bluuebunny/crossref_metadata_2025_split huggingface dataset:
Helminth-conditioned dendritic cells prime CD4+ T cells to IL4 production in vivo (IRC5P.469)
Authors: Ronchese Franca , Connor Lisa , Tang Shiau Choot , Camberis Mali , Le Gros Graham 
Published: January 2023

Abstract
Dendritic cells are critical for the initiation of immune responses, however, their role in priming IL4-producing Th2 cells is controversial and remains an area of intense investigation. We found that after skin injection of the nematode parasite Nippostrongylus brasiliensis (Nb), a strong inducer of Th2 responses, Nb material is taken up by a population of dermal dendritic cells in lymph node, and that this population is necessary and sufficient for IL4 priming. Blocking the migration of dermal dendritic cells to the lymph node also prevented the priming of IL4-producing cells, and chicken ovalbumin (OVA)-loaded dDC from Nb-treated mice were sufficient to sensitize OVA-specific CD4+ T cells to IL4 production in vivo. Nb-loaded dermal dendritic cells were CD11b+, CD326neg, CD103neg, PDL2hi and IRF4+, and modestly upregulated expression of OX40L. However, OX40L and TSLP were not required for Th2 priming. Thus, our data suggest that the appropriate stimuli can induce DC to express the unique signals that direct CD4+ T cells to Th2 differentiation.
Participation of c-FLIP in NLRP3 and AIM2 inflammasome activation (IRC5P.453)
Authors: Wu Yung-Hsuan , Kuo Wen-Chi , Lai Ming-Zong 
Published: January 2023

Abstract
Cellular FLICE-inhibitory protein (c-FLIP) is an inhibitor of caspase-8 and is required for macrophage survival. Recent studies have revealed a selective role of caspase-8 in noncanonical IL-1b production that is independent of caspase-1 or inflammasome. Here we demonstrated that c-FLIPL is an unexpected contributor to canonical inflammasome activation for the generation of caspase-1 and active IL-1b. Hemizygotic deletion of c-FLIP impaired ATP- and monosodium uric acid (MSU)-induced IL-1b production in macrophages primed through Toll-like receptors (TLRs). Decreased IL-1b expression was attributed to a reduced activation of caspase-1 in c-FLIP hemizygotic cells. In contrast, the production of TNF-a was not affected by downregulation in c-FLIP. c-FLIPL interacted with NLRP3 or procaspase-1. c-FLIP is required for the full NLRP3 inflammasome assembly, and c-FLIP is associated with NLRP3 inflammasome. c-FLIP downregulation also reduced AIM2 inflammasome activation. In contrast, c-FLIP inhibited SMAC mimetic-, FasL-, or Dectin-1-induced IL-1b generation that is caspase-8-mediated. Our results demonstrate a prominent role of c-FLIPL in the optimal activation of the NLRP3 and AIM2 inflammasomes, and suggest that c-FLIP could be a valid target for treatment of inflammatory diseases caused by overactivation of inflammasomes.
AMP-activated protein kinase suppresses autoimmune CNS disease by regulating M1 type macrophage-Th17 axis (IRC5P.474)
Authors: Giri Shailendra , Mangalam Ashutosh , Rattan Ramandeep , Denic Aleksandar , Viollet Benoit , Rodriguez Moses 
Published: January 2023

Abstract
AMPK is an energy-sensing metabolic switch implicating in various metabolic disorders. Here we report its new role in controlling the immunopathology of experimental autoimmune encephalitis by polarizing pro-inflammatory phenotype of Macrophage. AMPK alpha1 knockout (alpha1KO) developed severe EAE due to higher expression of IL17 with reduced IFNy and IL4. . AMPK alpha1KO also exhibited increased number of infiltrated mononuclear cells in CNS due to impaired blood brain barrier integrity. Macrophage from alpha1KO mice exhibited M1 type phenotype (IL10lowIL12/23high) with stronger capacity to induce allogenic and antigen-specific T-cell response and also heightened the encephalitogenic property of MOG35-55-primed CD4 T cells in B6 mice. Collectively, our data shows AMPK controls the clinical outcome of disease by regulating M1 phenotype-Th17 axis in animal model of MS.
TRIM22 enhances staurosporine-induced apoptosis in monocytes (IRM7P.492)
Authors: Shu Qiang , Chen Chi , Zhao Dongyan , Liu Xiwang , Chen QiXing , Fang XiangMing 
Published: January 2023

