A Novel Role for IL-6 Receptor Classic Signaling: Induction of RORγt+Foxp3+ Tregs with Enhanced Suppressive Capacity.

BACKGROUND: New therapies blocking the IL-6 receptor (IL-6R) have recently become available and are successfully being used to treat inflammatory diseases like arthritis. Whether IL-6 blockers may help patients with kidney inflammation currently remains unknown. METHODS: To learn more about the complex role of CD4+ T cell-intrinsic IL-6R signaling, we induced nephrotoxic nephritis, a mouse model for crescentic GN, in mice lacking T cell-specific IL-6Ra. We used adoptive transfer experiments and studies in reporter mice to analyze immune responses and Treg subpopulations. RESULTS: Lack of IL-6Ra signaling in mouse CD4+ T cells impaired the generation of proinflammatory Th17 cells, but surprisingly did not ameliorate the course of GN. In contrast, renal damage was significantly reduced by restricting IL-6Ra deficiency to T effector cells and excluding Tregs. Detailed studies of Tregs revealed unaltered IL-10 production despite IL-6Ra deficiency. However, in vivo and in vitro, IL-6Ra classic signaling induced RORγt+Foxp3+ double-positive Tregs (biTregs), which carry the trafficking receptor CCR6 and have potent immunoregulatory properties. Indeed, lack of IL-6Ra significantly reduced Treg in vitro suppressive capacity. Finally, adoptive transfer of T cells containing IL-6Ra-/- Tregs resulted in severe aggravation of GN in mice. CONCLUSIONS: Our data refine the old paradigm, that IL-6 enhances Th17 responses and suppresses Tregs. We here provide evidence that T cell-intrinsic IL-6Ra classic signaling indeed induces the generation of Th17 cells but at the same time highly immunosuppressive RORγt+ biTregs. These results advocate caution and indicate that IL-6-directed therapies for GN need to be cell-type specific.

Hepatic leptin receptor expression can partially compensate for IL-6Rα deficiency in DEN-induced hepatocellular carcinoma.

OBJECTIVE: The current obesity pandemic represents a major health burden, given that it predisposes to the development of numerous obesity-associated disorders. The obesity-derived adipokines not only impair systemic insulin action but also increase the incidence of hepatocellular carcinoma (HCC), a highly prevalent cancer with poor prognosis. Thus, worldwide incidences of HCC are expected to further increase, and defining the molecular as well as cellular mechanisms will allow for establishing new potential treatment options. The adipose tissue of obese individuals increases circulating leptin and interleukin-6 (IL-6) levels, which both share similar signaling capacities such as Signal Transducer and Activator of Transcription 3 (STAT3) and Phosphoinositide 3-kinase (PI3K)/Akt activation. While mouse models with deficient IL-6 signaling show an ameliorated but not absent Diethylnitrosamine (DEN)-induced HCC development, the morbid obesity in mice with mutant leptin signaling complicates the dissection of hepatic leptin receptor (LEPR) and IL-6 signaling in HCC development. Here we have investigated the function of compensating hepatic LEPR expression in HCC development of IL-6Rα-deficient mice. METHODS: We generated and characterized a mouse model of hepatic LEPR deficiency that was intercrossed with IL-6Rα-deficient mice. Cohorts of single and double knockout mice were subjected to the DEN-HCC model to ascertain liver cancer development and characterize metabolic alterations. RESULTS: We demonstrate that both high-fat diet (HFD)-induced obesity and IL-6Rα deficiency induce hepatic Lepr expression. Consistently, double knockout mice show a further reduction in tumor burden in DEN-induced HCC when compared to control and single LepRL-KO/IL-6Rα knock out mice, whereas metabolism remained largely unaltered between the genotypes. CONCLUSIONS: Our findings reveal a compensatory role for hepatic LEPR in HCC development of IL-6Rα-deficient mice and suggest hepatocyte-specific leptin signaling as promoter of HCC under obese conditions.

Obesity exacerbates colitis-associated cancer via IL-6-regulated macrophage polarisation and CCL-20/CCR-6-mediated lymphocyte recruitment.

Colorectal cancer (CRC) is one of the most lethal cancers worldwide in which the vast majority of cases exhibit little genetic risk but are associated with a sedentary lifestyle and obesity. Although the mechanisms underlying CRC and colitis-associated colorectal cancer (CAC) remain unclear, we hypothesised that obesity-induced inflammation predisposes to CAC development. Here, we show that diet-induced obesity accelerates chemically-induced CAC in mice via increased inflammation and immune cell recruitment. Obesity-induced interleukin-6 (IL-6) shifts macrophage polarisation towards tumour-promoting macrophages that produce the chemokine CC-chemokine-ligand-20 (CCL-20) in the CAC microenvironment. CCL-20 promotes CAC progression by recruiting CC-chemokine-receptor-6 (CCR-6)-expressing B cells and γδ T cells via chemotaxis. Compromised cell recruitment as well as inhibition of B and γδ T cells protects against CAC progression. Collectively, our data reveal a function for IL-6 in the CAC microenvironment via lymphocyte recruitment through the CCL-20/CCR-6 axis, thereby implicating a potential therapeutic intervention for human patients.

