The Beneficial Role of Probiotic Lactobacillus in Respiratory Diseases

Respiratory diseases cause a high incidence and mortality worldwide. As a natural immunobiotic, Lactobacillus has excellent immunomodulatory ability. Administration of some Lactobacillus species can alleviate the symptoms of respiratory diseases such as respiratory tract infections, asthma, lung cancer and cystic fibrosis in animal studies and clinical trials. The beneficial effect of Lactobacillus on the respiratory tract is strain dependent. Moreover, the efficacy of Lactobacillus may be affected by many factors, such as bacteria dose, timing and host background. Here, we summarized the beneficial effect of administered Lactobacillus on common respiratory diseases with a focus on the mechanism and safety of Lactobacillus in regulating respiratory immunity.


INTRODUCTION
Probiotics are defined as "live microorganisms" and confer a health benefit on the host when properly administered (1). Many probiotics have been shown the beneficial properties, including Lactobacillus spp., Bifidobacterium spp., Enterococcus spp., Streptococcus spp., Propionibacterium spp., Bacillus cereus, Saccharomyces boulardii, and several specific strains of Escherichia coli (1,2). Among probiotics, the most widely used are microorganisms of the genus Lactobacillus, which contains more than 200 species bacteria (3,4). Lactobacillus spp. are gram-positive, facultative anaerobic bacterium that ferments carbohydrates to produce lactic acid (5), and has a high economic value due to the use in biotechnology, food as well as therapeutic application (4,6,7). In human hosts, Lactobacillus spp. exist in the gastrointestinal tract, vagina, oral cavity, respiratory tract and skin. They account for 6% and 95% of the total bacteria in the intestinal and vaginal tracts, respectively (8).
As natural immunobiotics, Lactobacilli have excellent immunomodulatory function, and their ability in improving gastrointestinal, oral and vaginal disorders is already well known (6,(9)(10)(11)(12). Meanwhile, emerging evidence shows that Lactobacillus can also modulate respiratory immunity (13)(14)(15)(16). Administration of Lactobacillus confer a beneficial role in respiratory diseases including respiratory tract infections (RTIs), asthma, lung cancer, cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) (17)(18)(19)(20)(21). Administration of Lactobacillus may be an alternative strategy to alleviate respiratory diseases. In this review, we focus on the beneficial effect of probiotic Lactobacillus in promoting respiratory health and discuss its potential mechanism and safety.

