bluuebunny/crossref_metadata_2025 huggingface dataset:
CD163/WC1 functions as a pattern recognition receptor and co-receptor for the γδ TCR (113.18)
Authors: Hsu Haoting , Cheng Chuang , Baldwin Cynthia , Telfer Janice
Published: January 2023

CD163/WC1 proteins, specifically expressed by γδ T cells, are members of the scavenger receptor cysteine-rich (SRCR) domain family. Phosphorylation of a tyrosine in the WC1 cytoplasmic domain upon co-ligation of WC1 and TCR is required for the WC1 co-receptor activity. Two out of fourteen WC1 proteins contribute to the γδ T cell response to the spirochete Leptospira. Other closely related SRCR family members (e.g. DMBT1, CD6 and CD163A) bind to bacteria, suggesting that WC1 acts a signaling co-receptor in γδ T cell activation via Leptospira ligation. Jurkat cells expressing the chimeric protein CD4/WC1cyt, CD4/WC1cyt mutated on a dileucine endocytic motif, and CD4/WC1cyt mutated on two serines upstream of the dileucine motif were treated with PMA, resulting in endocytosis of wild type protein. The leucine mutants reduced PMA-induced endocytosis and enhanced IL-2 production stimulated by co-crosslinking of CD3 and CD4/WC1cyt, suggesting that sustained membrane co-ligation with CD3 increases WC1 signaling. The serine mutation had no effect on endocytosis, but did decrease IL-2 production, suggesting that serine phosphorylation of the cytoplasmic domain of WC1 is involved in T cell signaling. The N-terminal SRCR domain of the WC1 protein previously implicated in the Leptospira response binds strongly and specifically to Leptospira, supporting a model in which WC1 acts both as a pathogen recognition receptor (PRR) and a membrane proximal signaling co-receptor to the γδ TCR.
Identification of a novel immunosuppressive dendritic cell population derived from conventional DC (113.5)
Authors: Mikhailova Anastassia , Diao Jun , Cattral Mark
Published: January 2023

Dendritic cells (DC) play a central role in the initiation and regulation of immune responses. Conventional DC (cDC) arise from circulating immediate precursors (pre-cDC) and are currently thought to be terminally differentiated. Here we show, however, that cDC are capable of generating progeny that lost all characteristic features of cDC; and investigate the functional properties of this novel population. Sorted bone marrow pre-cDCs were cultured on a stromal monolayer in the presence/absence of granulocyte-macrophage colony stimulating factor (GM-CSF). On day three, proliferating CD11c+ MHCII+ cDC developed in both cultures. However, in the absence of GM-CSF, these cells gave rise to a homogeneous population of CD11clow MHClow cells (DC-regs) on day 8-10 of culture. In contrast to cDCs, DC-regs failed to up-regulate MHCII and co-stimulatory molecules in response to DC maturation stimuli. DC-regs displayed higher antigen capture capacity but were poor stimulators in T cell proliferation assays. Further, DC-regs not only induced T cell anergy, but also suppressed T cell proliferation in cultures containing immuno-stimulatory DC. Co-transfer of DC-regs with DCs in vivo inhibited T cell proliferation. DC-regs expressed know immunosuppressive molecules such as iNOS, arginase, ICOSL and PD-L1. Our findings challenge the prevalent view that cDC are terminally differentiated, and reveal their potential to generate a regulatory DC population with potent immunosuppressive properties.
Regulation of skin inflammation and T cell activity by annexin A1 (114.3)
Authors: Yang Yuan , Deane James , Song Wuqi , Hickey Michael , Morand Eric
Published: January 2023

Annexin A1 (Anxa1) is a mediator of the anti-inflammatory actions of glucocorticoids. We explored the role of Anxa1 in regulation of the T cell-mediated contact sensitivity (CS) model of skin inflammation. CS was induced in Anxa1-/- and wild type mice by sensitisation to oxazolone at day 0 and subsequent challenge with oxazolone at day 5 (first challenge) and day 7 (second challenge). Deficiency of Anxa1 significantly increased CD4+ T cell proliferation in the draining LN after the first challenge, although there was no significant difference in CS-induced skin edema between WT and Anxa1-/- mice. In contrast, a significant increase in skin edema was observed in Anxa1-/- mice after a second CS challenge, accompanied by increased antigen-specific proliferation of CD4+ and CD8+ T cells obtained from spleen and skin. Anxa1-/- mice displayed significantly increased RORγT, and IL-17A mRNA in LN after a second challenge, indicating Anxa1 regulation of T cell activity in CS related to the Th17 driven immune response. Intravital microscopy studies showed that Anxa1 deficiency was associated with significantly increased CD4+ adhesion in dermal venules after the first challenge, and CD4+ and CD8+ adhesion after the second challenge. These data demonstrate that endogenous Anxa1 is a potent inhibitory regulator of T cell activity in skin inflammation, potentially via an inhibitory effect on development of the Th17 axis.
IL-2 controls the stability of Foxp3 expression in TGF-β-induced Foxp3+ T cells in vivo (115.11)
Authors: Chen Qian
Published: January 2023

