bluuebunny/crossref_metadata_2025 huggingface dataset:
Recombinant modification of surfactant protein D (SP-D) increases neutralizing and opsonizing activity for influenza virus (68.19)
Authors: Hartshorn Kevan , Seaton Barbara , White Mitchell , McCormack Francis , Crouch Erika
Published: January 2023

Human SP-D inhibits seasonal, but not pandemic, influenza A virus (IAV). We modified key residues in the carbohydrate recognition domain (CRD) of SP-D to increase antiviral activity. Isolated neck and CRD (NCRD) preparations of SP-D were used. The NCRD of wild type human SP-D did not inhibit IAV (although the full length molecule does). We reported that the substitutions D325A and R343V in the NCRD markedly increase anti-IAV activity in vitro and in vivo. We now report similar activity of a double mutant containing D325S and R343V changes. These double mutants (called D325A+R343V or D325S+R343V) also inhibit pandemic IAV strains. Crystals of D325A+R343V show distinct interactions with viral glycans. D325A+R343V causes marked viral aggregation and increase in IAV uptake by neutrophils and monocytes while wild type SP-D NCRD does not. D325A+R343V inhibits viral protein synthesis in monocytes and suppresses IAV induced tumor necrosis factor (TNF) production. Surprisingly, the wild type human SP-D NCRD also suppresses IAV-induced TNF production without altering viral replication in monocytes. Hence, modification of residues surrounding the lectin site of SP-D markedly increases viral neutralizing and aggregating activity and also broadens the spectrum of inhibited strains. Inhibition of IAV-induced TNF production by monocytes is at least in part mediated by a direct effect on these cells independent of viral neutralization.
Transcriptional factor E2A regulates the differentiation of Th17 cells (69.10)
Authors: Nakatsukasa Hiroko , Maruyama Takashi , Chen Wanjun
Published: January 2023

The molecular mechanisms that direct transcription of the gene encoding the transcription factor RORγt, a master regulator of Th17 cells, remain ill-defined. We previously showed that transcription factor E2A is enriched at the Foxp3 promoter and regulates the differentiation of regulatory T cells in response to TGF-β. Since TGF-β is also required to generate Th17 cells, we hypothesized that E2A might also be involved in the differentiation of Th17 cells. We utilized Id3 deficient mice, as Id3 is reported to inhibit E2A binding to DNA, and found that Id3 deficient mice had a much greater frequency and absolute number of Th17 cells in the lamina propria and Peyer’s patches. In vitro study showed that Id3 deficient CD4(+)CD25(-) naïve T cells showed greater differentiation into Th17 cells in response to TGF-β1 plus IL-6. Treatment with TGF-β1 substantially enriched E2A at the Rorc promoter in naïve CD4(+) T cells. In addition, E2A knockdown by siRNA resulted much less Rorc transcription and IL-17 production in CD4(+) T cells stimulated with TGF-β1 plus IL-6. These data suggest that E2A plays an important role in promoting the RORγt expression through enrichment to Rorc promoter and the consequent Th17 differentiation. We plan to investigate how IL-6-mediated signaling pathway is integrated with E2A-mediated Rorc activation in directing IL-17 production in CD4(+) T cells.
How does the age-dependent loss of central tolerance relate to the regulation of ectopic gene expression? (69.6)
Authors: Michel Chloe , Kyewski Bruno
Published: January 2023

Aging is accompanied by an increased susceptibility to autoimmune diseases and a heightened cancer incidence. It has been suspected that age-related T cell-mediated autoimmunity is causally connected to the involution of the thymus. Thymic involution is characterised by a reduction of T cell output to the periphery. While it is well established that the heterogeneous thymic stroma is essential for T cell development, our knowledge of stromal cell function in aging is still limited. Here we report how aging affects: (i) the composition of the epithelial compartment in relation to other thymic cell types; (ii) the promiscuous expression of self-antigens in the medullary thymic epithelial cells (mTECs). Our results show that among mTECs, the mature subset decreased more rapidly with age than the immature subset. The expression of self-antigens per cell was not significantly different in old as compared to young animals, a finding compatible with their rapid turnover. Since the tolerance process is limited by available niches provided by mTECs, the efficacy of this process will also depend on the ratio of thymocytes to mTECs. Interestingly the number of single-positive thymocytes vs. mature mTECs was already increased 2 fold in 12 month-old animals when compared to their young counterparts. These findings document age-related changes in the thymic microenvironment, which collectively are conducive to subvert central tolerance induction.
Tim3/Galectin-9 interactions regulate innate and adaptive immunity to Mycobacterium tuberculosis (70.6)
Authors: Jayaraman Pushpa , Sada-Ovalle Isabel , Anderson Ana , Kuchroo Vijay , Behar Sam
Published: January 2023

In vivo control of Mycobacterium tuberculosis (Mtb) reflects the balance between host-immunity and bacterial evasion strategies. Effector TH1 cells that mediate protective immunity by depriving the bacterium of its intracellular niche are regulated in many ways to prevent over-exuberant inflammation. Although Tim3 is generally recognized to down regulate TH1 responses and mediate T cell exhaustion, we recently described that its interaction with Galectin-9 (Gal9) expressed by Mtb infected macrophages (Mϕ) stimulates IL-1β secretion, which is essential for host resistance to Mtb. IL-1β signaling is both necessary for Tim3 mediated Mtb control and sufficient to directly kill Mtb in murine and human Mϕ. The molecular mechanism by which IL-1β restricts bacterial growth is not known. We report that IL-1β kills Mtb through the recruitment of other antimicrobial effector molecules. IL-1β directly augments TNF signaling through increased TNF secretion and TNFR cell surface expression. Furthermore, Tim3/Gal9 and IL-1β activates the executioner caspase-3. Thus, Tim3/Gal9, via IL-1β and downstream TNF production lead to apoptosis and restriction of intracellular Mtb growth. We propose that Tim3/Gal9 interaction acts as a bidirectional molecular rheostat that activates pathways to clear intracellular pathogens in innate immune cells while fine-tuning termination of TH1 responses to prevent immunopathology.
Host resistance to a gastrointestinal parasite regulated by a SNP modulating SMAD3 binding to a TGFβ-regulated transcriptional enhancer of Mina, encoding an immunoregulatory member of the Jmjc protein family (70.9)
Authors: Bix Mark , Lian Shangli , Pillai Meenu
Published: January 2023

