The intricate interplay among microbiota, mucosal immunity, and viral infection in the respiratory tract

Abstract

The mucosal system serves as the primary barrier against respiratory diseases and plays a crucial role in combating viral infections through mucosal immunity. The resident microbial community constitutes the main component of the mucosal system and exerts a significant inhibitory impact on the invasion of exogenous agents. However, the precise relationship between resident microbiota, mucosal immunity, and viral infections remains incomplete. This review aims to summarize the regulatory interactions between the resident microbiota of the mucosal system and innate immune components such as mucosal immunity and trained immunity. By clarifying these complex relationships, this review seeks to identify potential targets for augmenting respiratory disease prevention strategies and developing novel vaccine formulations. Furthermore, we propose the possibility of integrating the fields of microbiome-based therapeutics and vaccine development to create multifunctional vaccine formulations capable of targeting mucosal immunity induction. Such an approach holds great potential in offering novel pathways and strategies for the prevention and treatment of respiratory diseases.

Background

The respiratory tract functions as a vital interface for gas exchange and environmental interaction, simultaneously serving as a primary entry portal for pathogenic viruses responsible for infectious diseases such as influenza, rhinoviruses, SARS-CoV-2 (COVID-19), respiratory syncytial virus (RSV), and adenoviruses [1,2,3]. The respiratory mucosa constitutes the frontline defense mechanism, integrating physical, chemical, and immunological barriers against invading pathogens. This mucosal layer harbors a sophisticated defense system known as the respiratory mucosal system (RMS), a critical subsystem of the mucosal immune system (MIS). The RMS encompasses diverse cellular components (e.g., macrophages, dendritic cells, T cells, B cells), secretory immunoglobulin A (sIgA), microbiota, secretions, and metabolites [4]. This intricate system effectively combats pathogens while maintaining the immune homeostasis in the host respiratory tract [5, 6].

Recent advances in mucosal immunology have highlighted the pivotal role of RMS in antiviral defense. For instance, during the COVID-19 pandemic, mucosal-inducing strategies have displayed promising clinical potential for vaccines [7,8,9]. Thorough investigations into respiratory mucosal immunity are crucial for the effective prevention and treatment of viral infections.

Advances in high-throughput sequencing technologies have unveiled the complexity of respiratory microbial ecosystems, collectively termed the respiratory microbiota [10, 11]. The mucosal interface provides a niche for microbial colonization through physicochemical support and nutrient availability [12, 13]. Dysregulation of this microbial equilibrium correlates with respiratory pathologies, including asthma, chronic obstructive pulmonary disease (COPD), and recurrent infections. Critically, the microbiota engages in bidirectional crosstalk with the host immune system, modulating pathogen-specific immune responses [14, 15]. This symbiotic relationship between mucosal immunity and microbiota ensures respiratory homeostasis. As reviewed by Benjamin G. Wu et al., dynamic shifts in lung microbiota composition during disease states directly influence mucosal immune phenotypes [16]. Microbes complement the immune system by assisting in training and modulating immune responses while also regulating the microbial community to maintain a balanced and healthy environment.

Emerging evidence positions respiratory microbiota as a key regulator of viral pathogenesis. Specific bacterial taxa within the upper respiratory microbiome—such as Streptococcus spp. and Neisseria spp. correlate with prolonged influenza viral shedding [17], whereas streptococcal-derived proteins in the lower respiratory tract alter epithelial antiviral responses to influenza infection [18]. Conversely, viral infections or environmental stressors disrupt microbial homeostasis, promoting pathogen adhesion and exacerbating respiratory infections [19]. This duality underscores the intricate interplay between mucosal immunity, resident microbiota, and viral pathogens: while certain bacteria facilitate viral entry and replication, others exert protective effects against viral invasion [20,21,22].

A comprehensive understanding of microbiota, mucosal immunity, and viral infections is imperative for devising innovative strategies against respiratory viruses and advancing targeted therapies. This review systematically discusses the mechanistic interactions among these components, with a focus on harnessing MIS and microbiota networks to combat viral infections across all pathological stages.

Mucosal immunity in the respiratory tract

The mucosal immune system in the respiratory tract is an integral part of the body’s defense against foreign pathogens, providing both physical and chemical barriers and immune functions through chemical, cellular, and tissue components. Mucosal immunity comprises inductive and effector sites [23]. Inductive sites, which include mucosal epithelial cells [24], microfold cells (M cells) [25], and immune tissue networks such as bronchus-associated lymphoid tissue (BALT) [26] and nasopharynx-associated lymphoid tissue (NALT) [23], are responsible for antigen capture, processing, and presentation, as well as the activation of immune cells and induction of immune responses. Effector sites, composed of dispersed immune cells like lamina propria lymphocytes (LPL) [5] and innate lymphoid cells (ILCs) [27], are primarily responsible for responding to antigens. The dynamic process of respiratory mucosal immunity can be categorized into three functional components: physical and chemical barrier defenses, innate immune responses, and adaptive immune regulation (Fig. 1).

Fig. 1

Formation of the mucosal barrier in the respiratory tract and its functions in the immune system. When pathogens invade, respiratory mucosal immunity can impede pathogen invasion and prevent further dissemination through three stages: physicochemical barrier, innate immune responses, and adaptive immune responses. I. The physicochemical barrier is composed of mucus and epithelial cells. Mucins and sIgA in the mucus can neutralize pathogens and expel them from the respiratory tract through the clearance action of ciliated cells. Pathogens that breach the mucus layer interact with epithelial cells, which release cytokines or engage in direct cell-to-cell contact (gap junctions, exosomes, etc.) to activate innate immune cells. II. After penetrating the physicochemical barrier, pathogens enter the lamina propria. Professional cells in the lamina propria (DCs, macrophages, mast cells) recognize PAMPs to initiate signal cascades, recruiting more innate immune cells to participate in the antiviral response. PRRs activate adapter proteins, triggering cascades that activate IRFs and NF-κB. These factors translocate to the nucleus, initiating interferon gene transcription and enhancing immune defense. III. During the adaptive immune phase, T cells and B cells play dominant roles. DCs that have encountered pathogens present antigens and transmit stimulatory signals to T cells and B cells. Antigen-presenting cells promote the maturation and proliferation of naïve CD4⁺ and CD8⁺ T cells through T cell receptor (TCR) engagement and co-stimulation (CD80/CD86). After antigen recognition, B cells differentiate into antibody-secreting plasma cells and memory B cells with the help of cognate CD4⁺ Tfh cells. IV. Effector cells migrate from the induction site to the effector site to exert their functions. DCs, dendritic cells; ILCs, innate lymphoid cells; IRFs, interferon regulatory factors; NF-κB, nuclear factor-kappa B; IFNs, interferons; Th cell, T helper cell; M cell, microfold cell; MUC5AC, mucin 5AC; MUC5B, mucin 5B; IgA, immunoglobulin A; sIgA, secretory Immunoglobulin A; CTL, cytotoxic T lymphocyte; T_RM, tissue-resident memory T cell

