bluuebunny/crossref_metadata_2025 huggingface dataset:
Suppressor T cell exosomes via miR-150* and antigen specific Ig light chains. (50.13)
Authors: Askenase Philip
Published: January 2023

Tolerance in contact sensitivity (CS) is due to CD8+ suppressor T cell (Ts) released factor (TsF) that is phenol chloroform extractable (PCE), in RNA fraction of Qiagen DNA/RNA separation column, sensitive to RNase A, not DNase nor trypsin, and also sensitive to RNase III; a property of double stranded RNA (dsRNA) like miRNA. Sizing electrophoresis indicated the active inhibitory RNA was about 75bp; the size of pre-miRNA. TsF RNA also inhibited in vitro IL-2 responses of T cell lines. We concluded that the small suppressive dsRNA was in the miRNA family TsF RNA is transported systematically in vivo in exosomes in the blood plasma of Ts donors. Remarkably, the exosomes act antigen (Ag) specifically due to surface coat of Ag-specific immunoglobulin free light chains, likely derived from activated B-1 B cells, and absorbed to receptors on the exosome surface. Ag specificity of the exosomes that eluted from the Ag column provided enriched exosome RNA for cDNA cloning and deep sequencing to identify candidate miRNA-150*. We postulate that the suppressive Ag-specific exosomes act systemically in an endocrine manner, via the blood, to target Ag specific effector T cells at distant sites, and inhibit their function via transfer of the inhibitory miRNA. This is the first demonstration of systemic immunoregulation by exosomes delivering inhibitory miRNA Ag specifically from donor suppressor T cells to distant target effector T cells in an endocrine manner.
Bariatric surgery regulates tissue specific inflammation in high fat diet-induced obese mice (50.14)
Authors: Yin Dengping , Gao Qiang , Ma Lian Li , Yan Wenwei , Williams Phillip , Wasserman David , Abumrad Naji
Published: January 2023

Visceral fat deposition induces chronic low grade inflammatory reactions and insulin resistance. However, surgical removal of visceral fat fails to reverse metabolic disease, suggesting that intrahepatic fat, not visceral fat, may be linked with metabolic complications of obesity. Using gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB) mouse models, we examined whether the effects of bariatric surgery in the treatment of obesity and insulin resistance were associated with the regulation of inflammation in visceral adipose tissue (VAT) and liver in DIO mice. SG produced weight loss and improved glucose tolerance in the short term, and RYGB, but not SG, resulted in sustained prevention of weight gain and impaired glucose tolerance and decrease in phosphorylation of IRS-1/Ser307 in liver and muscle. RYGB, SG and caloric restriction inhibited NF-kappaB activation in VAT and muscle. RYGB and SG decreased circulating insulin, inhibited proinflammatory cytokine transcription in VAT and reduced CD11b macrophages in liver and in VAT. However, RYGB, but not SG, inhibited CD11c+ cells (-53%) and promoted CD206+ cells (+79%) in liver but not in VAT. RYGB and SG inhibited CD4+/IFNgamma+ subset (-50% and -44%) in DIO VAT, and only RYGB enhanced CD4+/IL-10+ subset (+100%) in DIO liver. We conclude that the improvements in insulin resistance associated with RYGB and SG appear to be modulated by the inhibition of inflammation in a tissue specific manner.
TLR4-activated B cells out-compete TLR9-activated B cells to establish peripheral tolerance (50.16)
Authors: Matheu Melanie , Su Yan , Greenberg Milton , Parker Ian , Scott David , Cahalan Michael
Published: January 2023

Antigen presentation by B cells can generate either long-lived antigen-specific CD4+ T cell tolerance or activate T cells for additional B cell help during the humoral response. In our model, both naïve and LPS-activated B cells mOVA B cells expressing ovalbumin induced long-lived peripheral T cell tolerance, suppressing OTII T cell proliferation and T-cell-dependent antibody production in vivo. In contrast, CpG-activated mOVA B cells did not induce tolerance to OVA. Using two-photon microscopy we compared T-B cell interactions in the lymph node during tolerance induction or priming of OTII T cells mOVA B cells. Naïve, LPS- or CpG-activated mOVA B cells were individually capable of establishing long-lived interactions with OTII-T cells. Disruption of CTLA-4 / B7 interactions prevented establishment of long-lived T-B cognates and inhibited tolerance induced by LPS-treated B cells in vivo. In competition experiments with co-transferred LPS- and CpG-activated B cells, LPS- activated B cells dominated cell-cell interactions and were capable of establishing tolerance. In addition, tolerogenic LPS-activated B cells were capable of physically disrupting pre-established CpG-activated mOVA T-B pairs, exhibiting directional motility towards pre-established CpG-OTII pairs prior to disruption. Our findings demonstrate that LPS-activated B cells can act early in an immune response to establish tolerogenic dominance in the presence of priming CpG-activated B cells.
GRAIL targets CDC37 to maintain CD4 T cell unresponsiveness (50.9)
Authors: Whiting Chan , Su Leon , Lin Jack , Lineberry Neil , Fathman C.
Published: January 2023

