bluuebunny/crossref_metadata_2025 huggingface dataset:
β-Catenin induces T-cell transformation by promoting genomic instability (HEM4P.237)
Authors: Emmanuel Akinola , Dose Marei , Keerthivasan Shilpa , Aghajani Katayoun , Gounari Fotini
Published: January 2023

Deregulated activation of β-catenin has been correlated with genomic instability in cancer. During thymocyte development β-catenin activates transcription in partnership with Tcf-1, an essential T-cell specific DNA-binding protein. We previously reported that targeted activation of β-catenin in thymocytes (CAT mice) induces lymphomas that depend on recombination-activating-gene (RAG) and c-Myc activities. Here we show that these lymphomas have recurring Tcra/Myc translocations that resulted from illegitimate RAG-recombination events and resembled oncogenic translocations in ponies. We therefore used the CAT animal model to obtain mechanistic insights into the transformation process. Interestingly, ChIP-seq analysis uncovered a link between Tcf-1 and RAG2 showing that the two proteins shared binding sites marked by trimethylated histone-3 lysine-4 (H3K4me3) throughout the genome, including sites near the translocation. Pre-transformed CAT thymocytes had increased DNA damage at the translocating loci and showed altered repair of RAG induced DNA double strand breaks (DSBs). Importantly these cells were able to survive despite DNA damage as activated β-catenin promoted an anti-apoptosis gene expression profile. Thus, activated β-catenin promotes genomic instability that leads to T-cell lymphomas and this is associated with compromised DSB repair and increased survival of thymocytes with damaged DNA.
Defects in pro-apoptotic genes, Bim or Bax/Bak, cause accumulation of TCRβhi DN4 cells in thymus (HEM4P.241)
Authors: Li Kun-Po , Roy Eron , Hildeman David
Published: January 2023

Regulated induction of apoptosis during thymocyte development is critical to prevent the generation of self-reactive T cells whilst maintaining a repertoire of T cells that can promote protective immunity. Although the pro-apoptotic Bcl-2 family member Bim has been shown to promote apoptotic negative selection of self-reactive thymocytes, the role of Bim in this process is controversial. Here, we have investigated the role of Bim and downstream mediators Bax and Bak on thymocyte selection. Surprisingly, the absence of Bim results in a striking accumulation of DN4 cells with surface TCRβhi feature in the thymus. Similar results were observed in mice with a T cell specific loss of Bax in a Bak knock out background. DN4 cells from Bim-/- or LckCreBaxfl/flBak-/- mice cultured with OP9-DL1 cells largely failed to generate DP cells relative to WT DN4 cells. Further, using a dLckCre system, in which Cre is expressed late in the DP stage, revealed that conditional knock out of Bim or Bax did not result in an accumulation of DN4-TCRβhi cells. Combined, these data suggest that accumulation of DN4-TCRβhi cells in either Bim-/- or LckCreBaxfl/flBak-/- are the result of cells having failed negative selection that downregulate CD4 and CD8 coreceptors. Such a strategy would inherently limit their TCR avidity and reduce their potential for autoreactivity. Our current studies are examining the persistence and location of DN-TCRβhi cells in the periphery of Bim-/- or LckCreBaxfl/flBak-/- mice.
Metabolic inhibition by inositol-tetrakisphosphate delays thymocyte β-selection and renders it notch-dependent (HEM4P.227)
Authors: Sauer Karsten , Huang Yina , Rigaud Stephanie , Conche Claire , Nosaka Lyn'Al , Siegemund Sabine , Sternberg Luise
Published: January 2023

In β-selection, surface-expression of a pre-T cell receptor (pre-TCR) induces thymocyte metabolic activation, proliferation, survival and differentiation. This pivotal αβ T cell fate-determining event requires co-stimulatory Notch signals. Here, we show that this Notch dependence is enforced through antagonistic signaling by inositol-trisphosphate 3-kinase B (Itpkb) and phosphoinositide 3-kinase (PI3K). Itpkb produces soluble inositol-tetrakisphosphate which competes with the PI3K lipid-product PIP3 for binding to the effector-kinase Akt. Itpkb-/- thymocytes are pre-TCR hyperresponsive, hyperactivate Akt, downstream mTOR and metabolism, undergo an accelerated β-selection and can develop to CD4+CD8+ cells without Notch. This is reversed by inhibition of Akt, mTOR or glucose-metabolism. Thus, Itpkb restricts pre-TCR induced metabolic activation to enforce coincidence-detection of pre-TCR expression and Notch-engagement, and to prevent premature thymocyte maturation.
Convergence of NF-κB-mediated inflammatory and HNF4α-mediated metabolic signaling networks in age-related thymic involution (HEM4P.229)
Authors: Weinberg Kenneth , Min Dullei , Venkatasubrahmanyam Shivkumar , Chung Brile , Yang Yujun , Goetz Christine , Winston Brent , Krampf Mark , Karamchandani Jason , Blazar Bruce , Butte Atul
Published: January 2023

Thymic epithelial cell loss results in age-related thymic involution and immune deficiency. We investigated mechanisms of TEC loss during chronological aging (6 weeks, 7 months, 15 months) of normal C57BL6/J mice. Aged TEC showed replicative senescence with increased apoptosis, decreased proliferation, decreased telomerase activity and shortened telomere length. A subpopulation of aged TEC expressed Fas in response to pro-inflammatory cytokines (TNFα,IL-1β) made by aged thymocytes. Fas signaling and caspase-8 activation were induced by FasL selectively expressed by aged intrathymic memory T cells. Combined pharmacological inhibition of Caspase-8 and TEC growth stimulation by KGF restored thymopoiesis to neonatal levels. Aged TEC differentially expressed a 175-gene set predicted to be dually regulated by two transcription factors, NF-κB and HNF4α, which are activated by inflammatory cytokines and fatty acids, respectively. NF-κB and HNF4α signaling in aged TEC was confirmed by ChIP assays demonstrating binding to promoters of age-related genes, gene reporter assays, and synergistic induction of TEC apoptosis by inflammatory cytokines and fatty acids. TEC aging is mediated by the convergence of different TFs responsive to inflammatory and metabolic signals on shared promoters. Thymic regeneration in aging will require mitigation of the effects of non-autonomous inflammatory and metabolic signals, e.g., from infection and obesity, which drive TEC death and thymic involution.
Protein tyrosine kinase 7 promotes PI3K/AKT signaling and survival in primary and neoplastic CD4+ T-lineage cells. (HEM4P.231)
Authors: Acharya Swati , Dionis-Petersen Kira , Jackson Peter , Admas Christopher , Lewis David
Published: January 2023

