Interactions between the lung microbiome and host immunity in chronic obstructive pulmonary disease

Abstract Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease and the third leading cause of death worldwide. Developments in next‐generation sequencing technology have improved microbiome analysis, which is increasingly recognized as an important component of disease management. Similar to the gut, the lung is a biosphere containing billions of microbial communities. The lung microbiome plays an important role in regulating and maintaining the host immune system. The microbiome composition, metabolites of microorganisms, and the interactions between the lung microbiome and the host immunity profoundly affect the occurrence, development, treatment, and prognosis of COPD. In this review, we drew comparisons between the lung microbiome of healthy individuals and that of patients with COPD. Furthermore, we summarize the intrinsic interactions between the host and the overall lung microbiome, focusing on the underlying mechanisms linking the microbiome to the host innate and adaptive immune response pathways. Finally, we discuss the possibility of using the microbiome as a biomarker to determine the stage and prognosis of COPD and the feasibility of developing a novel, safe, and effective therapeutic target.


| INTRODUCTION
Chronic obstructive pulmonary disease (COPD) is a common, preventable, and treatable chronic lung disease that poses a substantial burden on global health and the economy. According to the results of a 27-year global cohort study published in The Lancet, the top three causes of death worldwide in 2017 were cardiovascular disease (31.8% of all death causes), cancer (17.1% of all death causes), and chronic respiratory disease (7.0% of all death causes). COPD is the most common cause of death from chronic respiratory diseases. 1 As the third leading cause of death, COPD was responsible for 3.23 million deaths globally in 2019. 2 Additionally, COPD is commonly associated with the presence of comorbidities, and as the global population ages, more people will die from COPD complications than from the disease itself.
Although the etiology and pathogenesis of COPD remain unknown, it is speculated that COPD results from the interaction between various environmental factors and host factors over time. Tobacco exposure is generally considered to be the primary environmental factor causing COPD. 3 Other risk factors include occupational exposure to organic dust or chemicals; indoor air pollution from biomass fuel or coal combustion 4 ; early life events that hinder lung development and maturation, such as intrauterine dysplasia, preterm birth, and lower respiratory tract infections 5 ; infections caused by bacteria, fungi, viruses (such as SARS-CoV-2), mycoplasma, and other microbial infections [6][7][8] ; and α-1 antitrypsin deficiency. 9 Microbial infection is an important factor in the pathogenesis of COPD. The lung was considered sterile for a long time owing to the limitations of culture techniques and the barrier function of the pharynx, which prevented isolation of the oropharynx and trachea. In 2010, Hilty et al. used 16S rRNA sequencing to detect microbiota in the lower respiratory tract of patients with asthma and COPD. Their findings confirmed the presence of a characteristic microbiota, challenging the traditional medical dogma regarding the sterility of the lower respiratory tract. 10 Knowledge regarding the interactions between the lung microbiome and the host immunity is crucial to determine the role of the lung microbiome in the pathogenesis of COPD.

| HEALTHY LUNG MICROBIOME
The microbiome refers to the entire microbial community (including bacteria, fungi, viruses, protozoa, and archea) as well as their genomes, metabolites, and interactions with the host's internal environment, which are present in a specific habitat at a specific time. 11,12 As the largest and most intimate window through which the body interacts with the external environment, the respiratory tract is exposed to the surrounding environment when the airway is open. A study measuring the internal surface area of the lungs found that the internal surface area of a normal lung with a lung volume of 3000 mL ranges from 29 to 40 m 2 , which is dozens of times greater than the surface area of the body. 13 According to Hilty et al., at least 2000 bacterial genomes exist per square centimeter of the respiratory epithelium. 10 A healthy lung harbors an abundant microbiome, which primarily comprises bacteria. Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria are the dominant phyla, whereas Pseudomonas, Streptococcus, Prevotella, Fusobacteria, and Veillonella are dominant genera in the lung microbiome. 14 Mycobiomes are also present in healthy lungs. An observational study revealed that Candida, Malassezia, and Sarocladium are the most common fungi in healthy lungs. 15 However, another comparative study showed that pulmonary fungi are primarily composed of environmental agents, such as Davidiellaceae, Cladosporium, and some Aspergillus with low abundance. 16 Additionally, a certain load of rhinovirus, coronavirus, and bocavirus can be detected in the lungs of healthy people. 17 If the human body is compared to an ecosystem, different anatomical regions of the body are equivalent to different regions of the ecosystem, and each microbe in the human body is equivalent to a species in the ecosystem. In a healthy state, the microbiome composition in a specific area of the body is primarily determined by the migration and elimination of microorganisms, whereas in a diseased state, the local microenvironment and bacterial reproduction rate dominate the species richness. Microaspiration from the oral cavity is the main source of microorganisms in the lungs, and the composition of the microbiota in the lungs is in a dynamic balance between the entry and selective elimination of the transient microbiota. 18 Factors such as anatomical structure, physiological distance, oropharyngeal microbiome load, cough reflex, mucociliary clearance, and immune response can affect the migration and elimination of microbiota, thereby altering the composition of the lung microbiome. 