Respiratory Tract Infections
RTIs are the major source of incidence and mortality in the world (22). Although many RTIs are of mild and self-limited nature, they caused 4 million deaths worldwide each year (23,24). In 2017, influenza virus has infected 54.5 million people worldwide, resulting in about 145,000 deaths (25). Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is raging globally. Effective vaccines are not available for many respiratory pathogens, and the increase in drug-resistant microbe makes the effective treatment of RTIs extremely challenging. Therefore, it is important to find a safe and effective method to reduce the risk of RTIs. Recently, the probiotic Lactobacillus has been used to fight against a variety of RTIs, including virus and bacterial infection ( Table 1). Oral administration of Lactobacillus can improve symptoms caused by poly(I:C) treatment (49). Many Lactobacillus species can prevent influenza virus infection, including L. rhamnosus GG, L. casei Shirota, L. plantarum DK119, L. paracasei MCC1849, L. gasseri SBT2055, L. fermentum CJl-112 and L. kunkeei YB38 (34,(50)(51)(52)(53)(54)(55). Moreover, Lactobacilli also have excellent role as a vaccine or adjuvant in preventing influenza virus infection due to its safety and biotechnological advantage (56)(57)(58). It is worth pointing out that oral supplementation with L. johnsonii (strain not shown) in pregnant BALB/c mice can reduce Th2 type cytokines and lung inflammation in Respiratory Syncytial Virus (RSV)-infected newborn mice (59). It means Lactobacillus may have preventive effect for RTIs of offspring when administered to the mother. Notably, supplementation with live and inactivated bacteria of the same Lactobacillus strain (such as L. rhamnosus CRL1505) generally have similar effect (39,40), suggesting that viability is not necessary for Lactobacillus achieve the protective immunoregulatory effect. However, nasally administration of, viable but not heat-killed L. rhamnosus CRL1506 can provide complete protection against RSV infection in mice (28). Although the reason needs further investigation, it shows that Lactobacilli efficacy is strain-dependent. Therefore, the selection of a Lactobacillus strain with potent immunomodulatory ability is crucial.
Although vaccines for SARS-CoV-2 are available, the rapidly SARS-CoV-2 mutating makes the effectiveness of these vaccines challenging (60). Notably, in vitro experiment has demonstrated that L. fermentum 90 TC-4 pretreatment increases the activity of SARS-CoV-2-infected Vero E6 cells (grass monkey kidney cells) (61). Pretreatment of L. plantarum MPL16 and CRL1506 can also inhibit the proliferation of SARS-CoV-2 in human lung epithelial cell line Calu-3 (62). In Central Europe, it has been reported that low death rate of COVID-19 patients is associated with the consumption of fermented vegetables containing many Lactobacilli species (63). Therefore, Lactobacilli may have the potential to become an adjuvant for treating SARS-CoV-2.
However, there are some limitations in the therapeutic effect of administering Lactobacillus. For example, neonatal C57BL/6 mice pre-treated with L. rhamnosus GG maintain 100% survival rate post influenza virus infection; however, the survival rate is only 10% when mice treated with L. rhamnosus GG at 48h post influenza virus infection (29). Similarly, the survival rate of BALB/c mice treated with L. plantarum at 24h post Pneumonia virus infection is 100%, but all mice die when L. plantarum administered at 72h post infection (3,64). These results show that Lactobacillus administration post infection cannot exert an obvious protective immunoregulatory effect.
Lactobacilli can also provide resistance to respiratory bacterial infections. Administration of Lactobacilli such as L. rhamnosus CRL1505, L. casei CRL 431 and L. pentosus B240 increases resistance of mice to Streptococcus pneumoniae infection (41)(42)(43). Intranasal inoculation of L. rhamnosus CRL1505 is beneficial in S. pneumoniae infected-immunodeficient mice (39). Interestingly, peptidoglycan from L. rhamnosus CRL1505 shows a similar protective effect with the whole bacteria in preventing S. pneumoniae infection (40). Moreover, nasal administration of L. rhamnosus CRL1505 can also reduce pathogen load and lung damage of infant mice with RSV infection and secondary S. pneumoniae infection (65,66).
In clinical trials, Lactobacilli are generally given in the form of tablets, capsules, powders, fermented yogurt or dairy products, and mainly used for preventive purposes ( Table 2). For instance, oral L. rhamnosus GG in adults can reduce rhinovirus infection (83), and the combination of oral L. paracasei (strain not shown), L. casei CRL 431 and L. fermentium PCC also reduces rhinovirus-induced common and influenza-like infection (78). In addition, oral mixed probiotic (mainly Lactobacilli) can decrease the risk of respiratory failure in COVID-19 patients by 8-fold and reduce the rate of transfer to Intensive Care Unit and mortality (84). In patients with severe COVID-19, oral tablet (live B. longum, live L. bulgaricus and live S. thermophilus, strains not shown) also shortens the time to reach a negative nucleic acid test of SARS-CoV-2 and decreases blood C-reactive protein and procalcitonin (81). Moreover, clinically administration of many Lactobacillus species such as L. rhamnosus GG, L. paracasei N1115 and L. plantarum L-137 can decrease the total incidence as well as shorten the duration of RTIs (26,27,81). Therefore, the prospect of Lactobacillus clinical application in reducing the risk of RTIs is promising. and b2 agonists, there is still no specific treatment for asthma and healthcare is expensive during exacerbation (86). Emerging evidence suggests that respiratory asthma symptoms can be ameliorated when probiotic Lactobacillus is administered. Oral administration of many Lactobacillus species has shown an effective preventive role for asthma in animal studies, including L. rhamnosus GG, L. plantarum K37, L. reuteri (ATCC No. 23272), L. casei Shirota, L. paracasei HB89 and L. salivarius (strain not shown) (18,(87)(88)(89)(90)(91). Some Lactobacillus species such as L. rhamnosus GG, L. paracasei (strain not shown) and L. fermentum (strain not shown) are orally used in the clinical and have beneficial effect on asthma in children (92,93). Interestingly, the acute asthma is closely associated with RTIs, especially rhinovirus infection (94). Consequently, the role of Lactobacillus administration in preventing RTIs may be useful in relieving exacerbation of acute asthma. Based on the "hygiene hypothesis," exposure to specific microbial components early in life can decrease the susceptibility to asthma and allergic diseases (95). Therefore, oral administration of probiotic Lactobacillus in the early life may also play an important role in preventing asthma.

