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The successful treatment of certain autoimmune conditions with the humanized anti-IL-6 receptor (IL-6R) antibody tocilizumab has emphasized the clinical importance of cytokines that signal through the β-receptor subunit glycoprotein 130 (gp130). In this Review, we explore how gp130 signaling controls disease progression and examine why IL-6 has a special role among these cytokines as an inflammatory regulator. Attention will be given to the role of the soluble IL-6R, and we will provide a perspective into the clinical blockade of IL-6 activity in autoimmunity, inflammation, and cancer.

Plasma IL-6 is elevated after myocardial infarction (MI) and is associated with increased morbidity and mortality. Which cardiac cell type preferentially contributes to IL-6 expression and how its production is regulated are largely unknown. Here, we studied the cellular source and purinergic regulation of IL-6 formation in a murine MI model. We found that IL-6, measured in various cell types in post-MI hearts at the protein level and by quantitative PCR and RNAscope, was preferentially formed by cardiac fibroblasts (CFs). Single-cell RNA-Seq (scRNA-Seq) in infarcted mouse and human hearts confirmed this finding. We found that adenosine stimulated fibroblast IL-6 formation via the adenosine receptor A2bR in a Gq-dependent manner. CFs highly expressed Adora2b and rapidly degraded extracellular ATP to AMP but lacked CD73. In mice and humans, scRNA-Seq revealed that Adora2B was also mainly expressed by fibroblasts. We assessed global IL-6 production in isolated hearts from mice lacking CD73 on T cells (CD4-CD73-/-), a condition known to be associated with adverse cardiac remodeling. The ischemia-induced release of IL-6 was strongly attenuated in CD4-CD73-/- mice, suggesting adenosine-mediated modulation. Together, these findings demonstrate that post-MI IL-6 was mainly derived from activated CFs and was controlled by T cell-derived adenosine. We show that purinergic metabolic cooperation between CFs and T cells is a mechanism that modulates IL-6 formation by the heart and has therapeutic potential.

Cytokines of the IL-12 family show structural similarities but have distinct functions in the immune system. Prominent members of this cytokine family are the pro-inflammatory cytokines IL-12 and IL-23. These two cytokines share cytokine subunits and receptor chains but have different functions in autoimmune diseases, cancer and infections. Accordingly, structural knowledge about receptor complex formation is essential for the development of new therapeutic strategies preventing and/or inhibiting cytokine:receptor interaction. In addition, intracellular signaling cascades can be targeted to inhibit cytokine-mediated effects. Single nucleotide polymorphisms can lead to alteration in the amino acid sequence and thereby influencing protein functions or protein–protein interactions. To understand the biology of IL-12 and IL-23 and to establish efficient targeting strategies structural knowledge about cytokines and respective receptors is crucial. A highly efficient therapy might be a combination of different drugs targeting extracellular cytokine:receptor assembly and intracellular signaling pathways.

Histological analysis with 2,3,5-triphenyltetrazolium chloride (TTC) staining is the most frequently used tool to detect myocardial ischemia/reperfusion injury. However, its practicality is often challenged by poor image quality in gross histology, leading to an equivocal infarct-boundary delineation and potentially compromised measurement accuracy. Here, we introduce several crucial refinements in staining protocol and sample processing, which enable TTC images to be analyzed with light microscopy. The refined protocol involves a two-step TTC staining process (perfusion and immersion) and subsequent Zamboni fixation to differentiate myocardial viability and necrosis, and use of Coomassie brilliant blue to label area-at-risk. After the duo-staining steps were completed, the heart sample was embedded and sliced transversally by a cryostat into a series of thin sections (50 µm) for microscopic analysis. The refined TTC (redTTC) assay yielded remarkably high-quality images with striking color intensity and sharply defined boundaries, permitting unambiguous and reliable delineation of the infarct and area-at-risk. In the same animals, the redTTC assay showed good agreement with the in-vivo gold standard measurements (LGE and MEMRI). Meanwhile, redTTC imaging allows tracking of viable cardiomyocytes at cellular resolution, and with this enhanced capability, we convincingly demonstrated the pro-survival action of stem cells based-therapy. Therefore, the redTTC assay represents a significant technical advance that permits precise detection of the true extent of cardiac injury and cardiomyocyte viability. This approach is cost-effective and may be adapted for use in diverse applications, making it highly appealing to many laboratories performing ischemia/reperfusion injury experiments.

