bluuebunny/crossref_metadata_2025 huggingface dataset:
The two main microvesicle subtypes are significantly different in construction and have different physiological roles (174.18)
Authors: Inal Jameel , Stratton Dan
Published: January 2023

Microvesicles or MVs exist as two distinct sub-types, cMVs and sMVs, which exhibit radically different sizes and calcium contents. The further characterisation of these sub-types was needed to assess significant differences in their biological roles as hitherto reported in the literature. cMVs are heavier then sMVs, containing many proteins reminiscent of the parent cell, including an abundance of surface receptors. They have high carbohydrate levels and have been shown to contain DNA sequences. Conversely, sMVs are lighter, and whilst containing a representative assortment of receptors and surface proteins, contain fewer proteins from their parental cells. They contain low carbohydrate levels and contain very little DNA. Furthermore, whereas cMVs may have various specific roles, sMV release acts as a mechanism to postpone apoptosis by exporting stress agents from the cell, allowing the cell to recover. Furthermore, sMVs have a tendency to offer protection against complement-mediated lysis for both mammalian and bacterial cell types.
The E3 ubiquitin ligase adaptor Ndfip1 regulates TH17 differentiation by limiting the production of pro-inflammatory cytokines (175.11)
Authors: Beal Allison , Ramon Hilda , Worthen George , Oliver Paula
Published: January 2023

Ndfip1 is an adaptor that promotes polyubiquitylation by the E3 ubiquitin ligase Itch. Mice that lack either Ndfip1 or Itch develop a severe TH2-mediated inflammatory disease at sites of environmental antigen exposure, including the lung. Here we demonstrate that lungs from Ndfip1-/- mice showed increased numbers of neutrophils and TH17 cells. This was not because Ndfip1-/- T cells are intrinsically biased towards TH17 differentiation. In fact, fewer Ndfip1-/- T cells differentiated into TH17 cells in vitro due to the overproduction of IL-4 production. Rather, TH17 differentiation in vivo was increased in Ndfip1-/- mice due to elevated numbers of IL-6 producing eosinophils. Interestingly, mice lacking both Ndfip1 and IL-4 had levels of IL-6 comparable to WT controls and these Ndfip1-/- IL-4-/- mice had fewer TH17 cells in their lungs compared to Ndfip1-/- IL-4+/+ mice. These results indicate that TH2 inflammation, such as that observed in Ndfip1-/- mice, can increase TH17 differentiation by recruiting IL-6 producing eosinophils into secondary lymphoid organs and tissues. This may explain why TH17 cells develop within an ongoing TH2 inflammatory response, such as asthma.
Transcriptional profiling of steady-state and inflamed lymph node stroma reveals potential hematopoietic-stromal cross-talk pathways and suggests an active role for stroma during ongoing immune responses. (176.22)
Authors: Malhotra Deepali , Fletcher Anne , Astarita Jillian , Lukacs-Kornek Veronika , Elpek Kutlu , Turley Shannon , (ImmGen) Immunological Genome Project Consortium
Published: January 2023

Lymph node stromal cells (LNSCs) closely regulate immunity and self-tolerance, yet key aspects of their biology remain poorly illuminated. Little is known about what factors these cells produce beyond a short list of critical molecules, or how these subsets respond to inflammation. Comparative transcriptomic analyses of steady-state murine LNSC subsets revealed expression of important immune mediators and growth factors. Further, pairwise analyses of ligand and cognate receptor expression by hematopoietic and stromal subsets suggested a complex web of cross-talk. Transcriptional profiling of steady-state LNSC subsets also suggested a readiness of these cells to respond to inflammatory and infectious triggers. Accordingly, fibroblastic reticular cells (FRCs), blood endothelial cells (BECs), and lymphatic endothelial cells (LECs) isolated from inflamed lymph nodes upregulated expression of numerous genes involved in the acute phase response, immune cell recruitment, and the MHC II antigen processing and presentation pathway, among others. Flow cytometric analysis demonstrated elevated levels of surface MHC II on FRCs, BECs, and LECs isolated from skin-draining lymph nodes 18 hours after the onset of inflammation. LNSC subsets from inflamed lymph nodes also produced the antimicrobial molecule lipocalin 2. Together these data offer insight into the transcriptional identities of murine LNSC subsets, while suggesting that they may be active players during ongoing inflammation.
Uric acid acts as a natural adjuvant by promoting Th17 cell differentiation via an inflammasome-dependent mechanism (176.28)
Authors: Mortellaro Alessandra , Conforti-Andreoni Cristina , Spreafico Roberto , Ricciardi-Castagnoli Paola
Published: January 2023

Non-microbial molecules released from damaged cells act as natural adjuvants capable of promoting an efficient immune response mediated by innate cells, including macrophages and dendritic cells (DCs). Among these molecules, uric acid (UA) in its crystallized form has been found to trigger inflammation through the activation of NLRP3 inflammasome, which leads to secretion of the proinflammatory cytokines IL-1β and IL-18. UA modulation of innate immune responses has been extensively studied, but the impact of this damage-associated molecular pattern on adaptive responses remains largely undocumented. As one of the most ubiquitous sterile danger signals in mammals, and in view of the key role played by UA in alum adjuvanticity, we sought to determine how UA crystals shape adaptive immune responses. In the present study, we focused on adaptive CD4+ T cell polarization since adjuvants that are able to direct appropriately polarized responses will be key to the success of next-generation vaccination strategies. In the presence of NF-κB signaling delivered by muramyl dipeptide, UA crystals were capable of stimulating DCs to promote the release of cytokines associated with Th17 polarization. Accordingly, UA crystals potently induced Th17 responses both in vitro and in vivo. These effects were dependent on the inflammasome-related cytokines IL-1α/β and IL-18, as well as on ASC and caspase-1. Accordingly, NLRP3 deficiency significantly impaired Th17 polarization, at least in vitro.
Effect of AhR activation by TCDD on the immunosuppressive CTLA-4 - IFNγ - IDO pathway (176.5)
Authors: Rohlman Diana , Pennington Jamie , Bradford Sam , Kerkvliet Nancy
Published: January 2023

