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IntroductionEosinophils are critical in allergic disorders, and promoting eosinophil death effectively attenuates allergic airway inflammation. Ferroptosis is a recently described novel form of cell death; however, little is known about ferroptosis in eosinophils and related diseases. This study aimed to investigate the effects of ferroptosis-inducing agents (FINs) on eosinophil death and allergic airway inflammation, and to explore their potential synergistic effect with glucocorticoids (GCs).MethodsEosinophils isolated from the peripheral blood of humans or mice were incubated with FINs, and eosinophil ferroptosis was assessed. The in vivo effects of FINs alone or in combination with dexamethasone (DXMS) were examined in a mouse model of allergic airway inflammation. Bronchoalveolar lavage fluid and lung tissue were collected to examine airway inflammation.ResultsTreatment with FINs time and dose dependency induced cell death in human and mouse eosinophils. Interestingly, FINs induced non-canonical ferroptosis in eosinophils, which generated morphological characteristics unique to ferroptosis and was iron dependent but was independent of lipid peroxidation. The antioxidants glutathione and N-acetylcysteine significantly attenuated FIN-induced cell death. Treatment with FINs triggered eosinophil death in vivo and eventually relieved eosinophilic airway inflammation in mice. Furthermore, FINs exerted a synergistic effect with DXMS to induce eosinophil death in vitro and to alleviate allergic airway inflammation in vivo.ConclusionsFINs induced ferroptosis-like cell death of eosinophils, suggesting their use as a promising therapeutic strategy for eosinophilic airway inflammation, especially due to the advantage of their synergy with GCs in the treatment of allergic disorders.

Background and objective: Some types of T lymphocytes, especially cytotoxic T-cells (Tc1) and T-helper (Th17) cells, play a pivotal role in cigarette smokeinduced lung diseases. However, whether Tc17 cells are involved remains largely unknown.We investigated Tc17 involvement using a cigarette smoke-exposure model. Methods: Groups of mice were exposed to cigarette smoke or filtered air. At weeks 2, 8, 12 and 24, mice were sacrificed to observe histological changes by HE stain and/or immunohistochemical staining.The frequency of T cell subsets in the lung and spleen were detected by flow cytometry. In addition, the expression levels of T cellrelated factors were measured by real-time polymerase chain reaction or enzyme-linked immunosorbent assay. Results: Cigarette smoke caused substantial inflammatory cell infiltration and led to emphysema. Cigarette smoke exposure promoted the expression of interferon-gamma (IFN)-γ and interleukin (IL)-17A at the messenger ribonucleic acid and protein levels. In addition to Tc1 and Th17 cells, pulmonary and splenic Tc17 cells increased, which was accompanied by the upregulation of cytokines IL-6, transforming growth factor beta (TGF)-β) and transcriptional factors Stat3 and RAR-related orphan receptor gamma. Compared with untreated mice, γH2AX-positive cells were more frequently observed in mice exposed to cigarette smoke. Conclusions: Long-term cigarette smoke exposure induced Tc17 cell expansion both locally and distally, which was associated with emphysema and deoxyribonucleic acid damage. As an important source of IL-17A, this T cell subset may be a potential target for chronic obstructive pulmonary disease therapy.

Ozone is a common environmental air pollutant leading to respiratory illness. The mechanisms regulating ozone-induced airway inflammation remain poorly understood. We hypothesize that ozone-triggered inflammasome activation and interleukin (IL)-1 production regulate neutrophilic airway inflammation through IL-17A. Pulmonary neutrophilic inflammation was induced by extended (72 h) low-dose (0.7 ppm) exposure to ozone. IL-1 receptor 1 (Il1r1)−/−, Il17a−/− mice and the caspase-1 inhibitor acetyl-YVAD-chloromethylketone (Ac-YVAD-cmk) were used for in vivo studies. Cellular inflammation and protein levels in bronchial alveolar lavage fluid (BALF), cytokines, and IL-17A-producing γδT-cells, as well as mitochondrial reactive oxygen species (ROS), mitochondrial DNA (mtDNA) release, and inflammasome activation in lung macrophages were analyzed. Ozone-induced neutrophilic airway inflammation, accompanied an increased production of IL-1β, IL-18, IL-17A, Granulocyte-colony stimulating factor (G-CSF), Interferon-γ inducible protein 10 (IP-10) and BALF protein in the lung. Ozone-induced IL-17A production was predominantly in γδT-cells, and Il17a-knockout mice exhibited reduced airway inflammation. Lung macrophages from ozone-exposed mice exhibited higher levels of mitochondrial ROS, enhanced cytosolic mtDNA, increased caspase-1 activation, and higher production of IL-1β. Il1r1-knockout mice or treatment with Ac-YVAD-cmk decreased the IL-17A production and subsequent airway inflammation. Taken together, we demonstrate that ozone-induced IL-17A and neutrophilic airway inflammation is orchestrated by the caspase-1-IL-1 cascade.

IL-17 is known to play important roles in immune and inflammatory disease, such as in asthma, but its functions in allergic airway inflammation are still controversial, and the molecular mechanisms mediating these functions remain unclear. Increased production of eosinophils in bone marrow and their emergence in the airway have been linked to the onset and progression of allergic asthma. In this study, we investigated the effects of exogenous IL-17 on allergic airway inflammation and explored the underlying molecular mechanisms through eosinophil generation. Exogenous IL-17 significantly attenuated the features of allergic inflammation induced by ovalbumin in mice. It inhibited eosinophil differentiation both in vivo and in vitro, accompanied by down-regulated expression of CC chemokine receptor 3, GATA binding protein 1 (GATA-1), and GATA binding protein 2 (GATA-2), as well as reduced formation of common myeloid progenitors and eosinophil progenitors, but without influencing eosinophil apoptosis. IL-17 also significantly decreased the number of eosinophils in IL-5-transgenic mice, although it notably increased the levels of IL-3, IL-5, and granulocyte/macrophage colony-stimulating factor. In addition, IL-17 had little effect on secretion of the inflammatory cytokines by eosinophils. Neutralization of endogenous IL-17 significantly augmented eosinophil recruitment in the airways. Together, these findings suggest that exogenous IL-17 protects against allergic airway inflammation, most likely through inhibition of the eosinophil differentiation in bone marrow.