Protein tyrosine kinase 7 (PTK7), a catalytically inactive receptor tyrosine kinase (RTK) that is highly expressed in intrathymic development, is a novel marker for human CD4+ recent thymic emigrants (RTEs), and is also highly expressed on some T-lineage thymomas, e.g., Jurkat cells. The function of PTK7 in normal human T-cell development and tumors remains unclear. Here, using RNAi-mediated gene silencing in T-lineage tumor cells, primary human peripheral T-cells and thymocytes, we found that targeting PTK7 consistently decreased cell survival by augmenting caspase-3 activation of apoptosis. The PTK7 knockdown also decreased AKT phosphorylation and PI3 kinase activity, suggesting an essential role for PTK7 in survival of RTEs and developing thymocytes involving the PI3K/AKT pathway. Using mass spectrometry of we identified insulin-like growth factor-1 (IGF-1) receptor as an active kinase partner of PTK7. Knockdown of PTK7 reduced IGF-1 receptor function in primary T-lineage cells, confirming the biologic importance of this physical association. This role for PTK7 in promoting cell survival via its effects on cell survival via the PI3K/AKT pathway may not only be relevant to normal T-cell development, including the RTE compartment, but also to the oncogenesis of T-lineage and epithelial cell tumors.
B cells support a dominant Th17 cytokine signature in type 2 diabetes (HEM4P.255)
Authors: Belkina Anna , DeFuria Jason , Jagannathan-Bogdan Madhumita , Hasson Brooke , Kuchibhatla Ramya , McDonnell Marie , Apovian Caroline , Istfan Nawfal , Xiao Qiang , Nikolajczyk Barbara
Published: January 2023

T cell inflammation plays critical roles in the development of obesity-associated type 2 diabetes (T2D). B cells support T cell-mediated inflammation in T2D, but the mechanisms underlying lymphocyte cross-talk and the relative importance of T cell inflammation in human T2D remain untested. Using peripheral blood mononuclear cells (PBMC) and/or purified cells from T2D and non-diabetic (ND) subjects, we show that B cell contact upregulates a pro-inflammatory CD4+ Th17 T cell program in T2D. Neutralization of either B cell CD80/86 or the downstream production of T cell IL-17A/F significantly decreased production of classical diabetogenic cytokines (TNFα, IL-6) in samples from T2D subjects. Although monocytes, B cells and T cells produced TNFα in response to treatment of PBMCs with αCD3/CD28, monocyte TNFα production was independent of IL-17A/F, while IL-17F was critical for maximal TNFα production by both T and B cells. IL-17A/F neutralization also reduced IL-17A and IL-17F production by CD4+ T cells, indicating Th17 function is auto-regulated. We conclude that human B cell co-stimulation broadly supports Th17 cells, which in turn stimulate lymphocytes (but not monocytes) to produce the classical diabetogenic cytokine TNFα.
The rare IL-13R105Q variant, strongly enhances IL-13 protein activity (HYP6P.268)
Authors: Rosenbaum David , Hawkins Gregory , Zhu Lingxiang , Chen Yin , Bleecker Eugene , Meyers Deborah , Vercelli Donata
Published: January 2023

Because common genetic variants account for only a modest proportion of human complex disease risk, rare variants are currently under investigation as potential sources of phenotypic variance. Focusing on IL13, one of the most replicated asthma/allergy susceptibility genes, we sought to assess the contribution of rare coding variants to disease risk by coupling sequencing-based variant discovery with in vitro studies of protein activity. Sequencing of IL13 exons in DNA from 96 non-Hispanic White and 96 African American asthmatics identified a rare non-synonymous variant (rs140828306, minor allele frequency 1.6% in African Americans) in exon 4 that results in a non-conservative arginine (R) /glutamine (Q) substitution at amino acid position 105, immediately upstream of a conserved IL-13 receptor binding domain. For functional studies, recombinant WT IL-13 and IL-13R105Q were expressed in eukaryotic cells and quantified by ELISA adjusting for differential antibody recognition of the variant. IL-13R105Q was significantly more potent than WT IL-13 in inducing STAT6 phosphorylation (16-fold) and CD23 expression (5-fold) in human monocytes, and transcription of allergy-associated genes in primary human epithelial cells grown at an air-liquid interface (3-43-fold). Neither protein acted on human T cells. These results show that IL-13R105Q is a gain-of-function variant that is likely to significantly enhance IL-13-dependent events in vivo.
Differences in macrophage polarization in allergic inflammation (HYP6P.271)
Authors: Spears Nicole , Fang Jessie , Heller Nicola
Published: January 2023

