Elevated CD4+ T Cell Senescence Associates with Impaired Immune Responsiveness in Severe COVID-19.

Aging is a critical risk factor for unfavorable clinical outcomes among COVID-19 patients and may impact vaccine efficacy. However, whether the senescence of T cells is associated with severe COVID-19 outcome in elderly individuals is unclear. Using flow cytometry, we analyzed the frequency of senescent T cells (Tsens) in peripheral blood from 100 hospitalized elderly COVID-19 patients and compared differences between those with mild/moderate and severe/critical illness. We also assessed correlations between the percentage of Tsens and the quantity and quality of spike-specific antibodies by ELISA, neutralizing antibody test kit, and ELISPOT assay respectively, the cytokine production profile of COVID-19 reactive T cells, and plasma soluble factors by cytometric bead array (CBA). Our study found a significantly elevated level of CD4+ Tsens in patients with severe/critical disease compared to those with mild/moderate illness. Patients with a higher level of CD4+ Tsens (>19.78%) showed a decreased survival rate compared to those with a lower level (≤19.78%). This is more pronounced among patients with breakthrough infections. The percentage of CD4+ Tsens was negatively correlated with spike-specific antibody titers, neutralization ability, and COVID-19 reactive IL-2+CD4+ T cells. In addition, spike-specific antibody levels were positively correlated with IL-2 producing T cells and plasma IL-2 amount. Mechanistically, with defective CD40L, T cells from patients with CD4+ Tsens >19.78% were unable to support B cell proliferation and differentiation. Our data demonstrate that the percentage of CD4+ Tsens in peripheral blood may serve as a reliable biomarker for the prognosis of severe COVID-19 patients, especially in breakthrough infections. Therefore, restoring the immune response of CD4+ Tsens may be key to preventing severe illness and improving vaccine efficacy in older adults.

Causal relationship between frailty and chronic obstructive pulmonary disease or asthma: A two sample bidirectional Mendelian randomization study.

BACKGROUND: Observational studies have established a strong association between frailty and obstructive lung diseases. However, the causal nature of this association remains unclear. To address this gap, we conducted a bidirectional Mendelian randomization (MR) study to investigate the causal relationship between frailty, as measured by the frailty index (FI), and chronic obstructive pulmonary disease (COPD) or asthma. METHODS: The latest meta-analysis of genome-wide association studies for FI, which included individuals of European ancestry from UK Biobank and TwinGene (N = 175,226), yielded the genetic instruments for frailty and outcome summary statistics. The genetic instrument for COPD and asthma, as well as the outcome summary data, were derived from the GWAS conducted on individuals of European ancestry from the FinnGen, with a sample size of 16,410 cases and 283,589 controls for COPD, and 37,253 cases and 187,112 controls for asthma. The analysis of MR was conducted employing the inverse-variance weighted (IVW) method, complemented by the weighted median method, MR-Egger regression, and MR pleiotropy residual sum and outlier (MR-PRESSO) test. RESULTS: Our results showed that genetically predicted higher FI was significantly associated with increased risk of COPD (odds ratio [OR] 1.75, 95 % confidence interval [CI] 1.29-2.36) and asthma (OR 2.10, 95 % CI 1.44-3.16). In the reverse direction analysis, genetic liability to both COPD (beta 0.06, 95 % CI 0.01-0.10) and asthma (beta 0.08, 95 % CI 0.06-0.11) showed significant associations with a higher FI. CONCLUSIONS: Our research has reinforced the existing evidence supporting a reciprocal causal relationship between frailty and obstructive lung diseases. A deeper comprehension of this interconnection is imperative for the prevention and treatment of obstructive lung diseases.

Risk factors and biomarkers for post-tuberculosis lung damage in a Chinese cohort of male smokers and non-smokers: protocol for a prospective observational study.