It has long been appreciated that CD4 T cells are held in G1/S cell cycle arrest. How this is initially achieved, and then lost upon activation, is not well understood. Recent studies from our lab have demonstrated that the E3 ligase, GRAIL (gene related to anergy in lymphocytes, also known as RNF128), is expressed only in late stage QA2 positive thymocytes upon their exit from the thymus, and that its expression holds CD4 T cells in cell cycle arrest. GRAIL is not only expressed in late stage thymic migrants, but in resting CD4 T cells as well as in anergic CD4 T cells suggesting that GRAIL might be involved in holding CD4 T cells in cell cycle arrest. Using a novel E3 substrate screen, we identified cdc37 (a G1-specific cyclin) as a target of GRAIL mediated ubiquitination. Consistent with this idea, GRAIL expression leads to diminished cdc37 expression and maintained cell cycle arrest upon activation. Inversely, primary CD4 T cells lacking GRAIL have higher levels of cdc37 compared to wildtype T cells in resting conditions and allow full activation with TCR stimulation in the absence of costimulation. These data suggest that GRAIL ubiquinates and targets cdc37 for degradation and thus, maintains CD4 T cells in G1/S cell cycle arrest. These data also highlight a role for cdc37 in GRAIL-mediated cell cycle regulation and CD4 T cell proliferation. The activated CD4 T cells reacquire GRAIL expression and return to the resting G1/S interphase by unknown mechanisms.
Innate immune agonists conjugated to nanolipoproteins elicit robust inflammatory responses in mouse macrophages: implications for host-based therapeutics. (52.10)
Authors: Rasley Amy , Blanchette Craig , Fischer Nicholas , El-Etr Sahar , Loots Gabriela , Corzett Michele , Thomas Cindy , Urbin Salustra
Published: January 2023

Modulation of the innate immune system has the potential to provide transient, non-specific protection from a variety of infectious organisms. We hypothesize that combinatorial formulations of synthetic innate immune agonists may significantly enhance protection over single-agonist formulations. To readily incorporate multiple, chemically diverse agonists onto a single particulate platform for co-localized delivery, nanolipoprotein particles (NLPs) will be used. NLPs are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins, analogous to reconstituted HDLs. NLPs assembled with human apolipoproteins have been used for numerous biotechnology applications, including membrane protein solubilization, drug delivery, and diagnostic imaging. NLPs can be functionalized to incorporate myriad agonists, including CpG-containing oligonucleotides, monophosphoryl Lipid A, muramyl dipeptide, flagellin, and an LL-37 analog. Development of NLPs incorporating CpGs and MPLA will be described, as well as preliminary results from in vitro analyses, including quantitative inflammatory cytokine secretion, cellular cytotoxicity, and cellular localization. Our data indicate that agonist:NLP constructs are internalized by macrophages, are immunostimulatory, and induce low levels of cellular cytotoxicity.
The lipid raft hinders antibodies from binding to the external segment of the membrane-anchor peptide of mIgA (52.19)
Authors: Hung Alfur , Chen Jiun-Bo , Lu Chien-Sheng , Chen Nien-Yi , Yu Hui-Ming , Chang Tse Wen
Published: January 2023

The mIgA is associated with Igα/Igβ as the B cell receptor (BCR) complex on mIgA-expressing B cells. The α chain of mIgA (mα), comparing to the α chain of secreted IgA, contains an extra C-terminal membrane-anchor peptide, which encompasses an extracellular, a transmembrane, and an intracellular segments. The extracellular segment, referred to as mIg isotype-specific (migis-α) segment or extracellular membrane proximal domain (EMPD), has been proposed as a specific antigenic site for isotype-specific targeting of mIgA-expressing B cells by antibodies. In this study, we developed several anti-migis-α mAbs, such as MAb 29C11, which were specific to a peptide segment toward the N-terminal of migis-α. The mAbs bound strongly to synthetic peptides of migis-α and various recombinant proteins containing migis-α in ELISA. However, the mAbs did not bind to mIgA on B cell lines strongly in fluorescence flow cytometric analysis. After disrupting the lipid raft integrity of the B cells by a typical cholesterol extraction procedure, the mAbs could then bind to the treated B cells strongly. Immunopricipitation analysis with the mAbs indicated only when the B cell lines were solubilized by sufficiently strong detergents, such as sodium dodecyl sulfate (SDS), the mIgA could be pulled down by the mAbs. These results suggest that the migis-α region of mIgA in the BCR is associated with the lipid raft. Future studies will investigate inhibitory effects of mAb 29C11 on mIgA-expressing B cells.
Amplification of long-lived polyfunctional memory CD8 T cells by prime boost vaccination is dependent on high precursor frequencies. (52.29)
Authors: Fraser Kathryn , Schenkel Jason , Vezys Vaiva , Masopust David
Published: January 2023

Memory CD8 T cell quantity and quality are related to their capacity to protect against infection. We have shown that heterologous prime boost vaccination results in abundant effector memory CD8 T cells due to minimized contraction of tertiary effector cells. Critical questions include, 1) why are tertiary effector CD8 T cells so efficiently recruited into the memory pool, and 2) does repeated boosting result in functionally exhausted memory cells? We demonstrate that enhanced recruitment into the tertiary memory pool is dependent upon high antigen-specific precursor frequencies prior to boosting and correlates with retention of CD127 and Bcl2 during clonal expansion and a more modest number of cell divisions. Despite enrichment of an effector memory phenotype at the population level, resulting tertiary memory cells were highly functional, as measured by cytokine secretion potential, antigen sensitivity, and proliferative potential. However, reducing the precursor frequency prior to boosting resulted in effectors that undergo a greater number of divisions and experience large changes in global gene expression patterns, reduced expression of CD127 and Bcl2, reduced probability of recruitment into the memory pool, and reduced functionality of resulting memory. These results give insight into the parameters that regulate memory CD8 T cell development and define conditions under which immense quantity can be achieved while either preserving or diminishing protective potential.
Innate immune responses to açaí polysaccharides (52.31)
Authors: Holderness Jeff , Freedman Brett , Schepetkin Igor , Kemoli Sharon , Hedges Jodi , Jutila Mark
Published: January 2023