The aryl hydrocarbon receptor (AhR) is a promising target for the treatment of immune-mediated diseases. Activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in potent immunosuppression in a murine graft versus host (GVH) model, concurrent with a CD4+ regulatory phenotype characterized by increased expression of CTLA-4. CTLA-4 is associated with induction of indoleamine 2,3-dioxygenase (IDO) via IFNγ, resulting in decreased T-cell proliferation and immunosuppression. Treatment with TCDD has also been shown to induce IDO, suggesting that the TCDD-mediated up-regulation of CTLA-4 on donor cells may be responsible for the induction of IDO. Our results show that increased expression of CTLA-4 on donor CD4+ T-cells was concurrent with increased IFNγ, and significant up-regulation of the genes encoding IDO, Ido and Ido2. However, antibody blockade of CTLA-4 did not prevent the suppression of the CTL response by TCDD, suggesting that increased expression of CTLA-4 was not essential for the immunosuppressive effects. A direct role for increased IDO expression in TCDD-mediated immunosuppression is currently under investigation.
Chronic IL-33 exposure induces a hypo-responsive mast cell phenotype (177.13)
Authors: Jung Mi-Yeon , Smrz Daniel , Desai Avanti , Bandara Geethani , Ito Tomonobu , Kang Jeong-Han , Beaven Michael , Metcalfe Dean , Gilfillan Alasdair
Published: January 2023

Antigen-mediated mast cell (MC) activation, and the ensuing release of inflammatory mediators, is a pivotal event in the initiation of such conditions as asthma and anaphylaxis. IL-33 is elevated in asthmatic lungs and thus has been hypothesized to play a role in the pathogenesis of atopic diseases. However, we observed that IL-33 by itself did not induce anaphylaxis in a mouse model. Indeed, peritoneal mast cells taken from chronically IL-33-treated mice, appeared less sensitive to antigen challenge. We therefore examined the direct effects of IL-33 on mouse and human MCs in culture. Cells exposed to IL-33 for >72 h had dramatically reduced antigen-mediated degranulation and generation of specific cytokines. This was not a consequence of reduced expression of the high affinity IgE receptor, FcϵRI, or KIT but reflected altered signaling in the IL-33-treated cells. In this respect, the required antigen-mediated calcium signal and cytoskeletal reorganization were diminished in IL-33-treated mast cells, and this was respectively associated with IL-33-dependent down-regulation of PLCγ1 and Hck expression. These data thus reveal that MCs can be reprogrammed to produce a hypo-responsive phenotype and furthermore suggest that IL-33 may have a protective, rather than a causative role, in allergic disorders. The ability to manipulate MC activation in this manner may provide novel opportunities for therapeutic intervention of MC-driven disease.
The unfolded protein response induces the resistance of hepatoma cells to NK cells’ cytotoxicity (178.13)
Authors: Chen Lihua , Gong JiuYu , Liu Rongrong , Fang Liang , Zhuang Ran , Zhang Yun , Jin Boquan
Published: January 2023

NK cells, as a critical components of anti-tumor immunity, recognize and kill malignant cells through the specific NK cell activating receptors. CD226 and NKG2D are known as important NK cell activating receptors whose ligands are CD112, CD155 and MICA/B, respectively. It has been proven that CD112, CD155 and MICA/B highly express on a series of tumors and directly affect the cytotoxicity of NK cells to the malignant cells. Here we showed that the unfolded protein response induced by tunicamycin could decrease the expression levels of CD112, CD155 and MICA/B on hepatoma cells via the ATF6 and IRE1 pathway and induce the resistance of hepatoma cells to NK cells’ cytotoxicity. Furthermore, the IRE1 pathway increased the expression of the ER associated degradation (ERAD) related molecule HRD1 and facilitated the degradation of CD112, CD155 and MICA/B. Moreover, we demonstrated that the expression levels of CD112, CD155 and MICA/B in hepatoma tissues were inversely correlated with the expression level of GRP78 and the TMN grade of hepatocellular carcinoma in vivo. The results suggest that the production of HRD1 is up-regulated to facilitate the hepatoma cells escape from the NK cells’ cytotoxicity by degrading the ligands expression of the NK cells’ activating receptors.
Oral administration of Capsaicin, a ligand of VR1, can modulate dendritic cell population and induces CD4+FoxP3- Tr-1 cells in the GALT (178.14)
Authors: Acharya Nandini , Basu Sreyashi
Published: January 2023

VR1 is a non-selective cation channel, highly expressed by sensory neurons and cells of the immune system e.g. dendritic cells (DCs) and macrophages. Capsaicin (CP), the pungent component of chili peppers is a chemical ligand of VR1. We have recently reported that oral administration of CP inhibits proliferation and activation of auto-reactive T cells and protects mice from development of Type-1 diabetes (T1D) in a dose dependent manner. Here we demonstrate that orally administered CP can lead to an increase in IL-27 producing DCs, and IL10 producing CD4+/FoxP3- T cells (Tr1) but decrease in the frequency of CD4+/IL17+ T cells (TH17) in the pancreatic lymph node (PLN). We also demonstrate that splenocytes and PLN cells when adoptively transferred from capsaicin fed mouse to a naïve NOD mouse leads to protection of the recipient mouse from T1D. Our results thus demonstrate that engagement of VR1 on immune cells can have profound effect on gut mediated immune tolerance and autoimmunity.
The Cytomegalovirus mimic of Herpesvirus entry mediator avoids CD160 and inhibits IL-2 signaling through B and T lymphocyte attenuator restricting Natural Killer cell function (178.20)
Authors: Sedy John , Bekiaris Vasileios , Smith Wendell , Nemcovicova Ivana , Norris Paula , Benedict Chris , Zajonc Dirk , Ware Carl
Published: January 2023