Asthma
For the purpose of asthma prevention, the general route of administration of Lactobacillus species is oral delivery. However, a study indicates that although oral administration of L. paracasei NCC2461 could provide effective protection for female BALB/c mice with asthma, the efficacy of intranasal delivery is better (96). Nevertheless, two studies show that oral administration of L. rhamnosus GR-1 prevents the worsening of asthma in male BALB/c mice, but nasal administration has little effect on the improvement of asthma symptoms in male BALB/c mice (97,98). This may be caused by the lower dose and duration of nasal administration. Interestingly, oral administration of L. paracasei NCC2461 in perinatal mice provides prevention for allergic airway inflammation in the offspring (99). Similarly, in clinical trials, oral L. rhamnosus GG has been shown to prevent atopic diseases in children in both prenatal and postnatal (100). Thus, Lactobacillus may have a preventive effect against offspring asthma when administered to the mother. Furthermore, supplementation with Lactobacillus GG also has a therapeutic rather than only a preventive effect on mice suffering from asthma (101). Additionally, animal studies have found that oral Lactobacillus can also reduce other airway allergic reactions such as allergic rhinitis. For example, oral L. rhamnosus GG, L. gasseri TMC0356, L. plantarum IM76, L. plantarum CJLP133 and CJLP243 can effectively improve the symptoms of allergic rhinitis (102)(103)(104). In clinical trials. L. gasseri KS-13, L. casei Shirota and L. acidophilus L-92 have been used to effectively prevent seasonal allergic rhinitis (105)(106)(107). Thus, the administration of Lactobacillus may have excellent preventive effect on airway allergic reaction.

Lung Cancer
Lung cancer is a malignant tumor with a high incidence and mortality rate in the world (108). In recent years, immunotherapy has sparked a new surge in tumor treatment. Although immune The duration of diarrhea is shortened. Significantly shorter time to nucleic acid negativity and significantly lower inflammatory markers such as calcitoninogen and C-reactive protein Combination of L. plantarum KABP022, KABP023, KAPB033, and P. acidilactici KABP021 Patients with COVID-19 The reduction in nasopharyngeal viral load, pulmonary infiltration, and duration of digestive and non-digestive symptoms. check inhibitors (ICIs) such as anti-PD-1/PD-L1 and anti-CTLA 4 antibodies has become the first-line clinical treatment for tumors, the application of ICIs increases T-cell activity and removes the "braking" of the immune system, and these drugs are likely been associated with immune-related adverse events, especially when used in combination (109). Therefore, the search for natural immunobiotics with immunomodulatory properties to assist in the treatment of cancer patients may be a direction. It has been reported that probiotics show the same degree of anti-tumor ability as PD-L1 inhibitors, while simultaneous combination therapy with PD-L1 inhibitors virtually eliminated tumor growth in mice (110). Moreover, in clinical trials, oral administration of yogurt with probiotics provides a potential protective effect against lung cancer (111).
Lactobacillus is one of the most widely studied probiotic involved in the treatment of lung cancer. Administration of Lactobacillus can inhibit the metastasis of tumor cells to the lung. For example, nasal administration of L. rhamnosus GG can inhibit the metastasis of melanoma B16 to the lung in C57BL/6 mice (112). L. casei (strain not shown) has significant inhibitory effect on the proliferation of A549 lung cancer cells in vitro (113). Furthermore, intravenous and intradermal injection of L. casei YIT 9018 can increase the anti-tumor activity against Lewis lung carcinoma in C57BL/6 mice (114). In addition, oral administration of L. casei CRL 431 also reduces the side effects of chemotherapy (115), which may improve the prognosis of lung cancer patients. These suggest that some Lactobacillus species may have potential to become an effective adjuvant to treat lung cancer.

