A network map of Interleukin-10 signaling pathway

Interleukin-10 (IL-10) is an anti-inflammatory cytokine with important immunoregulatory functions. It is primarily secreted by antigen-presenting cells such as activated T-cells, monocytes, B-cells and macrophages. In biologically functional form, it exists as a homodimer that binds to tetrameric heterodimer IL-10 receptor and induces downstream signaling. IL-10 is associated with survival, proliferation and anti-apoptotic activities of various cancers such as Burkitt lymphoma, non-Hodgkins lymphoma and non-small scell lung cancer. In addition, it plays a central role in survival and persistence of intracellular pathogens such as Leishmania donovani, Mycobacterium tuberculosis and Trypanosoma cruzi inside the host. The signaling mechanisms of IL-10 cytokine are not well explored and a well annotated pathway map has been lacking. To this end, we developed a pathway resource by manually annotating the IL-10 induced signaling molecules derived from literature. The reactions were categorized under molecular associations, activation/inhibition, catalysis, transport and gene regulation. In all, 37 molecules and 76 reactions were annotated. The IL-10 signaling pathway can be freely accessed through NetPath, a resource of signal transduction pathways previously developed by our group.     

A knowledgebase resource for interleukin-17 family mediated signaling

Interleukin-17 (IL-17) belongs to a relatively new family of cytokines that has garnered attention as the signature cytokine of Th17 cells. This cytokine family consists of 6 ligands, which bind to 5 receptor subtypes and induce downstream signaling. Although the receptors are ubiquitously expressed, cellular responses to ligands vary across tissues. The cytokine family is associated with various autoimmune disorders including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, asthma and psoriasis in addition to being implicated in the pathogenesis of cancer. In addition, this family plays a role in host defense against bacterial and fungal infections. The signaling mechanisms of the IL-17 family of proinflammatory cytokines are not well explored. In this study, we present a resource of literature-annotated reactions induced by IL-17. The reactions are catalogued under 5 categories, namely; molecular association, catalysis, transport, activation/inhibition and gene regulation. A total of 93 molecules and 122 reactions have been annotated. The IL-17 pathway is freely available through NetPath, a resource of signal transduction pathways previously developed by our group.     

Syk negatively regulates TLR4-mediated IFNβ and IL-10 production and promotes inflammatory responses in dendritic cells

BackgroundWhile Syk has been shown to associate with TLR4, the immune consequences of Syk–TLR interactions and related molecular mechanisms are unclear.MethodsGain- and loss-of-function approaches were utilized to determine the regulatory function of Syk and elucidate the related molecular mechanisms in TLR4-mediated inflammatory responses. Cytokine production was measured by ELISA and phosphorylation of signaling molecules determined by Western blotting.ResultsSyk deficiency in murine dendritic cells resulted in the enhancement of LPS-induced IFNβ and IL-10 but suppression of pro-inflammatory cytokines (TNFα, IL-6). Deficiency of Syk enhanced the activity of PI3K and elevated the phosphorylation of PI3K and Akt, which in turn, lead to the phospho-inactivation of the downstream, central gatekeeper of the innate response, GSK3β. Inhibition of PI3K or Akt abrogated the ability of Syk deficiency to enhance IFNβ and IL-10 in Syk deficient cells, confirmed by the overexpression of Akt (Myr–Akt) or constitutively active GSK3β (GSK3 S9A). Moreover, neither inhibition of PI3K–Akt signaling nor neutralization of de novo synthesized IFNβ could rescue TNFα and IL-6 production in LPS-stimulated Syk deficient cells. Syk deficiency resulted in decreased phosphorylation of IKKβ and the NF-κB p65 subunit, further suggesting a divergent influence of Syk on pro- and anti-inflammatory TLR responses.ConclusionsSyk negatively regulates TLR4-mediated production of IFNβ and IL-10 and promotes inflammatory responses in dendritic cells through divergent regulation of downstream PI3K–Akt and NF-κB signaling pathways.General significanceSyk may represent a novel target for manipulating the direction or intensity of the innate response, depending on clinical necessity.     

