Relationship of influenza virus to inflammatory factors and immune function in elderly patients with COPD: A retrospective analysis

Abstract

Objective

The aim of this retrospective study was to investigate the relevance of influenza A virus (IAV) in acutely exacerbating airway inflammatory response and disrupting immune function in elderly COPD patients.

Methods

The group conducted a pre-test: using multiplex combined real-time PCR detection kits Multiple real⁃time PCR was used to detect twenty-four pathogens, 385 patients clinically diagnosed with COPD were tested for viral nucleic acid in throat swabs. At the same time, peripheral blood leukapheresis was collected from both groups of patients, and their IL-6, IL-8, IL-1β, and TNF-α levels were detected, along with the levels of T-cell differentiation markers CD4 and CD8, to assess the influence of influenza virus on the immune function of elderly COPD patients and its relevance to the acute exacerbation of airway inflammatory response in elderly COPD patients.

Results

Results showed that the expression of inflammatory cytokines IL-6, IL-8, IL-1β and TNF-α was significantly higher in the viral group compared with the non-infected group (P < 0.05, P < 0.01). The levels of T cell differentiation type markers CD4 and CD8 were significantly lower in the infected group compared with the uninfected group.

Conclusion

Influenza virus further exacerbated airway inflammatory response and decreased the immune function of T cells by activating intrinsic immune molecules such as IL-6, IL-8, IL-1β and TNF-α.

Keywords

Influenza virus elderly chronic obstructive pulmonary disease inflammatory response immune function

Introduction

Chronic obstructive pulmonary disease (COPD) is an incurable global disease characterized by persistent airflow obstruction and persistent emphysema or chronic bronchitis. Common symptoms include persistent chronic cough and sputum, shortness of breath or frequent breathlessness, wheezing, chest tightness, and intermittent fatigue. Smoking is the leading cause of COPD, accounting for more than 90% of COPD cases in developed countries.^1,2 COPD patients have different symptoms at different stages of the disease.³ According to the 2018 World Health Organization classification method and criteria, lung volume was used to determine the stage of COPD, and it was divided into 4 stages, which corresponded to different degrees of lung function lesions.⁴ These four stages correspond to different degrees of lung function. Other studies have shown that chronic respiratory diseases have become the top three diseases in the global mortality rate^5,6 COPD is the leading cause of mortality worldwide. Although the clinical attention to COPD has been increasing in recent years, the research on its pathogenesis and treatment still needs to be further deepened.

Influenza virus, short for influenza virus, is the etiologic agent of the zoonotic infectious disease that causes influenza. Influenza viruses are categorized into influenza A, B, and C viruses based on antigenic differences in their nucleoprotein properties. The viral surface antigens Hemagglutinin (HA) and Neuraminidase (NA) are further divided into 16 subtypes of HA (H1-H16) and 9 subtypes of NA (N1-N9), and further combinations of HA and NA can be used to synthesize multiple subtypes of influenza A. Influenza B and C viruses are no longer divided into subtypes. Influenza B and C viruses are no longer subtyped.⁷ In 2019, influenza activity in mainland China significantly increased compared to the previous three years, with the virus types still dominated by H3N2 and H1N1.^8,9 H3N2 and H1N1 viruses continue to dominate. On the one hand, because influenza viruses are prone to antigenic mutation, A revised version could be: “the population is generally susceptible to mutated strains, and there is a shortage of influenza vaccinations in China. Therefore, we must attach great importance to the high morbidity and mortality rates of influenza viruses, which are climbing year by year. According to the latest influenza disease burden survey in China, there are 88,100 influenza-associated excess respiratory deaths per year on average nationwide, with an excess respiratory mortality rate of 6.5/100,000, and about 80% of influenza-associated excess respiratory deaths occur in middle-aged and elderly people aged 60 years and above. The excess respiratory mortality rate varies among influenza virus types/subtypes, with type A (H3N2) predominating.¹⁰ Meanwhile, influenza virus HA and NA proteins undergo antigenic mutation at a point mutation frequency of 1% per year.¹¹ The rate of antigenic mutation in the HA and NA proteins of influenza virus is 1% per year. Therefore, it is urgent to study the molecular mechanism of influenza virus-induced chronic acute exacerbations, especially the pathogenesis of seasonal influenza A virus (IAV/H3N2).