INTRODUCTION: Post-tuberculosis lung damage (PTLD) refers to the residual pulmonary impairment following the completion of antituberculosis (TB) therapy, characterised by persistent respiratory symptoms and abnormal pulmonary function. The risk factors and biomarkers for PTLD have been scarcely investigated. More importantly, whether and to what extent cigarette smoking is involved in PTLD remain to be known. METHODS AND ANALYSIS: This prospective observational study will enrol 400 male smoking or non-smoking patients aged 25-65 years, with newly confirmed active TB between 2022 and 2024, from the Department of Respiratory and Critical Care Medicine at Peking University Third Hospital and the Tuberculosis Department at Beijing Geriatric Hospital. Because females rarely smoke in China, we will enrol only males in this study. Demographic data, smoking history and amount, clinical symptoms, lung function, and chest CT findings will be prospectively collected. Respiratory questionnaires, lung function measurements and chest CT examinations will be performed immediately after, and 1 year, 2 years and 3 years after the completion of TB treatment. Peripheral blood samples will be obtained at baseline and at the end of anti-TB therapy, and a Luminex xMAP-based multiplex immunoassay will be used to measure inflammatory mediators and cytokines in serum. The collected data will be analysed to determine the incidence and factors/biomarkers of PTLD according to smoking status. ETHICS AND DISSEMINATION: The study was approved by the Ethics Committee of Peking University Third Hospital (approval number: (2022)271-03; approval date: 8 June 2022). The research results will be disseminated through scientific and medical conferences and will be published in an academic journal. TRIAL REGISTRATION NUMBER: NCT04966052.

More severe lung lesions in smoker patients with active pulmonary tuberculosis were associated with peripheral NK cell subsets.

BACKGROUND: Both pulmonary tuberculosis (PTB) and cigarette smoke (CS) exposure may lead to lung damage. The potential impact of CS exposure on tuberculosis-associated lung damage and the disturbance of immune cells and mediators involved, need to be further elucidated. METHODS: We firstly evaluated the chest X-ray (CXR) scores of a retrospective cohort of male patients with active PTB, followed for 6 months, and compared the scores between smoker (≥10 pack-years) and non-smoker patients. In a cross-sectional study, we measured the peripheral blood NK cell subsets and plasma inflammatory cytokines in male smoker and non-smoker patients with active PTB before anti-tuberculosis therapy, and the proportions of NK cell subsets and the levels of cytokines were analyzed for correlation with the CXR scores. RESULTS: In the retrospective cohort, male smoker patients with active PTB showed a higher CXR score, characterized by more cavitary lesions, enlarged lymph nodes and emphysema, as compared to non-smokers. The cross-sectional study revealed that the CXR score in smoker patients was correlated inversely with the percentages of blood CD107a+, NKP46+, and TIGIT+ NK cells. CONCLUSION: In patients with active PTB, CS exposure was associated with more severe lung lesions, which were correlated with peripheral NK cell subsets.

IL-17A Promotes Epithelial ADAM9 Expression in Cigarette Smoke-Related COPD.