Lung macrophages (alternatively activated or “M2” macrophages) are critical modulators of the severity of pulmonary inflammation. M2 macrophages differentiate from mature macrophages upon exposure to Th2 cytokines, IL-4 and IL-13, abundant during allergic lung inflammation. Greater numbers of M2 macrophages were found in the lungs of female mice than male mice and in patients with asthma compared to healthy controls. SOCS1 and SOCS3 are negative regulators that control M1/M2 polarization of macrophages and they may be regulated by estrogen. We hypothesize that monocytes from asthmatic females are more responsive to IL-4/IL-13 than those from asthmatic men and that estrogen increases and androgen diminishes monocyte responses to IL-4 and IL-13. To compare sex differences in IL-4/IL-13 responses in monocytes, we purified and stimulated monocytes from male and female allergic and healthy donors with IL-4 and IL-13. We found high levels of SOCS3 in monocytes from allergic donors compared to healthy controls after IL-4 stimulation. Interestingly, expression of ERα mRNA was higher in allergic compared to healthy human macrophages, whereas ERβ and AR expression was lower. Differences in SOCS induction will provide clues to understanding sensitivity of monocyte-macrophages from allergic individuals to IL-4 and IL-13. The role of ERα, β and AR to IL-4-induced M2 macrophage polarization will help us understand why the prevalence of asthma in adult women is higher than in men.
ILC2 + DC = Th2, group 2 innate lymphoid cell derived IL-13 is essential for efficient T helper 2 cell differentiation in allergic lung inflammation. (HYP6P.258)
Authors: Halim Timotheus , Steer Catherine , Matha Laura , Gold Matthew , Martinez-Gonzalez Itziar , McNagny Kelly , McKenzie Andrew , Takei Fumio
Published: January 2023

Naïve CD4+ T cell differentiation into distinct subsets of T helper (Th) cells is a pivotal process in the initiation of the adaptive immune response. Allergens predominantly stimulate Th2 cells, causing allergic inflammation. However, why allergens induce Th2 cell differentiation is not understood. Here we show that Group 2 Innate Lymphoid Cells (ILC2) are required to mount a robust Th2 cell response to the protease-allergen papain. Intranasal administration of papain stimulates ILC2 and Th2 cells, causing allergic lung inflammation and elevated IgE titre. This process is severely impaired in ILC2-deficient mice. More careful analysis reveals that ILC2 derived IL-13 is important for Th2 cell differentiation. Adoptive transplant of WT but not IL-13KO ILC2s restores the Th2 response. Recombinant IL-13 administration also rescues the Th2 defect in ILC2-deficient mice. Notably, intranasal IL-13 + OVA injection efficiently drives Th2 skewing of OTII transgenic T cells. Mechanistically, we show that IL-13 has a profound effect on homing of dendritic cells to the draining lymph-node, where they prime naïve CD4+ T cells. Lung explant from IL-13KO mice cultured in a transwell system show severely impaired DC migration to lymph-node homing chemokine gradients. Moreover, these processes require IL-33-dependent ILC2 activation, which suggests a common pathway in the initiation of the Th2 response to allergens.
T helper 9 (Th9) cellular proliferation and cytokine production is regulated by cAMP responsive element modulator (Crem) (HYP7P.289)
Authors: Hufford Matthew , Jabeen Rukhsana , Kaplan Mark
Published: January 2023

Th9 cells, a CD4+ T cell subset characterized by their robust production of interleukin (IL)-9, can promote allergic inflammation in mice and are associated with atopic disease in humans. Because it might provide targets for therapeutic intervention, it is important to understand the molecular controls governing overall Th9 development and their subsequent effector function. To understand the Th9 transcriptional profile, we recently performed microarray analysis to highlight gene expression enriched in Th9 cells compared to other Th subsets. Crem, which expresses a basic leucine zip domain protein with roles in activating and repressing genes, was among a number of genes with increased expression in Th9 cells. Analysis of mRNA and protein expression among in vitro differentiated Th subsets confirmed Crem was enriched in Th9 cells compared to other subsets. Interestingly, ectopic expression of Crem in Th9 cells repressed their capacity to produce IL-9 upon CD3 re-stimulation while promoting overall cellular proliferation. Crem ectopic expression also promoted cellular expansion in other cultured Th subsets. Conversely, acute siRNA knockdown of Crem mRNA in Th9 differentiated cells resulted in reduced cellular proliferation and enhanced IL-9 production upon CD3 restimulation. Therefore, Crem potentially serves a role in modifying Th9 effector activities upon T cell receptor signaling during allergic disease.
BATF dependency of Th9-associated genes (HYP7P.290)
Authors: Jabeen Rukhsana , Awe Olufolakemi , Kaplan Mark
Published: January 2023

Th9 cells are a recently described T helper subset that develops in the presence of TGF-β and IL-4. Th9 cells promote allergic inflammation, anti-tumor immunity, and may contribute to the regulation of auto inflammatory disease. Th9 cells are specialized for IL-9 production and this requires transcription factors including STAT6, PU.1 and IRF4. We have recently described that BATF promotes Th9 development in both mouse and human cells and BATF is required for T cells to promote the development of allergic inflammation. BATF-transduced Th9 cells demonstrated a potent ability to promote allergic inflammation in vivo compared to control transduced Th9 cells. To begin to define the transcriptional network controlling genes in Th9 cells, we characterized the dependence of Th9-enriched genes on each transcription factor, including BATF. Multiple genes including transcription factors, cytokines, and cytokine receptors were BATF-dependent. Unique functional properties of Th9 cells are suggested by the Th9-enriched expression of IL-33R, IL-25R, granzyme A, ephrin A5, and the salt-sensitive serum glucocorticoid kinase. BATF is required for 14/19 genes studied in detail thus far, suggesting that it is a major controller of the Th9 genetic program. Further defining of the module of BATF-dependent genes will aid in integrating the circuitry of Th9 development and function.
Maternal and postnatal dietary probiotic supplementation enhances splenic regulatory T helper cell population and reduces ovalbumin allergen-induced hypersensitivity responses in mice (HYP7P.305)
Authors: Toomer Ondulla , Ferguson Martine , Pereira Marion , Do Andrew , Bigley Elmer , Gaines Dennis , Williams Kristina
Published: January 2023