This retrospective study focuses on the effect of influenza A virus on airway inflammation in elderly COPD patients. With the increasing understanding of the intrinsic immune response, it is likely that influenza virus infection induces AECOPD because it affects the body's intrinsic immune response, which in turn exacerbates the inflammatory response in COPD. Influenza virus infection in COPD patients does not directly cause respiratory symptoms, but rather activates the immune system through the replication of influenza virus in the patient's body, causing systemic inflammation. It has been reported in the literature that, compared with AECOPD without influenza virus infection, influenza virus-positive patients with AECOPD have higher clinical symptom scores, more inflammatory cytokine expression in the blood, longer disease recovery time, and higher inflammatory cytokine expression in the blood. amounts, longer disease recovery times and more frequent acute exacerbations than AECOPD without influenza virus infection. This study aims to explore the pathogenic mechanisms and clinical manifestations of influenza virus in COPD patients, providing new insights into the prevention and management of influenza in this population.

Materials and methods

Case selection

Our hospital successfully collected pharyngeal swabs from 385 AECOPD inpatients from April 1, 2021 to April 30, 2023, of which 184 were confirmed to be infected with the virus and 201 were not infected. At the end of the pre-examination, we tested 184 endogenously positive patients and found that 104 patients tested positive for respiratory viruses. We will analyze the infection type of these patients. Approved by the Ethics Committee (No.BFHHZS20240299)

Inclusion and exclusion criteria

Inclusion criteria: (1) age ≥60 years; (2) clinically diagnosed to meet the diagnostic criteria related to COPD, and after inhaling 400 mg of salbutamol for 15 min, the ratio of the forced expiratory volume in one second (FEV1) to the forced vital capacity (FVC) ratio < 70%, all of which met the diagnostic criteria of the 2017 GOLD guidelines. (3) The patient's clinical data were complete.

Exclusion criteria: (1) those with severe cardiac, hepatic, and pulmonary diseases; (2) those with incomplete clinical data; (3) those with infections at other sites; and (4) those with a history of treatment with glucocorticoids and other medications.

Influenza virus detection

Respiratory samples, including sputum, tracheal aspirates, and throat swabs, were collected for viral nucleic acid detection. Sputum and tracheal aspirates were digested using Sputasol™ solution (Thermo Fisher Scientific, USA), and viral nucleic acids were extracted from all processed samples using the QIAamp Viral RNA Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. Multiplex combined real-time PCR was performed in a 25 μL reaction system consisting of 17.2 μL enzyme mix, 0.8 μL enzyme mix, and 5 μL primer pool. The cycling conditions were as follows: 42°C for 10 min for reverse transcription, 94°C for 10 s for initial denaturation, followed by 40 cycles of 94°C for 5 s, 56°C for 50 s, and 72°C for 15 s, with fluorescence signals collected during the annealing step on the ABI 7500 Real-Time PCR System (Applied Biosystems, USA). The assay simultaneously detected Influenza A virus, Influenza B virus (including FluB-V and FluB-Y), Parainfluenza virus types 1–4, human coronaviruses (HCoV-229E, HCoV-HKU, HCoV-NL63, HCoV-OC43, SARS-CoV-2), Adenovirus, Human Parvovirus, Respiratory Syncytial Virus, Enterovirus, and Rhinovirus. Positive and negative controls provided by the Kit were included in each run to ensure the accuracy of results.

Peripheral blood sample collection and detection

Peripheral blood samples were collected in heparinized anticoagulation tubes, and single nucleated cells were isolated using Ficoll density gradient centrifugation. Specifically, Ficoll-Paque Plus (Amersham Biosciences, UK) was used to separate peripheral blood. After centrifugation (400 × g, 20 min, 21°C), the middle white mist layer was carefully aspirated, while cells at the bottom layer were collected. These cells were washed twice with Hanks’ balanced salt solution (centrifugation: 300 × g, 5 min, 4°C).

The levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in cell supernatants were quantified using enzyme-linked immunosorbent assay (ELISA). Flow cytometry was employed to detect T-cell differentiation markers CD4 and CD8 in peripheral blood mononuclear cells (PBMCs). These markers were analyzed to assess the effects of influenza virus on immune function and its relevance to the inflammatory response during acute exacerbations of airway inflammation in elderly patients with chronic obstructive pulmonary disease (COPD).

Statistics

Results were analyzed using GraphPad Prism 9 (San Diego, California) and SPSS 21.0 (IBM,Armonk, NY, USA). Data were analyzed at x ± Multiple sample mean comparisons were performed using one-way ANOVA, and two-by-two comparisons were performed using the SNK-q test. Differences were statistically significant when P < 0.05 and significant when P < 0.01.

Results

Comparison of pre-test basic patient information

Among all the samples, 184 cases were virally infected, and 201 cases were non-virally infected. AECOPD was mainly male, accounting for 65.17% (120/184); the age distribution of the population was wide, ranging from middle-aged people over 60 years old to super-elderly people over 96 years old, mainly concentrated in the age group of 60 to 79 years old. Among them, there were 120 males and 64 females. The main clinical manifestations of patients were cough and sputum, shortness of breath and dyspnea after activities, and mMRC grade 4 was the main symptom, accounting for 78.73% (145/184). AECOPD patients with respiratory viral infection had more obvious palpitations than those without respiratory viral infection. Comprehensive analysis revealed that respiratory viral infections were significantly correlated with fever, palpitations, length of hospitalization, glucocorticoid treatment, lipid metabolism and cardiac failure in AECOPD patients. Compared with the uninfected respiratory virus group, combined respiratory viral infection had no significant effect on the typical symptoms of COPD, but the number of fever cases and palpitation symptoms caused by viral infection in COPD patients were significantly higher than those in other groups (P < 0.05). Compared with the analysis, with P value respiratory virus is more likely to be infected in patients with abnormal lipid metabolism, and at the same time, viral infection is more likely to cause the occurrence of cardiac function abnormalities compared with those without respiratory virus infection (P < 0.05). Moreover, respiratory virus infection resulted in a longer duration of illness in AECOPD patients (16.87 ± 8.89) than in the viral infection group (12.52 ± 11.87 days) (P < 0.05), leading to the combination of respiratory diarrhea, abdominal pain, and other obvious gastrointestinal symptoms (Table 1).

Table 1.

Basic clinical characteristics of all AECOPD patients.

Considerations	State	Viral Infection (n = 184)	Virus-Free (n = 201)	Statistic	P-value
Gender	Male	120	149		0.0324
Gender	Female	64	52
Age (mean ± SD)		75 ± 9.4	71.3 ± 9.2	1.6423	0.0756
Cough with sputum	Yes	103	156		0.8973
Cough with sputum	No
Phlegm	Yes	81	45
Phlegm	No
MMRC grading	3	39	48	−1.2976	0.3021
MMRC grading	4	145	153
Dizziness	Yes	75	145	0.8564	0.3745
Dizziness	No	109	56
Abnormal gastrointestinal symptom	Yes	68	134	2.3578	0.1184
Abnormal gastrointestinal symptom	No	116	67
Chest tightness and discomfort	Yes	124	135	0.8142	0.3627
Chest tightness and discomfort	No	60	66
Palpitate	Yes	89	20	74.526	<0.0001
Palpitate	No	95	181
Breathing rate	>25/min	93	87
	21–25/min	58	70
	16–20/min	33	44
Application of inhaled glucocorticosteroids:	Yes	103	168	3.2536	0.0354
Application of inhaled glucocorticosteroids:	No	81	33

Comparison of immune and inflammatory indicators in AECOPD patients

The differences in the levels of IL-6, IL-8, IL-1β and TNF-α were statistically significant when comparing the two groups (P < 0.05). The levels of IL-6, IL-8, IL-1β and TNF-α were significantly higher in the infected group compared to the uninfected group (P < 0.01) (Figures 1, 2, 3 and 4).

Figure 1.

Comparison and scatter plot of IL-1β content. Note: VI: viral infection; VF: virus-free; ** P < 0.001.