Background: It has been reported that a disintegrin and metalloproteinase 9 (ADAM9) is involved in the pathogenesis of cigarette smoke (CS)-associated chronic obstructive pulmonary disease (COPD). But how CS exposure leads to upregulation of ADAM9 remains unknown. Methods: Patients who underwent lobectomy for a solitary pulmonary nodule were enrolled and divided into three groups: non-smokers with normal lung function, smokers without COPD and smoker patients with COPD. Immunoreactivity of interleukin (IL)-17A and ADAM9 in small airways and alveolar walls was measured by immunohistochemistry. Wild-type and Il17a -/- C57BL/6 mice were exposed to CS for six months, and ADAM9 expression in the airway epithelia was measured by immunoreactivity. In addition, the protein and mRNA expression levels of IL-17A and ADAM9 were assessed in CS extract (CSE) and/or IL-17A-treated human bronchial epithelial (HBE) cells. Results: The immunoreactivity of ADAM9 was increased in the airway epithelia and alveolar walls of patients with COPD compared to that of the controls. The expression of IL-17A was also upregulated in airway epithelial cells of patients with COPD and correlated positively with the level of ADAM9. The results from the animal model showed that Il17a -/- mice were protected from emphysema induced by CS exposure, together with a reduced level of ADAM9 expression in the airway epithelia, suggesting a possible link between ADAM9 and IL-17A. Consistently, our in vitro cell model showed that CSE stimulated the expression of ADAM9 and IL-17A in HBE cells in a dose- and time-dependent manner. Recombinant IL-17A induced ADAM9 upregulation in HBE cells and had a synergistic effect with CSE, whereas blocking IL-17A inhibited CSE-induced ADAM9 expression. Further analysis revealed that IL-17A induced c-Jun N-terminal kinase (JNK) phosphorylation, thereby increasing ADAM9 expression. Conclusion: Our results revealed a novel role of IL-17A in CS-related COPD, where IL-17A contributes to ADAM9 expression by activating JNK signaling.

Complement component 3 protects human bronchial epithelial cells from cigarette smoke-induced oxidative stress and prevents incessant apoptosis.

The complement component 3 (C3) is a pivotal element of the complement system and plays an important role in innate immunity. A previous study showed that intracellular C3 was upregulated in airway epithelial cells (AECs) from individuals with end-stage chronic obstructive pulmonary disease (COPD). Accumulating evidence has shown that cigarette smoke extract (CSE) induces oxidative stress and apoptosis in AECs. Therefore, we investigated whether C3 modulated cigarette smoke-induced oxidative stress and apoptosis in AECs and participated in the pathogenesis of COPD. We found increased C3 expression, together with increased oxidative stress and apoptosis, in a cigarette smoke-induced mouse model of COPD and in AECs from patients with COPD. Different concentrations of CSEinduced C3 expression in 16HBE cells in vitro. Interestingly, C3 knockdown (KD) exacerbated oxidative stress and apoptosis in 16HBE cells exposed to CSE. Furthermore, C3 exerted its pro-survival effects through JNK inhibition, while exogenous C3 partially rescued CSE-induced cell death and oxidative stress in C3 KD cells. These data indicate that locally produced C3 is an important pro-survival molecule in AECs under cigarette smoke exposure, revealing a potentially novel mechanism in the pathogenesis of COPD.

NK Cells in the Pathogenesis of Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a prevalent chronic airway disease with varied frequencies of acute exacerbations, which are the main cause of morbidity and mortality of the disease. It is, therefore, urgent to develop novel therapies for COPD and its exacerbations, which rely heavily on understanding of the pathogenesis and investigation for potential targets. Current evidence indicates that natural killer (NK) cells play important roles in the pathological processes of COPD. Although novel data are revealing the significance of NK cells in maintaining immune system homeostasis and their involvement in pathogenesis of COPD, the specific mechanisms are largely unknown. Specific and in-depth studies elucidating the underlying mechanisms are therefore needed. In this review, we provided a brief overview of the biology of NK cells, from its development to receptors and functions, and outlined their subsets in peripheral blood and lungs. Then we reviewed published findings highlighting the important roles played by NK cells in COPD and its exacerbations, with a view of providing the current state of knowledge in this area to facilitate related in-depth research.

RANKL Mediates Muscle Atrophy and Dysfunction in a Cigarette Smoke-induced Model of Chronic Obstructive Pulmonary Disease.

