Th1 Cells Alter the Inflammatory Signature of IL-6 by Channeling STAT Transcription Factors to Alu-like Retroelements.

Cytokines that signal via STAT1 and STAT3 transcription factors instruct decisions affecting tissue homeostasis, antimicrobial host defense, and inflammation-induced tissue injury. To understand the coordination of these activities, we applied RNA sequencing, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing to identify the transcriptional output of STAT1 and STAT3 in peritoneal tissues from mice during acute resolving inflammation and inflammation primed to drive fibrosis. Bioinformatics focused on the transcriptional signature of the immunomodulatory cytokine IL-6 in both settings and examined how profibrotic IFN-γ-secreting CD4+ T cells altered the interpretation of STAT1 and STAT3 cytokine cues. In resolving inflammation, STAT1 and STAT3 cooperated to drive stromal gene expression affecting antimicrobial immunity and tissue homeostasis. The introduction of IFN-γ-secreting CD4+ T cells altered this transcriptional program and channeled STAT1 and STAT3 to a previously latent IFN-γ activation site motif in Alu-like elements. STAT1 and STAT3 binding to this conserved sequence revealed evidence of reciprocal cross-regulation and gene signatures relevant to pathophysiology. Thus, we propose that effector T cells retune the transcriptional output of IL-6 by shaping a regulatory interplay between STAT1 and STAT3 in inflammation.

Cross-talk between IL-6 trans-signaling and AIM2 inflammasome/IL-1ß axes bridge innate immunity and epithelial apoptosis to promote emphysema.

Pulmonary emphysema is associated with dysregulated innate immune responses that promote chronic pulmonary inflammation and alveolar apoptosis, culminating in lung destruction. However, the molecular regulators of innate immunity that promote emphysema are ill-defined. Here, we investigated whether innate immune inflammasome complexes, comprising the adaptor ASC, Caspase-1 and specific pattern recognition receptors (PRRs), promote the pathogenesis of emphysema. In the lungs of emphysematous patients, as well as spontaneous gp130F/F and cigarette smoke (CS)-induced mouse models of emphysema, the expression (messenger RNA and protein) and activation of ASC, Caspase-1, and the inflammasome-associated PRR and DNA sensor AIM2 were up-regulated. AIM2 up-regulation in emphysema coincided with the biased production of the mature downstream inflammasome effector cytokine IL-1ß but not IL-18. These observations were supported by the genetic blockade of ASC, AIM2, and the IL-1 receptor and therapy with AIM2 antagonistic suppressor oligonucleotides, which ameliorated emphysema in gp130F/F mice by preventing elevated alveolar cell apoptosis. The functional requirement for AIM2 in driving apoptosis in the lung epithelium was independent of its expression in hematopoietic-derived immune cells and the recruitment of infiltrating immune cells in the lung. Genetic and inhibitor-based blockade of AIM2 also protected CS-exposed mice from pulmonary alveolar cell apoptosis. Intriguingly, IL-6 trans-signaling via the soluble IL-6 receptor, facilitated by elevated levels of IL-6, acted upstream of the AIM2 inflammasome to augment AIM2 expression in emphysema. Collectively, we reveal cross-talk between the AIM2 inflammasome/IL-1ß and IL-6 trans-signaling axes for potential exploitation as a therapeutic strategy for emphysema.

Cytosolic DNA sensor AIM2 promotes KRAS-driven lung cancer independent of inflammasomes.

