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[H.sub.2] has been clinically demonstrated to provide antioxidant and anti-inflammatory effects, which makes it an attractive agent in exercise medicine. Although exercise provides a multiplicity of benefits including decreased risk of disease, it can also have detrimental effects. For example, chronic high-intensity exercise in elite athletes, or sporadic bouts of exercise (i.e., noxious exercise) in untrained individuals, result in similar pathological factors such as inflammation, oxidation, and cellular damage that arise from and result in disease. Paradoxically, exercise-induced pro-inflammatory cytokines and reactive oxygen species largely mediate the benefits of exercise. Ingestion of conventional antioxidants and antiinflammatories often impairs exercise-induced training adaptations. Disease and noxious forms of exercise promote redox dysregulation and chronic inflammation, changes that are mitigated by [H.sub.2] administration. Beneficial exercise and [H.sub.2] administration promote cytoprotective hormesis, mitochondrial biogenesis, ATP production, increased [NAD.sup.+]/NADH ratio, cytoprotective phase II enzymes, heat-shock proteins, sirtuins, etc. We review the biomedical effects of exercise and those of [H.sub.2], and we propose that hydrogen may act as an exercise mimetic and redox adaptogen, potentiate the benefits from beneficial exercise, and reduce the harm from noxious exercise. However, more research is warranted to elucidate the potential ergogenic and therapeutic effects of [H.sub.2] in exercise medicine. Key words: reactive oxygen species, antioxidants, molecular hydrogen, free radicals, exercise, inflammation, redox dysregulation, anti-inflammatory, hormesis, mitochondria. Des donnees cliniques ont montre que l'[H.sub.2] a des effets antioxydants et anti-inflammatoires, ce qui en fait un agent attrayant en medecine de l'exercice physique. Bien que l'exercice physique offre une multitude de bienfaits, y compris une diminution du risque de maladie, il peut aussi avoir des effets deleteres. Par exemple, les effets de l'exercice physique de haute intensite de maniere prolongee chez les athletes d'elite, ou de pointes sporadiques d'exercice physique--c'est-a-dire de l'exercice physique nocif--chez des personnes non entrainees, se resument a des facteurs pathologiques similaires comme l'inflammation, l'oxydation et des dommages cellulaires, lesquels entrainent des maladies ou en sont le resultat. Paradoxalement, les cytokines pro-inflammatoires et les derives reactifs de l'oxygene produits pendant l'exercice physique contribuent largement a la mediation des bienfaits de l'exercice physique. En outre, l'ingestion d'antioxydants et d'anti-inflammatoires classiques nuit souvent aux modes d'action d'adaptation a l'entrainement mis en jeu pendant l'exercice physique. De fait, la maladie et les formes nocives d'exercice physique favorisent le dereglement redox et l'inflammation chronique, changements pourtant attenues par l'administration d'[H.sub.2]. Effectivement, l'exercice physique benefique et l'administration d'[H.sub.2] favorisent l'hormese cytoprotectrice, la biogenese mitochondriale, la production d'ATP, l'augmentation du ratio [NAD.sup.+]/NADH, les enzymes cytoprotecteurs de phase II, les proteines de choc thermique, les sirtuines, etc. En resume, nous offrons une synthese des effets biomedicaux de l'exercice physique et de ceux de l'[H.sub.2], et nous proposons que l'hydrogene puisse agir en tant que mimetique de l'exercice physique et d'adaptogene redox, potentialiser les bienfaits de l'exercice physique benefique et attenuer les dommages causes par l'exercice physique nocif. Il serait cependant justifie de proceder a plus de recherches en vue d'elucider les effets ergogenes et therapeutiques de l'[H.sub.2] en medecine de l'exercice physique. [Traduit par la Redaction] Mots-cles: derives reactifs de l'oxygene, antioxydants, hydrogene moleculaire, radicaux libres, exercice physique, inflammation, dereglement redox, anti-inflammatoire, hormese, mitochondrie., Introduction Hydrogen gas may have potential as an ergogenic and therapeutic molecule for sports athletes. The biomedical interest in molecular hydrogen has grown exponentially since the publication of a paper [...]

Author(s): Charlotte Cleasby [sup.1], Tim Marshall [sup.1] [sup.2], Anthony C. Gordon [sup.1], David B. Antcliffe [sup.1], Charlotte Cleasby, Tim Marshall, Josie K. Ward, Claire Bradley, Farah Al-Beidh, Anthony C. Gordon, [...]

Beclin 2 plays a critical role in metabolic regulation and obesity, but its functions in innate immune signaling and cancer development remain largely unknown. Here, we identified Beclin 2 as a critical negative regulator of inflammation and lymphoma development. Mice with homozygous ablation of BCL2-interacting protein 2 (Becn2) developed splenomegaly and lymphadenopathy and markedly increased ERK1/2 and NF-[kappa]B signaling for proinflammatory cytokine production. Beclin 2 targeted the key signaling kinases MEKK3 and TAK1 for degradation through an ATG9A-dependent, but ATG16L/ Beclin 1/LC3-independent, autophagic pathway. Mechanistically, Beclin 2 recruited MEKK3 or TAK1 through ATG9A to form a complex (Beclin 2-ATG9A-MEKK3) on [ATG9A.sup.+] vesicles upon ULK1 activation. Beclin 2 further interacted with STX5 and STX6 to promote the fusion of MEKK3- or TAK1-associated [ATG9A.sup.+] vesicles to phagophores for subsequent degradation. Importantly, Becn2-deficient mice had a markedly increased incidence of lymphoma development, with persistent STAT3 activation. Myeloid-specific ablation of MEKK3 (Map3k3) completely rescued the phenotypes (splenomegaly, higher amounts of proinflammatory cytokines, and cancer incidence) of Becn2-deficient mice. Hence, our findings have identified an important role of Beclin 2 in the negative regulation of innate immune signaling and tumor development through an ATG9Adependent, but ATG16L/Beclin 1/LC3-independent, autophagic pathway, thus providing a potential target for the treatment of inflammatory diseases and cancer., Introduction Autophagy is an essential cellular process for maintaining cell homeostasis and attenuating cell stresses through a 'self-eating' mechanism. Many autophagy-related (ATG) proteins have been identified as functioning in autophagy [...]

