activation of tumor-specific CD8 T, CD4 T and iNKT cells, and greater memory CD8 T cell expansion capable of producing the effector cytokines IFN-γ and TNF-α in response to tumor. These studies shed light on how whole cell vaccines targeting iNKTs can be utilized in PCa immunotherapy.
Targeted chemotherapeutic delivery to disseminated lymphoma tumors via adoptive transfer of drug nanoparticle-carrying T cells (53.25)
Authors: Huang Bonnie , Irvine Darrell
Published: January 2023

Despite being sensitive to a wide variety of cytotoxic drugs, lymphomas often require high doses of systemically administered chemotherapy to eliminate disseminated tumors, causing toxicity. We hypothesized that the efficacy of chemotherapy could be enhanced while greatly reducing off-target toxicity if autologous lymphocytes were used as carriers to target drugs to the lymphoid tissue sites where lymphomas home. To test this concept, we prepared controlled release lipid nanoparticles (NPs) loaded with the potent topoisomerase I poison SN-38. These NPs were covalently conjugated to the plasma membrane of in vitro-activated primary T-cells that retained CD62L expression for lymph node homing. In vitro co-culture of T-cells and murine Eu-myc lymphoma cells with SN-38-releasing NPs showed that lymphoma cells were much more sensitive to SN-38 than T-cells. T-cells with plasma membrane-conjugated SN-38-releasing NPs were then used as live vectors to transport NPs systemically into lymphoid organs where lymphoma cells are enriched. NP-decorated T-cells retained and delivered NPs to Eu-myc lymphoma tumors in vivo as early as one day post-adoptive transfer. Therapy studies in a transplanted Eu-myc lymphoma model indicated that high numbers of SN-38 NP-functionalized T cells can rapidly reduce tumor burden in multiple anatomical sites. These results suggest that autologous lymphocytes may be useful as chaperones for targeted delivery of chemotherapy-loaded NPs to lymphoid tumors.
CϵmX peptide-carrying HBcAg virus-like particles induced antibodies that down-regulate mIgE-B cells (53.7)
Authors: Lin Chien-Jen , Nien-Yi Chen Nien-Yi , Chen Jiun-Bo , Lu Chien-Sheng , Hung Alfur Fu-Hsin , Shiung Yu-Yu , Wu Pheidias C. , Pan Rong-Long , Chang Tse Wen
Published: January 2023

Type-I hypersensitivity reactions play a critical role in the pathogenesis of various allergic diseases. The successful development of the anti-IgE antibody, omalizumab, has validated IgE as an effective therapeutic target for the treatment of various IgE-mediated allergic diseases. Two research groups have reported that mAbs specific for certain parts of CϵmX, a domain of 52 aa residues in human membrane-bound IgE (mIgE), can cause the lysis of mIgE-B cells by apoptosis and antibody-dependent cellular cytotoxicity (ADCC). Herein, we explore the virus-like particles (VLPs) formed by hepatitis B virus core antigen (HBcAg) that harbors the entire CϵmX peptide or its fragments as immunogens for inducing anti-CϵmX antibodies. Mice immunized subcutaneously with some of these immunogens produce antibodies that bind to recombinant CϵmX-containing human IgE.Fc in ELISA and Western blot analysis and to genetically engineered human mIgE-expressing Ramos B cell line in fluorescence flow cytometric assays. The IgG antibodies purified from the sera of immunized mice could cause the apoptosis of mIgE-expressing Ramos cells through a BCR-dependent caspase pathway. Furthermore, the IgG mediated ADCC on human mIgE-expressing murine B cell lymphoma A20. These results suggest that HBcAg-CϵmX peptide immunogens warrant further investigation as a therapeutic modality for modulating IgE in patients with IgE-mediated allergic diseases.
Engineering an RNA virus as a vector for targeted cellular immunity (53.8)
Authors: Pavelko Kevin , Bell Michael , Allen Kathleen , Hansen Michael , Pease Larry
Published: January 2023