Stimulation of naïve mouse CD4+Foxp3- T cells in the presence of TGF-β results in the induction of Foxp3 expression and T suppressor function. However, Foxp3 expression in these induced Treg (iTreg) is unstable raising the possibility that iTreg would not be useful for treatment of autoimmune diseases. To analyze the factors that control the stability of Foxp3 expression in iTreg, we generated OVA-specific iTreg from OT-II Foxp3-GFP knock in mice. Following transfer to normal C57BL/6 mice, OT-II GFP+ cells maintained high levels of Foxp3 for 8 days. However, they rapidly lost Foxp3 expression upon stimulation with OVA in IFA in vivo. This unstable phenotype was associated with a strong methylation of the Treg-specific demethylated region (TSDR) within the Foxp3 locus. Administration of IL-2/anti-IL-2 complexes expanded the numbers of transferred Foxp3+ iTreg in the absence of antigen challenge. Notably, when the iTreg were stimulated with antigen, treatment with IL-2/anti-IL-2 complexes stabilized Foxp3 expression and resulted in enhanced demethylation of the TSDR. Conversely, neutralization of IL-2 diminished Foxp3 expression resulting in decreased suppressor function of the iTreg in vivo. Our data suggest that stimulation with TGF-β in vitro is not sufficient for imprinting T cells with stable Foxp3 expression. Administration of IL-2 in vivo stabilizes Foxp3 expression and may prove to be a valuable adjunct for the use of iTreg for the treatment of autoimmune diseases.
Therapeutic effects of TGF-β-induced iTregs on established autoimmune diseases (115.3)
Authors: Lan Qin , Wang Julie , Brand David , Stohl William , Zou Hejian , Horwitz David , Liu Zhongmin , Zheng Song Guo
Published: January 2023

While recent studies reveled injection of nTregs has less therapeutic effects on established autoimmune diseases, it is less clear if iTregs can treat these diseases. We now reveal that unlike nTregs, injection of iTregs can markedly ameliorate established autoimmune arthritis and lupus. Both antigen-specific and polyclonally induced iTregs suppressed the established CIA. CIA mice given iTregs had significantly lower clinic scores than control groups. We found nTregs but not iTregs were converted into Th1/Th17 cells in vitro and in vivo in the inflammatory milieu, injection of iTregs to naïve and immune deficient mice displayed similar levels of Foxp3 stability as comparing with nTregs. Of note, the stability of Foxp3 expression was only found in iTregs during established CIA. iTregs suppressed Th17 and osteoclast differentiation. Injection of iTregs to established lupus decreased levels of anti-dsDNA and proteinuria, and prolonged lupus survival. Anti-TGF-β Ab or TGF-βRI inhibitor, or anti-IL-10R Ab abolished the therapeutic effects of iTregs on lupus. We further observed that DC isolated from lupus received iTregs expressed lower levels of B7.1/7.2 and adoptive transfer of these DCs to another lupus mouse can suppress the disease development. We suggest that iTregs are stable and able to target DC in the inflammatory milieu, these DC then have become tolerogenic DC and further suppress disease progression via its direct or indirect effect in autoimmune disease settings.
Regulatory T cell perturbation in biliary atresia (115.4)
Authors: Tucker Rebecca , Brindley Stephen , Mack Cara
Published: January 2023

Biliary atresia (BA) is an inflammatory disease of the biliary system which leads to cirrhosis and the need for liver transplantation in the majority of children. The etiology of BA is unknown and one theory is that bile duct injury is due to a virus-induced, subsequent autoreactive T cell-mediated inflammatory insult. Perturbation of the quantity or function of regulatory T cells (Tregs) in BA could result in unchecked autoimmunity. The aim of this study is to determine if Tregs are diminished or dysfunctional in murine and human BA. Murine BA results from infection of BALB/c neonates with Rhesus rotavirus (RRV). Mice infected with RRV on day 1 that received either Tregs or control T cells on day 5 led to a difference in disease status as early as 4 days post-transfer. This trend continued and biliary disease occurred in 67% of controls vs. 39% of Tregs recipients at 17 days of life. Secondly, ablation of functional Tregs in 4 day-old mice by treatment with a CD25-depleting antibody followed by RRV infection 3 days later results in increased bile duct infiltrate and disease incidence at 3 weeks of age. Finally, analysis of human PBMCs revealed a decreased percentage of FoxP3+ Tregs in BA patients (1.5±0.3%) compared to age-matched controls (4.1±0.7%). These studies suggest that lack of or dysregulation of Tregs is present in the murine model of BA and that diminished numbers of Tregs may be implicated in human BA progression.
Asp299Gly polymorphism decreases TLR4-elicited activation of MyD88- and TRIF-dependent signaling pathways (116.17)
Authors: Figueroa Leandra , Medvedev Andrei
Published: January 2023

Innate immune cells detect Gram-negative bacterial lipopolysaccharide (LPS) via Toll-like receptor (TLR) 4 that employs myeloid differentiation primary response gene (MyD) 88 and TIR-domain containing adapter-inducing IFN-β (TRIF) pathways to activate NF-κB, IRF3, and induce pro-inflammatory cytokines and type I IFNs, respectively. Asp299Gly and Thr399Ile TLR4 polymorphisms have been associated with an increased risk of Gram-negative sepsis and certain bacterial infections. The mechanisms by which these mutations impact TLR4 signal transduction are incompletely understood. We used transfection-based complementation of TLR4-/- HEK293 cells with CD14, MD2, wild-type (WT) or polymorphic YFP-TLR4s to determine the impact of the Asp299Gly and Thr399Ile polymorphisms on LPS binding and TLR4-elicited activation of MyD88 and TRIF pathways. In contrast to strong LPS responses induced by WT TLR4, the Asp299Gly variant elicited decreased phosphorylation of p38 and IRF3, activation of NF-κB- and IFN-β-driven reporters, and expression of IL-8 mRNA. 293/MD2 cells expressing WT or polymorphic TLR4s showed comparable LPS binding, and had similar total levels of transfected YFP-TLR4s and Flag-MD2, as well as similar expression of total p38 and β-tubulin. Thus, the D299G polymorphism compromises TLR4-mediated activation of MyD88 and TRIF-dependent pathways, and these signaling deficiencies are not due to lower expression levels of the TLR4 receptor complex.
Sensing bacterial viability for host defense (116.23)
Authors: Sander Leif , Davis Michael , Boekschoten Mark , Dascher Christopher , Ryffel Bernard , Amsen Derk , Swanson Joel , Mueller Michael , Blander Julie
Published: January 2023