Activation of the vascular endothelin-1 (ET-1) system is a key abnormality in vascular dysfunction of human obesity, especially in patients developing complications, such as the metabolic syndrome, diabetes, and atherosclerosis. Vascular insulin resistance, an increased insulin-stimulated endothelial production of ET-1 combined with impaired nitric oxide availability, is the hallmark of obesity-related vasculopathy, but dysregulated adipokine release from obese adipose tissue may contribute to the predominance of ET-1-dependent vasoconstriction. ET-1, in turn, might determine unhealthy obese adipose tissue expansion, with visceral and perivascular adipose tissue changes driving the release of inflammatory cytokines and atherogenic chemokines. In addition, ET-1 might also play a role in the development of the metabolic complications of obesity. Studies have shown inhibition of lipoprotein lipase activity by ET-1, with consequent hypertriglyceridemia. Also, ET-1 in pancreatic islets seems to contribute to beta cell dysfunction, hence affecting insulin production and development of diabetes. Moreover, ET-1 may play a role in nonalcoholic steatohepatitis. Recent clinical trials using innovative design have demonstrated that antagonism of ET-type A receptors protects against some complications of obesity and diabetes, such as nephropathy. These findings encourage further investigation to evaluate whether targeting the ET-1 system could afford better protection against other consequences of the obesity epidemic. Key words: endothelin, adipose tissue, adipokines, obesity, prediabetes, NASH, dyslipidemia. L'activation du systeme de l'endotheline vasculaire 1 (ET-1) est une anomalie cle dans le dysfonctionnement vasculaire retrouve avec l'obesite chez l'homme, particulierement chez les patients presentant des complications, comme le syndrome metabolique, le diabete et l'atherosclerose. La resistance vasculaire a l'insuline, augmentation de la production d'ET-1 stimulee par l'insuline associee avec une disponibilite diminuee de l'oxyde nitrique, constitue la marque de la vasculopathie liee a l'obesite, mais le dereglement de la liberation des adipokines du tissu adipeux des personnes atteintes d'obesite pourrait aussi contribuer a la predominance de la vasoconstriction dependante de l'ET-1. De fait, l'ET-1 pourrait etre a la base de l'expansion de mauvais tissu adipeux en situation d'obesite, avec des variations dans les tissus adipeux visceral et perivasculaire menant a la liberation de cytokines inflammatoires et de chemokines atherogenes. De plus, l'ET-1 pourrait aussi jouer un role dans l'apparition de complications metaboliques de l'obesite. Des etudes ont montre une inhibition de l'activite de la lipoproteine lipase par l'intermediaire de l'ET-1, avec une hypertriglyceridemie consecutive. En outre, dans les ilots pancreatiques, l'ET-1 semble contribuer au dysfonctionnement des cellules beta, et donc affecter la production d'insuline, avec presentation du diabete. De surcroit, l'ET-1 pourrait jouer un role dans la steatohepatite non alcoolique. De recents essais cliniques suivant de nouveaux concepts ont montre que l'inhibition des recepteurs de l'ET de type A est associee a une protection contre certaines complications de l'obesite et du diabete, comme la nephropathie. Ces resultats viennent encourager la mise en reuvre de plus amples recherches en vue d'evaluer si en ciblant le systeme ET-1 on pourrait obtenir une meilleure protection contre d'autres consequences de l'epidemie d'obesite. [Traduit par la Redaction] Mots-cles: endotheline, tissu adipeux, adipokines, obesite, prediabete, SHNA, dyslipidemie., Introduction The incidence of obesity and type 2 diabetes is still increasing worldwide at an alarming rate, in relation predominantly to lack of regular physical activity and consumption of high-fat/sugar [...]

BACKGROUND. Undifferentiated systemic autoinflammatory diseases (USAIDs) present diagnostic and therapeutic challenges. Chronic interferon (IFN) signaling and cytokine dysregulation may identify diseases with available targeted treatments. METHODS. Sixty-six consecutively referred USAID patients underwent underwent screening for the presence of an interferon signature using a standardized type-I IFN-response-gene score (IRG-S), cytokine profiling, and genetic evaluation by next-generation sequencing. RESULTS. Thirty-six USAID patients (55%) had elevated IRG-S. Neutrophilic panniculitis (40% vs. 0%), basal ganglia calcifications (46% vs. 0%), interstitial lung disease (47% vs. 5%), and myositis (60% vs. 10%) were more prevalent in patients with elevated IRG-S. Moderate IRG-S elevation and highly elevated serum IL-18 distinguished 8 patients with pulmonary alveolar proteinosis (PAP) and recurrent macrophage activation syndrome (MAS). Among patients with panniculitis and progressive cytopenias, 2 patients were compound heterozygous for potentially novel LRBA mutations, 4 patients harbored potentially novel splice variants in IKBKG (which encodes NF-[kappa]B essential modulator [NEMO]), and 6 patients had de novo frameshift mutations in SAMD9L. Of additional 12 patients with elevated IRG-S and CANDLE-, SAVI- or Aicardi-Goutieres syndrome-like (AGS-like) phenotypes, 5 patients carried mutations in either SAMHD1, TREX1, PSMB8, or PSMG2. Two patients had anti-MDA5 autoantibody-positive juvenile dermatomyositis, and 7 could not be classified. Patients with LRBA, IKBKG, and SAMD9L mutations showed a pattern of IRG elevation that suggests prominent NF-[kappa]B activation different from the canonical interferonopathies CANDLE, SAVI, and AGS. CONCLUSIONS. In patients with elevated IRG-S, we identified characteristic clinical features and 3 additional autoinflammatory diseases: IL-18-mediated PAP and recurrent MAS (IL-18PAP-MAS), NEMO deleted exon 5autoinflammatory syndrome (NEMO-NDAS), and SAMD9L-associated autoinflammatory disease (SAMD9L-SAAD). The IRG-S expands the diagnostic armamentarium in evaluating USAIDs and points to different pathways regulating IRG expression. TRIAL REGISTRATION. ClinicalTrials.gov NCT02974595. FUNDING. The Intramural Research Program of the NIH, NIAID, NIAMS, and the Clinical Center., Introduction The discovery of Mendelian defects that cause immune-dysregulatory and autoinflammatory diseases rapidly expanded our ability to diagnose pediatric patients with systemic sterile inflammation and provided insights into pathogenic mechanisms [...]

