bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamic capping of CD25 occurs at point of contact prior to delivery of IL-2 from dendritic cell to Treg hybridoma. (50.12)
Authors: Field Elizabeth , Kulhankova Katarina , Rouse Todd , Nasr Mohamed
Published: January 2023

CD4+CD25+ regulatory cells (Tregs) depend on external IL-2 for expansion, homeostasis and function, but the process of IL-2 delivery to Treg is poorly understood. We reported that IL-2 producing dendritic cells (DCs) control Treg function in vitro and that IL-2 delivery to Treg requires cell:cell contact and CD25. To further explore DC delivery of IL-2 we demonstrated that IL-2 producing dendritic cells similarly controlled suppressor function of CD4+CD25+ Treg hybridoma cells RD6, then studied the fate of DC-derived material and CD25 using live cell imaging and confocal microscopy of dye-labeled or IL-2mCherry transfected DCs and dye-labeled or CD25-AcGFP transfected RD6. Conjugate formation between DCs and RD6 was independent of IL-2 and CD25. However, the uptake of DC-derived material into RD6 cells required both IL-2 sufficient DCs and CD25. RD6 internalized DC-derived material from dye-labeled WT DCs and IL-2mCherry-expressing IL-2 KO DCs but not mCherry-expressing IL-2 KO DCs. Anti-CD25 blocked internalization of DC-derived material. RD6 exhibited dynamic capping of CD25 to the point of contact with IL-2 sufficient DCs, but showed no redistribution of CD25 after contact with IL-2 deficient DCs. CD25 capping preceded the release of IL-2 from the DC. The data indicate that IL-2 delivery from DC to RD6 involves membrane remodeling with spatial redistribution of CD25 to the DC contact site, raising the possibility for actin-cytoskeletol control of IL-2 delivery in Tregs.
Lymphoma resulting from β-catenin stabilization are sensitive to mTOR inhibition: a unit on cancer and the immune system (51.8)
Authors: Lo Oanh , Gounari Fotini , Dose Marei
Published: January 2023

The Wnt/β-Catenin pathway is an important cell-signaling pathway that affects the development of T-cells. If certain signals are not working properly, lymphomas may arise and drugs can be used as treatment. β-Catenin stabilization in mice results in thymic lymphomas which suggest that there are parallels to human leukemias and T-cell lymphomas. Recently, the immunosuppressive drug rapamycin has been under investigation for its potential in cancer therapy. This lab experience inspired me to create a unit that can be used in any type of high school biology, anatomy, or health class in understanding the role of the immune system in controlling cancer. First, students will learn about the anatomy of the immune system. They will gain an understanding of how cancers arise and the role of the immune system and cancer. Students will also participate in a laboratory activity that allows them to differentiate between normal and cancer cells by viewing slides under a microscope in a blood-based cancer diagnosis lab activity. The unit will conclude with an on-line research component allowing students to investigate how cancer cells are treated with immunosuppressive drugs.
CpG-induced inflammatory DCs promote strong Th1/CTL responses to particulate antigens (52.11)
Authors: De Koker Stefaan , De Geest Bruno G. , Willart Monique , Remon Jean Paul , Lambrecht Bart , Grooten Johan
Published: January 2023

Increasing safety issues are now driving the vaccine field to develop vaccines composed of recombinant antigens. Most protein antigens are however poorly immunogenic and require the addition of immunepotentiators to evoke immunity. Moreover, compared to particulate antigens, uptake and presentation of soluble antigens by DCs is far inferior. By encapsulating antigens into polyelectrolyte-based particles modified with TLR ligands, we aim to create entirely synthetic but non-pathological mimics of micro-organisms. Encapsulating OVA in these particles strongly increased antigen presentation by DCs via MHCI and MHCII. Although antigen encapsulation in these particles was sufficient to elicit immunity, the strength of the induced Th1 and CTL response was dramatically increased by functionalizing the particles with the TLR9 ligand CpG, yielding protective immunity against B16-OVA melanoma. Importantly, the particulate formulation was also far superior to a mixture of soluble antigen and CpG. Injection of CpG resulted in a strong recruitment of monocyte-derived inflammatory DCs in the draining lymph nodes, which were totally absent in CCR2-/- mice. Although immune responses to soluble antigen and CpG remained unaffected in CCR2-/- mice, the strong Th1/CTL response observed in wild type mice to particulate antigen/CpG was almost totally abrogated in the CCR2-/- mice, pinpointing to important differences in how T-cell responses are initiated to particulate and soluble antigens.
Investigation of TLR ligand-generated inflammatory signals for the induction of high magnitude memory CD8+ T lymphocyte responses (52.14)
Authors: Nelson Michelle , Johnson Alison , Milligan Gregg
Published: January 2023

Most currently used vaccines elicit antibody and CD4+ T cells, but protection against some pathogens also require a CD8+ T cell response. CD8+ T cells can be readily elicited using live attenuated vaccines or non-viable vaccine antigens in combination with adjuvants. However, little is known about how adjuvants affect the elicited CD8+ T cell response. We examined the ability of different adjuvant stimuli (TLR ligands CpG, MPL and Poly(I:C)) to elicit critical signals in bone marrow-derived dendritic cells (BMDC) necessary for appropriate development of antigen-specific CD8+ memory T cells. Mice vaccinated with MPL- or CpG-stimulated, peptide-pulsed BMDC exhibited increased CTL activity compared to those given Poly(I:C)-treated BMDC. Greater numbers of antigen-specific memory cells were detected in the spleen and iliac LN of the MPL-stimulated BMDC group. While minimal variation was observed in the expression of chemokine receptors on the surface of CD8+ T cells among the various immunization groups, there were differences observed in cytokine production. Less TNF-α was generated by CD8+ T cells from CpG-stimulated BMDC vaccinated mice. In summary, we determined that various TLR ligands differentially affected the magnitude, homing, and effector functions of memory CD8+ T lymphocytes. The results of these studies suggest that specific TLR ligand adjuvants may affect the quality of vaccine-elicited CD8+ T cells which may, in turn, impact vaccine-mediated protection.
Distinct populations of CD4+ helper T cells generate CD4 and CD8 memory responses to infection (52.23)
Authors: Weber K. Scott , Li Qi-Jing , Persuad Steve , Campbell Jeff , Davis Mark , Allen Paul
Published: January 2023