CF
CF is an autosomal recessive, monogenic disease with lesions affecting the lung, intestine, and other organ, but lung disease is the leading cause of morbidity and mortality in people with CF (116). CF is closely related to intestinal flora and significantly reduces intestinal flora diversity (117,118). A study indicates that oral L. rhamnosus GG improves disorders of intestinal flora in children with CF (119). Moreover, clinical trial shows oral administration of L. rhamnosus GG reduces pulmonary exacerbation and hospitalization rate in CF patients (120). This suggests administration of L. rhamnosus GG may be able to alleviate the symptoms of CF. In another clinical trial, however, oral administration of L. rhamnosus GG did not promote respiratory health in patients of CF (121). The main reason may be due to different inclusion criteria for clinical trial subjects. The clinical efficacy of applying a single Lactobacillus strain on CF is unstable, and the combined administration of multiple probiotic Lactobacillus species may be more effective. For example, intestinal supplementation with probiotic capsules (consisting mainly of Lactobacillus species) significantly reduces the incidence of lung deterioration and improves the quality of life in patients with CF (19,122). However, these are single-center clinical trials. Multicenter and expansion of the number of subjects are needed to further clarify the efficacy of Lactobacillus administration on CF in clinical trials.

Other Respiratory Diseases
Several studies have reported the potential of Lactobacillus in improving COPD. The commonly known causes of COPD are cigarettes and air pollutants (123). In vitro experiment has demonstrated that L. rhamnosus NutRes1 can reduce inflammatory mediators produced by cigarette-activated human macrophages (124). Moreover, oral feeding of L. rhamnosus (strain not shown) increases levels of IL-10 as well as SOCS3 and TIMP1/2, and attenuates lung injury of COPD induced by cigarette in C57BL/6 mice (21). Additionally, the development of COPD is associated with reduction of Lactobacillus spp. in lung (125). In addition, a study indicates that a decrease in intestinal Lactobacillus may contribute to the development of pulmonary arterial hypertension (126). In clinical trial, intestinal supplementation with L. casei Shirota can reduce the incidence of Ventilator-Associated Pneumonia in hospitalized patients (127). Thus, Lactobacillus may also be beneficial in other respiratory diseases and need more studies.

POTENTIAL MECHANISM OF PROBIOTIC LACTOBACILLUS IN IMPROVING RESPIRATORY DISEASES
As mentioned above, many members of the genus Lactobacillus have immunomodulatory properties. Notably, not only the whole bacteria, but also their components can exert immunomodulatory function, including peptidoglycans, extracellular polysaccharides, surface proteins and metabolites (short-chain fatty acids, SCFAs) and inorganic polyphosphate liquids (40,(128)(129)(130)(131). The mechanism of health-promoting effect of Lactobacillus on respiratory tract is complex. The same bacteria, when administered orally or nasally, or even administered live and inactivated bacteria, may not have the same effect on promoting respiratory health (49,50,52). The potential mechanisms include gut-lung axis and enhancement of the local mucosal immunity of the respiratory tract.

The Gut-Lung Axis
In recent years, many studies have reported that there is a crosstalk between gut and lung, and this connection described as gut-lung axis seems to be bidirectional (132,133). Oral administration of some probiotics, especially Lactobacilli, could promote respiratory health via gut-lung axis (17,30,134). Although the exact mechanisms by which Lactobacillus enterica affects the lung immunity via gut-lung axis are not fully understood, there are three main aspects (Figure 1).