Neutrophil extracellular traps (NETs) are extracellular reticular fibrillar structures composed of DNA, histones, granulins and cytoplasmic proteins that are delivered externally by neutrophils in response to stimulation with various types of microorganisms, cytokines and host molecules, etc. NET formation has been extensively demonstrated to trap, immobilize, inactivate and kill invading microorganisms and acts as a form of innate response against pathogenic invasion. However, NETs are a double-edged sword. In the event of imbalance between NET formation and clearance, excessive NETs not only directly inflict tissue lesions, but also recruit pro-inflammatory cells or proteins that promote the release of inflammatory factors and magnify the inflammatory response further, driving the progression of many human diseases. The deleterious effects of excessive release of NETs on gut diseases are particularly crucial as NETs are more likely to be disrupted by neutrophils infiltrating the intestinal epithelium during intestinal disorders, leading to intestinal injury, and in addition, NETs and their relevant molecules are capable of directly triggering the death of intestinal epithelial cells. Within this context, a large number of NETs have been reported in several intestinal diseases, including intestinal infections, inflammatory bowel disease, intestinal ischemia-reperfusion injury, sepsis, necrotizing enterocolitis, and colorectal cancer. Therefore, the formation of NET would have to be strictly monitored to prevent their mediated tissue damage. In this review, we summarize the latest knowledge on the formation mechanisms of NETs and their pathophysiological roles in a variety of intestinal diseases, with the aim of providing an essential directional guidance and theoretical basis for clinical interventions in the exploration of mechanisms underlying NETs and targeted therapies. Keywords: Intestinal diseases, Neutrophils, Neutrophil extracellular traps, Infection, Inflammation, Colitis, Therapeutics, Colorectal neoplasms, Author(s): Feng Chen[sup.1], Yongqiang Liu[sup.1,2], Yajing Shi[sup.1], Jianmin Zhang[sup.1], Xin Liu[sup.1,2], Zhenzhen Liu[sup.1], Jipeng Lv[sup.1] and Yufang Leng[sup.1,2] Background Neutrophils are the most abundant immune cells and the fastest recruitment [...]

Induction of memory [CD8.sup.+] T cells is important for controlling infections such as malaria and HIV/AIDS and for cancer immunotherapy. Accurate assessment of antigen-specific (Ag-specific) [CD8.sup.+] T cells is critical for vaccine optimization and for defining correlates of protection. However, conditions for determining Ag-specific [CD8.sup.+] T cell responses ex vivo using intracellular cytokine staining (ICS) may be variable, especially in humans with complex antigens. Here, we used an attenuated whole parasite malaria vaccine model in humans and various experimental infections in mice to show that the duration of antigenic stimulation and timing of brefeldin A (BFA) addition influence the magnitude of Ag-specific and bystander T cell responses. Indeed, after immunization with an attenuated whole sporozoite malaria vaccine in humans, significantly higher numbers of IFN-[gamma]-producing memory [CD8.sup.+] T cells comprising Ag-specific and bystander responses were detected when the duration of Ag stimulation prior to addition of BFA was increased. Mechanistic analyses of virus-specific [CD8.sup.+] T cells in mice revealed that the increase in IFN-[gamma]-producing [CD8.sup.+] T cells was due to bystander activation of Ag-experienced memory [CD8.sup.+] T cells, and correlated with the proportion of Ag-experienced [CD8.sup.+] T cells in the stimulated populations. Incubation with anti-cytokine antibodies (e.g., IL-12) improved accuracy in detecting bona fide memory [CD8.sup.+] T cell responses, suggesting this as the mechanism for the bystander activation. These data have important implications for accurate assessment of immune responses generated by vaccines intended to elicit protective memory [CD8.sup.+] T cells., Introduction [CD8.sup.+] T cells have a role in mediating protection in humans against diverse pathogens impacting public health, such as HIV, influenza, and Plasmodium, the causative agent of malaria, and [...]