In a hunt for Th2-bias regulatory loci in mice, we have used interval-specific congenic mapping to positionally clone a gene called Mina, encoding an immunoregulatory member of the JmjC protein family. Th2-bias is a genetic trait defined as the propensity of naïve T helper cells to develop in vitro under neutral conditions into T helper 2 (Th2) effector cells. Th2 cells play a critical role in host protection from multicellular parasites such as intestinal helminths and when disregulated contribute to allergic diseases such as asthma. The Th2-bias trait is known to vary across different inbred mouse strains and correlates inversely with the relative abundance of intracellular Mina protein. Adding further support for a causal link between Mina protein level and Th2-bias, Mina was shown to act as a dose-dependent transcriptional corepressor of the gene encoding interleukin-4 (IL4), a key regulator of Th2 development. We have now identified a SNP that explains differential Mina expression level in high and low Th2-biased strains such as BALB/c and C57BL/6. Most interestingly, this SNP acts by modulating SMAD3 binding to a TGFβ-regulated intronic Mina enhancer. We are currently tying this into its physiological role in pulmonary inflammation and intestinal helminth infection using Mina conditional KO mice, which exhibit dramatic phenotypes in both models.
Role of Regulatory T cells and TGF-β in an Experimental Model of Psoriasis in mice (71.8)
Authors: Zanvit Peter , Chen Wanjun
Published: January 2023

Psoriasis is inflammatory disease with active interactions between the immune system and the skin, which affects approximately 2% of the world’s population and attacks about 10 million people in North America. The pathogenesis of this disease is not completely understood. Regulatory T cells (Tregs) are typically considered to be inhibitors of autoimmune responses, but the role of Tregs in pathogenesis of psoriasis remains largely unclear. Previous works have suggested that overexpression of transforming growth factor beta (TGF-β) is involved in severe skin inflammation similar to psoriasis, but the molecular mechanism by which TGF-β functions in this autoimmune disease remains to be determined. By utilizing an experimentally induced model of psoriasis using Imiquimod (TLR7/8 ligand and potent immune activator), we observed that topical aplication of Imiquimod can induce psoriasis through the IL-23/IL-17 axis in mice. We also found that TGF-β signaling and frequency of Tregs are decreased in this model of psoriasis. To further understand the molecular mechanism of TGF-β in psoriasis we have created transgenic mouse model K14.TGF-β1glo that overexpress TGF-β in epidermal keratinocytes. We are actively investigating the interplay between TGF-β and Treg cells in skin in the pathogenesis of psoriasis.
The E3 ubiquitin ligase Smurf2 suppresses c-myc expression and B cell lymphoma in mice (74.4)
Authors: Gerstein Rachel , Ramkumar Charusheila , Kong Yahui , Cui Hang , Hao Suyang , Jones Stephen , Zhang Hong
Published: January 2023

The NCI estimates in 2012 there will be 70,130 new cases and 18,940 deaths from non-Hodgkin’s lymphoma (NHL) in the US. Diffuse large B-cell lymphoma (DLBCL) is the most common NHL. This aggressive B-cell lymphoma is heterogeneous, with significant variation in response to treatment. Gene expression profiling identifies biologically distinct subtypes of DLBCL, which correlate with both response to treatment and survival. This suggests the critical importance of sub-type-specific targets in the diagnosis and treatment of DLBCL. The E3 ubiquitin ligase Smurf2 induces senescence in human cells, suggesting it is a tumor suppressor. We discovered that ~30% of mice deficient in Smurf2 develop NHLs with a post-germinal center phenotype resembling human DLBCL. B cells from healthy Smurf2-deficient mice exhibit enhanced proliferation and elevated expression of the oncogene c-myc. We find that Smurf2 mediates ubiquitination of a key transcription factor, which, when stabilized in Smurf2-deficient cells increases c-myc expression. This Smurf2-c-Myc axis represents a novel mechanism by which Smurf2 suppresses cell proliferation and lymphomagenesis. Analysis of gene expression databases reveals a significant decrease in Smurf2 expression in human DLBCL compared to normal B cells. Importantly, low level Smurf2 expression correlates with inferior overall survival in DLBCL patients. These data suggest that Smurf2 is a novel target in the diagnosis and potential treatment of human DLBCL.
No neurorestorative effect of IVIg in a mouse model of Parkinson Disease (75.6)
Authors: St-Amour Isabelle , Bousquet Mélanie , Paré Isabelle , Drouin-Ouellet Janelle , Bazin Renée , Calon Frederic
Published: January 2023