Physical and chemical barriers of RMS

The epithelial layer and mucus layer form the physicochemical barrier of RMS, serving as the primary line of defense against respiratory pathogens. The epithelial layer comprises ciliated cells, club cells, goblet cells, basal cells, and other rare cell types [24]. Airway mucus (97% water, 3% solids) contains mucins, salts, lipids, and cellular debris [28]. Two strongly expressed mucins in the airways, MUC5AC and MUC5B, are antimicrobial mediators produced by secretory cells or submucosal glands, which clear pathogens by participating in the mucociliary clearance mechanism [29]. sIgA is produced by plasma cells beneath the epithelium and neutralizes pathogens through antibody activity as a non-mucin component [30]. When pathogens invade, respiratory epithelial cells rely on mucus secretion and tight junctions between epithelial cells to form a physicochemical barrier that prevents airway infection by certain pathogens [24, 31]. Respiratory mucus secreted by mucus-producing cells acts as a “scavenger” and is expelled from the respiratory tract through coordinated motion facilitated by ciliated cells [32, 33]. When various receptors on epithelial cells sense external stimuli, these cells can coordinate local immune cells by releasing soluble mediators (inflammatory factors, chemokines, defensins and antimicrobial peptides, growth factors, etc.) [34,35,36,37], and through cell-cell contact mechanisms (antigen presentation, direct contact, etc.) [38, 39] to initiate downstream innate and adaptive immune responses.

Innate immune responses in RMS

When pathogens penetrate the mucus-epithelial barrier, they enter the lamina propria beneath the epithelial layer. Dendritic cells (DCs), macrophages, and mast cells residing in this layer are specialized in detecting pathogens that have crossed the epithelial barrier [40]. This process involves the recognition of pathogenic components by pattern recognition receptors (PRRs), which include Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and NOD-like receptors (NLRs). Upon activation, these receptors coordinate a complex signaling cascade that results in the secretion of various cytokines and chemokines. These signaling molecules are crucial for recruiting diverse immune cells such as macrophages, natural killer cells (NKs), neutrophils, and ILCs to the site of infection [5, 41]. The engagement of viral components by PRRs specifically initiates the interferon (IFN) signaling pathway, leading to the substantial secretion of proinflammatory cytokines, chemokines, and numerous IFN-stimulated genes (ISGs), ultimately establishing robust innate immune defense against viral infection [42]. When the pathogen breaches the physical barrier and enters the alveoli, alveolar macrophages (AMs) rapidly recognize and engulf pathogens, releasing cytokines, such as interleukins (ILs), tumor necrosis factor (TNF), to recruit more neutrophils and monocytes to the wound site. These cytokines are instrumental in recruiting additional neutrophils and monocytes to the site, which amplifies the production of cytokines and bolsters the inflammatory response [5, 31]. Macrophages, act as “sentinels” in mucosal tissues, orchestrating immune responses by presenting antigens and secreting cytokines, not only directly eliminating pathogens but also secreting type I IFN (IFNα/β) by recognizing RLR. Conversely, IFN-γ, IL-17, and IL-22 enhance the phagocytosis of macrophages [43]. Collectively, the innate immune response, equipped with the above-specialized cells, plays acritical role in immune defense by producing cytokines and chemokines that modulate inflammatory responses and recruit other immune cells.

Adaptive immune responses in RMS

The adaptive immune response plays a leading role in defending against viral infections within the RMS. B cells and T cells are the primary functional components at this stage. During the adaptive immune phase, mature tissue-resident DCs migrate into the draining lymph nodes, carrying virus-derived antigens and contributing to the initiation of T cell-mediated responses [44]. Additionally, in NALT and BALT, specialized epithelial cells known as M cells capture antigens through a process called “phagocytosis” and deliver them to antigen-presenting cells (APCs) located beneath the epithelium, thereby initiating mucosal immunity [43, 45]. Antigen recognition triggers B cell differentiation, leading to the migration of B cells to the edges of lymphoid follicles and their proliferation. CD4⁺ helper T cells can recognize major histocompatibility complex (MHC) class II antigens presented by B cells, and the interaction with CD4⁺ helper T cells results in the isotype class switching of immunoglobulins from IgM to IgG or IgA [46]. IgA can be divided into serum IgA and sIgA. sIgA is mediated by the secretion of the polymeric immunoglobulin receptor (pIgR), which is internalized by epithelial cells through endocytosis and transported to the epithelial surface via intracellular vesicles to neutralize antigens [47]. In addition to the protective role played by antibodies produced by B cells, T cell-mediated cellular immunity can directly clear viruses. With the involvement of the T cell receptor (TCR) and appropriate co-stimulatory signals, T cells rapidly differentiate. CD8⁺ T cells acquire cytotoxic activity, recognizing viral peptides presented by MHC class I molecules. CD4⁺ T cells recognize viral peptides presented by MHC class II molecules and differentiate into Th1, Th2, Th17, and follicular helper T cells (Tfh), among others, to exert their effects [5, 44]. This section summarizes the cellular subtypes involved in mucosal immunity along with their respective functions, aiming to enhance the understanding of the immunological microenvironment within the mucosal system (summarized in Table 1).

Table 1 Differential cells attended in mucosal immune response

The commensal respiratory tract microbiota-mucosal immunity axis

Distinctive microbiota has evolved in the respiratory tract, and its stability is related to various diseases, such as asthma, COPD, pulmonary fibrosis, and acute respiratory distress syndrome (ARDS) [10, 14, 48, 49]. The respiratory system has a unique microbiota that co-occurs within the pulmonary respiratory mucosal system, which is essential for maintaining a healthy respiratory tract [50]. Resident microbiota colonizes the mucosal barrier and settles in the respiratory tract, playing integral roles in initiating an immune response from maintaining homeostasis to responding to infectious challenges [11]. Growing attention is being directed toward the intricate interactions between the commensal microbiota of the respiratory tract and the host’s mucosal immune system, a complex relationship that forms the ‘respiratory tract microbiota-mucosal immunity axis’ and plays a crucial role in defending against pathogen invasion, maintaining immune homeostasis, and influencing the development of respiratory diseases. This section provides an overview of known respiratory tract resident microbial communities and their associations with mucosal immunity.

Overview of the commensal respiratory tract microbiota and its diversity

The human respiratory tract extends from the nostrils to the alveoli and harbors a diverse microbiota. Due to the selective clearance of microorganisms by ciliated epithelial cells in the respiratory mucosa, there are variations in the types and quantities of microorganisms in different parts of the respiratory tract. The density and diversity of microorganisms in the respiratory tract decrease gradually with increasing distance from the oropharynx, while the mucosal density of lung microbiota decreases as it moves away from the central airway [14, 51]. Microaspiration is likely the primary route for microbial entry into the respiratory tract. Research has shown that the core microbiota in the nasal cavity is mainly composedofPropionibacterium, Corynebacterium,Staphylococcus [52]. Prevotella, Veillonella, Streptococcus, Fusobacterium, Neisseria, Haemophilus spp. are predominant in the oral cavity [53, 54]. Meanwhile, in the lung, Prevotella, Streptococcus, Veillonella, Fusobacterium and Haemophilus spp. are the main microbiota [14, 20]. The composition of the respiratory microbiome obtained through sequencing may vary under different conditions [55,56,57,58,59].