Constitutively active KRAS mutations are among the major drivers of lung cancer, yet the identity of molecular co-operators of oncogenic KRAS in the lung remains ill-defined. The innate immune cytosolic DNA sensor and pattern recognition receptor (PRR) Absent-in-melanoma 2 (AIM2) is best known for its assembly of multiprotein inflammasome complexes and promoting an inflammatory response. Here, we define a role for AIM2, independent of inflammasomes, in KRAS-addicted lung adenocarcinoma (LAC). In genetically defined and experimentally induced (nicotine-derived nitrosamine ketone; NNK) LAC mouse models harboring the KrasG12D driver mutation, AIM2 was highly upregulated compared with other cytosolic DNA sensors and inflammasome-associated PRRs. Genetic ablation of AIM2 in KrasG12D and NNK-induced LAC mouse models significantly reduced tumor growth, coincident with reduced cellular proliferation in the lung. Bone marrow chimeras suggest a requirement for AIM2 in KrasG12D-driven LAC in both hematopoietic (immune) and non-hematopoietic (epithelial) cellular compartments, which is supported by upregulated AIM2 expression in immune and epithelial cells of mutant KRAS lung tissues. Notably, protection against LAC in AIM2-deficient mice is associated with unaltered protein levels of mature Caspase-1 and IL-1ß inflammasome effectors. Moreover, genetic ablation of the key inflammasome adapter, ASC, did not suppress KrasG12D-driven LAC. In support of these in vivo findings, AIM2, but not mature Caspase-1, was upregulated in human LAC patient tumor biopsies. Collectively, our findings reveal that endogenous AIM2 plays a tumor-promoting role, independent of inflammasomes, in mutant KRAS-addicted LAC, and suggest innate immune DNA sensing may provide an avenue to explore new therapeutic strategies in lung cancer.

Interleukin-6 signaling drives fibrosis in unresolved inflammation.

Fibrosis in response to tissue damage or persistent inflammation is a pathological hallmark of many chronic degenerative diseases. By using a model of acute peritoneal inflammation, we have examined how repeated inflammatory activation promotes fibrotic tissue injury. In this context, fibrosis was strictly dependent on interleukin-6 (IL-6). Repeat inflammation induced IL-6-mediated T helper 1 (Th1) cell effector commitment and the emergence of STAT1 (signal transducer and activator of transcription-1) activity within the peritoneal membrane. Fibrosis was not observed in mice lacking interferon-γ (IFN-γ), STAT1, or RAG-1. Here, IFN-γ and STAT1 signaling disrupted the turnover of extracellular matrix by metalloproteases. Whereas IL-6-deficient mice resisted fibrosis, transfer of polarized Th1 cells or inhibition of MMP activity reversed this outcome. Thus, IL-6 causes compromised tissue repair by shifting acute inflammation into a more chronic profibrotic state through induction of Th1 cell responses as a consequence of recurrent inflammation.

ADAM17 Deficiency Protects against Pulmonary Emphysema.

Pulmonary emphysema is the major debilitating component of chronic obstructive pulmonary disease (COPD), which is a leading cause of morbidity and mortality worldwide. The ADAM17 (A disintegrin and metalloproteinase 17) protease mediates inflammation via ectodomain shedding of numerous proinflammatory cytokines, cytokine receptors, and adhesion molecules; however, its role in the pathogenesis of emphysema and COPD is poorly understood. This study aims to define the role of the protease ADAM17 in the pathogenesis of pulmonary emphysema. ADAM17 protein expression and activation was investigated in lung biopsies from patients with emphysema, as well as lungs of the emphysematous gp130F/F mouse model and an acute (4 d) cigarette smoke (CS)-induced lung pathology model. The Adam17ex/ex mice, which display significantly reduced global ADAM17 expression, were coupled with emphysema-prone gp130F/F mice to produce gp130F/F:Adam17ex/ex. Both Adam17ex/ex and wild-type mice were subjected to acute CS exposure. Histological, immunohistochemical, immunofluorescence, and molecular analyses as well as lung function tests were performed to assess pulmonary emphysema, inflammation, and alveolar cell apoptosis. ADAM17 was hyperphosphorylated in the lungs of patients with emphysema and also in emphysematous gp130F/F and CS-exposed mice. ADAM17 deficiency ameliorated the development of pulmonary emphysema in gp130F/F mice by suppressing elevated alveolar cell apoptosis. In addition, genetic blockade of ADAM17 protected mice from CS-induced pulmonary inflammation and alveolar cell apoptosis. Our study places the protease ADAM17 as a central molecular switch implicated in the development of pulmonary emphysema, which paves the way for using ADAM17 inhibitors as potential therapeutic agents to treat COPD and emphysema.

Constitutive STAT3 Serine Phosphorylation Promotes Helicobacter-Mediated Gastric Disease.

