Blockade Of Interleukin-6 Signaling Research Articles

Blockade of IL-6 signaling alleviates atherosclerosis in Tet2-deficient clonal hematopoiesis.

Clonal hematopoiesis (CH) increases the risk of atherosclerotic cardiovascular disease possibly due to increased plaque inflammation. Human studies suggest that limitation of interleukin-6 (IL-6) signaling could be beneficial in people with large CH clones, particularly in TET2 CH. Here we show that IL-6 receptor antibody treatment reverses the atherosclerosis promoted by Tet2 CH, with reduction of monocytosis, lesional macrophage burden and macrophage colony-stimulating factor 1 receptor (CSF1R) expression. IL-6 induces expression of Csf1r in Tet2-deficient macrophages through enhanced STAT3 binding to its promoter. In mouse and human Tet2-deficient macrophages, IL-6 increases CSF1R expression and enhances macrophage survival. Treatment with the CSF1R inhibitor PLX3397 reversed accelerated atherosclerosis in Tet2 CH mice. Our study demonstrates the causality of IL-6 signaling in Tet2 CH accelerated atherosclerosis, identifies IL-6-induced CSF1R expression as a critical mechanism and supports blockade of IL-6 signaling as a potential therapy for CH-driven cardiovascular disease.

Abstract CT565: Clinical and translational results of a phase I study, tocilizumab plus gemcitabine/nab-paclitaxel in patient with gemcitabine/nab-paclitaxel-refractory metastatic pancreatic cancer

Abstract Background: Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway inhibition may overcome chemoresistance of metastatic pancreatic cancer (MPC) refractory to gemcitabine + nab-paclitaxel (GN) due to suppression of cancer associated fibroblast (CAF) and increase of drug infiltration in tumor. We sought to determine the safety, recommended dose of tocilizumab (TCZ), an IL-6 receptor monoclonal antibody, and biological correlates of tumor shrinkage in patients with GN-refractory MPC. Methods: This phase 1, dose finding trial was conducted following preclinical study of IL-6 signaling blockade in genetically engineered mouse model of pancreatic cancer (pGEMM) and patients-derived CAF, enrolled 10 patients with MPC that had progressed after GN. During the dose-finding part to determine dose-limiting toxicity (DLT), patients initially received TCZ 8 mg/kg on Day 1 and nab-paclitaxel 100 mg/m2 + gemcitabine 750 mg/m2 on Days 2, 9, and 16. The subsequent dose-expansion part used the TCZ dose identified in the dose-finding study. Before Cycle 1 and at the end of Cycle 1, biopsy of liver metastasis was performed 3 to 5 hours after levofloxacin (LVXF) administration to measure LVFX infiltration in tumor using matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI). Results: In pGEMM tumor, mouse IL-6 receptor antibody plus GN led pathological response. The growth of CAFs from patients with pancreatic cancer was inhibited by TCZ. In this phase I study, no DLTs occurred and the recommended dosage of TCZ was determined to be 8 mg/kg. Treatment-emergent adverse events (TEAEs) occurred in 80% of patients (Grade ≥3 in 60%) with decreased neutrophil count (n=5, 7 events) being the most common TEAE. Disease control rate was 80·0% (95%CI: 44·4-97·5) and tumor reduction during cycle 1 was observed in 4 patients who were defined as responder. In paired-biopsied samples, decrease of phosphorylated (p) STAT3 expression in tumor was observed in 7 of 8 patients (88%). Responder-related biological activities were increase of cleaved PARP expression of tumor nuclei (P = 0.01), decrease of proliferative CAF (P = 0.08), and increase of LVFX infiltration in tumor (P = 0.04). Decrease of pSTAT3 expression (P = 0.02) was favor to increase of LVFX infiltration against increase of gamma-H2AX, an index of gemcitabine exposure (P = 0.20). Conclusion: TCZ+GN-rechallenge had a manageable safety profile and showed preliminary activity via inhibition of CAF and gain of intratumoral drug infiltration in MPC. Citation Format: Shuichi Mitsunaga, Masafumi Ikeda, Kazunori Aoki, Kyoko Yamaguchi, Noriaki Sawada, Etsuko Fujii, Masanobu Nishidate, Takashi Fujitomo, Hideaki Mizuno, Yoko Kayukawa, Atsuhiko Kato, Mayu Makikawa, Hiroshi Imaoka, Mitsuhito Sasaki, Kazuo Watanabe, Hiroyuki Tsunoda, Kimio Terao, Atsushi Ochiai. Clinical and translational results of a phase I study, tocilizumab plus gemcitabine/nab-paclitaxel in patient with gemcitabine/nab-paclitaxel-refractory metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT565.

A Novel Humanized Anti-Interleukin-6 Antibody HZ0408b With Anti-Rheumatoid Arthritis Therapeutic Potential.