Helper T cells are critical for protective immunity, CD8 T cell memory, and CD4 recall responses required for effective vaccines. Despite the importance of CD4 T cells in immune responses, studies examining T cell responses to Listeria monocytogenes have focused primarily on CD8 T cells. We have developed two CD4 T cell transgenic mouse lines specific for an immunodominant L. monocytogenes epitope, providing a novel means of examining CD4 T cell activation and memory formation. These mice, called LLO118 and LLO56, differ by only 14 amino acids in their TCR sequences and have identical responses to peptide and L. monocytogenes infection in vitro. Comparison of the in vivo CD4 T cell responses to L. monocytogenes infection revealed LLO118 T cells proliferate much more strongly in a primary infection and cause a more robust CD8 response to secondary infection than LLO56. Surprisingly, we found that LLO56 has a superior CD4 recall response to secondary infection. CD5 expression and TCR downregulation correlate with the discordant primary and secondary responses. CD5 is higher in LLO56 prior to infection whereas LLO118 TCR levels downregulate more after the primary infection. Thus, a strong initial proliferation results in a better CD8 recall response, but a decreased CD4 recall response due to downregulated TCR levels. These findings imply that there are two populations of helper T cells: one that works best in primary responses and another in the secondary response.
CD8+ T cells adversely effect their own memory potential via the regulation of the IL-12/IL-15Rα axis (52.32)
Authors: Kohlhapp Frederick , Zloza Andrew , McCracken James , O'Sullivan Jeremy , Jagoda Michael , Lacek Andrew , Guevara-Patiño José
Published: January 2023

Current paradigms establish that CD8+ T cell memory potential is adversely affected by the degree of inflammation during an effector response. However, the regulation of this balance between inflammation and memory potential remains poorly understood. Here we demonstrate that CD8+ T cells, themselves, limit their own memory potential through direct regulation of inflammatory cytokine production by antigen presenting cells (APCs) during vaccination. By depleting CD8+ T cells during vaccination, we show augmented formation of memory precursor effector cells (MPECs), which results in the enhanced quantity (antigen-specific CD8+ T cell number) and quality (cytolytic function) of the resulting memory responses. Furthermore, we show that CD8+ T cells promote inflammation and shift the balance towards terminal effector CD8+ T cell responses by increasing IL-12 production and decreasing IL-15Rα expression by APCs in an IFN-γ-dependent manner. These results identify an unrecognized role for CD8+ T cells in the regulation of their own effector differentiation fate and a previously uncharacterized relationship between the balance of inflammation (IFN-γ, IL-12) and memory formation (IL-15Rα).
Quantitative evaluation of biomarkers to assess efficacy of immunotherapy in inflammatory diseases (52.7)
Authors: Meno-Tetang Guy , Berges Alienor , Gisbert Sophie , Austin Daren
Published: January 2023

Background and aims: Clinical endpoints of disease activity, such as the Crohn’s disease activity index or the ulcerative colitis activity index are highly subjective and variable scales. To our knowledge, no systematic analysis of the quantitative relationship between degrees of alteration of biomarkers and the likelihood of relapse and remission following drug treatment is available in the literature. The objective of this work was to identify biomarkers that are predictors of drug efficacy and that could be used to optimize combination therapies. This work focused on immunomodulatory agents that target T-lymphocytes and cytokines. Methods and outcome: Several databases were searched up to early 2011. We limited our search to randomized clinical trials lasting as least 8 weeks for induction of remission and 52 weeks for prevention of relapse. Time-course of biomarkers in serum or urine and clinical endpoints were digitized in order to retrieve data. A quantitative analysis was performed only for drug that affected biomarkers and clinical endpoints. We investigated the correlation between the level of modulation of biomarkers and clinical scores. Techniques such as principal component analysis, multiple regression, linear and nonlinear regression were applied. In this systematic review of more than 150 articles, we present information relating to the quantitative predictability of biomarkers for relapse and remission of treatment with small molecules and biologics.
Naturally occurring MHC class I restricted TCRs effectively redirect human T regulatory cells to suppress high affinity, polyfunctional CD8 T cell responses (52.8)
Authors: Riley James , Plesa Gabriela , Lingjie Zheng , Zhao Yangbing , Jakobsen Bent
Published: January 2023

Because most autoimmune diseases exist in the periphery, we investigated whether MHC class I restricted TCRs could be used to re-direct expanded human Tregs in order to better target engineered Tregs to the site of the aberrant immune response. By taking advantage of a series of HLA-A2 restricted TCRs that recognize the same peptide-MHC class I (pMHC) with varying affinities, we observed augmenting TCR affinity had no effect on the ability of Tregs to function. In fact, MHC class I restricted TCRs that had limited activity in CD4+ T effector cells (Teff) were fully able to suppress the expansion of highly functional CD8 T cells expressing a high affinity TCR. These studies demonstrate that Tregs expressing naturally occurring, introduced MHC class I restricted TCRs have potent suppressive activity and further highlight the fundamental differences that exist between effector and regulatory T cells.
Novel antigen discovery by proteome-wide screening of CD4 and CD8 T cell epitopes using P. falciparum as a model (53.14)
Authors: Doolan Denise , Douradinha Bruno , Roddick Joanne , Robinson Leanne , Taraika Jack , Mark Danga , Rarau Patricia , Mueller Ivo , Peters Bjoern , Sidney John , Sette Alessandro
Published: January 2023