The açaí fruit has become a popular nutritional supplement with anecdotal claims of increasing immunity. Previous works by others show that the procyanidin fraction of this supplement induces immune activation. We expand upon this work to identify an endotoxin-free, purified polysaccharide fraction in Açaí fruit that can also stimulate immune responses. Treatment with Açaí polysaccharides results in activation of the innate immune system, particularly γδ T cell and monocyte cell activation. Responses include: TNFα, IL-6, and IL-10 cytokine production, γδ T cell activation (CD69/IL-2Rα expression), ROS production, as well as myeloid cell recruitment/activation and IL-12 secretion in the lung after i.t. delivery. This response to Açaí polysaccharide is at least partially mediated by the polysaccharide receptor, Dectin-1. Unlike fungal polysaccharide recognition, which typically utilizes TLR2 as a co-receptor, our studies suggest that the lung immune response to Açaí polysaccharides is TLR2-independent and instead may require TLR4.
Targeting of peptide coupled dendrimers to human dendritic cells leads to autologous T cell activation (53.15)
Authors: Gert Philipp , Assenmacher Mario , Jähn Peter
Published: January 2023

Targeted delivery of vaccines to dendritic cells (DC) has been shown to be an effective approach to induce antigen-specific T cell immunity in vitro and in vivo. Peptides are used for ex vivo pulsing of DC, but not suitable to target dendritic cells in vivo. To overcome this limitation we have covalently attached 15 mers peptides covering the whole CMV pp65 amino acid sequence onto polyamidoamine (PAMAM) dendrimers and coupled them to an anti biotin antibody. Using this construct we were able to specifically target large amounts of peptides via CD36 biotin antibody to blood DC for effective stimulation of autologous CMV specific T cells analyzed by intracellular interferon gamma production. Appropriate activation of targeted DC with Toll like receptor ligands proved to be essential for effective T cell stimulation. Peptide pulsed DC were not sensitive to the antigen processing inhibitor chloroquine, whereas PAMAM-peptide targeted DC were and failed to stimulate T cells. Moreover T cell stimulation with PAMAM-peptide showed a substantial CD4 T cell response, but only weak CD8 response. Taken together this demonstrates that targeted PAMAM-peptides are internalized and processed before presentation via MHC class II molecules. In vivo targeting of PAMAM-peptides may represent a promising perspective for effective induction of DC derived immune responses against defined antigen epitopes.
Use of Ehrlichia Hsp60 and p28 in diagnostics and vaccines to protect against Ehrlichia infection (53.16)
Authors: Thomas Sunil , Valdes Patricia , Thirumalapura Nagaraja , Crossley Emily , Walker David
Published: January 2023

The obligately intracellular bacterium Ehrlichia chaffeensis that resides in mononuclear phagocytes is the etiologic agent of human monocytotropic ehrlichiosis (HME). HME is an emerging, highly prevalent, and often life-threatening tick-transmitted infectious disease in the United States. As yet there are no vaccines against ehrlichiosis. We had recently demonstrated that Ehrlichia Hsp60 and p28 are the major antigens of Ehrlichia, and both the proteins are post-translationally modified as determined by eastern blotting. Based on these observations we designed peptides specific to Ehrlichia Hsp60 and p28-19. The designed peptide sequences were highly conserved in Ehrlichia. In initial studies we tested the peptides as probes to detect antibodies to Ehrlichia. Both the Ehrlichia Hsp60 and p28 could detect antibodies stimulated by E. chaffeensis, E. canis, E. muris, and E. ewingii. However, the p28-19 peptide was more sensitive than Ehrlichia Hsp60 in diagnosis of ehrlichiosis. Both Ehrlichia peptides-Hsp60 and p28-19-could function as a vaccine. The bacteria were cleared more rapidly within 14 days in vaccinated mice. The p28-19 peptide elicited higher levels of IgG1, IgG2b, and IgG3 antibodies compared to Ehrlichia Hsp60. The experiments demonstrate that Ehrlichia antigenic peptides Hsp60 and p28-19 are good candidates to be used in diagnostics and also could function as vaccines to protect against ehrlichiosis.
Nano-antigen presenting cells for the development of therapeutic vaccines (53.2)
Authors: Liu Mengya , Miao Tizong , Li Su-ling , Wang Ping
Published: January 2023

Antigen presenting cells (APC) initiate immune responses against pathogens by presenting antigen and costimulatory signals to T lymphocytes. APCs loaded with antigens in vitro have been used as therapeutic vaccines to induce antiviral or tumour immune responses in chronic infectious diseases or cancer. However, the live APCs are unstable in vivo and cannot overcome the immune tolerance induced by persistent viral infection or tumour microenvironment. We have developed nano-sized APCs that are at the size of ~ 500nM and can present antigen, costimulatory molecules and bioengineered IL-2 to antigen specific T cells directly. The nano-APCs are prepared from isolated endoplasmic reticulum membranes of APCs engineered with an ER-retained IL-2. Assembled with specific antigenic peptides, the nano-APC effectively induce responses of CMV or HBV specific CD4 and/or CD8 T cells responses in peripheral blood lymphocytes (PBL) of CMV serum converted or chronic HBV infected patients. With selected antigens, the bio-engineered nano-APCs will be used as therapeutic vaccines for the treatment of chronic HBV, HCV or HIV infection and also cancer.
Dynamics of memory T cells clearing a persistent CNS infection in the absence of severe tissue injury (53.9)
Authors: Herz Jasmin , McGavern Dorian
Published: January 2023