Trans-presentation of IL-6 by dendritic cells is required for the priming of pathogenic TH17 cells.

The cellular sources of interleukin 6 (IL-6) that are relevant for differentiation of the TH17 subset of helper T cells remain unclear. Here we used a novel strategy for the conditional deletion of distinct IL-6-producing cell types to show that dendritic cells (DCs) positive for the signaling regulator Sirpα were essential for the generation of pathogenic TH17 cells. Using their IL-6 receptor α-chain (IL-6Rα), Sirpα+ DCs trans-presented IL-6 to T cells during the process of cognate interaction. While ambient IL-6 was sufficient to suppress the induction of expression of the transcription factor Foxp3 in T cells, trans-presentation of IL-6 by DC-bound IL-6Rα (called 'IL-6 cluster signaling' here) was needed to prevent premature induction of interferon-γ (IFN-γ) expression in T cells and to generate pathogenic TH17 cells in vivo. Our findings should guide therapeutic approaches for the treatment of TH17-cell-mediated autoimmune diseases.

Differential T Cell Cytokine Receptivity and Not Signal Quality Distinguishes IL-6 and IL-10 Signaling during Th17 Differentiation.

How a large number of cytokines differentially signal through a small number of signal transduction pathways is not well resolved. This is particularly true for IL-6 and IL-10, which act primarily through STAT3 yet induce dissimilar transcriptional programs leading alternatively to pro- and anti-inflammatory effects. Kinetic differences in signaling, sustained to IL-10 and transient to IL-6, are critical to this in macrophages. T cells are also key targets of IL-6 and IL-10, yet how differential signaling in these cells leads to divergent cellular fates is unclear. We show that, unlike for macrophages, signal duration cannot explain the distinct effects of these cytokines in T cells. Rather, naive, activated, activated-rested, and memory CD4(+) T cells differentially express IL-6 and IL-10 receptors in an activation state-dependent manner, and this impacts downstream cytokine effects. We show a dominant role for STAT3 in IL-6-mediated Th17 subset maturation. IL-10 cannot support Th17 differentiation because of insufficient cytokine receptivity rather than signal quality. Enforced expression of IL-10Rα on naive T cells permits an IL-10-generated STAT3 signal equivalent to that of IL-6 and equally capable of promoting Th17 formation. Similarly, naive T cell IL-10Rα expression also allows IL-10 to mimic the effects of IL-6 on both Th1/Th2 skewing and Tfh cell differentiation. Our results demonstrate a key role for the regulation of receptor expression rather than signal quality or duration in differentiating the functional outcomes of IL-6 and IL-10 signaling, and identify distinct signaling properties of these cytokines in T cells compared with myeloid cells.

Modular organization of Interleukin-6 and Interleukin-11 α-receptors.

Interleukin (IL)-6 and IL-11 are the only canonical members of the IL-6 family of cytokines that induce signaling through a homodimer of the common ß-receptor glycoprotein (gp)130. A pre-requisite for signal transduction is the initial binding of the cytokines to their unique α-receptors, IL-6R and IL-11R. The cell-type specific expression of the two receptors determines the target cells of IL-6 and IL-11, because gp130 is ubiquitously expressed. However, ciliary neurotrophic factor (CNTF) and IL-27p28/IL-30 have been described as additional ligands for the IL-6R, underlining a remarkable plasticity among the cytokines of the IL-6 family and their receptors. In this study, we show that neither IL-6 nor IL-11 can bind to and signal through the α-receptor of the respective other cytokine. We further create eight chimeric IL-6/IL-11 receptors, which are all biologically active. We find that the domains D1 to D3, which contain the cytokine binding module (CBM), determine which cytokine can activate the chimeric receptor, whereas the stalk region, the transmembrane region, or the intracellular region do not participate in the ligand selectivity of the receptor and are therefore interchangeable between IL-6R and IL-11R. These results suggest a modular organization of the IL-6R and IL-11R, and a similar signal transduction complex of the two cytokines.

T Follicular Helper Cell-Dependent Clearance of a Persistent Virus Infection Requires T Cell Expression of the Histone Demethylase UTX.

