Contribution of Lactobacillus iners to Vaginal Health and Diseases: A Systematic Review

1The vaginal microbiome plays an important role in determining human vaginal health. Using high-throughput metagenomic and 16S rRNA sequencing, over 250 bacterial species have been identified in the human vagina. Among them, Lactobacillus is the most frequently detected microorganism in the healthy vagina, and this includes Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri. For decades, Lactobacillus species have been regarded as beneficial to the vaginal econiche by preventing the invasion of pathogens through the production of organic acids, hydrogen peroxide (H2O2), bacteriocin, and other antimicrobial compounds. Predisposing factors, such as menstruation, pregnancy, sexual practices, vaginal douching, and uncontrolled use of antibiotics, can rapidly alter the microbial community. A disruption of the vaginal ecosystem is characterized by the depletion of Lactobacillus species and the overgrowth of non-Lactobacillus microbes. Typically, the overgrowth of anaerobic bacteria can result in aberrant conditions, such as bacterial vaginosis (BV) and sexually transmitted infections (STIs), as well as pregnancy-related complications.

2Since the advent of metagenome sequencing techniques, L. iners has been recognized as the most prevalent Lactobacillus species in the vaginal econiche. This species has been initially overlooked in past bacteriologically-based studies because of its fastidious requirements and inability to grow on de Man-Rogosa-Sharpe agar (MRS), a selective culture medium that isolates vaginal Lactobacilli. Furthermore, L. iners has very unique characteristics compared with other symbiotic Lactobacillus species in the vaginal econiche. Most vaginal Lactobacillus species exert a protective effect and play a role in the resistance of the vaginal tract to colonization by pathogens. However, the relationship between L. iners and vaginal health is somewhat complicated and ambiguous. This review aims to present the overall characteristics, an overview of different arguments, and the dual roles of L. iners in the vaginal econiche.

3L. iners was first described by Falsen et al. in 1999 in vaginal and urinary tract specimens. This species had escaped our attention for a long time due to its inability to grow on MRS agar under the same culture conditions as other Lactobacillus species. Nevertheless, L. iners is characterized by small, smooth, circular, translucent, and non-pigmented colonies after 24 h of anaerobic incubation on blood agar. Indeed, most L. iners isolates can grow on MRS agar upon the addition of 1–5% sheep and human blood. In addition, Yoshimura et al. demonstrated that L. iners can grow on MRS agar under anaerobic conditions for a period of at least 7 days, which is evidently longer than that of other Lactobacillus species. In MRS broth with 0.5% cysteine as the reducing agent, which created the anaerobic conditions, L. iners grows slowly to its highest concentration of only 107 CFU/ml and then growth decreases after 12 h.

4L. iners was initially believed to be a Gram-positive, rod-shaped, non-spore-forming, and facultative anaerobic bacterium. However, several studies have reported that, unlike other Lactobacillus species, L. iners does not always clearly stain as Gram-positive, and it seems to have a coccobacillary rather than a bacillary morphology. Yoshimura et al. reported that L. iners was mostly Gram-negative with a very short rod shape and weak acid resistance, as it was non-viable in pH 3 medium. This may be the reason why L. iners was initially overlooked by culture and microscopy methods. By transmission electron microscopy, Kim et al. revealed that the peptidoglycan (PG) layer in the cell wall of L. iners was thin enough to give an apparent Gram-negative morphology. This morphological characteristic and Gram-staining property of L. iners are clinically very important to consider, as Nugent scoring, which is based on the Gram-staining of vaginal smears, remains a common diagnostic tool in the assessment of vaginal health. The Gram-negative property of L. iners masks the fact that it is a Lactobacillus species and this may lead to the misdiagnosis of BV, which is a condition characterized by the depletion of Lactobacillus species in Gram-stained vaginal smears under microscopy. This may help explain why as many as 50% of women diagnosed with BV by the Nugent score are asymptomatic.