Figure 2.

Comparison and scatter plot of TNF-α content. Note: ** P < 0.001.

Figure 3.

Comparison and scatter plot of IL-6 levels. Note: ** P < 0.001.

Figure 4.

Comparison and scatter plot of IL-8 levels. Note: ** P < 0.001.

Comparing the differences in CD4 and CD8 levels in the two groups, the ratio between the two groups was statistically significant (P < 0.05). Compared with the uninfected group, CD4 and CD8 levels were significantly lower in the infected group (P < 0.01) (Figures 5 and 6).

Figure 5.

Comparison and scatter plot of CD4 content. Note: ** P < 0.001.

Figure 6.

Comparison and scatter plot of CD8 content. Note: ** P < 0.001.

Infection type analysis

104 of the 184 endogenous positive patients tested positive for respiratory viruses. 68 patients had a single virus infection, 27 had a mixture of two viruses, and 9 had a mixture of three viruses (Figure 7).

Figure 7.

Multiple respiratory infections virus situation.

A total of 104 respiratory viruses tested positive. They were influenza A FluA (50/104), influenza B FluB (8/104), neo-H1N1 (3/104) H3N (3/104), coronavirus SRAS-Cov-2 (2/104), HCoV-OC43 (1/104), HCoV-229E (4/104), HCoV-HKU (3/104), HCoV- NL63 (2/104), Rhinovirus RHV (1/104), Adenovirus ADV (4/104), Enterovirus EV.(2/104), Respiratory Syncytial Virus RSV.(3/104), Human Parvovirus HMPV (4/104), Parainfluenza 1 PIV1.(3/104), Parainfluenza 2 PIV2.(1/104), Parainfluenza 3 PIV3.(2/104), Parainfluenza 4 PIV4.(6/104), and Influenza B virus FluB-V (1/104) FluB-Y (1/104) (Figure 8).

Figure 8.

Composition of respiratory viral infections in AECOPD patients.

The identification of pathogenic bacteria indicated that 65 patients had Gram-negative infections in the respiratory tract, of which 37 had combined respiratory viral infections and 28 had no respiratory viral infections; 47 patients had mixed infections of Gram-negative and Gram-positive bacteria, of which only 26 were infected with respiratory viral infections; 21 had Gram-positive bacterial infections, and 7 had respiratory viral infections and 14 had no respiratory viral infections. Respiratory viruses were not detected in 14 patients; 16 patients with respiratory viral infections and 35 patients with undetectable respiratory viral infections did not have any obvious pathogenic organisms on multiple smears during hospitalization (Figure 9).

Figure 9.

Respiratory viral co-infections with bacteria.

Discussion

Current studies have shown that influenza virus infection has a wide range of pathological effects on COPD patients, involving aggravation of airway inflammation, further destruction of airway structure, acute decline in lung function, and other aspects.¹² These pathological changes will not only aggravate the symptoms of COPD patients, but may also lead to worsening of the disease, increasing the risk of hospitalization and death. Therefore, for COPD patients, prevention of upper respiratory tract infections and early intervention are crucial.¹³

The results of the present study also concluded that influenza virus can promote the expression of IL-6. There are many types of ILs with different mechanisms of action, and regardless of the mode of action, they are all related to the condition of COPD.¹⁴ At present, further studies are needed on the mechanism of action of ILs and their influence on the course of respiratory diseases, but we can conclude that ILs can be used as serum markers for the inflammation of airways associated with acute exacerbation of COPD.¹⁵ Serologic indicators of acute exacerbation of airway inflammation.