Abstract
Background TRIM22 was first identified as an interferon-induced gene, and constitutively expressed in peripheral blood leukocytes and lymphoid tissues, especially expressed at high levels in monocytes. Researches suggest that TRIM22 may have an impact on monocyte fate in immune response. However, the direct evidence of TRIM22 on monocyte apoptosis remains absent. Methods TRIM22 was constructed and transfected into a monocytic cell line THP-1 cell. Apoptosis of transfected cells was induced by STS and tested by flow cytometry. The caspase pathways involved in TRIM22-related apoptosis were illustrated by inhibitory experiments. Proapoptotic/antiapoptotic protein were measured by immunoblot. Results Overexpression of TRIM22 in THP-1 cells caused extensive apoptosis induced by STS; The activation of caspase-9 and caspase-3, and the release of cytochrome c were increased in TRIM22-overexpressing cells; Inhibition of caspase activation protected the cells from apoptosis augmented by TRIM22; Bak was upregulated in TRIM22 overexpressing monocytes. Conclusions Overexpression of TRIM22 in human acute monocytic leukemia cell THP-1 caused extensive apoptosis induced by STS both in absence and presence of lipopolysaccharide, which was associated with elevated cleavage of caspase-9 and caspase-3, and release of cytochrome c. Z-VAD-fmk, a pan caspase inhibitor, prevented TRIM22-induced apoptosis. Western blot found that Bak was upregulated in TRIM22 overexpressing monocytes.
Staphylococcal enterotoxin B (SEB)-induced miR-155 targets SOCS-1 to promote acute inflammatory lung injury. (IRM7P.481)
Authors: Rao Roshni , Nagarkatti Prakash , Nagarkatti Mitzi 
Published: January 2023

Abstract
Staphylococcal enterotoxin B (SEB) initiates an exaggerated inflammatory response characterized by immune cell infiltration, the release of inflammatory cytokines and tissue damage. However, the role of microRNAs (miR) associated with the damaging effects of SEB have not yet been delineated. In this study, we show that inhalation exposure to SEB results in acute inflammatory lung injury accompanied by altered miR expression profile in lung infiltrating cells. Amongst the miRNA that were significantly elevated, miR-155 is the most over-expressed. In contrast, miR-155-/- mice were protected from SEB-mediated inflammation. Interestingly, there was a positive correlation between miR-155 levels and IFN-γ secretion, suggesting a functional link between SEB-induced miR-155 and pro-inflammatory cytokine IFN-γ. Through the use of bioinformatics tools, suppressor of cytokine signaling -1 (SOCS1), a negative regulator of IFN-γ, was identified as a potential target of miR-155. While miR-155-/- mice displayed increased SOCS1, the overexpression of miR-155 led to its suppression, thereby enhancing IFN-γ levels. Additionally, the inhibition of miR-155 and IFN-γ resulted in restored SOCS1 expression. Our data, therefore, confirm an important role of miR-155 in facilitating SEB-mediated inflammation and proposes that miR-155 may be an important target in the treatment of acute lung inflammation.
Novel orally active ligands of lanthionine synthetase C-like protein 2 ameliorate influenza-related inflammation and immunopathology (IRM7P.482)
Authors: Philipson Casandra , Lu Pinyi , Bassaganya-Riera Josep , Vento Salvador , Carbo Adria , Viladomiu Monica , Kale Shiv , Zhang Xiaoying , Uren Aykut , Kallarakal Abraham , Xu Bin , Hontecillas Raquel 
Published: January 2023

Abstract
The use of broad-based drugs that target the host inflammatory response and facilitate recovery has been proposed for treating influenza. We previously identified lanthionine synthetase C-like protein 2 (LANCL2) as a new therapeutic target for inflammatory diseases, predicted in silico that NSC61610 and abscisic acid (ABA) bind to LANCL2 and demonstrated their therapeutic efficacy in mouse models of colitis. Here, we use surface plasmon resonance to demonstrate for the first time that NSC61610 binds to LANCL2, promotes cAMP accumulation and protein kinase A (PKA) phosphorylation in RAW macrophages. To examine the therapeutic efficacy of NSC61610 in vivo, we infected mice with influenza A H1N1pdm virus and determined its impact on influenza-related morbidity, mortality, lung inflammatory profiles and histopathology. Our results demonstrate that oral administration of NSC61610 (20 mg/kg/day) ameliorates influenza by down-modulating pulmonary TNF-α and MCP-1 mRNA expression and reducing the infiltration of neutrophils and pro-inflammatory monocytes in the lungs. The therapeutic actions and signaling of NSC61610 were abrogated following gene silencing by LANCL2 siRNA administration further validating our findings. Thus, binding of NSC61610 to LANCL2 and signaling through cAMP and PKA represents a novel mechanism for modulating pulmonary host responses and improving disease resolution in influenza virus-infected mice.
The role of GCN2 in type-2 mucosal inflammation within the lung (IRM7P.483)
Authors: Bradley Jillian , Liu Haiyun , Ravishankar Buvana , Huang Lei , Perdue Aja , Urban Joseph , McGaha Tracy 
Published: January 2023