Many viruses can infect the mammalian central nervous system (CNS), some with devastating consequences, and others with little or no overt pathology. Immunotherapy is a successful approach to treat persistent infections that has shown promise in humans. Our laboratory studies a murine model of persistent lymphocytic choriomeningitis virus (LCMV) infection to understand the mechanism(s) by which immunotherapy with virus-specific memory T cells cleanses the infected CNS with minimal cellular damage and blood brain barrier breakdown. Using two-photon microscopy, we observed that therapeutic memory T cells invade the meninges and brain parenchyma, scanning both areas systemically for viral antigen. Interestingly, T cell surveillance of the persistently infected brain was not associated with massive recruitment of peripheral myelomonocytic cells - innate cells known to cause severe vascular pathology during LCMV meningitis. Instead, we revealed using CD11c-YFP reporter mice that immunotherapeutic T cells transformed microglia into a DC phenotype, inducing CD11c expression on microglia throughout the brain parenchyma. Microglia also participated in immunotherapy by directly releasing chemokines such as CCL5. We propose that successful clearance of a persistently infected brain can be achieved without induction of severe tissue injury by tailoring chemokine release to limit myelomonocytic cell recruitment and by converting microglia into local antigen presenting cells.
Lung dysfunction in a mouse model of inflammation is dependent on T-cell-independent IL-13 expression (54.18)
Authors: Jacobsen Elizabeth , Sergei Ochkur , Wen Li , Zellner Katie , Colbert Dana , Protheroe Cheryl , Lee James , Lee Nancy
Published: January 2023

Chronic asthma is characterized by pulmonary remodeling and sustained lung dysfunction. The expression of IL-13 has been demonstrated as sufficient and necessary to induce these pathologies. Despite the common assumption that T cells are the primary source of IL-13 in the lung following allergen provocation, the mechanistic events leading to IL-13 expression are unclear, including the potential importance of other cells (i.e., non-T cells). Methods: Co-expression of IL-5 and eotaxin-2 induces pulmonary histopathologies and lung dysfunction that are eosinophil-dependent (i.e., they are absent in triple transgenic I5/hE2/PHIL) animals. IL-13 knockout and CD4 knockout mice were crossed with I5/hE2 mice to assess the effects of these deficiencies on the induced pathologies occurring in this double transgenic model. Conclusions: The Th2 pulmonary pathologies associated with I5/hE2 mice included recruitment of pulmonary effector T cells and increased brochoalveolar Th2 cytokine levels. Conversely, I5/hE2/CD4-/- had only a ~50% reduction in IL-13 levels as compared to I5/hE2 mice, suggesting that a significant portion of IL-13 is non-T cell derived in these mice. I5/hE2/IL-13-/- mice are improved in lung dysfunction and mucus production as compared to I5/hE2 mice. All mice had similar levels of eosinophil degranulation. These results demonstrate a role for non-T cell derived IL-13 that contributes to mucus production and lung dysfunction in a mouse model of chronic severe asthma.
Cholinergic activation by galantamine suppresses obesity-associated inflammation in mice (54.4)
Authors: Pavlov Valentin , Satapathy Sanjaya , Ochani Mahendar , Dancho Meghan , Hudson LaQueta , Rosas-Ballina Mauricio , Valdes-Ferrer Sergio , Olofsson Peder , Harris Yael , Chavan Sangeeta , Roth Jesse , Tracey Kevin
Published: January 2023

Low-grade systemic inflammation is a central event in obesity and mediates insulin resistance and other complications. Previously we discovered a role for neural cholinergic signaling in controlling inflammation (Nature, 2000, 405, 6785; Proc Natl Acad Sci USA, 2006, 5219) and demonstrated that the acetylcholinesterase inhibitor galantamine suppresses systemic lethal inflammation (Brain Behav Immun, 2009, 4; 1). Here we tested the efficacy of galantamine in alleviating obesity-associated inflammation. C57BL/6J mice with high fat-diet induced (for 8 weeks) obesity were treated with either galantamine (4 mg/kg daily, i.p.), or saline for 4 weeks while on the high-fat diet. Galantamine treatment of obese mice resulted in lower body weight and abdominal adiposity as compared to saline treatment. Galantamine significantly decreased systemic levels of characteristic pro-inflammatory cytokines/adipokines, including IL-6, MCP-1, resistin and leptin and increased adiponectin levels, accompanied by alleviated hyperglycemia, hyperinsulinemia, insulin resistance and hepatosteatosis. These results demonstrate that galantamine reduces obesity-associated inflammation and alleviates obesity-related complications and provide a rationale for further mechanistic studies and novel therapeutic implications. Translational aspects of these studies will be additionally facilitated by the fact that galantamine is a FDA approved (for the treatment of Alzheimer’s disease) cholinergic agent.
Aloe complex improves insulin sensitivity via inhibition of obesity induced inflammation in diet-induced obese type II diabetes mellitus (54.5)
Authors: Shin Seulmee , Shin Eunju , Do Seon-Gil , Park Yoonhee , Kim Seulah , Lee Chong-Kil , Cho Kyunghea , Kim Kyungjae
Published: January 2023