For many complex pathogens, the targets of cellular immunity are largely unknown and correlates of protection after exposure or immunization are unclear. A major challenge for rational vaccine development is to identify the key antigens and epitopes targeted by protective immune responses. Using malaria as a model, we are addressing this problem using an integrated approach that incorporates bioinformatic predictions, HLA supertype considerations, high-throughput MHC peptide binding assays, and cellular assays. Antigens and epitopes can be prioritized on the basis of frequency and magnitude of immune responses, and responses to novel proteins ranked against characterized antigens already in clinical development. This strategy has allowed us to identify a number of novel immunoprevalent target antigens from P. falciparum that are recognized frequently and in the context of multiple genetic restrictions. Many of these antigens are more immunogenic than antigens identified historically, and at least one has been confirmed as a target of cross-species protection. These antigens and their epitopes represent promising candidates for next generation vaccine development. Additionally, data provide important information on the patterns of immunodominance following exposure to a complex pathogen.
Heterologous immunization induces potent anti-SIV immunity with qualitative and quantitative improvements (53.22)
Authors: Tang Xian , Chen Zhiwei
Published: January 2023

Background: The live replication-competent modified vaccinia virus Tiantan (MVTT) is an attractive vaccine vector. In this study, we constructed a recombinant MVTT expressing SIV Gag-Pol and Env (MVTT-SIV) and compared its efficacy for inducing antigen-specific immunity when administrated in different routes with or without rAd5-based vaccine encoding the same antigens (rAd5-SIV). Methods: MVTT-SIV was tested for its immunogenicity in mice administered homologously by a prime-boost strategy or heterologously in combination with rAd5-SIV. The prime routes included intranasal (IN), sublingual (SL), oral, mixed (mix of the previous three musocal routes) or intramuscular (IM) injection. All groups received IM boost. Result: Mice given heterologous boost produced significantly greater SIV specific T cell and Gag-specific antibody responses than homologous boost groups. Besides, heterologous immunization generated much higher level of neutralizing antibodies, despite of the lower titers of Env-specific binding antibodies according to Elisa assay. Moreover, the immunogenicity of MVTT-SIV given by mixed route followed by rAd5-SIV intramuscular boost was unique in terms of anti-SIV cellular and humoral immunity. Conclusion: Heterologous immunization induces potent anti-SIV Immunity with improvements in quality and quantity. The combination of MVTT-SIV mixed prime and rAd5-SIV intramuscular boost may be an effective and protective vaccination strategy in further studies.
Generation of human CD154 BAC transgenic mice to explore therapy for X-linked Hyper-IgM syndrome (53.6)
Authors: Cron Randy , Genin Anna
Published: January 2023

XHIM is a congenital immunodeficiency from inadequate CD154 expression on activated CD4 T cells. The only curative therapy is bone marrow transplantation. Recently, lentiviral gene therapy approaches for XHIM have been explored. However, beyond the CD154 transcriptional promoter, the regulatory cis-elements required for cell type-specific, activation dependent, and time-limited expression of CD154 are unclear. We generated a hCD154 BAC transgenic mouse spanning ~143 kb upstream, ~11 kb of the intact hCD154 gene, and ~20 kb of downstream DNA sequence. This BAC contains all known CD154 regulatory elements (promoter, enhancers, 3’ UTR). Confirmation of the intact BAC sequence was determined by DNA sequencing at intervals throughout the BAC. hCD154 transgene expression was detected by flow cytometry. After polyclonal activation of splenocytes, hCD154 is restricted to CD4 T cells (absent on B and CD8 T cells). Activated CD4 T cells co-express mCD154 and hCD154. Like mCD154, hCD154 expression is activation dependent but displays a slightly delayed and prolonged expression. Human CD4 T cells activated for 6 hours and irradiated to prevent division stimulate mCD40 expressing B cells (EdU incorporation), and this is inhibited by anti-hCD154 antibody. By deletion or mutation of known cis-regulatory elements in the hCD154 BAC construct, we will test the minimal requirements for optimal hCD154 expression to stimulate CD40+ cells. This will help design gene therapy vectors for XHIM.
In vivo molecular imaging for a nasal botulism vaccine in mice and nonhuman primates (53.8)
Authors: Yuki Yoshikazu , Nochi Tomonori , Harada Norihiro , Katakai Yuko , Mejima Mio , Tokuhara Daisuke , Kurokawa Shiho , Takahashi Yuko , Shibata Hiroaki , Kohda Tomoko , Kozaki Shunji , Tsukada Hideo , Kiyono Hiroshi
Published: January 2023

Nasal administration is an effective route for a needle-free vaccine. However, safety concerns were raised by the potential of nasally administered antigens to reach the central nervous system. We tested the real-time quantitative tracking of a nasal vaccine candidate for botulism, which is a recombinant nontoxic fragment of botulinus toxin A (BoHc/A), by using a newly established in vivo imaging method, [18F]-labeled BoHc/A-positron emission tomography (PET). This method provides results that are consistent with direct counting of [18F] radioactivity or the traditional [111In]-radiolabel method in dissected tissues of mice and non-human primates. In addition, no deposition of BoHc/A in the cerebrum and olfactory bulb was found after nasal administration of [18F]-BoHc/A in mice and non-human primates. We also established a real-time quantitative profile of elimination of this nasal vaccine candidate and demonstrated that it induces highly protective immunity against botulism in non-human primates. These results demonstrate a new way to use in vivo imaging for safety evaluation of nasal vaccines.
Effect of chronic ingestion of a histamine H1 receptor antagonist on the progression of atherosclerosis in the apolipoprotein-E null mouse (54.1)
Authors: Vimala Raveendran Vineesh , Tan Xiaoyu , Sweeney Matthew , Stechschulte Daniel , Dileepan Kottarapat
Published: January 2023