Resonance assignments of Ca2+-bound human S100A11

The S100 family belongs to the EF-hand calcium-binding proteins regulating a wide range of important cellular processes via protein–protein interactions. Most S100 proteins adopt a conformation of non-covalent homodimer for their functions. Calcium binding to the EF-hand motifs of S100 proteins is essential for triggering the structural changes, promoting exposure of hydrophobic regions necessary for target protein interactions. S100A11 is a protein found in diverse tissues and possesses multiple functions upon binding to different target proteins. RAGE is a multiligand receptor binding to S100A11 and the interactions at molecular level have not been reported. However, the three-dimensional structure of human S100A11 containing 105 amino acids is still not available for further interaction studies. To determine the solution structure, for the first time we report the ¹H, ¹⁵N and ¹³C resonance assignments and protein secondary structure prediction of human S100A11 dimer in complex with calcium using a variety of triple resonance NMR experiments and the chemical shift index (CSI) method, respectively.     

An integrated signal transduction network of macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) is a glycosylated multi-functional protein that acts as an enzyme as well as a cytokine. MIF mediates its actions through a cell surface class II major histocompatibility chaperone, CD74 and co-receptors such as CD44, CXCR2, CXCR4 or CXCR7. MIF has been implicated in the pathogenesis of several acute and chronic inflammatory diseases. Although MIF is a molecule of biomedical importance, a public resource of MIF signaling pathway is currently lacking. In view of this, we carried out detailed data mining and documentation of the signaling events pertaining to MIF from published literature and developed an integrated reaction map of MIF signaling. This resulted in the cataloguing of 68 molecules belonging to MIF signaling pathway, which includes 24 protein-protein interactions, 44 post-translational modifications, 11 protein translocation events and 8 activation/inhibition events. In addition, 65 gene regulation events at the mRNA levels induced by MIF signaling have also been catalogued. This signaling pathway has been integrated into NetPath (http://www.netpath.org), a freely available human signaling pathway resource developed previously by our group. The MIF pathway data is freely available online in various community standard data exchange formats. We expect that data on signaling events and a detailed signaling map of MIF will provide the scientific community with an improved platform to facilitate further molecular as well as biomedical investigations on MIF.     

A modular map of Bradykinin-mediated inflammatory signaling network

Bradykinin, a member of the kallikrein-kinin system (KKS), is associated with an inflammatory response pathway with diverse vascular permeability functions, including thrombosis and blood coagulation. In majority, bradykinin signals through Bradykinin Receptor B2 (B2R). B2R is a G protein-coupled receptor (GPCR) coupled to G protein family such as Gα_qs, Gα_q/Gα_11, Gα_i1, and Gβ1_γ2_. B2R stimulation leads to the activation of a signaling cascade of downstream molecules such as phospholipases, protein kinase C, Ras/Raf-1/MAPK, and PI3K/AKT and secondary messengers such as inositol-1,4,5-trisphosphate, diacylglycerol and Ca²⁺ ions. These secondary messengers modulate the production of nitric oxide or prostaglandins. Bradykinin-mediated signaling is implicated in inflammation, chronic pain, vasculopathy, neuropathy, obesity, diabetes, and cancer. Despite the biomedical importance of bradykinin, a resource of bradykinin-mediated signaling pathway is currently not available. Here, we developed a pathway resource of signaling events mediated by bradykinin. By employing data mining strategies in the published literature, we describe an integrated pathway reaction map of bradykinin consisting of 233 reactions. Bradykinin signaling pathway events included 25 enzyme catalysis reactions, 12 translocations, 83 activation/inhibition reactions, 11 molecular associations, 45 protein expression and 57 gene regulation events. The pathway map is made publicly available on the WikiPathways Database with the ID URL: https://www.wikipathways.org/index.php/Pathway:WP5132. The bradykinin-mediated signaling pathway map will facilitate the identification of novel candidates as therapeutic targets for diseases associated with dysregulated bradykinin signaling.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00652-0.     