19 When the host is sick, the internal environment of the lungs may be suitable for or inhibit the survival of certain microorganisms, causing the corresponding microorganisms to colonize, contract, or expand. significant reduction was observed in the relative abundances of Bacteroidetes and some common anaerobic bacteria, such as Prevotella, Veillonella, and Actinomyces, in patients with COPD. 22 Wang et al. analyzed sputum samples from healthy subjects and patients with different COPD states and found that the relative abundance of Hemophilus in the lungs of smokers and patients with COPD was greater than that in the lungs of nonsmokers and healthy subjects, and the relative abundances of Moraxella, Streptococcus, and Actinomyces in the lungs of patients with stable COPD significantly increased. 24,25 Sze et al. compared lung biopsy samples from patients with severe COPD (GOLD4 stage) and healthy controls and found that the proportions of Proteobacteria and Actinobacteria in the lungs of patients with COPD were slightly higher than those in the lungs of healthy individuals, whereas the proportions of Firmicutes and Bacteroidetes decreased. 26 Some studies have reported that the major pulmonary fungi in patients with COPD are Candida, yeast, Campylobacter, and Aspergillus. 15,27 Adenoviruses, respiratory syncytial viruses (RSV), and multiple respiratory viruses can often be observed in the lungs of patients with stable COPD. 28 Acute exacerbation of COPD (AECOPD) is closely linked to the microbiome. The relative abundance of proteobacteria in the lungs of patients in the exacerbation phase is higher than that in the stable phase. 29 The relative abundances of Hemophilus, Moraxella, Streptococcus pneumoniae, and Pseudomonas increases, whereas the α diversity of their microbiomes decreases. 30,31 Patients with COPD having predominant proportions of Aspergillus, Penicillium, and Curvularia tend to have more frequent exacerbations and higher mortality and show a more severe systemic immune response to these fungi. 32 Indoor and outdoor fungal sensitization factors can also contribute to frequent COPD exacerbations. 33,34 Additionally, colonization by Pneumocystis jiroveci also aggravates chronic small airway inflammation and airway remodeling. 35 A systematic review involving 19 studies and 1728 patients demonstrated that AECOPD is primarily associated with the presence of rhinoviruses, RSV, and influenza viruses, all of which had a detection rate of more than 5%, whereas coronaviruses, parainfluenza viruses, adenoviruses, metapneumoviruses, and bocaviruses had a detection rate of 5%. 36 The results of this study were consistent with those of several other studies. 34,37 Notably, coinfection with multiple viruses, such as rhinovirus, RSV, and adenovirus, tends to occur in patients with severe COPD. 38 Overall, there is no remarkable difference in the types of lung microbiome in patients with COPD versus healthy people; however, the diversity and distribution of the microbial community are considerably decreased in patients with COPD and further decreased with the severity and exacerbation of COPD. The relative abundance of commensal bacteria decreases, the proportion of opportunistic pathogens increases, and some foreign pathogens appear. The results of different studies vary, possibly owing to the bias caused by specimen selection, detection method, and the source of patients with COPD. The major composition of lung microbes in patients with COPD is summarized in Table 1. Changes in the lung microbiome can also be used as a biological indicator for the prevention, diagnosis, and prognosis of COPD.

| ANTI-INFECTION MECHANISM OF THE LUNG
Normal people inhale large amounts of air every day, and dust, poisons, and pathogens are high-risk factors for various diseases. The lungs possess a strong defense system involving mucus, structural cells, immune cells, and extracellular matrix that clears or inactivates causative agents. 51 Different pathogenic factors lead to different host immune responses. In this section, we focused on the anti-infection mechanism of the lungs. The immune defense mechanisms of the lungs consist of innate and adaptive immunity, and mucociliary clearance is the first line of defense in innate immunity. When a pathogen enters the respiratory tract along with air, part of it is discharged from the body through expiratory movement. The other part is deposited on the surface of the respiratory tract or alveolar epithelium, binds to mucin in the mucus, and is transported to the pharynx with the movement of cilia and turbulence of the mucous layer before being coughed up or swallowed. 52 The bulk of mucus is a network of water and a variety of mucins as well as a variety of functional proteins with antibacterial activity, such as secreted immunoglobulin A, lysozyme, lactoferrin, leukocyte protease inhibitor, and phospholipase A2. 53 These antibacterial substances carry out preliminary removal or inactivation of pathogens. In addition, the body excretes foreign bodies and excess mucus through the cough and sneeze reflexes. 54 Alveolar epithelial cells, macrophages, and dendritic cells bind to pathogenassociated molecular patterns (PAMPs) on their surfaces through pattern recognition receptors (PRRs) when a pathogen breaks through the immune defenses of the airway epithelial surfaces and recognizes the corresponding pathogen. Currently, the main PRRs include the toll-like receptor (TLR), C-type lectin receptor (CLR), retinoic acid-inducible gene I-like receptor (RLR), and Nod-like receptor (NLR). 55 TLR-2/4 is the most extensively studied PRRs targeting pathogens in the lungs. When different components of microorganisms combine with TLR, two signaling pathways, the MyD88dependent signaling pathway and the TRIF-dependent signaling pathway, are primarily triggered. Various proinflammatory cytokines and type I interferon are induced. 56 T A B L E 1 Summary of studies utilizing either cultured or noncultured methods to describe the microbiome in COPD.
Year Participants Type of sample was the most significant difference between healthy individuals and GOLD stage 4 COPD patients [29] 2015 Twenty-eight healthy subjects.