Compared to the upper respiratory tract (URT), the lower respiratory tract (LRT) composed of the trachea and lungs exhibits relatively lower biomass due to factors such as higher oxygen levels in the external environment, coughing mechanism along with mucociliary clearance, and innate immune system. It is widely accepted that LRT microbiota is mainly derived from URT microbiota migration [53]. In early life, the sequence and dynamics of respiratory tract microbiota can influence the development of normal respiratory mucosal immunity [54]. The microbial community in the respiratory tract acts as gatekeepers, providing resistance against colonization by respiratory pathogens and playing a crucial role in training and developing the host immune system [54, 60]. To maintain a symbiotic relationship with the microbial community, the host immune system continuously evolves and employs various mechanisms to achieve homeostasis between “microbes-host.” In other words, the interactions between the microbiota and the host mucosal immune system are bidirectional. Disruption of this equilibrium or alterations in the composition of the microbial community, known as dysbiosis, can contribute to various respiratory diseases [13], such as asthma, COPD, and pulmonary fibrosis. A variety of factors, including social, environmental, and physiological factors, can influence the stability of the microbiota [11]. Therefore, maintaining a balanced respiratory microbiota is essential for optimal functioning of the host immune system (Fig. 2).

Fig. 2

Factors affecting the homeostasis of the respiratory microbiota. These factors can drive the microbiota either toward a balanced and stable state that resists pathogen proliferation or, conversely, toward an unstable state susceptible to infection and inflammation

Role of respiratory tract commensal microbiota in modulating mucosal immunity

The respiratory commensal microbiota plays a significant role in promoting the maturation of respiratory immune function, maintaining mucosal immune homeostasis, and regulating mucosal immune responses through their interactions with the mucosal immune system.

Promotion of respiratory immune maturation

The development of the respiratory system continues after birth, during which the respiratory tract is colonized by commensal microorganisms that assist in the establishment of the defense system. The evolution of dominant microbiota and the abundance of the microbiome during the early colonization process in early life is crucial for the development of mucosal immune function and resistance to respiratory infections. If microbial colonization is lacking in early life, such as through the disruption of the microbiome by antibiotics and other drugs, or in germ-free mice, the susceptibility of the body to viruses and allergens will increase [61]. In early life, the presence of dominant microbiota in the nasopharynx undergoes a temporal shift from being predominantly Staphylococcus aureus to dominance of Gram-positive commensal bacteria such as Corynebacterium and Dolosigranulum, and then to a predominance of Moraxella spp. [62, 63]. A premature transition from a Staphylococcus into a Moraxella-dominated profile, characterized by diminished and less-prolonged establishment of Corynebacterium and Dolosigranulum spp., is related to more frequent respiratory tract infections [62]. Compared to healthy individuals, infants who frequently experience lower respiratory tract infections show significant differences in the composition of their nasopharyngeal microbiota at 24 months of age, exhibiting low diversity and a high susceptibility to pathogen colonization [64]. These studies indicate that the normal colonization and diversity of the respiratory tract microbiota over time play an important role in the establishment and proper functioning of respiratory immune function.

Maintenance of mucosal immune homeostasis

The coordination of the types, proportions, and anatomical structures of the resident respiratory tract microbiota across different species is an important factor in maintaining mucosal immune homeostasis. In various respiratory diseases, dysbiosis of the resident respiratory tract microbiota affects the normal function of mucosal immunity through multiple pathways, thereby impacting the normal function of the respiratory tract. In patients with COPD, a disturbance in the homeostasis of the respiratory tract microbiota has been observed to be associated with a decline in forced expiratory volume in 1 s (FEV1). Further studies using human multi-omics and mouse models have shown that chronic carriage of Staphylococcus aureus in the airway induces a shift from neutrophil apoptosis to NETosis via the AKT1-S100A8/A9 axis, thereby exacerbating inflammation, tissue damage, and decline in lung function [65]. The URT microbiota of COVID-19 fatality patients is significantly different from that of recovered patients. In recovered patients, a microbiota dominated by Streptococcus is more abundant, exhibiting high temporal stability and resistance to pathogens, while the microbiota of patients who died becomes more deviated from the normal state after admission [66]. In lung cancer patients, the dysbiosis of the lower respiratory tract microbiota is associated with poor tumor prognosis. In mice, a microbial disturbance dominated by the enrichment of Veillonella parvula leads to the upregulation of the ERK/MAPK, IL-1, IL-6, and inflammasome signaling pathways, as well as the activation of immune checkpoint inhibitor markers [67]. The balance of the resident microbiota in the respiratory tract can serve as a target for maintaining immune homeostasis, regulating inflammatory damage, and immune suppression.

Regulation of the mucosal immune responses

The resident microbiota of the respiratory tract regulates the function of mucosal immune cells, further modulating innate and adaptive immunity to achieve immune defense against pathogens. In mouse experiments, it has been shown that Prevotella in the airway can increase TLR2-dependent, serine protease-mediated killing of S. pneumoniae by lung neutrophils during the early stages of pneumococcal infection and activate related pulmonary pro-inflammatory responses to clear S. pneumoniae; later, it can regulate pulmonary inflammation through the induction of IL-10, achieving a protective effect on the lungs [68]. In mice colonized with Corynebacterium, it has also been observed that there is an early clearance of Streptococcus pneumoniae from the airway, a reduction in pulmonary inflammatory factors and inflammatory cells, and a limitation of lung damage [69]. In the lower respiratory tract of mice, episodic aspiration with oral commensals over 14 days can lead to an increase in a range of T cell subsets, including activated CD4⁺ and CD8⁺ T cells, CD4⁺ and CD8⁺ T cells expressing PD-1, regulatory T cells (Tregs), and produce a transient inflammatory response. On day 14, there is a significant increase in Th1-like, Th17, and γδ T cells. After the inhaled microbes are rapidly cleared, a prolonged Th17 response can decrease susceptibility to S. pneumoniae, achieving an immune protective effect [70].

Trained immunity: a Bridge of mucosal immunity with microbiota

Trained immunity is directly initiated by the stimulus of PRR from endogenous or exogenous sources, subsequently proceeding with metabolic reprogramming, and enhancing functional responses upon secondary stimuli [71]. Unlike the adaptive immune system, which has a specific memory relying on B and T cells, trained immunity involves epigenetic reprogramming and metabolic changes in innate immune cells like NKs, DCs, ILCs, neutrophils, stromal and epithelial cells [72]. Innate immune cells express a diverse array of pattern recognition receptors (PRRs) that detect pathogen-associated molecular patterns (PAMPs) from exogenous pathogens and damage-associated molecular patterns (DAMPs) [73]. These primary stimuli orchestrate dynamic epigenetic remodeling and metabolic reprogramming within the innate immune cells, which potentiates their responsiveness to secondary challenges.

