Repopulating Microglia Promote Brain Repair in an IL-6-Dependent Manner.

Cognitive dysfunction and reactive microglia are hallmarks of traumatic brain injury (TBI), yet whether these cells contribute to cognitive deficits and secondary inflammatory pathology remains poorly understood. Here, we show that removal of microglia from the mouse brain has little effect on the outcome of TBI, but inducing the turnover of these cells through either pharmacologic or genetic approaches can yield a neuroprotective microglial phenotype that profoundly aids recovery. The beneficial effects of these repopulating microglia are critically dependent on interleukin-6 (IL-6) trans-signaling via the soluble IL-6 receptor (IL-6R) and robustly support adult neurogenesis, specifically by augmenting the survival of newborn neurons that directly support cognitive function. We conclude that microglia in the mammalian brain can be manipulated to adopt a neuroprotective and pro-regenerative phenotype that can aid repair and alleviate the cognitive deficits arising from brain injury.

Origin and Function of Stress-Induced IL-6 in Murine Models.

Acute psychological stress has long been known to decrease host fitness to inflammation in a wide variety of diseases, but how this occurs is incompletely understood. Using mouse models, we show that interleukin-6 (IL-6) is the dominant cytokine inducible upon acute stress alone. Stress-inducible IL-6 is produced from brown adipocytes in a beta-3-adrenergic-receptor-dependent fashion. During stress, endocrine IL-6 is the required instructive signal for mediating hyperglycemia through hepatic gluconeogenesis, which is necessary for anticipating and fueling "fight or flight" responses. This adaptation comes at the cost of enhancing mortality to a subsequent inflammatory challenge. These findings provide a mechanistic understanding of the ontogeny and adaptive purpose of IL-6 as a bona fide stress hormone coordinating systemic immunometabolic reprogramming. This brain-brown fat-liver axis might provide new insights into brown adipose tissue as a stress-responsive endocrine organ and mechanistic insight into targeting this axis in the treatment of inflammatory and neuropsychiatric diseases.

Activation of naïve CD4+ T cells re-tunes STAT1 signaling to deliver unique cytokine responses in memory CD4+ T cells.

The cytokine IL-6 controls the survival, proliferation and effector characteristics of lymphocytes through activation of the transcription factors STAT1 and STAT3. While STAT3 activity is an ever-present feature of IL-6 signaling in CD4+ T cells, prior activation via the T cell antigen receptor limits IL-6's control of STAT1 in effector and memory populations. Here we found that phosphorylation of STAT1 in response to IL-6 was regulated by the tyrosine phosphatases PTPN2 and PTPN22 expressed in response to the activation of naïve CD4+ T cells. Transcriptomics and chromatin immunoprecipitation-sequencing (ChIP-seq) of IL-6 responses in naïve and effector memory CD4+ T cells showed how the suppression of STAT1 activation shaped the functional identity and effector characteristics of memory CD4+ T cells. Thus, tyrosine phosphatases induced by the activation of naïve T cells determine the way activated or memory CD4+ T cells sense and interpret cytokine signals.

The persistence of interleukin-6 is regulated by a blood buffer system derived from dendritic cells.

The interleukin-6 (IL-6) membrane receptor and its circulating soluble form, sIL-6R, can be targeted by antibody therapy to reduce deleterious immune signaling caused by chronic overexpression of the pro-inflammatory cytokine IL-6. This strategy may also hold promise for treating acute hyperinflammation, such as observed in coronavirus disease 2019 (COVID-19), highlighting a need to define regulators of IL-6 homeostasis. We found that conventional dendritic cells (cDCs), defined in mice via expression of the transcription factor Zbtb46, were a major source of circulating sIL-6R and, thus, systemically regulated IL-6 signaling. This was uncovered through identification of a cDC-dependent but T cell-independent modality that naturally adjuvants plasma cell differentiation and antibody responses to protein antigens. This pathway was then revealed as part of a broader biological buffer system in which cDC-derived sIL-6R set the in-solution persistence of IL-6. This control axis may further inform the development of therapeutic agents to modulate pro-inflammatory immune reactions.

Inflammatory networks underlying colorectal cancer.

Inflammation is emerging as one of the hallmarks of cancer, yet its role in most tumors remains unclear. Whereas a minority of solid tumors are associated with overt inflammation, long-term treatment with non-steroidal anti-inflammatory drugs is remarkably effective in reducing cancer rate and death. This indicates that inflammation might have many as-yet-unrecognized facets, among which an indolent course might be far more prevalent than previously appreciated. In this Review, we explore the various inflammatory processes underlying the development and progression of colorectal cancer and discuss anti-inflammatory means for its prevention and treatment.