The innate immune system safeguards the host from invading microorganisms by eliciting carefully measured responses. For example, weaker responses are mounted to dead than to viable microorganisms, a property with important implications for vaccination and host defense. Pathogen associated molecular patterns (PAMPs), which serve to alert the immune system, are present in dead and viable bacteria, raising the question as to how the immune system discriminates between the two. Here we show that the innate immune system can directly sense bacterial viability through detection of a special class of viability-associated PAMPs (vita-PAMPs). We identify prokaryotic messenger RNA (mRNA) as a vita-PAMP present only in viable bacteria, recognition of which elicits a unique innate immune response. Detection of vita-PAMPs in phagocytosed bacteria induces robust production of IFN-β and activation of the NLRP3 inflammasome in various innate immune cells. All responses require the Toll-like receptor adaptor protein TRIF. Importantly, recognition of vita-PAMPs in vivo induces host protective antibody mediated immunity. Thus, the immune system actively gauges the infectious risk by scouring PAMPs for signatures of microbial life and thus infectivity. Detection of vita-PAMPs triggers an alert mode not warranted for dead bacteria. Vaccine formulations that incorporate vita-PAMPs could combine the superior protection of live vaccines with the safety of dead vaccines.
Ribozyme mediated alternative poly-adenylation of the inhibitory NK-ligand Clec2d. (116.36)
Authors: Horan Lucas
Published: January 2023

Structured RNAs that are embedded in mature mRNAs have been shown to effectively regulate gene expression in a variety of single-celled organisms. Our discovery of conserved mammalian hammerhead ribozymes located in the 3‘-untranslated regions (UTR) of certain C-type lectin type II (CLEC2) genes suggests that a similar form of regulation may also be operative in more complex organisms. The ribozyme-associated Clec2d gene codes for a broadly expressed cell-surface ligand, CLEC2D, that is involved in suppressing natural killer cell-mediated lysis of CLEC2D expressing cells. Published data show that CLEC2D is downregulated in infected cells and cells exposed to various forms of stress. We hypothesize that the location of the fast cleaving ribozyme in the 3‘UTR of a dynamically expressed gene implicates the ribozyme in regulating CLEC2D expression. Previously described regulatory ribozymes (both naturally occurring and engineered) induce loss of gene expression through ribozyme cleavage and stabilize gene expression by preventing ribozyme cleavage. Surprisingly we do find that cells expressing CLEC2D protein predominantly contain transcripts that are poly-adenylated at the ribozyme cleavage site. This directly implicates the mClec2d ribozyme in an alternative poly-adenylation mechanism and suggests ribozyme cleavage can be involved in stabilizing protein expression.
Evolving function of Toll-like receptor pathways (116.6)
Authors: Charette Jeremy , Phennicie Ryan , Pietraszewski Matthew , Lage Christopher , Smalley David , Kim Carol
Published: January 2023

The innate and adaptive arms of immunity work synergistically to mount robust host defenses against invading pathogens. The zebrafish lacks a fully functional adaptive immune system for the first four to six weeks of development, making it an ideal model for studying many aspects of innate immunity, including the Toll-like receptor (TLR) pathways. In mammals, TLRs recognize specific pathogen associated molecular patterns and, acting through adaptor molecules such as MYD88 and TICAM1, elicit an array of pro- and anti-inflammatory responses. In two recent studies we described differences in TLR ligand recognition and downstream signaling in the zebrafish, and proved that sequence homology does not necessarily indicate functional homology. These data support the notion that alternative signaling pathways may have evolved in the zebrafish. To characterize this system, ligands of the zebrafish TLRs and the intracellular protein-protein interactions were studied. Here we report the identification of unique interactors and targeted morpholino knock-down of TLR-associated genes to analyze the impact on signaling of each each protein-protein interaction within zebrafish TLR pathways. The discovery of novel protein interactions in the zebrafish highlights the potential of this model system for studies of innate immune function and further elucidates the evolution of vertebrate immunity.
Genetic deletion of chemokine receptor Ccr6 decreases atherogenesis in ApoE-deficient mice (117.2)
Authors: Wan Wuzhou , Lim Jean , Lionakis Michail , Rivollier Aymeric , McDermott David , Kelsall Brian , Farber Joshua , Murphy Philip
Published: January 2023

Background—The chemokine receptor CCR6 and its ligand CCL20 are both expressed in human atheroma; however, their functional roles in atherogenesis remain undefined. Here, we addressed this question in the apolipoprotein E-deficient (ApoE-/-) mouse model of atherosclerosis. Methods and Results—Both Ccr6 and Ccl20 are expressed in atherosclerotic aorta from ApoE-/- mice. Atherosclerotic lesion area in the aorta in Ccr6-/-ApoE-/- mice was ~40% and ~30% smaller than in Ccr6+/+ApoE-/- mice at 16 and 24 weeks of age, respectively. In addition, lesions of Ccr6-/-ApoE-/- mice had 44% less macrophage content compared to Ccr6+/+ApoE-/- mice. Since monocytes are the source of lesional macrophages, we tested whether monocytes express Ccr6 and the effect of Ccr6 deletion on monocyte function. We found that Ccr6 was expressed on a subset of primary mouse monocytes and on the mouse monocyte/macrophage cell line RAW 264.7. The receptor was functional, as defined by Ccl20-induced chemotaxis of primary monocytes from wild type but not Ccr6-/- mice; moreover, Ccl20 induced monocytosis in ApoE-/- mice in vivo. Consistent with this, we observed 30% fewer monocytes in the circulating blood of Ccr6-/-ApoE-/- mice, mainly due to fewer CD11b+Ly6Chigh inflammatory monocytes. Conclusions—We conclude that Ccr6 promotes atherosclerosis in ApoE-deficient mice. This may be due in part to Ccr6 support of normal monocyte levels in blood in the model, as well as direct Ccr6-dependent monocyte migration.
Acute exercise alters the Type 1/Type 2 cytokine balance in both low (CD27+) and highly (CD27-) differentiated subsets of CD4+ and CD8+ T cells (117.23)
Authors: LaVoy Emily , Lowder Thomas , Simpson Richard
Published: January 2023