Natural killer (NK) cells express multiple activating receptors that, upon engagement, result in the rapid release of cytolytic and antiviral effectors required for host defense, notably against herpesviruses. The host is protected from the powerful inflammatory mediators produced by NK cells through the action of inhibitory receptors that typically associate with major histocompatibility class I proteins. To develop into fully functional effector cells and to maintain homeostasis, NK cells depend upon common γ chain signaling. Here, we show that the ORF UL144 mimic of herpesvirus entry mediator (HVEM, TNFRSF14) from the β-herpesvirus cytomegalovirus (CMV), binds exclusively to B and T lymphocyte attenuator (BTLA) but not to CD160, resulting in inhibition of IL-2-mediated activation of NK cells. HVEM and the two Ig-superfamily member receptors that bind HVEM, CD160 and BTLA, are all expressed on NK cells. Mechanistically, UL144 association with BTLA results in dephosphorylation of JAK1 and STAT5, resulting in decreased strength and duration of IL-2 receptor signaling. Subsequently, inhibition of IL-2 receptor signaling by UL144-BTLA suppresses NK cell activation and the ability of NK cells to express antiviral cytokines. Taken together, our results show a novel mechanism by which CMV can control the activation of NK cell antiviral effector functions via UL144-BTLA interactions that result in inhibition of IL-2 receptor signaling.
Competing positive and negative feedbacks mediated by PH domain ligand interactions regulate Itk activation kinetics in T Cells (178.3)
Authors: Das Jayajit , Mukherjee Sayak , Rigaud Stephanie , Seok Sang-Cheol , Fu Guo , Prochenka Agnieszka , Dworkin Michael , Gascoigne Nicholas , Vieland Veronica , Sauer Karsten
Published: January 2023

Inositol phosphate second messengers often regulate crosstalk between receptor signaling and lipid metabolic networks critically affecting cell decision processes, however, molecular mechanisms underlying such cross-regulation are poorly understood. Pairing mathematical modeling and experiments we elucidate these mechanisms in thymocyte activation initiated by T cell receptor (TCR) and antigen interactions. Thymocyte activation is carefully controlled by transient activation kinetics of the Tec-family protein tyrosine kinase Itk generated by TCR signaling, and, production of the membrane lipid phosphatidylinositol(3,4,5)trisphosphate (PIP3) and soluble inositol(1,3,4,5) tetrakisphosphate (IP4). By combining PLC-g (substrate of active Itk) activation kinetics in experiments with maximum entropy based computational approaches we show multiple possible in-silico models describing different modes of molecular interactions between Itk, PIP3, and IP4, can be distinguished. We show that models displaying maximum robustness share a cooperative-allosteric mode of Itk regulation by IP4 involving oligomeric Itk PH domains which induces dueling positive and negative feedbacks in Itk activation. Models lacking the feedbacks or containing monomeric Itk are significantly less robust. We also elucidate key mechanisms regulating the "shape" of the transient Itk kinetics that can be manipulated in experiments for developing therapeutic strategies targeting TCR signaling.
Src and Syk family kinases phosphorylate distinct tyrosines of RIAM (178.4)
Authors: Sari Duygu , Patsoukis Nikolaos , Boussiotis Vassiliki
Published: January 2023

RIAM is a novel adaptor involved in actin polymerization, cell adhesion and PLC-γ1 activation. RIAM contains two coiled-coil regions in the N-terminus, a small proline-rich N-terminus region, a Ras-association (RA) domain, a pleckstrin homology (PH) domain and a large proline-rich C-terminus region. Via its domains RIAM mediates distinct interactions and serves different functions. It was previously determined that RIAM is a substrate of the TCR proximal Src family kinases Fyn and Lck and the Syk family kinase ZAP-70. In the present study we sought to determine the precise tyrosine(s) of RIAM, which are phosphorylated by these kinases, by co-expressing RIAM deletion mutants (N-terminus, RA-PH, and C-terminus) along with active forms of each kinase in COS cells. Immunoprecipitations and immunoblot assays revealed that Src family kinases mediated robust and selective tyrosine phosphorylation of RIAM within the RA-PH domain. Mass spectrometry identified that tyrosine 340 within the PH domain was the specific and predominant target of Fyn and Lck. In contrast, ZAP-70 did not mediate phosphorylation in the RA-PH domain but, instead, induced robust phosphorylation in RIAM C-terminus downstream of the PH domain. The distinct tyrosine phosphorylation of RIAM mediated by Src family kinases and ZAP-70 suggests unique roles of these kinases in regulating RIAM interactions and functions. The role of the distinct phosphorylation events in these outcomes will be discussed.
Proto-oncoprotein ZBTB2 represses transcription activation of RELA gene by Sp1 (178.6)
Authors: Hur Man-Wook , Kim Min-Young
Published: January 2023

Recently, we found that ZBTB2, a POK family transcription factor, is a proto-oncogenic control gene of the p53 pathway and is a transcription repressor of the p21 by inhibiting p53 and Sp1. NF-κB is a protein complex that controls the transcription. NF-kB is found in almost all animal cell types and is involved in cellular responses to stimuli such as stress, cytokines, free radicals, and ultraviolet irradiation. Incorrect regulation of NF-kB has been linked to cancer, inflammatory diseases, viral infection, and improper development. Surprisingly little is known how transcription of the genes encoding p65or p50 is regulated. Interestingly, we found that ZBTB2 represses transcription activation of pNF-kB-Luc reporter by NF-kB, and knock-down of ZBTB2 expression derepresses transcription. Our data suggested that ZBTB2 represses transcription of NF-kB responsive gene by repressing expression of the genes encoding NF-kB subunit p65. Sp1 plays a key role in the expression of RELA gene and ZBTB2 repressed transcription of the gene by acting on the three proximal Sp1 binding elements. Co-IP/western blot and GST fusion protein pull-down assays revealed direct protein-protein interaction between Sp1 and ZBTB2. EMSA, ChIP, and oligonucleotide pull-down assays revealed that ZBTB2 represses transcription of RELA gene by inhibiting Sp1 binding by protein-protein interaction. Functional significances and the details of regulatory mechanism are under investigation..
Immune regulation of tissue-expressed B7x and B7-H1 (179.10)
Authors: Zang Xingxing
Published: January 2023