Interleukin-6 (IL-6), a pleiotropic cytokine that regulates immune responses and inflammatory reactions, plays a pivotal role in the development of rheumatoid arthritis (RA). Blockade of IL-6 signaling with the monoclonal antibody (mAb) represents an important advancement in RA treatment. Although two IL-6 receptor antibodies are already available in the clinic, there is no mAb specifically targeting the human IL-6 to block IL-6 signaling for RA treatment. In this study, we have developed a novel humanized anti-IL-6 mAb HZ-0408b with potent binding and neutralizing activity to human IL-6. We demonstrated that HZ-0408b has a high species specificity and low cross-reactivity. Moreover, HZ-0408b showed a more potent inhibitory effect on IL-6 signaling than Siltuximab, an FDA-approved anti-IL-6 chimeric mAb. HZ-0408b is comparable to Olokizumab, a humanized mAb against IL-6 that is already in phase III studies. We observed that HZ-0408b is well tolerated at doses that can achieve therapeutic serum levels in cynomolgus monkey. Most importantly, we proved that HZ-0408b treatment significantly ameliorated joint swelling after the onset of arthritis and dramatically reduced plasma C-reactive protein (CRP) levels in a monkey collagen-induced arthritis (CIA) model. Collectively, our findings using non-human primates indicate that humanized anti-IL-6 mAb HZ-0408b has excellent safety and efficacy profiles for RA therapy.

Tocilizumab As a Novel Immunomodulatory Regimen for Hemophilia Gene Therapy

Hemophilia is an X-linked bleeding disorder typically resulting in deficiency of factor VIII (FVIII) or factor IX (FIX) due to mutations in the F8 or F9 genes, respectively. Data from clinical trials have shown that the investigational delivery of functional F8 or F9 gene by recombinant adeno-associated virus (AAV) to hepatocytes can substantially eliminate the need for infusions of clotting factor in hemophilia patients. Despite major advances, the durability and redosing of these investigational gene therapies have been limited by the host immune response against the AAV capsid (Verdera et al. Mol Ther 2020). Currently, an oral corticosteroid, prednisone, is commonly used to prevent cytotoxic T cells from killing AAV-transduced hepatocytes and to sustain the production of transgenic clotting factor. However, in some instances, transgene expression was lost despite prednisone administration and prolonged use of prednisone can be associated with adverse effects. Previously, we demonstrated that the anti-capsid humoral response depends on interleukin 6 (IL-6) secretion from human monocyte-related dendritic cells (Kuranda et al. JCI 2018). IL-6 signaling in response to AAV was also observed in human non-parenchymal liver cells in vitro, animal gene transfer models and AAV-based gene therapy trial for hemophilia B (Konkle et al. Blood 2021).Here, we investigated the effects of the IL-6 signaling blockade as a possible targeted approach to modulate AAV vector immunogenicity in hemophilia gene therapy using a non-human primate (NHP) model. Spk100 AAV capsid was used to deliver the human F9 gene. To prevent IL-6 signaling, we used a monoclonal antibody, tocilizumab (TCZ), which blocks the IL-6 receptor. TCZ is currently approved for use in several forms of arthritis and cytokine release syndrome. Ten male cynomolgus monkeys received an intravenous injection of Spk100-hFIX vector (4x10 12 vg/kg) and 5 of those animals received a single dose of TCZ (8 mg/kg) the day prior to vector administration and were monitored for 13 weeks.As assessed by an array of clinical and anatomic pathology parameters, the investigational use of gene therapy combined with prophylactic TCZ administration was safe and well-tolerated. TCZ did not interfere with vector biodistribution, liver transduction or the transgenic FIX production. Spk100-hFIX alone modestly increased IL-6 secretion from NHP peripheral blood mononuclear cells (PBMC) in vitro but, following the vector infusion in animals, plasma IL-6 levels did not change significantly. Overall, cytokine secretion in response to Spk100 capsid and hFIX protein was lower in PBMCs isolated from TCZ-treated animals compared to cells obtained from control animals. As expected, animals administered Spk100-hFIX alone developed anti-capsid antibodies. The administration of TCZ was associated with a lower level of anti-Spk100 IgM, IgG and neutralizing antibodies (NAb). While the blockade of IL-6 signaling was effective for about for 14 days post-vector infusion, the lower level of anti-Spk100 antibodies persisted for the entire study duration. In the TCZ group, 4 out of 5 animals had NAb titers equal to or below 1:10, theoretically compatible with vector readministration. In contrast, 4 out of 5 animals in the control group developed high titer NAbs following vector infusion. Importantly, TCZ reduced the detection of the capsid-specific TNFα-positive T cells that were observed in animals after vector infusion. Finally, livers from sacrificed animals were used to prepare liver non-parenchymal cells for ex vivo phenotyping. Compared to the control animals, liver-resident immune cells from the TCZ-treated group had increased basal IL-10 secretion, while liver sinusoidal endothelial cells (albumin-CD45-CD31+CD146+MHCII+) had lower surface levels of MHC class II molecules, suggesting an anti-inflammatory milieu in the TCZ-treated livers.Our results show that short-term prophylactic blockade of IL-6 signaling was safely used in NHPs and has the potential to reduce immune responses commonly observed post AAV vector infusion. These results support the continued investigation of tocilizumab as a targeted immunomodulatory regimen in liver gene therapy for hemophilia. DisclosuresKuranda: Spark Therapeutics: Current Employment. Weisshaar: Spark Therapeutics: Current Employment. Chen: Spark Therapeutics: Ended employment in the past 24 months. Smith: Spark Therapeutics: Current Employment. Beck: Spark Therapeutics: Current Employment. Kahle: Spark Therapeutics: Current Employment. Mingozzi: Spark Therapeutics: Current Employment.