OW, NS, BALF, and gastric aspirate samples 16S rRNA gene sequencing In healthy individuals, the microbiome of the lung overlapped those found in the mouth but were found at lower concentrations, with lower membership and a different community composition. And the nasal microbiome contributed little to the lung microbiome [46] 2017 Subjects with moderate-to-severe COPD (n = 73) and severe asthmatic patients (n = 32)

Sputum mediators
Factor and cluster analyses, 16S rRNA gene sequencing Revealed three subgroups of asthma and COPD exacerbations, Cluster 1 increased proportions of the bacterial phylum Proteobacteria. Cluster 2 increased proportions of the bacterial phylum Bacteroidetes. Cluster 3 increased proportions of the phyla Actinobacteria and Firmicutes. [23] 2017 Adults with COPD (n = 18), smokers with no airway disease (n = 8) and healthy individuals (n = 11) BALF Molecular detection-Illumina MiSeq sequencing Microbiome in the lower airways of patients with COPD is significantly different to that found in smokers and nonsmokers. There was more pseudomonas in the lower airway of patients with COPD, Bacteroidetes were more common in the control group. Community diversity (α and β) was significantly lower in COPD group than in healthy group. [24] 2017 Various tissue-resident lymphocytes in the lungs secrete different cytokines stimulated by antigens, recruit effector cell subsets, and provoke corresponding immune responses, thereby eliminating pathogens. 57 All three innate lymphoid cells (ILCs) were detected in the lungs. 45 Group 1 ILCs principally consist of NK cells and noncytotoxic ILC1, which can secrete IFNγ. Group 2 ILCs mainly consist of ILC populations that generate TH2 cell-associated cytokines (IL-5 and IL-13), whose maturation depends on GATA binding protein 3 and retinoic acid-related orphan receptor-α. Group 3 ILCs contain all ILCs that produce IL-17 or IL-22, and their maturation depends on the transcription factor retinoic acid-related orphan receptors-γt. 58 Upon stimulation, these lymphocyte subsets generate signals that activate downstream effector cells and eliminate pathogens. For example, IL-22 regulates IL-2/IL-2R22 production in the lungs and reduces the expression of SARS-COV-2 entry receptors, such as ACE2 and TMPRSS2. 59 Adaptive immunity includes cellular immunity mediated by T cells and humoral immunity mediated by B cells. The innate immune system is essential for adaptive immunity. PRRs expressed by antigenpresenting cells (especially dendritic cells) bind to PAMPs to activate the adaptive immune response, 60 and cytokines secreted by other innate immune cells, such as interferon, can induce the proliferation and differentiation of T cells or B cells. 61 CD8 + T cells are cytotoxic. Activated by antigen-presenting cells, CD8 + T cells prolifically differentiate into cytotoxic effector T cells, which kill infected cells and release cytokines, such as granzyme B, IFN-γ, IL-12, IL-2, and perforin. 62 CD4 + Th cells can be divided into different subpopulations, including Th1, Th2, Th17, and regulatory T cells. After being stimulated by antigens, CD4 + Th cells rapidly differentiate and release related cytokines. 63 Th1 cells mainly secrete inflammatory factors, such as IL-2, IFN-y-, and TNF-P, which mediate immune responses related to cytotoxicity and inflammation and activate other immune cells. Th2 cells secrete cytokines such as IL-4, IL-5, IL-9, IL-10, IL-13, IL-25, and amphiregulin to activate and maintain humoral immune responses against extracellular pathogens, allergens, and toxins. 64 Th17 cells secrete cytokines such as IL-17A, IL-17F, IL-21, and IL-22, which mediate immune responses to bacteria and fungi. 47,65,66 Follicular helper T cells can promote the maturation of germinal centers and regulate their functions by mediating protective immunity against pathogens. 67 Regulatory T cells (Treg) suppress T cell immune responses, regulate immune tolerance, and prevent autoimmune diseases through cytokine (IL-10, TGF-β, and IL-35) or contact-dependent pathways. 68 B lymphocytes secrete various antibodies, bind to surface proteins necessary for pathogens to enter cells to neutralize infection 69 or bind to Fc receptors to exert antibody-dependent cytotoxicity or activate complement responses. 70 The most important antibodies in Year Participants Type of sample Test method Main findings References 2020 Five hundred and ten COPD patients from UK sites of the BEAT-COPD, COPDMAP, and AERIS cohorts.
One thousand seven hundred and six sputum samples were analyzed using COPDMAP and AERIS as a discovery data set and BEAT-COPD as a validation data set Sputum differential cell counts and 16S rRNA gene sequencing The lung microbiome can stratify COPD into neutrophilic Haemophilus-predominant, neutrophilic balanced microbiome, and eosinophilic subgroups. [9] 2020 Healthy individuals (n = 47), stable COPD (n = 337), acute exacerbation of COPD (n = 66), and a longitudinal COPD cohort (n = 34).
Sputum and serum samples DNA extraction, mycobiome sequencing, and specific-IgE assays The lung mycobiome in COPD is characterized by Aspergillus, Penicillium, and Curvularia, which associated with exacerbations and increased mortality, but not for antibiotics or corticosteroids. [49] 2020 Two hundred severe COPD patients from Europe and North America and followed longitudinally for 3 years.
Sputum samples collected at stable, acute exacerbation and follow-up visits.
Nucleic acid detection and 16 S ribosomal RNA gene sequencing Geographic and longitudinal differences in the lung COPD microbiota were correlated with diverse outcomes. Moraxella and Haemophilus were 5-fold and 1.6-fold more likely to be increased during an exacerbation event. Human rhinovirus (13.1%), coronavirus (5.1%), and influenza virus (3.6%) were the most common virus in AECOPD. the lungs are dimeric SIgA and IgG, which are produced by airway epithelial cells. 71 Furthermore, there are some multi-reactive natural IgM autoantibodies that can not only resist the invasion of microorganisms but also regulate excessive inflammation caused by various factors to prevent autoimmune diseases. 72 In addition to secreting antibodies, B lymphocytes can also present antigens and secrete cytokines including IL-2, IL-4, IL-6, IL-10, IL-12, TGF-β1, TNF, and IFN-γ. 73 It is worth mentioning that, like the symbiotic microbiota of the gut, the normal flora in the lungs also plays a protective role for the host (Figure 1).