Pleiotropy and Specificity: Insights from the Interleukin 6 Family of Cytokines.

Since the molecular cloning of interleukin-6 (IL-6) in 1986, many other cytokines have been found to share the same signal transducer, gp130, in their receptor complexes. Thus, the IL-6 family of cytokines now consists of ten members. Although some of the family members' functions are redundant as a result of the expression of gp130, there are also functional distinctions between members. The mechanisms that determine functional redundancies and distinctions are not completely understood. Yet, research has clarified the role of IL-6 family cytokines in autoimmune diseases and has led to effective therapies that target them. Here, we review the IL-6 family of cytokines in autoimmune diseases, with a particular focus on the prototypical member IL-6, from the viewpoints of their structure, signaling, and biological features and discuss possible mechanisms of their functional pleiotropy.

Targeting Interleukin-6 Signaling in Clinic.

Interleukin-6 (IL-6) is a pleiotropic cytokine with roles in immunity, tissue regeneration, and metabolism. Rapid production of IL-6 contributes to host defense during infection and tissue injury, but excessive synthesis of IL-6 and dysregulation of IL-6 receptor signaling is involved in disease pathology. Therapeutic agents targeting the IL-6 axis are effective in rheumatoid arthritis, and applications are being extended to other settings of acute and chronic inflammation. Recent studies reveal that selective blockade of different modes of IL-6 receptor signaling has different outcomes on disease pathology, suggesting novel strategies for therapeutic intervention. However, some inflammatory diseases do not seem to respond to IL-6 blockade. Here, we review the current state of IL-6-targeting approaches in the clinic and discuss how to apply the growing understanding of the immunobiology of IL-6 to clinical decisions.

cGAS-STING drives the IL-6-dependent survival of chromosomally instable cancers.

Chromosomal instability (CIN) drives cancer cell evolution, metastasis and therapy resistance, and is associated with poor prognosis1. CIN leads to micronuclei that release DNA into the cytoplasm after rupture, which triggers activation of inflammatory signalling mediated by cGAS and STING2,3. These two proteins are considered to be tumour suppressors as they promote apoptosis and immunosurveillance. However, cGAS and STING are rarely inactivated in cancer4, and, although they have been implicated in metastasis5, it is not known why loss-of-function mutations do not arise in primary tumours4. Here we show that inactivation of cGAS-STING signalling selectively impairs the survival of triple-negative breast cancer cells that display CIN. CIN triggers IL-6-STAT3-mediated signalling, which depends on the cGAS-STING pathway and the non-canonical NF-κB pathway. Blockade of IL-6 signalling by tocilizumab, a clinically used drug that targets the IL-6 receptor (IL-6R), selectively impairs the growth of cultured triple-negative breast cancer cells that exhibit CIN. Moreover, outgrowth of chromosomally instable tumours is significantly delayed compared with tumours that do not display CIN. Notably, this targetable vulnerability is conserved across cancer types that express high levels of IL-6 and/or IL-6R in vitro and in vivo. Together, our work demonstrates pro-tumorigenic traits of cGAS-STING signalling and explains why the cGAS-STING pathway is rarely inactivated in primary tumours. Repurposing tocilizumab could be a strategy to treat cancers with CIN that overexpress IL-6R.

LEF-1 and TCF-1 orchestrate T(FH) differentiation by regulating differentiation circuits upstream of the transcriptional repressor Bcl6.

Follicular helper T cells (T(FH) cells) are specialized effector CD4(+) T cells that help B cells develop germinal centers (GCs) and memory. However, the transcription factors that regulate the differentiation of T(FH) cells remain incompletely understood. Here we report that selective loss of Lef1 or Tcf7 (which encode the transcription factor LEF-1 or TCF-1, respectively) resulted in T(FH) cell defects, while deletion of both Lef1 and Tcf7 severely impaired the differentiation of T(FH) cells and the formation of GCs. Forced expression of LEF-1 enhanced T(FH) differentiation. LEF-1 and TCF-1 coordinated such differentiation by two general mechanisms. First, they established the responsiveness of naive CD4(+) T cells to T(FH) cell signals. Second, they promoted early T(FH) differentiation via the multipronged approach of sustaining expression of the cytokine receptors IL-6Rα and gp130, enhancing expression of the costimulatory receptor ICOS and promoting expression of the transcriptional repressor Bcl6.

