bluuebunny/crossref_metadata_2025 huggingface dataset:
M1 macrophage induction in severely burned mice treated with CCL1 antisense ODN (111.9)
Authors: Asai Akira , Herndon David , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

We have reported that M2bMϕ, an inhibitor cell for M1Mϕ generation, are generated in mice 1 to 3 weeks after severe burn injury. CCL1 released from M2bMϕ acts as an autocrine signaler for keeping their suppressor cell properties. In this study, we tried to induce M1Mϕ in burned mice treated with CCL1 antisense ODN. BALB/c burned mice (25% TBSA, 3rd degree flame burn) were treated s.c. with 10 μg/mouse of CCL1 antisense ODN twice a day for 2 days beginning 13 days after burn injury. F4/80+ peritoneal Mϕ isolated from mice 12 hrs after the final treatment were tested for their M2bMϕ properties based on the expression of LIGHT mRNA and intracellular IL-10. These burned mice were then stimulated with heat-killed MRSA for M1Mϕ induction. Mϕ were considered as M1Mϕ if they expressed iNOS mRNA and intracellular IL-12. CCL1 was not detected in sera of burned mice treated with CCL1 antisense ODN. Mϕ from these mice did not express LIGHT mRNA and IL-10, while LIGHT+ IL-10+ Mϕ were isolated from burned mice treated with scrambled ODN. Further, iNOS+ IL-12+ Mϕ were induced by the bacterial antigen in burned mice subjected to the gene therapy. These results indicate that M2bMϕ are eliminated by CCL1 antisense ODN in severely burned mice, and M1Mϕ are induced in these mice stimulated with the antigen. CCL1 antisense ODN gene therapy may improve host’s antibacterial innate immunity severely suppressed in burned individuals.
Molecular dissection of T-cell differentiation defects in the old CD8 T cell (113.12)
Authors: Renkema Kristin , Rudd Brian , Li Gang , Samadder Partha , Smithey Megan , Nikolich-Zugich Janko
Published: January 2023

One in eight people are 65 years and older in the United States; this population is expected to double within the next 50 years. Alarmingly, infectious diseases remain amongst the leading causes for death in people over 65 years old. Widespread defects in the immune system have been found to increase with age, including qualitative and quantitative decline with adaptive immunity. CD8 T cells have been found to decrease in both number and function in aged mice. We recently found that in response to systemic Listeria monocytogenes (Lm) infection, old mice mobilize fewer Lm-specific CD8 T cells. Moreover, these cells exhibited lower effector cytokine production, and are less polyfunctional. We evaluated the expression of key transcription factors crucial to effector CD8 T cell development in adult and old CD8 T cells in the mouse. We found that in response to in vivo stimulation, old CD8 T cells exhibited altered expression of T-homeobox transcription factors , correlating with lower expression of interferon-gamma (IFNγ), tumor necrosis factor alpha (TNFα) and granzyme B. Altered transcriptional activation may constitute a common denominator underlying suboptimal function of old CD8 T cells in response to primary infection. Experiments are in progress to correct these defects and improve function of aged CD8 T cells.
Lipopolysaccharide-induced molecular interactions of the TLR4 signaling complex and the BK potassium channel (113.16)
Authors: Davis Ryan , Kozina Carol , Branda Steve , Rempe Susan
Published: January 2023

TLR4 signaling plays a central role in pathogen recognition and is fundamental to innate immunity. However, positive feedback in the systemic inflammatory response to TLR4 stimulation, manifest as sepsis, is a significant concern in managing infection. Thus pharmacological targets for modulating feedback in the immune response are desirable as next-generation therapies. Ion channels provide a ubiquitous mechanism for influencing cellular activation by controlling electrochemical polarization, and may be useful for amelioration of septic shock. Correspondingly, a recent report by Scheel and coworkers suggests that TLR activation of macrophages requires the function of BK potassium channels. On the molecular level, the formation of the LPS-induced TLR4 signaling complex requires intimate interactions of the co-receptors CD14 and MD2. However, our recent in vivo studies of TLR4 diffusion prior to and following LPS-stimulation in HEK293 suggest that several components in addition to these fundamental units may also be involved. In fact, increased FRET efficiency observed in LPS-stimulation of cells co-transfected with fluorescent constructs of TLR4 and the BK potassium channel suggest direct interactions of the signaling complex with this ion channel. Finally, we present results detailing the impact of association with the TLR4 signaling complex on calcium-activation of the BK channel. These data suggest that the BK channel may play an important role TLR4 feedback.
Control of T cell metabolism and regulatory T cell generation by a DRAK2/p70S6K1 signaling axis (113.19)
Authors: Hernandez Jeniffer , Michalek Ryan , Weist Brian , Newton Ryan , Limon Jose , Carrillo Mayra , Paterson Elyse , Nguyen Long , Fruman David , Rathmell Jeffrey , Walsh Craig
Published: January 2023

The delicate balance between effector T cells (Teff cells) and regulatory T cells (Treg cells) is crucial for preventing autoimmunity while maintaining efficient immune function. Mice deficient in the serine/threonine kinase, Dap Related Apoptosis inducing Kinase 2 (DRAK2), are resistant to T cell-mediated organ-specific autoimmune diseases yet retain antiviral immunity. Here we show that DRAK2 dictates the fate of a naïve T cells by regulating their metabolism. This is achieved through amplifying the activity of p70S6K1 in a parallel manner to mTORC1, which leads to an increase in glycolytic metabolism, a process required for Teff cell survival. In addition, we find that the inhibition of the DRAK2/p70S6K1 axis promotes the generation of Treg cells similar to mTORC1 inhibition with rapamycin. By regulating T cell metabolism and Treg cell generation, the DRAK2/p70S6K1 signaling axis orchestrates the balance between Teff cells and Treg cells. Blockade of DRAK2, which is most highly expressed in lymphocytes, may offer an alternative and selective approach vs. rapamycin in the suppression of autoimmunity.
The protein tyrosine phosphatase, SHP-1, is an intrinsic central regulator of dendritic cell function (113.8)
Authors: Levitt Jonathan , Ramachandran Indu , Spencer David
Published: January 2023