Interleukin (IL)-2 is a pleiotropic cytokine that is necessary to prevent chronic inflammation in the gastrointestinal tract.sup.1-4. The protective effects of IL-2 involve the generation, maintenance and function of regulatory T (T.sub.reg) cells.sup.4-8, and the use of low doses of IL-2 has emerged as a potential therapeutic strategy for patients with inflammatory bowel disease.sup.9. However, the cellular and molecular pathways that control the production of IL-2 in the context of intestinal health are undefined. Here we show, in a mouse model, that IL-2 is acutely required to maintain T.sub.reg cells and immunological homeostasis throughout the gastrointestinal tract. Notably, lineage-specific deletion of IL-2 in T cells did not reduce T.sub.reg cells in the small intestine. Unbiased analyses revealed that, in the small intestine, group-3 innate lymphoid cells (ILC3s) are the dominant cellular source of IL-2, which is induced selectively by IL-1[beta]. Macrophages in the small intestine produce IL-1[beta], and activation of this pathway involves MYD88- and NOD2-dependent sensing of the microbiota. Our loss-of-function studies show that ILC3-derived IL-2 is essential for maintaining T.sub.reg cells, immunological homeostasis and oral tolerance to dietary antigens in the small intestine. Furthermore, production of IL-2 by ILC3s was significantly reduced in the small intestine of patients with Crohn's disease, and this correlated with lower frequencies of T.sub.reg cells. Our results reveal a previously unappreciated pathway in which a microbiota- and IL-1[beta]-dependent axis promotes the production of IL-2 by ILC3s to orchestrate immune regulation in the intestine. A microbiota- and IL-1[beta]-dependent axis of IL-2 production by group-3 innate lymphoid cells is shown in a mouse model to be necessary to maintain immunological homeostasis and regulatory T cells in the small intestine., Author(s): Lei Zhou [sup.1] [sup.2] [sup.3] , Coco Chu [sup.1] [sup.2] [sup.3] , Fei Teng [sup.1] [sup.2] [sup.3] , Nicholas J. Bessman [sup.1] [sup.2] [sup.3] , Jeremy Goc [sup.1] [sup.2] [...]

Genetic variants at the PTPN2 locus, which encodes the tyrosine phosphatase PTPN2, cause reduced gene expression and are linked to rheumatoid arthritis (RA) and other autoimmune diseases. PTPN2 inhibits signaling through the T cell and cytokine receptors, and loss of PTPN2 promotes T cell expansion and CD4- and [CD8.sup.- ]driven autoimmunity. However, it remains unknown whether loss of PTPN2 in [FoxP3.sup.+] regulatory T cells (Tregs) plays a role in autoimmunity. Here we aimed to model human autoimmune-predisposing PTPN2 variants, the presence of which results in a partial loss of PTPN2 expression, in mouse models of RA. We identified that reduced expression of Ptpn2 enhanced the severity of autoimmune arthritis in the T cell-dependent SKG mouse model and demonstrated that this phenotype was mediated through a Treg-intrinsic mechanism. Mechanistically, we found that through dephosphorylation of STAT3, PTPN2 inhibits IL-6-driven pathogenic loss of FoxP3 after Tregs have acquired ROR[gamma]t expression, at a stage when chromatin accessibility for STAT3-targeted [IL-17.sup.-]associated transcription factors is maximized. We conclude that PTPN2 promotes FoxP3 stability in mouse [ROR[gamma]t.sup.+] Tregs and that loss of function of PTPN2 in Tregs contributes to the association between PTPN2 and autoimmunity., Introduction Rheumatoid arthritis (RA) is a chronic autoimmune, systemic inflammatory disorder that primarily affects diarthrodial joints (1). To date, various genome-wide association studies have identified over 100 risk loci for [...]

Immune recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors often activates proinflammatory NF-[kappa]B signalling.sup.1. Recent studies indicate that the bacterial metabolite d-glycero-[beta]-d-manno-heptose 1,7-bisphosphate (HBP) can activate NF-[kappa]B signalling in host cytosol.sup.2-4, but it is unclear whether HBP is a genuine PAMP and the cognate pattern recognition receptor has not been identified. Here we combined a transposon screen in Yersinia pseudotuberculosis with biochemical analyses and identified ADP-[beta]-d-manno-heptose (ADP-Hep), which mediates type III secretion system-dependent NF-[kappa]B activation and cytokine expression. ADP-Hep, but not other heptose metabolites, could enter host cytosol to activate NF-[kappa]B. A CRISPR-Cas9 screen showed that activation of NF-[kappa]B by ADP-Hep involves an ALPK1 (alpha-kinase 1)-TIFA (TRAF-interacting protein with forkhead-associated domain) axis. ADP-Hep directly binds the N-terminal domain of ALPK1, stimulating its kinase domain to phosphorylate and activate TIFA. The crystal structure of the N-terminal domain of ALPK1 and ADP-Hep in complex revealed the atomic mechanism of this ligand-receptor recognition process. HBP was transformed by host adenylyltransferases into ADP-heptose 7-P, which could activate ALPK1 to a lesser extent than ADP-Hep. ADP-Hep (but not HBP) alone or during bacterial infection induced Alpk1-dependent inflammation in mice. Our findings identify ALPK1 and ADP-Hep as a pattern recognition receptor and an effective immunomodulator, respectively.The bacterial metabolite ADP-heptose activates NF-[kappa]B in host cells via alpha-kinase 1 and the TIFA-TRAF signalling pathway., Author(s): Ping Zhou [sup.1] , Yang She [sup.1] [sup.2] [sup.3] , Na Dong [sup.4] , Peng Li [sup.1] , Huabin He [sup.1] , Alessio Borio [sup.5] , Qingcui Wu [sup.1] [...]