An overview of the oxytocin-oxytocin receptor signaling network

Oxytocin, a nine amino acid long neuropeptide hormone, is synthesized in the hypothalamus and stored and released from the neural lobe of the pituitary gland. Although commonly known for its central role in the regulation of parturition and lactation, oxytocin signaling also plays a key role in modulating social behavior, evoking contentment, initiating maternal behavior, inducing trust, generosity and bonding in humans and animals. Oxytocin signaling can prove to be of great importance in therapeutics and drug targeting because of its diverse range of actions. However, a well annotated map of oxytocin signaling pathway is currently lacking in the publicly available pathway resources. Therefore, we systematically curated the available signaling information of oxytocin from published literature and collated the data to develop a more complete map. We cataloged 66 molecules belonging to oxytocin signaling pathway, which included 9 protein-protein interactions, 39 post-translational modifications, 14 protein translocation events and 22 activation/inhibition events. Further, Oxytocin signaling network data is made freely available to academic fraternity by integrating this into NetPath (http://www.netpath.org/), a freely available human signaling pathway resource developed previously by our group.     

Ingested (oral) tocilizumab inhibits EAE

BackgroundBlocking the activity of IL-6 can inhibit autoimmune diseases such as rheumatoid arthritis and Crohn’s disease.ObjectiveWe examined whether an antibody against IL-6, tocilizumab (TCZ) (Actemra®), used clinically in rheumatoid arthritis (RA) would have similar anti-inflammatory effects in EAE after oral administration.Design/methodB6 mice were immunized with MOG peptide 35–55 and gavaged with control saline or TCZ during ongoing disease. Splenocytes, CD4⁺ T cells or macrophages/monocyte lineage cells (CD11b⁺) from control fed or TCZ fed mice were adoptively transferred into active MOG peptide 35–55 immunized recipient mice during ongoing disease. Actively fed and recipient mice were examined for disease inhibition, inflammation, and cytokine responses.ResultsIngested (oral) TCZ inhibited ongoing disease and decreased inflammation. Adoptively transferred cells from TCZ fed donors protected against actively induced disease and decreased inflammation. There was a decrease in IL-6 in actively treated spleen, decrease in TNF-α, Th1-like cytokine IL-12 and increase in Th2-like cytokine IL-10 in active fed and adoptively treated recipients.ConclusionsIngested (orally administered) TCZ can inhibit disease, CNS inflammation, decrease pro-inflammatory Th1-like cytokines and increase Th2-like anti-inflammatory cytokines.     

Production of the PET isotope 11C in hadron therapy via neutron knockout

PurposePositron emitting isotopes such as ¹¹C and ¹⁰C can be used for vital dose verification in hadron therapy. These isotopes are produced when the high energy ¹²C primary beam particles undergo nuclear reactions within the patient.MethodsWe discuss a model for calculating cross sections for the production ¹¹C in ¹²C + ¹²C collisions, applicable at hadron therapy energies.ResultsGood agreement with the available cross section measurements is found for ¹²C(−1n), though more detailed, systematic measurements would be very valuable.ConclusionsNuclear structure plays a crucial role in the reactions of light nuclei, particularly when those reactions are peripheral and involve only a few nucleons. For such reactions, nuclear structure has a strong influence on the energy and angular distribution of the cross section, and is an important consideration for reliable dose verification using ¹¹C in hadron therapy.     