One limitation to the development of targeted tumor immunity is the availability of safe vectors that can drive strong T-cell responses capable of eradicating tumors. We have designed viral vectors using Theiler’s murine encephalomyelitis virus (TMEV) that effectively inhibit melanoma outgrowth. By cloning the TMEV genome into a eukaryotic expression vector that can easily be manipulated we were able to generate several novel tumor specific virus vaccines. We find that incorporation of defined MHC class I epitopes into sites within the leader protein of the virus can drive strong CD8+ T-cell responses against these target peptides. Further, we find that the site of incorporation can change the elicited cytokine profile and the virulence of the virus. Incorporation of the ovalbumen peptide after the third amino acid of the L protein (p3) generates a strong IFNβ response, whereas insertion within the downstream XhoI restriction site elicits a strong IL6 response similar to wild-type TMEV. Further, the p3 virus is not lethal to RAG-deficient mice, where the XhoI and wild-type TMEV virus are. However, p3 virus persists and kills IFNα/β receptor-deficient mice, suggesting that innate immune responses are responsible for viral clearance. In spite of these differences the p3 and XhoI viruses can stimulate antigen specific T-cell responses and inhibit tumor outgrowth equivalently, demonstrating the efficacy of TMEV and its potential as a therapeutic intervention.
Platelet-associated CD40/CD154 mediates remote tissue damage after mesenteric ischemia/reperfusion injury (54.1)
Authors: Kannan Lakshmi , Lapchak Peter , Ioannou Antonis , Rani Poonam , Dalle Lucca Jurandir , Tsokos George
Published: January 2023

Several innate and adaptive immune cell types participate in ischemia/reperfusion induced tissue injury. Amongst them, platelets have received little attention as contributors in the process of tissue damage after ischemia reperfusion (I/R) injury. It is currently unknown whether platelets participate through the immunologically important molecules including, CD40 and when activated, CD154 (CD40L), in the pathogenesis of I/R injury. We hypothesized that constitutive expression of CD40 and activation-induced expression of CD154 on platelets mediate local mesenteric and remote lung tissue damage after I/R injury. Wild type (WT; C57BL/6J), CD40 and CD154 deficient mice underwent mesenteric ischemia for 30 minutes followed by reperfusion for 3 hours. WT mice subjected to mesenteric I/R injury displayed both local intestinal and remote lung damage. In contrast, there was significantly less intestinal damage and no remote lung injury in CD40 and CD154 deficient mice when compared to WT mice. Platelet-depleted WT mice transfused with platelets from CD40 or CD154 deficient mice failed to reconstitute remote lung damage. In contrast, when CD40 or CD154 deficient mice were transfused with WT platelets lung tissue damage was re-established. Together, these findings suggest that multiple mechanisms are involved in local and remote tissue injury and also identify platelet-expressed CD40 and/or CD154 as mediators of remote tissue damage.
Human astrocytes express a novel NLRP2 inflammasome complex activated by extracellular ATP (54.12)
Authors: Minkiewicz Julia , de Rivero Vaccari Juan , Keane Robert
Published: January 2023

Spinal cord injury/brain trauma involves extensive central nervous system loss due to the mechanical insult and to an innate inflammatory response that is, in part, in response to activation by extracellular ATP. The innate immune response is regulated by pattern recognition receptors (PRRs), which include NOD-like receptors (NLR). However, the PRR receptors and signaling cascades regulating innate glial responses to CNS injury remain largely undefined. Here we show for the first time that human astrocytes express NLRP2 inflammasome proteins and these proteins form an inflammasome complex. Treatment of human astrocytes with ATP resulted in a dose-dependent activation of the NLRP2 inflammasome leading to inflammatory caspase-1 cleavage and interleukin-1β (IL-1β) production. ATP induction of the NLRP2 inflammasome was blocked by the pannexin 1 inhibitor, probenecid and by treatment with the P2X7 receptor antagonist, brilliant blue G (BBG). siRNA knockdown of NLRP2 significantly decreased NLRP2 inflammasome activation and caspase-1 activation in response to ATP treatment. Our findings suggest that the NLRP2 inflammasome in astrocytes is an important component of the CNS inflammatory response and may be a therapeutic target to inhibit inflammation induced by CNS injury.
The receptor tyrosine kinase Ron attenuates obesity-induced atherosclerosis progression and liver injury by down-regulating inflammation in ApoE-/- mice (54.16)
Authors: Yu Shan , Xia Mingcan , Xiong Na , Hankey Pamela
Published: January 2023