Histamine (HIS) is a known modulator of vascular functions and its levels are elevated in coronary arteries in ischemic heart disease and variant angina. We have shown that HIS, acting via H1 receptors (H1R), stimulates the expression of IL-6, COX2, TLR2 and TLR4 in human endothelial cells. The study tested the effect of chronic ingestion of cetirizine (CET), an H1R antagonist, on atherosclerosis progression in ApoE-/- mice. CET containing water (5 mg/L) or plain water was supplied ad libitum to male ApoE-/- mice for 3 and 5 months, after which 24h urine, blood, and aorta were collected. The lesion coverage in the descending aorta and intima/media thickness at aortic root were quantified. The aortic tissue was also analyzed for COX1, COX2, H1R, IL10, Egr1 and MCP2 mRNA expression and for macrophages (MAC3+), T lymphocytes (CD3+) and mast cells (CD117+). A significant increase in plaque thickness was noted in CET-treated mice at 3 months but not at 5 months. Aortic lesion coverage between control and CET groups remained unchanged at 3 and 5 months. PGI2 and TXA2 indices in urine at 3 months showed reduction in PGI2/TXA2 ratio. RT- PCR data showed significantly decreased expression of COX1 and H1R but no changes in other genes. The lesion coverage, urinary prostanoid levels, gene expression, serum lipids, and MAC3+, CD3+ and CD117+ cell numbers were unchanged at 5 months. The results suggest that long-term use of CET does not alter atherosclerosis progression in ApoE-/- mice.
Identification of novel small molecule inducers of endothelium-driven innate immunity (54.10)
Authors: Kibbie Jon , Whang Zhiming , Kwon Ohyun , Modlin Robert , Cruz Daniel
Published: January 2023

The endothelium plays a critical role in promoting inflammation in cardiovascular disease and other chronic inflammatory conditions. Conversely, an augmented immune response can be protective against microbial pathogens and in cancer immunotherapy. Many small molecule screens have sought to identify agents that prevent endothelial cell activation. Yet, small molecule screens to identify agents that induce endothelial cell activation have not been reported. In this regard, a bioassay was developed which identifies activated endothelium by its capacity to trigger MIP1 beta from primary monocytes. Subsequently, a 642 compound library of 39 distinctive scaffolds generated by diversity oriented synthesis was screened for small molecules that activated the endothelium. From the identified inflammatory compounds, 96 analogs of one particular class of compounds were efficiently prepared by solid phase split-and-pool synthesis. Structure-function analysis combined with transcriptional profiling of active and inactive analogues identified inflammatory gene networks induced exclusively by the active compound. The identification of a family of chemical probes that augment innate immunity through endothelial cell activation provides a framework for understanding gene networks involved in endothelial inflammation as well as the development of novel endothelium-driven immunotherapeutic agents.
Liver sinusoidal endothelial cells undergo apoptosis during sepsis, leading to organ dysfunction. (54.13)
Authors: Hutchins Noelle , Borgerding Joshua , Chung Chun-Shiang , Ayala Alfred
Published: January 2023

Sepsis is the leading cause of death in critically ill patients, and treatment options are limited. Our laboratory studies polymicrobial sepsis in murine models, and uses the liver as a model organ to investigate the disease’s pathophysiology. The liver, characterized as an immune tolerant organ, is susceptible to septic induced inflammation. We have previously reported that there is a twofold increase in the number of liver CD8+ T cells, 24 hours following cecal ligation and puncture (CLP—a surgical procedure that generates sepsis in mice). Since activated T lymphocytes must transverse the endothelium to reach the site of infection, it is possible that they directly interact with LSECs, and mediate liver injury through Fas-mediated apoptosis. This led us to hypothesize that as CD8+ T cells directly interact with LSECs, endothelial cell dysfunction occurs. Our preliminary results indicate that septic liver CD8+ T cells have cytotoxic effects on endothelial monolayer integrity, and release pro-inflammatory cytokines when directly co-cultured with CRL-2167, a murine endothelial cell line. We have also observed through flow cytometry that LSECs (CD146+/CD45- and CD31+/CD45- populations) have an increased expression of CD54 (ICAM-1), Fas, and Annexin V staining in vivo. These data suggest that CD8+ T cells directly interact with LSECs, alter the tolerance capacity of LSECs, and potentially kill LSECs through the Fas-FasL system, ultimately leading to liver organ injury in sepsis.
CD11b+/Gr-1int monocytic myeloid derived suppressor cells contribute to high-fat induced inflammation and delayed tolerance in mouse liver (54.15)
Authors: Xie Qifa , Zhang Jingwen , Ghare Smita , Barve Shirish , McClain Craig
Published: January 2023