Abstract
Infection with the parasitic roundworm Nippostrongylus brasiliensis causes lung damage associated with an inflammatory response dominated by alternative (type-2) inflammation. Macrophages are requisite mediators of lung inflammation and pathology resolution in the N. brasiliensis infection model. Metabolic stress resulting from enzymatic consumption of amino acids engages the GCN2 arm of the integrated stress response, which profoundly alters the inflammatory potential of antigen presenting cells, causing them to suppress type-1 cytokine expression and promote type-2 cytokine expression. Thus, we hypothesized that GCN2 activation, as a result of infection driven nutrient stress, may be a key mechanism promoting M2 macrophage differentiation and type-2 inflammatory processes. When we examined cytokine expression in the lungs of GCN2KO mice infected with N. brasiliensis, we found significantly decreased levels of IL-10 relative to WT controls. This was associated with increased lung infiltration of macrophages that expressed reduced expression of FIZZ1 and YM-1, indicative of a skewing towards an M1 phenotype. Moreover, we observed dramatic increases in lung pathology associated with increased cellular infiltrate and fibrosis in infected GCN2KO mice suggestive of severe helminth-driven lung inflammation. Thus, our findings suggest a novel regulatory role for GCN2 kinase during type-2 inflammatory processes in the lung.
ATAD5 deficiency decreases B cell division and Igh recombination (IRM8P.700)
Authors: Zanotti Kimberly , Gearhart Patricia 
Published: January 2023

Abstract
Mammalian ATAD5 and its yeast homolog ELG1 are responsible for unloading PCNA from newly synthesized DNA. Prior work in HeLa and yeast cells showed that a decrease in ATAD5 protein levels resulted in accumulation of chromatin-bound PCNA and slower cell division. We used B cells from heterozygous (Atad5+/m) mice to examine the effects of PCNA buildup and decreased cell proliferation on antibody diversity. ATAD5 haploinsufficiency did not affect the frequency or spectrum of somatic hypermutation in antibody genes from Peyer’s patch B cells, indicating that PCNA accumulation did not alter DNA repair or DNA polymerase η usage at the Igh locus. However, following ex vivo stimulation, Atad5+/m B cells were delayed during S-phase cell cycle progression and had reduced cell proliferation compared to wild type cells. These cells had a significant decline in activation-induced deaminase expression, resulting in decreased double-strand breaks in the switch region and inter-chromosomal translocations. Class switch recombination was also reduced, although the types of end-joining pathways were not affected. Our results describe a defect in DNA replication that directly affects Igh recombination via reduced cell division.
B cell-intrinsic mTOR signaling regulates the generation of germinal center B cells (IRM8P.716)
Authors: Jones Derek , Chernova Irene , Allman David 
Published: January 2023

Abstract
Germinal centers (GCs) are sites enriched for proliferative B cells undergoing class switch, somatic hypermutation, and selection for clones expressing high-affinity antigen-specific receptors. GCs are a main source of long-lived plasma cells (PCs) and memory B cells, and many B-lineage cancers including multiple myeloma (MM), a malignancy of PCs, also originate in GCs. However, little is known about the biochemical pathways regulating GC B cells or GC-derived effectors. Thus, we sought to define the role of mammalian target of rapamycin (mTOR), a serine/threonine kinase known to regulate several biochemical pathways needed for cell growth, division, and survival, in the maintenance of GC B cells and GC-derived cells. To address the role of mTOR in GC B cell generation, we treated NP-CGG-immunized mice with rapamycin, an mTOR inhibitor. Rapamycin treatment caused a near-complete loss of NP-specific GC B cells in the spleen, and a selective reduction of newly formed bone marrow PCs. We next generated Raptorf/f x CD20-ER-Cre mice to induce B cell-specific deletion of the mTOR-requisite protein Raptor; Raptor deletion caused the ablation of antigen-specific GCs, as well as secondary antibody responses mediated by memory B cells. The data indicate that GC B cells utilize mTOR signaling to regulate metabolic pathways required for cell division and/or survival. Future studies will address the notion that malignant PCs responsible for MM must be replenished by ongoing GC responses.
Immunoglobulin isotype switching during early B cell development (IRM8P.702)
Authors: Kumar Satyendra , Kenter Amy 
Published: January 2023