New BBB Model Reveals That IL-6 Blockade Suppressed the BBB Disorder, Preventing Onset of NMOSD.

Background and ObjectivesTo evaluate the pathophysiology of neuromyelitis optica spectrum disorder (NMOSD) and the therapeutic mechanism and levels of interleukin-6 (IL-6) blockade (satralizumab), especially with respect to blood-brain barrier (BBB) disruption with the new in vitro and ex vivo human BBB models and in vivo model.MethodsWe constructed new static in vitro and flow-based ex vivo models for evaluating continued barrier function, leukocyte transmigration, and intracerebral transferability of neuromyelitis optica-immunoglobulin G (NMO-IgG) and satralizumab across the BBB using the newly established triple coculture system that are specialized to closely mimic endothelial cell contact of pericytes and endfeet of astrocytes. In the in vivo study, we assessed the effects of an anti–IL-6 receptor antibody for mice (MR16-1) on in vivo BBB disruption in mice with experimental autoimmune encephalomyelitis in which IL-6 concentration in the spinal cord dramatically increases.ResultsIn vitro and ex vivo experiments demonstrated that NMO-IgG increased intracerebral transferability of satralizumab and NMO-IgG and that satralizumab suppressed the NMO-IgG–induced transmigration of T cells and barrier dysfunction. In the in vivo study, the blockade of IL-6 signaling suppressed the migration of T cells into the spinal cord and prevented the increased BBB permeability.DiscussionThese results suggest that (1) our triple-cultured in vitro and in ex vivo BBB models are ideal for evaluating barrier function, leukocyte transmigration, and intracerebral transferability; (2) NMO-IgG increased the intracerebral transferability of NMO-IgG via decreasing barrier function and induced secretion of IL-6 from astrocytes causing more dysfunction of the barrier and disrupting controlled cellular infiltration; and (3) satralizumab, which can pass through the BBB in the presence of NMO-IgG, suppresses the BBB dysfunction and the infiltration of inflammatory cells, leading to prevention of onset of NMOSD.

Spinal interleukin-1β induces mechanical spinal hyperexcitability in rats: Interactions and redundancies with TNF and IL-6.

Both spinal tumor necrosis factor (TNF) and interleukin-6 (IL-6) contribute to the development of "mechanical" spinal hyperexcitability in inflammatory pain states. Recently, we found that spinal sensitization by TNF was significantly reduced by blockade of spinal IL-6 signaling suggesting that IL-6 signaling is involved in spinal TNF effects. Here, we explored whether spinal interleukin-1β (IL-1β), also implicated in inflammatory pain, induces "mechanical" spinal hyperexcitability, and whether spinal IL-1β effects are related to TNF and IL-6 effects. We recorded the responses of spinal cord neurons to mechanical stimulation of the knee joint in vivo and used cellular approaches on microglial and astroglial cell lines to identify interactions of IL-1β, TNF, and IL-6. Spinal application of IL-1β in anesthetized rats modestly enhanced responses of spinal cord neurons to innocuous and noxious mechanical joint stimulation. This effect was blocked by minocycline indicating microglia involvement, and significantly attenuated by interfering with IL-6 signaling. In the BV2 microglial cell line, IL-1β, like TNF, enhanced the release of soluble IL-6 receptor, necessary for spinal IL-6 actions. Different to TNF, IL-1β caused SNB-19 astrocytes to release interleukin-11. The generation of "mechanical" spinal hyperexcitability by IL-1β was more pronounced upon spinal TNF neutralization with etanercept, suggesting that concomitant TNF limits IL-1β effects. In BV2 cells, TNF stimulated the release of IL-1Ra, an endogenous IL-1β antagonist. Thus, spinal IL-1β has the potential to induce spinal hyperexcitability sharing with TNF dependency on IL-6 signaling, but TNF also limited IL-1β effects explaining the modest effect of IL-1β.

Current Evidence of Interleukin-6 Signaling Inhibitors in Patients With COVID-19: A Systematic Review and Meta-Analysis.

Background: Interleukin-6 (IL-6) is known to be detrimental in coronavirus disease 2019 (COVID-19) because of its involvement in driving cytokine storm. This systematic review and meta-analysis aimed to assess the safety and efficacy of anti-IL-6 signaling (anti-IL6/IL-6R/JAK) agents on COVID-19 based on the current evidence. Methods: Studies were identified through systematic searches of PubMed, EMBASE, ISI Web of Science, Cochrane library, ongoing clinical trial registries (clinicaltrials.gov), and preprint servers (medRxiv, ChinaXiv) on August 10, 2020, as well as eligibility checks according to predefined selection criteria. Statistical analysis was performed using Review Manager (version 5.3) and STATA 12.0. Results: Thirty-one studies were included in the pooled analysis of mortality, and 12 studies were identified for the analysis of risk of secondary infections. For mortality analysis, 5630 COVID-19 cases including 2,132 treated patients and 3,498 controls were analyzed. Anti-IL-6 signaling agents plus standard of care (SOC) significantly decreased the mortality rate compared to SOC alone (pooled OR = 0.61, 95% CI 0.45–0.84, p = 0.002). For the analysis of secondary infection risk, 1,624 patients with COVID-19 including 639 treated patients and 985 controls were included, showing that anti-IL-6 signaling agents did not increase the rate of secondary infections (pooled OR = 1.21, 95% CI 0.70–2.08, p = 0.50). By contrast, for patients with critical COVID-19 disease, anti-IL-6 signaling agents failed to reduce mortality compared to SOC alone (pooled OR = 0.75, 95% CI 0.42–1.33, p = 0.33), but they tended to increase the risk of secondary infections (pooled OR = 1.85, 95% CI 0.95–3.61, p = 0.07). A blockade of IL-6 signaling failed to reduce the mechanical ventilation rate, ICU admission rate, or elevate the clinical improvement rate. Conclusion: IL-6 signaling inhibitors reduced the mortality rate without increasing secondary infections in patients with COVID-19 based on current studies. For patients with critical disease, IL-6 signaling inhibitors did not exhibit any benefit.