| PATHOGENESIS AND IMMUNOPATHOLOGY OF COPD
COPD develops as a result of a combination of multiple factors, 74 chronic inflammation of the airway, pulmonary parenchyma, pulmonary blood vessels, imbalance of the protease-antiprotease system, 75 oxidative stress, 76 autonomic dysfunction, 77 nutritional disturbances, 78 and other mechanisms that jointly cause small airway inflammation and emphysema changes. These pathological changes work together, resulting in COPD. 79 Among them, the core is chronic inflammation of the small airways, in which the aggregation and activation of neutrophils, macrophages, T lymphocytes, and other inflammatory cells is a key link. 80 According to different predisposing factors, pathogenesis, clinical phenotype, and severity, COPD can be divided into different types; however, there is no uniformly accepted classification. Factor and cluster analyses were used to analyze the cytokine profiles of patients, and COPD was divided into three inflammatory endotypes: neutrophilic inflammation, eosinophilic inflammation, and mixed cellular inflammation. 81 Among these, neutrophil inflammation is the most common inflammatory endotype in COPD. When pathogenic factors contact the airway epithelium, the expression of various neutrophil chemokines (IL-17, LTB, CXCL1, CXCL5, and CXCL8) increases, and neutrophils are recruited and activated in large numbers in the small airway, releasing neutrophil mediators such as neutrophil elastase and matrix metalloproteinases (MMPs), activating ILC3s, and mediating pathogen clearance. However, chronic excessive neutrophil inflammation and protease system imbalance can also lead to mucus hypersecretion, small airway damage, and airway remodeling. 20,82,83 Neutrophils can also cause oxidative stress, which further aggravates airway inflammation. In addition, the formation of neutrophil extracellular traps (NETs) can be observed in the respiratory tract of patients with COPD in a positive association with the severity and frequency of progression. 84 Once activated, neutrophils release a variety of proteases, peroxidases, and chromatin, which assemble outside the cell to form a fibrous trap to capture and kill microorganisms and release high concentrations of antimicrobial peptides to degrade bacterial virulence factors. 85 The phagocytic function of neutrophils is in a state of competition with their ability to form Nets. When phagocytic function is impaired, neutrophils will turn to form many Nets to maximize the killing of pathogens. 86 Macrophages are gatekeepers of the lungs. They can not only phagocytose foreign pathogenic factors and necrotic cell components in the host body, but also act as antigen-presenting cells and secrete a large number of cytokines. 31 Macrophages can be roughly divided into types M1 and M2, which mediate Th1-dominated pro-inflammatory responses and Th2related anti-inflammatory responses, respectively. 87 The number of M2 macrophages in the lungs of patients with COPD decreases as the number of M1 macrophages increases, indicating that the function of macrophages gradually transforms from anti-inflammatory to proinflammatory during the long-term phagocytosis of foreign bodies. 88 In addition, the ability of macrophages to phagocytose pathogens and necrotic cells is weakened in patients with COPD, 89 leading to the immune escape of pathogens and the persistence of local inflammatory responses, further exacerbating the progression of COPD. The eosinophilic inflammatory endotype is also a rare type of COPD, but is associated with better corticosteroid prognosis, 90 and Th2-related immune microenvironments are more common in the lungs of these patients. 39 Lymphocyte infiltration into the small airway wall increases with the progression of COPD, among which CD8 + T cells and B cells increase the most. 91 Grumelli et al. found that emphysemarelated pathological changes in patients with COPD were strongly correlated with Th1-related immunity, 43 whereas the Th17 cell population and its related cytokines, IL-17 and IL-22, were strongly associated with the generation and exacerbation of COPD. 66,92,93 On the one hand, IL-17 and IL-22 play a pivotal role in the clearance of pathogens. 94 On the other hand, Th17 cells are closely associated with the development of small airway obstruction and emphysema. In mouse models of cigarette-induced COPD, IL-22 knockout mice had significantly lower lung function impairment than normal mice. 47 This functional contradiction may be related to the different expression patterns of the two in time and space ( Figure 2).

| ROLE OF LUNG MICROBIOME IN THE PATHOGENESIS AND HOST IMMUNE ALTERATION IN COPD
Healthy lungs possess an extremely rich microbiome, and during the development of COPD, there is an imbalance in the original microbiome in the lungs and the invasion of foreign pathogens. These two conditions overlap to a certain extent, blurring the dividing line The lungs possess a strong defense system of mucus, structural cells, immune cells, and extracellular matrix that clears or inactivates causative agents. The immune defense mechanism of the lungs consists of innate immunity and adaptive immunity, and mucociliary clearance is the first line of defense in innate immunity. Cells such as alveolar epithelial cells, macrophages, and dendritic cells bind to PAMPs on their surfaces through PRRs when a pathogen breaks through the immune defenses of airway epithelial surfaces and recognize the corresponding pathogen. Various tissue-resident lymphocytes in the lungs secrete different cytokines stimulated by antigens, recruit effector cell subsets and provoke corresponding immune responses, eliminating pathogens. Adaptive immunity mainly includes cellular immunity mediated by T cells and humoral immunity mediated by B cells. The normal flora in the lungs also plays a good protective role for the host. They can also contend for ecological niches with opportunistic pathogens and foreign pathogens, decreasing the likelihood of pathogenic infection colonization. GM-CSF, granulocyte-macrophage colony stimulating factor; IFN-γ, interferon-γ; IL, interleukin; IL-12p70, Interleukin-12p70; MyD88, myeloid differentiation factor 88; PAMP, pathogen-associated molecular patterns; PRR, pattern recognition receptors; TNF, tumor necrosis factor; TRIF, Tir domaincontaining adaptor inducing interferon-beta. between colonization and infection. Changes in the microbiome interact with the host's immune response, leading to a vicious cycle in the process of COPD; that is, multiple pathogenic factors damage the lung's defense mechanism, making the lung environment more suitable for pathogen infection or colonization, exacerbating inflammation in the small airways, which further deepens the damage to the lung's immune mechanism and expands the imbalance of the lung microbiome. This can lead to the exacerbation of COPD. 95 In the field of ecology, there exists the concept of core and hub microbiota, where "core microbiota" refers to the constant presence of certain microbiota in almost all related communities of a particular host. "Hub microbiota" refers to those microorganisms that have strong interactions with the host and other microorganisms, and even a minor change in their abundance is sufficient to alter the entire microenvironment. 96 A similar core and hub microbiota is present in the lungs. 44,97 At the phylum level, the core microbiota of the lungs includes Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria, and includes Pseudomonas, Streptococcus, Prevotella, Fusobacterium, Hemophilus, Veillonella, and Porphyromonas at the genus level. Numerous published studies have shown that the diversity of the lung microbiome is decreased, and the ratio of the core microbiota is broken in patients with COPD compared to healthy individuals. For example, the abundance of Bacteroidetes decreases while that of Proteobacteria increases, and the abundance of opportunistic pathogens such as Hemophilus, Moraxella, Streptococcus, and Pseudomonas significantly increases. 24,30,31 At the same time, some foreign pathogens can also be observed in the lungs of some patients with COPD, such as P. jiroveci and SARS-COV-2. 8,98 Numerous functional redundancies are generated in the interaction between the microbiomes and the host, and thus the concept of functional microbiome has been introduced to classify microbiomes with analogous functions. 99 Here, we focus on species that have a greater impact on the progression of COPD.