Highly differentiated T cells are preferentially mobilized into the peripheral blood in response to exercise, which may explain some of the observed changes in immune function that occur with strenuous exercise. Although acute exercise has been shown to alter the type1/type2 cytokine balance, no study has attempted to document if this is due to increased frequency of highly differentiated T cells in the periphery. Eight trained male cyclists (Age: 31±6.1 yrs) cycled for 60-min at 95% maximal steady state. Blood samples collected before, immediately after, and 1h after exercise were prepared for cell culture and flow cytometry analysis. Proportions of highly differentiated (KLRG1+CD28-;CD45RA+CD27-) T cells increased immediately after exercise. This was accompanied by decreased intracellular expression of IL-2, IL-4, IL-6, TNF-α, and IFN-γ, and increased IL-10 in T cells following mitogen stimulation. Cytokine changes with exercise were similar in both CD27+ (low differentiated) and CD27- (highly differentiated) cells. Independently of exercise, CD27+ cells produced more IL-2 and IL-6 but less TNF-α and IFN-γ than CD27- cells. These data indicate that acute exercise alters the type1/type2 cytokine balance in response to mitogen stimulation, but independently of altered proportions of low and highly differentiated T-cells. Future work will attempt to establish mechanisms for this response and the role that type 1/type 2 cytokine shifts might play in athlete infection risk.
Paracrine IL-33 stimulation enhances lipopolysaccharide-mediated macrophage activation (117.30)
Authors: Ohno Tatsukuni , Nakae Susumu , Azuma Miyuki
Published: January 2023

BACKGROUND: IL-33, a member of the IL-1 family of cytokines, provokes Th2-type inflammation accompanied by accumulation of eosinophils through IL-33R, which consists of ST2 and IL-1RAcP. We previously demonstrated that macrophages produce IL-33 in response to LPS. Some immune responses were shown to differ between ST2-deficient mice and soluble ST2-Fc fusion protein-treated mice. Even in anti-ST2 antibody (Ab)-treated mice, the phenotypes differed between distinct Ab clones, because the characterization of such Abs (i.e., depletion, agonistic or blocking Abs) was unclear in some cases. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the precise role of IL-33, we newly generated neutralizing monoclonal Abs for IL-33. Exogenous IL-33 potentiated LPS-mediated cytokine production by macrophages. That LPS-mediated cytokine production by macrophages was suppressed by inhibition of endogenous IL-33 by the anti-IL-33 neutralizing mAbs. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that LPS-mediated macrophage activation is accelerated by macrophage-derived paracrine IL-33 stimulation.
Distribution of cells and cytokines in the spleen of dogs naturally infected with Leishmania chagasi (117.31)
Authors: Silva Joselli , Andrade Alan , Santana Claudia , Tavares Veras Patricia , Vassalo José , Conrado-Dos Santos Washington
Published: January 2023

Visceral leishmaniasis is associated with architectural disorganization of the splenic tissue in human and in dogs. In this study we estimate the changes in the number of Ki-67+ (proliferating cells), CD3+ (T cells), α-CD79+ (cells B) and S100+ (follicular dendritic cells) and in the expression of CXCL13, LTα, TNF, IFN-gamma, IL10, TGFbeta, CCR7, CCL19, CCL21 in the spleens of dogs from an endemic area of visceral leishmaniasis, with organized or disorganized splenic architecture. The number of T cells, B cells, proliferating cells and follicular dendritic cells was lower in the lymphoid follicle of the animals with disorganized splenic tissue than in animals with organized splenic tissue (P < 0.01). The number of B cells was also reduced in the MZ of animals with disorganized splenic tissue (P < 0.01). B cells (P < 0.01) and proliferating cells (P < 0.05) density was lower in the lymphoid follicles of animals with disorganized splenic tissue. CXCL13 (P < 0.02) and LT-α (P < 0.05) expressions were lower in the spleens of animals with disorganized splenic tissue than with organized splenic tissue. There is a correlation between the disorganization of the splenic tissue and abnormal cell distribution and expression of cytokines involved in architectural organization of the splenic lymphoid tissue. These alterations of the spleen may impair leukocyte cooperation against infection by ,Leishmania and other pathogens.
Trans-signaling mediates some key actions of interleukin-6 in the central nervous system (117.6)
Authors: Campbell Iain , Lim Sue , Frausto Ricardo , Rose-John Stefan , Scheller Jürgen
Published: January 2023