Gastric cancer is associated with chronic inflammation (gastritis) triggered by persistent Helicobacter pylori (H. pylori) infection. Elevated tyrosine phosphorylation of the latent transcription factor STAT3 is a feature of gastric cancer, including H. pylori-infected tissues, and aligns with nuclear transcriptional activity. However, the transcriptional role of STAT3 serine phosphorylation, which promotes STAT3-driven mitochondrial activities, is unclear. Here, by coupling serine-phosphorylated (pS)-STAT3-deficient Stat3SA/SA mice with chronic H. felis infection, which mimics human H. pylori infection in mice, we reveal a key role for pS-STAT3 in promoting Helicobacter-induced gastric pathology. Immunohistochemical staining for infiltrating immune cells and expression analyses of inflammatory genes revealed that gastritis was markedly suppressed in infected Stat3SA/SA mice compared with wild-type mice. Stomach weight and gastric mucosal thickness were also reduced in infected Stat3SA/SA mice, which was associated with reduced proliferative potential of infected Stat3SA/SA gastric mucosa. The suppressed H. felis-induced gastric phenotype of Stat3SA/SA mice was phenocopied upon genetic ablation of signaling by the cytokine IL-11, which promotes gastric tumorigenesis via STAT3. pS-STAT3 dependency by Helicobacter coincided with transcriptional activity on STAT3-regulated genes, rather than mitochondrial and metabolic genes. In the gastric mucosa of mice and patients with gastritis, pS-STAT3 was constitutively expressed irrespective of Helicobacter infection. Collectively, these findings suggest an obligate requirement for IL-11 signaling via constitutive pS-STAT3 in Helicobacter-induced gastric carcinogenesis.

The ADAM17 protease promotes tobacco smoke carcinogen-induced lung tumorigenesis.

Lung cancer is the leading cause of cancer-related mortality, with most cases attributed to tobacco smoking, in which nicotine-derived nitrosamine ketone (NNK) is the most potent lung carcinogen. The ADAM17 protease is responsible for the ectodomain shedding of many pro-tumorigenic cytokines, growth factors and receptors, and therefore is an attractive target in cancer. However, the role of ADAM17 in promoting tobacco smoke carcinogen-induced lung carcinogenesis is unknown. The hypomorphic Adam17ex/ex mice-characterized by reduced global ADAM17 expression-were backcrossed onto the NNK-sensitive pseudo-A/J background. CRISPR-driven and inhibitor-based (GW280264X, and ADAM17 prodomain) ADAM17 targeting was employed in the human lung adenocarcinoma cell lines A549 and NCI-H23. Human lung cancer biopsies were also used for analyses. The Adam17ex/ex mice displayed marked protection against NNK-induced lung adenocarcinoma. Specifically, the number and size of lung lesions in NNK-treated pseudo-A/J Adam17ex/ex mice were significantly reduced compared with wild-type littermate controls. This was associated with lower proliferative index throughout the lung epithelium. ADAM17 targeting in A549 and NCI-H23 cells led to reduced proliferative and colony-forming capacities. Notably, among select ADAM17 substrates, ADAM17 deficiency abrogated shedding of the soluble IL-6 receptor (sIL-6R), which coincided with the blockade of sIL-6R-mediated trans-signaling via ERK MAPK cascade. Furthermore, NNK upregulated phosphorylation of p38 MAPK, whose pharmacological inhibition suppressed ADAM17 threonine phosphorylation. Importantly, ADAM17 threonine phosphorylation was significantly upregulated in human lung adenocarcinoma with smoking history compared with their cancer-free controls. Our study identifies the ADAM17/sIL-6R/ERK MAPK axis as a candidate therapeutic strategy against tobacco smoke-associated lung carcinogenesis.

Hyperactive gp130/STAT3-driven gastric tumourigenesis promotes submucosal tertiary lymphoid structure development.