Immunotherapies have been proposed as a therapeutic strategy for the treatment of Parkinson disease (PD). Intravenous immunoglobulin preparation (IVIg) is used for the treatment of immunodeficiency and autoimmune diseases. Here, we investigated the effect of IVIg treatment on the dopaminergic (DAergic) nigrostriatal system after MPTP-induced denervation in mice. Mice received 4 MPTP injections (15 mg/Kg) at 2-hour intervals followed by a 14 days treatment with IVIg. IVIg injections led to a 21,6% increase in TReg cells in vehicle group (p<0.05) and minimal specific immune response. As expected, MPTP insult induced a significant 80% and 84% depletion of striatal dopamine content (p<0.01), as well as a 31% and 45% nigral DAergic neuron loss (p<0.001) in the Control and IVIg group, respectively. Moreover, two-way ANOVA analyses revealed a significant downregulating effect of IVIg on striatal tyrosine hydroxylase protein level (-16%, p<0.05) and on the number of nigral DAergic neurons (-29%, p<0.05), paralleling the toxic effect of MPTP. Collectively, our results provide no evidence of a neurorestorative effect of IVIg on the nigrostriatal system of the MPTP-treated mouse, and suggest that we need to proceed with caution before treating PD patients with IVIg.
TLR3 signaling is either protective or pathogenic for the development of TMEV-IDD depending on the time of viral infection (76.5)
Authors: Jin Young-Hee , Kaneyama Tomoki , Kang Hyun Seok , Koh Chang-Sung , Kim Byung S.
Published: January 2023

We have previously shown that toll-like receptor 3 (TLR3)-mediated signaling plays an important role in the induction of innate cytokine responses to Theiler’s murine encephalomyelitis virus (TMEV) infection. In addition, cytokine levels produced after TMEV infection are significantly higher in the glial cells of susceptible SJL mice compared to those of resistant C57BL/6 mice. However, it is not known whether TLR3-mediated signaling plays a protective or pathogenic role in the development of demyelinating disease. In this study, we investigated the role of TLR3-mediated signaling in the protection and pathogenesis of TMEV-induced demyelinating disease. Our results indicate that TLR3-mediated signaling plays an important role in protecting mice from the development of disease during viral infection, as a lack of TLR3 signals in TLR3-deficient mice exacerbates the disease. However, activation of TLR3 with poly IC prior to viral infection also exacerbated disease development, whereas administration of poly IC after viral infection restrained disease development. These results suggest that TLR3-mediated signaling during viral infection protects against demyelinating disease by reducing the viral load and modulating immune responses. In contrast, premature activation of TLR3 signal transduction during early viral infection may lead to pathogenesis via over-activation of the pathogenic immune response.
LAMP5 distribution in and on plasmacytoid dendritic cells (P5019)
Authors: Zhang Hong , Pahuja A. , Lin Xiao , Li Sky , Ransom John , Yang Xi Feng
Published: January 2023

The brain and dendritic cell (BAD)-associated lysosome-associated membrane protein (LAMP)-like molecule, also known as BAD-LAMP, c20orf103, UNC-46, is a newly identified LAMP family member, which has been recently officially designated as LAMP5. It is a transmembrane glycosylated protein localized in the ER-Golgi intermediate compartment (ERGIC). LAMP5 shares sequence and structural homology with other LAMP family members. In mouse, LAMP5 is expressed in postnatal cortical neurons of particular layers, where it is enriched in defined zones along neuronal projections. In human, like its murine homolog, LAMP5 is principally expressed in brain, and it was recently found in primary plasmacytoid dendritic cells (pDCs). However, LAMP5 expression profile and function have not been extensively studied in pDCs. By using anti-human LAMP5 monoclonal antibody (clone 124-40B), we report that LAMP5 is specifically expressed in peripheral blood pDCs which are characterized by surface expression of CD303+/CD304+/CD123+/HLA-DR+/CXCR3+/CD11c-/CD14-/CD209-, but is not expressed by GM-CSF plus FL3 or IL-4 induced monocyte-derived CD303+/CD123+/HLA-DR+/CD209+/CD14+/CXCR3dim pDCs or CD209+/CD123dim/CXCR3dim/HLA-DR+/CD11c+/CD14+ mDCs. Like other LAMP members, LAMP5 is rapidly relocated on the cell surface of peripheral blood pDCs after 24 hours stimulation by IL-3. These data suggest that LAMP5 may be a useful marker for studying pDC development and activation.
Inhibition of the CD8 T cell response and cytotoxicity by human intravenous immunoglobulin. (P5023)
Authors: Trepanier Patrick , Bazin Renée
Published: January 2023

Intravenous immunoglobulin (IVIg) is made of plasma-derived human IgG and exerts anti-inflammatory effects in several autoimmune disorders when administered at high doses. The constant increase in its use exposes providers and patients to considerable risks of shortage. Understanding its complex mechanisms of action is required in order to develop potent substitutes. Our group previously demonstrated that IVIg inhibits the in vitro and in vivo activation of CD4 T cells (Aubin et al, Blood 2010), as well as the in vitro CD8 T cell activation (Trépanier et al, Blood 2012). In the present work, we investigated whether IVIg could also interfere with the CD8 T cell response, which contribute to the persistence and severity of many autoimmune conditions. Bone marrow-derived DC from C57BL/6 mice were treated with IVIg and used to cross-present ovalbumin to OVA-specific OT-I CD8 T cells. Proliferation and cytokine production were measured after 72 hours. Cytotoxicity was measured using SIINFEKL-pulsed EL-4 target cells in presence of therapeutic doses of IVIg. Results showed a reduced (50%) CD8 T cells activation in the presence of IVIg and a concomitant decrease in IL-2 and IFN-gamma secretion. In addition, IVIg-treated CD8 T cells are less cytotoxic, as measured by specific lysis of target cells. These results support a novel immunomodulatory mechanism by which IVIg decrease CD8 T cell response. The importance of these results in vivo are currently being investigated.
Effect of cellular activation on the antigen processing machinery of primary CD4 T cells. (P5029)
Authors: Boucau Julie , Madouasse Julien , Carlin Christopher , Zhu Tom , Xu Yang , Shimada Mariko , Le Gall Sylvie
Published: January 2023