| ALTERATIONS IN THE MICROBIOME AS A RESULT OF EXPOSURE TO RISK FACTORS
When the host is exposed to cigarette smoke, it attacks the peripheral immune system, resulting in damage to immune cells, making it difficult to maintain the effect of clearing infection. In addition, exposure to cigarette smoke may increase biofilm formation by specific bacteria, facilitating the reproduction and immune escape of pathogenic bacteria. Cigarette smoke also affects the microenvironment (e.g., oxygen, pH, and acids) in the lungs. 46 The interaction of these mechanisms leads to changes in the composition of the microbiome in the lungs, with some anti-inflammatory commensal flora being inhibited and pathogenic bacteria, such as Pseudomonas aeruginosa, expanding. 100 The overuse of antibiotics also changes the diversity of the lung microbiome. 101 For example, beta-lactam antibiotic exposure is strongly associated with reduced microbial diversity in the lung, 102 105 Prevotella mainly binds to TLR2 to stimulate antigen-presenting cells and airway epithelial cells to release IL-23, IL-1, IL-8, IL-6, CCL20, and other cytokines; recruit neutrophils; and activate Th17-related immune responses, which are involved in establishing lung tolerance and improving respiratory tract anti-infection ability. 49,106 In the lungs of patients with COPD, Prevotella competes with Streptococcus, Hemophilus, and Moraxella. 107 The relative abundance of Prevotella decreases, and the inhibitory effect on opportunistic pathogenic bacteria is weakened, which also promotes the progression of COPD. Rothia mucilaginosa, a gram-positive obligate anaerobe, performs a similar function. It not only inhibits the levels of IL-6, IL-8, GM-CSF, and monocyte chemoattractant protein-1 (MCP-1) produced by pathogenic bacteria such as P. aeruginosa after invading lung epithelial cells, but also downregulates the expression of nuclear factor-κB (NF-κB) in epithelial cells through gene expression, dephosphorylation, and inactivation, thus suppressing inflammation of the small airways. 108 Einarsson et al. found that the relative abundance of obligate anaerobes (Prevotella, Veillonella, and actinomycetes) was lower in the lungs of patients with COPD than in normal controls. 22 These resident obligate anaerobes in the lungs participate in the production of short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, which can REVIEW OF LUNG MICROBIOME IN COPD | 113 promote the recovery of airway barrier function, reduce the pathological changes associated with emphysema, and inhibit the proliferation of opportunistic pathogens. 78 Commensal bacteria can also compete for ecological niches with opportunistic and foreign pathogens, decreasing the likelihood of pathogenic infection and colonization. 109

| POTENTIAL PATHOGENIC MICROORGANISMS IN THE LUNGS OF PATIENTS WITH COPD
The relative abundance of proteobacteria gradually increases in the lungs of patients with COPD at the stable and exacerbation stages, among which the most common species are H. influenzae, Moraxella, S. pneumoniae, and P. aeruginosa. [24][25][26]110 Interestingly, the bacterial load of H. influenzae, Moraxella, or S. pneumoniae in the lungs must reach a certain number to be able to cause COPD exacerbation, whereas lowload P. aeruginosa can cause COPD exacerbation. 111 H. influenzae is a facultative anaerobic gramnegative bacillus, one of which, nontypeable H. influenzae (NTHI), is the most dominant pathogen in most studies of COPD microbiome. 112 H. influenzae can also be observed in the lungs of healthy individuals, indicating that it is an opportunistic pathogen. When the respiratory tract is stimulated by cigarette smoke, biological contamination, or poisons, a variety of immunomodulatory pathways in the lungs are impaired, especially the ability to kill microorganisms, and H. influenzae changes from a colonizing to a pathogenic state, thus leading to the progression of COPD. 41 Pettigrew et al. performed 15-year whole-genome sequencing of NTHI isolates from the lungs of patients with COPD. They found that NTHI alters its simple sequence repeats in the genome by slipped-strand mispairing, thereby modulating its key functions in adapting to the changing lung environment. 113 Four genetic islands, G2, G6, G8, and G10, are the most common in COPD strains, encoding potential transcriptional regulators and other small ORFG2, the entire urease operon, aspartate-semialdehyde dehydrogenase, and ABC transporter, respectively. They regulate the formation of the corresponding products to facilitate the survival of NTHI in an acidic environment, maintain membrane integrity, and enhance drug resistance. In addition, it damages the respiratory epithelium via ammonia toxicity. 114 For example, NTHI inhibits intracellular E-cadherin production by inducing the expression of fibroblast growth factor 2, disrupting integrity and barrier functions in lung epithelial cells. 42 NTHI can also express a variety of structural and functional proteins, enhance oxidative resistance and stress reactivity, increase nutrient uptake ability, improve adhesion activity, resist killing by antimicrobial peptides, and promote biofilm formation. 112 Biofilms are how multiple microorganisms, including H. influenzae, colonize. It is composed of numerous extracellular polymeric substances and bacterial communities. Normally, the flow of gas and mucus in the lungs and the flapping of cilia cells limit biofilm formation. 