5L. iners has the smallest genome of ∼1.3 Mbp on a single chromosome among the Lactobacillus species identified so far, with its pangenome count of 2300 genes and average GC content of ~33.3%. This low genome size is comparable to those of human symbionts and parasites, and is strongly indicative of a more parasitic, host-dependent lifestyle. The genome of L. iners seems to have undergone rapid evolution events that resulted in large-scale gene loss and genome reduction, as well as the acquisition of genes, such as iron-sulfur genes, for specific adaption to the vaginal econiche.

6Comparative genome analysis revealed that L. iners had a severely reduced number of genes related to carbohydrate and amino acid metabolism, whereas it maintained conserved genes for largely core metabolic proteins and membrane transport genes for essential compounds from the host or the community. Three potential core genes (inerolysin, ZnuA, and hsdR) were identified to be closely related to the specific adaption of L. iners to the vaginal environment. Among them, inerolysin is an unusual pore-forming cholesterol-dependent cytolysin that is active in the acidic vaginal environment and creates aqueous pores within the cell membrane. It may be one of the essential L. iners genes required to stably obtain nutrients from the host. High-affinity zinc uptake requires the binding protein ZnuA type I (ZnuA), which is essential for metal ion homeostasis in L. iners. ZnuA may be a key mediator of strong adhesion of L. iners to vaginal epithelial cells. Type I restriction enzyme R protein (hsdR) was suggested to be involved in the defense mechanism against bacteriophage infection during BV. In addition, L. iners contains genes that encode all enzymes directly involved in PG synthesis and hydrolysis. The unique and thin PG layer of L. iners cell membranes may absorb nutrients or secrete proteins more easily than other Lactobacillus species, which can provide essential nutrients or respond to rapid changes in the vaginal environment.

7Lactobacillus species are the main lactic acid-producing bacteria in the vagina, and they reduce the vaginal pH and restrict the growth of potentially harmful bacteria. Nevertheless, this lactic acid-producing ability is different from the main Lactobacillus species found in the vaginal microbiome. L. crispatus, L. gasseri, and L. jensenii can produce D- and L-lactic acid by fermenting glycogen, whereas L. iners can produce only L-lactic acid because it lacks the gene that codes for D-lactate dehydrogenase in its genome. Because of the almost complete absence of D-lactic acid, the L/D lactic acid ratio is highest in L. iners. Witkin et al. reported that the isomers of lactic acid have different effects on the host immune system. The L/D lactic acid ratio in the vagina may elevate extracellular matrix metalloproteinase inducer (EMMPRIN) and subsequently activate matrix metalloproteinase-8 (MMP-8), which facilitates the breakdown of the extracellular matrix, helps bacteria transverse the cervix, and initiates upper genital tract infections. Additionally, D-lactic acid has been reported to have a greater inhibitory effect on exogenous bacteria than L-lactic acid. Therefore, it seems that L-lactic acid renders L. iners less effective in preventing the invasion of pathogens.

8Srinivasan et al. reported that microbiomes abundant in L. crispatus were consistently strongly associated with low vaginal pH, but this was not the case for women with L. iners overgrowth. In fact, L. iners was very weak in a vaginal environment with low pH, failing to maintain intravaginal acidity. A high vaginal pH is a characteristic of BV, a condition in which Gardnerella vaginalis and L. iners are generally found in the vaginal econiche but other Lactobacillus species are rarely found. In addition, L. iners does not have the molecular and cellular machinery to produce H2O2 through pyruvate oxidation. The production of H2O2 is considered to be one of the mechanisms by which Lactobacillus species can prevent anaerobic bacteria from colonizing the vagina. As such, when pathogenic bacteria challenge the vaginal environment, L. iners cannot resist the overgrowth of pathogenic bacteria and the increase of pH, whereas it may persist in dysbiosis.