Suppression of Interleukin-11–mediated bone resorption by cyclooxygenases inhibitors

We previously found that human melanoma (A375M) and human breast cancer (MDA-MB-231) cells formed osteolytic bone metastasis in vivo. These cancer cells produced interleukin-11 (IL-11) by themselves and stimulated its production from osteoblasts. Interleukin-11 could increase the number of osteoclasts and raise the calcium concentration in the medium of neonatal murine calvaria organ culture, indicating bone resorption in vitro. Therefore, IL-11 could play an important role in the promotion of osteolysis at the site of bone metastasis. In the present study, we used the calvaria culture system to try to clarify the mechanisms of IL-11–mediated bone resorption. The murine calvaria expressed both the specificity-determining α subunit and the signal–transducing β subunit (gp130) of the IL-11 receptor. When IL-11 was added to the calvaria culture, the concentrations of prostaglandin E₂ (PGE₂) was elevated. Pretreatment of calvaria with cyclooxygenases inhibitors (e.g., indomethacin, NS-398, and dexamethasone) suppressed the production of PGE₂ and the bone resorption induced by IL-11. Addition of exogenous PGE₂ overcame the inhibitory effect of cyclooxygenases inhibitors and promoted bone resorption. These results indicate that IL-11 promotes bone resorption through a PGE₂ synthesis–dependent mechanism and that cyclooxygenases inhibitors could be interesting drugs to suppress IL-11–mediated osteolytic bone metastasis of cancer cells. J. Cell. Physiol. 175:247–254, 1998. © 1998 Wiley-Liss, Inc.     

H4R activation utilizes distinct signaling pathways for the production of RANTES and IL-13 in human mast cells

Context: The histamine H4 receptor functionally expressed on human mast cells and their signaling pathways for the production of IL-13 and RANTES have never been analyzed side by side in a directly comparable manner.

Objective: Therefore, the aim of the study was to investigate signaling transduction pathways of H4R via ERK1/2, Akt and NFκB leading to the induction of inflammatory cytokine expression.

Materials and methods: In the present study, HMC-1 cells and CBMCs were pretreated individually with H4R antagonist JNJ7777120, H1R antagonist mepyramine and signaling molecule inhibitors PD 98059, LY294002, Bay 117082 followed by stimulation was done with or without histamine or 4-MH. Furthermore, the siRNA mediated H4R gene silencing effects are studied at the H4R protein expression level and also signal transduction level.

Results: We found that the pretreatment with JNJ7777120 and H4R gene silencing decreased histamine, 4-MH induced phosphorylation of ERK1/2, Akt and NFκB-p65. Moreover, PD 98059, LY294002 and Bay 117082, which respectively inhibited the histamine and 4-methylhistamine induced phosphorylation of ERK1/2, Akt and NFκB-p65 respectively. We also found that the activation of H4R caused the release of IL-13 and RANTES on human mast cells. The MEK inhibitor PD98059 blocked H4R mediated RANTES/CCL5 production by 20.33?pg/ml and inhibited IL-13 generation by 95.71?pg/ml. In contrast, PI3 kinase inhibitor LY294002 had no effect on 4-MH induced RANTES/CCL5 production but blocked IL-13 generation by 117.58?pg/ml.

Discussion and conclusion: These data demonstrate that the H4R activates divergent signaling pathways to induce cytokine and chemokine production in human mast cells.     

Mechanistic insights into high mobility group box-1 (HMGb1)-induced Toll-like receptor 4 (TLR4) dimer formation

Abstract

Supplemental data for this article can be accessed here.High mobility group box-1 (HMGb1), an endogenous danger-associated molecular pattern protein (DAMP) whose extracellular release has been associated with sterile injury and various inflammatory diseases and conditions, has been shown to be a valuable clinical drug target. Elucidation of the specific interactions with the HMGb1 receptor, Toll-like receptor 4 (TLR4) and adaptor protein myeloid differentiation factor-2 (MD-2), will lead to more precisely targeted therapeutics. We sought to examine detailed interactions and dynamics of the HMGb1 A-box and B-box fragments, as well as the intact protein using in silico protein–protein docking (ZDOCK, ZRANK) and molecular dynamics (Schrödinger Desmond, New York, NY). Mutagenesis and SPR-binding studies allowed us to draw further conclusions regarding the details of the HMGb1–TLR4–MD2 interaction and shed light on the reasons for the opposing biological activities of HMGb1 A-box and B-box fragments. From our findings, we hypothesize that disulfide A-box fragment binds as an anchor toward the TLR4–MD-2 but does not facilitate the TLR4 dimer formation, thereby competing with the HMGb1-binding site and preventing HMGb1-induced signaling and downstream inflammation, whereas the pro-inflammatory B-box fragment retains the MD-2 active conformation and binds to both TLR4 proteins in the complex to aid TLR4 dimer formation, which activates the intracellular signaling for downstream inflammatory pathways and cytokine release.