Some B7 family members are expressed in peripheral cells but their in vivo functions remain largely unknown. Here we showed that B7x protein (B7-H4 or B7S1) was not expressed in human or murine immune cells, but was expressed in some peripheral cells such as pancreatic β cells and lung epithelial cells. Using knock-out and transgenic mice, we found that B7x in pancreatic β cells inhibited autoimmune CD8 or CD4 T cell-mediated diabetes, whereas B7x in lung epithelial cells contributed to tumor metastasis and lethal pulmonary infection. B7-H1 (PD-L1) is widely expressed in immune cells and is important in the coinhibition of T cell function via binding its receptor PD-1. We showed that both human and mice intestinal epithelium expressed B7-H1 and mice deficient for B7-H1 were highly susceptible to gut injury and associated mortality and morbidity. Defective B7-H1 led to loss of epithelial integrity and systemic dispersion of commensal bacteria during intestinal inflammation. B7-H1 expressed on parenchymal cells but not on hematopoietic cells controlled intestinal inflammation, which was not dependent on PD-1, B7-1, and the adaptive immunity. Taken together, all these findings suggest that tissue-expressed B7x and B7-H1 critically control immune response and inflammation.
Tr17: a unique subset of T regulatory cells that produce IL-17 and display potent suppressive function (179.11)
Authors: Li Lequn , Patsoukis Nick , Petkova Victoria , Boussiotis Vassiliki
Published: January 2023

Immune suppression by T regulatory (Treg) cells is required for peripheral T cell tolerance. Treg cells display plasticity and are subjected to regulation by other immune cells. We determined previously that co-culture of CD4+CD25- cells and Treg cells in the presence of antigen presenting cells induced Treg cells producing IL-17 (named Tr17 cells). In the present study, we investigated the conditions required for the generation of Tr17 cells and we analyzed their molecular and functional properties. We determined that both IL-1β and IL-2 were required for the development of Tr17 cells. Compared to IL-17- Treg cells, Tr17 cells expressed increased levels of RORγt and Runx1 while retaining expression of Foxp3. To gain insight into how IL-17 transcription is regulated in Treg cells, we performed CHIP assays and found that Runx1 was able to bind to the IL-17 promoter and enhancer. Tr17 cells displayed more potent suppressive activity compared to IL-17- Treg and responder cells co-cultured with Tr17 cells were more prone to cell death than those co-cultured with IL-17- Treg. Tr17 cells expressed increased levels of perforin and granzyme B relative to IL-17- Treg and used this pathway to induce killing of responder cells as determined by degranulation assay. Our findings suggest that Tr17 cells represent a subset of Treg cells that is generated under inflammatory conditions and may have a unique role in suppressing inflammatory responses by inducing effector cell killing.
TGF-β-induced CD4+ regulatory T cells but not nTregs ameliorate autoimmune arthritis by shifting the balance between Tregs and Th17 cells In vivo (179.14)
Authors: Zheng Song Guo , Kong Lin , Lan Qin , Chen Maogen , Wang Julie , Horwitz David , Brand David , Ryffel Bernhard
Published: January 2023

Transferred CD4+CD25+Foxp3+ regulatory cells (Tregs) can prevent autoimmune disease, but generally fail to ameliorate established disease. Here we demonstrate that antigen-specific Tregs induced with IL-2 and TGF-β ex-vivo (iTregs), but not equivalent expanded thymus-derived nTregs can prevent progression of established collagen-induced arthritis. This was because following transfer, nTregs exhibited decreased Foxp3 and Bcl-2 expression, decreased suppressive activity, and many converted to Th17 cells. By contrast, transferred iTregs were more numerous, retained their suppressive activity in the presence of IL-6 and were resistant to conversion. Remarkably, ten days after transfer iTregs shifted the predominance from Th17 to Treg cells in draining LNs. These findings provide evidence that transferred TGF-β-induced iTregs are more stable and functional than nTregs in mice with established autoimmunity. Moreover, iTregs can have tolerogenic effects even in the presence of ongoing inflammation. The therapeutic potential of iTregs in subjects with chronic, immune-mediated inflammatory diseases deserves to be investigated.
Alternate function of Foxp3 in human CD4+ T cells (179.2)
Authors: Martin Sunil , Riley James
Published: January 2023

Foxp3 is the master regulator of T-reg phenotype and function. Unlike murine T cells, human T cells express two versions of Foxp3 of which the smaller form is missing exon 2 (Δ2). Previously we showed that Foxp3Δ2 fails to up-regulate the Pim 2 kinase and others have reported that members of the ROR family bind to exon 2 of Foxp3. Nonetheless, it is not clear if these isoforms differentially affect regulatory and nonregulatory T cell function. This study demonstrate that transduction of Foxp3 and Foxp3Δ2 into primary human CD4+CD45RA+CD25- naïve T cells induced suppressive activity that was identical to natural T-regs. However, in CD4+CD45RA-CD25- memory T cells, the full-length Foxp3 construct was unable to introduce a suppressive phenotype, whereas Foxp3Δ2 retained its ability to promote suppressive activity. Furthermore, memory cells transduced with Foxp3 abrogated the suppressive functions of naïve cells transduced with Foxp3 but not with Foxp3Δ2.Cytokine profiling indicated a 3 fold increase in the IL-2+IFN-γ+ cells within the memory cells transduced with Foxp3. Altogether these observations reveal an isoform dependent alternate function of Foxp3 in the CD4+ memory T cell compartment. These studies support the use of naïve T cells for strategies that transduce effector T cells with Foxp3 expressing vectors as a means to rapidly generate T-regs for use in adoptive T cell therapy against autoimmune diseases.
Alpha7 cholinergic-agonist prevents systemic inflammation by Sepsis. (179.3)
Authors: Thompson-Bonilla Maria del , Vida Gergely , Pena Geber , Ulloa Luis
Published: January 2023