9The adherence of vaginal Lactobacillus species to host cells is believed to play a role in the exclusion of pathogenic microorganisms through a mechanism that involves the blocking of their binding sites on vaginal epithelial cells. Although L. iners lacks most of the main adhesion molecules of Lactobacillus species, it still shows a strong adhesive ability to vaginal epithelial cells. Fibronectin is an insoluble glycoprotein in the extracellular matrix of the vaginal epithelium. The L. iners genome encodes a fibronectin-binding protein that contains a motif (fibronectin-binding protein A) common to pathogenic strains of Staphylococcus aureus, thereby mediating the adhesion and the invasion of S. aureus to host cells. McMillan et al. demonstrated that L. iners bound significantly stronger to human fibronectin than other Lactobacillus species at a more neutral pH, which may contribute to the persistence of L. iners in the vagina despite the presence of pathogens or treatment with antibiotics. An in vitro study reported that L. iners may increase the adhesion of BV-causing G. vaginalis. It was also demonstrated that L. iners produces inerolysin, a pore-forming protein typically found in pathogenic bacteria, which can enhance the adhesive ability. These findings indicate that the unique adhesive function of L. iners reduces the protection of the healthy vaginal microbiome from pathogenic bacteria.

10L. iners has an unusually small genome with reduced metabolic capabilities, but it contains a broader array of genes that was probably acquired from foreign sources. The nutrient requirements of this species are more complex than those of other vaginal Lactobacillus species, thereby allowing L. iners to adapt to the diverse niche in the vagina. The fluctuation of hormones and other factors may affect the vaginal environment, resulting in changes in mucus and glycogen production, pH, and microbial species, which may provide essential nutrients for L. iners. Genome analysis has indicated a higher dependence of L. iners on exogenous sources of amino acids. Furthermore, L. iners has the molecular and cellular machinery to ferment glucose, maltose, trehalose, and mannose, among which glucose and maltose are common glycogenolysis products. Macklaim et al. reported that L. iners genes for the uptake of mannose and maltose, genes for glycogen decomposition, as well as genes for mucin and glycerol transport, were strongly up-regulated in BV. Although no iron uptake system has been identified in L. iners, ferrochelatase, which is capable of catalyzing ferrous ion and binding protoporphyrin IX to form heme, was detected in L. iners.

11The ability of L. iners to produce inerolysin may be one of the most important factors influencing its ability to acquire nutrients from the vaginal environment. L. iners is the only Lactobacillus species known to code for inerolysin, which is related to intermedilysin (69.2% similarity) and vaginolysin (68.4% similarity) produced by Streptococcus intermedius and G. vaginalis, respectively. Over 10% of genes coding for inerolysin in L. iners are more highly expressed in dysbiosis than in balanced microbial environments. This cytolysin can liberate resources directly from host tissues or cells, which necessitates that L. iners acquire its nutrients from the host in a symbiotic way. In other words, this characteristic may give L. iners a competitive advantage in the vaginal environment when nutrients are scarce, especially under potentially adverse conditions, such as BV, when other Lactobacillus species cannot colonize the vagina.

12Compared with intestinal microflora, a typical feature of the vaginal microbial environment in healthy individuals is its extremely low bacterial diversity. There are five major community-state types (CSTs) in healthy premenopausal women, namely, L. crispatus-dominated CST I, L. gasseri-dominated CST II, L. iners-dominated CST III, and L. jensenii-dominated CST V, whereas CST IV is characterized by the absence of Lactobacillus species. Vaginal dysbiosis, which is defined by a high bacterial diversity and a mixture of anaerobic bacteria, is frequently associated with a variety of gynaecological diseases.

13L. iners can be predominant in the vagina of healthy women, or in those with vaginal dysbiosis, such as BV, or even in those receiving antimicrobial therapy. Many studies have reported that the presence of L. crispatus in the vagina is associated with good health, whereas communities dominated by L. iners fail to provide sufficient protection against vaginal dysbiosis. The existence of L. iners is related to higher levels of proinflammatory factors, such as interleukin-1α, interleukin-18, macrophage migration inhibitory factor, and tumor necrosis factor-α, which are responsible for the activation of an inflammatory response in the vagina. L. iners is even believed to play a role in the onset of vaginal dysbiosis, although the precise role of L. iners remains debated. However, it seems that the abundance of L. iners remains relatively constant, and L. iners is not easily displaced by pathogens or infectious conditions. In cases of BV, L. iners, rather than L. crispatus, usually coexists with other potentially harmful bacteria that colonize the vagina. The ability of L. iners to adapt to dysbiosis, despite its small genome, may be related to its changes in genes involved in metabolism and cytolysis, as well as antibacteriophage defense genes, to changing conditions in the vagina. The remarkable ability of L. iners to survive under various conditions indicates that this species may be an important member of the host’s defense and may be a persistent symbiotic Lactobacillus species that can maintain and restore the vaginal microbiome.