115 When COPD occurs, NTHI expresses a variety of attachment proteins, such as type IV pilus protein and adhesion protein OMP P1/P5, resulting in a large amount of mucous production, which impairs mucociliary clearance and biofilm form. 30 Biofilms not only preserve bacteria for a long time, enhance their immune escape and antibiotic resistance, but also induce chronic inflammation in the host, further exacerbating the progression of COPD. 115 NTHI can also express peroxiredoxin-glutaredoxin and catalase to resist oxidative stress and escape killing by NETs. 116 NTHI outer membrane protein 6 (P6) binds to TLR2 and activates p38 mitogen-activated protein kinase (MAPK) or nuclear factor kappa B to induce the expression of endogenous anti-inflammatory mediators such as cyclooxygenase 2 and prostaglandin E2. 117 Hexa-acylated lipid A lipopolysaccharides of NTHI bind to TLR4 and stimulate the production of numerous inflammatory cytokines. Jeppe et al. found that, owing to the high biological activity of hexa-acylated lipopolysaccharide, cytokines such as IL-23, IL-12p70, and IL-10 induced by pathogens such as Hemophilus and Moraxella in patients with COPD are 3-5 times that of commensal bacteria. 104 NTHI can also bind to NLR, stimulate caspase-1 expression, and secrete numerous IL-1 family cytokines (IL-1β and IL-18), aggravating the pathological changes in COPD. 50 All inflammatory mediators work together with NTHI to form positive feedback loops with the NF-κB pathway as the core, which continuously enhances chronic airway inflammation and leads to COPD exacerbation. 41 Moraxella is a facultative anaerobic gram-negative bacillus belonging to the phylum Proteobacteria. Among the common pathogenic bacteria in the airways of patients with COPD, the colonization rate of Moraxella is second only to that of Streptococcus and H. influenzae. 107 Moraxella binds to and adheres to collagen VI on airway epithelial surfaces. In COPD, the expression of this adhesion target is upregulated to recruit more Moraxella to colonize and adhere to the airway surfaces. 118 Colonization is primarily associated with severe or exacerbated COPD. There are various functional proteins on the surface of Moraxella, the most important of which are ubiquitous surface protein A molecules (UspAs), which mediate the adhesion, invasion, nutrient acquisition, and immune escape of the bacteria. 48 Among these, the ability to escape from complement immunity has the greatest influence on the pathogenesis of COPD. Complement is activated by the classical pathway, alternative pathway, or lectin pathway/mannose-binding lectin, forming membraneattack complexes and killing microorganisms. Moraxella UspA2 inhibits the activation of classical and alternative pathways by binding to the complement inhibitor C4bp and absorbing serum C3, respectively, and decreases the activity of membrane attack complexes. 119 In addition, Moraxella UspA1 binds to CEACAM1, an epithelial cell adhesion molecule, and inactivates the PI (3)K-NF-κB signal transduction pathway through phosphorylation to escape from the clearance by the immune system. 120 Like other opportunistic pathogens in the lungs, Moraxella stimulates airway epithelial cells to release inflammatory factors such as IL-8 and GM-CSF via MAPK and NF-κB activation, and promotes the production of IL-8 by acetylating histones H3 and H4. 121 S. pneumoniae, an aerobic or facultative anaerobic gram-positive coccus in the phylum Firmicutes, is another major opportunistic pathogen in the lungs after Hemophilus. 107 Polysaccharide capsules and pneumolysins are the main pathogenic factors of Streptococcus. The negatively charged polysaccharide capsule generates electrostatic repulsion with mucopolysaccharides, helping Streptococcus escape mucous immune clearance and colonize the airway epithelial surface. The presence of a polysaccharide capsule can also help S. pneumoniae escape macrophage phagocytosis. 122 Pneumolysins can interact with the host in various ways. First, it can activate the host complement system through the classical pathway. Second, it interacts with epithelial cells to form pores, causing "osmotic sensing" and activating downstream p38 MAPK and NF-κB. Third, it binds to TLR4 and secretes chemokine IL-8, recruiting neutrophils. Pneumolysin downregulates the expression of host antiproteases and combines with hydrogen peroxide generated by bacteria to induce apoptosis in host cells. 123 P. aeruginosa, a gram-negative obligate anaerobe in the phylum proteobacteria, can be isolated from patients with COPD during exacerbation or recurrent hospitalization. 124 In the early colonization stage, the emergence of P. aeruginosa is always accompanied by lower cytotoxicity, motility, and protease expression levels and the formation of more biofilms, which is the mechanism of its long-term colonization in the lungs. 125 It exhibits extremely high variability and can vary in different microenvironments. In diverse environments, P. aeruginosa possesses LPS with different structures, such as penta-acylated or hexa-acylated lipid A, which leads to changes in its immunogenicity. 126 P. aeruginosa releases quorum-sensing signal molecules to promote mucus secretion and biofilm formation and inhibits the activity of immune cells, forming immune tolerance to the host. 127 Neutrophils are the main defensive force against P. aeruginosa infections. On the one hand, P. aeruginosa stimulates airway epithelial cells and immune cells to synthesize chemokines such as CD11a (b)/18, which drives neutrophils to migrate to the lungs. 