Aloe is known to have various medical effects. These include healing damaged skin, as well as anti-diabetic, anticancer, and anti-arthritis activity. This study was performed to investigate the therapeutic effect of Aloe complex on diabetic symptoms in die-induced obesity (DIO) C57BL/6N mice. Aloe complex was orally administrated to diabetic mice for 8 weeks and the diabetic clinical markers, including blood glucose level, body weight, and insulin level were measured. The oral administration of Aloe complex in DIO mice significantly decreased of blood glucose and body weight level including total WATs compared with diabetic control group. The levels of serum insulin, tissue insulin, GTT, and ITT were improved after Aloe complex treatment. Moreover, both serum and tissue adiponectin increased whereas serum leptin decreased by Aloe complex. Aloe exerted beneficial effects on lipogenesis related transcription factor genes, PPARγ and LXRα, and aP2 expression in WAT. The gene expression of pro-inflammatory cytokines, IL-1β, IL-6, and TNF-α, and Th1 cytokines, IFN-γ, IL-12, protein levels were suppressed via inhibiting nuclear NF-κB by Aloe complex. In contrast, protein levels of Th2 cytokines, IL-4 and IL-10, in WAT and AMPK activity in muscles were increased. The results suggest that Aloe complex has hypoglycemic effects resulting the attenuation of glucose and lipogenesis and that Aloe-containing diet or drugs may give a benefit for controlling type II diabetes patients.
Cleavage of CD23 by ADAM proteins is regulated by beta-2 adrenergic receptor engagement on primed B cells (55.11)
Authors: Padro Caroline , Sanders Virginia
Published: January 2023

Allergic asthma severity may be associated with IgE levels. We have shown that beta-2 adrenergic receptor (β2AR) engagement on CD40L/IL-4-primed B cells increases the level of IgE produced per cell, without affecting class switch recombination. β2AR agonists alleviate bronchoconstriction by targeting the β2AR expressed on bronchiolar smooth muscle cells to trigger airway relaxation, but these drugs also target the β2AR on primed B cells, potentially producing an IgE effect that may be counterproductive to the drugs intended use. We reported that surface CD23 expression remained constant on primed B cells exposed to a β2AR agonist, while CD23 mRNA, total CD23 protein, and soluble CD23 (sCD23) increased. Thus, if sCD23 positively regulates IgE production, an increase due to β2AR engagement may worsen bronchoconstriction, albeit relieving it in the short term. We hypothesized that the primary sheddase of CD23, ADAM10, is regulated by β2AR engagement on a primed B cell. Although we found that β2AR engagement was unable to affect the level of ADAM10 protein or mRNA produced compared to primed cells alone, our recent data indicated that the β2AR-induced increase in CD23 shedding may be mediated by increased localization of mature ADAM10 with CD23. Thus, ADAM10 may be a molecular target that can be blocked during β2AR drug therapy to prevent increased sCD23 and IgE, which may counteract the therapeutic bronchorelaxation.
2-linoleoylglycerol inhibits allergic response and expression of inflammatory cytokines in mast cells (55.19)
Authors: Ahn Eun-Kyung , Lee Jae Yeon , Hong Seong Su , Oh Joa Sub
Published: January 2023

Mast cell-mediated allergic disorder is associated with many diseases such as allergic asthma, atopic dermatitis and allergic rhinitis. In this study, we investigated the effect of the 2-linoleoylglycerol isolated from Adenophora triphylla on the Immunoglobulin E (IgE)-mediated allergic response and expression of inflammatory cytokines in mast cells. We observed that 2-linoleoylglycerol significantly inhibited β-hexosaminidase release in a dose-dependent manner in antigen-stimulated RBL-2H3 mast cells. 2-linoleoylglycerol also suppressed the expression of TNF-α, IL-4, IL-10 and COX-2 mRNA in mast cells. In addition, we showed that 2-linoleoylglycerol attenuated the activating phosphorylation of AKT, p38MAPK, JNK and ERK1/2. Our findings suggest that 2-linoleoylglycerol inhibits mast cell-derived allergic response and expression of inflammatory cytokines and that this effect is related by inhibition of AKT, p38MAPK, JNK and ERK1/2 phosphorylations.
LPS-induced CD11b+Gr1intF4/80+ myeloid cells suppress allergic airway inflammation (55.3)
Authors: Arora Meenakshi , Poe Stephanie , Oriss Tim , Krishnamorthy Nandini , Yarlagadda Manohar , Ray Anuradha , Ray Prabir
Published: January 2023

The bacterial product lipopolysaccharide (LPS) has been associated with protection from allergic diseases such as asthma in humans. However, differential effects of LPS based on the dose have been noted in the mouse models of allergic asthma. Our recent work has described the ability of LPS to increase the frequency of CD11b+Gr1intF4/80+ cells in the lung tissue of mice in a dose-dependent fashion that is dependent on TLR4 and MyD88. Although the Gr1int cells are readily detectable in the lung tissue of LPS-treated mice, they are not detected in the lung-draining lymph nodes or in the airway lumen which causes selective enrichment of these cells over dendritic cells (DCs) in the tissue. The Gr1int cells were found to blunt the ability of lung DCs to upregulate GATA-3 or to promote STAT5 activation in primed Th2 cells, both transcription factors having critical roles in Th2 effector function. Suppression of Th2 effector function was reversed by anti-interleukin-10 or inhibition of arginase 1. Adoptive transfer of LPS-induced Gr1int cells suppressed allergen-induced airway inflammation suggesting that these myeloid cells may have regulatory functions in allergic asthma. Thus, a complete understanding of the generation and regulation of the Gr1int cells would provide new avenues to either promote or delete these cells for disease-specific immunoregulation.
Interleukin-3-deficient mice have increased susceptibility to Plasmodium berghei infection (56.2)
Authors: Saunders Bryan , Roth Kenneth , Howe Lauren , Lantz Chris
Published: January 2023