Neonatal to early childhood is the critical period for establishing a balance of T helper 1 (Th1) versus T helper 2 (Th2) mucosal immunity, which is strongly influenced by the source and establishment of gut microflora. Probiotic administration has been shown to attenuate Th2-biased cellular immunity and predisposition to food allergies. To test this hypothesis we fed a probiotic-supplemented or control diet to lactating dams with suckling pups and weaned pups until 10 weeks of age. Weaned mice were sensitized with egg allergen ovalbumin, saline or adjuvant at 6, 8 and 10 weeks of age. At 3, 6, 8 and 10 weeks, fecal samples were collected for microbial analysis, while blood samples were analyzed for ovalbumin and total plasma IgE levels. At termination, splenic T helper population subtypes were determined by FACS analysis and gene expression by PCR array. At 21 days of age, pups suckled by lactating dams fed the probiotic supplemented diet had significantly enhanced Lactobacillus acidophilus fecal counts compared to controls. Moreover, mice fed the probiotic supplemented diet had enhanced splenic naturally occurring and induced regulatory T cell populations, enhanced TGFβ gene expression and reduced expression of allergic mediator IL13 compared to controls. These results provide evidence that early probiotic supplementation may provide host protection from hypersensitivity reactions to food allergens by attenuating food allergen inflammatory responses.
Transplacental passage of IgG anti-IgE/IgE immune complexes (HYP7P.306)
Authors: Matson Adam , Bundhoo Arvin , Paveglio Sara , Blumberg Richard , Rafti Ektor
Published: January 2023

Maternal IgG mediates the transplacental passage of several antigens; however, it is unclear if maternal IgE may be transferred across the placenta as IgG anti-IgE/IgE immune complexes. In this study, we determined maternal and cord blood serum concentrations of IgG anti-IgE/IgE immune complexes in a cohort of allergic and non-allergic mother/infant dyads. We found a strong correlation (r = 0.88) between maternal and cord serum concentrations, which is strongly suggestive of placental transmission. Subclass analysis demonstrated the majority of IgG anti-IgE/IgE immune complexes in maternal sera were of the IgG1 and IgG4 subclasses, while those in cord blood were of the IgG1 subclass. Levels of IgG1 anti-IgE/IgE immune complexes were significantly higher in allergic vs. non-allergic pregnant women. To investigate the role of FcRn in the transplacental passage of IgG anti-IgE/IgE immune complexes, we used MDCK cells stably transfected with hFcRn. IgG, IgG anti-IgE, and IgG anti-IgE/IgE immune complexes bound to hFcRn-expressing MDCK cells in a pH-dependent manner whereas IgE alone did not. The addition of an FcRn blocking antibody effectively inhibited the binding of IgG anti-IgE/IgE immune complexes. Ongoing experiments are being conducted using the MDCK cells to demonstrate FcRn-mediated transcytosis. The ability of maternal IgG anti-IgE/IgE immune complexes to bind fetal myeloid dendritic cells and regulate FcεRI-dependent pro-inflammatory responses is being investigated.
Specialized proresolving lipid mediators (SPMs) decrease human B cell IgE production: implications for allergy and asthma (HYP7P.309)
Authors: Phipps Richard , Kim Nina , Ramon Sesquile , Sime Patricia , Zehr Lindsey
Published: January 2023

Resolution of inflammation is a biologically active process regulated by newly identified specialized proresolving lipid mediators (SPMs). These endogenous lipid-derived mediators promote the resolution of inflammation and maintain homeostasis by regulating the immune system. Little is known about the functions of SPMs on adaptive immune cells, such as B cells. Our laboratory has discovered that certain omega-3 fatty acid derived SPMs promote human B cell IgM and IgG antibody production in vitro. Human B cells also produce IgE antibodies, which play an essential role in the onset of inflammatory diseases, such as seasonal allergies and asthma. Here, we show that certain SPMs decrease human B cell IgE production in vitro. Omega-3-derived SPMs, 17-HDHA (17-hydoxydocosahexaenoic acid) and RvD1 (resolvin D1), strongly decreased the IgE production in B cells, as well as the number of IgE-secreting cells in peripheral blood of healthy and asthmatic human donors. In addition, 17-HDHA and RvD1 also reduced IgE production by the spontaneously IgE secreting multiple myeloma B cell line, U266. These results show that SPMs are non-immunosuppressive endogenous mediators that could specifically dampen IgE production and have potential as therapeutics for asthma. Moreover, SPMs might also be effective on other IgE-mediated inflammatory diseases such as atopic dermatitis.
The inhibitory cytokine IL-35 in Treg-mediated control of allergic diseases (HYP7P.285)
Authors: Sawant Deepali , Corry David , Vignali Dario
Published: January 2023

Regulatory T cells (Tregs) represent a critical suppressive T cell population that maintains immune homeostasis and tolerance. Increasing evidence suggests reduced Treg numbers or compromised Treg function in atopic and asthmatic individuals. Treg derived suppressive cytokines, IL-10 and TGFβ, alleviate Th2 inflammation through allergen-specific immunotherapy. Studies on experimental asthma highlight an undisputed role for Tregs in allergic diseases; however, they fail to pinpoint a single/predominant mechanism employed by Tregs to control asthmatic inflammation. Our lab has identified a novel Treg-derived suppressive cytokine IL-35 (a heterodimer of Ebi3 and Il12a/p35) that is essential for their maximal suppressive activity. IL-35 also induces the conversion of naïve T cells into an IL-35-dependent suppressive population termed iTr35 cells. We hypothesize that IL-35-secreting Tregs and iTr35 cells are potent suppressors of Th2 allergic responses. We demonstrate that allergic inflammation induces the IL-35-mediated regulatory milieu (IL-35-expressing Tregs and iTr35 cells) in two distinct models of asthma (OVA/Alum and fungal protease). Further, IL-35 neutralization exacerbates asthmatic inflammation. Current studies using IL-35 reporter and gene-deficient mouse strains are directed at evaluating the expression, function and mechanisms of IL-35-mediated Treg control of allergic inflammation and have the potential to lead to novel IL-35-based therapeutic regimens.
Receptor-interacting protein 3 (RIP3) deficiency inhibits immune cell infiltration and attenuates organ injury in sepsis (INC6P.344)
Authors: Sharma Archna , Matsuo Shingo , Yang Weng-Lang , Wang Zhimin , Wang Ping
Published: January 2023