Sarilumab monotherapy or in combination with non-methotrexate disease-modifying antirheumatic drugs in active rheumatoid arthritis: A Japan phase 3 trial (HARUKA)

Objectives: To determine long-term safety and efficacy of sarilumab as monotherapy or with non-methotrexate (MTX) conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) in Japanese patients with active rheumatoid arthritis (RA).Methods: In this double-blind, randomized study (NCT02373202), patients received subcutaneous sarilumab 150 mg q2w (S150) or 200 mg q2w (S200) as monotherapy or with non-MTX csDMARDs for 52 weeks. The primary endpoint was safety.Results: Sixty-one patients received monotherapy (S150, n = 30; S200, n = 31) and 30 received combination therapy (S150 + csDMARDs, n = 15; S200 + csDMARDs, n = 15). Rates of treatment-emergent adverse events (TEAEs) were 83.3%/90.3%/93.3%/86.7% for S150/S200/S150 + csDMARDs/S200 + csDMARDs, respectively. Nasopharyngitis and neutropenia were the most frequently reported TEAEs. One serious infection was reported in each monotherapy group and in the S200 + csDMARDs group. There were no cases of grade 4 neutropenia; no patients with grade 3 neutropenia experienced associated serious infection. Improvements in ACR20/50/70 response rates were generally similar between the two monotherapy groups and between the two combination groups; improvements in physical function (Health Assessment Questionnaire-Disability Index, HAQ-DI) and DAS28-CRP were observed at weeks 24 and 52 (all groups).Conclusion: The safety profile of sarilumab was consistent with known class effects of interleukin-6 signaling blockade therapeutics. Sarilumab as mono- or combination therapy improved clinical signs/symptoms and physical function in Japanese RA patients.

Immunogenicity of Sarilumab Monotherapy in Patients with Rheumatoid Arthritis Who Were Inadequate Responders or Intolerant to Disease-Modifying Antirheumatic Drugs.

IntroductionThis open-label study evaluated the immunogenicity, safety, and efficacy of sarilumab monotherapy in patients with active, moderate-to-severe rheumatoid arthritis (RA) and inadequate response or intolerance to prior conventional synthetic disease-modifying antirheumatic drugs.MethodsAdults with RA (n = 132) were randomized to receive subcutaneous sarilumab (150 [n = 65] or 200 mg [n = 67]) every 2 weeks (q2w) for 24 weeks. Endpoints included incidence of antidrug antibodies (ADAs) at week 24, safety, and efficacy.ResultsPersistent ADAs occurred in eight patients (12.3%) receiving sarilumab 150 mg q2w, seven of whom (10.8%) had neutralizing antibodies (NAbs), and in four patients (6.1%) receiving sarilumab 200 mg q2w, two of whom (3.0%) had NAbs; all exhibited low antibody titers. Infections and neutropenia were the most common adverse events (AEs). There were three serious AEs, no reports of anaphylaxis, and few hypersensitivity reactions (e.g., rash) with no notable differences in hypersensitivity reactions in ADA-positive patients relative to ADA-negative patients. Changes in absolute neutrophil count, alanine aminotransferase level, and platelet count were consistent with interleukin-6 signaling blockade and in agreement with previous observations. At week 24, overall American College of Rheumatology 20%/50%/70% improvement criteria responses were 73.8%/53.8%/29.2%, respectively, with sarilumab 150 mg q2w and 71.6%/50.7%/29.9% with sarilumab 200 mg q2w. No patients with an ADA-positive response showed loss of efficacy.ConclusionsADA titers were low and persistent ADAs and NAbs occurred relatively infrequently in both sarilumab dose groups. ADA did not meaningfully impact the safety or efficacy of either dose of sarilumab over 24 weeks.Trial RegistrationClinicalTrials.gov, identifier NCT02121210.FundingSanofi Genzyme and Regeneron Pharmaceuticals, Inc.Plain Language SummaryPlain language summary available for this article.Electronic Supplementary MaterialThe online version of this article (10.1007/s40744-019-0157-3) contains supplementary material, which is available to authorized users.