Tertiary lymphoid structures (TLSs) display phenotypic and functional characteristics of secondary lymphoid organs, and often develop in tissues affected by chronic inflammation, as well as in certain inflammation-associated cancers where they are prognostic of improved patient survival. However, the mechanisms that govern the development of tumour-associated TLSs remain ill-defined. Here, we observed tumour-associated TLSs in a preclinical mouse model (gp130F/F ) of gastric cancer, where tumourigenesis is dependent on hyperactive STAT3 signalling through the common IL-6 family signalling receptor, gp130. Gastric tumourigenesis was associated with the development of B and T cell-rich submucosal lymphoid aggregates, containing CD21+ cellular networks and high endothelial venules. Temporally, TLS formation coincided with the development of gastric adenomas and induction of homeostatic chemokines including Cxcl13, Ccl19 and Ccl21. Reflecting the requirement of gp130-driven STAT3 signalling for gastric tumourigenesis, submucosal TLS development was also STAT3-dependent, but independent of the cytokine IL-17 which has been linked with lymphoid neogenesis in chronic inflammation and autoimmunity. Interestingly, upregulated lymphoid chemokine expression and TLS formation were also observed in a chronic gastritis model induced by Helicobacter felis infection. Tumour-associated TLSs were also observed in patients with intestinal-type gastric cancer, and a gene signature linked with TLS development in gp130F/F mice was associated with advanced clinical disease, but was not prognostic of patient survival. Collectively, our in vivo data reveal that hyperactive gp130-STAT3 signalling closely links gastric tumourigenesis with lymphoid neogenesis, and while a TLS gene signature was associated with advanced gastric cancer in patients, it did not indicate a favourable prognosis.

Therapeutic Targeting of the IL-6 Trans-Signaling/Mechanistic Target of Rapamycin Complex 1 Axis in Pulmonary Emphysema.

RATIONALE: The potent immunomodulatory cytokine IL-6 is consistently up-regulated in human lungs with emphysema and in mouse emphysema models; however, the mechanisms by which IL-6 promotes emphysema remain obscure. IL-6 signals using two distinct modes: classical signaling via its membrane-bound IL-6 receptor (IL-6R), and trans-signaling via a naturally occurring soluble IL-6R. OBJECTIVES: To identify whether IL-6 trans-signaling and/or classical signaling contribute to the pathogenesis of emphysema. METHODS: We used the gp130F/F genetic mouse model for spontaneous emphysema and cigarette smoke-induced emphysema models. Emphysema in mice was quantified by various methods including in vivo lung function and stereology, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to assess alveolar cell apoptosis. In mouse and human lung tissues, the expression level and location of IL-6 signaling-related genes and proteins were measured, and the levels of IL-6 and related proteins in sera from emphysematous mice and patients were also assessed. MEASUREMENTS AND MAIN RESULTS: Lung tissues from patients with emphysema, and from spontaneous and cigarette smoke-induced emphysema mouse models, were characterized by excessive production of soluble IL-6R. Genetic blockade of IL-6 trans-signaling in emphysema mouse models and therapy with the IL-6 trans-signaling antagonist sgp130Fc ameliorated emphysema by suppressing augmented alveolar type II cell apoptosis. Furthermore, IL-6 trans-signaling-driven emphysematous changes in the lung correlated with mechanistic target of rapamycin complex 1 hyperactivation, and treatment of emphysema mouse models with the mechanistic target of rapamycin complex 1 inhibitor rapamycin attenuated emphysematous changes. CONCLUSIONS: Collectively, our data reveal that specific targeting of IL-6 trans-signaling may represent a novel treatment strategy for emphysema.

Nucleotide oligomerization domain 1 enhances IFN-γ signaling in gastric epithelial cells during Helicobacter pylori infection and exacerbates disease severity.

Virulent Helicobacter pylori strains that specifically activate signaling in epithelial cells via the innate immune molecule, nucleotide oligomerization domain 1 (NOD1), are more frequently associated with IFN-γ-dependent inflammation and with severe clinical outcomes (i.e., gastric cancer and peptic ulceration). In cell culture models, we showed that H. pylori activation of the NOD1 pathway caused enhanced proinflammatory signaling in epithelial cells in response to IFN-γ stimulation through the direct effects of H. pylori on two components of the IFN-γ signaling pathway, STAT1 and IFN regulatory factor 1 (IRF1). Specifically, H. pylori activation of the NOD1 pathway was shown to increase the levels of STAT1-Tyr(701)/Ser(727) phosphorylation and IRF1 expression/synthesis in cells, resulting in enhanced production of the NOD1- and IFN-γ-regulated chemokines, IL-8- and IFN-γ-induced protein 10, respectively. Consistent with the notion that heightened proinflammatory signaling in epithelial cells may have an impact on disease severity, we observed significantly increased expression levels of NOD1, CXCL8, IRF1, and CXCL10 in human gastric biopsies displaying severe gastritis, when compared with those without gastritis (p < 0.05, p < 0.001, p < 0.01, and p < 0.05, respectively). Interestingly, NOD1, CXCL8, and IRF1 expression levels were also significantly upregulated in gastric tumor tissues, when compared with paired nontumor samples (p < 0.0001, p < 0.05, and p < 0.05, respectively). Thus, we propose that cross-talk between NOD1 and IFN-γ signaling pathways contribute to H. pylori-induced inflammatory responses, potentially revealing a novel mechanism whereby virulent H. pylori strains promote more severe disease.