Obesity and its associated metabolic diseases are leading causes of death worldwide. Obesity increases the likelihood of atherosclerosis, diabetes and hepatic steatosis, which are caused largely by macrophage activation at aorta, adipose tissue and liver, respectively. Macrophages are classified as classical activated macrophages(M1), which are associated with increased inflammatory mediators, and alternative activated macrophages(M2), which are associated with decreased inflammation and tissue repair. Recent studies suggest M2 predominate in non-inflamed tissue in lean animals, while either a switch from M2 to M1 or direct recruitment of M1 in aorta and adipose tissue promotes atherosclerosis and insulin resistance progression. Our previous studies show that the Ron receptor tyrosine kinase, expressed primarily on tissue-resident macrophages, inhibits hallmarks of M1 activation and skews populations towards an M2 phenotype both in vitro and in vivo. Here, we show that the absence of Ron results in increased severity of atherosclerosis and liver injury, which is accompanied by elevated expression of inflammatory markers in aortas and livers in ApoE-/- X Ron-/- mice on a high cholesterol diet. However, we failed to detect significant differences in glucose tolerance or fatty acid metabolism. Our results indicate Ron plays a central role in limiting obesity-induced inflammation and plays a protective role in the progression of atherosclerosis and hepatic steatosis.
Fibrocytes contribute to the increased levels of fibrosis observed in Th2 pulmonary granulomas in TLR9-/- mice (54.18)
Authors: Saito Fumitake , Ito Toshihiro , Burdick Marie , Allen Ronald , Hogaboam Cory , Lukacs Nicholas , Strieter Robert , Kunkel Steven
Published: January 2023

Despite many recent advances in the field, the underlying mechanisms governing pulmonary fibrosis remain poorly understood. We have used an experimental system of fibrotic granuloma development via the embolization of Schistosoma mansoni eggs to the lungs. Egg embolization results in the release of highly antigenic glycoproteins (Schistosoma egg antigen,SEA) that promote a dominant Th2 immune response. We previously demonstrated that in comparison to wild-type mice, TLR9-/- mice showed increased granuloma size and augmented collagen deposition. In the present study, we investigated the mechanisms underlying the fibrotic change of this Th2-driven pulmonary granuloma system, and the contribution of TLR9 signals to this phenomenon. The chemokine receptor/ligand pair CXCR4/CXCL12 has been shown to play an important role in the homing of fibrocytes; these cells express CXCR4 and migrate in response to CXCL12 in a model of bleomycin-induced pulmonary fibrosis. Levels of CXCL12 were higher in the lungs of mice treated with S. mansoni eggs, but there were no difference between wild-type and TLR9-/- mice. Flow cytometry data indicated that TLR9-/- mice showed increased number of CD45+Col I+CXCR4+ fibrocytes in the lungs of TLR9-/- mice treated with S. mansoni eggs in comparison to wild-type mice. Thus, our results suggested that increased fibrocytes may contribute to the increased levels of fibrosis observed in TLR9-/- mice.
TRAF-6 dependent signaling pathway is essential for TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation (54.21)
Authors: Hsu Ping-Ning , Liao Hsiu-Jung , Tsai Hwei-Fang
Published: January 2023

Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Recent evidence indicates that in addition to triggering apoptosis, the TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation. To understand TRAIL-mediated signal transduction mechanism in osteoclastogenesis, we demonstrated that TRAIL induces osteoclast differentiation via a Tumor necrosis factor receptor-associated factor 6 (TRAF-6)-dependent signaling pathway. TRAIL-induced osteoclast differentiation was significantly inhibited by treatment with TRAF-6 siRNA and TRAF6 decoy peptides in both human monocytes and murine RAW264.7 macrophage cell lines, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. Moreover, TRAIL-induced osteoclast differentiation was also abolished in TRAF6 knockout bone marrow macrophages. In addition to induction of NFATc1, treatment of TRAIL also induced ubiquitination of TRAF6 in osteoclast differentiation. Thus, our data demonstrate that TRAIL induces osteoclastic differentiation via a TRAF-6 dependent signaling pathway. This study suggests TRAF6-dependent signaling may be a central pathway in osteoclast differentiation, and that TNF superfamily molecules other than RANKL may modify RANK signaling by interaction with TRAF6-associated signaling.
Glycogen synthase kinase-3 facilitates Con A-induced IFN-γ-mediated immune hepatic injury (54.24)
Authors: Tsai Cheng-Chieh , Huang Wei-Ching , Chen Chia-Ling , Hsieh Chia-Yuan , Lin Yee-Shin , Chen Shun-Hua , Yang Kao-Chi , Lin Chiou-Feng
Published: January 2023

IFN-γ-mediated inflammation followed by hepatic cell death determines Con A-induced immune hepatic injury. Glycogen synthase kinase (GSK)-3, which acts proapoptotically and is proinflammatory, is also important for facilitating IFN-γ signaling. We hypothesized a pathogenic role for GSK-3 in Con A hepatic injury. Con A stimulation caused GSK-3 activation in the livers of C57BL/6 mice. Inhibiting GSK-3 reduced Con A hepatic injury, including hepatic necrosis and apoptosis, inflammation, infiltration of T cells and granulocytes, and deregulated expression of adhesion molecule CD54. Con A induced hepatic injury in an IFN-γ receptor 1-dependent manner. Con A/IFN-γ induced activation and expression of STAT1 in a GSK-3-dependent manner. GSK-3 facilitated IFN-γ-induced inducible NO synthase, but had limited effects on CD95 upregulation and CD95-mediated hepatocyte apoptosis in vitro. Notably, inhibiting GSK-3 decreased Con A-induced IFN-γ production in both wild-type and IFN-γ receptor 1-deficient C57BL/6 mice. In Con A-activated NKT cells, GSK-3 was also activated and was required for nuclear translocation of T-box transcription factor Tbx21, a transcription factor of IFN-γ, but it was not required for CD95 ligand expression or activation-induced cell death. These results demonstrate the dual and indispensable role of GSK-3 in Con A hepatic injury by facilitating IFN-γ-induced hepatopathy.
The role of macrophages and neutrophils in pulmonary Francisella infection (55.24)
Authors: Steiner Donald , Metzger Dennis
Published: January 2023

Francisella tularensis is the causative agent of tularemia. Due to its ease of dissemination, high lethality, and low infectious dose required for respiratory infection, this bacterium poses a risk as an agent of bioterrorism. Though F. tularensis is believed to be an intracellular pathogen that requires replication within macrophages during respiratory infection, we have found that depletion of alveolar macrophages by intranasal treatment with liposomal Clodronate reduced mean time to death and total survival rates of infected mice. Furthermore, mice with a macrophage-specific insensitivity to interferon-γ (MIIG mice) similarly show heightened sensitivity to pulmonary Francisella infection. Such mice exhibited reduced expression of tumor necrosis factor-α, a potent neutrophil chemoattractant, in lung tissue, and systemic neutrophil depletion resulted in reduced mean time to death and total survival rates. Exogenous interleukin-12 promoted survival and bacterial clearance in mice, but this effect was lost upon neutrophil depletion and in IFN-γ-/- mice. The results suggest that rather than being a required reservoir for bacterial replication, alveolar macrophages are necessary for defense against F. tularensis infection by recruiting neutrophils to the lung, which are the primary cells responsible for clearing the bacterium.
High M-CSF and IL-13 levels are associated with foamy cell expansion in experimental actinomycetoma by Nocardia brasiliensis. (55.27)
Authors: Meester Irene , Rosas-Taraco Adrian , Salinas-Carmona Mario
Published: January 2023