Direct Immigration of Immune Cells and Cytokines From the Intestine to the Respiratory Tract Through the Circulation
Recognition of Lactobacillus species or their components by pattern recognition receptors (PRRs, such as TLRs or NLRs, etc.) on immune cells in the intestinal mucosa can result in the activation of innate immune cells which could migrate to lung tissue via circulation. For instance, innate lymphoid group 3 cells (ILC3s) can migrate from the gut to the lung where IL-22 + ILC3 exhibits resistance to pneumonia (135). Oral delivery of L. paracasei CNCM I-1518 also significantly increases ILC3 in lung tissue and enhances resistance to influenza virus (36). Intestinal supplements with L. paracasei MCC1849 can provide protection against influenza virus, which may be associated with an increased proportion of IgA + B cells and follicular helper T cells in Peyer's patches and significantly increases IgA secretion in lung (53). Villena suggests that Oral delivery of L. rhamnosus CRL1505 induces Th1 response in the gut-associated lymphoid tissue and promotes the migration of Th1 cell to the lung tissue where Th1 cells produces IFN-g which induces activation of alveolar macrophages, natural killer cells, CD103 + and CD11b high dendritic cell (DC) (49). Additionally, a study indicates that intestinal supplementation with L. rhamnosus GG can inhibit pulmonary inflammation, which is associated with an increase in Tregs of the intestinal tract (136). Moreover, oral administration of L. murinus (strain not shown) to antibiotictreated mice can also increase Tregs in lung (137). Tregs not only inhibit inflammation, but also inhibit Th2 type immune response (138). As controlling airway inflammation is crucial in preventing asthma progression (94), migration of Tregs to lung tissue may alleviate asthma symptoms.
Cytokines secreted in the mucosa of the gastrointestinal tract by Lactobacillus can reach the circulation and interact with other mucosal tissues, such as lung (139). Intestinal supplementation with L. rhamnosus CRL1505 can increase TNF-a, IFN-a, IFN-b, IFN-g in bronchoalveolar lavage fluid which exert a significant antiviral effect in the respiratory tract, and these cytokines show a similar increased trend in the intestinal fluid (49).

Influence of Lactobacillus Metabolites on Pulmonary Immunity via the Circulation
In parallel to promoting the migration of intestinal immune cells and cytokines, members of Lactobacillus genus produce metabolites that can modulate host respiratory immunity. The most important intestinal bacteria metabolites affecting pulmonary immunity is SCFAs, such as propionate, butyrate and acetate (140). Studies show that intestinal supplementation with many Lactobacillus species can increase SCFAs in the intestine and blood (141)(142)(143)(144)(145). The unmetabolized SCFAs enter the circulation and may affect pulmonary immunity in two ways. The first way is that SCFAs enter the bone marrow through the circulation enhancing generation of macrophage and DC progenitors (MDPs) (146), and meanwhile SCFAs can increase the conversion of MDPs into Ly6Cmonocytes (147,148); then bone marrow Ly6Cmonocytes migrate into lung tissue where they differentiate into alternatively activated macrophages (149). The second way is that SCFAs directly enter into the lung tissue where they may activate G proteincoupled receptors (GPCRs) or inhibit histone deacetylase (150)(151)(152). For example, butyrate can not only promote the generation of Tregs and IL-10 production by activating GPR109A (153), but also restore IL-10 in the lung by inhibiting histone deacetylase in mice (154). Another metabolite of Lactobacillus enterica, lithocholic acid, can also enhance Tregs function while inhibit FIGURE 1 | Potential mechanisms of Lactobacilli to modulate respiratory immunity via the gut-lung axis (1). Migration of activated immune cells and cytokines from mesenteric lymph nodes (MLNs) and intestinal lamina propria to the lung through the circulation (2). Some endocrine cytokines (such as TNF-a, IL-6) may migrate to the lung tissue through the circulation, and then alter immune environment of the lung (3). SCFAs affect bone marrow hematopoiesis and promote the conversion of macrophage and DC progenitors (MDPs) into Ly6C-monocytes, which reaches lung tissue and differentiates into anti-inflammatory alternatively activated macrophages (AAMs); AAMs inhibit chemokine CXCL1 production thus leading to reduced neutrophils recruitment in lung tissue (4). In the intestinal lumen, Lactobacilli or their components and production of metabolites (such as SCFAs) are taken up by intestinal epithelial cells and then enter to the lung via the circulation (5). Lactobacilli or their components from the intestinal lumen reach lung directly via microbreathing or esophageal reflux.
Th17 response (155). Other Lactobacillus intestinal metabolites (such as lactic acids, polyamines and indole derivatives) with immunomodulatory properties are involved in intestinal homeostasis (156)(157)(158). Nevertheless, it remains to be studied whether these metabolites can impact respiratory health via the gut-lung axis. In summary, Lactobacillus metabolites, such as SCFAs (especially butyrate) and lithocholic acid have excellent anti-inflammatory capacity, which may contribute to alleviate the development of RTIs, asthma, COPD. In addition to antiinflammatory effect, butyrate and propionate can induce apoptosis of lung cancer cells and inhibit proliferation of lung cancer cells in vitro (159)(160)(161). Therefore, oral administration of some lactobacilli may provide benefit for the treatment of lung cancer by producing butyrate and propionate.