Interleukin-3 (IL-3), which can be derived from T cells and other sources, is well documented at promoting the in vitro differentiation and proliferation of murine hematopoietic progenitors, yielding multipotential blast cells, mast cells, and mature cells of the myeloid and erythroid lineages. Although IL-3-deficient (knockout [KO]) mice display intact steady-state hematopoiesis, administration of IL-3 to mice and humans can promote hematopoiesis and influence the effector functions of mature hematopoietic cells in vivo. Because of these biological activities of IL-3, the contribution of IL-3 to resistance to blood-stage malaria was investigated by infecting KO mice intraperitoneally with Plasmodium berghei-infected RBCs. We found that KO mice were more susceptible to infection than wild-type (WT) mice, as evidenced by markedly higher peak parasitemia. P. berghei-infected KO mice also had significantly greater anemia and splenomegaly compared to similarly infected WT mice. Our results indicate that IL-3 contributes to resistance to P. berghei infection.
The role of JAK/STAT pathway in the synthesis of prostaglandin E2 by macrophages infected with Francisella tularensis (56.24)
Authors: Studt Ashley , Bryan Joshua , Woolard Matthew
Published: January 2023

Francisella tularensis is the causative agent of the disease tularemia. Respiratory tularemia is the most serious form of the disease. F. tularensis induces the synthesis of prostaglandin E2 (PGE2) from infected bone marrow-derived macrophages. The synthesis of PGE2 inhibits the generation of beneficial adaptive immune responses during respiratory tularemia resulting in increased severity of disease. We lack knowledge of the signal transduction events that are required for F. tularensis to induce PGE2 synthesis from macrophages. The JAK/STAT pathway has been documented to be important in the macrophage response to F. tularensis. We wanted to determine if JAK/STAT pathways were involved in F. tularensis induced PGE2 synthesis. We demonstrated that STAT1-/- macrophages and macrophages treated with Ag490 (a JAK2 inhibitor) were unable to synthesize PGE2 in response to F. tularensis. These data suggest the involvement of the JAK/STAT pathway in the synthesis of PGE2.
Trypanosoma cruzi regulates the defensin alpha-1 interactome network and defensin alpha-1 causes trypanosome membrane pore formation to control the early process of infection. (56.30)
Authors: Johnson Candice , Kleshchenko Yulia , Furtak Vyachslav , Nde Pius , Pratap Siddharth , Madison Nia , Lima Maria , Villalta Fernando
Published: January 2023

The interactome networks of innate immune molecules operating in the early process of Trypanosoma cruzi infection of human cells are unknown. Here we show that T. cruzi regulates the defensin alpha-1 interactome network and that human defensin alpha-1 plays a fundamental role in the initiation of cellular responses to T. cruzi. We demonstrate that human epithelial cells respond to early infection by up regulating the expression and secretion of defensin alpha-1, which causes trypanosome membrane pore formation and depolarization to inhibit T. cruzi motility, which prevents cellular infection. The pores formed in the flagellar membrane of the invasive trypanosomes cause the detachment of the flagellum from the trypanosome body to abolish trypanosome motility, which is required for trypanosome cell entry. This is the first report elucidating the defensin alpha-1 interactome network and showing that defensin alpha-1 disrupts the T. cruzi flagellum to compromise trypanosome motility and prevent cellular infection. Thus, defensin alpha-1 possesses remarkably distinct modes of activity against protozoan parasites than bacteria and viruses, and its actions may provide insights for the development of new anti-trypanosomal strategies. This work was supported by NIH grants AI080580 and AI007281.
Ecto-5′-nucleotidase (CD73) decreases mortality and organ injury in sepsis (56.31)
Authors: Németh Zoltán , Csóka Balázs , Koscsó Balázs , Spolarics Zoltán , Rolandelli Rolando , Haskó György
Published: January 2023

The extracellular concentrations of adenosine are elevated during sepsis and adenosine receptors regulate the host’s response to sepsis. Here, we investigated the role of the adenosine generating ectoenzyme, ecto-5'-nucleotidase (CD73) in regulating immune and organ function during sepsis. Polymicrobial sepsis was induced by subjecting CD73 knockout (KO) and wild type (WT) mice to cecal ligation and puncture. CD73 KO mice showed increased mortality in comparison with WT mice, which was associated with increased bacterial counts and elevated inflammatory cytokine and chemokine concentrations in the blood and peritoneum. CD73 deficiency promoted lung injury as indicated by increased myeloperoxidase activity and neutrophil infiltration, and elevated pulmonary cytokine levels. CD73 KO mice had increased apoptosis in the thymus, as evidenced by increased activation of caspase-3, poly(ADP-ribose) polymerase and NF-κB. Septic CD73 KO mice had higher blood urea nitrogen levels and elevated cytokine levels in the kidney, indicating increased renal dysfunction. The increased kidney injury of CD73 KO mice was associated with augmented activation of p38 MAPK and decreased phosphorylation of Akt. Pharmacological inactivation of CD73 in WT mice using AMPCP augmented cytokine levels in the blood and peritoneal lavage fluid. These findings suggest that CD73-derived adenosine may be beneficial in sepsis.
SOCS molecules are upregulated during IL-4-induced inhibition of Type I interferon responses in murine myeloid dendritic cells. (57.23)
Authors: Sriram Uma , Xu Jun , Varghese Linda , Bennett Heather , Shivers Debra , Gallucci Stefania
Published: January 2023