14BV is the most common type of vaginitis in women of childbearing age. It is characterized by a significant reduction or disappearance of Lactobacillus species, accompanied by the emergence of more diverse microbiota dominated by anaerobic and facultative bacteria such as Gardnerella species, Prevotella species, and A. vaginae. However, L. iners is usually the only vaginal Lactobacillus species coexisting with BV-associated bacteria that can be detected during BV. It can persist under the drastically changing vaginal environment of BV due to its ability to respond and regulate its genomic functions. The increased gene expression of L. iners may lead to the production of succinate and other short-chain fatty acids and the increase in the pH value in the BV environment. To adapt to the BV environment, L. iners can increase the expression of inerolysin and mucin, and promote the production of glycerol and the expression of related metabolic enzymes, which ensures its acquirement of nutrients from foreign sources. In addition, bacteriophages were one of the reasons for the sudden decrease of Lactobacillus species during BV, whereas L. iners can upregulate defense systems such as the type I RM system and CRISPR, as well as its specific hsdR gene, to resist bacteriophage invasion during BV. Nevertheless, a recent study discovered three active peptides of bacteriocin produced by a human intestinal strain named Lactobacillus paragasseri. These bacteriocins have strong selective inhibitory activity against L. iners, whereas L. crispatus, L. jensenii, and L. gasseri were only slightly inhibited, indicating that these Lactobacillus-derived effective inhibitors of L. iners can be combined with metronidazole to improve the current BV treatments.

15As the coexistence of L. iners in BV is different from that of other Lactobacillus species, the prevalence of L. iners can be used as a microbial indicator to predict the onset of BV or the intermediate BV status. Furthermore, L. iners is metronidazole-resistant, and it was found to be the predominant Lactobacillus species, even after treatment of BV with metronidazole. Compared with other more protective Lactobacillus species, which hardly exist during BV, L. iners showed a stronger competitive advantage and coexisted in the disrupted microbiome. It has been proposed that L. iners facilitates the transition between BV and non-BV states. Interestingly, it has been reported that even after BV treatment, the vaginal microbiome does not change from the L. iners-dominant state to the L. crispatus-dominated state. Therefore, the persistence of L. iners may lead to long-term vaginal dysbiosis, especially after repeated treatment cycles of BV. Further studies are needed to clarify whether this species is only a biomarker of the vaginal microbiota transition or a contributing factor of BV.

16Biofilms are bacterial structures tightly attached to a surface, and they are known to be more resistant to the host immune response and antibiotic therapy than planktonic cells. It has been shown that biofilm formation on vaginal epithelial cells is strongly associated with vaginal infections. There is sufficient evidence that BV associates with the presence of a dense polymicrobial biofilm, in which G. vaginalis is the dominant bacterial strain on the vaginal epithelium. It has been hypothesized that Gardnerella spp. initiate biofilm formation, which supports the attachment of other BV-associated bacteria (BVAB) to the vaginal epithelium, further enhancing the biofilm thickness. Moreover, Gardnerella biofilms serve as barriers to antibiotics and function to protect other BVAB by preventing the penetration of antibiotics. It is generally believed that the high rate of BV recurrence is due to the formation of biofilms that protect the bacteria from antibiotic treatment, and even serve as a reservoir for pathogen regrowth.