Dendritic cells (DCs) initiate proinflammatory or regulatory T cell responses, depending on their activation state. Despite extensive knowledge of DC-activating signals, mechanisms of DC-inhibitory signals are less well understood. We show that the SH2-domain-containing protein tyrosine phosphatase, SHP-1, is an important inhibitor of DC signaling that targets multiple activation pathways. Downstream of TLR4, SHP-1 showed increased interactions with several proteins including IRAK-4, and modulated LPS signaling through the inhibition of NF-κB, AP-1, ERK and JNK activity, while enhancing p38 activity. In addition, SHP-1 inhibited pro-survival signaling modulated through AKT activation. Further, SHP-1 inhibited CCR7 protein levels and CCR7 mediated cytokine expression. Inhibition of SHP-1 in DCs enhanced proinflammatory cytokines, IL-6, IL-12 and IL-1β, promoted survival and increased the rate DC migration from the periphery to draining lymph nodes. In vivo administration of SHP-1-inhibited DCs loaded with antigen, significantly induced the expansion of antigen-specific cytotoxic T cells and inhibited Foxp3+ regulatory T cell proliferation compared to unmodified DCs. When used in a vaccination protocol, SHP-1-inhibited DCs significantly reduced progression of pre-established mouse melanoma and prostate tumors. Taken together, these data demonstrate that SHP-1 is an intrinsic global regulator of DC function, controlling many facets of T cell-mediated immune responses.
Regulation of thymocyte positive selection and motility by GIT2 (113.9)
Authors: Phee Hyewon , Dzhagalov Ivan , Mollenauer Marianne , Wang Yana , Irvine Darrell , Robey Ellen , Weiss Arthur
Published: January 2023

Thymocytes are highly motile cells that migrate under the influence of chemokines in distinct thymic compartments as they mature. The motility of thymocytes is tightly regulated; however, the molecular mechanisms that control thymocyte motility are not well understood. Herein, we report that G protein-coupled receptor kinase-interactor 2 (GIT2) is required for efficient positive selection. Interestingly, GIT2-/- double positive (DP) thymocytes display increased Rac activation, actin polymerization and migration towards SDF-1 and CCL25 in vitro. Using two-photon laser scanning microscopy, we found that scanning activity of GIT2-/- thymocytes is severely compromised in the thymic cortex. Moreover, the GIT2-/- thymocytes accumulated at discrete small regions throughout the cortex including areas adjacent to SDF-1 positive blood vessels and interacted with them extensively. The impaired scanning activity of GIT2-/- thymocytes was accompanied by impaired positive selection in TCR transgenic GIT2-/- mice, suggesting GIT2 plays a key role in controlling chemokine-mediated motility of DP thymocytes and ultimately regulates positive selection.
Regulation of the development of acute hepatitis by IL-23 through IL-22 and IL-17 production (114.13)
Authors: Mingli Xu , Noriko Morishima , Izuru Mizoguchi , Yukino Chiba , Koji Fujita , Masahiko Kuroda , Yoichiro Iwakura , Daniel J Cua , Koji Yasutomo , Junichiro Mizuguchi , Takayuki Yoshimoto
Published: January 2023

IL-23 plays a critical role in the expansion of highly pro-inflammatory Th17 cells secreting IL-17 and IL-22. Recently, we demonstrated that Notch signaling drives IL-22 secretion through the aryl hydrocarbon receptor (AHR) and plays a protective role in Con A-induced hepatitis. In the present study, we investigated a role for IL-23 in the hepatitis using IL-23p19- and IL-17-deficient mice. In WT mice, injection of Con A induced up-regulation of various cytokines including IL-23p19, IL-22 and IL-17. In IL-23p19-deficient mice, exacerbated hepatitis was observed and serum IL-22 and IL-17 levels were greatly reduced, while IL-17-deficient mice showed ameliorated hepatitis. Injection of exogenous IL-22 protected p19-deficient mice from the hepatitis, whereas injection of exogenous IL-23 greatly enhanced serum levels of not only IL-22 but also IL-17, and much less effectively protected from the hepatitis in IL-17-dependent and -independent manners. Finally, it was revealed that STAT3, STAT4 and Notch contributed to the production of both cytokines and that AHR was important for only IL-22 production in response to Con A and IL-23 in liver mononuclear cells. These results suggest that IL-23 plays a protective role in the hepatitis through IL-22 production and also a pathological role via IL-17-dependent and -independent mechanisms.
Chronic mucocutaneous candidiasis in humans with inborn errors of interleukin-17 immunity (114.16)
Authors: Puel Anne , Cypowyj Sophie , Bustamante Jacinta , Wright Jill , Liu Luyan , Lim Hye Kyung , Migaud Mélanie , Israel Laura , Chrabieh Maya , Audry Magali , Gumbleton Matthew , Toulon Antoine , Bodemer Christine , El-Baghdadi Jamila , Whitters Matthew , Paradis Theresa , Brooks Jonathan , Collins Mary , Wolfman Neil , Al-Muhsen Saleh , Galicchio Miguel , Abel Laurent , Picard Capucine , Casanova Jean-Laurent
Published: January 2023