128 On the other hand, it can activate NLRC4 inflammasome-induced neutrophil apoptosis. 129 Fungi also play a significant role in the development of COPD. In a healthy state, fungi in the lungs can regulate host immune function and protect the airway mucosa. When the host suffers from COPD, the composition of the pulmonary fungal community changes and the relative abundance of Aspergillus changes most dramatically. 130 Aspergillus and other allergens are associated with poorer lung function and more severe GOLD stage, and cause elevated IgE levels in nearly half of patients with COPD. 131 The most important risk factors for Aspergillus infection are low host immunity and neutropenia; neutrophils are the main forces of the host that kill Aspergillus. 132 Neutrophils synthesize IL-17A via dectin-1-and IL-23dependent pathways, providing a protective immune response to COPD complicated by invasive pulmonary aspergillosis. 133 Furthermore, Aspergillus fumigatus induces neutrophil apoptosis by synthesizing secondary metabolites, such as gliotoxin, thereby evading clearance by the lung's immune response. 134 When combined with TLR2/4 or non-TLR PRRs dectin-1 on the surface of pulmonary macrophages, Aspergillus upregulates the expression of high mobility group box 1, which stimulates macrophages to release TNF-α, IL-1β, IL-6, IL-33, and other inflammatory factors, leading to the exacerbation of COPD. 135 In addition, the roles of other fungi, such as P. jiroveci, in the progression of COPD should not be neglected. 98 P. jirovecii can secrete a variety of matrices either by itself or by inducing the airway. This disrupts the balance of the proteinaseantiproteinase system in the body, resulting in aggravation of airway obstruction. 136 It can also activate macrophages through an NF-κB-dependent pathway to release TNF-α, IL-6, IL-8, and other cytokines. This pathway is mediated by the β-glucan-rich cell wall of P. jirovecii and occurs more slowly and persistently than the same response induced by bacterial LPS. 137 The host upregulates the expression of INF-γ, CXCL9, CXCL10, and CXCL11-related genes and mediates the clearance of P. jirovecii through Th1-related immunity, which also aggravates inflammation of the small airways. 138 Exacerbation of COPD is closely related to respiratory viral infection. In a comparative study, half of the stable COPD cases are associated with bacteria (54.7%) or viruses (48.4%), compared with 17.2% and 42.2% in acute exacerbations, respectively. 139 Rhinovirus is the most common virus in the lungs of patients with COPD. After entering the lungs, rhinovirus primarily invades airway epithelial cells and replicates in large quantities. 140 Replication of viral RNA leads to durable expression of CXCL-1, induces multiple immune cells to aggregate in the airways, binds to multiple PRRs in the host, activates transcription factors such as interferon regulators and NF-κB, and then induces transcription of type I and III interferons and other cytokines. 141 However, the activation of this antiviral immunity is delayed in patients with COPD compared to that in healthy individuals. Rhinovirus-induced innate immunity peaks at 48 h in healthy subjects and is delayed to 96 h or even later in patients with COPD. 140 Rhinovirus infection can also impair the innate immunity of the airway epithelium by inhibiting antimicrobial peptides and increasing oxidative stress in the lungs, further increasing the susceptibility of patients with COPD to pathogens. 142 This result was also demonstrated in another study, in which Molyneaux et al. inoculated subjects with rhinoviruses in both patients with COPD and healthy individuals. Compared with the control group, the COPD group had a six-fold increase in pulmonary bacterial load after 15 days, and a 16% increase in the proportion of Proteobacteria sequences dominated by Hemophilus that lasted up to 6 weeks. 143 In a large prospective study involving 1099 adults hospitalized with RSV, patients with COPD were 3-13 times more likely to be infected than those without COPD. 144 A few studies have shown that RSV infection can decrease type I IFN levels, increase IL-17 and IL-23 secretion, upregulate mucin gene expression (MUC5AC and GOB5), and destroy ciliated epithelial cells in COPD models, leading to mucus retention and alveolar cavity enlargement. 145 In addition, RSV infection increases the synthesis of MMP-2 and MMP-9, facilitating small airway remodeling and alveolar wall destruction. 146 In addition to bacteria, fungi, and viruses, atypical pathogens are associated with the occurrence of COPD. A randomized controlled trial in Korea found that approximately 5.6% of patients with COPD showed serological evidence of acute infection with Mycoplasma pneumoniae. 147 Similar results were observed for Chlamydia pneumoniae, and macrophages released more cytokines, such as TNF-α and IL-8, after phagocytosis of C. pneumoniae. 148 However, a Turkish study found no significant difference in C. pneumoniae infection between COPD and healthy individuals and no significant association between serological markers of Chlamydia infection and COPD severity. 149 At present, there are few related studies, and some research results are quite different. The different results in these studies may result from the heterogeneity of COPD on the one hand, and the differences in experimental protocols, including specimen sources and detection methods, on the other hand. However, atypical pathogens may also induce COPD.