The killing of HIV-infected CD4 T cells by specific CD8 T cells (CTL) requires the presentation of peptide-MHC-I complexes produced during the intracellular degradation of viral proteins by the proteasome and other peptidases, to their cognate T cell receptors. HIV infection induces general cellular activation, which renders cells more susceptible to infection. Whether the activation state of cells alters the expression and activity of the antigen processing machinery and the kinetics and nature of epitopes displayed by MHC-I is not known despite its potential role in altering the efficiency of recognition of infected cells by CTL. We compared the expression and peptidase activities of the antigen processing machinery of primary CD4 T cells upon activation with anti-CD3/CD28 beads or with phytohemagglutinin. In primary CD4 T cells from HIV seronegative individuals, the hydrolytic activities of the proteasome, the expression of several of its subunits and of the peptidase TPP2 significantly increased upon activation. The mass spectrometry analysis of in vitro degradation of 2 long epitope-containing HIV peptides in extracts from CD3/CD28-activated CD4 T cells showed production of smaller and less antigenic peptides than in the non-activated samples. Reduced presentation of peptides by activated cells might lead to reduced recognition and slower clearance of activated infected cells by CTL thus allowing more time for viral replication and spread.
Immunodominant and subdominant WNV epitopes are differentially presented (P5005)
Authors: McMurtrey Curtis , Kim Sojung , Bardet Wilfried , Schafer Fredda , Diamond Michael , Weidanz Jon , Hansen Ted , Hildebrand William
Published: January 2023

There are many factors that are thought to contribute to T-cell immunodominance hierarchies including timing and levels of antigen presentation to T-cells. One of the most potent tools for studying antigen presentation for a specific determinant are monoclonal antibodies that are specific for not only MHC but a specific ligand, similar to a T cell receptor. In this study we utilize these antibodies to study levels and timing of antigen presentation of immunodominant (SVG9), sub-dominant (SLF9), and super sub-dominant (YTM9) T-cell epitopes during West Nile virus (WNV) infection. In this study, we demonstrate that these ligands are differentially expressed on the surface of a cell infected with WNV. Specifically, the super sub-dominant ligand (YTM9) was presented earlier and at 20 times the levels of the immunodominant (SVG9) and subdominant (SLF9) ligands. Transfection of ICP47 in the infected cells show that all three epitopes are TAP dependent. Using these highly specific reagents we show that there is differential ligand presentation within the restrictive allele. High levels of peptide HLA complexes on the surface of an infected do not correspond with immunodominance hierarchies as the super subdominant epitope YTM9 was presented at higher levels than the most dominant epitope SVG9. This differential in presentation was not due the TAP dependence of the ligands.
Initiation of TCR phosphorylation and signal transduction. (P5041)
Authors: Casas Javier , Brzostek Joanna , Hoerter John , Fu Guo , Ampudia Jeanette , Gascoigne Nicholas
Published: January 2023

Recognition of antigen by the T cell receptor (TCR) is the key event for the T cell activation. The earliest molecular events in T cell recognition have not yet been fully described, and the TCR triggering mechanism remains a subject of controversy. By using supported lipid bilayers to present peptide MHC class I complexes to CD8+ cells, we can monitor the molecular events occurring at the immune synapse. The lipid bilayer technology mimics the signaling environment for T cell activation as judged by Ca2+ flux, total phospho-Tyr levels, and IL-2 secretion. Using TIRF/FRET microscopy we have observed an early (> 1 min) interaction between CD3ζ and the coreceptor CD8 that is independent of the MHC-CD8 binding, but requires CD8 association with Lck. Later (< 10 min) CD3ζ-CD8 interactions require CD8-MHC binding. This suggests that, upon antigen recognition, TCR may be initially phosphorylated by Lck not associated with coreceptor, followed by MHC dependent recruitment of CD8-Lck complexes. We have now additional evidences that free Lck is the responsible for the initial TCR phosphorylation. We are currently working on elucidating the proposed different roles of unbound and coreceptor associated Lck during the early stages of antigen recognition.
MAIT cell recognition of MR1 bound by non-peptide antigen on bacterially infected and uninfected cells. (P5053)
Authors: Young Mary , U'Ren Lance , Kappler John , Marrack Philippa , Gapin Laurent
Published: January 2023

Mucosal-associated invariant T cells are a unique population of T cells that express a semi-invariant αβ TCR and are restricted by the MHC class I-related molecule MR1. MAIT cells recognize uncharacterized ligand(s) presented by MR1 through the cognate interaction between their TCR and MR1. To understand how the MAIT TCR recognizes MR1 at the surface of APCs cultured both with and without bacteria, we undertook extensive mutational analysis of both the MAIT TCR and MR1 molecule. We found differential contribution of particular amino acids to the MAIT TCR-MR1 interaction based upon the presence of bacteria or bacterially-derived compounds, supporting the hypothesis that the structure of the MR1 molecules with the microbial-derived ligand(s) differs from the one with the endogenous ligand(s). Furthermore, we demonstrate that microbial-derived ligand(s) is unlikely proteinaceous or lipidaceous, arguing that a new class of antigen(s) is most likely recognized by this unique lymphocyte population.
Satiated-efferocytosis: A novel functional property for resolution-phase macrophages regulated by glucocorticoids, resolvins, galectin-1 and the chemokine-scavenging receptor D6 (P4157)
Authors: Ariel Amiram , Serhan Charles , Rabinovich Gabriel , Pashover-Schallinger Ester , Gross Nufar , Aswad Miran , Rostoker Ran , Assi Simaan , Yaseen Hiba , Schiff-Zuck Sagie
Published: January 2023