The alpha7 nicotinic acetylcholine receptors (α7nAChR) play a critical role in modulating systemic inflammation in sepsis (1). However, α7nAChR-agonists have failed to provide significant advantages in several pilot studies with septic patients. Since we recently reported that the vagus nerve prevents sepsis by activating regulatory lymphocytes (2), we reasoned that the massive apoptosis of lymphocytes during sepsis may prevent the therapeutic effects of the α7nAChR-agonists. Our results indicate that α7nAChR-agonists failed to prevent systemic inflammation in lymphocyte-deficient nude mice. The transfer of typical regulatory CD3+CD4+CD25+ suppressor/regulatory lymphocytes failed to reestablish the benefits of α7nAChR-agonists. But, the transfer of CD3+CD4+CD25- modulatory lymphocytes reestablished the anti-inflammatory potential of the α7nAChR-agonists. Likewise, α7nAChR-agonist prevented systemic inflammation in polymicrobial sepsis when administered before sepsis. The anti-inflammatory potential and benefits of the α7nAChR-agonists vanishes as the treatment is delayed after the onset of the infection. However, the adoptive transfer of CD3+CD4+CD25- modulatory lymphocytes reestablished the anti-inflammatory potential of the α7nAChR-agonists, even if the treatment is started long after the infection. These results suggest that a new set of CD25- modulatory lymphocytes can provide clinical advantages for the treatment of infections and inflammatory disorders.
Role of class IA PI3K in regulatory T cell function (179.5)
Authors: Fujii Hideki , Matsuda Satoshi , Ohtani Masashi , Koyasu Shigeo
Published: January 2023

PI3K (phosphoinositide 3-kinase) family members control variety of cellular responses, such as cell growth, survival, cytoskeletal remodelling and the trafficking of intracellular organelles, in many cell types, including T lymphocytes. The PI3K pathway has emerged as a key regulator of regulatory T cell (Treg) development and homeostasis and may be required for Treg-mediated suppression. To identify the role of regulatory subunits of class IA PI3K in Treg suppressive function, we compared WT and p85α KO Tregs in their function. WT but not p85α KO Tregs showed suppressive capacity in co-cultures with CD4+CD25- T cells stimulated with anti-CD3-coated APCs in vitro. To determine whether p85α KO Tregs were functional in vivo, we conducted experiments in which Tregs offer protection from colitis caused by the transfer of CD4+CD62L high T cells into RAG2-/- recipients. WT Tregs suppressed the capacity of CD4+CD62L high T cells to cause colitis 2 weeks after transfer. In contrast, mice co-injected with p85α KO Tregs developed colitis 2 weeks after transfer that was indistinguishable from that in mice injected with CD4+CD62L high cells alone. Although the precise mechanism of Treg-mediated suppression is unclear, we suppose that p85α functions through cell-cell contact-dependent manner rather than cytokine-dependent manner. S.K. is a consultant for Medical and Biological Laboratories, Co. Ltd. The authors otherwise have no financial conflicts of interest.
Inhibition of TLR Signaling by TIRAP/Mal-derived Decoy Peptides (180.14)
Authors: Toshchakov Vladimir , Couture Leah , Piao Wenji , Ru Lisa , Vogel Stefanie
Published: January 2023

TIRAP/Mal is an adapter protein that facilitates recruitment of MyD88 to TLR4 and TLR2 signaling complexes. We have previously generated a library of cell-permeating TLR4 TIR-derived decoy peptides fused to the translocating segment of Drosophila antennapedia homeodomain and examined each peptide for the ability to inhibit TLR4 signaling. To expand previous studies we now have generated TIRAP-derived peptides and tested them for the ability to inhibit TLR2 and TLR4 signailng. Five TIRAP peptides, TR3, TR5, TR6, TR9, and TR11, inhibited LPS-induced cytokine mRNA expression and MAPK activation. Inhibition was confirmed at the protein secretion level, as select peptides abolished the LPS-induced cytokine production measured in macrophage supernatants collected 24 h after LPS stimulation. Two TIRAP inhibitory peptides, TR3 and TR6, also inhibited cytokine production induced by a TLR2/TLR1 agonist, S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-Ser-Lys4-OH (P3C); however, a higher peptide dose was required to achieve TLR2 inhibition. TR3 and TR6 did not block macrophage activation induced by TLR2/6 agonist, S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-[R]-Cys-Ser-Lys4-OH (P2C). This study has identified novel peptides that block TLR signaling at low micromolar concentrations. Comparison of TLR4 inhibition by TLR4 TIR- and TIRAP-derived peptides suggests that structurally diverse regions mediate functional interactions of TIR domains.
Development of inhibitory peptide to control TLR9 signaling pathway. (180.17)
Authors: Singh Sujay , Lee Hyun-Ku , Singh Gita , Luo Yi Feng , Chitta Sriram , Rosenberg Jonathan
Published: January 2023