Myeloid derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells with universal expansion in nearly all inflammatory conditions. This study investigated the accumulation of MDSC in chronic inflammation and tolerance of obesity-related fatty liver disease. We found that the expansion of MDSC, specifically associated with the monocytic MDSC (M-MDSC): CD11b+Gr-1intLy6G-Ly6Chigh in the liver of B6 mice fed a high-fat diet contributes to liver inflammation and tolerance. M-MDSCs isolated from the liver of obese mice are more easily activated by way of Toll-like receptor (TLR4) stimulation resulting in inflammatory cytokine expression, and were functional MDSCs, readily inhibiting T cell proliferation in vitro dependent on cell-cell contact between MDSCs and T cells. The rapid accumulation of M-MDSC also contributed to delayed ConA tolerance in obesity-related fatty liver disease. M-MDSCs isolated from the liver of obese mice developed with ConA tolerance induce less liver regulatory T (Treg) cell expansion and less suppressing T cell proliferation in vitro. Experiments using mice depleted of Gr-1, or M- MDSC adoptive transfer demonstrated the important role of M-MDSC in liver inflammation and tolerance. These data unveil that M-MDSCs play an essential negative feedback function in liver immune homeostasis.
Role for NADPH oxidase in macrophage clearance of apoptotic cells (54.2)
Authors: Zeng Melody , Dinauer Mary
Published: January 2023

Ingestion of apoptotic neutrophils by macrophages (efferocytosis) is important for both neutrophil (PMN) homeostasis and resolution of tissue inflammation following infection or injury. In this study, we demonstrated that apoptotic PMN triggered activation of the NADPH oxidase in engulfing mouse macrophages, leading to oxidant production in phagosomes containing PMNs (also called efferosomes). Consistently, immunofluorescent staining revealed accumulation of gp91phox, a membrane-associated subunit of the oxidase, on the efferosome membrane. Examination of MyD88-/- macrophages illustrated a partial requirement for MyD88 in apoptotic PMN-mediated activation of the oxidase. To delineate the role of the NADPH oxidase in degradation of PMNs by macrophages, we observed that in macrophages from gp91 phox -/- mice, which do not generate oxidants during efferocytosis, lysosome-efferosome fusion occurred at a slower rate, thus implicating the importance of oxidants in efferosome maturation. Degradation of ingested PMNs within macrophages was further monitored by staining PMN myeloperoxidase (MPO), which was much more prominent in gp91phox-/- macrophages 6 hours after efferocytosis compared to WT macrophages, suggesting slower degradation in the absence of oxidants. Taken together, our data support that, in addition to its well-characterized anti-microbial function, macrophage NADPH oxidase may be an integral player in PMN clearance to facilitate the resolution of inflammation.
Recovery of wound healing in immunosuppressed mice by L-Arginine topical treatment (54.6)
Authors: Jeronimo Marcio , Nóbrega Yanna , Souza Ana Camila , Siqueira Isaque , Souza Nathalia , Amorim Rivadávio , Cavalcanti Neto Florencio , Borim Maria , Bocca Anamélia
Published: January 2023

The wound healing is a coordinated mechanism involving soluble and insoluble mediators and immune response activation. Some pathological conditions are related with this mechanism, like immunosuppression, which interferes with important mechanisms of tissue repair like healing phase. This study aims to evaluate the capacity of immunosuppressed mice to wound healing after L-arginine supplementation. Swiss mice received 25 mg/kg of hydrocortisone for two consecutive days intraperitoneally. To evaluate the wound healing, a lesion was made with a 5 mm diameter biopsy punch. The animals received daily topical 10% L-arginine in glycerin and were killed 7 days post lesion (dpl). Animals treated only with the vehicle were used as control. Fragments of the healing process area were collected for immunohistochemistry analysis for CD31, TGF-b and iNOS. It was also performed a qRT-PCR analysis of the healing tissue for IL-1α, TNF-α and CCR5 genes. We found an increased expression of CD31 molecule in the animals treated with L-arginine and decreased of iNOS expression compared with the control group. The transcriptions of CCR-5, IL-1 and TNF-α in treated animals were increased. These results indicate enhanced neovascularization and cell migration, promoting the inflammatory phase recoverment and improving the repair in the group receiving the treatment. Our work demonstrates the modulation by L-arginine in the wound healing process; it can be a new approach as an adjuvant treatments.
TNFRSF25 mediated in vivo expansion of regulatory T-cells reduces immunopathology in a chronic model of allergic lung inflammation (55.4)
Authors: Tsai Matthew , Schreiber Taylor , Wolfe Dietlinde , Podack Eckhard
Published: January 2023

TNF receptor super family member 25 (TNFR25, DR3) signaling by agonistic antibody (4C12) has been shown to effectively expand CD4+FoxP3+ regulatory T-cells (Treg) in vivo, and prevent acute allergic lung inflammation, a model for asthma. Here we investigate if Treg expansion by TNFR25 stimulation can reduce lung immunopathology associated with chronic allergic lung inflammation. After antigen priming (Ovalbumin/ALUM) and three weeks of antigen aerosol exposure (Ovalbumin in Saline), TNFR25 agonistic antibody given during an aerosol-free period is able to expand Tregs in the peripheral blood and in the lungs as previously reported. After two additional weeks of antigen aerosol exposure, and at a time when TNFR25-expanded Tregs have almost contracted to baseline levels, 4C12 treated animals remained protected from chronic lung immunopathology. Specifically, TNFR25 agonistic antibody treated mice, as compared to IgG treated controls, showed reduced eosinophils in the bronchoalveolar lavage fluid, reduced expression of Th2 cytokines (IL-4, IL-5, IL-13), CCL11 (eotaxin-1), and TNF-a, and reduced mucus production in the bronchial epithelium. These results indicate that TNFR25 mediated expansion of Tregs after established pulmonary inflammation, is able to generate a sustained suppression of chronic lung immunopathology, which may be mediated by antigen-specific Tregs.
Innate imprinting of resident alveolar macrophages by a preceding allergic bronchial inflammation increases the inflammatory TLR-reactivity through an enhanced MyD88-independent signaling and the type I interferon, IFN-β. (55.5)
Authors: Naessens Thomas , Vander Beken Seppe , De Koker Stefaan , Lienenklaus Stefan , Van Rooijen Nico , Grooten Johan
Published: January 2023