Interleukin 6 Signaling Blockade Exacerbates Acute and Late Injury From Focal Intestinal Irradiation

To evaluate the acute changes in leukocyte populations after focal irradiation and to assess the role of interleukin 6 (IL-6) in acute and late radiation injury. Mice were surgically implanted with a radiopaque marker on the surface of the small intestine. Mice were then imaged with cone beam computed tomography to locate the marker and irradiated with 18Gy of 5×5mm collimated x-rays onto the marked intestine using the Small Animal Radiation Research Platform. Intestinal sections and blood were harvested 1, 3.5, 7, and 14days and 2months postirradiation (post-IR) for histology and complete blood count, respectively. Immune cell populations were assessed by immunofluorescence in the acute phase. Collagen deposition was assessed 2months post-IR. IL-6-/- intestinal sections were assessed post-IR for morphology, EdU, Ki67, and TUNEL in comparison to IL-6+/+ mice. Furthermore, a set of IL-6+/+ mice were treated with anti-IL-6R to assess the role of IL-6 in late intestinal injury. Intestinal radiation damage peaked 14days post-IR, and fibrosis had developed by 60days post-IR. There was a marked infiltration of immune cells into the irradiated intestine, with increased neutrophils, macrophages, B-cells, and CD4+ T cells maintained from 3.5 to 14days post-IR. CD8+ T cells were decreased from days 7 to 14 post-IR. Systemically, leukocytes were increased in the peripheral blood 14days post-IR with anemia being maintained from 14days to 2months. IL-6 was significantly increased in the serum post-IR. IL-6-/- mice demonstrated worsened intestinal injury acutely post-IR. Moreover, anti-IL-6R-treated mice presented with worsened intestinal fibrosis 2months post-IR. Focal irradiation of the intestine produced a significant increase in immune cells in the irradiated area and systemic inflammation and anemia. Blockade of IL-6 signaling was found to exacerbate acute intestinal injury and late intestinal injury after focal irradiation.

Simultaneous blockade of IL-6 and CCL5 signaling for synergistic inhibition of triple-negative breast cancer growth and metastasis

BackgroundMetastatic triple-negative breast cancer (TNBC) is a heterogeneous and incurable disease. Numerous studies have been conducted to seek molecular targets to treat TNBC effectively, but chemotherapy is still the main choice for patients with TNBC. We have previously presented evidence of the important roles of interleukin-6 (IL-6) and chemokine (C-C motif) ligand 5 (CCL5) in TNBC tumor growth and metastasis. These experiments highlighted the importance of the crosstalk between cancer cells and stromal lymphatic endothelial cells (LECs) in tumor growth and metastasis.MethodsWe examined the viability and migration of MDA-MB-231-LN, SUM149, and SUM159 cells co-cultured with LECs when treated with maraviroc (CCR5 inhibitor) and tocilizumab (anti-IL-6 receptor antibody). To assess the anti-tumor effects of the combination of these two drugs in an athymic nude mouse model, MDA-MB-231-LN cells were implanted in the mammary fat pad and maraviroc (8 mg/kg, orally daily) and cMR16-1 (murine surrogate of the anti-IL-6R antibody, 10 mg/kg, IP, 3 days a week) were administrated for 5 weeks and effects on tumor growth and thoracic metastasis were measured.ResultsIn this study, we used maraviroc and tocilizumab to confirm that IL-6 and CCL5 signaling are key pathways promoting TNBC cell proliferation and migration. Further, in a xenograft mouse model, we showed that tumor growth was dramatically inhibited by cMR16-1, the mouse version of the anti-IL6R antibody. The combination of maraviroc and cMR16-1 caused significant reduction of TNBC tumor growth compared to the single agents. Significantly, the combination of maraviroc and cMR16-1 abrogated thoracic metastasis.ConclusionTaken together, these findings show that IL-6 and CCL5 signaling, which promote crosstalk between TNBC and lymphatic vessels, are key enhancers of TNBC tumor growth and metastasis. Furthermore, these results demonstrate that a drug combination inhibiting these pathways may be a promising therapy for TNBC patients.

Disease modifying actions of interleukin-6 blockade in a rat model of bone cancer pain.

Metastasis of cancer to the skeleton represents a debilitating turning point in the lives of patients. Skeletal metastasis leads to moderate to severe ongoing pain along with bone remodeling that can result in fracture, events that dramatically diminish quality of life. Interleukin-6 (IL-6) levels are elevated in patients with metastatic breast cancer and are associated with a lower survival rate. We therefore determined the consequences of inhibition of IL-6 signaling using a novel small molecule antagonist, TB-2-081, on bone integrity, tumor progression, and pain in a rodent model of breast cancer. Rat MAT B III mammary adenocarcinoma cells were injected and sealed within the tibia of female Fischer rats. Growth of these cells within the rat tibia elicited increased IL-6 levels both within the bone exudate and in the plasma, produced ongoing pain and evoked hypersensitivity, and bone fracture that was observed by approximately day 12. Systemic TB-2-081 delivered by subcutaneous osmotic minipumps starting at tumor implantation prevented tumor-induced ongoing bone pain and evoked hypersensitivity without altering tumor growth. Remarkably, TB-2-081 infusion significantly reduced osteolytic and osteoblastic bone remodeling and time to fracture likely by decreasing osteoclastogenesis and associated increase in bone resorption. These findings indicate that blockade of IL-6 signaling may represent a viable, disease-modifying strategy to prevent tumor-induced bone remodeling allowing for stabilization of bone and decreased fractures as well as diminished ongoing pain that may improve quality of life of patients with skeletal metastases. Notably, anti-IL-6 antibodies are clinically available allowing for rapid testing of these possibilities in humans.