BackgroundThe therapeutic efficacy of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in advanced EGFR-mutant lung squamous cell carcinoma (SCC) patients remains uncertain. Furthermore, the factors underlying the responsiveness have not been fully investigated. We therefore investigated the link between genomic profiles and EGFR-TKI efficacy.Material and MethodsWe consecutively enrolled stage IV, EGFR-mutant, and EGFR-TKI–treated patients with SCC. Patients with EGFR wild-type lung SCC and EGFR-mutant lung adenocarcinoma were consecutively enrolled as controls, and next-generation sequencing (NGS) was performed.ResultsIn total, 28 EGFR-mutant lung SCC, 41 EGFR-mutant lung adenocarcinoma, and 40 EGFR wild-type lung SCC patients were included. Among the patients with EGFR mutations, shorter progression-free survival (PFS) was observed in SCC compared to adenocarcinoma (4.6 vs. 11.0 months, P<0.001). Comparison of the genomic profiles revealed that EGFR-mutant SCC patients had similar mutation characteristics to EGFR-mutant adenocarcinoma patients, but differed from those with EGFR wild-type SCC. Further exploration of EGFR-mutant SCC revealed that mutations in CREBBP (P = 0.005), ZNF217 (P = 0.016), and the Wnt (P = 0.027) pathway were negatively associated with PFS. Mutations in GRM8 (P = 0.025) were associated with improved PFS.ConclusionsEGFR-mutant lung SCC has a worse prognosis than EGFR-mutant adenocarcinoma. Mutations in other genes, such as CREBBP, ZNF217, GRM8, or Wnt that had implications on PFS raise the possibility of understanding mechanisms of resistance to EGFR-TKI in lung SCC, which will aid identification of potential beneficial subgroups of patients with EGFR-mutant SCCs receiving EGFR-TKIs.

Eosinophil infiltration is considered a hallmark in allergic airway inflammation, and the blockade of eosinophil differentiation may be an effective approach for treating eosinophil-related disorders. Mammalian target of rapamycin (mTOR) is a vital modulator in cell growth control and related diseases, and we have recently demonstrated that rapamycin can suppress eosinophil differentiation in allergic airway inflammation. Considering its critical role in haematopoiesis, we further investigated the role of mTOR in eosinophil differentiation in the context of asthmatic pathogenesis. Intriguingly, the inhibition of mTOR, either by genetic deletion or by another pharmacological inhibitor torin-1, accelerated the eosinophil development in the presence of IL-5. However, this was not observed to have any considerable effect on eosinophil apoptosis. The effect of mTOR in eosinophil differentiation was mediated by Erk signalling. Moreover, myeloid specific knockout of mTOR or Rheb further augmented allergic airway inflammation in mice after allergen exposure. Ablation of mTOR in myeloid cells also resulted in an increased number of eosinophil lineage-committed progenitors (Eops) in allergic mice. Collectively, our data uncovered the differential effects of mTOR in the regulation of eosinophil development, likely due to the distinct functions of mTOR complex 1 or 2, which thus exerts a pivotal implication in eosinophil-associated diseases.

BackgroundImmune checkpoint inhibitor (ICI) monotherapy remains the standard of care for patients with previously treated non-small cell lung cancer. However, few reports have compared the clinical benefits of second-line ICIs alone with those of ICIs combined with other therapies, including anti-angiogenesis therapy or chemotherapy.MethodsPatients with previously treated advanced non-small cell lung cancer who received ICIs were retrospectively reviewed. The progression-free survival (PFS), overall survival, objective response rate, disease control rate, and safety were assessed. Complete blood cell counts and serum lactate dehydrogenase (LDH) levels were measured before and after ICI treatment.ResultsOf 120 patients, 75 were treated with ICI monotherapy, 26 with ICIs plus anti-angiogenic therapy (ICI+A), and 19 with ICIs plus chemotherapy (ICI+C). The objective response rate was significantly higher in the ICI+C group (57.9%) than ICI monotherapy (26.3%) and ICI+A (31.8%) groups. The depth of response was significantly greater in the ICI+C (-35.1%) than ICI+A (−2.04%) and ICI monotherapy (3.963%) groups. ICI+C afforded a better PFS compared with the ICI monotherapy and ICI+A groups (8.5 vs. 4.6 and 4.1 months, respectively). Notably, the pre- and post-treatment peripheral neutrophil/lymphocyte ratios and serum LDH levels were negatively correlated with the PFS of the entire cohort. More importantly, the pretreatment lung immune prognostic index (neutrophil/lymphocyte ratio ≥ 4 and LDH level ≥ upper limit of normal) satisfactorily predicted the responses to ICI-based strategies. Adverse events (AEs) occurred in 65.3%, 92.3%, and 94.7% of patients in the ICI monotherapy, ICI+A, and ICI+C groups, respectively. Grade 3–5 AEs were more common in the combination therapy groups (ICI+A, 19.2%; ICI+C, 21%; ICI monotherapy, 4%).ConclusionIn second-line settings and beyond, ICIs combined with chemotherapy prolonged survival, with tolerable AEs. Addition of anti-angiogenic agents to ICIs did not afford any additional benefits. Further prospective studies are warranted.