Resident alveolar macrophages (rAM) play an important regulatory role in limiting excessive inflammatory responses in the respiratory tract. In a mouse model of allergic asthma we identified a new autofluohigh CD11c+ CD11b+ DEC205+ F4/80+ rAM population residing in the airways following the clearance of a prior allergic bronchial inflammation. Compared to naïve rAM, post-inflammation rAM feature a reduced basal phagocytosis along with an increased in vitro inflammatory response to LPS. Especially genes dependent for expression on the MyD88-independent signaling pathway and the type I interferon, IFN-β, showed strongly increased expression levels compared to the naïve rAM. A preferential enhancement of MyD88-independent signaling in post-inflammation rAM was also observed after in vivo LPS-challenge and resulted in a differential response pattern to TLR-3 and TLR-7 ligands. Using CD45 chimeric mice and intratracheal instilled fluorescent microparticles to distinguish resident from elicited AM, we observed a rapid and near complete disappearance of naïve rAM during allergen challenges. Furthermore, we showed that this inflammation-induced rAM turnover is a crucial event in the development of the post-inflammation rAM phenotype. This novel mechanism of innate imprinting may contribute to the increased sensitivity of the post-inflammation lung to inflammatory insults.
New mechanism of mucosal immunity in oral candidiasis: insights from Hyper-IgE (STAT3-deficient) patients (56.1)
Authors: Gaffen Sarah , Conti Heather , Freeman Alexandra , Baker Olga , Li Rui , Jang Woong , Holland Steven , Edgerton Mira
Published: January 2023

Oropharyngeal candidiasis (OPC, thrush) is an opportunistic infection caused by the commensal fungus Candida albicans. OPC is an AIDS-defining illness, and is also common in patients on chemotherapy and those with congenital immunodeficiencies. An understanding of immunity to Candida has recently begun to unfold with the identification of fungal pattern recognition receptors such as dectins, which trigger protective Th17 responses in the mucosa. Hyper-IgE syndrome (HIES/Job’s Syndrome) is a rare congenital immunodeficiency characterized by dominant-negative mutations in STAT-3. STAT-3 is downstream of the Th17-inductive cytokines IL-6 and IL-23, and hence HIES patients exhibit dramatic Th17 defects. Striingly, HIES patients develop oral and mucocutaneous candidiasis, supporting a protective role for Th17 cells. However, the Th17-dependent mechanisms of immunity in OPC are poorly defined. An often-unappreciated aspect of oral immunity is saliva, which is rich in anti-microbial proteins (AMPs) and exerts direct antifungal activity. We found that both Th17-deficient mice and HIES patients show significant impairment in salivary candidacidal activity. In HIES, this was linked to reduction in AMPs, such as β-defensin 2 and Histatins. Moreover, IL-17 induces histatins in cultured salivary gland cells. This is the first demonstration that the Th17 axis of immunity is associated with salivary activity, and provides a mechanism for the severe susceptibility of HIES patients to OPC.
Altered activation of neonatal alveolar macrophages affects the immune response to Pneumocystis. (56.10)
Authors: Kurkjian Cathryn , Hollifield Melissa , Murphy Brian , Garvy Beth
Published: January 2023

Postnatal lung development renders a different lung environment and composition in neonates as compared to adults, making the neonatal immune response to lung pathogens, such as Pneumocystis (PC), less efficient than adults. Alveolar macrophages (AMs) are the major effector cells in the immune response to PC, and a proper AM activation phenotype is imperative for clearance of infection. Following peak infection, alternative markers (arginase, mannose receptor and CD23) are increased on adult AMs, suggesting a role for this phenotype in PC resolution. A more robust increase in these alternative markers is observed in neonates following peak infection. Additionally, naïve neonatal AMs constitutively express FIZZ1 and Ym1 at levels comparable to infected counterparts. It is possible that adult AMs develop a classical phenotype in the initial response to PC, and that this phenotype cannot be obtained by neonatal AMs early in infection due to increased expression of alternative markers. Additionally, increases in these alternative markers on neonatal AMs late in infection may suppress an efficient response to PC, thus resulting in delayed clearance as compared to adults. We hypothesize that delayed AM activation and increased expression of alternative markers during resolution results in an insufficient immune response to PC infection in neonates.
Mechanisms of Dectin-1 dependent IL-17A production during invasive fungal infection. (56.14)
Authors: Werner Jessica , Gessner Melissa , Lilly Lauren , Nelson Michael , Metz Allison , Horn Dawn , Deshane Jessy , Chaplin David , Brown Gordon , Weaver Casey , Steele Chad
Published: January 2023

In the current work, we show that mice deficient in the beta-glucan receptor Dectin-1 demonstrate significantly reduced levels of IL-17A in the lungs 48 h after Aspergillus fumigatus (AF) challenge. Culturing cells from enzymatic lung digests ex vivo further demonstrated Dectin-1 dependent IL-17A production, providing a sensitive method to identify mechanisms associated with lung IL-17A production after AF challenge. Neutralization of IL-23, but not IL-1β or IL-6, in WT lung digest cell cultures reduced IL-17A production by 60%. Lung digest cells from AF-challenged Rorc-/- deficient mice had attenuated IL-17A production, however, Rorc and Rora mRNA, and RORγt protein, expression was not impaired in lung digest cells from Dectin-1 KO mice. Ahr, Irf4 and Card9 mRNA expression was also unchanged. Intracellular cytokine staining revealed that CD11b+ and CD11b+ Ly-6G+ cells were sources of IL-17A, although only CD11b+ Ly-6G+ cells produced IL-17A in a Dectin-1 dependent manner. In turn, Ly-6G+ cells purified from the lungs of AF-challenged Dectin-1 KO mice displayed a 2-fold reduction in Il17a mRNA expression, yet increased Rorc and Rora mRNA expression. IL-23R+ cells in lung digest were predominantly CD11b+ Ly-6G+ cells. In summary, Dectin-1 dependent innate IL-17A production in the lungs during invasive fungal infection is mediated in part by CD11b+ Ly-6G+ neutrophils, which is partially dependent on IL-23. Dectin-1 deficiency does not, however, compromise induction of Rorc.
Differential intracellular trafficking of Francisella tularensis strains in human dendritic cells. (56.15)
Authors: El-Etr Sahar , Vergez Lisa , Rasley Amy
Published: January 2023