ERK-dependent IL-6 autocrine signaling mediates adaptive resistance to pan-PI3K inhibitor BKM120 in head and neck squamous cell carcinoma.

Hyperactivation of phosphatidylinositol 3-kinase (PI3K) pathway occurs frequently in head and neck squamous cell carcinoma (HNSCC). However, clinical outcomes of targeting the PI3K pathway have been underwhelming. In present study, we investigated the resistant mechanisms and potential combination therapeutic strategy to overcome adaptive resistance to PI3K inhibitor in HNSCC. Treatment of NVP-BKM120, a pan-PI3K inhibitor, led to upregulation of interleukin-6 (IL-6) and subsequent activation of either extracellular signal-regulated kinase (ERK) or signal transducers and activators of transcription 3 (STAT3), causing modest antitumor effects on the growth of HNSCC cells. Blockade of autocrine IL-6 signaling with siRNA or neutralizing antibody for IL-6 receptor (IL-6R) completely abolished NVP-BKM120-induced activation of ERK and STAT3 as well as expression of c-Myc oncogene, which resulted in enhanced sensitivity to NVP-BKM120. Moreover, when compared with a pharmacologic inhibitor or silencing of STAT3, trametinib, a MEK inhibitor, in combination with NVP-BKM120 yielded more potent anti-proliferative effects by inhibiting S phase transition, arresting cells at G0/G1 phase, and downregulating IL-6 and c-Myc expression. Furthermore, as compared with either agent alone, combination of NVP-BKM120 with trametinib or tocilizumab, a humanized anti-IL-6R antibody, significantly suppressed tumor growth in NVP-BKM120-resistant patient-derived tumor xenograft (PDTX) models, which was also confirmed in PDTX-derived cell lines. Collectively, these results suggested that IL-6/ERK signaling is closely involved in adaptive resistance of NVP-BKM120 in HNSCC cells, providing a rationale for a novel combination therapy to overcome resistance to PI3K inhibitors.

CTLA4-Ig suppresses development of experimental autoimmune uveitis in the induction and effector phases: Comparison with blockade of interleukin-6

Recently, a number of biologics have been used in the treatment of autoimmune diseases. However, in the treatment of severe autoimmune uveitis, only TNF-alpha inhibitors are preferably used and the effect of other biologics such as interleukin-6 (IL-6) signaling blockade or cytotoxic T-lymphocyte antigen-4–immunoglobulin fusion protein (CTLA4-Ig) has not been well studied. Previously, we reported that IL-6 blockade effectively suppresses the development of experimental autoimmune uveitis (EAU), a mouse model for uveitis, by inhibiting Th17 cell development. In this study, we investigated the effect of CTLA4-Ig on EAU development and compared it with the effect of anti-IL-6 receptor monoclonal antibody (MR16-1). C57BL/6J mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) and treated once with CTLA4-Ig or MR16-1. Both CTLA4-Ig and MR16-1 administered in the induction phase (the same day as immunization) significantly reduced the clinical and histopathological scores of EAU. Fluorescence-activated cell sorting studies using draining lymph node (LN) cells from EAU mice 10 days after immunization showed that CTLA4-Ig can suppress early T-helper cell activation. CTLA4-Ig administered in the effector phase of the disease (one week after immunization), when IRBP-reactive T cells have been primed, also significantly reduced the clinical and histopathological scores of EAU. In contrast, MR16-1 administered in the effector phase did not ameliorate EAU. To investigate the differences between these biologics in the effector phase, in vitro restimulation analysis of LN cells obtained from EAU mice one week after immunization was performed and revealed that CTLA4-Ig, but not MR16-1, added to culture media could inhibit the proliferation of IRBP-specific CD4+ T cells which possessed capacities of producing IFN-gamma and/or IL-17. Collectively, CTLA4-Ig ameliorated EAU through preventing initial T-cell activation in the induction phase and suppressing proliferation of IRBP-specific T cells in the effector phase. Blockade of IL-6 signaling did not have such inhibitory effects after T-cell priming. CTLA4-Ig may have therapeutic effects on human chronic uveitis.

Upregulated interleukin-6 expression contributes to erlotinib resistance in head and neck squamous cell carcinoma

Despite the role of epidermal growth factor receptor (EGFR) signaling in head and neck squamous cell carcinoma (HNSCC) development and progression, clinical trials involving EGFR tyrosine kinase inhibitors (TKIs) have yielded poor results in HNSCC patients. Mechanisms of acquired resistance to the EGFR TKI erlotinib was investigated by developing erlotinib-resistant HNSCC cell lines and comparing their gene expression profiles with their parental erlotinib-sensitive HNSCC cell lines using microarray analyses and subsequent pathway and network analyses. Erlotinib-resistant HNSCC cells displayed a significant upregulation in immune response and inflammatory pathways compared to parental cells. Interleukin-6 (IL-6) was one of thirteen genes that was significantly differentially expressed in all erlotinib-resistant HNSCC cell lines, which was validated using RT-PCR and ELISA. Blockade of IL-6 signaling using the IL-6 receptor antagonist tocilizumab, was able to overcome erlotinib-resistance in erlotinib-resistant SQ20B tumors in vivo. Overall, erlotinib-resistant HNSCC cells display elevated IL-6 expression levels compared to erlotinib-sensitive HNSCC cells and blockade of the IL-6 signaling pathway may be an effective strategy to overcome resistance to erlotinib and possibly other EGFR TKIs for HNSCC therapy.