The engulfment of apoptotic leukocytes (efferocytosis) by macrophages during the resolution of inflammation is essential for homeostasis and results in macrophage reprogramming/immune-silencing. Here, we show CD11bhigh macrophages convert to CD11blow ones and stop efferocytosing apoptotic PMN after reaching an engulfment threshold in vivo. In addition, CD11blow macrophages are distinct from either M1 or M2 in their protein expression profile and display pro-resolving properties, such as diminished responses to different TLR ligands ex vivo and propensity to emigrate from resolving inflammation sites to lymphoid organs. Of interest, we found the pro-resolving lipid mediators resolvin E1 and D1, as well as the glucocorticoid dexamethasone (Dex) and the lectin galectin-1 enhance satiated-efferocytosis and consequently CD11blow macrophage conversion from their CD11bhigh counterparts. Deficiency in the atypical chemokine receptor D6 resulted in delayed satiation and reduced immune-silencing of macrophages, and inhibits their departure from resolving inflammation sites. In sum, satiated-efferocytosis is a novel phagocyte property of CD11blow macrophages that is regulated by pro-resolving mediators. Moreover, satiated-efferocytosis is required for CD11blow macrophage emigration from resolving inflammation sites and the return of tissue homeostasis. Thus, satiated-efferocytosis is essential for the completion of timely- and spatially-coordinated resolution of acute inflammation.
Adenylyl cyclase 6 activation negatively regulates TLR4 signaling through lipid raft-mediated endocytosis (P4169)
Authors: Xu Congfeng , Cai Wei , Feng Kuan , Zhao Xiaonan , Jin Min , Qian Liu , Ostrom Rennolds , Zhang Yanyun
Published: January 2023

Proper intracellular localization of Toll-like receptors is essential for their signaling and biological functions. Endocytosis constitutes a key step in protein turnover as well as maintenance of localization, and thus provides important points to regulate their signaling. Here we demonstrated that adenylyl cyclase activation attenuated membrane Toll-like receptor 4-dependent NF-κB signaling and IRF activation in the murine macrophage cell line RAW264.7 and in bone marrow-derived macrophages stimulated with lipopolysaccharide. We further showed that the adenylyl cyclase 6 isoform distributed in lipid raft-enriched domains played key roles in the negative regulation of Toll-like receptor 4 signaling by promoting its degradation. Toll-like receptor 4 is normally endocytosed through the clathrin-mediated pathway, which is a critical step in signal transduction, while adenylyl cyclase 6 activation initiated its colocalization with Toll-like receptor 4 and their interaction, and shifted clathrin-mediated endocytosis of Toll-like receptor 4 to lipid raft-mediated endocytosis, leading to accelerated degradation of Toll-like receptor 4 and consequently suppressed downstream signaling. Our studies have unveiled a new mechanism of negative regulation of Toll-like receptor 4 signaling through adenylyl cyclase 6-mediated endocytosis. Adenylyl cyclase 6-specific activation might provide a novel therapeutic approach for limiting inflammatory and autoimmune diseases.
SIRPα-dependent phagocytosis of tissue cells by macrophages (P4171)
Authors: Bian Zhen , Liu Yuan
Published: January 2023

Macrophage surface SIRPα, a ITIMs-containing inhibitory signaling receptor, plays a pivotal role in controlling macrophage phagocytosis of host cells, including healthy self-cells, cancerous and apoptotic cells, through interactions with CD47 expressed on target cells. Utilizing SIRPα-/- and CD47-/- mice, we further investigated SIRPα-dependent macrophage phagocytosis and found that, while ligation of macrophage SIRPα by CD47 triggering inhibitory signaling prohibits phagocytosis, depletion of SIRPα does not immediately render/enhance macrophage phagocytosis. In contrast, SIRPα-/- mice, and also CD47-/- mice, display no signs of macrophage-mediated tissue-destruction or anemia. In adoptive transfer experiments, SIRPα-/- mice demonstrated phagocytic suppression and failure of clearance of CD47-/- RBC, while WT mice hastily cleared CD47-/- RBC in the spleen. We found that treating SIRPα-/- macrophages or mice with inflammatory cytokines especially IL-17, but not IFNγ, releases such functional suppression resulting in significantly augmented macrophage phagocytosis (splenomegaly) and anemia in vivo. IL-17 also largely promotes SIRPα-dependent phagocytosis of macrophages derived from WT mice. Remarkably, we observed that IL-17-treated SIRPα-/- macrophages aggressively ingest cancer cells in vitro, and IL-17-adminitrated SIRPα-/- and WT mice completely cleared and largely delayed, respectively, implanted Lewis lung tumors and B16 melanoma in vivo.
Human P2X7 receptor activation induces ADAM10-mediated shedding of CD23 and CXCL16 (P4137)
Authors: Pupovac Aleta , Wang Bin , Foster Christopher , Sluyter Ronald
Published: January 2023

Activation of the damage-associated molecular pattern receptor P2X7 by extracellular ATP induces the shedding of cell surface molecules, including CD23, from leukocytes. The mechanisms involved in this process however remain poorly understood. Using a flow cytometric assay, we demonstrated that ATP-induces the rapid shedding of CD23 from the surface of human multiple myeloma RPMI 8226 and peripheral blood B cells with a t1/2 of 7 min. The P2X7 antagonist, AZ10606120, impaired ATP-induced CD23 shedding by 89%. Moreover, P2X7-induced CD23 shedding was impaired by a broad-spectrum metalloprotease inhibitor, BB-94, by 63% and the ADAM10 inhibitor, GI254023X, by 67%. RT-PCR confirmed the expression of ADAM10 in B cells. P2X7-induced CD23 shedding was not dependent on changes in extracellular Na+, K+ and Ca2+ concentrations, and was unaffected by a panel of enzyme inhibitors targeting various kinases and phospholipases previously implicated in P2X7-mediated signaling events. ATP also induced the rapid shedding of the ADAM10 substrate, CXCL16, with a t1/2 of 1 min. AZ10606120 impaired ATP-induced CXCL16 shedding by 86%. Moreover, P2X7-induced CXCL16 shedding was impaired by BB-94 by 77% and GI254023X by 87%. This data indicates that human P2X7 activation induces the rapid shedding of CD23 and CXCL16, and that this process involves ADAM10. Thus, P2X7 may represent a novel target in disorders involving CD23 or CXCL16.
CD40L induces networks of branching tunneling nanotube-like structures exclusively in type 1 polarized dendritic cells (P4187)
Authors: Zaccard Colleen , Mailliard Robbie , Kalinski Pawel , Ayyavoo Velpandi , Salter Russel , Watkins Simon , Rinaldo Charles
Published: January 2023