Foamy cells (FC) have been described in various infectious diseases, and can be usurped by intracellular pathogens for survival. In a mouse model of experimental actinomycetoma induced by Nocardia brasiliensis, FC have been observed at day 3 after inoculation, remaining constant during acute inflammation, and expanding in surface area during chronic disease. These FC seem to be monocytes/macrophages and/or dendritic cells (DC) that accumulate lipid droplets upon infection. Our aim was to elucidate the levels of cytokines known to be involved in the differentiation of macrophages or DC (IL-4, IL-13, M-CSF, and GM-CSF). Female BALB/c mice (8-12 wk) were infected with N. brasiliensis in the left hind foot pad and sacrificed at 0, 0.5, 1, 3, 7, 15, 30 and 60 days of infection (n=5). Biopsies of the left foot pads were homogenized in PBS or Trizol to determine the cytokine concentrations at both the protein and the transcript level by means of ELISA and RT-PCR. We found the highest M-CSF and IL-13 levels during chronic inflammation, when the major expansion of CF occurs. M-CSF and IL-13 have been related with the alternative activation of macrophages. In conclusion: high M-CSF and IL-13 levels coincide with the expansion of CF in an experimental model of actinomycetoma.
CXCR3 ligand blockade ameliorates the experimental autoimmune cystitis (EAC) in mice (56.3)
Authors: Singh Udai , Singh Narendra , Singh Balwan , Price Robert , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

Interstitial cystitis (IC) is a complex disease associated with chronic bladder inflammation that primarily affects women. The symptoms of IC include frequent urinary urgency accompanied by discomfort or pain in the bladder and lower abdomen. The autoimmune mechanisms have an important role in IC pathogenesis and triggered lots of attention after development of autoimmune phenotype experimental autoimmune cystitis (EAC) model, similar to IC with functional and histological alterations confined to the urinary bladder. We report that the serum levels of CXCR3 ligand and local T helper type 1 (Th1) cytokines are elevated in patients with IC as well as in the murine EAC model of IC. We also show that CXCL10 blockade can attenuate the overall clinical score, reverse the development of IC-associated pathogenesis, decrease the local as well as systemic cytokines and CXCR3 and its ligands level. Urinary bladder CD4+ T cell, mast cell, and neutrophil infiltrates are also reduced following anti-CXCL10 antibody (Ab) treatment to mice with IC. We demonstrate that anti-CXCL10 Ab treatment reversed the up-regulated level of CXCR3 ligand mRNA at sites in the urinary bladder. Moreover, the decreased number and percentage of systemic CD4+ T cells in mice with IC reversed back to normal after anti-CXCL10 Ab treatment. Taken together, the present study provides new and important information on a useful animal model for IC and cellular mechanism of CXCR3 ligand mediated amelioration of IC.
Tolerance induction by monoclonal CD3 antibodies is dependent on indoleamine 2,3 dioxygenase (IDO) (56.5)
Authors: Kuhn Chantal , You Sylvaine , Valette Fabrice , Bach Jean-François , Waldmann Herman , Chatenoud Lucienne , Grohmann Ursula
Published: January 2023