Abstract
During B cell lymphopoiesis in the bone marrow (BM), V(D)J joining assembles immunoglobulin (Ig) VH and VL genes mediated by RAG recombinase. Mature splenic B cells undergo class switch recombination (CSR) and somatic hypermutation initiated by activation induced deaminase (AID). However, the strict ontological separation of these processes may breakdown under some circumstances. Several immunodeficiencies including Omenn syndrome display increased serum IgE despite undetectable numbers of B cells. We hypothesized that CSR occurs in BM pro- or pre-B cells. Earlier, we showed that Rag1 and Mb1 deficient pro-B cells can be induced to undergo robust CSR, albeit to a restricted subset of IgH isotypes, including IgG2b and IgE. Immunization of Rag1-/- mice with LPS, promoted IgG2b switching in BM pro-B cells. We demonstrated that V(D)J joining can be induced to follow CSR. We found a unique stage specific chromatin conformation of Igh locus in pro-B cells that may repress IgG3 and IgG1 switching (Genes Dev. 27:2439). Here we show that CSR is inducible in WT pro-B cells. Using an elegant mouse model to irreversibly mark AID expressing cells, a knockin carrying AIDcre/+RosaYFP (Nat Imm. 10:1292), we assess the frequency of AID expression in BM pro-B cells activated in vitro and in vivo with LPS. These findings have implications for the hyper-IgE phenotype in Omenn syndrome, autoimmune repertoire development and leukemagenesis via the co-expression of the RAG and AID recombinases.
Assessing B lymphocyte clonal diversity and expansion by high-throughput sequencing of rearranged IgH loci from naïve and memory repertoires (IRM8P.708)
Authors: Emerson Ryan , DeWitt William , Lindau Paul , Sherwood Anna , Williamson David , Rieder Mark , Chung Moon-Wook , Carlson Christopher , Robins Harlan 
Published: January 2023

Abstract
Diversity in B cell antigen specificity arises from structural diversity of the B cell receptor generated by somatic rearrangement of the immunoglobulin heavy (IgH) and light chain loci during B cell development. Antigen-experienced (memory) B cells are stimulated to proliferate upon successful binding of antigen, whereas the extent to which antigen-inexperienced (naïve) cells proliferate, and therefore the total diversity of circulating naïve B cells, is unclear. High-throughput sequencing of rearranged IgH CDR3 regions was performed for sorted memory and naïve B cell samples from each of 3 adults. For each sample, approximately 5 million B cells were split among 188 libraries for multiplex PCR amplification of IgH CDR3 segments, allowing us to estimate clone abundances using the number of libraries occupied by each distinct CDR3 segment. Modeling sample clone abundances as a superposition of homogeneous Poisson processes, memory repertoire clone abundance was characterized. Data from naïve samples suggest extreme diversity with more than 98% of clones occupying only one library (i.e., almost surely present in only one cell in the sample). A measure-theoretic upper bound on mean clone abundance in the naive repertoire was computed based on Monte Carlo simulation, indicating that naïve B cells typically undergo little, if any, expansion. We estimate the diversity of circulating naive B cells to be on the order of 100 million in healthy adults.
Immunohistochemical and multicolor flow cytometric analysis of STAT1 (pY701) expression in human and rat cells (IRM9P.733)
Authors: Ernst David , Ji Xiang Dong , Gao Guo-Jian , Wang Xiao , Berry Jody 
Published: January 2023

Abstract
Signal Transducer and Activator of Transcription 1 (STAT1) is phosphorylated at tyrosine 701 (pY701) upon cellular activation by interferons. This site-specific phosphorylation activates the signaling activity of STAT1 that translocates to the nucleus and regulates target gene transcription, which determines cellular growth, proliferation, and effector functions. An antibody that specifically recognizes STAT1 (pY701) was developed to study STAT1 roles played in experimental systems. Human tonsil sections, briefly cultured with a phosphatase inhibitor, revealed enhanced immunohistochemical (IHC) staining of nuclear STAT1 (pY701) in lymphocytes and epithelial cells when compared with untreated or phosphatase-treated controls. Similarly, in an in vivo model, liver sections prepared from rats treated with a phosphatase inhibitor revealed enhanced IHC staining of nuclear STAT1 (pY701) in hepatocytes compared with controls. The Ab also served as a specific probe in multicolor flow cytometric analyses designed to characterize the nature of lymphoid cells that express STAT1 (pY701) in response to cytokines. Exogenous interferon-alpha treated CD4+ and CD8+ T cells, B cells, monocytes and NK cells all were induced to express STAT1 (pY701) as determined by multicolor flow cytometric analysis. The applications of this Ab, as well as additional Abs specific for phosphorylated STATs 3-6, will be presented and discussed in terms of their use for cell analysis across multiple applications.
SAP facilitates recruitment and activation of LCK at NTB-A receptors during restimulation-induced cell death. (IRM10P.734)
Authors: Katz Gil , Krummey Scott , Larsen Sasha , Stinson Jeffrey , Snow Andrew 
Published: January 2023