Prolonged exposure to microbial products such as lipopolysaccharide can induce a form of innate immune memory that blunts subsequent responses to unrelated pathogens, known as lipopolysaccharide tolerance. Sepsis is a dysregulated systemic immune response to disseminated infection that has a high mortality rate. In some patients, sepsis results in a period of immunosuppression (known as 'immunoparalysis').sup.1 characterized by reduced inflammatory cytokine output.sup.2, increased secondary infection.sup.3 and an increased risk of organ failure and mortality.sup.4. Lipopolysaccharide tolerance recapitulates several key features of sepsis-associated immunosuppression.sup.5. Although various epigenetic changes have previously been observed in tolerized macrophages.sup.6-8, the molecular basis of tolerance, immunoparalysis and other forms of innate immune memory has remained unclear. Here we perform a screen for tolerance-associated microRNAs and identify miR-221 and miR-222 as regulators of the functional reprogramming of macrophages during lipopolysaccharide tolerization. Prolonged stimulation with lipopolysaccharide in mice leads to increased expression of miR-221 and mir-222, both of which regulate brahma-related gene 1 (Brg1, also known as Smarca4). This increased expression causes the transcriptional silencing of a subset of inflammatory genes that depend on chromatin remodelling mediated by SWI/SNF (switch/sucrose non-fermentable) and STAT (signal transducer and activator of transcription), which in turn promotes tolerance. In patients with sepsis, increased expression of miR-221 and miR-222 correlates with immunoparalysis and increased organ damage. Our results show that specific microRNAs can regulate macrophage tolerization and may serve as biomarkers of immunoparalysis and poor prognosis in patients with sepsis. The microRNAs miR-221 and miR-222 regulate the reprogramming of macrophages during the development of lipopolysaccharide tolerance, and increased expression of these microRNAs is associated with immunosuppression and poor prognosis in patients with sepsis., Author(s): John J. Seeley [sup.1] , Rebecca G. Baker [sup.1] , Ghait Mohamed [sup.2] , Tony Bruns [sup.2] [sup.3] , Matthew S. Hayden [sup.1] [sup.4] , Sachin D. Deshmukh [sup.2] [...]

Infections by pathogens that contain DNA trigger the production of type-I interferons and inflammatory cytokines through cyclic GMP-AMP synthase, which produces 2'3'-cyclic GMP-AMP (cGAMP) that binds to and activates stimulator of interferon genes (STING; also known as TMEM173, MITA, ERIS and MPYS).sup.1-8. STING is an endoplasmic-reticulum membrane protein that contains four transmembrane helices followed by a cytoplasmic ligand-binding and signalling domain.sup.9-13. The cytoplasmic domain of STING forms a dimer, which undergoes a conformational change upon binding to cGAMP.sup.9,14. However, it remains unclear how this conformational change leads to STING activation. Here we present cryo-electron microscopy structures of full-length STING from human and chicken in the inactive dimeric state (about 80 kDa in size), as well as cGAMP-bound chicken STING in both the dimeric and tetrameric states. The structures show that the transmembrane and cytoplasmic regions interact to form an integrated, domain-swapped dimeric assembly. Closure of the ligand-binding domain, induced by cGAMP, leads to a 180° rotation of the ligand-binding domain relative to the transmembrane domain. This rotation is coupled to a conformational change in a loop on the side of the ligand-binding-domain dimer, which leads to the formation of the STING tetramer and higher-order oligomers through side-by-side packing. This model of STING oligomerization and activation is supported by our structure-based mutational analyses. Cryo-electron microscopy structures of full-length STING show that cyclic GMP-AMP induces a half-turn rotation of the ligand-binding domain relative to the transmembrane domain, forming a tetramer and higher-order oligomers for signalling., Author(s): Guijun Shang [sup.1] , Conggang Zhang [sup.2] , Zhijian J. Chen [sup.2] [sup.3] [sup.4] , Xiao-chen Bai [sup.5] [sup.6] , Xuewu Zhang [sup.1] [sup.5] Author Affiliations: (1) Department of [...]

The cytokine interferon-[gamma] (IFN[gamma]) is a central coordinator of innate and adaptive immunity, but its highly pleiotropic actions have diminished its prospects for use as an immunotherapeutic agent. Here, we took a structure-based approach to decoupling IFN[gamma] pleiotropy. We engineered an affinity-enhanced variant of the ligand-binding chain of the IFN[gamma] receptor IFN[gamma]R1, which enabled us to determine the crystal structure of the complete hexameric (2:2:2) IFN[gamma]-IFN[gamma]R1-IFN[gamma]R2 signalling complex at 3.25 Å resolution. The structure reveals the mechanism underlying deficits in IFN[gamma] responsiveness in mycobacterial disease syndrome resulting from a T168N mutation in IFN[gamma]R2, which impairs assembly of the full signalling complex. The topology of the hexameric complex offers a blueprint for engineering IFN[gamma] variants to tune IFN[gamma] receptor signalling output. Unexpectedly, we found that several partial IFN[gamma] agonists exhibited biased gene-expression profiles. These biased agonists retained the ability to induce upregulation of major histocompatibility complex class I antigen expression, but exhibited impaired induction of programmed death-ligand 1 expression in a wide range of human cancer cell lines, offering a route to decoupling immunostimulatory and immunosuppressive functions of IFN[gamma] for therapeutic applications. The X-ray structure of the hexameric complex of interferon-[gamma] bound to its receptors is solved at 3.25 Å resolution, providing a basis for engineering variants of interferon-[gamma] that enable decoupling of its immunomodulatory functions., Author(s): Juan L. Mendoza [sup.1] [sup.2] [sup.7] , Nichole K. Escalante [sup.3] [sup.4] , Kevin M. Jude [sup.1] [sup.2] , Junel Sotolongo Bellon [sup.5] , Leon Su [sup.1] [sup.2] , [...]