Background In the current medical landscape, synthetic cytokine receptors have emerged as a pivotal component in the development of novel therapeutic interventions. Interleukin-23 (IL-23) is the dominant regulatory cytokine in a cluster of immune-mediated inflammatory diseases induced due to the increased expression of the IL-23 receptor (IL-23R) on pathogenic TH17 cells. The modulation of IL-23 signaling by altering IL-23R presents a promising avenue for further investigation. Chimeric cytokine receptors expressing the extracellular domain (ECD) of one protein and the intracellular domain (ICD) of another have been used to isolate the effects of ligand binding from signaling. Methods We designed chimeric IL-23Rs that comprise the extracellular and transmembrane domains of IL-23R, as well as various parts of the intracellular region of gp130, the IL-6 signal transducing receptor. To characterize signaling properties by these synthetic cytokine receptors analysis of the activation of the signaling proteins ERK1/2 and STAT3, and SOCS3 was combined with multiplexed single-cell flow cytometry data and information theoretic approaches. Results Simply transferring the SOCS3-binding site from the IL-6 signal transducing receptor gp130 to IL-23R was not enough to make IL-23 signaling sensitive to SOCS3. However, an iterative transfer process identified a region of gp130 that bound SOCS3 and rendered IL-23 signaling sensitive to SOCS3 regulation. Opposingly, SOCS3-independent gp130 hyper-signaling was achieved by transferring a minimal IL-23R ERK activation motif to gp130. Notably, this motif retained gp130-dependent ERK activation without negative SOCS3 feedback. Conclusions In summary, this study identifies the WLYEDIPN motif in IL-23R as an indispensable motif for IL-23R signal transduction and emphasizes the complexity of IL-23-induced signaling pathways and the function of various tyrosines in the intracellular part of cytokine/IL-23 receptor(s). Plain English Summary Cellular communication is facilitated by soluble molecules that transmit information from one cell to another. Cytokines are important signaling mediators in the immune system. They are released from sender cells and interact with their respective receptors on receiver cells inducing intracellular signaling cascades resulting in biological responses. Most cytokines bind to membrane spanning receptors that are linked to Janus kinases via their intracellular domain. Binding of a cytokine to the extracellular part of these receptors activates the JAK-STAT signaling pathway, which plays a role in maturation of blood cells, developing the immune system, growing and developing sexually, and turning fat cells into other types of cells. Any receptor that activates intracellular signaling pathways is equipped with negative feedback systems ensuring that the activated intracellular signaling pathways and downstream transcription factors are only activated temporarily. Suppressor of cytokine signaling (SOCS) proteins stop the action of many, but not all, JAK-STAT activating receptors. Here, we show that IL-23 signaling is insensitive to SOCS3 in contrast to IL-6 signaling. By changing the intracellular part of the IL-23R inside the cell we have created a novel artificial cytokine receptor that can be controlled by SOCS3. Additionally, we were able to show that a specific motif in IL-23R is important for its unique signal transduction. However, introducing this IL-23R motif in the IL-6 signal transducing receptor gp130 led to a SOCS3-insensitive receptor. In the future, we might be able to use receptors with modified signal transduction to treat certain diseases more effectively.

Although Interleukin (IL)-6-type cytokine signaling is critical for maintaining the body's homeostasis, aberrant signaling has been observed in numerous diseases including autoimmunity and cancer. Currently, all approved biologics that inhibit IL-6-type cytokines specifically target the key pro-inflammatory mediator IL-6 or its receptor (IL6R). Historically, direct inhibition of glycoprotein 130 (gp130)-the shared transmembrane receptor for IL-6-type cytokines-was avoided due to concerns that broad suppression might cause more harm than benefit. However, this view is being reconsidered in light of the clinical success of Janus kinase (JAK) inhibitors, which broadly disrupt cytokine signaling, including pathways mediated by gp130. Here we developed four single domain antibodies (sdAb), consisting out of a camelid-derived nanobody and a human Fc-fragment, and characterized them by direct protein interaction analysis, epitope binding, epitope binning, as well as inhibition of cytokine-induced stimulation and proliferation of appropriate Ba/F3 cell lines and trans-migration in HT-29 cells. The four sdAb-Fc constructs GP01-, GP11- GP13- and GP20-Fc bind directly to gp130 in the cytokine binding module (CBM) and largely inhibit IL-6-type cytokine signaling by interfering with the high-affinity binding site of IL-6, IL-11, CLCF1, CT1, CNTF, OSM and LIF. Furthermore, we functionally demonstrate the inhibitory effect of the selected nanobodies in cell-based transmigration assays of the human colorectal cancer cell line HT-29. In summary, our study has identified and characterized four novel inhibitory high-affinity gp130 nanobodies with potential for use in cytokine-dependent autoimmunity or cancer therapy.

Recently, we have shown that after partial hepatectomy (PHx), an increased hepatic blood flow initiates liver growth in mice by vasodilation and mechanically-triggered release of angiocrine signals. Here, we use mass spectrometry to identify a mechanically-induced angiocrine signal in human hepatic endothelial cells, that is, myeloid-derived growth factor (MYDGF). We show that it induces proliferation and promotes survival of primary human hepatocytes derived from different donors in two-dimensional cell culture, via activation of mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3). MYDGF also enhances proliferation of human hepatocytes in three-dimensional organoids. In vivo, genetic deletion of MYDGF decreases hepatocyte proliferation in the regenerating mouse liver after PHx; conversely, adeno-associated viral delivery of MYDGF increases hepatocyte proliferation and MAPK signaling after PHx. We conclude that MYDGF represents a mechanically-induced angiocrine signal and that it triggers growth of, and provides protection to, primary mouse and human hepatocytes. After partial liver resection, the remaining liver blood vessels receive more blood flow and get mechanically stretched. Here the authors show that MYDGF is released from the stretched cells of these liver vessels, and that it is required and sufficient to promote liver regeneration and cell proliferation.