The results of the present study showed that IAV infection of airway epithelial cells activated the TLR7/NF-κB signaling pathway, which led to an increase in the expression of TLR7 and NF-κB proteins, and an increase in the content of IL-6 and TNF-α in the cell culture supernatant. The results of the present study also showed that the expression of TLR7 and NF-κB proteins as well as the expression of IL-6 and TNF-α in the culture supernatant of the COPD group was higher than that of the NHBE group, which was consistent with the results of the study by He ZH et al.¹⁶ This is consistent with the findings of He ZH et al. This may be due to the fact that COPD is a chronic inflammatory airway disease, and repeated respiratory infections promote the expression of inflammatory factors. Clinically, after influenza virus infects airway epithelial cells, the TLR7/NF-κB inflammatory signaling pathway is activated, the synthesis and release of inflammatory factors and inflammatory mediators are increased, and the cascade of airway inflammatory responses is amplified, which destroys the normal structure of the lung tissues, and the proliferation of mesenchymal fibrous tissues around bronchioles and blood vessels leads to a large, solid shadow in the lungs, and even the appearance of clinically known as “white lungs The proliferation of interstitial fibrous tissue around the bronchi and blood vessels leads to large solid shadows in the lungs, and even the appearance of what is clinically known as “white lungs”, leading to the aggravation of clinical symptoms and rapid progression of the disease.¹⁷ This leads to aggravation of clinical symptoms and rapid progression of the disease.

The interleukin (IL) family is an acute-phase inflammatory response factor that is produced by a variety of cytokines and can act on a wide range of cells. They can play an important role in acute phase immunomodulation and acute phase inflammatory response.^18,19

IL-8, as a pro-inflammatory apoptotic factor, binds to its specific receptor and induces a specific inflammatory response, while IL-32, as an immunoreactive apoptotic factor that regulates apoptosis, is involved in inflammation by activating and inhibiting a variety of apoptotic factors, including IL-1, IL-6, and IL-8.²⁰ It is worth noting that the study by Donald A Mahler et al. confirmed the therapeutic value of IL-8 monoclonal antibody in COPD.²¹ IL-6 can be produced by various cytokines, and viral and bacterial infections can directly induce an increase in the production of IL-6.²² Some researchers have also pointed out that in elderly patients with acute exacerbation of chronic obstructive pulmonary disease, viral infections are associated with more severe respiratory symptoms than non-viral infections, and higher levels of inflammatory factors, such as IL-6, worsen the condition and prolong hospitalization.²³

TNF-α is a multifunctional cytokine that participates in inflammatory response and immune regulation.²⁴ Current studies have shown that this cytokine can participate in the progression of COPD by inducing inflammatory response, promoting cell apoptosis, regulating immune response and remodeling airways.^25,26 It is worth noting that TNF-α-mediated inflammatory response often forms a complex inflammatory cascade with a variety of inflammatory mediators (such as IL-1, IL-6, etc.). Therefore, the regulation of TNF-α is currently regarded as a potential target for COPD treatment. In addition, existing studies have also pointed out that TNF-α can further damage lung tissue by activating oxidative stress response.²⁷ Oxidative stress plays an important role in the pathogenesis of COPD, which can trigger inflammatory response, promote protease dysregulation and destroy the antioxidant defense system.²⁸ In general, TNF-α plays a key role in the onset and progression of COPD, and promotes lung inflammation, tissue damage and airway remodeling through multiple mechanisms. This makes TNF-α a potential target in the treatment of COPD, and anti-TNF-α therapy is also believed to have a certain effect on alleviating COPD symptoms and delaying disease progression.

The study has several limitations. Its retrospective design may introduce biases related to patient selection and data collection. While the sample size offers some statistical power, it may not be sufficient to generalize the findings across all elderly COPD patients, given the variability in individual health conditions and responses to influenza. Moreover, the focus on a specific population (elderly COPD patients) limits the applicability of the results to other age groups or individuals with different respiratory conditions. Lastly, the study does not address the long-term effects of influenza infection on COPD progression, which could provide valuable insights for chronic disease management.

In summary, influenza virus increases the release of inflammatory factors IL-6 and TNF-α through activation, exacerbating the acute airway inflammatory response in COPD, leading to rapid progression of the disease and increased mortality.

Conclusion

Influenza virus further aggravated the airway inflammatory response in elderly chronic obstructive pulmonary disease (COPD) by activating the intrinsic immune molecules, such as IL-6, IL-8, IL-1β, and TNF-α, which produced inflammatory responses in the lung epithelial cells, and decreased the immune function of T cells.

Footnotes

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: National Science and Technology Major Project(2021ZD0111000).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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