IL-6 as a keystone cytokine in health and disease.

Interleukin 6 (IL-6) has a broad effect on cells of the immune system and those not of the immune system and often displays hormone-like characteristics that affect homeostatic processes. IL-6 has context-dependent pro- and anti-inflammatory properties and is now regarded as a prominent target for clinical intervention. However, the signaling cassette that controls the activity of IL-6 is complicated, and distinct intervention strategies can inhibit this pathway. Clinical experience with antagonists of IL-6 has raised new questions about how and when to block this cytokine to improve disease outcome and patient wellbeing. Here we discuss the effect of IL-6 on innate and adaptive immunity and the possible advantages of various antagonists of IL-6 and consider how the immunobiology of IL-6 may inform clinical decisions.

Gp130-HIF1α axis-induced vascular damage is prevented by the short-term inhibition of IL-6 receptor signaling.

Protection against endothelial damage is recognized as a frontline approach to preventing the progression of cytokine release syndrome (CRS). Accumulating evidence has demonstrated that interleukin-6 (IL-6) promotes vascular endothelial damage during CRS, although the molecular mechanisms remain to be fully elucidated. Targeting IL-6 receptor signaling delays CRS progression; however, current options are limited by persistent inhibition of the immune system. Here, we show that endothelial IL-6 trans-signaling promoted vascular damage and inflammatory responses via hypoxia-inducible factor-1α (HIF1α)-induced glycolysis. Using pharmacological inhibitors targeting HIF1α activity or mice with the genetic ablation of gp130 in the endothelium, we found that inhibition of IL-6R (IL-6 receptor)-HIF1α signaling in endothelial cells protected against vascular injury caused by septic damage and provided survival benefit in a mouse model of sepsis. In addition, we developed a short half-life anti-IL-6R antibody (silent anti-IL-6R antibody) and found that it was highly effective at augmenting survival for sepsis and severe burn by strengthening the endothelial glycocalyx and reducing cytokine storm, and vascular leakage. Together, our data advance the role of endothelial IL-6 trans-signaling in the progression of CRS and indicate a potential therapeutic approach for burns and sepsis.

Signaling by IL-6 promotes alternative activation of macrophages to limit endotoxemia and obesity-associated resistance to insulin.

Obesity and resistance to insulin are closely associated with the development of low-grade inflammation. Interleukin 6 (IL-6) is linked to obesity-associated inflammation; however, its role in this context remains controversial. Here we found that mice with an inactivated gene encoding the IL-6Rα chain of the receptor for IL-6 in myeloid cells (Il6ra(Δmyel) mice) developed exaggerated deterioration of glucose homeostasis during diet-induced obesity, due to enhanced resistance to insulin. Tissues targeted by insulin showed increased inflammation and a shift in macrophage polarization. IL-6 induced expression of the receptor for IL-4 and augmented the response to IL-4 in macrophages in a cell-autonomous manner. Il6ra(Δmyel) mice were resistant to IL-4-mediated alternative polarization of macrophages and exhibited enhanced susceptibility to lipopolysaccharide (LPS)-induced endotoxemia. Our results identify signaling via IL-6 as an important determinant of the alternative activation of macrophages and assign an unexpected homeostatic role to IL-6 in limiting inflammation.

An HNF4α-miRNA inflammatory feedback circuit regulates hepatocellular oncogenesis.

Hepatocyte nuclear factor 4α (HNF4α) is essential for liver development and hepatocyte function. Here, we show that transient inhibition of HNF4α initiates hepatocellular transformation through a microRNA-inflammatory feedback loop circuit consisting of miR-124, IL6R, STAT3, miR-24, and miR-629. Moreover, we show that, once this circuit is activated, it maintains suppression of HNF4α and sustains oncogenesis. Systemic administration of miR-124, which modulates inflammatory signaling, prevents and suppresses hepatocellular carcinogenesis by inducing tumor-specific apoptosis without toxic side effects. As we also show that this HNF4α circuit is perturbed in human hepatocellular carcinomas, our data raise the possibility that manipulation of this microRNA feedback-inflammatory loop has therapeutic potential for treating liver cancer.

Engineered interleukin-6-derived cytokines recruit artificial receptor complexes and disclose CNTF signaling via the OSMR.