Toll-like receptors (TLRs) play an important role in sensing microbial molecules. Upon recognition of microbial components, TLRs quickly engage with their adaptors and initiate innate immune signals by sequentially recruiting downstream signaling mediators, which provide immediate host protection and stimulate the host’s adaptive immune response. Despite the fact that TLR-mediated innate immune signals are required to combat invading pathogens, when signaling is not well regulated, uncontrolled activation can result in the disruption of host homeostasis leading to chronic inflammation and septic shock. Although there are naturally occurring negative regulators of TLR signaling that function as part of cell homeostasis, these are not adequate in all situations. Hence development of a targeted therapeutic means or molecule that can effectively control TLR-mediated chronic inflammation is crucial. In response to this, we have developed a synthetic peptide based on a CpG-binding motif in the extracellular domain of TLR9, which can inhibit TLR9 signaling. Using TLR9/NF-κB/SEAPorter™ HEK 293 cell line, the inhibitory peptide was validated together with a control peptide in which conserved residues were modified. The inhibitory peptide significantly suppressed CpG-mediated TLR9 activation while the control peptide did not affect TLR9 activation. Test of another peptide with differential residue modification further revealed the key residues involved in CpG-mediated TLR9 activation.
R753Q polymorphism compromises TLR2 signaling functions by altering TLR6-TLR2 hetero-dimerization, TLR2 tyrosine phosphorylation and MyD88 recruitment (180.2)
Authors: Xiong Yanbao , Song Chang , Medvedev Andrei
Published: January 2023

TLR2 senses lipoproteins expressed by Gram positive bacteria, mycoplasma, mycobacteria and fungi. The Arg753Gln (R753Q) polymorphism in the TIR domain of TLR2 has been linked to increased incidence of tuberculosis, leprosy and other diseases. But the molecular mechanisms by which it affects TLR2 functions are unclear. We studied the impact of complementation of HEK293 cells and mouse TLR2-/- macrophages with wild-type (WT) vs. R753Q TLR2 on TLR2 expression, interactions with TLR6, tyrosine phosphorylation, recruitment of MyD88, and functional responses. Transfected WT and R753Q TLR2 variants had comparable expression, and even 100-fold increase in input amounts of R753Q vs. WT TLR2 did not overcome deficiency of the mutant receptor. R753Q TLR2 exhibited deficient agonist-induced tyrosine phosphorylation, hetero-dimerization with TLR6 and recruitment of MyD88. R753Q TLR2 mediated impaired activation of p38, NF-κB, and induction of IL-8 mRNA in response to inactivated Mycobacterium tuberculosis, mycobacteria cell wall, culture filtrate fractions, lipoarabinomannan and Pam3Cys. In contrast, stimulation with TNF-α led to similar NF-κB reporter activation and IL-8 gene expression in cells expressing wild-type or R753Q TLR2. These results revealed deficient TLR2 tyrosine phosphorylation, hetero-dimerization with TLR6 and recruitment of MyD88 as key features underscoring signaling deficiency of the R753Q TLR2.
Enhanced Glycogen Synthase Kinase 3β phosphorylation modulates leukocytes Toll-like receptor-mediated cytokine secretion in obese people. (180.7)
Authors: Yen Chia-Liang , Chang Chin-Sung , Wu Chih-Hsing , Shieh Chi-Chang
Published: January 2023

Obesity is an increasing modern disease which leads to type II diabetes mellitus and cardiovascular diseases. However, the impacts of obesity on immune function and infectious diseases have not been fully understood. To clarify the role of obesity in the immune responses, we investigated the relationship between insulin-induced signaling and Toll-like Receptors (TLRs)-induced leukocyte cytokine secretion in obese subjects. We found decreased TLR-induced IFN-γ, IL-6 secretion and elevated TLR-induced IL-1β, IL-10 secretion by leukocytes isolated from obese subjects when compared with normal leukocytes. Peripheral blood mononuclear cells (PBMCs) from obese subjects showed enhanced basal Akt/Glycogen Synthase Kinase 3β (GSK3β) phosphorylation which was not further increased by insulin and lipopolysaccharide (LPS). Leukocytes isolated from obese subjects also showed increased IκB level. We further investigated the role of GSK3β on LPS-induced cytokine secretion and NF-κB activation in THP-1 cells under hyperinsulinemic and hyperlipidemic conditions. We found that insulin and palmitate stimulation can increase GSK3 phosphorylation and IκB level. We also found that insulin stimulation and GSK3β siRNA knockdown can increase IκB level and IL-10 secretion. We conclude that enhanced leukocyte GSK3β phosphorylation may contribute to aberrant immune response in obese subjects.
Pivotal roles of both Wiskott-Aldrich syndrome protein (WASP) and N-WASP in the activation and attenuation of the B cell receptor (P1093) (42.1)
Authors: Liu Chaohong , Sharma Shruti , Miller Heather , Upadhyaya Arpita , Dahlberg Carin , Westerberg Lisa , Snapper Scott , Song Wenxia
Published: January 2023

Lymphocytes express both Wiskott-Aldrich syndrome protein (WASP) and N-WASP. WASP deficiency, the cause of WAS, generates more severe defects in T-cells than B cells, suggesting a strong compensatory role of N-WASP in B cells. Using knockout mouse models, this study shows that WASP knockout (WKO) reduces B cell spreading, B cell receptor (BCR) aggregation, tyrosine phosphorylation, Btk phosphorylation and F-actin accumulation at the B cell surface induced by membrane-associated antigen, and WASP/N-WASP double knockout nearly abolished these activities. However, in B cells with N-WASP conditional knockout (cNKO), B cell spreading and F-actin accumulation are enhanced, while the merge of BCR aggregates and attenuation of tyrosine and Btk phosphorylation are inhibited. Additionally, cNKO has a much stronger inhibitory effect on BCR endocytosis than WKO. In contrast to Btk-dependent activation of WASP, N-WASP phosphorylation is upregulated by Btk-deficiency but downregulated by SHIP KO. Furthermore, the phosphorylation of WASP and N-WASP is inversely regulated by each other. These results reveal that the combined function of WASP and N-WASP is required for the optimal activation of the BCR and its signaling attenuation, but these two proteins function distinctively: WASP and N-WASP play dominant roles in the initiation and attenuation of BCR activation respectively. Thus, the integrated action of WASP and N-WASP provide a critical regulatory mechanism for BCR activation.
Transcriptional regulation by 2,3,7,8-tetrachlorodibenzo-ρ-dioxin within the human polymorphic hs1,2 enhancer (42.7)
Authors: Liu Jing , Ochs Sharon , Sulentic Courtney
Published: January 2023