Components of the microbiota, such as lipopolysaccharides (LPS) and other microbial-associated molecular patterns (MAMPs), are recognized by PRRs on innate immune cells. This interaction can lead to epigenetic changes and enhanced metabolic activity in these cells, essentially “training” them to mount a quicker and stronger immune response in the future. Upon stimulation by stimulants such as BCG, β-glucan, LPS, and other stimulants, immune cells can undergo epigenetic reprogramming through the mTOR pathway to increase the production of pro-inflammatory factors and achieve the protective effect of immune cells against infection. Simultaneously, immune cells activation leads to increased energy demand accompanied by elevated aerobic glycolysis, glutamine catabolism, and fatty acid synthesis. This metabolic shift results in an accumulation of key intermediates including acetyl-CoA, fumaric acid, succinic acid, nicotinamide adenine dinucleotide phosphate (NADPH), and mevalonate that further modulates epigenetic levels and achieves the role of training the immune to produce a rapid and efficient response during a second strike.

Understanding how the microbiota trains mucosal immunity will promote the development of more effective mucosal vaccines, which are designed to enhance local immune responses and establish immunity where pathogens typically enter the body. The interplay between trained immunity and mucosal immunity in the context of the microbiota is complex and multifaceted (Fig. 3). It is clear that the microbiota plays a crucial role in priming and training the immune system at mucosal surfaces, providing a robust first line of defense against pathogens. Further research is needed to fully understand the mechanisms and to harness these interactions for preventive and therapeutic purposes.

Fig. 3

Relationships between “trained immunity-mucosal immunity- microbiota”. Upon initial host exposure to the resident microbiota, MAMPs such as β-glucan and LPS interact with PRRs on innate immune cells. This interaction modulates the TCA cycle and glycolysis via the mTOR pathway. The resulting metabolic intermediates directly mediate epigenetic changes through histone modifications, leading to increased accessibility of open-chromatin regions containing inflammatory genes and regulatory elements, thus enhancing cytokine transcription. These epigenetic modifications persist even after immune cells return to baseline, enabling a rapid and efficient response upon re-exposure to similar stimuli. MAMPs, microbial-associated molecular patterns; PRRs, pattern recognition receptors; mTOR, mammalian target of rapamycin; TCA, tricarboxylic acid cycle

Reprograming metabolism by respiratory tract microbiota and MIS

As previously mentioned, processes such as aerobic glycolysis, glutamine catabolism, and fatty acid synthesis affect the process of metabolic reprogramming. This section will focus on exploring the relationship between respiratory microbiota and metabolic reprogramming in mucosal immunity (Fig. 4).

Fig. 4

Reprogramming metabolism by respiratory tract microbiota and MIS. The respiratory microbiota interacts with host immunity through metabolic modulation, influencing immune-metabolic reprogramming via three core pathways:I. Glycolysis: Staphylococcus epidermidis AIT01 enhances TNF-α production in neutrophils via secreted GAPDH. β-glucan activates the Akt-mTOR-HIF-1α axis, boosting glycolytic intermediates that drive epigenetic immune regulation. Conversely, Senecavirus exploits glycolysis-derived lactate to disrupt MAVS-RIG-I interactions, suppressing type I interferon responses for immune evasion. II. Amino Acid Metabolism: SARS-CoV-2 alters airway microbiota composition and induces distinct amino acid metabolic disorders in COVID-19 patients. Targeting amino acid metabolism reduces inflammatory cytokine release in PBMCs, suggesting therapeutic potential for mitigating hyperinflammation. III. Fatty Acid Metabolism: In COPD, Lactobacillus salivarius and Lactobacillus oris elevate IAA by modulating K01426 gene abundance, reducing airway IL-1β, IL-6, and IL-17A. Gut-derived SCFAs further mediate lung immunoprotection via the gut-lung axis, highlighting cross-organ metabolic-immune regulation. Collectively, microbiota-derived metabolites dynamically reshape immune responses through metabolic intermediates, offering insights into host-microbe synergy in health and disease. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RIG-I, retinoic acid-inducing gene I; MAVS, mitochondrial antiviral-signaling protein; ISGs, interferon-stimulated genes; PBMC, peripheral blood mononuclear cells; SCFAs, short chain fatty acids; IAA, indole-3-acetic acid

Relation to glycolysis

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic pathway that converts glyceraldehyde-3-phosphate to glycerol 1,3-diphosphate. Kim Gwanghee et al. analyzed the secreted proteome of Staphylococcus epidermidis AIT01 using a two-dimensional gel. They found that secreted GAPDH significantly enriches TNF-α-producing neutrophils, which in turn enhances the mechanism of respiratory immunity to protect the host from lethal viral infections [74]. This study provides the direct basis for the resident respiratory flora to influence the host immune response by producing glycolysis-related products. Although there are limited studies demonstrating the role of certain intermediates of glycolysis produced by respiratory resident flora in mucosal immunity, there have been more studies targeting trained immunity showing that β-glucan on the surface of resident flora in contact with immune cells can affect the tricarboxylic acid cycle via the Akt-mTOR-HIF-1α pathway, triggering trained immunity and leading to the activation of immune cells. In contrast, Huizi Li et al.'s study on Senecavirus (SVA) showed that SVA infection reprogrammed host cellular glucose metabolism, shifting ATP production from aerobic oxidation to glycolysis. This process promotes lactic acid production through glycolysis which attenuates the interactions between the mitochondrial antiviral signaling proteins (MAVS) and retinoic acid-inducible gene I (RIG-I), ultimately inhibiting type I IFN and achieves immune escape [75]. The study suggests that the interaction between the flora and immune cells leads to the development of trained immunity. Moreover, at the level of glycolysis, it has been observed that the flora exerts an inhibitory effect on IFN antiviral inhibition against viruses. In addition, Samuel Philip Nobs et al. demonstrated in diabetic mice that under hyperglycaemic conditions, elevated levels of acetyl coenzyme A in lung DCs of hyperglycaemic mice increased their susceptibility to respiratory infections, and that antiviral adaptive immunity in the lungs was also extensively impaired [76]. From a pathological perspective, it has been proven that influencing metabolites associated with glucose metabolism can impact normal immunity.

Relation to amino acid metabolism

Several studies have shown significant disparities in the abundance of different species of respiratory microbiota between COVID-19 and non-COVID-19 patients [66, 77]. In the viral high-load group, there was a decreased prevalence of 12 bacterial groups including Corynebacterium, Staphylococcus, Granilucatella, Neisseria, and Prevotella, which were less abundant [77]. These diminished flora species exhibit an overlapping relationship with the resident respiratory flora. At the same time, it has been shown that COVID-19 patients have a unique disorder of amino acid metabolism [77, 78], including arginine, tryptophan, or purine metabolism. An experiment using the rhesus monkey model demonstrated that interfering with arginine, tryptophan, or purine metabolism effectively inhibited the release of inflammatory factors in peripheral blood mononuclear cells (PBMC) during COVID-19 infection [78]. Changes in respiratory microbiota and amino acid profiles in COVID-19 patients indicate that both microbial communities and amino acid metabolism may influence MIS function. However, direct causal relationships need further investigation.