Migration of Lactobacillus and Their Components to the Lung
There may be two main routes for transfer of intestinal bacteria to the lung tissue: the first is intestinal bacteria or bacterial components enter into the circulation through the mesenteric lymphatic system and then reach the lung tissue (162)(163)(164); the second is the migration of intestinal bacteria or bacterial components to the lung tissue through microbreathing and oropharyngeal reflux (165,166). Therefore, Lactobacillus or components of Lactobacillus in the intestine may be directly transferred to lung tissue and thus modulate lung immunity.

Enhancement of the Mucosal Immunity of the Respiratory Tract
Intestinal supplementation with Lactobacillus has been shown to promote respiratory health, but direct action of immunobiotic Lactobacillus on the respiratory mucosa may modulate local immunity of the respiratory tract. It has been found that intranasal administration of Lactobacillus can induce better respiratory immune response than oral administration (3,50,167). Nasal administration of Lactobacillus does not generally produce SCFAs due to the absence of substrate. The potential mechanisms by which they regulate respiratory immunity mainly have two aspects.
The first one is that some components of Lactobacillus can be recognized by PRRs in the respiratory tract and then activate downstream pathways. For example, nasal priming with peptidoglycan from L. rhamnosus CRL1505 increases TNF-a and IL-10 levels of lung and upregulates TLR2 and TLR9 expression in alveolar macrophages, which is similar to intranasal administration of whole bacteria (40). Meanwhile, other studies have shown that nasal priming with peptidoglycan from L. rhamnosus CRL1505 can enhance the TLR3/RIG-Itriggered antiviral immune response by increasing IFN-g and NK cell activity, thus contributing to higher viral clearance and reducing lung tissue damage (28,65,168). In addition, lung peptidoglycan can also be recognized by peptidoglycan recognition proteins (PGRPs), a type of PRRs, which mediates bactericidal effect (169). For instance, activated PGRP2 could promote neutrophil recruitment in lung tissue of S. pneumoniae infected mice (170). Of note, not all peptidoglycan of Lactobacillus species has the same protective effect. Nasal administration the peptidoglycan from L. rhamnosus CRL534 does not enhance resistance to S. pneumoniae infection in immunodeficient mice (41). This strongly suggests that the protective effect provided by Lactobacillus is strain-specific. Importantly, nasal administration of Lactobacillus may activate PRRs by multiple pathways. Even if one PRR is blocked, another pathway can be activated to provide protection in a compensatory manner. For example, studies have shown that only when both NOD2 and TLR2 are knocked out can L. plantarum BAA-793 lose its role in protecting against pneumonia virus infection (3,64,171). Therefore, components of Lactobacillus to activate the PRRs may be an important part of the protective role performed by intranasal Lactobacillus delivery.
The second one is that Lactobacillus can bind to host cells to antagonize adhesion or binding of pathogen. For the bacteria, experiments have demonstrated the ability of Lactobacillus to directly inhibit the adhesion of bacteria to respiratory epithelial cells. L. rhamnosus Kx151A1, L. reuteri PTA-5289, and L. salivarius LMG9477 can inhibit the adhesion of S. pyogenes to pharyngeal epithelial cells (172). Moreover, intranasal administration of L. murinus CNCM I-5314, a eubacterium of the murine lung, can provide a barrier function against the colonization of S. pneumoniae in the lung tissue (173). In the case of viruses, Lactobacillus binds competitively to viral receptor molecule to prevent viral entry into the host cell. For instance, lipopeptides released by L. curvatus, L. sakei and L. lactis (strains not shown) can bind to the receptor molecule (angiotensinconverting enzyme 2) of SARS-CoV-2 spike glycoprotein, and may prevent virus entry into host cells (174,175). In addition to inhibiting the adhesion and binding of pathogenic bacteria, Lactobacillus directly displays antibacterial activity. Some Lactobacillus spp. exhibit antibacterial effect against group A Streptococcus in vitro (176). Similarly, L. rhamnosus Kx151A1 and L. reuteri PTA-5289 significantly inhibit hemolytic activity of S. pyogenes in vitro (172). Additionally, some proteins secreted by Lactobacillus have antimicrobial activity. For example, reuterin secreted by L. reuteri has broad-spectrum antibacterial effect (2). However, whether it will alter lung microbial composition and affect lung homeostasis remains further investigation.