Experimental data demonstrated that a short treatment with CD3 antibodies (CD3 Ab) promotes immune tolerance in primed hosts. Successful clinical transfer of this strategy has been achieved in autoimmune diabetes, and on this basis, CD3 Abs are currently under pharmaceutical development. The tolerogenic effect of CD3 Ab evolves in two consecutive phases: a first phase of “induction” during Ab treatment, involving transient T cell depletion as well as antigenic modulation and a second phase of “maintenance” that persists after CD3 Ab clearance and that correlates with the presence of active and transferable tolerance involving TGF-β dependent Tregs. We focused on the causal links relating the “induction” and “maintenance” phases of tolerance using spontaneously diabetic NOD mice and human CD3ϵ transgenic NOD mice, a novel preclinical model that we established. It is well known that abnormalities in dendritic cells (DCs) contribute to autoimmune diabetes in NOD mice, e.g. indoleamine 2,3 dioxygenase (IDO) induction in CD8+ DCs of NOD mice by IFN-γ is defective. Our results show that depletion of DCs or inhibition of IDO during CD3 Ab therapy abrogates the tolerogenic effect. TGF-β has been shown to induce IDO and we reported that remission from diabetes after CD3 Ab therapy is TGF-β-dependent. We propose that CD3 Ab mediated tolerance ensues from a local immune privilege in the pancreas driven by induction of TGF-β and tolerogenic DCs expressing IDO.
Asthma-associated IL13 single nucleotide polymorphisms (SNPs) upregulate human IL4 expression in mice carrying a human Th2 locus BAC transgene (57.10)
Authors: Pivniouk Vadim , Bailey Tom , Pivniouk Oksana , Rosenbaum David , Mobley Sara , Stern Debra , Halonen Marilyn , Vercelli Donata
Published: January 2023

A block of 9 SNPs in human (h) IL13 is strongly associated with increased susceptibility to allergy and asthma in multiple human populations. To study the functional significance of these SNPs in vivo, we generated mice that carry a 160 kb BAC5 transgene (TG) encompassing a non-polymorphic hTh2 cytokine locus (HapI). A HapII TG was engineered by introducing the asthma-associated IL13 SNPs into BAC5. hTh2 cytokine expression was faithfully regulated in HapI and HapII mice. To evaluate the effects of asthma-associated SNPs, we compared Th2 cytokine expression by polarized Th2 cells from Hap I and Hap II mice. Mouse Th2 cytokine expression was unaffected. hIL13 secretion was marginally upregulated in Hap II mice but, surprisingly, hIL4 protein and mRNA levels were dramatically increased. Increased hIL4 expression in carriers of IL13 SNPs was confirmed in humans. To elucidate the mechanisms underlying the effects of hIL13 SNPs on hIL4 expression, we performed a high-resolution analysis of DNA methylation at the Th2 cytokine locus in HapI and HapII Th2 cells. Several differentially methylated regions were identified. Notably, the HSIV element that is located ~25 kb 3’ of the IL13 SNPs and acts as an IL4 silencer in mice, was significantly hypermethylated at three consecutive CpG sites in HapII Th2 cells. Collectively, our results point to novel long-range effects of the IL13 SNPs on HSIV-dependent IL4 silencing, which may increase susceptibility to Th2-dependent allergic disease.
Interferon regulatory factor 4 (IRF4) directs dendritic cells to promote Th2 responses (57.11)
Authors: Williams Jesse , Clay Bryan , Tjota Melissa , Decker Donna , Singh Harinder , Sciammas Roger , Sperling Anne
Published: January 2023