The ability of dendritic cells to mediate CD40L-dependent CD4+ T cell help for the induction and support of effector CD8+ cytotoxic T lymphocyte responses greatly depends on activation signals they received during maturation. In addition to being 'programmed' to produce high levels of IL-12p70, we found that DC matured under type 1 polarizing conditions also form intricately branched tunneling nanotube-like structures upon CD40L stimulation. These formations can create extensive interconnected networks between cells, a process we termed 'reticulation'. Importantly, this response was found to occur exclusively in mature type 1 polarized DC and was uniquely induced by recombinant CD40L or CD40L-expressing cells. Using live-cell time-lapse confocal imaging, we were able to determine that these structures can support the transfer of endosomes, HIV-1 virus-like particles, and nano-beads representing antigen from 'donor' to 'recipient' type 1 polarized DC. This induction of the process of DC reticulation potentially represents a novel helper function of CD40L, as it likely enhances intercellular communication and contributes to the superior capacity of type 1 polarized DC to induce cellular immune responses. This active immune cell process may also be utilized by viruses such as HIV-1 for cell-to-cell spread.
p62/SQSTM1 enhances NOD2-mediated signaling and cytokine production through stabilizing NOD2 oligomers (P4189)
Authors: Kim Sung Ouk , Park Sangwook , Ha Soon-Duck , Coleman Macon , Meshkibaf Shahab
Published: January 2023

NOD2 is a cytosolic pattern-recognition receptor that senses muramyl dipeptide of peptidoglycan that constitutes the bacterial cell wall, and plays an important role in maintaining immunological homeostasis in the intestine. To date, multiple molecules have shown to be involved in regulating NOD2 signaling cascades. p62 (sequestosome-1; SQSTM1) is a multifaceted scaffolding protein involved in trafficking molecules to autophagy, and regulating signal cascades activated by Toll-like receptors, inflammasomes and several cytokine receptors. Here, we show that p62 positively regulates NOD2-induced NF-kB activation and subsequent production of cytokines (IL-1b and TNF-a). p62 associated with the nucleotide binding domain of NOD2 through a bi-directional interaction mediated by either TRAF6-binding or ubiquitin-associated domains. NOD2 formed a large complex with p62 in an electron-dense area of the cytoplasm, which increased its signaling cascade likely through preventing its degradation. This study for the first time demonstrates a novel role of p62 in enhancing NOD2 signaling effects.
DAP12 inhibition of LPS signaling in macrophages is mediated by DOK3 (P4147)
Authors: Long Courtney , Peng Qisheng , Malhotra Shikha , Humphrey Mary
Published: January 2023

Toll like receptors serve as sentinels to the innate immune system by binding common pathological antigens and initiating host responses. These responses can be deleterious to the host if unchecked leading to chronic diseases including autoimmunity. DNAX-activating protein 12 (DAP12) is an adaptor protein containing an immunoreceptor tyrosine-based activation motif expressed in myeloid cells, NK cells, osteoclasts and microglia. In macrophages, DAP12 can couple with immunoreceptor triggering receptor expressed on myeloid cells 2 (TREM2) to negatively regulate TLR signaling through an as yet undefined mechanism. We have shown that downstream of kinase 3 (DOK3), through its phosphotyrosine domain, associates with DAP12 after low down LPS stimulation of bone marrow macrophages (BMM). DOK3-/- BMMs stimulated with LPS show increased phospoERK signaling at 20 and 40 minutes indicative of enhanced activation. Supernatants taken from these cells show significantly increased levels of proinflammatory cytokines TNFα, IL-6, and IL-12p40 compared to wild type cells. Upon LPS stimulation, DOK3 is phosphorylated in a DAP12- and Src kinase- dependent manner leading to DOK3 translocation to the plasma membrane. Despite the recruitment of SHIP1 to DOK3 and DAP12 after LPS stimulation; SHIP1 is not required for DAP12-dependent inhibition of LPS-induced ERK activation. These results reveal a new mechanism underlying DAP12 and TREM2 inhibition of TLR responses in macrophages.
Induction of CD70 is essential for TGF-β-mediated inhibition of T cells (P1019)
Authors: Yang Zhi-Zhang , Grote Deanna , Xiu Bing , Price-Troska Tammy , Ziesmer Steven , Hodge Lucy , Witzig Thomas , Novak Anne , Ansell Stephen
Published: January 2023

Although extensively studied, the exact mechanism by which TGF-β mediates T-cell inhibition is not entirely clear. When we examined the effect of TGF-β on the phenotype and function of T cells, we noted that CD70 was significantly up-regulated by TGF-β. This surprising finding led us to explore the underlying mechanism by which TGF-β upregulates CD70 expression on T cells and to define the function of CD70+ cells in B-cell NHL. Interestingly, we observed that TGF-β preferentially induced CD70 expression on effector memory T cells while it predominantly up-regulated Foxp3 on naïve T cells. CD70 induction is Smad3-dependent and involves Stat5 signaling. CD70+ T cells were highly susceptible to TGF-β-mediated apoptosis and that transfection of Jurkat cells with the CD70 gene led to a significantly reduced survival. TGF-β-induced CD70+ T cells had a diminished response to immune stimulation associated with downregulation of co-stimulatory molecules CD27 and CD28. CD70-expressing T cells from biopsy specimens of patients with B-cell NHL failed to produce cytokines and displayed no signal transduction when stimulated. CD70-expressing T cells were highly represented in follicular NHL biopsy specimens and increased numbers of CD70+ cells correlated with an inferior patient outcome. These novel findings not only revealed a biological role for a TGF-β/CD70 axis in mediating immune inhibition, but also may lead to therapeutic approaches in patients with B-cell NHL.
TL1A-Ig induces transplantation tolerance (P1003)
Authors: Khan Samia , Tsai Matthew , Schreiber Taylor , Wolf Dietlinde , Deyev Vadim , Podack Eckhard
Published: January 2023