Treatment with anti-interleukin-6 receptor antibody ameliorates intestinal polyposis in Apc(Min/+) mice under high-fat diet conditions.

The prevalence of colorectal malignancies is increasing in the world. The parallel increase of metabolic syndrome gives a speculation between these two conditions, although the precise mechanism is still unclear. Interleukin-6 (IL-6) is a cytokine known to correlate with obesity and serve as a proinflammatory adipokine. In the present study, we investigated the effect of IL-6 signaling blockade on intestinal polyp formation in obesity using a mouse model of adenomatous polyposis coli (Apc). Male C57BL/6J-Apc(Min/+) mice were fed a high-fat diet from 5 weeks of age, and the overweight mice thus obtained were given a weekly intraperitoneal injection of anti-mouse IL-6 receptor antibody (MR16-1) from 6 to 15 weeks of age, while control mice received IgG or phosphate-buffered saline (PBS). The total number of intestinal polyps was significantly decreased in the MR16-1-injected group (53.1 ± 6.8) relative to the control groups (PBS-injected, 81.3 ± 6.1; rat IgG-injected, 74.7 ± 4.8, p = 0.01), and in particular the number of polyps larger than 2 mm in diameter was markedly decreased. In addition, the mean diameter of polyps in the MR16-1-injected group was significantly smaller than that in the control groups. On the other hand, no significant differences in body weight, epididymal fat pad mass, or the plasma levels of glucose, insulin and triglyceride were observed among the three groups. Thus, treatment with anti-IL-6 receptor antibody suppressed polyp growth in obese Apc(Min/+) mice fed the high-fat diet. We suggest that IL-6 signaling may be responsible for the obesity-associated colorectal tumorigenesis.

Tumor-Associated Macrophages Produce Interleukin 6 and Signal via STAT3 to Promote Expansion of Human Hepatocellular Carcinoma Stem Cells

Cancer stem cells (CSCs) can contribute to hepatocellular carcinoma (HCC) progression and recurrence after therapy. The presence of tumor-associated macrophages (TAMs) in patients with HCC is associated with poor outcomes. It is not clear whether TAMs interact with CSCs during HCC development. We investigated whether TAMs affect the activities of CSCs in the microenvironment of human HCCs. HCCs were collected from 17 patients during surgical resection and single-cell suspensions were analyzed by flow cytometry. CD14(+) TAMs were isolated from the HCC cell suspensions and placed into co-culture with HepG2 or Hep3B cells, and CSC functions were measured. The interleukin 6 (IL6) receptor was blocked with a monoclonal antibody (tocilizumab), and signal transducer and activator of transcription 3 was knocked down with small hairpin RNAs in HepG2 cells. Xenograft tumors were grown in NOD-SCID/Il2Rg(null) mice from human primary HCC cells or HepG2 cells. CD44(+) cells from human HCCs and cell lines formed more spheres in culture and more xenograft tumors in mice than CD44(-) cells, indicating that CD44(+) cells are CSCs. Incubation of the CD44(+) cells with TAMs promoted expansion of CD44(+) cells, and increased their sphere formation in culture and formation of xenograft tumors in mice. In human HCC samples, the numbers of TAMs correlated with the numbers of CD44(+) cells. Of all cytokines expressed by TAMs, IL6 was increased at the highest level in human HCC co-cultures, compared with TAMs not undergoing co-culture. IL6 was detected in the microenvironment of HCC samples and induced expansion of CD44(+) cells in culture. Levels of IL6 correlated with stages of human HCCs and detection of CSC markers. Incubation of HCC cell lines with tocilizumab or knockdown of signal transducer and activator of transcription 3 in HCC cells reduced the ability of TAMs to promote sphere formation by CD44+ cells in culture and growth of xenograft tumors in mice. CD44(+) cells isolated from human HCC tissues and cell lines have CSC activities in vitro and form a larger number of xenograft tumors in mice than CD44(-) cells. TAMs produce IL6, which promotes expansion of these CSCs and tumorigenesis. Levels of IL6 in human HCC samples correlate with tumor stage and markers of CSCs. Blockade of IL6 signaling with tocilizumab, a drug approved by the Food and Drug Administration for treatment of rheumatoid arthritis, inhibits TAM-stimulated activity of CD44(+) cells. This drug might be used to treat patients with HCC.

