The Unfolded Protein Response Mediator PERK Governs Myeloid Cell-Driven Immunosuppression in Tumors through Inhibition of STING Signaling.

The primary mechanisms supporting immunoregulatory polarization of myeloid cells upon infiltration into tumors remain largely unexplored. Elucidation of these signals could enable better strategies to restore protective anti-tumor immunity. Here, we investigated the role of the intrinsic activation of the PKR-like endoplasmic reticulum (ER) kinase (PERK) in the immunoinhibitory actions of tumor-associated myeloid-derived suppressor cells (tumor-MDSCs). PERK signaling increased in tumor-MDSCs, and its deletion transformed MDSCs into myeloid cells that activated CD8+ T cell-mediated immunity against cancer. Tumor-MDSCs lacking PERK exhibited disrupted NRF2-driven antioxidant capacity and impaired mitochondrial respiratory homeostasis. Moreover, reduced NRF2 signaling in PERK-deficient MDSCs elicited cytosolic mitochondrial DNA elevation and, consequently, STING-dependent expression of anti-tumor type I interferon. Reactivation of NRF2 signaling, conditional deletion of STING, or blockade of type I interferon receptor I restored the immunoinhibitory potential of PERK-ablated MDSCs. Our findings demonstrate the pivotal role of PERK in tumor-MDSC functionality and unveil strategies to reprogram immunosuppressive myelopoiesis in tumors to boost cancer immunotherapy.

TGF-ß1-induced deposition of provisional extracellular matrix by tracheal basal cells promotes epithelial-to-mesenchymal transition in a c-Jun NH2-terminal kinase-1-dependent manner.

Epithelial cells have been suggested as potential drivers of lung fibrosis, although the epithelial-dependent pathways that promote fibrogenesis remain unknown. Extracellular matrix is increasingly recognized as an environment that can drive cellular responses in various pulmonary diseases. In this study, we demonstrate that transforming growth factor-ß1 (TGF-ß1)-stimulated mouse tracheal basal (MTB) cells produce provisional matrix proteins in vitro, which initiate mesenchymal changes in subsequently freshly plated MTB cells via Rho kinase- and c-Jun NH2-terminal kinase (JNK1)-dependent processes. Repopulation of decellularized lung scaffolds, derived from mice with bleomycin-induced fibrosis or from patients with idiopathic pulmonary fibrosis, with wild-type MTB cells resulted in a loss of epithelial gene expression and augmentation of mesenchymal gene expression compared with cells seeded into decellularized normal lungs. In contrast, Jnk1-/- basal cells seeded into fibrotic lung scaffolds retained a robust epithelial expression profile, failed to induce mesenchymal genes, and differentiated into club cell secretory protein-expressing cells. This new paradigm wherein TGF-ß1-induced extracellular matrix derived from MTB cells activates a JNK1-dependent mesenchymal program, which impedes subsequent normal epithelial cell homeostasis, provides a plausible scenario of chronic aberrant epithelial repair, thought to be critical in lung fibrogenesis. This study identifies JNK1 as a possible target for inhibition in settings wherein reepithelialization is desired.

Ablation of Glutaredoxin-1 Modulates House Dust Mite-Induced Allergic Airways Disease in Mice.

Protein S-glutathionylation (PSSG) is an oxidant-induced post-translational modification of protein cysteines that impacts structure and function. The oxidoreductase glutaredoxin-1 (Glrx1) under physiological conditions catalyzes deglutathionylation and restores the protein thiol group. The involvement of Glrx1/PSSG in allergic inflammation induced by asthma-relevant allergens remains unknown. In the present study, we examined the impact of genetic ablation of Glrx1 in the pathogenesis of house dust mite (HDM)-induced allergic airways disease in mice. Wild-type (WT) or Glrx1(-/-) mice were instilled intranasally with HDM on 5 consecutive days for 3 weeks. As expected, overall PSSG was increased in Glrx1(-/-) HDM mice as compared with WT animals. Total cells in bronchoalveolar lavage fluid were similarly increased in HDM-treated WT and Glrx1(-/-) mice. However, in response to HDM, mice lacking Glrx1 demonstrated significantly more neutrophils and macrophages but fewer eosinophils as compared with HDM-exposed WT mice. mRNA expression of the Th2-associated cytokines IL-13 and IL-6, as well as mucin-5AC (Muc5ac), was significantly attenuated in Glrx1(-/-) HDM-treated mice. Conversely, mRNA expression of IFN-γ and IL-17A was increased in Glrx1(-/-) HDM mice compared with WT littermates. Restimulation of single-cell suspensions isolated from lungs or spleens with HDM resulted in enhanced IL-17A and decreased IL-5 production in cells derived from inflamed Glrx1(-/-) mice compared with WT animals. Finally, HDM-induced tissue damping and elastance were significantly attenuated in Glrx1(-/-) mice compared with WT littermates. These results demonstrate that the Glrx1-PSSG axis plays a pivotal role in HDM-induced allergic airways disease in association with enhanced type 2 inflammation and restriction of IFN-γ and IL-17A.