Skeletal muscle dysfunction is one of the important comorbidities of chronic obstructive pulmonary disease (COPD); however, the underlying mechanisms remain largely unknown. RANKL (receptor activator of nuclear factor κB ligand), a key mediator in osteoclast differentiation, was also found to play a role in skeletal muscle pathogenesis. Whether RANKL is involved in COPD-related skeletal muscle dysfunction is as-of-yet unknown. We examined the expression of RANKL/RANK in skeletal muscles from mice exposed to cigarette smoke (CS) for 24 weeks. Grip strength and exercise capacity as well as muscular morphology were evaluated in CS-exposed mice with or without anti-RANKL treatment. The expressions of protein synthesis- or muscle growth-related molecules (IGF-1, myogenin, and myostatin), muscle-specific ubiquitin E3 ligases (MuRF1 and atrogin-1), and the NF-κb inflammatory pathway were also evaluated in skeletal muscles. The effect of CS extract on RANKL/RANK expression and that of exogenous RANKL on the ubiquitin-proteasome pathway in C2C12 myotubes were investigated in vitro. Long-term CS exposure induced skeletal muscle dysfunction and atrophy together with upregulation of RANKL/RANK expression in a well-established mouse model of COPD. RANKL neutralization prevented skeletal muscle dysfunction and atrophy. RANKL inhibition decreased expressions of myostatin and MuRF1/Atrogin1 and suppressed the NF-κb pathway in skeletal muscles from CS-exposed mice. In in vitro experiments with C2C12 myotubes, CS extract induced expression of RANKL/RANK, and exogenous RANKL induced activation of the ubiquitin-proteasome pathway and NF-κb pathway via RANK. Our results revealed an important role of the RANKL/RANK pathway in muscle atrophy induced by CS exposure, suggesting that RANKL may be a potential therapeutic target in COPD-related skeletal muscle dysfunction.

Administration route governs the therapeutic efficacy, biodistribution and macrophage targeting of anti-inflammatory nanoparticles in the lung.

BACKGROUND: Uncontrolled inflammation is a central problem for many respiratory diseases. The development of potent, targeted anti-inflammatory therapies to reduce lung inflammation and re-establish the homeostasis in the respiratory tract is still a challenge. Previously, we developed a unique anti-inflammatory nanodrug, P12 (made of hexapeptides and gold nanoparticles), which can attenuate Toll-like receptor-mediated inflammatory responses in macrophages. However, the effect of the administration route on its therapeutic efficacy and tissue distribution remained to be defined. RESULTS: In this study, we systematically compared the effects of three different administration routes [the intratracheal (i.t.), intravenous (i.v.) and intraperitoneal (i.p.)] on the therapeutic activity, biodistribution and pulmonary cell targeting features of P12. Using the LPS-induced ALI mouse model, we found that the local administration route via i.t. instillation was superior in reducing lung inflammation than the other two routes even treated with a lower concentration of P12. Further studies on nanoparticle biodistribution showed that the i.t. administration led to more accumulation of P12 in the lungs but less in the liver and other organs; however, the i.v. and i.p. administration resulted in more nanoparticle accumulation in the liver and lymph nodes, respectively, but less in the lungs. Such a lung favorable distribution was also determined by the unique surface chemistry of P12. Furthermore, the inflammatory condition in the lung could decrease the accumulation of nanoparticles in the lung and liver, while increasing their distribution in the spleen and heart. Interestingly, the i.t. administration route helped the nanoparticles specifically target the lung macrophages, whereas the other two administration routes did not. CONCLUSION: The i.t. administration is better for treating ALI using nanodevices as it enhances the bioavailability and efficacy of the nanodrugs in the target cells of the lung and reduces the potential systematic side effects.

Transient Receptor Potential Cation Channel Subfamily V Member 4 Mediates Pyroptosis in Chronic Obstructive Pulmonary Disease.