IL-6/Stat3-driven pulmonary inflammation, but not emphysema, is dependent on interleukin-17A in mice.

BACKGROUND AND OBJECTIVE: Pulmonary emphysema is linked to T cell-mediated autoimmune inflammation, although the pathogenic role of specific pro-inflammatory cytokines remains unclear. The Th17 type response, characterized by the production of the cytokine interleukin (IL)-17A, is modulated in part by the IL-6/signal transducer and activator of transcription (Stat)3 signalling axis and is associated with numerous autoimmune diseases. We therefore evaluated a causal role for IL-17A in the IL-6-driven gp130(F/F) mouse model for spontaneous pulmonary inflammation and emphysema. METHODS: The expression of Th17-related factors was quantified in the lungs of gp130(F/F) mice and emphysematous patients, and the degree of pulmonary inflammation and emphysema was measured in gp130(F/F) : Il17a-/- mice by immunohistochemistry, stereology and respiratory mechanics. RESULTS: In gp130(F/F) mice, lung gene expression of Il17a and other Th17-related factors was augmented compared with gp130+/+ (wild-type), gp130(F/F) : Il6-/- and gp130(F/F) : Stat3-/+ mice displaying normalized Stat3 activity and no lung inflammation. Importantly, genetic ablation of Il17a in gp130(F/F) : Il17a-/- mice prevented lung inflammation; however, emphysema still developed. Additionally, messenger RNA expression of inflammatory genes Cxcl1, Cxcl2, Ccl2 and Tnfα; as well as Il6 and the Stat3-target gene, Socs3, were upregulated in the lungs of gp130(F/F) mice compared with gp130(F/F) : Il17a-/- and gp130+/+ mice. Consistent with these findings, augmented IL17A expression was observed in emphysema patients presenting with inflammation compared with inflammation-free individuals. CONCLUSIONS: Collectively, our data suggest that the integration of IL-17A into the IL-6/Stat3 signalling axis mediates lung inflammation, but not emphysema, and that discrete targeting of IL-17A may alleviate pulmonary inflammatory-related diseases.

Deregulated Stat3 signaling dissociates pulmonary inflammation from emphysema in gp130 mutant mice.

Interleukin (IL)-6 is a potent immunomodulatory cytokine that is associated with emphysema, a major component of chronic obstructive pulmonary disease (COPD). IL-6 signaling via the gp130 coreceptor is coupled to multiple signaling pathways, especially the latent transcription factor signal transducer and activator of transcription (Stat)3. However, the pathological role of endogenous gp130-dependent Stat3 activation in emphysema is ill defined. To elucidate the role of the IL-6/gp130/Stat3 signaling axis in the cellular and molecular pathogenesis of emphysema, we employed a genetic complementation strategy using emphysematous gp130(F/F) mice displaying hyperactivation of endogenous Stat3 that were interbred with mice to impede Stat3 activity. Resected human lung tissue from patients with COPD and COPD-free individuals was also evaluated by immunohistochemistry. Genetic reduction of Stat3 hyperactivity in gp130(F/F):Stat3(-/+) mice prevented lung inflammation and excessive protease activity; however, emphysema still developed. In support of these findings, Stat3 activation levels in human lung tissue correlated with the extent of pulmonary inflammation but not airflow obstruction in COPD. Furthermore, COPD lung tissue displayed increased levels of IL-6 and apoptotic alveolar cells, supporting our previous observation that increased endogenous IL-6 expression in the lungs of gp130(F/F) mice contributes to emphysema by promoting alveolar cell apoptosis. Collectively, our data suggest that IL-6 promotes emphysema via upregulation of Stat3-independent apoptosis, whereas IL-6 induction of lung inflammation occurs via Stat3. We propose that while discrete targeting of Stat3 may alleviate pulmonary inflammation, global targeting of IL-6 potentially represents a therapeutically advantageous approach to combat COPD phenotypes where emphysema predominates.