Emerging mechanisms of glutathione-dependent chemistry in biology and disease.

Glutathione has traditionally been considered as an antioxidant that protects cells against oxidative stress. Hence, the loss of reduced glutathione and formation of glutathione disulfide is considered a classical parameter of oxidative stress that is increased in diseases. Recent studies have emerged that demonstrate that glutathione plays a more direct role in biological and pathophysiological processes through covalent modification to reactive cysteines within proteins, a process known as S-glutathionylation. The formation of an S-glutathionylated moiety within the protein can lead to structural and functional modifications. Activation, inactivation, loss of function, and gain of function have all been attributed to S-glutathionylation. In pathophysiological settings, S-glutathionylation is tightly regulated. This perspective offers a concise overview of the emerging field of protein thiol redox modifications. We will also cover newly developed methodology to detect S-glutathionylation in situ, which will enable further discovery into the role of S-glutathionylation in biology and disease.

Reducing protein oxidation reverses lung fibrosis.

Idiopathic pulmonary fibrosis is characterized by excessive deposition of collagen in the lung, leading to chronically impaired gas exchange and death1-3. Oxidative stress is believed to be critical in this disease pathogenesis4-6, although the exact mechanisms remain enigmatic. Protein S-glutathionylation (PSSG) is a post-translational modification of proteins that can be reversed by glutaredoxin-1 (GLRX)7. It remains unknown whether GLRX and PSSG play a role in lung fibrosis. Here, we explored the impact of GLRX and PSSG status on the pathogenesis of pulmonary fibrosis, using lung tissues from subjects with idiopathic pulmonary fibrosis, transgenic mouse models and direct administration of recombinant Glrx to airways of mice with existing fibrosis. We demonstrate that GLRX enzymatic activity was strongly decreased in fibrotic lungs, in accordance with increases in PSSG. Mice lacking Glrx were far more susceptible to bleomycin- or adenovirus encoding active transforming growth factor beta-1 (AdTGFB1)-induced pulmonary fibrosis, whereas transgenic overexpression of Glrx in the lung epithelium attenuated fibrosis. We furthermore show that endogenous GLRX was inactivated through an oxidative mechanism and that direct administration of the Glrx protein into airways augmented Glrx activity and reversed increases in collagen in mice with TGFB1- or bleomycin-induced fibrosis, even when administered to fibrotic, aged animals. Collectively, these findings suggest the therapeutic potential of exogenous GLRX in treating lung fibrosis.

Ablation of the Thiol Transferase Glutaredoxin-1 Augments Protein S-Glutathionylation and Modulates Type 2 Inflammatory Responses and IL-17 in a House Dust Mite Model of Allergic Airway Disease in Mice.

S-glutathionylation has emerged as an oxidant-induced post-translational modification of protein cysteines that affects structure and function. The oxidoreductase glutaredoxin-1 (Glrx1), under physiological conditions, catalyzes deglutathionylation and restores the protein thiol group. The involvement of Grx1/S-glutathionylation in allergic inflammation induced by asthma-relevant allergens remains unknown. In the present study we examined the impact of genetic ablation of Glrx1 for the pathogenesis of house dust mite (HDM)-induced allergic airway disease in mice. Wild-type (WT) or Glrx1(-/-) mice in the BALB/c background were instilled intranasally with 50 µg of HDM 5 consecutive days for 3 weeks and killed 72 hours post final exposure. As expected, overall protein S-glutathionylation was increased in Glrx1(-/-) mice exposed to HDM as compared with WT animals. Total cells in the bronchoalveolar lavage fluid were similarly increased in WT and Glrx1(-/-) HDM-treated mice compared with phosphate-buffered saline-treated control mice. However, in response to HDM, mice lacking Glrx1 demonstrated significantly more neutrophils but fewer eosinophils than HDM-exposed WT mice. mRNA expression of the Th2-associated cytokine IL-13, as well as MUC5ac, was significantly attenuated in Glrx1(-/-) HDM-treated mice compared with WT mice. Conversely, expression of IL-17A was increased in Glrx1(-/-) HDM mice compared with WT mice. Last, HDM-induced tissue damping and elastance were significantly attenuated in Glrx1(-/-) mice compared with WT littermates. These results demonstrate that the Grx1/S-glutathionylation redox status plays a pivotal role in HDM-induced allergic inflammation and airway hyperresponsiveness and suggest a potential role of Glrx1/S-glutathionylation in controlling the nature of the HDM-induced adaptive immune responses by promoting Type-2-driven inflammation and restricting IL-17A.