Communicated by Ramaswamy H. Sarma     

Functional expression of the interleukin-11 receptor alpha-chain and evidence of antiapoptotic effects in human colonic epithelial cells

A tissue-protective effect of interleukin-11 (IL-11) for the intestinal mucosa has been postulated from animal models of inflammatory bowel disease (IBD). Despite the fact that the clinical usefulness of the anti-inflammatory effects of this cytokine is presently investigated in patients with IBD, there are no data available regarding the target cells of IL-11 action and the mechanisms of tissue protection within the human colonic mucosa. IL-11 responsiveness is restricted to cells that express the interleukin-11 receptor alpha-chain (IL-11Ralpha) and an additional signal-transducing subunit (gp130). In this study, we identified the target cells for IL-11 within the human colon with a new IL-11Ralpha monoclonal antibody and investigated the functional expression of the receptor and downstream effects of IL-11-induced signaling. Immunohistochemistry revealed expression of the IL-11Ralpha selectively on colonic epithelial cells. HT-29 and colonic epithelial cells (CEC) constitutively expressed IL-11Ralpha mRNA and protein. Co-expression of the signal-transducing subunit gp130 was also demonstrated. IL-11 induced signaling through triggering activation of the Jak-STAT pathway without inducing anti-inflammatory or proliferative effects in colonic epithelial cells. However, IL-11 stimulation resulted in a dose-dependent tyrosine phosphorylation of Akt, a decreased activation of caspase-9, and a reduced induction of apoptosis in cultured CEC. In HLA-B27 transgenic rats treated with IL-11, a reduction of apoptotic cell numbers was found. This study demonstrates functional expression of the IL-11Ralpha restricted on CEC within the human colonic mucosa. IL-11 induced signaling through triggering activation of the Jak-STAT pathway, without inducing anti-inflammatory or proliferative effects. The beneficial effects of IL-11 therapy are likely to be mediated by CEC via activation of the Akt-survival pathway, mediating antiapoptotic effects to support mucosal integrity.     

Synthesis and evaluation of vitamin D3 analogues with C-11 modifications as inhibitors of Hedgehog signaling

Previous structure-activity relationship studies have provided potent and selective analogues of vitamin D3 as inhibitors of the Hedgehog (Hh) signaling pathway. These analogues contain both modified A- and seco-B ring motifs, and have been evaluated for anticancer therapeutic potential. To continue our studies on this scaffold, a new series of compounds were synthesized to explore additional interactions and spatial constraints. These compounds incorporate functional groups of varying size and hydrophobicity at the C-11 position. While large hydrophobic moieties (9c–e) resulted in significant loss of Hh inhibition, smaller or more flexible moieties (9a, 11) maintain anti-Hh activity. These results call for additional and continued studies to identify the binding pocket to better understand these structure-activity relationships.     