Relation to fatty acid metabolism

Zhengzheng Yan et al. discovered that dysregulated tryptophan metabolic pathways in the airway flora of chronic obstructive lung may lead to a decrease in indole-3-acetic acid (IAA), consequently leading to compromised protection against neutrophil inflammation and epithelial cell apoptosis. This was further supported by macro-genomic and KEGG enrichment analyses in chronic obstructive lung patients. Moreover, it was found that Lactobacillus salivarius and Lactobacillus oris can contribute to changes in the abundance of the IAA synthesis gene K01426. Inoculation with both Lactobacillus species simultaneously slowed the decline in lung function, tissue damage, and apoptosis, reducing airway levels of IL-1β, IL-6, and IL-17 A [79]. This study provides evidence for the secretion of metabolites by respiratory resident microbiota to modulate mucosal immunity under conditions of COPD. In addition, short-chain fatty acids (SCFAs) which are produced by microbiota that carry genes capable of fermenting fiber, also have an important role in regulating immune response through the lung-gut axis [80, 81].

Viral infections: shaping the microbiota mucosal immunity axis

Impact of respiratory viral infections on the microbiota composition

The homeostasis of respiratory microbiota is essential for the host to achieve normal immune function and to resist viral infections. Numerous studies have reported that viral infections can affect the composition and abundance of colonizing microbiota in the respiratory tract [82,83,84]. Under these conditions, pathogenic species may dominate the microbial community, potentially facilitating viral infection.

Respiratory viral infections can break nasopharyngeal microbiota homeostasis [85]. The composition of NP and OP flora in patients with IAV differed from that of healthy individuals. The NP of patients with IAV was lower in abundance than that of healthy children in the genera Moraxella, Staphylococcus, Corynebacterium, and Dolosigulum. Significant enrichment of Streptococcus and Phyllobacterium was commonly detected in the NP flora of patients. The genera with the highest abundance in the OP flora of patients showed a decreasing trend and included Streptococcus, Neisseria, and Haemophilus [86]. Symptomatic human rhinovirus(HRV) infection was associated with lower alpha diversity (Shannon diversity index [SDI]), higher bacterial density (PCR concentration), and beta diversity of the microbiota (Jaccard and Bray-Curtis indices) compared with asymptomatic HRV colonization [85]. A team of researchers analyzed the LRT microbiome, lung transcriptome, and bronchoalveolar lavage fluid (BALF) metabolome of mice intranasally inoculated with H1N1 simian influenza and found significant changes in microbial community composition and species diversity in the acute phase (7 days or dpi post-inoculation), the recovery phase (14 dpi), and the recovery phase (28 dpi). The dominant bacterial species shifted from the phylum Alpha Aspergillus to the phylum Gamma Aspergillus and the phylum Actinobacteria in infected mice. Moreover, differences in upper airway bacterial load, bacterial abundance, and within-group microbiome composition increased consistently with increasing severity of COVID-19 [87].

The mechanisms by which viruses affect the composition and abundance of respiratory microbiota may be derived from several aspects. Firstly, viral infections disrupt the epithelial barrier to promote the adhesion of bacterial pathogens [88,89,90]. Influenza virus infection promotes Streptococcus pneumoniae colonization by releasing host-derived nutrients and reducing the clearance function of mucosal cilia. Secondly, respiratory viruses can alter host congenital and adaptive immune responses, promoting bacterial colonization and infection. For example, respiratory viral infections can impair monocyte activity [91], induce prolonged desensitization of AMs to TLR ligands [92], hinder phagocytosis of AMs [93], as well as inhibit T helper cell-induced antimicrobial peptide production [94]. Additionally, respiratory mucosal epithelial cell TLRs and NLRs induce the release of antimicrobial peptides such as β-defensin 2 (BD-2) upon stimulation by pathogenic bacterial surface constituents, which may potentially influence the composition of the respiratory microbiota [95].

Bidirectional interactions: how the microbiota influences viral susceptibility and immune responses

The colonization of the respiratory tract with specific microbiota can modulate the host immune response in several ways, thereby enhancing the host antiviral immune response and suppressing the over-immune response induced by viral infections, which is beneficial for the maintenance of the body’s health upon viral challenge. For example, LPS-mediated activation of TLR4 on innate immune cells can initiate immunity and reduce the efficiency of influenza virus infection [96]. Previous studies have demonstrated that bacterial colonization inhibits influenza-mediated acute lung injury by inducing M2 AMs. Additionally, mice housed in specific pathogen-free (SPF) environments were more susceptible to influenza-mediated death than mice living in natural (non-SPF) environments when colonized with upper respiratory tract bacteria containing TLR2 ligands. This finding suggests that commensal microbiota in the respiratory tract protect against lethal inflammation of the lungs caused by influenza infection [97].

In addition, the respiratory microbiota can competitively or indirectly inhibit viral invasion and transmission through metabolites, thereby contributing to the maintenance of airway homeostasis. Hui-Wen Chen et al. identified a giant extracellular matrix-binding protein (Embp), which is a fibronectin-binding protein that is shed from the bacterial surface. They found that S. epidermidis bacterial particles and Embp could directly bind to the virus, filtering influenza viruses from the nostrils [98]. Keisuke Nishioka et al. showed that proteins produced by lower respiratory streptococci can alter the antiviral response of respiratory epithelial cells to the influenza virus [99].

Strategies employed by viruses to evade the respiratory immune response

Many viruses have evolved strategies to evade or antagonize the host’s innate immune response, which has been detailed in previous reviews [100,101,102]. Viral escape refers to the phenomenon in which the host’s immune system fails to respond to a virus. This process is also known as immune evasion or antigenic escape, which occurs during the evolution of viruses and helps them survive [103]. Viruses employ various mechanisms to elude respiratory mucosal immunity (Fig. 5). This section focuses on the ‘defense-escape’ strategy between viruses and the respiratory innate immune response.