Chronic mucocutaneous candidiasis disease (CMCD) is characterized by recurrent or persistent infections of the skin, nails, oral and genital mucosae caused by Candida albicans and, to a lesser extent, Staphylococcus aureus, in patients with no other infectious or autoimmune manifestations. We report two genetic etiologies of CMCD: autosomal recessive deficiency in the cytokine receptor, interleukin-17 receptor A (IL-17RA), and autosomal dominant deficiency of the cytokine IL-17F. IL-17RA deficiency is complete, abolishing cellular responses to IL-17A and IL-17F homo- and heterodimers. By contrast, IL-17F deficiency is partial, with mutant IL-17F-containing homo- and heterodimers displaying impaired, but not abolished activity. These experiments of nature indicate that human IL-17A and IL-17F are essential for mucocutaneous immunity against C. albicans, but otherwise largely redundant.
TNFαRII expressing FoxP3+ T reg cells have a reversible defect in their ability to suppress the autoimmune disease thyrocyte epithelial cell hyperplasia/proliferation (115.17)
Authors: Kayes Timothy , Braley-Mullen Helen
Published: January 2023

Most IFN-γ-/- NOD.H-2h4 mice given 0.05% NaI in their water develop an autoimmune thyroid disease characterized by infiltrating lymphocytes, expression of TNFα and TGFβ, production of anti-thyroglobulin antibodies, and uncontrolled proliferation of TEC, resulting in destruction of the gland and loss of function. IFN-γ-/- NOD.H-2h4 mice have elevated FoxP3+ CD4+ T reg in cervical LN and spleen (25-40%, and 25-35% of CD4+ T cells) dependent on age, presence of severe disease, and absence of IFN-γ. T reg cells from mice with severe TEC H/P exhibit a suppressive phenotype with expression of high levels of FoxP3, CD25, and TNFαRII (p75), and they suppress anti-CD3 mediated proliferation of T cells in vitro. Splenocytes from mice with severe TEC H/P transfer severe TEC H/P to IFN-γ-/- SCID recipients following 72 hr culture. During culture, percentages of T reg decrease, allowing transfer of severe TEC H/P. However, in recipient mice, percentages of T reg increase, and despite increased numbers of T reg in LN and spleen, the mice develop severe TEC H/P. This data suggests there may be a defect in the ability of these T reg to suppress TEC H/P, either because of an intrinsic defect in the T reg or because effector cells are resistant to suppression. The defect is reversible as addition of IL-2 (100 U/ml) to the culture increases T reg percentages, and inhibits transfer of TEC H/P, restoring the ability of T reg to suppress TEC H/P in recipient SCID mice.
Viral vector mediated tolerance induction in experimental autoimmune encephalomyelitis by dendritic cell targeted expression of a self-antigen (115.2)
Authors: de Andrade Pereira Bruna , Fraefel Cornel , Ackermann Mathias , Dresch Christiane
Published: January 2023

Multiple sclerosis (MS) is an autoimmune disease characterized by the presence of self-reactive T cells that target myelin components. Immunization of susceptible animals with myelin antigens induces experimental autoimmune encephalomyelitis (EAE), an animal model for MS. The aim of this project was to induce permanent, antigen-specific tolerance in EAE/MS and to address the mechanisms that would contribute to tolerance. The strategy includes the ex vivo modification of autologous hematopoietic stem cells (HSC) with lentiviral vectors that express myelin oligodendrocyte glycoprotein (MOG) under the control of a dendritic cell (DC) specific promoter. After re-infusion, the modified HSC will give rise to antigen expressing DCs. We demonstrated the effectiveness of this strategy for inducing MOG-specific tolerance in EAE, as 100% of the mice that received BM cells transduced with MOG-lentivector were protected. In addition, we show depletion of MOG specific T cells and generation of Foxp3+ regulatory T cells in chimeras that received HSC transduced with MOG-lentivector. As apposed to the protected animals, the diseased control mice showed high levels of IL-1, INF-γ and IL-17. The strategy presented here is particularly promising for clinical applications and important for addressing fundamental questions in immunity and tolerance.
Treatment of autoimmune demyelination by autoregulatory CD8+ T-cells (115.24)
Authors: Ortega Sterling , Tyler Andrew , Kashi Venkatesh , Mendoza Jason , Benagh Andrew , Karandikar Nitin
Published: January 2023

Immune dysregulation is believed to be the causative factor in the debilitating neurological disease Multiple Sclerosis (MS). We have recently shown that neuroantigen-specific auto-reactive regulatory (autoregulatory) CD8+ T-cells are capable of suppressing the murine model of MS, experimental autoimmune encephalomyelitis (EAE), a disease mediated by neuroantigen-specific CD4+ T-cells. We now show that deficiency of CD8+ T-cells correlates with more severe disease and an increase in IL-17+ and TCRVβ8.2+ CD4+ T-cell responses with reduction in IL-10+ CD4+ T-cells. Disease suppression by autoregulatory CD8+ T-cells is dependent on recognition of cognate antigen in vivo in the context of MHC Class I and requires IFN-γ and perforin production by CD8+ T-cells, suggesting a cytotoxic/suppressor mechanism. Suppression mediated by direct CD8-CD4 interactions is suggested by the ability of these cells to inhibit adoptively transferred disease and direct in vitro killing of neuroantigen-loaded CD4+ T cells. Finally, neuroantigen-specific CD8+ T-cells were able to traffic to the central nervous system (CNS) during ongoing inflammation, suggesting both a peripheral and central mechanism of action. These studies define a novel and unexpected immune regulatory function for tissue-reactive “autoregulatory” CD8+ T-cells that can be harnessed for the development of immunotherapeutic intervention in MS and other autoimmune diseases.
Indoleamine 2,3-dioxygenase 1 (IDO1) activity correlates with inflammation in systemic sclerosis (115.5)
Authors: Rutella Sergio , De Santis Maria , Bonanno Giuseppina , Procoli Annabella , Mariotti Andrea , Corallo Maria , Novarese Linda , Bosello Silvia , Scambia Giovanni , De Cristofaro Raimondo , Ferraccioli Gianfranco
Published: January 2023