IL-6 which is implicated in the pathogenesis of immunoinflammatory disorders of the CNS, signals via binding to either the membrane bound IL-6Rα (classical signaling) or soluble (s)IL-6Rα (trans-signaling) that then form a complex with gp130 to activate the JAK/STAT and SHP2/MAPK pathways. Here we determined the relative importance of classical- versus trans-signaling in mediating IL-6 actions in the CNS. Bigenic mice were generated with CNS-restricted, astrocyte-targeted production of IL-6 and the specific inhibitor of IL-6 trans-signaling, sgp130-Fc (termed GFAP-IL6/sgp130 mice). Transgene-encoded IL-6 mRNA levels were similar in the brain of GFAP-IL6 and GFAP-IL6/sgp130 mice. However, GFAP-IL6/sgp130 mice had decreased pY-STAT3 in the brain due to a marked reduction in the total number of pY-STAT3-positive cells and the absence of detectable pY-STAT3 in specific cell types. These changes in pY-STAT3 were associated with disproportionate reductions in various inflammatory markers as well as reduced vascular pathology and blood-brain barrier leakage. Degenerative changes in the cerebellum characteristic of GFAP-IL6 mice were almost absent in GFAP-IL6/sgp130 mice. The findings suggest that in the CNS: (i) sgp130-Fc is able to block IL-6 trans-signaling, (ii) trans-signaling is important for IL-6 cellular communication with selective cellular targets and, (iii) the blocking of trans-signaling may prove to be an effective means for alleviating the detrimental effects of IL-6.
In vivo consequences of liver-specific interleukin-22 expression: implications for human liver disease progression (117.7)
Authors: Park Ogyi , Wang Hua , Weng Honglei , Li Hai , Yin Shi , Feigenbaum Lionel , Ki Sung-Hwan , Yoo Seong Ho , Dooley Steve , Wang Fu-sheng , Young Howard , Gao Bin
Published: January 2023

Interleukin 22 (IL-22), which acts as either a pro-inflammatory or anti-inflammatory cytokine in various disease models, is markedly upregulated in chronic liver diseases, including viral hepatitis B and C infection. In this report, we demonstrate a strong correlation between IL-22 expression in the liver with active, inflammatory human liver disease. To clarify the role of IL-22 upregulation in the pathogenesis of liver diseases, liver specific IL-22 transgenic (IL-22TG) mice, under the control of albumin promoter, were developed. Despite elevated IL-22 serum levels ranging from 4000 to 7000 pg/ml, IL-22TG mice developed normally without obvious adverse phenotypes or evidence of chronic inflammation. Most interestingly, IL-22 TG mice were completely resistant to Concanavalin A-induced T cell hepatitis with minimal effect on liver inflammation and had accelerated liver regeneration after partial hepatectomy. Although they did not spontaneously develop liver tumors, IL-22 TG mice were more susceptible to diethylnitrosamine-induced liver cancer. Microarray analyses revealed that a variety of anti-oxidant, mitogenic, acute phase genes were upregulated in the livers from IL-22TG mice compared with those from wild-type mice. These findings indicate that localized production of IL-22 in the liver promotes hepatocyte survival and proliferation but primes the liver to be more susceptible to tumor development without significantly affecting liver inflammation.
IL-21R is required for the systemic accumulation of activated B and T lymphocytes in MRLlpr mice (117.9)
Authors: Andreyeva Tatyana , Rankin Andrew , Guay Heath , Herber Deborah , Carrier Yijun , Mayra Senices , Stedman Nancy , Ryan Mark , Bloom Laird , Medley Quintus , Collins Mary , Nickerson-Nutter Cheryl , Young Deborah , Dunussi-Jannopoulos Kyri
Published: January 2023

IL-21 is a pleiotropic cytokine that can influence the activation, differentiation and expansion of B and T cells. Lupus prone MRL/MpJ-Faslpr/lpr/J (MRLlpr) mice have been shown to develop renal pathology and autoantibodies in an IL-21 dependent manner. We generated MRLlpr mice deficient in IL-21 receptor (MRLlpr.IL-21R-/-) to assess the impact of loss of IL-21R signaling on the systemic B and T cell lymphoaccumulation and activation that occurs in MRLlpr mice. Consistent with therapeutic blockade of IL-21, MRLlpr.IL21R-/- mice developed reduced proteinuria, renal pathology and serum IgG autoantibody levels. Lymphoaccumulation was significantly reduced in MRLlpr.IL-21R-/- mice, as evidenced by decreased lymphadenopathy and splenomegaly. Importantly, spontaneous germinal center formation and plasma cell accumulation were absent in IL-21R deficient MRLlpr mice. In addition, activated CD4+ CD44+ CD62Llo T cells failed to accumulate. We also demonstrate that T extrafollicular helper cells, which are a subset of activated CD4+ T cells that are reported to be the primary inducers of antibody production in MRLlpr mice, require IL-21R for their generation. T helper cell differentiation was also found to be impacted by loss of IL-21R, as IFN-γ producing CD4+ T cells were significantly reduced in MRLlpr.IL21R-/- mice. Together, our data highlight that IL-21 promotes multiple pathogenic B and T cell effector responses in MRLlpr mice.
Autoimmunity-associated LYP regulates T cell receptor signaling and thymus development (148.21)
Authors: Wang Yaya , Sawatzke Kristy , Zhou Wenbo , Ellingson Sonja , Peterson Erik
Published: January 2023

A single nucleotide polymorphism (SNP) in the PTPN22 gene, which encodes the lymphoid tyrosine phophatase LYP, is associated with increased risk of Rheumatoid Arthritis and other autoimmune diseases. LYP inhibits T cell receptor (TCR) signaling by dephosphorylation of Lck and Zap70. The disease associated LYP R620W (LypW) protein caused by the missense C1858T SNP in PTPN22 appears to exert “gain-of-function” effects upon TCR activation. Here we show that LypW negatively regulates TCR-induced intracellular Ca2+ flux, tyrosine phosphorylation, MAPK activation, and NFAT response element transactivation in Jurkat cells. The effects of LYP on TCR signaling are dependent on its enzymatic function. Studies from ptpn22−/− mice shows that PTPN22 regulates thymocyte positive selection. To evaluate effects of LypW expression on lymphocyte selection and function, we generated BAC transgenic mice bearing either the major human PTPN22 allele (LypR) or LypW. In contrast to intact positive selection in LypW H-Y TCR transgenic female mice, we observed impaired negative selection in LypW, but not LypR, H-Y male mice. These findings suggest that susceptibility to autoimmune diseases in LypW-carrying people may result from enhanced presence of autoreactive T cell in the peripheral repertoire, due to impaired thymocyte deletion. Together, our data suggest a thymic developmental mechanism whereby a “gain of function” Lyp variant can predispose to autoimmune disease.
Immunogenic potential of mutations in mitochondrial DNA and possible implications in autoimmune disease (148.22)
Authors: Duvvuri Bhargavi , Duvvuri Venkata , Chen Lina , Wang Chao , Jamnik Roni , Grigull Jörg , Watts Tania , Wu Gillian
Published: January 2023