Fig. 5

Strategies employed by viruses to evade the respiratory immune response. A. Disruption of Physical Barriers: Viruses breach respiratory barriers by I. degrading mucus, II. disrupting ciliary motility, and III. damaging epithelial tight junctions. B. Blockade of Immune Signaling: Viruses evade immune detection by IV. inhibiting PRRs. They also V. upregulate host receptors or VI. induce apoptosis. C. Suppression of Immune Cell Function: VII. Viruses impair DCs maturation and induce macrophage necrosis. D. Interference with Bioactive Mediators: VIII. Viruses inhibit IFN production and disrupt IFN signaling. E. Viral Mutational Escape: IX. Antigenic drift and spike protein mutations in virus variants evade neutralizing antibodies and reduce vaccine efficacy. RSV, respiratory syncytial virus; CD31, cluster of differentiation 31; HLA I, human leukocyte antigen class I; ACE2, angiotensin-converting enzyme 2; SA, sialic acid; CX3CR1, C-X3-C-motif chemokine receptor 1; NS1, non-structural protein 1; Nsp1, non-structural protein 1; Nsp13, non - structural protein 13; TBK1, TANK-binding kinase 1; PLPro, papain-like protease; ISG15, interferon-stimulated gene 15; IRF3, interferon regulatory factor 3; IAV, influenza A virus; IFNAR1, interferon-alpha / beta receptor 1; JAK, janus kinase; TYK2, tyrosine kinase 2; STAT1, signal transducer and activator of transcription 1; STAT2, signal transducer and activator of transcription 2; IRF9, interferon regulatory factor 9; RIPK1, receptor-interacting protein kinase 1; RIPK3,receptor-interacting protein kinase 3; MLKL, mixed-lineage kinase domain-like

Interference with physical barriers

The viruses penetrate the physicochemical barrier by degrading mucus, disrupting ciliary movement, and destroying tight junctions in the epithelial layer. This leads to the destruction of the intrinsic structure of the epithelial layer and lamina propria. For example, the neuraminidase (NA) of the IAV breaks down sialic acid (SA) to facilitate viral release. The goblet cells in the respiratory epithelium express mucin glycoproteins, including MUC5AC, MUC5B, and MUC1 [104,105,106]. These SA-rich mucins function as viral receptor decoys, competitively inhibiting viral attachment to target epithelial cells. However, NA can degrade these mucins, thereby interfering with the normal function of the mucus layer [105]. Respiratory syncytial virus (RSV), rhinovirus, and influenza virus infections down-regulate the expression of key genes involved in cilia development, thus affecting the normal physiological function of cilia [107]. Various respiratory viruses can cause cilia defects through corresponding receptors such as CD31, human leukocyte antigen (HLA) class I, angiotensin-converting enzyme 2 (ACE2), SA, and CX3C chemokine receptor 1(CX3CR1), affecting cilia clearance of pathogens [108,109,110,111]. At the same time, respiratory viruses exert pathological effects on epithelial cells. They can directly damage epithelial cells by activating intrinsic protein hydrolase activity or indirectly by binding to protease-activated receptor-2 (PAR-2) expressed on the apical surface of bronchial epithelial cells [112]. Additionally, they can disrupt the barrier effect of epithelial cells by disrupting tight junctions in the epithelial layer. In vitro studies have shown that rhinovirus-infected cells result in loss of zona occludins(ZO)-1 in the junctional complex, leading to a decrease in transepithelial electrical resistance(TEER), suggesting barrier ‘leakage’ [88].Coxsackieviruses and rhinoviruses both affect occlusal proteins. While RSV causes disassembly of the junction complex [88, 113, 114].

Blocking immune signal transmission

Upon breaching the host's primary physical barriers, viral components encounter PRRs that initiate immune detection. The PRRs include Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and NOD-like receptors (NLRs) [111]. Respiratory viruses can achieve immune escape by evading recognition by PRRs in various forms. For example, the NS1 protein of influenza A virus in the form of sequestered viral dsRNA can evade the perception of PRRs [115]. SARS-CoV papain-like protease antagonizes the TLR7 signaling pathway by removing lys63-linked polyubiquitination of TNF receptor-associated factors to achieve escape from PRRs [116]. Furthermore, respiratory viruses can invade and destroy epithelial cells by upregulating the expression of receptors. For instance, in SARS-CoV-2-infected Vero cells, the expression of ACE2, the primary receptor molecule for SARS-CoV-2, is significantly activated [117]. Besides, viruses can evade mucosal immune surveillance by triggering host cell apoptosis. For example, the NS1 protein encoded by avian influenza A virus H5N1 induces apoptosis in human lung epithelial cells primarily through a caspase-dependent pathway, thereby reducing the immune effect of lung epithelial cells [118].

Inhibition of immune cell

The lamina propria is rich in lymphocytes and exerts an immune effect during pathogen infection. Viruses can achieve immune escape by interfering with the normal function of lamina propria cells. DCs play a crucial role in antigen presentation and cytokine production, and their dysfunction impairs host immune effects. It has been observed that SARS-CoV-2 may infect DCs, impede DC maturation, and limit T cell-mediated responses [119, 120]. Respiratory viruses can also achieve immune escape by interfering with the normal function of macrophages. AMs are the first immune cells to encounter pulmonary pathogens [121]. Evidence suggests that AM death plays a vital role in determining the pathogenesis of viral infections in the lungs [122]. Leonardo Duarte Santos et al. showed that replication-active RSV induces host cell autocrine TNF through the activation of receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), and mixed-lineage kinase domain-like (MLKL), which promotes necrosis in mouse macrophages and human monocytes. This necrotic pathway hinders early antiviral response to RSV infection and favors evasion from RSV immune [123].

Interfering with the production of biological modulators

Apart from destroying intrinsic structures, respiratory viruses can also achieve immune escape by disrupting critical immune regulators.production.SARS-CoV-2 NSP13 inhibits IFN-I production by degrading TANK-binding kinase 1 (TBK1) [125]. Nsp1 protein of SARS-CoV-2, significantly surpressretinoic acid-induced gene-dependent innate immune responses [126]. Additionally, papain-like protease of SARS-CoV-2 catalyzes the dissociation of ISG15 from IRF3 and reduces type I interferon response [127]. The NS1 protein in IAV is a major inhibitor of the host innate immune response and inhibits type I IFN production and signaling for viral escape by suppressing activation of IRF3, NF-κB, and AP-1 [105, 128,129,130]. These studies have demonstrated the ability of some respiratory viruses to interfere with the production and action of host IFN, leading to inhibition in immune cell proliferation and subsequent suppression in antiviral responses.

Additional means of achieving virus escape

In addition to interfering with the normal physiology and function of the respiratory mucosal immune system to achieve immune escape, viruses can also escape through mutations. Under host immune pressure, viruses are prone to genetic mutations, some of which can lead to antigenic variation and thus escape pre-existing immunity in the host, such as antigenic drift. For example, the surface glycoprotein HA of IAV contains antigenic epitopes critical for triggering the production of neutralizing antibodies by B cells. Therefore, the epitope of HA is a major determinant influencing the mechanism of mutation and recombination of the virus. The high variability of HA allows IAV to escape host immune surveillance and leads to seasonal influenza epidemics [105]. SARS-CoV-2, which has become prevalent in recent years, also has mutants with significant mutations in the S spiking protein, such as B.1.1.7 (Alpha), P.1 (Gamma), B.1.427/B.1.429 (Epsilon), B.1.351 (Beta)and B.1.617.2 (Delta). These mutations have reduced the efficacy of vaccines [111].

Mechanisms of microbiota-mucosal immunity axis in antagonizing viral infection

When a virus invades the respiratory mucosa, it first breaks through the mucus and cilia barrier. Subsequently, the virus binds to specific cell adhesion factors, such as ICAM-1 and SA. The interactions between infectious factors and epithelial cells induce PRRs, antimicrobial peptides exerting broad-spectrum antimicrobial activity, enzymes, reactive oxygen species, and secretion of various chemokines and growth factors. Simultaneously, intricate crosstalk between epithelial cells and both innate and adaptive immune cells orchestrates robust host defense mechanisms.