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD) inhibits Ig expression and secretion. Within the IgH gene, the 3’IgH regulatory region (3’IgHRR) has been identified as a transcriptional target of TCDD. TCDD inhibits mouse 3’IgHRR and induces aryl hydrocarbon receptor (AhR) binding to dioxin response elements (DREs) within the hs1,2 and hs4 enhancers. The human hs1,2 enhancer is polymorphic due to the presence of one to four invariant sequences (IS) which have been correlated with several autoimmune disorders. Interestingly, TCDD inhibits the transcriptional activity of the mouse hs1,2 enhancer, while the human polymorphic hs1,2 enhancer activity is increased. Several transcription factor binding sites are located within both the mouse and human hs1,2 enhancers, while others are only in the mouse or human. Thus, we hypothesize that the specific transcription factor within the mouse and the human enhancers lead to differential effects by TCDD. Our data show that insertion of Pax5, deletion of the whole IS, or mutation of the AP-1/Ets site outside the IS decreases overall transcriptional activity. However, mutation of the transcription factor binding sites within the IS or the Oct site outside the IS increase the transcriptional activity. These results underscore the complexity of the transcriptional regulation of the hs1,2 enhancer and suggest an interaction between multiple transcription factors, many of which have been shown to be modulated by TCDD.
Structurally identical capsular polysaccharide expressed by intact group B Streptococcus versus Streptococcus pneumoniae elicits distinct murine polysaccharide-specific IgG responses in vivo (43.10)
Authors: Arjunaraja Swadhinya , Paoletti Lawrence , Snapper Clifford
Published: January 2023

Our previous studies using intact Streptococcus pneumoniae type 14 (Pn14) (Gram-positive) and Neisseria meningitidis (Gram-negative) indicated distinct differences in the regulation of the associated polysaccharide (PS)-specific IgG responses. In light of potentially unique contributions of biochemically distinct capsular PS and their characteristic attachments to the underlying bacterium, it remains unresolved whether different bacterial sub-capsular domains can exert differential effects on PS-specific Ig responses. In this study, we utilized a mutant strain of group B Streptococcus (S. agalactiae) type III (GBS-III) that expresses desialylated IIIPS, biochemically identical to type 14 capsular PS (PPS14) of Pn14, to directly compare the PPS14-specific IgG responses to two distinct Gram-positive bacteria. Although both GBS-III and Pn14 elicited relatively rapid primary IgG anti-PPS14 responses that were dependent on CD4+ T cells, CD40L and CD28 costimulation, only GBS-III induced a highly boosted ICOS-dependent PPS14 specific IgG response. Of note, priming with Pn14 and boosting with GBS-III, although not isolated PPS14, elicited a similarly boosted PPS14-specific IgG response that was T cell- dependent. This indicates that Pn14 primes for memory but, unlike GBS-III, fails to elicit it. These data establish that structurally identical capsular PS expressed by two distinct Gram-positive extracellular bacteria can indeed elicit distinct PS specific IgG responses.
Th17, Th22 and regulatory T cells are essential for controlling parasite burden and mucosal inflammation during Toxoplasma gondii infection (43.11)
Authors: Fonseca Denise , Benevides Luciana , Souza Maria do Carmo , Bonfá Giuliano , Solano-Dias Murilo , Basso Alexandre , Silva Neide , Mineo Tiago , Ryffel Bernhard , Silva João
Published: January 2023

The Toxoplasma gondii infection induces intense intestinal inflammation and tissue damage in susceptible hosts. Here, we studied the regulation of immune response during intestinal mucosal inflammation induced by T. gondii, focusing on the role of Th17, Th22 and regulatory T cells (Tregs). C57BL/6-susceptible and BALB/c-resistant mice orally infected with T. gondii exhibited increased frequencies of Th17 and Th22 cells in Lamina Propria and Peyers patches during disease progression. However, whereas C57BL/6 mice exhibited higher frequency of Th17, BALB/c mice showed higher counts of Treg and Th22 cells. The infection also impaired the effector function of Treg cells in susceptible hosts, since Tregs obtained from C57BL/6-infected mice showed a reduced suppressive activity in vitro. The transfer of Foxp3+ cells sorted from non-infected mice, but not from infected mice, increased mice survival. In parallel, we evaluated the role of IL-17 and IL-22 in the disease and we observed that IL-17R-/- and IL-22-/- mice were more susceptible, presented higher parasite burden and intestinal inflammation and lower Treg frequency than C57BL/6. Moreover, IL-17R-/- showed a significant reduction of Th22 cells compared to C57BL/6 mice. Taken together, these findings suggest that IL-17 and IL-22 production are essential for resistance against T. gondii infection, by controlling parasite burden and along with IL-22, Tregs are important for controlling the intestinal inflammation.
In vivo blocking CD80 promotes Th17 polarization and amplifies autoimmune arthritis severity in mice infected with Mycoplasma arthritidis (43.15)
Authors: Mu Hong
Published: January 2023