Background. A contributory effect of IDO1 to inflammatory autoimmune pathologies such as rheumatoid arthritis and systemic lupus erythematosus has been recently described. We aimed to address whether IDO1-driven kynurenine (KYN) production correlates with degree of inflammation and disease extent in systemic sclerosis (SSc). Methods. Peripheral blood was obtained from 27 consecutive patients (median age 62 years, range 22-78; median disease duration = 10 months, range 1-35) with either limited cutaneous SSc (n=17) or diffuse cutaneous SSc (n=10). In 11 of the 27 patients, CD19+ B cells were magnetically purified to estimate IDO1 mRNA levels by quantitative PCR. Results. High serum KYN in SSc (3.17±0.82 μM compared with 1.82±0.37 μM in healthy controls; p<0.0001) positively correlated with serum CRP (r=0.42; p=0.03), an established marker of inflammation. We showed a trend towards higher KYN levels in diffuse SSc compared with limited SSc, although no correlations emerged between KYN release and serological features or lung fibrosis. Interestingly, purified CD19+ B cells expressed IDO1 mRNA, whose copy number correlated with serum CRP (r=0.58; p=0.048) and with the frequency of bona fide CD4+FoxP3+ Treg cells. Conclusions. The generation of KYN and expansion of Treg cells may reflect a counter-regulatory mechanism to limit excessive inflammation in SSc.
Ku70 is a novel cytosolic DNA sensor that induces a Type-III rather than Type-I IFN via activation of IRF-1 and IRF-7. (116.11)
Authors: Imamichi Tomozumi , Zhang Xing , Zhou Ming , Lempicki Richard , Baseler Michael , Veenstra Timothy , Young Howard , Lane H. Clifford
Published: January 2023

Recent studies have demonstrated that foreign cytoplasmic DNA is detected by cytosolic DNA sensors (DAI, AIM-2, LRRFIP1, RNA polymerase III, or IFI16) that induce Type-I IFN or IL-1β. We have previously found that HEK293 cell transfected with DNA induces a Type-III IFN (IFN-λ1) rather than Type-I IFN. In this study, utilizing a pull down assay followed by mass spectrometry, we identified the DNA sensor protein that specifically induces IFN-λ1. Changes in expression level of gene and protein were evaluated by real time RT-PCR and Western blot. Transfection of not only plasmid DNA but also PCR amplified DNA and bacteria DNA induced IFN-λ1 activation in cell lines, primary macrophages and dendritic cells. DNA virus (HSV-2) infection also induced IFN-λ1 production. The pull down assay using DNA-beads resulted in that DNA-binding proteins in cytosole were Ku70 and Ku80, a DNA repair protein, as playing potentially important role. Knockdown of Ku70, but not Ku80, by siRNA led to 90% and 70% reduction in the DNA- and the virus-mediated IFN-λ1 induction, respectively. Over expression of Ku70 induced activation of the IFN-λ1 promoter. DNA binding assay using NFκB-, ISRE- or PRDI-beads with nuclear extract from DNA transfected cells indicated that the DNA-mediated IFN-λ1 induction was associated with the activation of IRF-1 and IRF-7 rather than IRF-3 and NFκB. Taken together; these data demonstrated that Ku70 exerts a novel cytosolic DNA sensor that specifically activates IFN-λ1.
AID mediates innate immunity to endogenous retroelements (116.12)
Authors: Metzner Mirjam , Jäck Hans-Martin , Wabl Matthias
Published: January 2023

A member of the APOBEC protein family, activation-induced cytidine deaminase (AID) is a B-cell-specific mutator that deaminates cytosine to uracil in DNA. AID mediates somatic hypermutation and class switch recombination of immunoglobulin genes—processes important for the generation of high-affinity antibodies and changes in antibody effector functions, respectively. Recent data, including our own, suggest that AID may not only function in adaptive, but also in innate immunity. AID may inhibit retroelements—a function that is well established for APOBEC3 proteins. If unrestricted, endogenous retroelements accumulate in the cytoplasm and insert their DNA into the host genome, with the potential to cause disease ranging from cancer to autoimmunity. We found that AID inhibits the retrotransposition of LINE-1 (L1) retroelements in vitro. In activated B cells, AID forms cytoplasmic high molecular mass complexes with L1 mRNA and decreases the protein level of open reading frame 1 (ORF1) of L1 elements. We propose that AID protects B cells from the adverse effects of retroelements, such as insertional mutagenesis.
The zinc transporter SLC39A8 is a novel negative feedback regulator of the NF-κB pathway and innate immune activation (116.33)
Authors: Liu Mingjie , Bao Shengying , Gálvez-Peralta Marina , Pyle Charlie , Rudawsky Andrew , Nebert Daniel , Wewers Mark , Knoell Daren
Published: January 2023

Zinc is an essential micronutrient required for proper immune function. We previously reported that zinc deficiency increases inflammation and mortality in response to polymicrobial sepsis. Here we report that a specific zinc transporter, SLC39A8 (ZIP8), is a transcriptional target of NF-κB and functions as a negative regulator of NF-κB. Initially we observed that TNFα or LPS inducesSLC39A8 expression in human monocytes, macrophages and lung epithelial cells, leading to an increase in intracellular zinc. We then identified a functional NF-κB binding site in the promoter region that triggers ZIP8 expression. Silencing of ZIP8 expression significantly decreased intracellular accumulation of zinc and increased the inflammatory response, as determined by increased cytokine production and phosphorylation of IκBα, P65, ERK and Akt. We further revealed that zinc directly inhibits IKKβ in a ZIP8-dependent manner. In mice, endotoxin or cecal ligation and puncture resulted in increased ZIP8 expression and zinc uptake in circulating monocytes and lung tissue, contributing to hypozincemia. Consistent with these observations, mouse embryonic fibroblasts obtained from Slc39a8 hypomorphic mice were more responsive to TNFα and IL-1β compared to wild-type cells, confirming the function of ZIP8 as a negative regulator of NF-κB and innate immune activation. Taken together, our findings identify a novel inducible inhibitory feedback pathway for NF-κB and the pro-inflammatory response.
PKR is an essential regulator of HMGB1 release (116.38)
Authors: Lu Ben , Yang Huan , Chavan Sangeeta , Tracey Kevin
Published: January 2023