F. tularensis, the causative agent of tularemia, is an intracellular pathogen that replicates in macrophages and dendritic cells (DCs), where the bacteria escape the endosomal pathway and replicate in the cytoplasm. DCs act as sentries for the immune system and are often the first cells to engulf invading pathogens and trigger the activation of T-cells. F. tularensis inactivates DCs upon entry, but the mechanism of inactivation is not known. We hypothesize that F. tularensis inactivates DCs by disrupting intracellular trafficking and antigen presentation. Using immunofluorescence microscopy, we have performed a detailed characterization of intracellular trafficking of the F. tularensis laboratory strains Schu S4, LVS and novicida and two clinical type A strains in hDCs. Our data indicates that F. tularensis strains colocalize with EEA-1, LAMP-1 and Rab3 endosomal markers as early as 15 min post infection. In addition, we observe a significantly lower number of pathogenic F. tularensis colocalizing with the above markers than the nonpathogenic F. tularensis LVS and novicida strains. Very few bacteria colocalize with the late lysosomal marker Cathepsin D, and we do not observe differences among strains. These data indicate that pathogenic F. tularensis escape the endosomal pathway in hDCs much more rapidly and in greater numbers than nonpathogenic strains, suggesting that pathogenic F. tularensis actively disrupt intracellular trafficking and subsequent antigen presentation.
TCA cycle inactivation in Staphylococcus aureus alters nitric oxide production in RAW 264.7 cells (56.21)
Authors: Massilamany Chandirasegaran , Gangaplara Arunakumar , Gardner Donald , Steffen David , Somerville Greg , Reddy Jay
Published: January 2023

Inactivation of the Staphylococcus aureus tricarboxylic acid (TCA) cycle delays the resolution of cutaneous ulcers in a mouse soft tissue infection model. In the present study, we observed that cutaneous lesions in mice infected with wild-type or isogenic aconitase mutant S. aureus strains contained comparable inflammatory infiltrates, suggesting the delayed resolution was independent of the recruitment of immune cells. These observations led us to hypothesize that staphylococcal metabolism can modulate the host immune response. Using an in vitro model system involving RAW 264.7 cells, we observed that cells cultured with S. aureus aconitase mutant strains produced significantly lower amounts of nitric oxide (NO) and an inducible nitric oxide synthase as compared to those cells exposed to wild-type bacteria. Despite the decrease in NO synthesis, the expression of antigen-presentation and costimulatory molecules was similar in cells cultured with wild-type and those cultured with aconitase mutant bacteria. The data suggest that staphylococci can evade innate immune responses and potentially enhance their ability to survive in infected hosts by altering their metabolism. This may also explain the occurrence of TCA cycle mutants in clinical S. aureus isolates.
ESAT-6 from Mycobacterium tuberculosis inhibits T cell IFN-γ production through p38 MAP kinase pathway (56.28)
Authors: Samten Buka , Peng Hui , Wang Xisheng , Barnes Peter
Published: January 2023

We reported previously that ESAT-6 from Mycobacterium tuberculosis directly inhibits human T cell IFN-γ production and proliferation in response to stimulation with anti-CD3 plus anti-CD28. To determine the mechanism of this effect, we treated T-cells with kinase inhibitors before stimulation in the presence of ESAT-6. Only the p38 MAPK inhibitor, SB203580, abrogated ESAT-6-mediated inhibition of IFN-γ production in a dose-dependent manner. In contrast, SB203580 did not reverse ESAT-6-mediated inhibition of IL-17 and IL-10 production, suggesting a specific effect of SB203580 on IFN-γ production. SB203580 did not act through inhibition of AKT, as an AKT inhibitor did not affect ESAT-6 inhibition of T cell IFN-γ production and proliferation, ruling out the possible off target effect of SB203580 through AKT. ESAT-6 did not expand FoxP3+ T regulatory cells, suggesting that Tregs do not play a role in ESAT-6 inhibition of T cell IFN-γ production. Incubation of T cells with ESAT-6 induced phosphorylation and increased functional p38 MAPK activity, but not activation of ERK or JNK. Silencing of p38α MAPK with siRNA rendered T-cells resistant to ESAT-6 inhibition of IFN-γ production. Taken together, our results demonstrated that ESAT-6 inhibits T cell IFN-γ production in a p38 MAPK-dependent manner.
ESAT-6 inhibits IL-12, but promotes IL-23 production of human dendritic cells (56.9)
Authors: Wang Xisheng , Barnes Peter , Samten Buka
Published: January 2023