To seek the underlying cause of autoimmune diseases we tested the hypothesis that mutated self proteins from mitochondria are seen as foreign proteins. A total of 21 mitochondrial peptides were identified using epitope prediction tool, SYFPEITHI as likely HLA-A2 restricted peptides. In vitro T cell line assays were conducted based on MHC stabilization of the peptides on T2 cells. Ten peptides were identified and the significant correlations were observed between the insilico and in vitro binding affinities. To determine the frequency of T cellular memory for the identified mutated peptides, PBMCs from healthy donors were tested in INF gamma-ELISpot assays. Frequencies of T cells recognizing mutated self peptide ranged from as few as 10 in 200,000 to 90 in 200,000 of the PBMCs plated. Among A2-donors, significant T cell responses were seen with mean age of 61 years. Of the 21 peptides two peptides, named as P10 and P14 were identified to be immuno-dominant peptides based on the immunogenic response by most of the donors (both A2 and non-A2) tested. To further determine the phenotype of the responding T cells, T cell co-stimulation assays were done for selected peptides based on T2 binding and Elispot assay results. T cell co-stimulation assays further confirmed Elispot results and interestingly, both interferon gamma producing CD8+T cells and CD4+ T cells were identified. We propose and will discuss the role of these immunogenic peptides as “Enemies with-in”.
Up-regulation of MCP-1 and RANTES by cartilage (149.10)
Authors: Merly Liza , Smith Sylvia
Published: January 2023

Immunomodulation refers to changes in an organism’s immune response by factors that either activate or suppress immune function. In previous studies we have shown the presence of immunomodulatory factor(s) in commercial shark cartilage (SC) extracts. Cartilage extracts induce various inflammatory cytokines and a potent chemokine in human peripheral blood leukocytes (HPBL). The cytokine induction profile of SC-stimulated cells includes potent Th1 type cytokines, such as IL-2, TNFα, IFNγ, and IL-1β, two of which are characteristically involved in inflammatory responses and an inflammatory chemokine, IL-8. To further delineate the HPBL response to SC, supernatants from both short-term and long-term primary HPBL cultures were assayed for two potent mediators, macrophage chemoattractant protein 1(MCP-1) and RANTES, by ELISA. Within 4 hours, SC-treated leukocytes produced significantly (p < 0.05) elevated levels of MCP-1 when compared to cultures treated with medium alone and the response was similar to that seen in LPS-treated cultures. After 24 hours, however, the response to LPS was significantly reduced while the response to SC was consistently maintained through 96 hours. In contrast, the production of RANTES by both un-stimulated and stimulated HPBL was relatively consistent through the first 12 hours of culture. RANTES levels in leukocytes treated with medium alone decreased gradually after 24 hours, while those of LPS- and SC-treated cultures remain elevated.
Notch1 produces a deregulation of CXCR4/SDF-1 chemokine signaling in multiple myeloma cells (149.12)
Authors: Mirandola Leonardo , Apicella Luana , Colombo Michela , Carluccio Silvia , Platova Natalia , Comi Paola , Cobos Everardo , Kast W. , Chiriva Maurizio , Chiaramonte Raffaella
Published: January 2023

Notch deregulation occurs in several solid and hematopoietic tumors. Recently, the Notch receptor oncogenic role has been shown to be critical in multiple myeloma (MM) which frequently displays deregulation of the Notch ligand, Jagged2. MM is a malignant disorder of Ig-secreting plasma cells which accumulate in the bone marrow and cause bone lesions through stimulation of osteoclast activity. Several chemokines and their receptors (CRs) are critical in MM progression. Particularly, the axis SDF1-a/CXCR4 promotes MM cell growth, recruiting in the bone marrow compartment, apoptosis resistance, angiogenesis and osteolysis. This work aims to explore the effects of the Notch oncogenic pathway on the expression and function of a number of CRs and correspondent ligands which display a critical role in MM. Our results indicate that Notch pathway plays a regulative role on the expression and function of CXCR4/SDF-1α axis in different MM cell lines including RPMI-8226, KMS-12 and OPM-2.
Chemokine receptor expression on T and B cells in a murine model of ovalbumin-induced allergic airway disease (149.17)
Authors: Natarajan Prabitha , Guernsey Linda , Secor Eric , McNamara Jeffrey , Schramm Craig , Thrall Roger
Published: January 2023