TRPV4, a calcium permeable cation selective channel, was found to be involved in chronic obstructive pulmonary disease (COPD) through releasing ATP and IL-1ß. Pyroptosis, a newly discovered pro-inflammatory cell death, was induced by cigarette smoke (CS) in airway epithelial cells (AECs). More recent studies indicated that blocking Ca2+ influx effectively inhibited pyroptosis. Therefore, we asked whether TRPV4 mediated CS-induced pyroptosis of AECs and hence participated in the pathogenesis of COPD. We found that pyroptosis and TRPV4 were upregulated in AECs from patients with COPD and long-term CS-exposed mice. Moreover, pharmacological inhibition or knockdown of TRPV4 function alleviated CS extract (CSE)-induced pyroptosis by inhibiting NACHT, LRP, PYD domains-containing protein 3 (NLRP3) inflammasome/activated caspase-1/gasdermin D pathway, decreasing the number of PI positive cells and lactate dehydrogenase (LDH) release, decreasing the expression of pro- inflammatory interleukin gene (IL)-1ß, IL-8, and IL-18 expression, as well as increasing anti-inflammatory gene expression [NAD(P)H quinone dehydrogenase 1 (NQO1), superoxide dismutase 2 (mitochondrial) (MNSOD), and catalase, (CAT)]. Moreover, pharmacological inhibition or knockdown of TRPV4 function significantly relieved CSE-induced mitochondrial damage including decreased mitochondrial membrane potential, mitochondrial fusion protein (OPA1, MFN2) expression, and increased mitochondrial fission protein (DRP1, MFF) expression. Taken together, these findings indicate that TRPV4 mediates AEC pyroptosis via NLRP3/caspase-1/GSDMD pathway in COPD.

Enhanced Proinflammatory Cytokine Production and Immunometabolic Impairment of NK Cells Exposed to Mycobacterium tuberculosis and Cigarette Smoke.

Aim: Smoker COPD patients with chest radiological signs of prior tuberculosis (TB) showed more severe lung damage, but the mechanisms remain unclear. Emerging evidence has implicated NK cells in the pathogenesis of both COPD and TB. The purpose of this study was to delineate the profile and cytokine production of NK-cell subpopulations and their immunometabolic changes after exposure to both cigarette smoke (CS) and Mycobacterium tuberculosis(MTB). Methods: We profiled NK-cell subpopulations in terms of percentage and cytokine production by flow cytometry in smoker patients with pulmonary TB (PTB). In an in vitro coexposure model, we investigated proinflammatory cytokine production, glycolytic influx, and oxidative phosphorylation of NK cells under CS extract (CSE) and PPD costimulation. Results: Peripheral blood NK cells in smoker patients with active PTB (CS+PTB group) showed altered proportion of subpopulations and excessive proinflammatory cytokine expressions. In vitro, CSE- and PPD-coexposed NK-92 cells displayed enhanced proinflammatory cytokine production, concurrent with decreased glycolytic influx and oxidative phosphorylation. Conclusion: Smoker patients with active PTB showed enhanced proinflammatory cytokine expression within altered NK cell subpopulations. CSE and PPD coexposure induced heightened cytokine production concurrent with impaired cell metabolism in NK cells. These novel data suggest a potential role of NK cells in the pathogenesis of lung injury in subjects with coexposure to CS and TB.

Infection of Mycobacterium tuberculosis Promotes Both M1/M2 Polarization and MMP Production in Cigarette Smoke-Exposed Macrophages.