Respiratory dendritic cells (DC) are the primary initiator of T cells during lung allergic responses. DCs process inhaled allergen, traffic to the lung draining lymph node, present antigen to T cells, and guide the type of T-helper response initiated. However, the mechanisms by which DCs direct Th2 specific differentiation are poorly understood. We previously found that signaling through FcγRIII is able to modulate TLR4 signaling in DCs, and that these DCs now skew toward a predominant Th2 response. Microarray analysis identified IRF4 as a gene downstream of FcγRIII signaling in bone marrow derived DCs (BMDCs). In respiratory DCs, IRF4 was induced within 3 hrs of stimulation with known Th2-agonists. Using mice with conditional deletion of IRF4 in CD11c cells, we demonstrated reduced Th2 responses after house dust mite-induced allergic challenge, while DC trafficking and T cell activation remained intact. Direct effects between IRF4 in DCs and Th2 development was demonstrated using sorted pulmonary DCs from IRF4-deficient mice to skew T cells in vitro. Surprisingly, when IRF4-deficient DCs were used the resulting T cells produced decreased Th2 cytokines, but augmented IFNγ and IL-17. Further, we found that increasing IRF4 expression alone in BMDCs was sufficient to drive T cells toward a Th2 phenotype. Together these data identify IRF4 as a key transcriptional regulator in DCs necessary for the development of Th2 responses.
Th9 cells regulate mast cell recruitment in acute allergic lung inflammation (57.12)
Authors: Sehra Sarita , Yao Weiguo , Nguyen Evelyn , Kaplan Mark
Published: January 2023

Th2 cells are classically thought to contribute to the pathogenesis of asthma. IL-9, long described as a Th2 cytokine has also been shown to mediate allergic inflammation in both human and murine studies. Recently, we have identified an ETS-family transcription factor PU.1 required for generating the Th9 phenotype and in the development of allergic inflammation. Mice with a conditional deletion of PU.1 in T cells demonstrated attenuated allergic inflammation concomitant with decreased levels of IL-9. IL-9 is also well documented to play an important role in mast cell growth and differentiation. To determine if Th9 cells are required for the recruitment of mast cells in vivo, we examined mast cell infiltration in the lung in mice with blocking IL-9 antibodies as well as in mice with PU.1 deficient T cells. Both experiments with either blockade of IL-9 or PU.1 deficient T cells demonstrated diminished mast cell numbers in the lung. We further examined the effect of transferred Th2 and Th9 cells on mast cell recruitment. Transfer of differentiated Th2 or Th9 cells from D011.10 mice to wild type mice demonstrate that while Th2 cells increase mast cells, as detected by expression of mast cell proteases in the lung by 4-fold, recipients of Th9 cells had over 11-fold increased expression. Therefore, our studies suggest an important role for Th9 cells in mast cell recruitment in models of acute allergic inflammation.
TSLP promotes induction of Th2 differentiation but is not necessary during established allergen-induced pulmonary disease (57.13)
Authors: Jang Sihyug , Lukacs Nicholas
Published: January 2023

Thymic stromal lymphopoietin (TSLP) has been implicated in allergic inflammation by promoting a Th2-type response. Using in vitro cultures we demonstrated that TSLP played a critical role in the development of Th2 immune responses, but its role on an established Th2 immune environment was not significant. Adoptive transfer of naive DO11.10 ovalbumin (OVA)-specific T cells followed by exposure of OVA showed an early impairment of Th2 immune response in TSLP-/- mice compared to wild type (WT) mice. In contrast, transfer of differentiated Th2 cells into TSLP-/- mice did not change lung pathology or Th2 cytokine production compared to transfer into WT mice. Using a well-established allergen-induced Th2 model demonstrated that there was no difference between WT and TSLP-/- mice. Furthermore, when we treated allergic animals with established disease with a neutralizing anti-TSLP antibody there was no change in airway hyerreponsiveness (AHR) or Th2 cytokine production compared to the control antibody treated animals. Collectively, these studies suggest that in mice TSLP has an important role during the early development of Th2 immune responses. However, TSLP appears to have a minimal role at later stages of allergic disease suggesting that it may not be required for maintaining a persistent and established Th2 immune environment. These studies may offer important data for determining whether targeting TSLP would be beneficial for therapy in established Th2 mediated diseases.
Wide-scale and quantitative phosphoproteomic method for analysis of bidirectional signaling within interacting B and T cells (58.11)
Authors: Gu Tao , Ji Qinqin , Salomon Arthur
Published: January 2023