High-mobility group box 1 (HMGB1), a leader-less proinflammatory cytokine, is a necessary and sufficient mediator of inflammation during sterile and infectious injury. However, the mechanism of HMGB1 release by activated immune cells is largely unknown. Here we demonstrate that macrophages stimulated with Type 1 interferon release HMGB1 in a dose- and time- dependent manner. Consistently, inhibition of JAK-STAT pathway attenuated Type 1 interferon-induced HMGB1 release. Analysis of downstream effectors demonstrated that Interferon-inducible double-stranded RNA activated protein kinase R (PKR) is essential for Type 1 interferon mediated HMGB1 release. Inhibition of PKR activity by using specific inhibitor reduced Type 1 interferon-induced HMGB1 release in a dose dependent manner. Inhibition of PKR activity also blocked LPS- and Poly I:C- induced HMGB1 release from macrophages. Using immunohistochemical analysis, we have identified two important steps in HMGB1 release by activated macrophages. The first step is mediated by activation of JAK-STAT pathway by Type 1 interferon that culminates into translocation of HMGB1 from nucleus to cytoplasm. This is followed by PKR activation that in turn regulates HMGB1 release by macrophages. Our studies indicate that PKR is an essential regulator of HMGB1 release induced by multiple stimuli, such as Type 1 interferon, LPS and Poly I:C.
IL-34 produced by human synovial fibroblast in rheumatoid arthritis supports osteoclastogenesis (117.17)
Authors: Chang Eun-Ju , Kang Soon-Sook , Jeang Yeon-Ho , Kim Yong-Gil , Lee Chang Keun , Yoon Seung-Yong , Hwang Seung-Joon
Published: January 2023

Interleukin-34 (IL-34) has a similar function with M-CSF via M-CSF receptor, c-fms, which also involves in RANKL-osteoclastogenesis. In this study, we found that IL-34 was up-regulated in the synovial fluid of RA patients with diagnostic positive rheumatoid factors. IL-34 production was much higher in the synovial fibroblast of RA patients than in that of OA patients and which was enhanced by TNFa stimulation. The expression of IL-34 mRNA and protein in synovial fibroblasts of RA patient after TNFa stimulation were determined using real-time PCR and western blot. Interestingly, TNFa induced IL-34 expression but not M-CSF dependently on NF-kB activity. The conditioned medium of synovial fibroblasts of RA patient cultured with TNFa led to chemotactic migration of human monocytes, and which was blocked by anti-IL-34 antibodies. Moreover, blocking IL-34 activity hindered the RNAKL-induced osteoclastogenesis in same conditioned medium. These results demonstrate that the increase of biologically active IL-34 in the RA synovium induces the chemotactic migration of osteoclast precursor cells and subsequently supports osteoclastogenesis in the inflammed synovium. Together, the differential regulation of IL-34 and M-CSF expression levels by TNFa further implies discrete role of IL-34 in the inflammatory condition involving RA pathogenesis.
RSAD2 is a key requirement for Golgi trafficking of TNF-α in activated microglia (117.26)
Authors: Chung Eun Sook , Chung Hwansuck , Bae Hyunsu
Published: January 2023

Antiviral protein RSAD2 (radical S-adenosyl methionine domain-containing protein 2), also known as viperin, has been recognized as a highly inducible ER (endoplasmic reticulum) protein in response to a wide range of viruses and microbial products such as LPS and double-stranded RNA. However, the functions of RSAD2 other than its antiviral activity have remained unexplored. The present studies showed that RSAD2 mRNA and protein expression was highly induced by LPS treatment in both primary microglia and BV2 cells. To investigate the function of RSAD2 in activated microglia, BV2 cells were depleted RSAD2 by using a short hairpin RNA (shRNA) plasmid. TNF-α and IL-6 production in RSAD2 depleted cells was remarkably reduced after stimulation with LPS. However, mRNA expression of TNF-α and IL-6 did not reveal significant differences between RSAD2-shRNA transfected cells and cont-shRNA transfected cells after stimulation with LPS. The intracellular distribution and trafficking of TNF-α was demonstrated by immunofluorescence staining. Non-transfected control cells showed strong intracellular staining of TNF-α in a perinuclear Golgi complex after LPS treatment. However, depletion of RSAD2 inhibited the Golgi trafficking of TNF-α. These results suggest that RSAD2 play an important role in microglia activation, especially regulation of Golgi trafficking of secretary molecules such as TNF-α.
NLRP3 inflammasome regulates obesity induced systemic inflammation and insulin signaling (117.4)
Authors: Vandanmagsar Bolormaa , Youm Yun-Hee , Ravussin Anthony , Galgani Jose , Stadler Krisztian , Mynatt Randall , Ravussin Eric , Stephens Jacqueline , Dixit Vishwa
Published: January 2023

Activation of adipose tissue macrophages and T cells within fat depots is coupled with the development of obesity-induced proinflammatory state and insulin resistance. The release of IL-1β and IL-18 from macrophages is dependent on autocatalytic activation of procaspase-1 into active 20-kDa caspase-1. However, an upstream initiating event for obesity-induced immune cell activation in adipose tissue is not established. We found that Nlrp3 inflammasome, member of the Nod-like receptor (NLR) family of innate immune cell sensors is activated by obesity-associated lipotoxic factors. The obesity-induced macrophage activation and IL-1b production was lowered in absence of Nlrp3 signaling. Furthermore, elimination of Nlrp3 in obese mice reduced IL-18 and adipose tissue Interferon-g expression and improved insulin signaling. However, blocking the obesity-induced Nlrp3 inflamma¬some function in mice did not affect Treg cell homeostasis in adipose tissue but specifically reduced the numbers of effector memory cells and increased the naive CD4+ and CD8+ T cell populations in adipose tissue. Notably, weight loss associated improvement in type 2 diabetes in obese patients was associated with reduced NLRP3 expression and inflammation in adipose tissue. Our data establishes that Nlrp3 inflamamsome participates in origin of inflammation in obesity and development of insulin-resistance.
IL-4 suppresses prostaglandins production in human follicular dendritic cells by repressing COX-2 through JAK1-STAT6 (147.1)
Authors: Choe Jongseon , Cho Whajung
Published: January 2023