The molecular pathogenesis of STAT3-driven gastric tumourigenesis in mice is independent of IL-17.

Chronic activation of the gastric mucosal adaptive immune response is a characteristic trait of gastric cancer. It has recently emerged that a new class of T helper (Th) cells, defined by their ability to produce interleukin (IL)-17A (Th17), is associated with a host of inflammatory responses, including gastritis. However, the role of these Th17 cells in the pathogenesis of gastric cancer is less clear. To formally address this, we employed gp130(F/F) mice, which spontaneously develop gastric inflammation-associated tumours akin to human intestinal-type gastric cancer. At the molecular level, these tumours demonstrate hyper-activation of the latent transcription factor signal transducer and activator of transcription (STAT)3 via the IL-6 cytokine family member, IL-11. In gp130(F/F) mice, the generation of Th17 cells, as well as the gastric expression of IL-17a and other Th17-related factors (Rorγt, IL-23), were augmented compared to wild-type gp130(+/+) mice. Consistent with a role for IL-6 and STAT3 in regulating IL-17A, increased Th17 generation and gastric expression of Th17-related factors in gp130(F/F) mice were reduced to wild-type levels in gp130(F/F) :Stat3(-/+) mice displaying normalized STAT3 activity, and also in gp130(F/F) :IL-6(-/-) mice. Importantly, genetic ablation of IL-17A in gp130(F/F) :IL-17a(-/-) mice did not suppress the initiation and growth of gastric tumours. Furthermore, IL-17A and RORC gene expression was strongly increased in human gastric biopsies from patients with gastritis, but not gastric cancer. Collectively, our data suggest that increased expression of Th17-related factors does not correlate with the molecular pathogenesis of gastric tumourigenesis.

Toll-like Receptor 9 Promotes Initiation of Gastric Tumorigenesis by Augmenting Inflammation and Cellular Proliferation.

BACKGROUND & AIMS: Gastric cancer (GC) is strongly linked with chronic gastritis after Helicobacter pylori infection. Toll-like receptors (TLRs) are key innate immune pathogenic sensors that mediate chronic inflammatory and oncogenic responses. Here, we investigated the role of TLR9 in the pathogenesis of GC, including Helicobacter infection. METHODS: TLR9 gene expression was profiled in gastric tissues from GC and gastritis patients and from the spontaneous gp130F/F GC mouse model and chronic H felis-infected wild-type (WT) mice. Gastric pathology was compared in gp130F/F and H felis infection models with or without genetic ablation of Tlr9. The impact of Tlr9 targeting on signaling cascades implicated in inflammation and tumorigenesis (eg, nuclear factor kappa B, extracellular signal-related kinase, and mitogen-activated protein kinase) was assessed in vivo. A direct growth-potentiating effect of TLR9 ligand stimulation on human GC cell lines and gp130F/F primary gastric epithelial cells was also evaluated. RESULTS: TLR9 expression was up-regulated in Helicobacter-infected gastric tissues from GC and gastritis patients and gp130F/F and H felis-infected WT mice. Tlr9 ablation suppressed initiation of tumorigenesis in gp130F/F:Tlr9-/- mice by abrogating gastric inflammation and cellular proliferation. Tlr9-/- mice were also protected against H felis-induced gastric inflammation and hyperplasia. The suppressed gastric pathology upon Tlr9 ablation in both mouse models associated with attenuated nuclear factor kappa B and, to a lesser extent, extracellular signal-related kinase, mitogen-activated protein kinase signaling. TLR9 ligand stimulation of human GC cells and gp130F/F GECs augmented their proliferation and viability. CONCLUSIONS: Our data reveal that TLR9 promotes the initiating stages of GC and facilitates Helicobacter-induced gastric inflammation and hyperplasia, thus providing in vivo evidence for TLR9 as a candidate therapeutic target in GC.

Oncogenic dependency on STAT3 serine phosphorylation in KRAS mutant lung cancer.