Ciliary neurotrophic factor (CNTF) activates cells via the non-signaling α-receptor CNTF receptor (CNTFR) and the two signaling ß-receptors glycoprotein 130 (gp130) and leukemia inhibitory factor receptor (LIFR). The CNTF derivate, Axokine, was protective against obesity and insulin resistance, but clinical development was halted by the emergence of CNTF antibodies. The chimeric cytokine IC7 used the framework of interleukin (IL-)6 with the LIFR-binding site from CNTF to activate cells via IL-6R:gp130:LIFR complexes. Similar to CNTF/Axokine, IC7 protected mice from obesity and insulin resistance. Here, we developed CNTF-independent chimeras that specifically target the IL-6R:gp130:LIFR complex. In GIL-6 and GIO-6, we transferred the LIFR binding site from LIF or OSM to IL-6, respectively. While GIO-6 signals via gp130:IL-6R:LIFR and gp130:IL-6R:OSMR complexes, GIL-6 selectively activates the IL-6R:gp130:LIFR receptor complex. By re-evaluation of IC7 and CNTF, we discovered the Oncostatin M receptor (OSMR) as an alternative non-canonical high-affinity receptor leading to IL-6R:OSMR:gp130 and CNTFR:OSMR:gp130 receptor complexes, respectively. The discovery of OSMR as an alternative high-affinity receptor for IC7 and CNTF designates GIL-6 as the first truly selective IL-6R:gp130:LIFR cytokine, whereas GIO-6 is a CNTF-free alternative for IC7.

CD5: A New Partner for IL-6.

IL-6-STAT3 axis is known as a key factor of tumor progression. In this issue of Immunity, Yu and colleagues (2016) show that IL-6-binding CD5, rather than IL-6 receptor-α in B cells, amplifies STAT3 activation via JAK-STAT signaling to promote cancer.

CD5 Binds to Interleukin-6 and Induces a Feed-Forward Loop with the Transcription Factor STAT3 in B Cells to Promote Cancer.

The participation of a specific subset of B cells and how they are regulated in cancer is unclear. Here, we demonstrate that the proportion of CD5(+) relative to interleukin-6 receptor α (IL-6Rα)-expressing B cells was greatly increased in tumors. CD5(+) B cells responded to IL-6 in the absence of IL-6Rα. IL-6 directly bound to CD5, leading to activation of the transcription factor STAT3 via gp130 and its downstream kinase JAK2. STAT3 upregulated CD5 expression, thereby forming a feed-forward loop in the B cells. In mouse tumor models, CD5(+) but not CD5(-) B cells promoted tumor growth. CD5(+) B cells also showed activation of STAT3 in multiple types of human tumor tissues. Thus, our findings demonstrate a critical role of CD5(+) B cells in promoting cancer.

Mechanistic computational modeling of monospecific and bispecific antibodies targeting interleukin-6/8 receptors.

The spread of cancer from organ to organ (metastasis) is responsible for the vast majority of cancer deaths; however, most current anti-cancer drugs are designed to arrest or reverse tumor growth without directly addressing disease spread. It was recently discovered that tumor cell-secreted interleukin-6 (IL-6) and interleukin-8 (IL-8) synergize to enhance cancer metastasis in a cell-density dependent manner, and blockade of the IL-6 and IL-8 receptors (IL-6R and IL-8R) with a novel bispecific antibody, BS1, significantly reduced metastatic burden in multiple preclinical mouse models of cancer. Bispecific antibodies (BsAbs), which combine two different antigen-binding sites into one molecule, are a promising modality for drug development due to their enhanced avidity and dual targeting effects. However, while BsAbs have tremendous therapeutic potential, elucidating the mechanisms underlying their binding and inhibition will be critical for maximizing the efficacy of new BsAb treatments. Here, we describe a quantitative, computational model of the BS1 BsAb, exhibiting how modeling multivalent binding provides key insights into antibody affinity and avidity effects and can guide therapeutic design. We present detailed simulations of the monovalent and bivalent binding interactions between different antibody constructs and the IL-6 and IL-8 receptors to establish how antibody properties and system conditions impact the formation of binary (antibody-receptor) and ternary (receptor-antibody-receptor) complexes. Model results demonstrate how the balance of these complex types drives receptor inhibition, providing important and generalizable predictions for effective therapeutic design.

Treatment of type 2 diabetes with the designer cytokine IC7Fc.