We have previously reported that IL-4 suppresses the response of murine myeloid dendritic cells (mDCs) to Type I interferons (IFN). We are now investigating the molecular mechanisms of this inhibition in myeloid bone marrow-derived DCs (BMDCs). We have found that sub-optimal doses of IL-4 (down to 0.25ng/mL) can still suppress IFN-a induced gene expression and phosphorylation of STAT1 and STAT2, suggesting that IL-4 acts at the level or upstream of STAT molecules in the Type I IFN signaling pathway. IL-4 suppresses when administered before and also after Type I IFN stimulation and it inhibits the response of DCs to the autocrine IFN-b that is induced upon IFN-a stimulation, acting on its positive feedback loop. The inhibition of protein synthesis by Cyclohexamide (CHX) abrogated the suppressive effects of IL-4 indicating that IL-4 represses Type I IFN signaling through the upregulation of a protein. We then tested the expression of the SOCS molecules as immediate candidates that negatively regulate Type I IFN signaling and are regulated at the transcription level. The gene expression of SOCS-1was highly induced by IL-4 treatment and was synergistically upregulated in the presence of IL-4 and IFN-a; the levels drastically went down when DCs were pretreated with CHX, suggesting that SOCS-1 maybe one of the key players in suppressing the IFN responses. We are currently testing this hypothesis by silencing SOCS-1 expression in DCs.
PU.1 regulates chromatin modifications of the Il9 gene in Th9 cells (57.5)
Authors: Goswami Ritobrata , Kaplan Mark
Published: January 2023

IL-9-secreting Th9 cells are generated when naïve CD4+ T cells are cultured with IL-4 and TGF-β. The transcription factor, PU.1 is required for the development of Th9 cells. The expression of PU.1, which is abundantly expressed in Th9 cells, is dependent on TGF-β dose. Dose dependent increase in TGF-β leads to a concomitant increase in PU.1 expression and IL-9 production. Ectopic expression of PU.1 can convert Th2 cells to Th9 phenotype and enhance IL-9 production from Th9 cells. The DNA binding domain and the transactivation domains of PU.1 are required for PU.1 to activate Il9, suggesting that as it binds Il9, it recruits other factors to mediate gene activation. We hypothesized that PU.1 recruits specific chromatin modifying enzymes to the Il9 locus. H3 acetylation is decreased in PU.1-deficient Th9 cells on both the Il9 promoter and a second conserved non-coding sequence immediately after 3’ of the Il9 gene. H3K9/18 acetylation is also diminished in PU.1-deficient Th9 cells while H4 acetylation is not altered by PU.1-deficiency demonstrating specific PU.1-mediated histone modifications correlating with Il9 transcription. Overall these results indicate that PU.1 promotes expression of IL-9 by modifying specific histones at the Il9 locus.
Interferon-alpha/beta inhibit immunity towards HBV in a mouse model of HBV persistence (59.8)
Authors: Huang Miao-tzu , Chiang Bor-Luen , James Cassandra , Chen Pei-Jer
Published: January 2023

In humans, most HBV infection in the adult resolves after the acute disease, however, around ninety percent of the infection in neonates and childhood leads to chronic HBV persistence. Until today, causes for the age-dependent infection outcome of HBV are still unclear. By using hydrodynamic injection of replication-competent HBV DNA plasmid, we established a mouse model of in vivo HBV transfection in which HBV persistence was generated in young C57BL/6 mice of 4-6 w/o, but not in mice older than 10 w/o. In this system, we found that there was higher hepatic IFN-alpha/beta expression in the young mice after HBV transfection compared to adult mice. We speculate that differential IFN-alpha/beta immune regulation during early HBV infection leads to the age-dependent HBV persistence outcome. To address this, the adult mice were overexpressed with IFN-beta in the liver. HBV persistence rate of these mice was increased compared to controls. In addition, blockage of IFN-alpha/beta signaling in the young mice accelerated HBV clearance. The young mice expressed more IFN-alpha/beta-associated immune suppressor genes, Axl/Twist, after HBV transfection than the adult mice, suggesting that type-I IFN executed active immune suppression towards HBV immunity in the young mice. Our findings suggest a role for IFN-alpha/beta in suppressing effective anti-HBV immunity in the young mice and shed light on our understanding of the age-dependent immune function during early HBV infection.
Functional non-coding ncRNA in immunity and tolerance: what can be learned from structures and functions of transfer factors of delayed-type[-IV] hypersensititvity on genetics, epigenetics and recognition repertoires (62.12)
Authors: Wissler Josef
Published: January 2023

AIM: Immunity and [self-]tolerance are usually considered orchestrated by protein repertoires upon genetic [Mendelian] and epigenetic [non-Mendelian] variation mechanisms. All is still not fully understood, as evident from transfer factor and LeDouarin phenomena, environmental influences, peptidome complexity and diversity [Adv.Immunol.11,195-266,1969; Int.J.Dev.Biol.49,131-136,2005]. Endogenous ncRNA incl. microRNA mostly escaped consideration. METHODS: Ann.N.Y.Acad.Sci.1137,316-342,2008. RESULTS: Landsteiner-Chase-Lawrence transfer factors showed up as paradigmatic guides in some terra incognita of adaptive immune memory. Original crude preparations were found as paucidisperse solute systems of numerous functional small ncRNA [<200n] in interaction with proteins. Some environment-modified redox- and metalloregulated small hairpin shRNA [<200 bases] were sequenced. By 5'-CUG-3’-hairpin loops, they may address conserved homologous helix-nucleating domains shared in self and foreign proteofactors of tolerated growth, metabolism, vascularization, cancer and epigenome indexing. CONCLUSIONS: Immune cells are not restricted to known protein recognition systems. They have further repertoires of environment-adapted ncRNA as earliest outputs in transcription and translation. These are superior in diversity/specificity [≥10E17] to immune proteins repertoires [~10E13]. All attributes make them competent to integrate information flow on possible molecular shapes into genomic mechanisms.
The role of 2B4 on the generation of OVA-induced effector and memory CD8+ T-cells. (63.1)
Authors: Lee Jung Eun , Kim Seok-Ho , Kim Tae Woo , Lee You Jeong , Jung Kyeong Cheon , Kim Jae-Hong , Lee Kyung-Mi
Published: January 2023