| DISCUSSION
COPD is a chronic airway disease induced by numerous pathogenic factors that have been associated with multiple mechanisms. The role of microbiomes runs throughout the entire pathogenic process. In addition to the microorganisms themselves, the virulence factors released and downstream metabolites have an impact on COPD. After the combination of P. aeruginosa surface LPS with TLR4, the mitochondria of alveolar macrophages and neutrophils secrete massive succinic acid and reactive oxygen species, which cooperate with the activation of the inflammasome to induce the maturation of IL-1β and its release into the extracellular space. Staphylococcus aureus can synthesize a low metabolic small colony variant in the lungs, which can affect the glycolysis process of the host by degrading fumarate, consequently affecting host immunity. 150 As mentioned above, commensal microbiota in the lungs can strengthen host immunity and inhibit the proliferation of pathogens by synthesizing SCFAs. In addition, commensal microbiota provides numerous essential signals for the development and maturation of the host's innate and adaptive immune systems. 151 There is a mutually beneficial cooperative relationship between the host and commensal microbiota, and a mutually restrictive hostile relationship between the host and pathogen. This relationship exists not only between the host and the microbiome but also within vast microbiomes. Commensal microbiota competes with pathogens for the niche through nutrient uptake and metabolite production, preventing the colonization and multiplication of pathogens. However, when the host is stimulated by endogenous or exogenous factors, immune function is impaired, the local microenvironment changes, and it is no longer suitable for the survival of commensal microbiota. Then, the pathogens expand sharply and occupy the living space of the commensal microbiota. Interestingly, the presence of closely related species increases the chances of a new species entering the ecosystem, which is known as "like will to like" in the study of the gut microbiome. 152 A similar pattern was observed in the lungs. Wang et al. constructed a network of pulmonary microbiome interactions in patients with COPD exacerbations. They found that Hemophilus, Moraxella, and Streptococcus are exclusive, which is associated with reduced diversity of the lung microbiome during COPD exacerbations. 25 Jacobs et al. found that, in stable COPD, S. pneumoniae and H. influenzae had an interspecific synergistic relationship, whereas P. aeruginosa showed an interspecific competitive relationship with H. influenzae and Moraxella catarrhalis. In the acute exacerbation of COPD, the interspecific cocolonization relationship between S. pneumoniae and H. influenzae disappeared, whereas P. aeruginosa continued to inhibit the reproduction of H. influenzae and M. catarrhalis. 153 Similarly, the lungs have complex synergistic and antagonistic interactions with bacteria, fungi, viruses, and various atypical pathogens. Compared with infection alone, H. influenzae and rhinovirus coexposure synergize with the respiratory epithelium to stimulate CXCL8 and CCL20 production, leading to more severe airway inflammation. 154 These complicated interactions are closely related to the structure of pathogens, metabolites, and host immunity, creating the possibility of multiple infections in patients with COPD and further exacerbating the progression of the disease. However, these interactions can also be used to guide the treatment of COPD; for example, the rational use of narrow-spectrum antibiotics to specifically regulate the composition of the microbial community in the lungs, thereby indirectly affecting host immunity and improving the patient's condition.
The microbiomes in other parts of the host also influence COPD progression. The gut microbiome is closely related to the development of COPD, not only because the gut microbiomes can enter the lung through microaspiration, but also because both the gut and lung originate from the endoderm and have similar anatomical structures, such as microvilli and cilia. The interaction between gut microbiomes and host immunity can not only enhance the immune function of the lungs and bring benefits to patients with COPD, but may also exacerbate the progression of the disease. 155,156 Intestinal microorganisms are major producers of SCFAs in the body. SCFAs generated in the intestine can be transported to the lung in various ways to enhance the barrier function of the respiratory epithelium and pulmonary immunity function, which plays a protective role in patients with COPD. 78 One meta-analysis showed that patients with Helicobacter pylori infection were 100% more likely to develop COPD than healthy people. 157 In addition to gut microbiomes, oral microbiomes are associated with the occurrence of COPD. Recently, a few studies have shown that patients with stomatitis and periodontitis are more likely to develop COPD. 158,159 This may result from an increase in the number of pathogenic microorganisms in the unclean oral microenvironment, which increases the number of pathogen microaspirations into the lungs, ultimately leading to COPD.
During the development of COPD, the host immune system not only plays a role in resisting invasion, but also acts as a "destroyer." When pathogens invade the respiratory tract of the host, innate and adaptive immunity in the lungs are successively activated. They then synthesize numerous cytokines, which cooperate with the commensal microbiota in the lungs to prevent colonization and eliminate pathogens. When subjected to various endogenous or exogenous stimuli, pathogens escape or tolerate the host immunity. The immune system is continuously activated. Epithelial cells, neutrophils, macrophages, and lymphocytes secrete large amounts of inflammatory mediators that act on normal lung cells and tissues, leading to small airway inflammation and alveolar wall damage. The relationship between pathogens and host immunity forms a vicious cycle that amplifies the cascade of airway inflammation and immune dysfunction.

| CONCLUSION AND PERSPECTIVE
Owing to the development of next-generation sequencing, microbiome studies are no longer limited to culturedependent methods. Research on the microbiome in the host has expanded from the gut to the whole body. Sequence analysis of conserved 16S RNA and 18S RNA sequences for bacteria and fungi is more precise and reliable than the results obtained by phenotypic identifications. 40,160 Subsequently, metagenomic analysis was used to further refine the identification results. These rapidly evolving technologies facilitate our understanding of the role of the microbiome in the host body and how they relate to each other. COPD is a chronic respiratory disease caused by a combination of mechanisms, in which the microbiome runs through the entire development process of the disease. The study of the COPD microbiome and host immunity can help us gain a deeper understanding of its pathogenesis and find targeted therapeutics. It can also guide existing treatment methods and contribute to further enriching the consensus regarding the diagnosis and treatment of COPD. However, most current studies on the COPD microbiome are at the cross-sectional level, and owing to the heterogeneity of COPD, there are few long-term prospective cohort studies with large sample sizes; therefore, it is difficult to determine the causal relationship between the microbiome and host immunity. At present, we prefer a two-way causal relationship secondary to the trigger point, such as tobacco exposure. In addition, the human body is not solely spliced together by individual organs, but a complete microbiosphere. Networked connections exist between the microbiome and body in time and space. The environment in the body is in homeostasis, and endogenous or exogenous stimuli can disrupt this balance and cause a domino effect. Therefore, future studies on the interaction between the microbiome and host immunity in COPD should comprehensively measure the roles of other aspects, such as external stimuli (such as cigarette smoke, antibiotics, and glucocorticoids), microbiomes, and related metabolites in other parts of the body, as well as the interference of the autonomic nervous system.

AUTHOR CONTRIBUTIONS
Yixing Zhu collected the literature and wrote this review. De Chang guided the revision of the structure and content of this review.