Pulmonary tuberculosis (PTB) is a risk factor for COPD. Our previous study revealed more severe emphysema in COPD patients (mostly smokers) with prior tuberculosis. However, the mechanisms of interactions between cigarette smoke (CS) and Mycobacterium tuberculosis (Mtb) are unknown. In this study, we found that the frequencies of both M1 and M2 macrophages, and levels of MMP9 and MMP12 in bronchoalveolar lavage were increased in PTB patients with smoking. Between-group analysis showed that the frequency of M1 macrophages was higher in non-smoker PTB patients while more M2 macrophages were found in smokers without PTB, as compared to the non-smoker healthy controls. Bacille Calmette-Guérin (BCG) infection in CS extract (CSE)-incubated MH-S cells further enhanced secretion of M1-related (iNOS, IFN-γ and TNF-α) and M2-related (TGF-ß and IL-10) cytokines, reactive oxygen species (ROS) production and cellular apoptosis, concomitantly with up-regulation of MMP9 and MMP12, but not TIMP1. Moreover, BCG infection in acutely CS-exposed mice promoted macrophage polarization toward both M1 and M2 phenotypes, along with increased lung inflammatory infiltration. MMP9 and MMP12, but not TIMP1, were further up-regulated in lung tissues and BAL fluid after BCG infection in this model. Taken together, Mtb Infection promoted CS-exposed macrophages to polarize toward both M1 and M2 phenotypes, along with enhanced production of MMP9 and MMP12. These findings provide insights into the mechanistic interplay between CS exposure and tuberculosis in the pathogenesis of COPD.

Cigarette Smoke-Induced Lymphoid Neogenesis in COPD Involves IL-17/RANKL Pathway.

IL-17 is critical in lung lymphoid neogenesis in COPD, but the cellular and molecular mechanisms remain to be elucidated. Receptor activator of nuclear factor-κB ligand (RANKL) functions in lymphoid follicle formation in other organs, whether it is involved in IL-17A-dependent lymphoid neogenesis in COPD is unknown. To elucidate the expression and functional role of IL-17A/RANKL pathway in COPD. We first quantified and localized RANKL, its receptor RANK and IL-17A in lungs of patients with COPD, smokers and non-smokers. Next, IL-17A-/- and wild-type (WT) mice were exposed to air or cigarette smoke (CS) for 24 weeks, and lung lymphoid follicles and RANKL-RANK expression were measured. Lastly, we studied the in vitro biological function of RANKL pertaining to lymphoid neogenesis. We found that the expressions of RANKL-RANK and IL-17A, together with lymphoid follicles, were increased in lung tissues from patients with COPD. In WT mice exposed to CS, RANKL-RANK expressions were prominent in lung lymphoid follicles, which were absent in IL-17A-/- mice exposed to CS. In the lymphoid follicles, RANKL+ cells were identified mostly as B cells and RANK was localized in dendritic cells (DCs). In vitro IL-17A increased the expressions of RANKL in B cells and RANK in DCs, which in turn responded to RANKL stimulation by upregulation of CXCL13. Altogether, these results suggest that B lymphocyte RANKL pathway is involved in IL-17A-dependent lymphoid neogenesis in COPD.

Enhanced Anti-inflammatory Activity of Peptide-Gold Nanoparticle Hybrids upon Cigarette Smoke Extract Modification through TLR Inhibition and Autophagy Induction.

Overwhelming uncontrolled inflammation is the hallmark of pathophysiological features of many acute and chronic inflammatory diseases, such as sepsis and allergy and autoimmune disorders. It is important to develop potent pharmacological interventions to effectively control such detrimental inflammatory reactions in these diseases. Recently, we have developed a special class of peptide-gold nanoparticle hybrid system that can inhibit Toll-like receptor 4 (TLR4) signal transduction pathways and decrease its downstream inflammatory responses. Herein, we serendipitously discovered that a tiny amount of cigarette smoke extract (CSE, 1%) was able to significantly enhance the inhibitory activity of the hybrids on TLR4-mediated inflammatory responses. Mechanistically, it was found that active components in CSE were able to adsorb onto the hybrids and largely increased their cellular uptake in THP-1 cell-derived macrophages. Such high cellular uptake not only enhanced the inhibition on the endosomal acidification required for TLR4 activation but also contributed to autophagy induction and subsequent antioxidant protein expression. Consequently, this duel action strengthened the anti-inflammatory activity of the hybrids in cells and in an acute lung injury (ALI) mouse model. This work aids our fundamental understanding of nanoparticles regulating the innate immune responses. It also provides a new way to design potent anti-inflammatory nanotherapeutics for inflammatory diseases such as ALI.