Abstract
Upon TCR restimulation, activated, cycling T cells can undergo a self-regulatory form of apoptosis known as restimulation-induced cell death (RICD). We previously demonstrated that RICD is impaired in T cells from patients with X-linked lymphoproliferative disease (XLP), which lack SLAM-associated protein (SAP) expression. Both SAP and the specific SLAM receptor NK, T, and B cell antigen (NTB-A) are required for RICD, but the mechanism by which these molecules promote a strong, pro-apoptotic signal through the TCR remains unclear. Here we show that the Src-family kinase LCK, but not FYN, associates with NTB-A in activated human T cells. This association increased after TCR restimulation in a SAP-dependent manner, requiring both immunoreceptor tyrosine-based switch motifs (ITSMs) in the NTB-A cytoplasmic tail. Both NTB-A-associated LCK phosphorylation and kinase activity were enhanced in restimulated T cells, amplifying proximal TCR signaling. In contrast, TCR-induced LCK association with NTB-A, as well as phosphorylation and kinase activity, were reduced in XLP patient T cells or normal T cells transfected with SAP-specific siRNA, consistent with RICD resistance. Collectively, our data reveal how SAP nucleates a previously unknown signaling complex involving NTB-A and LCK to potentiate RICD of activated human T cells.
Analysis of human TCRβ repertoire by a RARE-sequencing method reveals distinct features of TCRβ diversity and distribution and their alterations with age (IRM10P.758)
Authors: Weng Nan-ping , Shi Alvin , Li Hoi-Ming , Hiroi Toyoko , Zhang Yongqing , Chen Guobing , Metter Jeffrey , Ferrucci Luigi 
Published: January 2023

Abstract
The T cell receptor (TCR) repertoire (diversity and distribution) is a vital parameter of the overall function of T cells. Reduction of TCR diversity and alteration of TCR distribution is believed to occur with age and acts as a major contributor for age-associated decline of immune function. However, neither the actual size of the TCR repertoire nor its precise age-related change have been directly determined. Applying a RACE-next generation sequencing (RAP-Seq) method, we assessed the TCRβ repertoire of blood CD4+ and CD8+ T cells of 13 adults (21-94 years old) including 6 adults with longitudinal samples. We found that TCRβ diversity of CD4+ T cells is 3-4 times greater than CD8+ T cells. TCRβ CDR3 was distinct between CD4+ and CD8+ T cells with preferential amino acid usage. Reduced TCRβ diversity and expanded highly abundant TCRβ clones occurred from middle to old subjects (45-75 years old) but not in very old subjects (80-94 years old). Together, these findings reveal the distinct features and actual age-associated changes of TCRβ repertoire, which could serve as a measure of the immune competency and a guide for the clinical intervention in older humans.
Co-expression of CD40 and EBV-associated latent membrane protein (LMP)1 results in altered downstream signaling responses in B cells (IRM11P.762)
Authors: Boody Christopher , La Porta James , Valentin-Acevedo Anibal , Covey Lori 
Published: January 2023

Abstract
The binding of CD40 on B cells to CD154 on activated CD4 T cells is essential for regulating humoral responses in antigen-primed B cells. The EBV transforming protein, LMP1 mimics many aspects of CD40 function through the binding of an overlapping set of TNF receptor-associated factor (TRAF) signaling molecules. Co-expression of LMP1 and CD40 occurs at distinct stages of EBV infection and in tumor cells in which EBV is an etiological agent. Although both molecules share functional homology, the signaling cross talk between LMP1 and CD40 is not well understood. Here we show that LMP1 directly increases both TRAF3 and CD40 in B cells through elevated RNA expression. Additionally, LMP1 enhances the association of CD40 and TRAF3 with lipid rafts. To better understand LMP1-specific changes in CD40 signaling we evaluated the activity of specific downstream targets in response to CD154 stimulation using phospho-specific flow cytometry. Stimulated B cells produced a greater signal for p38 and p65 than those constitutively expressing LMP1. Also, CD40 signaling reduced the activity of LMP1-induced pLCγ2 whereas LMP1 significantly lowered that of Akt in cells receiving both stimuli. Together, our results demonstrate that LMP1 increases expression of CD40 and TRAF3 and specifically modifies CD40 activation responses when both proteins are concurrently active. These changes have the potential of underlying differences in B cell activation, survival and differentiation.