17Vaginal indigenous Lactobacilli are believed to prevent the colonization of pathogenic bacteria through steric hindrance or receptor masking in the mucosa. Previous studies have used a Lactobacillus probiotic approach in an attempt to clear the polymicrobial biofilms, essentially impeding bacterial virulence and suppressing infection in the human vagina. Lactobacillus plantarum was reported to significantly reduce the adhesion of Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, and Pseudomonas aeruginosa in the HT-29 cell line, which made it a potential anti-biofilm agent for BV treatment. Saunders et al. reported that G. vaginalis biofilms grown in vitro were displaced with Lactobacillus reuteri RC-14, and to a limited extent with L. iners. Castro et al. demonstrated that L. crispatus drastically reduced the adhesion of G. vaginalis strains, both from a healthy woman and a woman with BV, to cervical epithelial cells. Interestingly, L. iners significantly reduced the adhesion of G. vaginalis strains from a healthy woman, but markedly enhanced pathogenic G. vaginalis adhesion, suggesting that L. iners can cohabitate with BV-associated G. vaginalis and may contribute to G. vaginalis-dominated biofilm formation. In addition, it is well known that Candida species, mainly C. albicans, can form thick and tough biofilms, which greatly increases the tolerance to antifungal drugs during the treatment of recurrent vulvovaginal candidiasis. Mckloud et al. reported the ability of various Lactobacillus species to inhibit C. albicans biofilm formation and biofilm-related gene expression when cocultured. Lactobacillus rhamnosus could down-regulate C. albicans biofilm-related gene expression. Conversely, coculture with L. iners resulted in an up-regulation of biofilm-related gene expression (ALS3 and ECE1), suggesting that the presence of L. iners may be indicative of a shift to vaginal dysbiosis; therefore, it should not be used as a probiotic intervention for C. albicans infection. A further understanding of the interactions between vaginal commensal Lactobacilli and the structure and function of biofilms is of extreme importance to identify novel treatment approaches for biofilm-associated infections.

18Previous studies have reported that L. crispatus-dominated vaginal microbiomes associate with a lower prevalence of STIs, whereas BV associates with an elevated risk of STIs such as infection with Chlamydia trachomatis, human immunodeficiency virus (HIV), Neisseria gonorrhoeae, cytomegalovirus, and herpes simplex virus-2. Van Houdt et al. reported that the vaginal microbiome dominated by L. iners at baseline significantly increased the risk of acquiring C. trachomatis infection one year later. A lack of D-lactic acid in the L. iners-dominated vaginal microbiome may increase the ability of HIV to transverse the cervicovaginal mucus by modulating cervical integrity. Interestingly, Mehta et al. and Reimers et al. demonstrated that the vaginal microbiome did not differ between HIV-positive and HIV-negative black women in the United States. However, Spear et al. conversely observed that the percentage of L. iners was significantly higher in HIV-negative African Americans than in HIV-positive African Americans. The reason for these inconsistent results may be differences in the genetic background or complicated social and behavioral factors in black women, as black women without BV were more likely to have vaginal microbiomes dominated by L. iners. The precise role of L. iners in HIV infection should be further examined. Many studies reported a higher diversity of vaginal microbes and a lower abundance of Lactobacillus species among HPV-positive women. Norenhag et al. showed that the vaginal microbiome dominated by L. iners was associated with high-risk HPV infection compared with L. crispatus. It can be speculated that vaginal dysbiosis may affect the host’s innate immunity against HPV infection, resulting in dysplasia/cervical cancer. These findings indicate that L. iners may exhibit rapid changes in the composition of the vaginal microbiome similar to BV and could be a valuable biomarker of the dynamic vaginal environment under STIs.

19It is especially important to maintain the natural and healthy balance of Lactobacillus species in the vaginal microbiome during pregnancy. Earlier studies confirmed that high estradiol levels and the consequent high glycogen levels in the vagina during pregnancy result in stronger vaginal acidification, thereby promoting the prevalence of Lactobacillus species as gestation progresses. However, many studies have indicated that the L. iners-dominated vaginal microbiome was more likely to shift towards dysbiosis during pregnancy. In our previous study, we found that the abundance of L. iners decreased significantly in the second and third trimester, whereas that of L. crispatus increased in the second trimester compared with the first trimester in healthy pregnant women. In addition, we observed that the increase in the abundance of L. iners, but not that of L. crispatus, was related to the increase in vaginal cleanliness and positive leukocyte esterase activity, which is consistent with the results of a previous study.