Cytokine-induced signal transduction is executed by natural biological switches, which among many others control immune-related processes. Here, we show that synthetic cytokine receptors (SyCyRs) can induce cytokine signaling using non-physiological ligands. High-affinity GFP- and mCherry-nanobodies were fused to transmembrane and intracellular domains of the IL-6/IL-11 and IL-23 cytokine receptors gp130 and IL-12Rβ1/IL-23R, respectively. Homo- and heterodimeric GFP:mCherry fusion proteins as synthetic cytokine-like ligands were able to induce canonical signaling in vitro and in vivo. Using SyCyR ligands, we show that IL-23 receptor homodimerization results in its activation and IL-23-like signal transduction. Moreover, trimeric receptor assembly induces trans-phosphorylation among cytokine receptors with associated Janus kinases. The SyCyR technology allows biochemical analyses of transmembrane receptor signaling in vitro and in vivo, cell-specific activation through SyCyR ligands using transgenic animals and possible therapeutic regimes involving non-physiological targets during immunotherapy. Cytokine-induced signaling acts as an ON/OFF switch dependent on the presence of ligands. Here the authors construct synthetic cytokine receptors responsive to synthetic ligands able to activate canonical signaling pathways.

The sirtuin SIRT4 has been implicated in the control of autophagy and mitochondrial quality control via mitophagy. However, the role of SIRT4 in regulating autophagy/mitophagy induced by different stressors is unclear. Here, we show that cells expressing SIRT4(H161Y), a catalytically inactive, dominant‐negative mutant of SIRT4, fail to upregulate LC3B‐II. These cells also exhibit a reduced autophagic flux upon treatment with different inducers of mitophagy/autophagy, that is, CoCl2‐triggered pseudohypoxia, CCCP (carbonyl cyanide 3‐chlorophenylhydrazone)/oligomycin‐mediated respiratory chain inhibition, or rapamycin treatment. Interestingly, SIRT4(H161Y) expression upregulated protein levels of HDAC6, which is involved in mitochondrial trafficking and autophagosome‐lysosome fusion, and inhibited the conversion of OPA1‐L to OPA1‐S, which is associated with increased mitochondrial fusion and decreased mitophagy. Both HDAC6 and OPA1 are SIRT4 interactors. However, the pharmacological inhibition of HDAC6 using Tubacin or of OPA1 using MYLS22 did not restore the stress‐induced upregulation of LC3B‐II levels upon autophagy/mitophagy treatment in SIRT4(H161Y)‐expressing cells. Remarkably, inhibition of autophagosome–lysosome fusion and thus disruption of late autophagic flux by BafA1 treatment also failed to restore LC3B‐II levels upon autophagy/mitophagy treatment, suggesting an inhibitory effect of SIRT4(H161Y) on the initiation/early phase of autophagy. Consistent with this, we demonstrate that SIRT4(H161Y) promotes the phosphorylation of ULK1 at S638 and S758 (mTORC1 targets), both of which mediate an important inhibitory regulation of autophagy initiation. Thus, our data suggest a positive regulatory function of SIRT4 in the ULK1‐dependent early regulation/initiation of stress‐induced autophagic flux, presumably via modulation of AMPK/mTORC1 signaling. Cells expressing SIRT4 (H161Y), a catalytically inactive mutant of the sirtuin SIRT4, fail to upregulate LC3B‐II and exhibit a reduced autophagic flux under stress conditions. Interestingly, SIRT4(H161Y) promotes phosphorylation of ULK1 at S638 and S758 that are associated with inhibition of autophagy initiation. Thus, SIRT4 may function in ULK1‐dependent early regulation/initiation of autophagic flux, presumably via modulation of AMPK/mTORC1 signaling.

Overexpression of TNF contributes to pathogenesis of multiple autoimmune diseases, accounting for a remarkable success of anti- TNF therapy. TNF is produced by a variety of cell types, and it can play either a beneficial or a deleterious role. In particular, in autoimmunity pathogenic TNF may be derived from restricted cellular sources. In this study we evaluated the feasibility of cell-type–restricted TNF inhibition in vivo. To this end, we engineered MYSTI (Myeloid-Specific TNF Inhibitor)—a recombinant bispecific antibody that binds to the F4/80 surface molecule on myeloid cells and to human TNF (hTNF). In macrophage cultures derived from TNF humanized mice MYSTI could capture the secreted hTNF, limiting its bioavailability. Additionally, as evaluated in TNF humanized mice, MYSTI was superior to an otherwise analogous systemic TNF inhibitor in protecting mice from lethal LPS/D-Galactosamine–induced hepatotoxicity. Our results suggest a novel and more specific approach to inhibiting TNF in pathologies primarily driven by macrophage-derived TNF.