In our biphasic Ovalbumin (OVA)-induced Allergic Airway Disease model, short-term exposure to OVA aerosols results in Allergic Airway Disease (AAD) whereas continued exposure leads to the resolution of disease and the development of Local Inhalational Tolerance (LIT). We have shown in adoptive transfer studies that Hilar Lymph Node (HLN) B cells of LIT mice suppress development of AAD. To understand trafficking of B cells within HLN, we assessed their expression of CXCR4 and CXCR5 chemokine receptors via flow cytometry in mice at various stages of the model: naïve, sensitized, AAD and LIT. B cell populations were divided into CD5+ and CD5- cells. Our data demonstrates that there was a significant increase in the number and percentage of total B cells expressing CXCR4 in the HLN of mice at AAD and LIT compared to naïve and sensitized mice. We also observed a significant increase in CXCR4 expression on CD5+ B cells compared to CD5- B cells. We also studied the chemokine receptor expression on T cells and observed that Foxp3+ T regulatory cells significantly increased the expression of CXCR4 at AAD and LIT. CXCR4 expression on B cells is required for germinal center development in lymphoid organs. It is unusual to find CD5+ B cells in lymph nodes. We hypothesize that these CD5+ B cells may be directly interacting with effector T cells in a regulatory manner converting them into regulatory T cells within germinal centers and hence contributing to the resolution of AAD.
A persistent loss of cytokine responsiveness defines CD8+ memory T cell function (150.2)
Authors: Perona-Wright Georgia , Kohlmeier Jacob , Mohrs Katja , Cookenham Tres , Woodland David , Mohrs Markus
Published: January 2023

Cytokines are soluble signaling molecules that orchestrate immune responses, regulating both immune protection and immunopathology. T cell differentiation is characterized by distinctive changes in cytokine production, but less is known about concomitant changes in cytokine responsiveness. Here we report that CD8+ T cell differentiation imposes permanent alterations in the cytokine receptor repertoire. We show that influenza virus-specific CD8+ memory T cells in both humans and mice lose responsiveness to interleukin (IL)-27. This loss is due to persistent downregulation of the shared gp130 cytokine receptor chain on effector and memory T cells and occurs as a direct consequence of antigenic stimulation. Loss of IL-27 responsiveness renders CD8+ memory T cells deficient in their expression of the immunosuppressive cytokine IL-10 during recall responses. Together our data reveal a previously unappreciated feature of CD8+ T cell differentiation, in which the selective and persistent loss of specific cytokine receptors defines the functional potential of memory cells during rechallenge. The altered cytokine responsiveness of effector and memory T cells has fundamental implications for the functional manipulation of these cells in vivo.
IL-2 production in human memory CD8+ T cells depends on ERK1 expression (150.7)
Authors: Kim Tae Kon , Benjamin Cara , Ma Qing , Eric Wieder , Komanduri Krishna
Published: January 2023

Recently, we showed that human late memory (CD45RA+CD27-) CD8+ T cells have a unique cytokine signature characterized by the lack of IL-2 production, and no IL-2/IFNγ co-production. Prior studies have suggested the importance of ERK1/2, in IL-2 production; however, little is known about the individual contributions of ERK isoforms to the functional signatures of human T cell subsets. We developed flow cytometric methods to study the differential expression of ERK1 and ERK2 in CD8+ T cell maturation subsets in association with their cytokine signatures. We found that the expression of total ERK1, but not ERK2 nor Ras/Raf/MEK1/MEK2, are diminished by ~80% (measured by flow cytometry and western blotting) in late memory CD8+ T cells, in comparison to early memory CD8+ T cells. Consequently, the diminished expression of total ERK1 resulted in decreased total and phosphorylated ERK1/2. In addition, total ERK1 expression in CD8+ T cells was bimodal on flow cytometry (ERK1high and ERK1low) and consistently identified the subset of memory CD8+ T cells capable (ERK1high) or incapable of IL-2 production (ERK1low) (mean IL-2 production in ERK1high vs. in ERK1low: 20% vs. 1.5%, (n=9, p=0.0039)). Interestingly, late memory cells transduced with lentivirus-expressing ERK1 cDNA, produced 2.5 times more IL-2 than control (n=3, p<0.05). Collectively, these data suggest that the capacity to produce IL-2 in human memory CD8+ T cells is determined by ERK1 expression.
Mast cell ADAM-10 expression is blocked by IL-10 and may regulate CD23 levels on B cells (151.12)
Authors: Faber Travis , Conrad Daniel , Pullen Nicholas , Ryan John
Published: January 2023

Asthma is an inflammatory lung disease that is caused in part by mast cell activation via IgE antibodies. IgE is produced by B cells, and suppressed when IgE binds to the B cell receptor CD23. Recent data have demonstrated that CD23 expression is blocked by the protease ADAM-10. We demonstrate that mast cells abundantly express ADAM-10 in vitro and in vivo. Surface ADAM-10 expression is suppressed by the cytokine IL-10 via a Stat3-dependent mechanism that also reduces ADAM-10 mRNA expression. Preliminary experiments indicate that ADAM-10 expression may allow mast cells to regulate CD23 levels on B cells. Our data support the hypothesis that ADAM-10 may function in feedback loops regulating mast cell activation.
Mast cell responsiveness to IL-10 is altered by genetic background (151.14)
Authors: Kolawole Motunrayo , Ryan John
Published: January 2023

Mast cell activation has been shown to be antagonized by the inhibitory cytokine IL-10. We have found that there are strain differences in IL-10 responsiveness, as BALB/c mast cell progenitors were resistant to IL-10-mediated apoptosis, while C57BL/6 mast cell progenitors were killed by IL-10. We now report that IL-10-mediated effects on the high affinity IgE receptor vary with genetic background. IL-10 enhanced FceRI expression on mast cells derived from Th2-prone BALB/c or 129/Sv mice, but not on C57BL/6 mast cells. We also found that IL-10 surprisingly enhanced IgE-mediated production of inflammatory cytokines MIP-1 alpha and IL-13 even on C57BL/6 mast cells. These data illustrate that cytokine-mediated immunoregulation may be altered by genetic background as well as the inflammatory factors being studied.
Mast cell deficient mice display a pulmonary Th2 polarization with an increase in alternatively activated alveolar macrophages (151.17)
Authors: Wang Xiaojia , Hilderbrand Susana , Brown Jared
Published: January 2023