A mutually beneficial relationship between hepatocytes and cardiomyocytes mitigates doxorubicin-induced toxicity

Use of doxorubicin (DOX) is limited by its toxicity in multiple organs. However, the relationship between different organs in response to DOX-induced injury is not well understood. We found that partial hepatectomy correlated with increased DOX-induced heart injury in vivo while supernatant prepared from DOX-treated hepatocytes mitigated DOX-induced cytotoxicity of cardiomyocytes in vitro. Meanwhile, the supernatant of DOX-treated cardiomyocytes mitigated DOX-induced cytotoxicity of hepatocytes. Investigation of the molecular mechanisms underlying these effects found that interleukin 6 (IL-6) was significantly up-regulated in DOX-treated tissues and cells, and supernatant from IL-6 treated cells had a similar effect to that from DOX-treated cells. Although the concentration of secreted IL-6 in supernatant from DOX-treated cells did not significantly differ, blockade of IL-6 signaling, by overexpressing SOCS3, suppressed expression of the downstream molecules trefoil factor family 3 (TFF3) and hepatocyte growth factor (HGF), impaired the mutually beneficial relationship between hepatocytes and cardiomyocytes. In conclusion, our study shows that a mutually beneficial relationship exists between hepatocytes and cardiomyocytes during the acute injury induced by DOX. Moreover, it demonstrates that this phenomenon may be indirectly caused by increased IL-6 expression and the activation of the downstream molecular mediators TFF3 and HGF in hepatocytes and cardiomyocytes, respectively.

Loss of p53 Attenuates the Contribution of IL-6 Deletion on Suppressed Tumor Progression and Extended Survival in Kras-Driven Murine Lung Cancer

Interleukin-6 (IL-6) is involved in lung cancer tumorigenesis, tumor progression, metastasis, and drug resistance. Previous studies show that blockade of IL-6 signaling can inhibit tumor growth and increase drug sensitivity in mouse models. Clinical trials in non-small cell lung cancer (NSCLC) reveal that IL-6 targeted therapy relieves NSCLC-related anemia and cachexia, although other clinical effects require further study. We crossed IL-6 -/- mice with Kras G12D mutant mice, which develop lung tumors after activation of mutant Kras G12D, to investigate whether IL-6 inhibition contributes to tumor progression and survival time in vivo. Kras G12D; IL-6 -/- mice exhibited increased tumorigenesis, but slower tumor growth and longer survival, than Kras G12D mice. Further, in order to investigate whether IL-6 deletion contributes to suppression of lung cancer metastasis, we generated Kras G12D; p53 flox/flox; IL-6 -/- mice, which developed lung cancer with a trend for reduced metastases and longer survival than Kras G12D; p53 flox/flox mice. Tumors from Kras G12D; IL-6 -/- mice showed increased expression of TNFα and decreased expression of CCL-19, CCL-20 and phosphorylated STAT3 (pSTAT3) than Kras G12D mice; however, these changes were not present between tumors from Kras G12D; p53 flox/flox; IL-6 -/- and Kras G12D; p53 flox/flox mice. Upregulation of pSTAT3 and phosphorylated AKT (pAKT) were observed in Kras G12D tumors with p53 deletion. Taken together, these results indicate that IL-6 deletion accelerates tumorigenesis but delays tumor progression and prolongs survival time in a Kras-driven mouse model of lung cancer. However, these effects can be attenuated by p53 deletion.

212: Innate neutrophil function is regulated locally through IL-6 control of prostaglandins

Interleukin-6 (IL-6) is vital for competent anti-microbial host defense. Mice deficient in IL-6 showed an inability to appropriately control bacterial peritonitis (5 × 108 cfu Staphylococcus epidermidis) and displayed both increased dissemination and reduced bacterial clearance. This response was reversed through local reconstitution of IL-6 trans-signaling using a chimeric sIL-6R-IL-6 fusion protein (termed HDS). Administration of HDS (10–1000 ng/ml) to infected IL-6−/− mice promoted bacterial clearance and reduced bacteremia in a dose-dependent fashion. This response was associated with improved resolution of the acute phase response and a more rapid normalization of body temperature. To define the mechanisms governing IL-6 involvement in anti-microbial immunity, we first developed an ex vivo flow cytometric assay to simultaneously monitor neutrophil respiratory burst and phagocytosis. Bacterial stimulation of circulating blood neutrophils from IL-6−/− and IL-6R−/− mice showed comparable effector function to that displayed by WT controls. Thus, IL-6 deficiency is not associated with an inherent defect in neutrophil function. However, evaluation of infiltrating neutrophils isolated by lavage from the peritoneal cavity of infected IL-6−/− and IL-6R−/− mice showed significantly reduced respiratory burst and phagocytic activity. Interleukin-6 therefore controls responses local to the site of infection and is consistent with the activation of non-hematopoietic stromal cells by IL-6 and sIL-6R. In human peritoneal mesothelial cells and peritoneal membranes from infected mice, IL-6 trans-signaling promoted cyclooxygenase-2 (COX2) expression. Blockade of IL-6 signaling in infected WT mice with the antagonist sgp130 or anti-IL-6R antibodies inhibited peritoneal COX2 expression. Mass spectrometry for the COX2 products, prostaglandin-E2 (PGE2) prostaglandin-D2 (PGD2) in lavage samples from infected mice showed that the generation of PGD2, but not PGE2, was impaired in IL-6 deficiency. Here, PGD2 levels correlated with the number of infiltrating neutrophils undergoing respiratory burst. These studies suggest IL-6 signaling regulates the effector characteristics of infiltrating neutrophils through local control of immuno-regulatory prostaglandin production.