The oncogenic potential of the latent transcription factor signal transducer and activator of transcription (STAT)3 in many human cancers, including lung cancer, has been largely attributed to its nuclear activity as a tyrosine-phosphorylated (pY705 site) transcription factor. By contrast, an alternate mitochondrial pool of serine phosphorylated (pS727 site) STAT3 has been shown to promote tumourigenesis by regulating metabolic processes, although this has been reported in only a restricted number of mutant RAS-addicted neoplasms. Therefore, the involvement of STAT3 serine phosphorylation in the pathogenesis of most cancer types, including mutant KRAS lung adenocarcinoma (LAC), is unknown. Here, we demonstrate that LAC is suppressed in oncogenic KrasG12D-driven mouse models engineered for pS727-STAT3 deficiency. The proliferative potential of the transformed KrasG12D lung epithelium, and mutant KRAS human LAC cells, was significantly reduced upon pS727-STAT3 deficiency. Notably, we uncover the multifaceted capacity of constitutive pS727-STAT3 to metabolically reprogramme LAC cells towards a hyper-proliferative state by regulating nuclear and mitochondrial (mt) gene transcription, the latter via the mtDNA transcription factor, TFAM. Collectively, our findings reveal an obligate requirement for the transcriptional activity of pS727-STAT3 in mutant KRAS-driven LAC with potential to guide future therapeutic targeting approaches.

Interleukin-6 promotes pulmonary emphysema associated with apoptosis in mice.

The IL-6 cytokine family, which signals via the shared gp130 coreceptor, is linked with the pathogenesis of emphysema. However, the definitive mechanisms by which these cytokines cause emphysema remain ill-defined. We took an in vivo genetic complementation approach to identify the specific IL-6 cytokine family members and gp130-regulated cellular processes that cause emphysema. We used gp130(F/F) mice homozygous for a subtle knock-in mutation in gp130 that deregulates intracellular signaling by the IL-6 cytokine family. The gp130(F/F) mice spontaneously develop emphysema by age 6 months. Within the IL-6 cytokine family, only IL-6 was significantly up-regulated in the lungs of gp130(F/F) mice, and the genetic targeting of IL-6 in gp130(F/F) mice (gp130(F/F):IL-6(-/-)) prevented emphysema. By contrast, the genetic ablation of receptor signaling via IL-11, which like IL-6 signals via a gp130 homodimer and uses the same signaling machinery, failed to ameliorate emphysema in gp130(F/F) mice. Among the disease-associated processes examined, emphysema strongly correlated with elevated alveolar cell apoptosis. Acute (4-day) exposure to cigarette smoke (CS) further augmented the expression of IL-6 in lungs of gp130(F/F) mice, and subchronic (6-week) exposure to CS exacerbated emphysematous and apoptotic changes in the lungs of gp130(F/F) but not gp130(F/F): IL-6(-/-) mice. IL-6 is the main causative agent of IL-6 cytokine family-induced emphysema, and operates to induce apoptosis in the lung. We propose that the discrete targeting of IL-6 signaling may provide an effective therapeutic strategy against human lung disease.

TLR2 activation promotes tumour growth and associates with patient survival and chemotherapy response in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis, and is plagued by a paucity of targeted treatment options and tumour resistance to chemotherapeutics. The causal link between chronic inflammation and PDAC suggests that molecular regulators of the immune system promote disease pathogenesis and/or therapeutic resistance, yet their identity is unclear. Here, we couple endoscopic ultrasound-guided fine-needle aspiration, which captures tumour biopsies from all stages, with whole transcriptome profiling of PDAC patient primary tumours to reveal enrichment of the innate immune Toll-like receptor 2 (TLR2) molecular pathway. Augmented TLR2 expression associated with a 4-gene "TLR2 activation" signature, and was prognostic for survival and predictive for gemcitabine-based chemoresistance. Furthermore, antibody-mediated anti-TLR2 therapy suppressed the growth of human PDAC tumour xenografts, independent of a functional immune system. Our results support TLR2-based therapeutic targeting for precision medicine in PDAC, with further clinical utility that TLR2 activation is prognostic and predictive for chemoresponsiveness.

IL-6 regulates neutrophil trafficking during acute inflammation via STAT3.