Epigenetic changes, including histone methylation, control T cell differentiation and memory formation, though the enzymes that mediate these processes are not clear. We show that UTX, a histone H3 lysine 27 (H3K27) demethylase, supports T follicular helper (Tfh) cell responses that are essential for B cell antibody generation and the resolution of chronic viral infections. Mice with a T cell-specific UTX deletion had fewer Tfh cells, reduced germinal center responses, lacked virus-specific immunoglobulin G (IgG), and were unable to resolve chronic lymphocytic choriomeningitis virus infections. UTX-deficient T cells showed decreased expression of interleukin-6 receptor-α and other Tfh cell-related genes that were associated with increased H3K27 methylation. Additionally, Turner Syndrome subjects, who are predisposed to chronic ear infections, had reduced UTX expression in immune cells and decreased circulating CD4(+) CXCR5(+) T cell frequency. Thus, we identify a critical link between UTX in T cells and immunity to infection.

The expression of interleukin-6 and its receptor in various brain regions and their roles in exploratory behavior and stress responses.

We examined the involvement of interleukin-6 (IL-6) and its receptor IL-6Rα on behavior and stress responses in mice. In the open field, both wild-type (WT) and IL-6 deficient mice displayed similar levels of locomotor activity; however, IL-6 deficient mice spent more time in the central part of the arena compared to control WT mice. After behavioral testing, mice were subjected to stress and then sacrificed. The levels of IL-6 and its receptor in their brains were determined. Immunohistochemical labeling of brain sections for IL-6 showed a high level of expression in the subventricular zone of the lateral ventricles and in the border zone of the third and fourth ventricles. Interestingly, 95% of the IL-6-expressing cells had an astrocytic phenotype, and the remaining 5% were microglial cells. A low level of IL-6 expression was observed in the olfactory bulb, hypothalamus, hippocampus, cerebral cortex, cerebellum, midbrain and several brainstem structures. The vast majority of IL-6-expressing cells in these structures had a neuronal phenotype. Stress increased the number of IL-6-immunoreactive astrocytes and microglial cells. The levels of the IL-6Rα receptor were increased in the hypothalamus of stressed mice. Therefore, in this study, we describe for the first time the distribution of IL-6 in various types of brain cells and in previously unreported regions, such as the subventricular zone of the lateral ventricle. Moreover, we provide data on regional distribution and expression within specific cell phenotypes. This highly differential expression of IL-6 indicates its specific roles in the regulation of neuronal and astrocytic functions, in addition to the roles of IL-6 and its receptor IL-6Rα in stress responses.

T cell-intrinsic role of IL-6 signaling in primary and memory responses.

Innate immune recognition is critical for the induction of adaptive immune responses; however the underlying mechanisms remain incompletely understood. In this study, we demonstrate that T cell-specific deletion of the IL-6 receptor α chain (IL-6Rα) results in impaired Th1 and Th17 T cell responses in vivo, and a defect in Tfh function. Depletion of Tregs in these mice rescued the Th1 but not the Th17 response. Our data suggest that IL-6 signaling in effector T cells is required to overcome Treg-mediated suppression in vivo. We show that IL-6 cooperates with IL-1ß to block the suppressive effect of Tregs on CD4(+) T cells, at least in part by controlling their responsiveness to IL-2. In addition, although IL-6Rα-deficient T cells mount normal primary Th1 responses in the absence of Tregs, they fail to mature into functional memory cells, demonstrating a key role for IL-6 in CD4(+) T cell memory formation.DOI: http://dx.doi.org/10.7554/eLife.01949.001.

Cutting edge: Inhibition of IL-6 trans-signaling protects from malaria-induced lethality in mice.

Circulating IL-6 levels correlate with the severity of blood-stage malaria in humans and mouse models, but the impact of IL-6 classic signaling through membrane IL-6Rα, as well as IL-6 trans-signaling through soluble IL-6Rα, on the outcome of malaria has remained unknown. In this study, we created IL-6Rα-deficient mice that exhibit a 50% survival of otherwise lethal blood-stage malaria of the genus Plasmodium chabaudi. Inducing IL-6 trans-signaling by injection of mouse recombinant soluble IL-6Rα in IL-6Rα-deficient mice restores the lethal outcome to malaria infection. In contrast, inhibition of IL-6 trans-signaling via injection of recombinant sGP130Fc protein in control mice results in a 40% survival rate. Our data demonstrate that IL-6 trans-signaling, rather than classic IL-6 signaling, contributes to malaria-induced lethality in mice, preceded by an increased inflammatory response. Therefore, inhibition of IL-6 trans-signaling may serve as a novel promising therapeutic basis to combat malaria.

Molecular characterization and expression analysis of the putative interleukin 6 receptor (IL-6Rα and glycoprotein-130) in rainbow trout (Oncorhynchus mykiss): salmonid IL-6Rα possesses a polymorphic N-terminal Ig domain with variable numbers of two repeats.