Tumor necrosis factor (TNF) superfamily member 15 (TL1A) is the natural ligand for TNFRSF25. We previously reported that TNFRSF25 stimulation with an agonist antibody 4C12 selectively expands pre-existing Tregs in vivo. To compare TL1A to 4C12, we generated a soluble mouse hexameric TL1A-Ig fusion protein. TL1A-Ig mediates proliferation of Foxp3+ Treg in vitro. TNFRSF25 stimulation by TL1A-Ig in vivo induces expansion of Tregs to 30-35% of all CD4+ T cells in the peripheral blood within 5 days of treatment. With daily injections of TL1A-Ig elevated levels of Tregs can be maintained for at least 20 days. TL1A-Ig expanded Tregs express high levels of the activation/memory markers KLRG1 and CD103 and are highly suppressive ex vivo. TL1A-Ig mediated Treg expansion in vivo is protective against allergic lung inflammation by reversing the ratio of Tconvs:Tregs in the lung and blocking eosinophil exudation into the bronchoaveolar fluid. We tested the potential of TNFRSF25 agonists in transplant tolerance in several models. (1) TL1A-Ig injections 12 days after injection of allogeneic splenocytes caused diminished alloantigen-specific T cell responses in mixed lymphocyte reactions as compared to mice treated with allogeneic splenocytes only. (2) TNFRSF25 mediated Treg expansion in vivo significantly prolonged allogeneic skin graft survival. Thus, TL1A-Ig fusion proteins are potent and tightly controllable agents that may provide therapeutic benefit in asthma and transplant tolerance.
PD-1 controls effectors but not the generation or function of natural or induced regulatory T-cells (P1011)
Authors: Ellestad Kristofor , Thangavelu Govindarajan , Ewen Catherine , Anderson Colin
Published: January 2023

During T cell activation, the balance of co-stimulatory and co-inhibitory interactions plays an important role in shaping the resulting T cell response. Co-inhibitory signals help maintain peripheral immune tolerance, and thus animals with defective co-inhibitory molecules, such as PD-1 KO mice, are predisposed to autoimmunity. Unlike PD-1 KO mice, which develop mild autoimmunity, adult Rag KO recipients of PD-1 KO hematopoietic stem cells (HSC) succumb to rapid and severe multi-organ autoimmune disease soon after T cells first emerge from the thymus, implicating PD-1 as a critical factor in the control of T cell self-reactivity during lymphopenia induced homeostatic proliferation (LIP). While we have found no reduction in the absolute number of FoxP3+ regulatory T cells (Treg) in diseased PD-1 KO vs. wild type (WT) HSC recipients, some studies have indirectly suggested that PD-1 deficiency may inhibit the development of induced Treg (iTreg) cells from conventional T cells in the periphery. We therefore investigated whether PD-1 deficiency results in an intrinsic inability of T cells to convert to iTreg in vivo, which could lead to disease in PD-1 KO HSC recipients. Surprisingly, our findings suggest that PD-1 deficiency does not inhibit the development of iTreg nor does it affect Treg suppressive function, and that in the context of LIP, co-inhibitor deficient effector T cells cause disease primarily by expanding beyond the ability of Treg to control them.
Strong systemic immune stimulation results in profound inhibition on the generation of subsequent primary responses (P3034)
Authors: Sckisel Gail , Bouchlaka Myriam , Mirsoian Annie , Monjazeb Arta , Murphy William
Published: January 2023

The immune system constantly maintains the intricate balance necessary to eliminate pathogens while preserving tolerance to self. We have previously shown that strong immune stimulation using agonistic αCD40/IL-2 immunotherapy (IT) results in the induction of robust, antigen-nonspecific CD8-mediated antitumor responses. In addition to proinflammatory reponses, a number of suppressive pathways become activated as well following IT. We sought to determine the ability of T cells to react to various stimuli immediately following strong immune stimulation. Studies revealed that while CD8 responses remained intact, CD4+ T cells exhibited markedly diminished ability to respond to TCR engagement including lack of CD25 upregulation and proliferation. Paralysis of the naïve CD4 population resulted in the inability to respond to primary antigen in vivo. Further analysis of the naïve CD4 population revealed concomitant upregulation of SOCS3, a negative regulator of JAK/STAT signaling, including STAT5. Consistent with this, STAT5 phosphorylation was diminished in CD4+ T cells restimulated following IT further suggesting a role for SOCS3 in the paralysis. These data have significant ramifications when attempting to apply IT in cancer. It also sheds light into the immune dysfunction documented during periods of strong immune stimulation such as acute infections or sepsis.
TLR-mediated modulation of micro-RNA profiling in human primary bronchial epithelial cells (P3003)
Authors: Vonakis Becky , Sirimalle Srinivas , Plunkett Beverly , Cheadle Christopher , Ceccarelli Michele , D’Angelo Fulvio , Cox Paul , Haddad El-Bdaoui , Stellato Cristiana
Published: January 2023