The successful resolution of inflammation is dependent upon the coordinated transition from the initial recruitment of neutrophils to a more sustained population of mononuclear cells. IL-6, which signals via the common receptor subunit gp130, represents a crucial checkpoint regulator of neutrophil trafficking during the inflammatory response by orchestrating chemokine production and leukocyte apoptosis. However, the relative contribution of specific IL-6-dependent signaling pathways to these processes remains unresolved. To define the receptor-mediated signaling events responsible for IL-6-driven neutrophil trafficking, we used a series of gp130 knockin mutant mice displaying altered IL-6-signaling capacities in an experimental model of acute peritoneal inflammation. Hyperactivation of STAT1 and STAT3 in gp130(Y757F/Y757F) mice led to a more rapid clearance of neutrophils, and this coincided with a pronounced down-modulation in production of the neutrophil-attracting chemokine CXCL1/KC. By contrast, the proportion of apoptotic neutrophils in the inflammatory infiltrate remained unaffected. In gp130(Y757F/Y757F) mice lacking IL-6, neutrophil trafficking and CXCL1/KC levels were normal, and this corresponded with a reduction in the level of STAT1/3 activity. Furthermore, monoallelic ablation of Stat3 in gp130(Y757F/Y757F) mice specifically reduced STAT3 activity and corrected both the rapid clearance of neutrophils and impaired CXCL1/KC production. Conversely, genetic deletion of Stat1 in gp130(Y757F/Y757F) mice failed to rescue the altered responses observed in gp130(Y757F/Y757F) mice. Collectively, these data genetically define that IL-6-driven signaling via STAT3, but not STAT1, limits the inflammatory recruitment of neutrophils, and therefore represents a critical event for the termination of the innate immune response.

ADAM17 selectively activates the IL-6 trans-signaling/ERK MAPK axis in KRAS-addicted lung cancer.

Oncogenic KRAS mutations are major drivers of lung adenocarcinoma (LAC), yet the direct therapeutic targeting of KRAS has been problematic. Here, we reveal an obligate requirement by oncogenic KRAS for the ADAM17 protease in LAC In genetically engineered and xenograft (human cell line and patient-derived) KrasG12D-driven LAC models, the specific blockade of ADAM17, including with a non-toxic prodomain inhibitor, suppressed tumor burden by reducing cellular proliferation. The pro-tumorigenic activity of ADAM17 was dependent upon its threonine phosphorylation by p38 MAPK, along with the preferential shedding of the ADAM17 substrate, IL-6R, to release soluble IL-6R that drives IL-6 trans-signaling via the ERK1/2 MAPK pathway. The requirement for ADAM17 in KrasG12D-driven LAC was independent of bone marrow-derived immune cells. Furthermore, in KRAS mutant human LAC, there was a significant positive correlation between augmented phospho-ADAM17 levels, observed primarily in epithelial rather than immune cells, and activation of ERK and p38 MAPK pathways. Collectively, these findings identify ADAM17 as a druggable target for oncogenic KRAS-driven LAC and provide the rationale to employ ADAM17-based therapeutic strategies for targeting KRAS mutant cancers.

IL6 Trans-signaling Promotes KRAS-Driven Lung Carcinogenesis.

Oncogenic KRAS mutations occur frequently in lung adenocarcinoma. The signaling pathways activated by IL6 promote Kras-driven lung tumorigenesis, but the basis for this cooperation is uncertain. In this study, we used the gp130(F/F) (Il6st) knock-in mouse model to examine the pathogenic contribution of hyperactivation of the STAT3 arm of IL6 signaling on KRAS-driven lung tumorigenesis. Malignant growths in the gp130(F/F):Kras(G12D) model displayed features of atypical adenomatous hyperplasia, adenocarcinoma in situ, and invasive adenocarcinoma throughout the lung, as compared with parental Kras(G12D) mice, where STAT3 was not hyperactivated. Among IL6 family cytokines, only IL6 was upregulated in the lung. Accordingly, normalization of pulmonary STAT3 activity, by genetic ablation of either Il6 or Stat3, suppressed the extent of lung cancer in the model. Mechanistic investigations revealed elevation in the lung of soluble IL6 receptor (sIL6R), the key driver of IL6 trans-signaling, and blocking this mechanism via interventions with an anti-IL6R antibody or the inhibitor sgp130Fc ameliorated lung cancer pathogenesis. Clinically, expression of IL6 and sIL6R was increased significantly in human specimens of lung adenocarcinoma or patient serum. Our results offer a preclinical rationale to clinically evaluate IL6 trans-signaling as a therapeutic target for the treatment of KRAS-driven lung adenocarcinoma.