Interleukin (IL)-6, the founding member of IL-6 family cytokines, plays non-redundant roles in hematopoiesis and acute phase responses. IL-6 signals via a specific private IL-6Rα and a common beta chain gp130. In this study, we have cloned both the IL-6Rα and gp130 in rainbow trout. The trout gp130 cDNA encodes 906 aa and is similar in size, extracellular domain structure (D1-D6) and presence of intracellular motifs important for signal transduction to tetrapod gp130s. The trout IL-6Rα cDNA encodes for 834 aa and is larger compared to tetrapod IL-6Rαs, as are other fish IL-6Rα molecules due to a large D1 domain. However, the cytokine-binding domain is well conserved across vertebrates, with four conserved cysteine residues in the N-terminal FNIII domain and a WSXWS motif in the C-terminal FNIII domain. Furthermore, a phylogenetic tree analysis confirmed that the reported fish IL-6Rα and gp130 molecules are orthologues to their tetrapod counterparts. The extra large D1 domain of the salmonid IL-6Rα molecules results partially from the insertions of two repetitive sequences of [TS]-[TF]-VSTTT-[ND]-TTSNG and TTVS-[AT]-IKD-[DG]-S-[KD]-N-[GR], respectively. Furthermore the numbers of repetitions of the two motifs were variable in different individuals and cell lines, and even in the same fish allelic polymorphism exists. Trout IL-6Rα was expressed at higher levels than gp130 in a number of tissues examined and the expression of both IL-6Rα and gp130 could be modulated by LPS and Poly I:C in the cell lines studied. The expression patterns of the receptors suggest that high level expression of IL-6Rα is critical for IL-6 responsiveness.

Glucose ingestion during endurance training in men attenuates expression of myokine receptor.

Glucose ingestion during exercise attenuates the release of the myokine interleukin-6 (IL-6) from working skeletal muscle, which results in a diminished increase in plasma IL-6. Interleukin-6 receptor alpha (IL-6Ralpha) expression in skeletal muscle is induced by acute exercise, mediated in part by an increased IL-6 concentration in the bloodstream. We hypothesized that endurance training would increase the density of IL-6Ralpha in skeletal muscle and that glucose ingestion would attenuate the effect. Nine subjects performed 10 weeks of one-legged knee-extensor training. They trained one leg (Glc-leg) while ingesting a glucose solution (Glc) and ingested a placebo (Plc) while training the other leg (Plc-leg). Endurance training increased peak power by 14% and reduced the exercise-induced gene expression of IL-6 and IL-6Ralpha in skeletal muscle and IL-6 plasma concentration. The IL-6Ralpha density increased to a lesser extent in the Glc-leg, suggesting that glucose ingestion attenuates the effect of training on IL-6Ralpha by blunting the IL-6 response. We conclude that glucose ingestion during endurance training attenuates the increase in IL-6Ralpha density.

Expression, purification and characterization of soluble human interleukin-6 receptor alpha-chain and its mutant protein in Escherichia coli.

To investigate the function of the N-terminal immunoglobulin (Ig)-like domain of the human interleukin-6 receptor alpha-chain (hIL-6R), we constructed a soluble human interleukin-6 receptor (shIL-6R) (named EC05, amino acids 20-354) and soluble variants of the shIL-6R lacking the Ig-like domain (named EC70, amino acids 105-354). The two extracellular portions of hIL-6R were expressed as soluble fusion proteins with thioredoxin in Escherichia coli and purified by using Ni-NTA agarose. Western blot showed that purified proteins were immunoreactive with the antibody against hIL-6R. They also possessed specific binding activity with human interleukin-6 (hIL-6) in ELISA analysis.

Changes in expression of interleukin-6 receptors in granulosa cells during follicular atresia in pig ovaries.

More than 99% of follicles undergo a degenerative process known as "atresia" in mammalian ovaries, and only a few follicles ovulate during follicular growth and development. Follicular selection predominantly depends on granulosa cell apoptosis. To reveal the molecular mechanisms of selective follicular atresia, we examined the changes in the levels of interleukin-6 (IL-6) receptors expressed in the granulosa cells of pig ovaries. The levels of IL-6 receptor (IL-6R)-alpha mRNA and protein in granulosa cells were quantified by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. IL-6R alpha mRNA and protein were highly expressed in the granulosa cells of progressed atretic follicles. Enzyme-linked immunosorbent assay showed that the expression of IL-6 soluble receptor (IL-6sR) protein in follicular fluid decreased during atresia. Moreover, we isolated porcine cDNA encoding an IL-6 signal transducer, gp130. Porcine gp130 (2,754 bp and 917 amino acids) was identified from a cDNA library prepared using follicular granulosa cells of pig ovaries. Porcine gp130 was highly homologous with human and murine gp130. RT-PCR analysis revealed that the level of gp130 mRNA also decreased during atresia. We presume that IL-6sR and gp130, but not IL-6R alpha, play important roles in regulation of granulosa cell survival.