Environmental genotoxic factors pose a challenge to the genomic integrity of epithelial cells at barrier surfaces that separate host organisms from the environment. They can induce mutations that, if they occur in epithelial stem cells, contribute to malignant transformation and cancer development.sup.1-3. Genome integrity in epithelial stem cells is maintained by an evolutionarily conserved cellular response pathway, the DNA damage response (DDR). The DDR culminates in either transient cell-cycle arrest and DNA repair or elimination of damaged cells by apoptosis.sup.4,5. Here we show that the cytokine interleukin-22 (IL-22), produced by group 3 innate lymphoid cells (ILC3) and [gamma][delta] T cells, is an important regulator of the DDR machinery in intestinal epithelial stem cells. Using a new mouse model that enables sporadic inactivation of the IL-22 receptor in colon epithelial stem cells, we demonstrate that IL-22 is required for effective initiation of the DDR following DNA damage. Stem cells deprived of IL-22 signals and exposed to carcinogens escaped DDR-controlled apoptosis, contained more mutations and were more likely to give rise to colon cancer. We identified metabolites of glucosinolates, a group of phytochemicals contained in cruciferous vegetables, to be a widespread source of genotoxic stress in intestinal epithelial cells. These metabolites are ligands of the aryl hydrocarbon receptor (AhR).sup.6, and AhR-mediated signalling in ILC3 and [gamma][delta] T cells controlled their production of IL-22. Mice fed with diets depleted of glucosinolates produced only very low levels of IL-22 and, consequently, the DDR in epithelial cells of mice on a glucosinolate-free diet was impaired. This work identifies a homeostatic network protecting stem cells against challenge to their genome integrity by AhR-mediated 'sensing' of genotoxic compounds from the diet. AhR signalling, in turn, ensures on-demand production of IL-22 by innate lymphocytes directly regulating components of the DDR in epithelial stem cells.Sporadic inactivation of the interleukin-22 receptor in the intestinal epithelium of the mouse shows that IL-22 is required for effective activation of the DNA damage response following DNA damage., Author(s): Konrad Gronke [sup.1] [sup.2] [sup.3] [sup.4] [sup.5] [sup.6] , Pedro P. Hernández [sup.5] [sup.6] [sup.7] , Jakob Zimmermann [sup.5] , Christoph S. N. Klose [sup.1] [sup.5] [sup.8] , Michael [...]

We describe a de novo computational approach for designing proteins that recapitulate the binding sites of natural cytokines, but are otherwise unrelated in topology or amino acid sequence. We use this strategy to design mimics of the central immune cytokine interleukin-2 (IL-2) that bind to the IL-2 receptor [beta][gamma].sub.c heterodimer (IL-2R[beta][gamma].sub.c) but have no binding site for IL-2R[alpha] (also called CD25) or IL-15R[alpha] (also known as CD215). The designs are hyper-stable, bind human and mouse IL-2R[beta][gamma].sub.c with higher affinity than the natural cytokines, and elicit downstream cell signalling independently of IL-2R[alpha] and IL-15R[alpha]. Crystal structures of the optimized design neoleukin-2/15 (Neo-2/15), both alone and in complex with IL-2R[beta][gamma].sub.c, are very similar to the designed model. Neo-2/15 has superior therapeutic activity to IL-2 in mouse models of melanoma and colon cancer, with reduced toxicity and undetectable immunogenicity. Our strategy for building hyper-stable de novo mimetics could be applied generally to signalling proteins, enabling the creation of superior therapeutic candidates.A hyper-stable de novo protein mimic of interleukin-2 computationally designed to not interact with a regulatory T-cell specific receptor subunit has improved therapeutic activity in mouse models of melanoma and colon cancer., Author(s): Daniel-Adriano Silva [sup.1] [sup.2] , Shawn Yu [sup.1] [sup.3] , Umut Y. Ulge [sup.1] [sup.3] , Jamie B. Spangler [sup.4] [sup.5] , Kevin M. Jude [sup.5] , Carlos Labão-Almeida [...]

Interferons (IFNs) are cytokines that play a critical role in limiting infectious and malignant diseases.sup.1-4. Emerging data suggest that the strength and duration of IFN signaling can differentially impact cancer therapies, including immune checkpoint blockade.sup.5-7. Here, we characterize the output of IFN signaling, specifically IFN-stimulated gene (ISG) signatures, in primary tumors from The Cancer Genome Atlas. While immune infiltration correlates with the ISG signature in some primary tumors, the existence of ISG signature-positive tumors without evident infiltration of IFN-producing immune cells suggests that cancer cells per se can be a source of IFN production. Consistent with this hypothesis, analysis of patient-derived tumor xenografts propagated in immune-deficient mice shows evidence of ISG-positive tumors that correlates with expression of human type I and III IFNs derived from the cancer cells. Mechanistic studies using cell line models from the Cancer Cell Line Encyclopedia that harbor ISG signatures demonstrate that this is a by-product of a STING-dependent pathway resulting in chronic tumor-derived IFN production. This imposes a transcriptional state on the tumor, poising it to respond to the aberrant accumulation of double-stranded RNA (dsRNA) due to increased sensor levels (MDA5, RIG-I and PKR). By interrogating our functional short-hairpin RNA screen dataset across 398 cancer cell lines, we show that this ISG transcriptional state creates a novel genetic vulnerability. ISG signature-positive cancer cells are sensitive to the loss of ADAR, a dsRNA-editing enzyme that is also an ISG. A genome-wide CRISPR genetic suppressor screen reveals that the entire type I IFN pathway and the dsRNA-activated kinase, PKR, are required for the lethality induced by ADAR depletion. Therefore, tumor-derived IFN resulting in chronic signaling creates a cellular state primed to respond to dsRNA accumulation, rendering ISG-positive tumors susceptible to ADAR loss. RNA editing enzyme ADAR1 is a therapeutic vulnerability in tumors with constitutive cell-autonomous production of interferon-stimulated genes, Author(s): Huayang Liu [sup.1] , Javad Golji [sup.1] , Lauren K. Brodeur [sup.1] , Franklin S. Chung [sup.1] , Julie T. Chen [sup.1] , Rosalie S. deBeaumont [sup.1] , Caroline [...]