Physiological interactions between T cells and antigen presenting cells (APC) induce the formation of the immunological synapse. The subsequent reversible tyrosine phosphorylation of a variety signaling proteins plays a pivotal role in regulation of T cell activation. Novel phosphoproteomic methods in mass spectrometry can facilitate and accelerate elucidation of cell signaling networks by providing a global picture of cellular phosphorylation. In this study a B and T cell line were labeled with stable isotopes of amino acids in cell culture, using the SILAC method. This labeling helped to differentiate and quantitate phosphopeptides from either type of cell through a timecourse of their interaction. Quantitative phosphoproteomic analysis revealed a wide array of newly discovered phosphorylation sites that changed significantly during B/T cell interaction. Phosphorylation sites were observed and quantified on the earliest upstream signaling components and the following downstream target proteins in both T cell and B cells (T cells: TCR-CD3 subunits, CD28, Erk, Fyn, Lck, P38, PI3K, PLCγ2, Zap-70 et al; B cells: B cell antigen receptor complex, liner protein, CD19, Fyn, Jnk, Lck, Lyn, P38, PI3K, PLCγ2, et al) .
R10K: an international collaborative project for biomarker discovery (58.7)
Authors: Han Jian , Wang Chunlin , Byrne-Steele Miranda , Yang Qunying , Myers Richard
Published: January 2023

The R10K Project is an international collaborative effort to sequence the immune repertoire (T and B cells) from 10,000 samples that cover 100 diseases. We have developed a novel multiplex PCR method, which can amplify targets from a complex genetic material, inclusively and semi-quantitatively. This easy-to-use technology has been successfully applied to amplify immune repertoire libraries from peripheral blood samples or other tissue samples. The natural duty of the adaptive immune system is to detect any internal and external threats and mount specific and measured reactions accordingly. Our pilot studies have demonstrated that immune repertoire sequencing can be used to measure this specific response and identify disease-related T cell CDR3s. Therefore, analyzing the immune repertoire of several disease states with high throughput sequencing may lead to the discovery of disease-specific biomarkers for diagnosis, prognosis, and treatment evaluations. The R10K project is organized and supported by the HudsonAlpha Institute for Biotechnology, a non-profit research institute specialized in genomic research. A Scientific Advisory Board has been established to help identify projects from online submitted proposals. The R10K data will be made public 6 months after it has been generated.
Engineering the Lymph Node Microenvironment via Intranodal Controlled Release Vaccine Depots (58.8)
Authors: Jewell Christopher , Bustamante Lopez Sandra , Irvine Darrell
Published: January 2023

Recent clinical trials of intra-lymph node (i.LN.) vaccine injection have generated promising results in cancer and autoimmunity. We hypothesized that controlled release biomaterials in the context of intranodal vaccination could sustain exposure of the LN to adjuvants or immunomodulatory drugs to augment vaccine responses. To test this idea we encapsulated the TLR3 agonist polyIC in biodegradable polymer particles designed to release this adjuvant within LNs over several days. Using a non-toxic tracer, we developed a non-surgical approach for i.LN. injection and injected polyIC in soluble form or encapsulated in microparticles (MP-polyIC) or nanoparticles (NP-polyIC). MP-polyIC conferred ~4-fold and 2-fold increases in polyIC retention in LNs compared with soluble polyIC or NP-polyIC, respectively; prolonged retention correlated with a dramatic increase in polyIC uptake by LN-resident DCs compared with soluble polyIC, which was quickly cleared from LNs. Mice vaccinated with antigen mixed with MP-polyIC induced antigen-specific CD8+ T cell expansion 4-fold greater than soluble polyIC, which could not be matched by even 10-fold greater doses of soluble polyIC. MP-polyIC vaccination also increased the percentage of IFN-γ and TNF-α secreting T-cells up to ~4-fold, indicating a polyfunctional vaccine enhancement. These results suggest that sustained release directly in the lymph node microenvironment can significantly alter and enhance the function of immunomodulatory compounds.