In a recent report, we demonstrated that IL-4 inhibits prostaglandins (PGs) production in follicular dendritic cell (FDC)-like cells, HK. To understand the inhibitory mechanisms of IL-4, its effects on the biosynthesis of enzymes in charge of PG production were assessed in this study. Although IL-4 did not affect COX-1 expression, it specifically inhibited LPS-induced COX-2 biosynthesis at the mRNA and protein levels. Protein expression of mPGES-1, a downstream enzyme of COX-2, was also markedly diminished by IL-4 but not by IL-10, maximizing the inhibitory activity. Next, we attempted to identify the early signaling molecules that led to this inhibition of COX-2 expression. Although IL-4 induced tyrosine phosphorylation of JAK1 and TYK2, RNA interference experiments revealed that only JAK1 was responsible for the IL-4-stimulated STAT6 phosphorylation. Knocking down JAK1 and STAT6 ablated the inhibitory effect of IL-4 on COX-2 expression and significantly reduced production of PGE2 and prostacyclin. These results stress the essential roles of JAK1 and STAT6 in the early signaling pathway of IL-4 leading to suppression of COX-2 expression and repression of PG production by HK cells. Our results suggest that T cells via IL-4 play a regulatory role in PG generation in FDC. IL-4 therapeutics may be applied to immune disorders where normal and ectopic expression of germinal center reactions needs to be regulated.
Monocytic myeloid-derived suppressor cells isolated from mice with colitis actively suppress CD4+ T cell functions in vitro. (147.11)
Authors: Kurmaeva Elvira , Berney Seth , Ostanin Dmitry
Published: January 2023

The inflammatory bowel diseases (IBD) are accompanied by extensive myelopoiesis and infiltration of myeloid cells into colonic lamina propria. Surprisingly, little is known about the contribution of these cells to the disease pathogenesis. In a mouse CD45RBhigh T cell transfer model we have identified a population of monocytic MDSC. When isolated from spleens and colons of colitic mice MDSC potently suppressed proliferation of ovalbumin (OVA)-specific CD4+ T cells (>95% inhibition at 1:1 ratio). Interestingly, suppression was also observed in an antigen-non-specific manner with in vitro stimulated WT CD4+ T cells. It was partially reversed by fixation of MDSCs and addition of nitric oxide synthase inhibitor L-NMMA. Correlating with the suppression we saw decreased levels of T cell-derived cytokines (GM-CSF, IFN-γ, IL-2, and IL-17), but increased levels of IL-10 in the cell culture supernatant. We did not observe enhanced T cell apoptosis associated with suppression. On the contrary, T cells cultured with MDSCs showed increased viability when compared to culturing of T cells alone. Taken together, our data indicate that myelopoiesis associated with chronic inflammation results in the increased release into the circulation and recruitment of anti-inflammatory monocytic MDSCs to the inflamed tissue. Better understanding of the mechanisms behind expansion of anti-inflammatory MDSC in chronic inflammatory diseases may prove beneficial for future therapies.
CD99 regulates CD1A expression during dendritic cells differentiation (147.14)
Authors: Bernard Ghislaine , Sghaier Mouna , Mahiddine Karim , Bzioueche Hanene , Mallavialle Aude , Bernard Alain
Published: January 2023

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that play a dominant role in the regulation of immune responses. CD1a has a unique expression pattern among Ag-presenting molecules, expressed specifically on cortical thymocytes and APCs. We have previously established that CD99, a 32kd transmembrane molecule expressed as two isoforms, could regulate classical MHC class I expression. We found that CD99LF triggers phosphorylation of p38 mitogen-activated protein kinase (MAPK) and two activating transcription factor (ATF)/CREB family members, CREB-1 and ATF-2 to inhibit CD1a expression in DC derived from monocytes CD14+. We also showed that this regulation is counteracted in the presence of CD99 short form (CD99SF).To confirm our results, we used CD99 deficient Jurkat cells and CD99pos transfected cells, CD99 silencing experiments and inhibitors/activators of cell-signaling pathways. Accumulating data have shown that the microenvironment of dendritic cells modulates subtype differentiation and CD1a expression, but the mechanisms by which exogenous factors confer these effects are poorly understood. This study strongly suggests a role for CD99 in regulation of differentiation of DCs subtype. In conclusion, we propose that cAMP is a novel pathway exploited by CD99 to regulate CD1a expression.
Role of host lipid metabolism and tissue macrophages in psoriasis (147.30)
Authors: Wharton David , Gharavi Nima , Navab Mohamad , Lee Sangderk , Berliner Judith , Fogelman Alan , Modlin Robert
Published: January 2023

Epidemiologic studies and clinical co-morbidity suggests a link between psoriasis and atherosclerosis. These conditions share common inflammatory mediators, including macrophage (Mp) signaling and the presence of oxidized low-density lipoprotein (LDL). We examined the role of lipid metabolism and tissue Mp in psoriasis. Using immunohistochemistry on psoriatic tissue, we identified a subpopulation of CD209+/CD163+ dermal Mp which have phagocytic properties and are capable of engulfing oxidized lipids. Next, using gene-expression arrays, we examined the effect of oxidized lipids on Mp signaling. Treatment of THP-1 Mp with a component of minimally oxidized LDL induced the expression of interleukins-1, -6, -15, -17, -23, cathelicidin and vascular endothelial growth factor, all of which play a role is psoriasis. Lastly, we examined the properties of high-density lipoprotein (HDL) in serum from psoriatic patients. In healthy patients, HDL is capable of reverse cholesterol transport and has potent anti-inflammatory properties. In patients with systemic inflammatory disease, including atherosclerosis, HDL loses these properties and becomes dysfunctional. Using a well-established model of monocyte chemotaxis as well as an in-vitro model of IL-23 production, both as markers of inflammation, we demonstrated that HDL from psoriatic patients was dysfunctional and had pro-inflammatory properties. Taken together, these data provide a role for tissue Mp and host lipid metabolism in psoriasis
Therapeutic modulation of experimental autoimmune encephalomyelitis by gamma secretase inhibition (148.15)
Authors: Cummings Matthew , Villegas Alicia , Karandikar Nitin , Eagar Todd
Published: January 2023