20Increasing evidence indicates that BV is one of the major etiological causes for adverse pregnancy outcomes, especially PTB. The L. iners-dominated vaginal microbiome, a so-called ‘intermediate microflora’ and a typical feature of BV, is speculated to be a risk factor for PTB. Petricevic et al. suggested that the prevalence of L. iners detected in vaginal smears of healthy women in early pregnancy can associate with PTB. This was also demonstrated by Kindinger et al., who reported that the vaginal microbiome dominated by L. iners at 16 weeks of gestation is a risk factor for both a short cervix and early PTB (<34 weeks), whereas L. crispatus dominance is protective against PTB in a more ethnically diverse cohort. Recent studies from different countries also showed a significant association between L. iners and an increased prevalence of PTB. However, several studies reported no significant association between L. iners and PTB. L. iners was also demonstrated to be the most abundant Lactobacillus species among pregnant black women. However, most studies did not identify a significant relationship between the Lactobacillus-dominant vaginal microbiome and PTB in pregnant black women. Conversely, three studies reported that L. iners was associated with a decreased risk of PTB. Therefore, the association between L. iners and PTB risk is controversial ( Table 1 ). Presently, it is believed that the vaginal microbiome in black women does not play an important role in the pathogenesis of PTB, as it does in Caucasians and Asians. Furthermore, the limited sample size, the time of sample collection, differences in the definition of PTB, ethnical and geographical variations, and differences in the methodology of strain identification, as well as complicated clinical conditions such as genetic abnormalities or a history of PTB, were confounding factors that impacted the results.

21According to the features of L. iners and its lack of protection against pathogens when it is the only Lactobacillus species in the vagina, some researchers have suggested that L. iners cannot be responsible for infections during pregnancy. Indeed, because the vaginal microbiome dominated by L. iners is relatively unstable, there is a tendency for transition to BV-associated CST-IV during pregnancy. In addition, the L. iners-dominated vaginal microbiome may increase the risk of PTB by modulating local tissue inflammation and cervical integrity, thereby disrupting chemical and mechanical mucosal protective barriers against ascending infections. Further studies are needed to clarify the potential mechanisms between the L. iners-dominated vaginal microbiome and PTB.

22Previous studies have reported that up to 40% of patients who failed assisted reproduction by in vitro fertilization (IVF) had an abnormal reproductive tract microbiome. Vaginal dysbiosis, including an elevated pH value, increased flora diversity, BV, vulvovaginal candidiasis, and trichomonal vaginitis, are recognized as risk factors for infertility. Campisciano et al. reported that the abundance of L. iners was associated with an increased infertility rate. Chen et al. also recently reported that the L. iners-dominated vaginal microbiome was associated with tubal infertility and C. trachomatis infection. As a transitional species, L. iners may facilitate the transition between an abnormal and a normal vaginal microbiome under treatment or artificially high estrogen levels that are needed for IVF. It is believed that the L. iners-dominated vaginal microbiome is an unfavorable factor for pregnancy.mk

23The Human Microbiome Project shows that in the microbial community for all body parts, including the vagina, within-subject variations over time are consistently lower than between-subject variations. The menstrual cycle is one of the most important factors disturbing the diversity of the vaginal microbiome. L. crispatus usually dominates the vagina of reproductive-age women, whereas L. iners overgrows and replaces L. crispatus during the menstruation cycle. A recent study reported that L. iners was the most recurrent microbe in the follicular phase; L. iners and CST IV (microbial diversity) were predominant in the periovulatory phase; and in the luteal phase, the most frequent type was CST IV. Indeed, the abundance of L. iners remarkably increases during menses, frequently in conjunction with an increase of G. vaginalis and/or Atopobium vaginae; however, they subsequently decrease after menses without intervention. As dynamic changes in the vaginal econiche were characterized at different time points in the menstruation cycle within the same individual, the moment of sampling relative to the menstrual cycle is very important for vaginal community analysis.

24L. iners is the most common and persistent vaginal symbiotic Lactobacillus species with good adaptability to the complex and dynamically changing vaginal environment. Under fluctuating environmental conditions, other Lactobacillus species may not survive, while L. iners persists with relatively constant abundance due to its ability to respond and regulate its genomic functions, such as specific carbohydrate uptake, fibronectin-binding protein activity, bacteriophage defense, and inerolysin synthesis. This remarkable ability to survive under a range of conditions contributes to L. iners’ being a dominant species when the microbiome is in a transitional stage, suggesting that L. iners may be an important member of the host defense mechanism as a persistent mutualistic lactobacilli, and even promote the restoration of a healthy vaginal microbiome.