The gp130 receptor cytokines IL-6 and CNTF improve metabolic homeostasis but have limited therapeutic use for the treatment of type 2 diabetes. Accordingly, we engineered the gp130 ligand IC7Fc, in which one gp130-binding site is removed from IL-6 and replaced with the LIF-receptor-binding site from CNTF, fused with the Fc domain of immunoglobulin G, creating a cytokine with CNTF-like, but IL-6-receptor-dependent, signalling. Here we show that IC7Fc improves glucose tolerance and hyperglycaemia and prevents weight gain and liver steatosis in mice. In addition, IC7Fc either increases, or prevents the loss of, skeletal muscle mass by activation of the transcriptional regulator YAP1. In human-cell-based assays, and in non-human primates, IC7Fc treatment results in no signs of inflammation or immunogenicity. Thus, IC7Fc is a realistic next-generation biological agent for the treatment of type 2 diabetes and muscle atrophy, disorders that are currently pandemic.

Effect of IL-6R blockade on plasma lipids and clinical outcomes among hospitalized patients with COVID-19 infection.

Plasma lipid levels are modulated by systemic infection and inflammation; it is unknown whether these changes reflect inflammatory responses or caused directly by pathogen presence. We explored the hypothesis that anti-inflammatory intervention via interleukin 6 receptor (IL-6R) blockade would influence plasma lipid levels during severe infection and evaluated the association of plasma lipid changes with clinical outcomes. Sarilumab (monoclonal antibody blocking IL-6R) efficacy was previously assessed in patients with coronavirus disease 2019 (COVID-19) (NCT04315298). This analysis determined whether strong inflammatory reduction by sarilumab in patients with COVID-19 pneumonia of increasing severity (severe, critical, multisystem organ dysfunction) affected plasma lipid changes between day 1 and day 7 of study therapy. Baseline lipid levels reflected the presence of acute systemic infection, characterized by very low HDL-C, low LDL-C, and moderately elevated triglycerides (TGs). Disease severity was associated with progressively more abnormal lipid levels. At day 7, median lipid levels increased more in the sarilumab versus placebo group (HDL-C +10.3%, LDL-C +54.7%, TG +32% vs. HDL-C +1.7%, LDL-C +15.4%, TG +8.8%, respectively). No significant association between lipid changes and clinical outcomes was observed. In conclusion, severe-to-critical COVID-19 pneumonia causes profound HDL-C depression that is only modestly responsive to strong anti-IL-6R inflammatory intervention. Conversely, LDL-C depression is strongly responsive to IL-6R blockade, with LDL-C levels likely returning to the predisease set point. These results advance our understanding of the complex relationship between serum lipids and infection/inflammation and suggest that HDL-C depression during acute contagious disease is driven by infection and not IL-6-mediated inflammation.

Bispecific soluble cytokine receptor-nanobody fusions inhibit Interleukin (IL-)6 trans-signaling and IL-12/23 or tumor necrosis factor (TNF) signaling.

At least 0.5% of people in the Western world develop inflammatory bowel disease (IBD). While antibodies that block tumor necrosis factor (TNF) α and Interleukin (IL-)23 have been approved for the treatment of IBD, IL-6 antibodies failed in the phase II clinical trial due to non-tolerable side effects. However, two clinical phase II studies suggest that inhibiting IL-6/soluble IL-6R (sIL-6R)-induced trans-signaling via the cytokine receptor gp130 benefit IBD patients with fewer adverse events. Here we develop inhibitors targeting a combination of IL-6/sIL-6R and TNF or IL-12/IL-23 signaling, named cs130-TNFVHHFc and cs130-IL-12/23VHHFc. Surface plasmon resonance experiments showed that recombinant cs130-TNFVHHFc and cs130-IL-12/23VHHFc bind with high affinity to IL-6/sIL-6R complexes and human TNFα (hTNFα) or IL-12/IL-23, respectively. Immunoprecipitation experiments have verified the higher ordered complex formation of the inhibitors with IL-6/sIL-6R and IL-12. We demonstrated that cs130-TNFVHHFc and cs130-IL-12/23VHHFc block IL-6/sIL-6R trans-signaling-induced proliferation and STAT3 phosphorylation of Ba/F3-gp130 cells, as well as hTNFα- or IL-23-induced signaling, respectively. In conclusion, cs130-TNFVHHFc and cs130-IL-12/23VHHFc represent a class of dimeric and bispecific chimeric cytokine inhibitors that consist of a soluble cytokine receptor fused to anti-cytokine nanobodies.