NK receptor 2B4 belongs to the signaling lymphocyte activation molecule (SLAM) family and has been shown to modulate NK cell responses. Previously, we and others have found that 2B4 became expressed on effector, not naive, CD8+ T cells and regulated their proliferation in vitro. However, the role of 2B4 in the Ag-specific CD8+ T cell responses in vivo remains elusive. To address this, we investigated the effector and memory CD8+ T cell generation using the 2B4-deficient mice. Upon primary OVA-Vaccinia virus infection of 2B4-/- mice, we observed significantly reduced numbers of OVA-specific effector and memory CD8+ T cells along with reduced IFN-γ, compared with WT controls. During secondary OVA-vaccinia challenge, 2B4-/- memory CD8+ T cells expanded to a much lesser extent than that seen in wild-type mice. These results demonstrate the participation of 2B4 signaling in the generation, expansion, and maintenance of Ag-specific CD8+ T cell during the course of OVA-vaccinia infection. Our data reveal previously unrecognized role of 2B4 on Ova-specific CD8+ T cells.
NF-κB-inducing kinase plays an essential T cell intrinsic role in graft-versus-host disease and OX40-mediated costimulation, and its overexpression causes lethal autoimmunity (63.3)
Authors: Murray Susan , Polesso Fanny , Rowe Alexander , Parker David
Published: January 2023

NF-κB-inducing kinase (NIK) is an essential upstream kinase in non-canonical, or alternative, NF-κB activation. TNF receptor family members such as BAFFR and LTβR depend on NIK for signaling, but little is known about the role of NIK downstream of TNF receptors in T cells. Here, we describe a T cell-intrinsic requirement for NIK in graft-versus-host disease (GvHD), wherein NIK-deficient alymphoplasia (aly) T cells transferred into MHC class II mismatched recipients fail to cause disease. We show that although NIK is not necessary for antigen receptor signaling, it is essential for costimulation through the TNF receptor family member OX40 (CD134). Notably, OX40 deficient T cells also fail to elicit GvHD. To assess whether NIK overexpression could recapitulate chronic OX40 activation, we conditionally overexpressed NIK in T cells in vivo using a Cre-inducible transgene. Mice with modest NIK overexpression limited to T cells suffered rapid fatal autoimmunity characterized by hyperactive effector T cells and poorly suppressive Foxp3+ regulatory T cells. Together, these data reveal a critical T cell-intrinsic role for NIK during immune responses, and suggest that tight regulation of the non-canonical NF-κB pathway is crucial to avoid autoimmunity.
Characterization of committed T cell progenitors within adult human bone marrow. (64.21)
Authors: Ramos Joseph , García-Ojeda Marcos
Published: January 2023

Hematopoietic stem cell transplantation (HSCT) remains the chief method for treating hematological and autoimmune disorders. However, HLA-matched donors are rare, leading to the use of allogeneic donors. Successful allo-HSCT relies on balancing the levels of donor T cells that facilitate engraftment and provide beneficial graft-versus-leukemia (GVL) effect without inducing graft-versus-host-disease (GVHD). A method for improving allo-HSCTs involves co-transplantation of hematopoietic stem cells (HSCs) with committed T cell progenitors (CTP). Mouse bone marrow contains a population of CTP that swiftly reconstitute T cells in allo-HSCT without inducing GVHD. We identified a CD34+CD38loCD7+CD10-Lin- population in human bone marrow using multi-color flow cytometry that we hypothesize is a human CTP. CD34+CD38loCD7+CD10-Lin- cells expresses CCR7, CXCR6, CCR9, and CD45RA. Using qPCR, we examined the gene expression profile of this population for the expression of T-, B-, and myeloid specific genes. We observed that CD34+CD38loCD7+CD10-Lin- cells have higher levels of lymphoid and T cell specific genes than CD34+CD38lo HSC or CD10+ BM cells. We examined myeloid development potential by methylcellulose cultures, and lymphoid potential using OP9 and OP9DL1 stroma. Our data indicates that the CD34+CD38loCD7+CD10-Lin- bone marrow population may be a true CTP. Future work would help us determine if this hCTP population can mitigate complications caused by allo-HSCT engraftments.
Transcription factor expression pattern differences are present in OP9 culture derived fetal liver origin early thymocytes when compared to fetal and adult thymocytes (64.22)
Authors: Scripture-Adams Deirdre , Georgescu Constantin , Rothenberg Ellen
Published: January 2023

The advent of the OP9 stromal culture system has tremendously improved our ability to replicate T cell development in vitro. While giving unprecedented access and numerical advantage, these cells may not mirror real thymocytes in their developmental capacity and transcription factor expression patterns. We have examined the pattern of expression of 65 developmentally regulated gene transcripts across the early thymocyte stages of DN1, DN2, DN3 and DN4, in fetal liver derived thymocytes (FLDN) derived from co-culture on OP9 DL-1 cells, and compared them to those found in fetal and adult thymocytes using real time quantitative PCR. Our assessment suggests an overall pattern of similarity between FLDN expression patterns and “real” freshly isolated thymocytes, but some classes of genes are not regulated similarly, and these may have important consequences for development and progression. We present a cluster analysis identifying transcription factors which are differentially regulated in FLDN relative to adult and fetal populations, and identify groups of factors which track with those differentially regulated transcripts. We additionally analyze the effect of long vs. short term culture on these changes in the transcription factor control network that drives T cell development.