SAFETY
As normal members of the human intestinal, vaginal, skin, oral and respiratory flora, Lactobacilli are low-toxicity commensal organisms and are mostly considered safe when taken as probiotics. In animal studies, long-term oral administration of L. plantarum PS128 had no bad side on the health in mice (177). In addition, nasal inoculation of L. reuteri F275 and L. rhamnosus GG, which are generally colonized in the intestinal tract. L. reuteri F275 is cleared in lung tissue less than 24 hours (178); live L. rhamnosus GG is detected on the nasal mucosa at 24 hours after intranasal administration, but not after 72 hours, and does not affect body weight or behavior in mice (97). In clinical trials, oral administration of some common Lactobacillus species such as L. reuteri DSM17938, L. casei Shirota and L. salivarius CECT5713 is safe in infants or children (2,(179)(180)(181). Moreover, with a long history of safe use, Lactobacilli are classified as GRAS (Generally Recognized as Safe) and QPS (Qualified Presumption of Safety) by the US Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA, 2021), respectively (182). Therefore, administration of some Lactobacillus species is generally safe.
However, when Lactobacillus is given intranasally, it can colonize the respiratory tract for long periods of time, and whether this could interfere lung microbial homeostasis or induce more severe inflammation or even lead to bacteremia is not clear. Although Lactobacillus bacteremia is a rare disease, it has been found in the clinical that L. rhamnosus GG and L. casei (strain not shown), which are generally considered safe, can also cause bacteremia when in a state of immunosuppression, prolonged hospitalization or surgical intervention (183). Even non-pathogenic bacteria of L. salivarius (strain not shown) in the oral cavity have been found to cause bacteremia, septic chest and diabetic ketoacidosis due to respiratory failure in the clinical (184). Moreover, it has been reported that pneumonia and pleural abscess are caused by mixed Lactobacillus infection in elderly people with esophageal cancer (185). Although it occurs being rare and almost exclusively in infants, children and immune-compromised populations, this also suggests that live Lactobacillus is not absolutely safe, especially when administered intranasally. Further, the presence of antibiotic resistance genes and virulence genes in Lactobacillus and their possible transfer to other microorganisms is also a concern.

CONCLUDING REMARKS
The use of Lactobacillus is a promising strategy for the prevention and treatment of respiratory diseases and is generally safe. The mechanism of Lactobacillus in regulating respiratory immunity includes the gut-lung axis and activation of mucosal immunity. The beneficial role of Lactobacillus on the respiratory tract is strain-dependent, and may change in different species of Lactobacillus and even subspecies of each Lactobacillus (32). Therefore, for clinical applications, the selection of effective Lactobacillus strains is crucial. Additionally, a single Lactobacillus is not always effective for all respiratory diseases and efficacy may be inconsistent even if the same Lactobacillus is administered due to many factors such as subject, dose and time of administration in clinical trials. The uncertainty of the effectiveness of Lactobacilli is one of the main reasons limiting their application in the clinic. For this problem, coadministration of multiple probiotic Lactobacillus or coadministration of probiotic Lactobacillus and other probiotics such as Bifidobacterium can provide more stable and better efficacy, which is also the trend of probiotic application. Furthermore, clinical application of the main active ingredients of Lactobacillus or inactivated bacteria can provide more safer effect and stable efficacy relative to live bacteria. Besides, Lactobacillus has preventive effect for offspring respiratory disease when administered to the mother, and it can greatly reduce the occurrence and alleviate the symptoms of respiratory diseases in infants and children if the stability and safety of Lactobacillus efficacy is fully established. Additionally, the economic burden of clinical application of Lactobacillus is low (186). Overall, administration of Lactobacillus is beneficial in improving pulmonary health and its application in treating respiratory diseases needs more clinical studies.

AUTHOR CONTRIBUTIONS
TD finished the original manuscript. NZ compiled table information. CZ and AL provided constructive comments and made critical revisions to the manuscript. All authors contributed to the article and approved the submitted version.