Intramembranous proteolysis by the γ-secretase complex is a key regulator of transmembrane protein turnover and intracellular signaling. Small molecule inhibitors of γ-secretase (γSI) have previously been shown to suppress Th1 differentiation and reduce the severity of EAE, presumably by affecting Notch1 signaling. In order to understand the mechanisms through which γSI affect T effector responses we examined helper T cell polarization and function in vitro and in EAE. In vitro γSI treatment reduced T cell production of the Th1 cytokine IFNγ. We found γSI do not change activation but have effects on proliferation. Additionally, expression of the Th1 transcription factor Tbet was also diminished in γSI treated T cells. Looking upstream of Tbet expression, we found that γSI modulated STAT1 phosphorylation induced by exposing the cells to IFNγ. These results suggest that γSI might work at preventing Th1 differentiation but do not address the affects γSI may have on fully differentiated Th1 cells. We found that treatment with a γSI reduced the severity of EAE induced by adoptive transfer of myelin-specific TCR-transgenic T cells. Antigen recall experiments demonstrated that there was a dramatic decrease in the production of IFNγ by T cells ex vivo. These results demonstrate that γSI act not just to diminish differentiation of myelin-specific T cells into autoimmune Th1 effectors but impair the ability of established Th1 cells to cause damage to the CNS.
Aire mediates peptide epitope-specific thymic deletion of self reactive T cells to prevent autoimmune uveitis (148.3)
Authors: Taniguchi Ruth , Devoss Jason , Moon James , Jenkins Marc , Anderson Mark
Published: January 2023

Aire is a transcription factor required for the expression of many tissue specific antigens in the thymus. In the absence of functional Aire protein, human patients and mice develop multi-organ autoimmune disease, including autoimmune uveitis, due to a defect in negative selection. The development of spontaneous uveitis in the Aire KO mouse model is dependent on the eye antigen, interphotoreceptor retinoid-binding antigen (IRBP), which has Aire-regulated expression the thymus. To study the mechanism by which Aire prevents IRBP-dependent autoimmune uveitis, two tetramer reagents were generated to measure the frequency and phenotype of IRBP specific CD4+ T cells in the polyclonal repertoire of WT and Aire KO mice. Analysis of the polyclonal T cell repertoire of WT and Aire KO mice showed that T cells specific for both IRBP tetramers proliferated in Aire KO mice when they developed uveitis. However, tetramer staining of thymocytes showed that only one of the two tetramer binding populations were deleted via an Aire-dependent mechanism. These studies demonstrate that two types of IRBP-specific T cells participate in spontaneous autoimmune uveitis in Aire KO mice, those that are deleted in an Aire-dependent manner and those that are not deleted by Aire.
Interleukin-17 exacerbates tumor necrosis Factor-α induced apoptotic cell death of oligodendrocytes (148.8)
Authors: Paintlia Manjeet , Paintlia Ajaib , Singh Avtar , Singh Inderjit
Published: January 2023

Pro-inflammatory cytokine-induced cell death of oligodendrocytes (OLs) is associated with axonal loss in neurodegenerative disorders including multiple sclerosis (MS) and that contributes to clinical exacerbation in affected individuals. Accumulating evidence suggest that both Th1 phenotype cytokine including tumor necrosis factor (TNF)-α and Th17 phenotype cytokine, interleukin (IL)-17 participate equally in MS pathogenesis. Here, we investigated the effect of IL-17 on OLs survival in the presence/absence of TNF-α in vitro. Our findings suggest that OL survival was not affected by IL-17 alone, but it exacerbated the TNF-α-induced OL apoptosis in a dose dependent fashion. Synergistic effect of these cytokines was ascribed to an inhibition of cell survival mechanisms, co-localization of Bid/Bax proteins in the mitochondrial membrane and caspase 8 activation mediated release of apoptosis inducing factor from mitochondria in OLs. In addition, cytokine treatment disturbed the mitochondrial membrane potential in OLs with corresponding increase in the generation of reactive oxygen species, which were attenuated by N-acetyl cysteine. Collectively, these data provide initial evidence that IL-17 exacerbates TNF-α-induced apoptotic cell death of OLs that antioxidant-based therapies have potential to limit CNS demyelination in MS or other related demyelinating disorders.
Functional expression of CCR9 by lung cancer (149.16)
Authors: Singh Rajesh , Grizzle William , Lillard Jr. James , Singh Shailesh
Published: January 2023