Sepsis is defined as a systemic inflammatory response syndrome (SIRS), leading to multiple organ dysfunction and lethality. RIP3-dependent necrosis is implicated in driving TNF-α- and sepsis-induced mortality in mice. However, it is unknown if RIP3 deficiency has any impact on immune cell trafficking, which attributes to organ damage in SIRS. To study this, male wild-type (WT) and RIP3-deficient (RIP3-KO) mice were subjected to cecal ligation and puncture (CLP), a sepsis model. Blood and tissue samples were collected at 20 h after CLP for analysis. In our severe sepsis model, the mean survival time of septic RIP3-KO mice was significantly extended from 41 h to 68 h, and their plasma levels of TNF-α, IL-6, AST, ALT and LDH were significantly decreased, compared to septic WT mice. In the lungs, septic RIP3-KO mice preserved a better integrity of microscopic structure with reduced apoptosis, and decreased the levels of IL-6, MIP2 and KC, compared to WT. In the liver, the levels of MIP2 and KC were also decreased in septic RIP3-KO mice. Particularly, the total number of neutrophils in the lungs and liver of septic RIP3-KO mice decreased by 64.6% and 52.2%, respectively, compared to septic WT mice. In addition, the number of natural killer (NK) and CD8T cells in the liver decreased by 63.9% and 50.3%, respectively, in septic RIP3-KO mice. Thus, targeting RIP3 may provide a therapeutic strategy in attenuating organ injury caused by excessive immune cell infiltration during sepsis.
Defining aspects of noncognate splenic iNKT cell help which contribute to humoral memory (INC6P.403)
Authors: Leadbetter Elizabeth , Vomhof-DeKrey Emilie , Lanthier Paula , Veerapen Natacha , Besra Gurdyal
Published: January 2023

Successful induction of pathogen-specific humoral B cell memory responses depends upon help from CD4+ T cells. We find that invariant natural killer T (iNKT) cells, glycolipid-specific, CD1d-restricted innate lymphocytes, can provide both cognate (direct) and noncognate (or indirect) helper signals to enhance B cell humoral responses. In fact, a subset of iNKT cells, termed iNKT follicular helper cells, are the cells which specifically act to promote germinal centers, facilitate optimal B cell memory, and plasma cell differentiation. While both cognate and noncognate iNKT cell help induce class-switched, antigen-specific humoral immune responses, only noncognate iNKT cell help drives formation of humoral memory and plasma cell development. We find that differences in the strength of the TcR signal received by iNKT cells, very early effector cytokine production by iNKT cells, T follicular helper cells, and T follicular regulatory cells all contribute to the observed differences in the ultimate humoral outcome. These findings help to identify the optimal requirements for iNKT induction of humoral memory, which has important implications for the application of glycolipid molecules as humoral adjuvants.
NK cell produced HMGB1 regulates DC maturation and Tim-3 expression (INC6P.339)
Authors: Kaus James , Guanqiao Li , Lotze Michael
Published: January 2023

NK cells critically mediate the maturation and activation of DCs in generating effective immune responses as well as their ability to produce cytokines, kill, and induce autophagy. Central to this maturation process is the NK cell mediated release of HMGB1, a pro-inflammatory cytokine, that plays an extracellular role in regulating immune responses as well as an intracellular role in promoting autophagy, a mechanism that allows stressed cells to survive mutagenic conditions. We examined the critical role of murine wild type (NKWT) and HMGB1 ablated (NKH) NK cells in stimulating DC maturation, using NK:DC co-cultures. In comparison with DCs cultured with NKWT cells, significant decreases in CD80, CD86, CD83, CD40, CCR7 and MHCII expressions were found using NKH co-cultures (p<.05). Furthermore, all-thiol rHMGB1 was able to restore the maturation of NKH to comparable levels (p>.05). Significant changes (p<.01) in the percent of cells expressing high levels of maturation markers CD80, CD86, CD83, MHCII were observed. Decreased expression of maturation markers under experimental conditions confirms that HMGB1 release by NK cells is necessary to induce DC maturation. In addition, anti-Tim-3 antibody coincubation increased CD86, CD80, and CD40 expression, even in the absence of HMGB1. These results demonstrate the importance of HMGB1 in stimulating DC maturation, triggering Tim-3 expression, and influencing DC function even with blockade of Tim-3 receptor.
Control of microtubule-dependent activation of nucleic acid sensors for antiviral host defenses (INM6P.419)
Authors: Chiang Hao-Sen , Zhao Yun , Song Joo-Hye , Liu Song , Wang Ninghai , Terhorst Cox , Sharpe Arlene , Basavappa Megha , Jeffrey Kate , Reinecker Hans-Christian
Published: January 2023