Early secreted antigenic target of 6 kD (ESAT-6) is a M. tuberculosis protein that induces protective immunity against tuberculosis in animal models and is a putative vaccine candidate. However, this protein also contributes to virulence and inhibits immune cells activation. In this study, we evaluated the effects of ESAT-6 on the function of human dendritic cells. Immature dendritic cells (DC) were induced from monocytes cultured with GM-CSF plus IL-4. ESAT-6 inhibited IL-12, but promotes IL-23 production of DC during maturation stimulated by LPS plus CD40L. This is correlated with downregulated IL-12p35 and upregulated IL-23p19 transcripts, respectively. ESAT-6 did not affect the induction of TNF-α, IL-6 and IL-8 from DC during maturation. ESAT-6 also inhibited upregulation of co-stimulatory molecules, CD80, CD86, HLA-DR, in both monocyte derived DC and myeloid DC freshly purified from human blood, in response to stimulation of LPS plus CD40L. DC pretreated with ESAT-6 induced less IL-2 and IFN-γ production in mixed lymphocyte reactions, and stimulated less IFN-γ, but more IL-17 production from M.tuberculosis responsive T cells compared to nontreated DC. Blocking TLR1/2 on DC or removing 20 amino acids in C-terminus did not ablate the inhibition of ESAT-6 on IL-12 production. We conclude that ESAT-6 enhances IL-23 production at the expense of IL-12 in a TLR2 independent mechanism, which favors Th17 over Th1 activation. (Supported by the NIH grants AI082335 and AI063514)
Bim is a component of the endosomal-lysosomal interface in Interleukin-7 dependent T cells (57.10)
Authors: Khaled Annette , Ruppert Shannon , Carlson Adina
Published: January 2023

Bim is a key protein in T cell homeostasis. Bim deficiency prevents activated T cell death, and protects splenic T cells in IL-7R/Bim double knockout mice through an undetermined mechanism. We propose a novel function for Bim in T cells involving lysosomal development in both apoptotic and non-apoptotic cells. Using IL-7 dependent T cell lines, Bim-containing and Bim-deficient (Bim-/-), we determined that the transcription of bim increased in the absence of IL-7 and the loss of Bim protected cells from apoptosis induced by IL-7 withdrawal. Treatment of Bim-containing cells with Bim siRNA confirmed these results. Bim-/- cells had increased endosomal and decreased lysosomal proteins, suggesting that Bim plays a role in endosomal to lysosomal trafficking and maturation. Subcellular organelle fractionation revealed that the BimL isoform associated with both endosomal and lysosomal fractions in non-apoptotic cells. However, during IL-7 withdrawal, the BimEL isoform associated with lysosomal fractions and less so with endosomal fractions. In fact, reduced co-localization of endosomes and lysosomes in the absence of Bim was observed by confocal microscopy, suggesting a defect in an endocytic compartment. Overexpression of the individual Bim isoforms restored acidic organelle distribution with BimL contributing the most in non-apoptotic cells. These results demonstrate that Bim has both apoptotic and non-apoptotic activities in T cells that affects lysosomal equilibrium.
Regulation of STAT-6 activation in ovalbumin stimulated murine splenocytes: mechanisms regulating biphasic induction (57.13)
Authors: Lee Soojin , Shan Jichuan , Fixman Elizabeth
Published: January 2023

T cell receptor (TCR) stimulation and IL-4 receptor signaling mediate STAT-6 activation and differentiation of naïve T cells into T helper type 2 (Th2) cells. However, the relationship between TCR stimulation and IL-4-induced STAT-6 activation is poorly understood. STAT-6 is tyrosine phosphorylated (pSTAT-6) in response to IL-4 as well as IL-13, and it also regulates gene expression of these Th2 cytokines. Our data demonstrate that pSTAT-6 is induced in a biphasic manner in splenocytes harvested from OVA-sensitized BALB/c mice and restimulated in vitro with OVA. pSTAT-6 was induced within 2-4h, lost at 12-24h and re-induced at 48-96h. IL-4, but not IL-13, followed similar biphasic kinetics. Inhibition of either IL-4 or the IL-4 receptor selectively during the first phase had no effect on secondary pSTAT-6 and production of IL-4 and IL-13. In addition, while biphasic pSTAT-6 was induced in splenocytes treated simultaneously with OVA and exogenous IL-4, stimulation of splenocytes with IL-4 alone did not induce secondary IL-4 production and pSTAT-6. These data demonstrate that the presence of IL-4 at early time points is neither required nor sufficient for IL-4 production and pSTAT-6 during the second phase. IL-4 production and pSTAT-6 during the second induction phase, on the other hand, are dependent upon OVA-induced TCR signaling. Studies are underway to define the cellular sources of IL-4 and pSTAT-6 as well as the cells required for each phase of the induction.
Targeted-glycosylation of "non-core region" of interleukin-11 modulates its biological function. (57.15)
Authors: Yanaka-Okada Saeko , Sano Emiko , Naruse Norio , Miura Kin-ichiro , Futatsumori-Sugai Mutsumi , Caaveiro Jose , Tsumoto Kouhei
Published: January 2023

Human interleukin-11 (hIL-11) is a pleiotropic cytokine administered to patients for recovering low platelet counts. From a structural point of view hIL-11 belongs to the long-helix cytokine superfamily, which is characterized by a conserved core motif consisting of four alpha-helices. We have investigated the region of hIL-11 that does not belong to the alpha-helical bundle motif, which we have termed “non-core region”. The interleukin was N- or O-glycosylated at specific positions within the non-core region, and the functional consequences of these modifications were examined in cell-based as well as biophysical assays. The data indicated that the non-core region modulates the function of hIL-11 in two ways. First, most of the muteins displayed enhanced cell-stimulatory properties (super-agonist behavior) in a glycosylation-dependent manner, suggesting that the non-core region is biologically designed to limit the cell-proliferative potential of hIL-11. Second, glycosylation of a predicted mini alpha-helix led to cytokine inactivation, demonstrating that this structural element interacts with a second gp130 receptor. These findings have unveiled new and unexpected elements modulating the biological activity of hIL-11, which may be exploited to develop more versatile medications based on this important cytokine. Yanaka S et al. J. Biol. Chem. in press. (2011)