Global changes in miRNA expression in airway epithelium, a cell type critical in the mucosal response to microbial invasion, allergens and pollutants, have been only partially investigated. Cell stimulation with a TLR3 agonist ± cytokines was used to model disease-specific miRNA changes. PBEC (n=5) were cultured under air-liquid interface and treated with medium or polyI:C (1-10 μg/ml) ±TNFα/IFNγ (10 ng/ml each) for 24 hr. Total RNA was used for miRNA profiling (Agilent) and real-time-PCR analysis of inflammatory genes (control). PBEC supernatants were assayed using antibody arrays. Genome Ontology analysis was performed by Ingenuity pathway analysis. Expression of miR409-3p and miR624 was downregulated >30-fold compared to control following polyI:C treatment. Expression of 16 miRNAs was downregulated following polyI:C/TNFα/IFNγ treatment, with >30-fold reduction for miR617 and miR136 (calculated as Fold Change ≥ 1.5, p< 0.05 after Robust Multichip Average normalization). In cells challenged with the combined treatment, 145 genes (including 16 transcription factors) were predicted with high confidence (by 6/6 programs) as molecular targets for 6 of 16 differentially expressed miRNAs. PolyI:C plus TNFα/IFNγ strongly synergized in upregulating the mRNA and protein expression of CXCL8, GM-CSF, IL-6 and CCL2. The identification of the miRNA signature may identify new mechanisms of posttranscriptional gene control and generate new anti-inflammatory strategies.
Cadmium suppresses IL-1β production and release in monocytes. (P3010)
Authors: Napolitano Jessica , Liu Mingjie , Bao Shengying , Knoell Daren
Published: January 2023

Tobacco smoke is the primary cause of chronic obstructive pulmonary disease (COPD) in the U.S. Immune dysfunction in the lung of chronic smokers increases the risk of respiratory infections. IL-1β is a vital cytokine in host defense and its expression is decreased in the lungs of these patients. Cadmium (Cd) is abundant in cigarette smoke and a major contributor to smoking-related lung disease. The purpose of this investigation is to determine whether Cd suppresses IL-1β in monocytes. THP-1 cells were differentiated into macrophages using PMA and then treated overnight with Cd, followed by combined LPS and ATP stimulation. Primary human monocytes were isolated from blood using CD14+ positive selection and subject to similar exposures. Analysis of THP-1 and monocyte cultures revealed that Cd suppresses IL-1β release following stimulation with LPS and ATP. Additionally, there was a decrease in IL-1β mRNA in Cd-treated LPS stimulated macrophages. We observed a decrease in phospho-p65 translocation into the nucleus of Cd-treated macrophages after LPS stimulation. Consistent with this, using a cell-free system, we observed that Cd inhibits IKKβ kinase activity (IC50 of ~100 nM). From these studies we conclude that Cd acts as an inhibitor of the canonical NF-κB pathway and inhibits IL-1β production. Accordingly, Cd may play a critical role in COPD pathogenesis by impairing monocyte immune function.
In vitro studies of interferon-alpha neurotoxicity (P3016)
Authors: Fritz-French Cari , Bassell Gary , Tyor William
Published: January 2023

Elevated levels of interferon-alpha (IFNα) in the central nervous system (CNS) are linked to cognitive dysfunction in patients with inflammatory CNS diseases such as HIV-associated dementia. Previous studies showed that IFNα treatment of neuronal cultures caused a dose dependent decrease in dendritic branching and length, that was prevented after pre-treatment with IFNα neutralizing antibodies. Antagonists to NDMA receptor were also found to be partially protective against IFNα induced neurotoxicity. To begin to determine the mechanism of IFNα induced neurotoxicity, the cell signaling pathway involving IFNα receptor (IFNAR) was evaluated. We demonstrate increased interferon stimulating gene 15 (ISG15) expression, an indicator for JAK-STAT cascade activation, after IFNα stimulation. Inhibiting IFNAR was found to partially protect neurons from IFNα neurotoxicity, but not as efficiently as neutralizing antibodies to IFNα. Our preliminary studies suggest that IFNα is in part acting through its receptor as well as NMDAR to reduce dendritic arborization in neurons. Further studies are needed to determine what other signaling pathways may be involved in IFNα induced neurotoxicity. Determining the mechanism of IFNα neurotoxicity could lead to therapies for cognitive dysfunction during neuroinflammation.
Nucleotide-binding and oligomerization domain 2 (NOD2) knock-in mice carrying a mutation associated with Blau syndrome show reduced amounts of NOD2 protein and decreased muramyl dipeptide (MDP)-induced inflammatory responses (P1254)
Authors: Dugan Jae , Griffiths Eric , Snow Paige , Rosenzweig Holly , Carr Daniel , Rosenbaum Jim , Davey Michael
Published: January 2023

Blau syndrome is an autosomal dominant disorder caused by mutations in NOD2 and characterized by arthritis, dermatitis and uveitis. NOD2 binds MDP and activates NF-kB and MAPK signaling cascades. Prior in vitro studies reported that NOD2 containing Blau mutations caused enhanced activation of NF-kB, suggesting a gain of function in mutated NOD2 caused Blau syndrome. We tested this hypothesis in vivo by creating a knock-in mouse where a point mutation resulted in a change of arginine [R] to glutamine [Q] at position 314 (R314Q) of NOD2 (position 314 in mice corresponds to 334 in humans). R314Q heterozygous (+/m) and homozygous (m/m) mice did not spontaneously develop arthritis or dermatitis. Bone marrow derived macrophages (BMDM) from R314Q mice showed a reduction in NOD2 protein levels compared to wild type (WT) mice despite comparable amounts of NOD2 mRNA. MDP treatment of BMDM showed reduced activation of NF-kB and p38 MAPK in +/m and m/m compared to WT mice that correlated with the copy number of mutated NOD2, with the greatest reduction in m/m mice. In response to ip MDP, reduced levels of IL-6 and KC were detected in the serum of +/m and m/m mice, also in manner correlating with the copy number of the mutation. These data indicate that R314Q-NOD2 mice do not demonstrate a gain of function of the NOD2 pathway. Rather, R314Q causes a deficiency of NOD2 and raises the possibility that Blau syndrome may fall within the spectrum of an immunodeficiency disease.