Viral targeting of dynein-based transport mechanisms plays an important role for intracellular movements and replication of viral pathogens. We show that the activation of the microtubule and dynein motor complex-associated guanine nucleotide exchange factor GEF-H1, encoded by Arhgef2, is essential for sensing of foreign RNA by RIG-I-like receptors. GEF-H1-deficient macrophages have a profound defect in the induction of IFN-β following detection of synthetic dsRNAs including HMW and LMW poly(I:C) and 5′ppp-dsRNA. The recognition of viral RNA and synthetic dsRNA in the MAVS pathway required the nucleotide exchange activity of GEF-H1. Furthermore, microtubule networks were required for the activation and interaction of GEF-H1 with TBK1 for IRF3 phosphorylation and subsequent induction of Ifnb1 gene expression. Generation of GEF-H1 deficient mice revealed a pronounced signaling defect that prevented antiviral host responses to encephalomyocarditis virus and influenza A virus. In conclusion, our findings identify GEF-H1 as an antiviral signaling component that directs utilization of TBK1 in the MAVS-dependent nucleic acid detection pathways for the sensing of ssRNA virus infection and induction of IFN-β expression and secretion.
Identification and characterization of potent, selective small molecule RIP2 kinase inhibitors (INM6P.422)
Authors: Lipshutz David , Haile Pamela , Cassillas Linda , Desai Biva , Capriotti Carol , Swift Barbara , Berger Scott , Sun Helen , Mahajan Mukesh , Nagilla Rakesh , Reilly Michael , Gough Peter , Bertin John , Votta Bart
Published: January 2023

NOD1 and NOD2 are cytoplasmic PRRs, which bind peptides derived from bacterial peptidoglycans. Signaling via these receptors involves recruitment and activation of RIP2 kinase, which results in NF-κB- and MAPK-dependent pro-inflammatory cytokine production. To enable a chemical biology approach to evaluate the role of RIP2 in inflammation we screened an in-house compound collection. Optimization of an aminoquinoline template led to the discovery of GSK’214 and related analogs. GSK’214 potently and reversibly inhibited human, rat and mouse RIP2 enzymatic activity (IC50 3-10 nM). Selectivity was demonstrated against a panel of 273 commercially-available kinases. NOD2-mediated signaling induced by muramyl dipeptide (MDP) was used to characterize functional activity, in vitro and in vivo. GSK’214 inhibited cytokine (TNFα, IL1β, IL-6 and IL-8) production (IC50 20 nM) by human monocytes stimulated by MDP, but not by ligands specific for TLR2, TLR4, TLR7, TLR9, IL1R and TNFR. Using an acute mouse peritonitis assay, orally administered GSK’214 inhibited neutrophil influx and cytokine release induced by MDP, but not LPS. Next, we used these inhibitors to probe the role of RIP2 in a murine TNBS-colitis model of intestinal inflammation. Selective inhibition of RIP2 kinase activity reduced disease severity to a degree comparable to prednisolone. These results demonstrate the potential of these inhibitors to elucidate the contribution of RIP2 kinase-dependent signaling in inflammation.
E3 ubiquitin ligase Cbl-b inhibits NLRP3 inflammasome activation by targeting NLRP3 for ubiquitination (INM6P.405)
Authors: Liu Qingjun , Guo Hui , Xiao Yun , Zhao Yixia , Zhang Jian
Published: January 2023

NLRP3 inflammasome has been shown to be involved in antiviral and antibacterial innate immunity, as well as in the response to adjuvants critical for adaptive immunity. However, inappropriate activation of NLRP3 contributes to deleterious inflammatory syndromes. Although recent studies show that NLRP3 undergoes ubiquitination, the mechanisms for negatively regulating the activation of the NLRP3 inflammasome in response to PAMPs or endogenous DAMPs are largely unknown. In this study, we found that Cbl-b selectively inhibits NLRP3 inflammasome activation, leaving the other key innate response pathways. Further indication of a negative regulation by Cbl-b on NLRP3 inflammasome activity is the finding that LPS injection induces a significant mortality in Cbl-b-/- mice compared to wild-type (WT) controls, and this increased mortality in Cbl-b-/- mice is blocked by an IL-1 receptor antagonist. At the molecular levels, Cbl-b binds to NLRP3 upon LPS priming and ATP stimulation independently of ASC. NLRP3 undergoes ubiquitination in WT macrophages upon NLRP3 inflammasome stimulation, whereas its ubiquitination is impaired in Cbl-b-/- macrophages. Interestingly, NLRP3 inflammasome stimulation also induces Cbl-b tyrosine phosphorylation, which is critical for its ubiquitin ligase activity, thus initiating a negative feedback that controls NLRP3 inflammasome activity. Therefore, we provide a previously-unappreciated function for Cbl-b in regulating NLRP3 inflammasome activation.
DNA sensing by innate immune receptors (INM6P.408)
Authors: Xiao Tsan , Jin Tengchuan , Chuenchor Watchalee , Ravilious Geoffrey , Jiang Jiansheng , Smith Patrick
Published: January 2023

Nucleic acids are amongst the most potent stimuli of host cells. Interferons and proinflammatory cytokines induced by nucleic acids have pleiotropic effects on the function of both innate and adaptive immune systems. Several families of innate immune receptors at different subcellular locations have evolved to recognize DNA and RNA molecules from both invading microbes and damaged host tissues. Recent findings on DNA sensing by cytosolic receptors AIM2, IFI16 and DHX36 helicase will be discussed. Structure-function studies of these receptors reveal a common mechanism of sequence-independent recognition through electrostatic attractions. As such, highly charged small molecules or peptides are potential candidates for therapeutic intervention of inflammatory conditions involving nucleic acid sensing. This work is supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA.
Profiling ileal immune response genes in obese rats induced by high-fat diet and adenovirus infection (INM7P.432)
Authors: Sang Yongming , Schade Andrew , Blecha Frank
Published: January 2023