As the first line of defence against pathogens, cells mount an innate immune response, which varies widely from cell to cell. The response must be potent but carefully controlled to avoid self-damage. How these constraints have shaped the evolution of innate immunity remains poorly understood. Here we characterize the innate immune response's transcriptional divergence between species and variability in expression among cells. Using bulk and single-cell transcriptomics in fibroblasts and mononuclear phagocytes from different species, challenged with immune stimuli, we map the architecture of the innate immune response. Transcriptionally diverging genes, including those that encode cytokines and chemokines, vary across cells and have distinct promoter structures. Conversely, genes that are involved in the regulation of this response, such as those that encode transcription factors and kinases, are conserved between species and display low cell-to-cell variability in expression. We suggest that this expression pattern, which is observed across species and conditions, has evolved as a mechanism for fine-tuned regulation to achieve an effective but balanced response.Comparison of transcriptomic data from immune-stimulated cells across different species sheds light on the architecture of the innate immune response., Author(s): Tzachi Hagai [sup.1] [sup.2] , Xi Chen [sup.1] , Ricardo J. Miragaia [sup.1] [sup.3] , Raghd Rostom [sup.1] [sup.2] , Tomás Gomes [sup.1] , Natalia Kunowska [sup.1] , Johan [...]

Primary liver cancer represents a major health problem. It comprises hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), which differ markedly with regards to their morphology, metastatic potential and responses to therapy. However, the regulatory molecules and tissue context that commit transformed hepatic cells towards HCC or ICC are largely unknown. Here we show that the hepatic microenvironment epigenetically shapes lineage commitment in mosaic mouse models of liver tumorigenesis. Whereas a necroptosis-associated hepatic cytokine microenvironment determines ICC outgrowth from oncogenically transformed hepatocytes, hepatocytes containing identical oncogenic drivers give rise to HCC if they are surrounded by apoptotic hepatocytes. Epigenome and transcriptome profiling of mouse HCC and ICC singled out Tbx3 and Prdm5 as major microenvironment-dependent and epigenetically regulated lineage-commitment factors, a function that is conserved in humans. Together, our results provide insight into lineage commitment in liver tumorigenesis, and explain molecularly why common liver-damaging risk factors can lead to either HCC or ICC.The tumour microenvironment determines which type of liver cancer develops, with transformed hepatocytes giving rise to intrahepatic cholangiocarcinoma or hepatocellular carcinoma depending or whether they are surrounded by cells undergoing necroptosis or apoptosis., Author(s): Marco Seehawer [sup.1] [sup.2] , Florian Heinzmann [sup.1] [sup.2] , Luana D'Artista [sup.1] [sup.2] , Jule Harbig [sup.1] [sup.2] , Pierre-François Roux [sup.3] [sup.4] [sup.5] , Lisa Hoenicke [sup.1] [...]

Although serum from patients with Parkinson's disease contains elevated levels of numerous pro-inflammatory cytokines including IL-6, TNF, IL-1[beta], and IFN[gamma], whether inflammation contributes to or is a consequence of neuronal loss remains unknown.sup.1. Mutations in parkin, an E3 ubiquitin ligase, and PINK1, a ubiquitin kinase, cause early onset Parkinson's disease.sup.2,3. Both PINK1 and parkin function within the same biochemical pathway and remove damaged mitochondria from cells in culture and in animal models via mitophagy, a selective form of autophagy.sup.4. The in vivo role of mitophagy, however, is unclear, partly because mice that lack either PINK1 or parkin have no substantial Parkinson's-disease-relevant phenotypes.sup.5-7. Mitochondrial stress can lead to the release of damage-associated molecular patterns (DAMPs) that can activate innate immunity.sup.8-12, suggesting that mitophagy may mitigate inflammation. Here we report a strong inflammatory phenotype in both Prkn.sup.-/- and Pink1.sup.-/- mice following exhaustive exercise and in Prkn.sup.-/-;mutator mice, which accumulate mutations in mitochondrial DNA (mtDNA).sup.13,14. Inflammation resulting from either exhaustive exercise or mtDNA mutation is completely rescued by concurrent loss of STING, a central regulator of the type I interferon response to cytosolic DNA.sup.15,16. The loss of dopaminergic neurons from the substantia nigra pars compacta and the motor defect observed in aged Prkn.sup.-/-;mutator mice are also rescued by loss of STING, suggesting that inflammation facilitates this phenotype. Humans with mono- and biallelic PRKN mutations also display elevated cytokines. These results support a role for PINK1- and parkin-mediated mitophagy in restraining innate immunity.Acute and chronic mitochondrial stress in mice require PINK1 and parkin to restrain STING-mediated innate immunity., Author(s): Danielle A. Sliter [sup.1] , Jennifer Martinez [sup.2] , Ling Hao [sup.1] , Xi Chen [sup.3] , Nuo Sun [sup.4] , Tara D. Fischer [sup.1] , Jonathon L. Burman [...]

Intratumoral stimulatory dendritic cells (SDCs) play an important role in stimulating cytotoxic T cells and driving immune responses against cancer. Understanding the mechanisms that regulate their abundance in the tumor microenvironment (TME) could unveil new therapeutic opportunities. We find that in human melanoma, SDC abundance is associated with intratumoral expression of the gene encoding the cytokine FLT3LG. FLT3LG is predominantly produced by lymphocytes, notably natural killer (NK) cells in mouse and human tumors. NK cells stably form conjugates with SDCs in the mouse TME, and genetic and cellular ablation of NK cells in mice demonstrates their importance in positively regulating SDC abundance in tumor through production of FLT3L. Although anti-PD-1 'checkpoint' immunotherapy for cancer largely targets T cells, we find that NK cell frequency correlates with protective SDCs in human cancers, with patient responsiveness to anti-PD-1 immunotherapy, and with increased overall survival. Our studies reveal that innate immune SDCs and NK cells cluster together as an excellent prognostic tool for T cell-directed immunotherapy and that these innate cells are necessary for enhanced T cell tumor responses, suggesting this axis as a target for new therapies. Cross-talk between innate immune cells helps to enhance the antitumor T cell response during checkpoint blockade therapy., Author(s): Kevin C. Barry [sup.1] [sup.2] , Joy Hsu [sup.1] [sup.2] , Miranda L. Broz [sup.1] [sup.2] , Francisco J. Cueto [sup.1] [sup.3] [sup.4] , Mikhail Binnewies [sup.1] , Alexis [...]