JNK inhibition reduces lung remodeling and pulmonary fibrotic systemic markers.

BACKGROUND: Lung remodeling and pulmonary fibrosis are serious, life-threatening conditions resulting from diseases such as chronic severe asthma and idiopathic pulmonary fibrosis (IPF). Preclinical evidence suggests that JNK enzyme function is required for key steps in the pulmonary fibrotic process. However, a selective JNK inhibitor has not been investigated in translational models of lung fibrosis with clinically relevant biomarkers, or in IPF patients. METHODS: The JNK inhibitor CC-930 was evaluated in the house dust mite-induced fibrotic airway mouse model, in a phase I healthy volunteer pharmacodynamic study, and subsequently in a phase II multicenter study of mild/moderate IPF (n = 28), with a 4-week, placebo-controlled, double-blind, sequential ascending-dose period (50 mg QD, 100 mg QD, 100 mg BID) and a 52-week open-label treatment-extension period. RESULTS: In the preclinical model, CC-930 attenuated collagen 1A1 gene expression, peribronchiolar collagen deposition, airway mucin MUC5B expression in club cells, and MMP-7 expression in lung, bronchoalveolar lavage fluid, and serum. In the phase I study, CC-930 reduced c-Jun phosphorylation induced by UV radiation in skin. In the phase II IPF study, there was a CC-930 dose-dependent trend in reduction of MMP-7 and SP-D plasma protein levels. The most commonly reported adverse events were increased ALT, increased AST, and upper respiratory tract infection (six subjects each, 21.4 %). A total of 13 subjects (46.4 %) experienced adverse events that led to discontinuation of study drug. Nine out of 28 subjects experienced progressive disease in this study. The mean FVC (% predicted) declined after 26-32 weeks at doses of 100 mg QD and 100 mg BID. Changes in MMP-7, SP-D, and tenascin-C significantly correlated with change in FVC (% predicted). CONCLUSIONS: These results illustrate JNK enzymatic activity involvement during pulmonary fibrosis, and support systemic biomarker use for tracking disease progression and the potential clinical benefit of this novel intervention in IPF. Trial registration ClinicalTrials.gov NCT01203943.

Attenuation of lung fibrosis in mice with a clinically relevant inhibitor of glutathione-S-transferase π.

Idiopathic pulmonary fibrosis (IPF) is a debilitating lung disease characterized by excessive collagen production and fibrogenesis. Apoptosis in lung epithelial cells is critical in IPF pathogenesis, as heightened loss of these cells promotes fibroblast activation and remodeling. Changes in glutathione redox status have been reported in IPF patients. S-glutathionylation, the conjugation of glutathione to reactive cysteines, is catalyzed in part by glutathione-S-transferase π (GSTP). To date, no published information exists linking GSTP and IPF to our knowledge. We hypothesized that GSTP mediates lung fibrogenesis in part through FAS S-glutathionylation, a critical event in epithelial cell apoptosis. Our results demonstrate that GSTP immunoreactivity is increased in the lungs of IPF patients, notably within type II epithelial cells. The FAS-GSTP interaction was also increased in IPF lungs. Bleomycin- and AdTGFß-induced increases in collagen content, α-SMA, FAS S-glutathionylation, and total protein S-glutathionylation were strongly attenuated in Gstp-/- mice. Oropharyngeal administration of the GSTP inhibitor, TLK117, at a time when fibrosis was already apparent, attenuated bleomycin- and AdTGFß-induced remodeling, α-SMA, caspase activation, FAS S-glutathionylation, and total protein S-glutathionylation. GSTP is an important driver of protein S-glutathionylation and lung fibrosis, and GSTP inhibition via the airways may be a novel therapeutic strategy for the treatment of IPF.