Recent studies have shown that an interaction among high-fat diet (HF), gut microbiota and the immune system evokes chronic inflammation in both intestinal and adipose tissues. To profile signature genes regulated by HF and intestinal infection, we induced obese rats by feeding a HF plus a human adenovirus 36 (Ad-36) infection. Compared to control groups, rats in the HF and HF plus Ad-36 groups showed significant increases in body weight, adiposity index, as well as intestinal permeability at 8 weeks post treatment. RNA-Seq analysis was used to examine the comparative transcriptome of ileal samples from animals of each group. Analysis of differentially expressed genes (DEGs) identified 3,998 genes significantly regulated among treated and control rats. Genome-wide examination of innate immune genes was used to cluster significantly up- and down-regulated DEGs, which might mediate chronic inflammation involved in obesity stimulated by HF and Ad-36 infection. In addition, we identified other prominent DEGs pertinent to transcription factors, steroid/cholesterol /lipid metabolism, as well as many functionally uncharacterized transcripts. Interestingly, pathway analysis suggested that the circadian rhythm and intestinal immune network for IgA production were significantly regulated pathways related to obesity. In summary, our findings identified early gene regulation occurring in the intestine during obesity and viral infection that may influence intestinal inflammation.
Diabetes exacerbates infection severity due to hyperinflammation rather than neutrophil defects (INM7P.425)
Authors: Bruhn Kevin , Pantapalangkoor Paul , Nielsen Travis , Tan Brandon , Bonomo Robert , Duncanson Frederick , Everson Michael , Doherty Terence , Lin Lin , Spellberg Brad
Published: January 2023

Patients with diabetes mellitus are at increased risk of dying from Gram negative bacterial infections. This excess mortality is often attributed to immune suppression, including neutrophil dysfunction. However, diabetic hosts also exhibit increased inflammatory responses to LPS. Here we show that lethal infections with Acinetobacter baumannii in diabetic mice are driven by LPS interactions with TLR4, as opposed to neutrophil defects. Mice made diabetic either by streptozotocin (Type 1) or diet induced obesity (Type 2) were hypersusceptible to lethality from A. baumannii bacteremia, whereas mice made neutropenic by anti-Ly6G administration were not more susceptible to infection. Diabetic infected mice developed severe septic shock, with hypothermia, medical acidosis, and marked elevations in inflammatory cytokines, compared to non-diabetic mice. Diabetic mice with impaired or absent TLR4 function, however, were protected against lethal infection and did not develop sepsis. Collectively, these results suggest that enhanced lethality of A. baumannii infection in diabetic mice is not driven by diabetes-related abrogation of neutrophil function. Rather, diabetic mice are more prone to progression to severe sepsis syndrome and are protected from this progression by impairment of TLR4 function. Short term abrogation of TLR4 signaling is a promising therapeutic approach to the treatment of A. baumannii, and possibly other Gram negative infections in diabetic hosts.
Aberrant insulin signaling results in mTOR suppression and immune dysfunction during diabetic infections. (INM7P.427)
Authors: Thurlow Lance , Richardson Anthony
Published: January 2023

Diabetics have defects in several aspects of immunity that result in increased infection rates; however, the underlying mechanisms of immune dysfunction are not well defined. Previous studies in this area concluded that infections in diabetics are hyper-inflamed as indicated by increased cytokine production. Conversely, despite high levels of pro-inflammatory cytokines, our research shows that innate immune cells from diabetics cannot produce inflammatory effectors such as reactive oxygen species (ROS) and nitric oxide (NO). ROS/RNS generation by immune cells is essential for killing bacteria and requires cells to undergo a metabolic shift reminiscent of Warburg metabolism. This shift is required for NADPH production, the sole reductant needed for ROS/RNS generation. Using murine infection models and cell culture, we show that Toll-like receptor signaling induces Warburg metabolism through activation of the mammalian target of rapamycin (mTOR). Moreover, we show that mTOR signaling is suppressed in diabetics and results in decreased ROS/RNS generation and worse infection outcomes. Finally, we demonstrate that mTOR inhibition in diabetes is caused by the activation of AMP-activated protein kinase (AMPK) as a result of deficient insulin signaling. Thus, diabetic infections are not hyper-inflamed. Instead, immune dysfunction in diabetic infections is caused by aberrant insulin signaling resulting in mTOR suppression and diminished antimicrobial effector production.
Type I IFN signaling promotes Coxiella burnetii infection (INM8P.446)
Authors: Hedges Jodi , Robison Amanda , Christensen Kelly , Ramstead Andrew , Jutila Mark
Published: January 2023

Coxiella burnetii is an obligate intracellular pathogen and the cause of Q fever. The strain Nine Mile Phase I (NMI) is virulent whereas a laboratory passaged avirulent form is referred to as Phase II (NMII). Interferon (IFN)-γ is critical for protection from C. burnetii, but a role for type I IFN has not been determined. Type I IFN supports host protection from a related pathogen, Legionella pneumophila, thus we hypothesized that it would be similarly protective in C. burnetii infection. When murine macrophages were treated with IFN-α in vitro at infection, replication of NMII decreased, however, treatment 24 hours post-infection enhanced replication. Infection of spleen cells from type I IFN receptor (IFNAR)-deficient mice in vitro yielded fewer NMII genomic equivalents compared to wild type, with no change in IFN-γ expression. Thus, type I IFN signaling early in infection protected against NMII replication, but later signaling promoted replication. In vivo studies using the virulent NMI similarly suggested that IFNAR signaling promoted pathogenesis. IFNAR-deficient mice infected with NMI were significantly protected from weight loss compared to wild-type mice, although they had larger spleens and similar C. burnetii genomic equivalents. The data suggest that type I IFN signaling promotes infection with C. burnetii, contrary to the case with L. pneumophila. These results support abrogation of this pathway as a potential therapeutic approach to Q fever.
Autophagy enhances NFκB activity and boosts early anti-fungal immunity (INM8P.447)
Authors: Kanayama Masashi , Inoue Makoto , He You-Wen , Shinohara Mari
Published: January 2023