Mesenchymal tumor subpopulations secrete pro-tumorigenic cytokines and promote treatment resistance.sup.1-4. This phenomenon has been implicated in chemorefractory small cell lung cancer and resistance to targeted therapies.sup.5-8, but remains incompletely defined. Here, we identify a subclass of endogenous retroviruses (ERVs) that engages innate immune signaling in these cells. Stimulated 3 prime antisense retroviral coding sequences (SPARCS) are oriented inversely in 3' untranslated regions of specific genes enriched for regulation by STAT1 and EZH2. Derepression of these loci results in double-stranded RNA generation following IFN-[gamma] exposure due to bi-directional transcription from the STAT1-activated gene promoter and the 5' long terminal repeat of the antisense ERV. Engagement of MAVS and STING activates downstream TBK1, IRF3, and STAT1 signaling, sustaining a positive feedback loop. SPARCS induction in human tumors is tightly associated with major histocompatibility complex class 1 expression, mesenchymal markers, and downregulation of chromatin modifying enzymes, including EZH2. Analysis of cell lines with high inducible SPARCS expression reveals strong association with an AXL/MET-positive mesenchymal cell state. While SPARCS-high tumors are immune infiltrated, they also exhibit multiple features of an immune-suppressed microenviroment. Together, these data unveil a subclass of ERVs whose derepression triggers pathologic innate immune signaling in cancer, with important implications for cancer immunotherapy. Retroelements located in antisense orientation within interferon-regulated genes are reactivated in a subset of cancer cells and initiate a STING- and MAVS-dependent feed-forward inflammatory loop, driving antitumor immunity and exhaustion., Author(s): Israel Cañadas [sup.1] , Rohit Thummalapalli [sup.1] , Jong Wook Kim [sup.1] [sup.2] , Shunsuke Kitajima [sup.1] , Russell William Jenkins [sup.1] [sup.3] , Camilla Laulund Christensen [sup.1] , [...]

The ubiquitously expressed non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, is involved in signal transduction downstream of multiple growth factor, cytokine and integrin receptors.sup.1. Its requirement for complete RAS-MAPK activation and its role as a negative regulator of JAK-STAT signaling have established SHP2 as an essential player in oncogenic signaling pathways.sup.1-7. Recently, a novel potent allosteric SHP2 inhibitor was presented as a viable therapeutic option for receptor tyrosine kinase-driven cancers, but was shown to be ineffective in KRAS-mutant tumor cell lines in vitro.sup.8. Here, we report a central and indispensable role for SHP2 in oncogenic KRAS-driven tumors. Genetic deletion of Ptpn11 profoundly inhibited tumor development in mutant KRAS-driven murine models of pancreatic ductal adenocarcinoma and non-small-cell lung cancer. We provide evidence for a critical dependence of mutant KRAS on SHP2 during carcinogenesis. Deletion or inhibition of SHP2 in established tumors delayed tumor progression but was not sufficient to achieve tumor regression. However, SHP2 was necessary for resistance mechanisms upon blockade of MEK. Synergy was observed when both SHP2 and MEK were targeted, resulting in sustained tumor growth control in murine and human patient-derived organoids and xenograft models of pancreatic ductal adenocarcinoma and non-small-cell lung cancer. Our data indicate the clinical utility of dual SHP2/MEK inhibition as a targeted therapy approach for KRAS-mutant cancers. The phosphatase SHP2 is required for mutant KRAS signaling in pancreatic and non-small-cell lung cancers and drives resistance to MEK inhibition., Author(s): Dietrich A. Ruess [sup.1] [sup.2] , Guus J. Heynen [sup.3] , Katrin J. Ciecielski [sup.1] , Jiaoyu Ai [sup.1] , Alexandra Berninger [sup.1] , Derya Kabacaoglu [sup.1] , Kivanc [...]

Patients with metastatic cancer experience a severe loss of skeletal muscle mass and function known as cachexia. Cachexia is associated with poor prognosis and accelerated death in patients with cancer, yet its underlying mechanisms remain poorly understood. Here, we identify the metal-ion transporter ZRT- and IRT-like protein 14 (ZIP14) as a critical mediator of cancer-induced cachexia. ZIP14 is upregulated in cachectic muscles of mice and in patients with metastatic cancer and can be induced by TNF-[alpha] and TGF-[beta] cytokines. Strikingly, germline ablation or muscle-specific depletion of Zip14 markedly reduces muscle atrophy in metastatic cancer models. We find that ZIP14-mediated zinc uptake in muscle progenitor cells represses the expression of MyoD and Mef2c and blocks muscle-cell differentiation. Importantly, ZIP14-mediated zinc accumulation in differentiated muscle cells induces myosin heavy chain loss. These results highlight a previously unrecognized role for altered zinc homeostasis in metastatic cancer-induced muscle wasting and implicate ZIP14 as a therapeutic target for its treatment. Accumulation of zinc in muscle cells resulting from transcriptional upregulation of metal transporter ZIP14 causes muscle atrophy and promotes cachexia in metastatic cancer., Author(s): Gang Wang [sup.1] , Anup K. Biswas [sup.1] , Wanchao Ma [sup.1] , Manoj Kandpal [sup.2] , Courtney Coker [sup.1] , Paul M. Grandgenett [sup.3] , Michael A. Hollingsworth [...]