In this process, the commensal microbiota colonizing the mucosal surface of the respiratory tract antagonizes viral infections and reduces viral damage to the organism through three main mechanisms, either directly or by interacting with the host’s innate or adaptive immunity. These specific mechanisms include direct obstruction of viral adherence, reduction of viral load, and attenuation of immune damage (Fig. 6).

Fig. 6

Mechanisms of microbiota-mucosal immunity axis in antagonizing viral infection. A. Viral invasion and host-microbiota interactions in the respiratory mucosa. I. Viral particles penetrate the mucus-cilia barrier and bind to cellular adhesion molecules (e.g., ICAM-1, sialic acid receptors). II. Epithelial activation triggers PRR signaling, releasing antiviral effector molecules. III. Cross-talk between mucosal epithelium and immune cells (innate/adaptive) establishes coordinated defense. IV. Commensal microbiota competitively inhibits viral pathogenesis through direct antagonism and immune modulation, forming the microbiota-mucosal immunity axis. B. Mechanisms of microbiota-mediated antiviral protection. I. Receptor competition: Resident bacteria sequester viral attachment receptors through molecular mimicry, reducing virion shedding and transmissibility. II. Immune priming: Microbial colonization stimulates interferon responses that suppress viral replication. III. Inflammatory regulation: Commensal-derived metabolites attenuate infection-induced cytokine storms and tissue pathology.NHNE, normal human nasal epithelial; PR8 influenza, A/PR/8/34 influenza

Impeding virus adhesion

Viral adhesion is the first step in the viral invasion of host cells. Impeding direct viral adhesion can exploit the competitive relationship between viruses and microbiota. For instance, Mila Brum Ortigoza et al. found that mice colonized with Streptococcus pneumoniae prior to IAV infection had a lower rate of infection with IAV and lower rates of viral shedding compared to mice without Streptococcus pneumoniae colonization, thus limiting the spread of IAV. This phenomenon may be due to the depletion of host SA from the epithelial surface of the mouse URT caused by serine enzymes expressed by colonized Streptococcus pneumoniae. Streptococcus pneumoniae can utilize free SA to meet its nutritional requirements, limiting infection by IAV, which requires SA for efficient attachment [131].

Reducing viral load

Activation of the innate immune response is a rapid and effective way to reduce viral load. The colonizing of the respiratory tract can rapidly suppress viral replication by activating the host’s innate immune response. For example, IFN-λ is a key immunomodulator against epithelial mucosal virus infections, and a rapid immune response against respiratory viruses can be obtained by activating IFN-λ. By treating normal human nasal epithelial (NHNE) cells with Streptococcus epidermidis N1 prior to in vitro inoculation with IAV, Hyun Jik Kim et al. found that nasal commensal Staphylococcus epidermidis induced the production and secretion of IFN-λ and promoted IFN-associated innate immune responses against IAV to inhibit IAV replication. This inhibitory effect induces IFN-λ expression by promoting phosphorylation of IRF7, which can be independent of the role of TLR2. Furthermore, in vivo experiments in mice inoculated with human nasal mucus-derived Staphylococcus epidermidis revealed that the inoculated mice exhibited lower levels of IAV PA mRNA, fewer pathological alterations, and significantly lower histological scores in their lung tissues as compared to uninoculated mice, suggesting that interferon-dependent lowering of viral load by human nasal commensal Staphylococcus epidermidis reduces organismal pathologic damage [132]. It should be noted that not all species of respiratory resident microbiota induce IFN-λ production and secretion. For example, pre-treatment with Haemophilus haemolyticus results in down-regulated several cytokines involved in the IAV response compared to IAV-infected mice alone, including IL-6, IFN-γ, IL-10, MCP-1 to reduce the inflammatory response [74].

Mitigating immune damage

In clinical studies of SARS-CoV-2, we have learned that virus-mediated host inflammatory storms threaten the recovery of organ function [133]. Therefore, mitigating immune damage cannot be ignored in the process of microbial co-host mucosal immunity antagonizing viral infection. Fortunately, the role of respiratory commensal microbiota in antagonizing host inflammatory immune damage has been demonstrated. Wang Jian et al. demonstrated that commensal bacteria in the airways prevented lethal inflammation of the lungs caused by influenza infection using an intranasal infection of mice with the PR8 strain of influenza virus. They found that S. aureus colonizing the upper airway surface induced AM polarization to M2 AM by triggering the production of IL-13. Furthermore, they observed that S. aureus infected WT mice with M2-type AMs expressed high levels of the anti-inflammatory cytokines IL-10 and TGF-β. It was also found that the number of macrophages increased and inflammatory cells were more sensitive to inflammatory diseases in the S. aureus-treated mice during PR8 infection. These treated mice showed an increase in the number of macrophages, a decrease in the number of inflammatory cells, and a reduction in CD8⁺ T cells, thus demonstrating the role of S. aureus customization in preventing influenza-mediated lung injury [134]. Ming-Shan Kao et al. showed that colonization of Staphylococcus epidermidis antagonized viral infection by activating the free fatty acid receptor (Ffar2), which attenuates the production of IL-6 induced by the nuclear coat protein of SARS-CoV-2 and facilitates suppression of inflammatory responses and lymphocyte exhaustion [135].

Approaches to prevent and treat viral infection

Strategies to combat respiratory viral infections can be based on the understanding of how viruses evade recognition by the mucosal immune system and interact with the respiratory tract microbiota (Fig. 7).

Fig. 7

Strategies to block the invasion of respiratory viruses. Blocking viral-receptor binding represents the critical first-line defense against viral invasion of host cells. This can be achieved through receptor antagonists or competitive inhibition by commensal microbiota occupying receptor sites, thereby preventing viral attachment and subsequent cellular entry. When viral penetration triggers immune activation, strategic modulation of relevant signaling pathways and cytokine responses can enhance antiviral efficacy while mitigating pathological hyperinflammation, ultimately improving clinical outcomes. Prophylactic strategies employing microbial component-based vaccines or intranasal immunization protocols may prime trained immunity in hosts, conferring protection against severe sequelae of viral infection. Notably, gut-lung axis modulation through intestinal microbiota intervention emerges as a promising approach to optimize systemic responses post-infection. Furthermore, preventing viral-bacterial co-infections is crucial for containing viral dissemination and replication by avoiding synergistic inflammatory cascades

Blocking virus from binding to receptor

Viral replication is particularly active in the early stages of viral infection. Thus, antiviral therapy can be most effective before the disease progresses to the systemic hyperinflammatory stage. Blocking the viral receptor binding domain is a critical step in antiviral approaches. However, currently available monoclonal antibody-based strategies are highly virus-specific and may develop resistance as the virus mutates. Employing a broader range of mechanisms to block viral-receptor binding may be therapeutic. For example, potential drugs could inhibit viral entry by targeting ACE2 receptors, such as Abidol and the drug sodium methanesulfonate foca, which targets TMPRSS2 to prevent viral entry [136]. Moreover, beneficial bacteria influence the invasion of viruses through blocking effects [137].