Inhibition of protein–protein interaction of HER2–EGFR and HER2–HER3 by a rationally designed peptidomimetic

Protein–protein interactions (PPI) play a crucial role in many biological processes and modulation of PPI using small molecules to target hot spots has therapeutic value. As a model system we will use PPI of human epidermal growth factor receptors (EGFRs). Among the four EGFRs, EGFR–HER2 and HER2–HER3 are well known in cancer. We have designed a small molecule that is targeted to modulate HER2-mediated signaling. Our approach is novel because the small molecule designed disrupts dimerization not only of EGFR–HER2, but also of HER2–HER3. In the present study we have shown, using surface plasmon resonance analysis, that a peptidomimetic, compound 5, binds specifically to HER2 protein extracellular domain and disrupts the dimerization of EGFRs. To evaluate the effect of compound 5 on HER2 signaling in vitro, Western blot and PathHunter assays were used. Results indicated that compound 5 inhibits the phosphorylation of HER2 kinase domain and inhibits the heterodimerization in a dose-dependent manner. Molecular modeling methods were used to model the PPI of HER2–HER3 heterodimer.     

Mesenchymal stromal cells impair the differentiation of CD14(++) CD16(-) CD64(+) classical monocytes into CD14(++) CD16(+) CD64(++) activate monocytes

Background aimsMesenchymal stromal cells (MSC) possess immunomodulatory activity both in vitro and in vivo. However, little information is available regarding their function during the initiation of immunologic responses through their interactions with monocytes. While many studies have shown that MSC impair the differentiation of monocytes into dendritic cells and macrophages, there are few articles showing the interaction between MSC and monocytes and none of them has addressed the question of monocyte subset modulationMethodsTo understand better the mechanism behind the benefit of MSC infusion for graft-versus-host treatment through monocyte involvement, we performed mixed leucocyte reactions (MLR) in the presence and absence of MSC. After 3 and 7 days, cultures were analyzed by flow cytometry using different approachesResultsMSC induced changes in monocyte phenotype in an MLR. This alteration was accompanied by an increase in monocyte counting and CD14 expression. MSC induced monocyte alterations even without contact, although the parameters above were more pronounced with cell–cell contact. Moreover, the presence of MSC impaired major histocompatibility complex (MHC) I and II, CD11c and CCR5 expression and induced CD14 and CD64 expression on monocytes. These alterations were accompanied by a decrease in interleukin (IL)-1β and IL-6 production by these monocytes, but no change was observed taking into account the phagocytosis capacity of these monocytesConclusionsOur results suggest that MSC impair the differentiation of CD14⁺⁺ CD16^? CD64⁺ classical monocytes into CD14⁺⁺ CD16⁺ CD64⁺⁺ activated monocytes, having an even earlier role than the differentiation of monocytes into dendritic cells and macrophages.     

Inhibition of TNF-induced IL-6 by the TWEAK-Fn14 interaction in rheumatoid arthritis fibroblast like synoviocytes

ObjectivesTNF-like weak inducer of apoptosis (TWEAK), a member of the TNF superfamily, has been shown to increase cytokine production by rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). In this study, we determined the effect of interaction between TWEAK and its receptor fibroblast growth factor-inducible-14 (Fn14) on cytokine expression in RAFLS.MethodsRAFLS were obtained from surgical synovial specimens and used at passage 5–10. Cytokine protein and mRNA expression were measured with ELISA and real time-PCR, respectively. Apoptotic cells were detected by TUNEL assay. RelB activation was detected by Western blot analysis.ResultsTWEAK inhibited IL-6 production from total synovial cells from RA. TWEAK weakly induced FLS IL-6 and IL-8, but in contrast TWEAK dose-dependently inhibited IL-6 and IL-8 production by TNFα-activated FLS. TWEAK did not induce apoptosis in FLS but inhibited proliferation of TNFα-activated FLS. TWEAK induced RelB activation and suppressed IL-6 mRNA expression in TNFα-activated FLS and both of these phenomenon were abolished by inhibition of new protein synthesis with cycloheximide.ConclusionsTWEAK has a previously unsuspected inhibitory effect on cytokine production by TNFα-activated RAFLS. This observation suggests that the effects of TWEAK on cytokine expression varies with the pro-inflammatory context, and that in TNFα-activated states such as RA TWEAK may have a net inhibitory effect.