To avoid excessive inflammation, molecular signaling that triggers immune responses has to be well restrained by various cellular and molecular regulatory mechanisms. However, in infections, such restraints must be removed to exert quick and efficient anti-microbial responses. Here, we report a novel role of autophagy in systemic Candida infection by enhancing early immune responses through increased nuclear factor kappa B (NFκB) activity. First, we found that Atg7 conditional knockout (CKO) mice with LysM-Cre were susceptible to Candida infection. Our ex vivo experiments showed that autophagy did not affect Candida-phagocytosis and inhibition of Candida expansion by macrophages and neutrophils. We also found that phagocytosed conidia were not contained in LC3-positive vesicles. The data suggested that it is unlikely that autophagy played a role in “direct” Candida killing by myeloid cells. Unexpectedly, instead, enhancement of NFκB activation was observed in a certain macrophage subset. Enhancement of NFκB activation was achieved by autophagy-mediated sequestration of an NFκB inhibitor. This allows the macrophages to release chemokines to effectively attract neutrophils to infected sites, resulting in better resistance to Candida in wild-type mice compared to Atg7 CKO mice. Thus, autophagy protects mice from systemic Candida infection by a mechanism distinct from those that protect mice from bacteria and viruses.
Targeting of the surfactant protein A receptor SP-R210L variant by influenza A virus in macrophages. (INM8P.440)
Authors: Yang Linlin , Hu Sanmei , Chroneos Zissis
Published: January 2023

Alveolar macrophages (AM) are critical for controlling IAV infection by limiting virus release and by initiating innate and adaptive immunity. Surfactant proteins A (SP-A) and D (SP-D) are a primary line of defense against IAV. Previous studies demonstrated that the SP-A receptor SP-R210 mediates clearance of SP-A-opsonized pathogens. Macrophages express two variants, SP-R210L and SP-R210S, though SP-R210L is predominant on AMs. Here, we studied the role of SP-R210 in IAV infection. We show that disruption of SP-R210L in vitro blocked IAV infection with H3N2 and H1N1 IAV strains. Flow cytometry and microscopy experiments showed that SP-R210L is required for endocytic trafficking of IAV to the nucleus. Furthermore, SP-R210L-mediated infection of macrophages was coupled to the production of TNFα. Interestingly, SP-R210L-deficient macrophages were hyper-responsive to the TLR7 ligand imiquimod. Accordingly, IAV inhibited SP-R210L expression in a time-dependent manner. To address these results in vivo, we studied transgenic mice with conditional disruption of SP-R210 in macrophages; SP-R210-deficient mice were significantly more susceptible to IAV infection as indicated by increased morbidity, decreased survival, and severe lung histopathology compared to WT littermates. Taken together, these findings support the model that by co-opting SP-R210L in AMs, IAV causes a functional ‘knock-down’, reducing the beneficial function of SP-R210L and leading to excessive lung inflammation.
Signaling and function of STAT3 in NK cells during murine cytomegalovirus infection (INM8P.442)
Authors: Tarrio Margarite , Kallal Lara , Sexl Veronika , Biron Christine
Published: January 2023

Cytokines induced during viral infection regulate NK cell functions to optimize antiviral defense and health. Type 1 IFNs (IFNαβ) and IL-12 employ STAT molecules to mediate their effects. In this study, we characterized STAT3 signaling and function within NK cells during infections of mice with murine cytomegalovirus (MCMV). Basally, NK cells activated STAT3 in response to IL-10, IFNα, and IL-12. During MCMV infection, with high circulating IL-12 and IFNα but not IL-10 levels on D1.5, NK cells had elevated STAT3 and both pSTAT4 and pSTAT3. The pathways for the changes were characterized as IFN-dependent induction of STAT3 with IL-12-activated pSTAT4 and pSTAT3. In comparison to wild-type (wt) littermate controls, mice with an NKp46-cell-specific STAT3 deletion within NK cells had comparable D1.5 responses and viral titers in spleens and livers through D4 of infection. In addition, NK cell numbers were maintained or increased as the infection progressed. Surprisingly, mice specifically lacking STAT3 in NK cells but not wt succumbed to high dose MCMV challenge conditions at D5.5. Thus, IL-12-dependent STAT3 activation is part of an ongoing response to infection, and STAT3 within NK cells plays a critical role in host survival. Ongoing studies are delineating the STAT3-dependent mechanism within NK cells functioning for host protection.
CD40 ligand-expressing T helper cells induce networks of tunneling nanotubes in dendritic cells activated by mediators of type-1 immunity (IRC5P.463)
Authors: Zaccard Colleen , Watkins Simon , Fecek Ronald , Kalinski Pawel , Salter Russell , Ayyavoo Velpandi , Rinaldo Charles , Mailliard Robbie
Published: January 2023

The ability of DC to mediate CD4+ T cell help for initiating cellular immunity greatly depends on instructive signals that they receive during maturation. Here we report that, in addition to producing high levels of IL-12p70, DC matured in the presence of type-1 associated inflammatory factors were also uniquely programmed to ‘reticulate’, or develop networks of tunneling nanotube-like structures in response to the CD4+ T helper cell derived signal, CD40L. Using high resolution live-cell imaging techniques, we demonstrated that tunneling nanotube networks are capable of facilitating the direct intercellular trafficking of endosome-associated vesicles, antigens, and HIV-1-like particles. Importantly, DC matured under non-type-1 polarizing conditions, such as those exposed to prostaglandin E2, failed to develop these membrane extensions. Moreover, we found that the T helper 1 associated cytokine IFN-γ plays a critical role in enhancing DC reticulation, while the T helper 2 cytokine IL-4 has a substantial inhibitory effect on this process. These data indicate that subsets of T helper cells have divergent abilities to modulate DC reticulation and subsequent intercellular communication. The induction of the reticulation process in DC represents a novel immunologic helper function of CD40L-expressing T cells, which can also be exploited by pathogens such as HIV-1 to promote direct cell-to-cell spread.