Regulating related signaling pathways and cytokines

Once the virus has breached the outermost immune barrier, the use of appropriate pharmacological interventions to protect the host ciliary structure can alleviate some aspects of the disease by modulating the appropriate signaling pathways, even if they do not restore the damage to the ciliary structure caused by the virus [99]. It is evident that down-regulation of IFN expression is present in the immune escape mechanisms of a wide range of respiratory viruses, and therefore, exogenous supplementation of IFN to modulate IFN levels in the mucosal immune system is expected to impede further viral invasion. The dysregulated cytokines produced by the virus-mediated inflammatory response in the host can be treated with immunotherapy. For example, immunomodulators can treat the dysregulated innate immune response to SARS-CoV-2 infection [130]. As the study of respiratory microbiota intensifies, there is a growing awareness of the antagonistic role of the resident respiratory flora against viral invasion. In particular, the training immunological effects of respiratory flora have been used in the field of disease treatment and vaccine development. MV130 is a multibacterial mucosal vaccine produced by Inmunotek, consisting of different proportions of heat-inactivated Gram-positive (90%) and Gram-negative (10%) bacteria, including Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, Catamorium, and Haemophilus influenzae, which induces training immunity against experimental viral respiratory infections [138]. In addition, available studies have shown that a variety of individually isolated respiratory resident flora have demonstrated antagonism of respiratory viral infections in in vitro and in vivo experiments [132, 139]. Intranasal delivery of one or more respiratory resident flora to induce an associated natural immune response to prevent and treat respiratory viral infections is a potential therapeutic approach. By administering effective probiotic resident flora prophylaxis before or during viral infection, medical care and antibiotic use can be reduced, resulting in fewer surgeries and lower antimicrobial resistance. A comprehensive investigation of how commensal bacteria induce the mucosal immune system to produce trained immunity is essential for developing vaccines and preventive drugs.

Using microbial antagonism against the virus

In recent years, increasing studies have shown that respiratory microbial-mucosal immune interactions are not independent of other mucosal immune systems. This interplay between the gut microbiota and the lung is known as the ‘gut-lung axis’. The gut microbiota influences the microbiological composition of the lung through direct seeding of the airways or the distribution of SCFAs. Junling Niu et al. showed that acetate, a metabolite produced by Enterobacteriaceae, helped mice resist influenza virus infection by enhancing NLRP3-mediated type I IFN production [81]. Consequently, targeting immune response modulation within the lung-gut axis offers novel approaches for combating respiratory viral infections. Furthermore, considering this crosstalk mechanism and the action of microorganisms on the mucosal immune system, modulation of pathological damage due to viral infections by oral probiotics may be a direction. Severe COVID-19 patients had higher chances of survival and a reduced risk of developing respiratory failure when administered with the probiotic formulation SLAB51 [140]. A study by Ha-Young Jeon et al. found that oral administration of probiotic-containing yogurt led to an increase in survival from viral infection, body weight, and levels of IFN-γ, IgG1, and IL-10, as well as a decrease in the inflammatory cytokines TNF-α and IL-6 in hamsters infected with H1N1 influenza. Although the number of SARS-CoV-2 replicates in the lungs of yoghurt-treated SARS-CoV-2-infected hamsters was not significantly reduced, their lung histopathological findings were modified [141]. Consequently, targeting immune response modulation within the lung-gut axis offers novel approaches for combating respiratory viral infections Although the interaction of multiple bacteria in the gut with host immunity may be similar in the lungs, the specificity of the flora in different body sites should be considered. In addition to oral administration, direct delivery of beneficial bacteria to the airway may become a more effective strategy [137].

Preventing viral and bacterial co-infections

Although many studies have shown that respiratory flora interacting with the mucosal system can antagonize viral infections, it should be noted that innocuous flora may become dysbiosis after the viral invasion and thus cause secondary infections. For example, highly susceptible viruses may cause excessive mucus production in the respiratory tract and lead to obstructive respiratory disease, allowing specific microorganisms to proliferate and exacerbate the disease [142]. It has been found that influenza A virus invasion triggers the release of pneumococci from biofilms, causing significant changes in their transcriptome and transforming them into more virulent phenotypes responsible for secondary airway infections. Studies have revealed [121]. Additionally, Ning Li et al. demonstrated that influenza virus NA activates TGF-β, which promotes the expression of cellular mucins, increasing bacterial colonization and facilitating viral-bacterial co-infections [143]. In such circumstances, attempts to eliminate the overly intense host inflammatory response induced during viral-bacterial co-infections, such as controlling bacterial replication and calming the host immune response, are considered to be improved therapeutic strategies [144]. Notably, combination treatment with azithromycin and dexamethasone significantly improves clinical outcomes, bacterial clearance, cytokine response, and immunopathology in a mouse model of dual infection [144].

Conclusion

Respiratory mucosal delivery has emerged as the preferred strategy for developing the next generation of vaccines. Respiratory mucosal administration induces immune responses by applying vaccines or drugs directly to the mucosal surface of the respiratory tract. Compared with the traditional intramuscular injection route, respiratory mucosal administration has unique advantages. First, the respiratory mucosa serves as a portal of entry for many pathogens, including respiratory viruses and bacteria, providing more direct protection against infection. In addition, it can stimulate mucosal immune memory, enabling a quicker and more effective response to reinfection. Consequently, this approach is considered an important direction in the next generation of vaccine strategies, particularly with great potential in responding to respiratory infectious diseases such as COVID-19 and improving vaccine efficacy. Both B and T cells are essential for maintaining healthy respiratory mucosal immunity. Training immunization enables some vaccines to prevent infections outside of specific targets. We propose that the interaction between microbiota and MIS may be mediated by some structural proteins or small molecule compounds responsible for maintaining immune balance. Identifying critical environmental factors that promote bacterial stability within the MIS could aid in developing mucosal vaccines capable of evading mucus barriers within this system. Although viral infection can disrupt the homeostasis of the mucosal system microbiota, there is still a lack of comprehensive understanding regarding the mechanisms underlying exogenous viral infection and resident microbiota interactions, as well as the precise dynamic balance point at which resident microbiota stimulate the mucosal immune system. This knowledge gap may partly stem from our limited understanding of the healthy respiratory microbiota. To better understand this complex relationship, more detailed longitudinal studies are required.

In future studies, we can focus on addressing the following questions: I. whether changes in the microbiome trigger mucosal immune responses to antiviral invasion; II. whether therapeutic interventions can restore microbiome equilibrium to modulate the progression of viral infections; III. whether other molecules or metabolites are produced in training immunity, which promote transboundary signaling and viral infections; IV. how viral infections disrupt the structure and balance of intricate microbial communities within the respiratory mucosal immune system, and which microorganisms are key targets for disrupting homeostasis?