Lung cancer is the leading cause of cancer-related deaths worldwide in both men and women. Metastasis is responsible for many of the lung cancer-related deaths; therefore, a better understanding of the metastatic processes and therapies to prevent the spread and growth of these tumors is greatly needed. The specific mechanisms that promote metastases have not been fully elucidated. Multiple chemokine receptors are involved in the multi-step process of metastasis and growth of cancer cells. For the first time, we show that non-small cell lung carcinoma (NSCLC) tissue express high levels of CCR9, which is the receptor for CCL25. It addition, serum levels of CCL25 which is natural ligand for CCR9 was significantly elevated in lung cancer patents compared to normal healthy donor. CCR9 was also highly expressed by NSCLC cell lines. To this end, CCR9 mRNA and protein levels were higher than similar levels expressed by small cell lung carcinoma (SCLC) cell lines. Functional CCR9 expression was demonstrated by CCL25-mediated lung cancer cell migration that was inhibited by anti-CCR9 antibody. The mechanisms underlying these clinically and biologically relevant findings need to be further explored. However, our studies elucidate a novel role for CCL25-CCR9 interactions in lung cancer progression.
IL-23 is required for long-term control of Mycobacterium tuberculosis and B cell follicle formation in the infected lung. (150.1)
Authors: Khader Shabaana , Guglani Lokesh , Rangel-Moreno Javier , Fountain Jeffrey , Martino Cynthia , Pearl John , Kolls Jay , Randall Troy , Cooper Andrea
Published: January 2023

IL-23 is required for effective IL-17 responses to aerosol infection with Mycobacterium tuberculosis (Mtb), however it does not appear to impact early control of bacterial growth in the lung. By extending the time course of infection using mice deficient for the p19 component of the IL-23 cytokine, we report that there is increased bacterial growth in the lungs of these mice compared to wild type infected mice. This increased bacterial growth is associated with decreased maintenance of B cell follicles within lungs of infected mice. While mRNA expression for molecules associated with limiting bacterial growth such as Interferon gamma (IFNγ) were equivalent between wild type and IL-23p19 deficient mice, reduced expression of both IL-17 and IL-22 mRNA was observed in IL-23p19 deficient Mtb-infected lungs. Importantly, we show that in mice deficient for IL-23p19, B cell follicle size was reduced during chronic Mtb infection and this was temporally associated with the increased bacterial burden. In mice deficient in the IL-17 receptor (IL-17R), B cell follicle generation was affected early in infection whereas the absence of IL-22 influenced the size of B cell follicles in the mid-term. Interestingly, the absence of IL-22 or IL-17R alone did not impact bacterial control. Thus, we show novel data that while IL-23 is required for long term control of Mtb growth in the lung, there is a compensatory effect of IL-22 and IL-17 during tuberculosis.
Memory CD8 T cell responses to IL-15 transpresentation are impaired in the absence of ICAM-1 (150.6)
Authors: Stonier Spencer , Acero Luis , Schluns Kimberly
Published: January 2023

Homeostatic proliferation is important for the long-term maintenance of memory CD8 T cells and is mediated by trans-presentation of IL-15. Since IL-15 transpresentation requires a cell-cell interaction, we asked whether ICAM-1 is important for IL-15 responses in memory CD8 T cells. After transfer into either Wt or ICAM-1-/- mice, Wt memory CD8 T cells displayed less homeostatic proliferation and decreased survival in the absence of ICAM-1. To determine whether the absence of ICAM-1 was directly affecting IL-15 transpresentation, IL-15 responses of isolated CD8 T cells were analyzed after culturing with DCs. IL-15-driven proliferation and STAT5 phosphorylation in memory CD8 T cells were completely abolished in cultures with ICAM-1-/- DCs or with blocking Ab against ICAM-1. Overall, this is the first report that establishes a link between ICAM-1 and IL-15 transpresentation and serves to demonstrate the importance of specific adhesion molecules in facilitating IL-15-mediated responses in memory CD8 T cells.
Role of inflammasome signaling in development and function of mast cells (151.11)
Authors: Mortaz Esmaeil , Folkerts Gert , Kraneveld Aletta , Kool Mirjam , Lambrecht Bart , Garssen Johan , Redegeld Frank
Published: January 2023

The inflammasome is a cytosolic molecular complex containing a ASC,caspase1 and NALP that mediate the maturation of the proinflammatory cytokines IL-1β by caspases-1. NALP3 is an intracellular receptor involved in the recognition of pathogen associated molecular patterns (PAMPs).The inflammasome signal transduction promotes the maturation of inflammatory cytokines 1L-1β.The levels of 1L-1β has been shown increased in lung disease like asthma and emphysema.Mast cells(MC)play as effector cells for IgE mediated allergic diseases.Besides, recent evidence indicates that MCs also function as important cells in immune surveillance. Not much is known about role of inflammasomes in biology and development of mast cells.In this study we investigated the role of NALP3/ASC in mast cells.Development and function of mast cells, derived from a culture of bone marrow cells of wild-type, ASC-/-, NALP3-/-mice was studied. Bone marrow cells were cultured in presence for 3 weeks. After 3 weeks the responsiveness of mast cells to IgE/Ag and LPS was studied.Results:We found that in ASC-/-,NALP3-/- mast cells,the cellular maturation and differentiation was decreased as evidenced by a lower expression of c-kit and FcϵRI. Degranulation and the production of TNF-α,IL-6 and IL-1β after stimulation by IgE/Ag or LPS was significantly lower in NALP3-/-,ASC-/- cells compared to control cells.Our study demonstrates that NALP3/Asc plays role in maturation,differentiation and function of mast cells in vitro.
Genetic analysis of basophil function in vivo (151.22)
Authors: Sullivan Brandon , Liang Hong-Erh , Bando Jennifer , Wu Davina , Cheng Laurence , McKerrow James , Allen Chris , Locksley Richard
Published: January 2023

Contributions by basophils to allergic and helminth immunity remain controversial. Using sensitive IL-4 reporter alleles, we demonstrate that basophil IL-4 production occurs by a CD4 T cell-dependent process restricted to affected peripheral tissues. We genetically marked and specifically deleted basophils and demonstrate that basophils do not mediate Th2 priming in vivo. Two-photon imaging confirmed that basophils do not interact with antigen-specific T cells in lymph nodes, but can engage in prolonged serial interactions with T cells in lung tissues. Although targeted deletion of IL-4/IL-13 in either CD4 T cells or basophils minimally impacted worm clearance, deletion from both lineages demonstrated a nonredundant role for basophil cytokines in primary helminth immunity.