25In fact, several studies have confirmed many probiotic characteristics of L. iners. It has many ecological functions, such as lactic acid production, that are similar to those of other Lactobacillus species. Although the genome of L. iners lacks most of the adhesion factors of other Lactobacillus species, it can still adhere strongly to vaginal epithelial cells.Thus, L. iners shows the similar phenotypic traits of colonization and host interaction, as well as excluding pathogens, as other vaginal Lactobacillus species. Shipitsyna et al. reported the loss of L. iners during BV and suggested that it was not the key pathogen causing the disease. L. iners can destroy or replace G. vaginalis to form biofilms in vitro. Macklaim et al. demonstrated that some specific functions of L. iners, such as the expression of cytokines, absorption of exogenous sources, and bacteriophage defense, facilitated the harsh conditions in the vagina, including BV. L. iners prevents harmful bacteria from obtaining important nutrients, such as iron, and inhibits their sustained growth by triggering the innate immune system in vaginal epithelial cells. In addition, the anti-inflammatory effects of L. iners were observed through specific molecular interactions between vaginal epithelial cells.

26The L. iners-dominated vaginal microbiome was associated with the induction of a stress response in the vaginal epithelium. Doerflinger et al. discovered that L. iners, but not L. crispatus, significantly upregulated the pattern-recognition receptor signaling pathway in human primary vaginal epithelial cells and increased the mRNA expression of tumor necrosis factor, indicating that the vaginal microbiome regulates the host immune response species-specifically. It has been suggested that, in response to stress, L. iners can activate the toll-like receptor signaling pathway in vaginal epithelial cells, increase heat shock protein 70 expression, and inhibit autophagy, which would destroy the homeostasis between vaginal epithelial cells and reduce the ability of these cells to recognize and respond to potential pathogens. Conversely, many compounds involved in the antimicrobial defense of vaginal epithelial cells, such as neutrophil gelatinase-associated lipocalin, calprotectin, and hyaluronan, were also preferentially induced by L. iners. These findings support the fact that L. iners can fight non-physiological threats, and maintain and promote the recovery to a healthier state, as well as exhibit proinflammatory qualities and act less like a commensal microbe under different conditions.

27In conclusion, L. iners is a unique and intriguing Lactobacillus species with extraordinary characteristics. Its small genome and concurrent nutrient dependency are conducive to its high adaptation to both the low and the high pH vaginal environment, as well as both BV-positive and BV-negative conditions. Therefore, L. iners is often classified as a transitional species that colonizes the vagina after an ecological disturbance. However, whether L. iners is beneficial or pathogenic for the host’s microbiome remains controversial. Most researchers are inclined to believe that L. iners offers limited protection against vaginal colonization by pathogens and may contribute to the onset and maintenance of vaginal dysbiosis. L. iners may also be a risk factor for sexually transmitted infections and adverse pregnancy outcomes. A greater understanding of the roles of L. iners in health and diseases in individuals of different races and ethnicities is warranted. In addition, further studies are required to clarify the role of L. iners in vaginal mucosal immune regulation, and to further clarify whether it can be used as a novel biomarker to detect the existence or prognosis of vaginal inflammation and guide subsequent clinical treatment.

28NZ, RG, JW, WZ, and ZL discussed the contents, wrote, reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.

29This present work was funded by the grants of the National Natural Science Foundation of China (81771724, 31700800, 81790631), the Taishan Scholar Foundation of Shandong Province (tsqn202103119), the Nutrition and Care of Maternal & Child Research Fund Project of Guangzhou Biostime Institute of Nutrition & Care (2019BINCMCF045), the National S&T Major Project of China (2018YFC2000500), and the Foundation of China’s State Key Laboratory for Diagnosis and Treatment of Infectious Diseases.

30The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

31All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.