Mycoplasma arthritidis, an agent that causes acute and chronic autoimmune arthritis and septic shock in rodents. Previously we demonstrated that the severity of septic shock induced in mice injected with M. arthritidis is associated with the up-regulation of co-stimulatory molecule CD80 on APCs and blockade of CD80 specifically enhances the severity of arthritis. In the present study, we report that anti-CD80 resulted in increased number of Th17 T cells and level of IL-23 expression by macrophages resulting in the increased expression and production of IL-17 and augmented Th17-associated transcriptional factors RORgt and Stat3 by CD4+ T cells. Also in arthritis susceptible mouse strain, injection of anti-TLR4 mAb significantly suppresses IL-23 response and IL-17 by CD4+ T cells and alleviate the arthritis. The inhibition of Th17 response by anti-TLR4 treatment is partially associated with the suppression of CD80 function. In addition, to elucidate the role of MAM, the superantigen secreted by M. arthritidis in Th17 polarization, mice injected with MAM-mutant M. arthritidis strains showed impaired IL-17 production, remarkably lower IL-23 and improved arthritis symptoms. Thus the contribution of MAM appears to be important for IL-17 contribution in the disease pathogenesis and is clearly involved in modulating pathways of immune responses leading to different disease outcomes and phenotypes in M. arthritidis-induced autoinflammatory disease.
Deficiency in interleukin 10 reverses the susceptibility phenotype of CBA/J mice during infection with Mycobacterium tuberculosis. (43.23)
Authors: Turner Joanne , Cyktor Joshua , Carruthers Bridget , Kominsky Rachel , Stromberg Paul , Beamer Gillian
Published: January 2023

Interleukin 10 is a known correlate of susceptibility to tuberculosis (TB) in man and similarly, elevated levels of IL-10 (either natural or induced) in mice can accelerate TB disease progression. The CBA/J mouse strain is a model that naturally produces abundant IL-10 and is also highly susceptible to infection with Mycobacterium tuberculosis (M.tb). We have generated CBA/J mice deficient in IL-10 (IL-10 KO) to determine the mechanisms of influence of IL-10 on TB disease progression in this highly susceptible mouse strain. In contrast to IL-10 KO on resistant mouse strain backgrounds where the lack of IL-10 has little impact on TB outcome, the CBA/J IL-10 KO was fully capable of limiting M.tb growth within the lung and spleen for an extended period of time, with over 2 Log fewer M.tb colony forming units (CFU), compared to CBA/J wild type. This reduction in bacterial growth correlated with a highly significant extension in survival, relative to CBA/J wild type mice. The ability to control M.tb infection was associated with an early detection of IFN-gamma within the lung and the detection of IFN-gamma producing antigen specific T cells throughout infection. Of particular significance was the finding that CBA/J IL-10 mice developed mature granulomas (encircled by collagen) with necrotic centers containing M.tb bacillus of variable acid-fast staining.
Identification of Plasmodium berghei novel liver stage CD8 T cell epitopes by caged MHC class I tetramer technology (43.30)
Authors: Pichugin Alexander , Ehrler Lindsey , Sehrawat Sharvan , Funk Theresa , Speake Cate , Duffy Patrick , Ploegh Hidde , Krzych Urszula
Published: January 2023

Studies conducted in mice and humans have shown that IFN-γ+ CD8 T cell are the key effectors against liver-stage (LS) malaria; however, owing to complexities associated with the development of Plasmodia parasites, specificities of CD8 T cell effectors have been difficult to establish. Direct detection of Ag-specific CD8 T cells in high-throughput manner became possible with the development of caged MHC-tetramer technology, based on conditional photo-cleavable MHC class I ligands that can be displaced during UV induced exchange with a peptide of interest. On the basis of previously identified P. falciparum (Pf) genes expressed exclusively during LS infection, we identified P. berghei (Pb) orthologues of Pf genes and validated their expression by qRT-PCR in Pb infected C57Bl/6 mice. Several Pb LS antigens, administered as DNA vaccines, significantly reduced LS parasite burden in mice infected with Pb sporozoites. Using sequences of the 28 novel Pb LS genes we screened ~400 peptides by the caged MHC class I tetramer approach. Our preliminary results demonstrate that 2 H-2Kb- and 1 H-2Db-restricted Pb peptides that correspond to protective Pb LS Ags form MHC class I tetramers that specifically bound to CD8 T cells from mice immunized with radiation-attenuated Pb sporozoites. Identification of these epitopes is crucial towards resolving the role of antigen-specific CD8 T cell responses and establishing these responses as correlates of protection to Plasmodia infection.
CCL19-Cre transgenics: targeting lymph node fibroblastic reticular cells in vivo (44.14)
Authors: Ludewig Burkhard , Chai Qian , Onder Lucas , Scandella Elke , Sparwasser Tim , Luther Sanjiv , Thiel Volker , Rülicke Thomas , Hehlgans Thomas
Published: January 2023

The paracortex of lymph nodes (LNs) harbors a sponge-like scaffold of stromal cells known as fibroblastic reticular cells (FRCs). A major function of FRCs is to build and enwrap the conduit system of collagen fibers that directs interstitial fluids from the afferent lymph through the T-cell zone to HEVs. Furthermore, FRCs regulate T-cell migration and survival by producing the chemokines CCL19 and CCL21. To gain further knowledge on the biology of FRCs and possibly other stromal cell subsets, we have generated a bacterial artificial chromosome (BAC)-transgenic mouse model that utilizes the CCL19 promoter to direct the Cre-recombinase to LN stromal cells. Crossing of CCL19-Cre mice to the R26-EYFP reporter revealed that transgene expression in LNs was almost exclusively confined to podoplanin+CD31- cells. Likewise, transgene activity in spleens and Peyer’s patches of CCL19-Cre mice was largely restricted to FRC-like cells, i.e. stromal cells within the T cell zone and the T-B border. Selective ablation of the lymphotoxin-beta receptor on CCL19-Cre-positive cells resulted in profound changes in the development and organization of secondary lymphoid organs. Taken together, stromal CCL19-Cre expression is well-suited (i) to characterize the development of LN FRCs in vivo, (ii) to molecularly dissect the contribution of stromal cells to lymphoid organogenesis, and (iii) to address the function of LN FRCs in the generation of innate and adaptive immune responses.