Macrophages are innate immune cells capable of coordination with the adaptive immune system during an immune response. The classical activation of macrophages is induced by IFN-γ, while the alternative activation of macrophages is triggered by IL-4 and IL-13. Mast cells are well recognized for their role in allergic inflammation, asthma and innate immunity. Due to a membrane tyrosine kinase mutation in c-Kit, B6.Cg-KitW-sh mice are unable to respond to stem cell factor and therefore do not develop mast cells. Thus, B6.Cg-KitW-sh mice do not develop IgE -dependent passive cutaneous anaphylaxis in the ear and joint as well as passive anaphylaxis. In the current study, we show that B6.Cg-KitW-sh mice display a Th2 phenotype with elevated levels of IL-4, IL-5 and IL-13 in serum and decreased IFN-γ levels in lung BAL fluid. This data led us to investigate whether alveolar macrophage phenotype is altered due to mast cell deficiency. We found an increase in the expression of genes associated with alternative activation of alveolar macrophages that included arginase-1 (Arg1), chitinases (Chi313), and FIZZ1 (Retnla) in BAL cells, when compared to background control mice (C57BL/6). In addition, these alternative activated macrophages display a > 25 fold change in class B scavenger receptor (Scarb2) expression. These data begin to suggest that mast cells may be important in regulation of alveolar macrophage phenotype.
Developing a passive sensitization procedure for basophils to diagnose IgE-mediated drug hypersensitivity (151.19)
Authors: Gentinetta Thomas , Hausmann Oliver , Kopac Peter , Colagiovanni Amira , Pichler Werner J.
Published: January 2023

Introduction: The basophil activation test is quite well established for protein allergens. It is normally done with the patients’ basophils and requires fresh cells and rapid processing. It would be a particularly interesting test for drug allergy, as only very few drug specific in vitro IgE assays exist and it seems to be a highly relevant read out system. Objective: To set up a passive sensitization protocol to test IgE-mediated drug hypersensitivity. Methods: PMBCs of basophil donors are incubated with lactic acid (pH=3.9) to remove surface bound IgE. Afterwards, re-sensitization with patient serum and subsequent stimulation with serial dilutions of drugs. Degranulation can be measured by flow cytometry (anti-CCR3-APC/anti-CD63-PE). Results: We were able to establish a well functional procedure for protein allergens and for some drug allergens (cephalosporines, NMBAs). Passive sensitization assays using serum of patients with well-documented IgE-mediated amoxicillin allergy were reproducibly false negative. Conclusion: Using defined donor basophils for passive sensitizations with serum would have several advantages over direct assays, as it would facilitate diagnosis, as patient’s sera is easy to store and as it would provide a direct proof of an IgE-mediated hypersensitivity reaction. However, a better understanding of how small drugs are actually able to cross-link cell surface bound IgE seems to be a prerequisite to improve this assay and to widen its application.
Mast cells induce a novel pro-inflammatory function of myeloid-derived suppressor cells (151.2)
Authors: Morales Johanna , Faber Travis , Saunders Bryan , Graham Laura , Bear Harry , Lantz Chris , Ryan John
Published: January 2023

Mast cells and myeloid-derived suppressor cells (MDSC) are both increased at sites of growing tumors. Like mast cells, MDSC have recently been suggested to function in parasitic or bacterial infection. We have tested the effects of mast cell-MDSC interactions in vitro and in a mouse model of parasitic infection. Surprisingly, co-culture of these cells resulted in a drastic increase in pro-inflammatory mediator release, which was attributed entirely to MDSC. MDSC depletion from mice infected with Nippostrongylus brasiliensis resulted in delayed worm expulsion and a drastically increased intestinal worm burden reminiscent of mast cell-deficient mice. Mast cells can therefore reprogram MDSC to a pro-inflammatory phenotype, suggesting that the evolutionary purpose of these suppressive cells may be to potentiate inflammatory Th2 responses necessary for parasite expulsion.
MTORC1 and mTORC2 control normal and neoplastic mast cell homoeostasis through regulation of cell division rather than maintenance of survival (151.20)
Authors: Smrz Daniel , Kim Mi-Sun , Silhankova Sarka , Wilson Todd , Metcalfe Dean , Gilfillan Alasdair
Published: January 2023

Mature mast cells are terminally differentiated and non-dividing. However, a pathologic increase in mast cells is associated with the myeloproliferative disorder mastocytosis which has been linked to expression of an activating mutation (D816V) in the mast cell growth factor receptor, KIT. We found that in primary human mast cells developed from peripheral blood CD34+ progenitor cells, KIT activation led to activation of mTORC1 and mTORC2 pathways through phosphorylation of mTOR (mammalian target of rapamycin) at S2481 and that these pathways were constitutively activated in both D816V positive (HMC-1.2) and negative (HMC-1.1, LAD2) neoplastic human mast cells. A dual acting inhibitor of the mTORC1 and mTORC2 pathways, Torin1, and a more selective mTORC1 inhibitor, Rapamycin, abolished proliferation of the malignant mast cells regardless of the presence of the D816V mutation. However, little reduction in numbers of the non-dividing primary mast cells was observed. The effects of the inhibitors on cell proliferation correlated to downregulation of mTORC1 and mTORC2 pathways which was verified by shRNA. Furthermore, no evidence of apoptosis in response to the inhibitors was observed in either neoplastic or primary mast cells. Collectively, our data demonstrate that by targeting the mTORC1 and mTORC2 pathways, it may be possible to inhibit the proliferation of rapidly dividing mast cell populations associated with mastocytosis while leaving normal mast cells largely unaffected.