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Lactobacillus gasseri LGV03-derived indole-3-lactic acid ameliorates immune response by activating aryl hydrocarbon receptor

Microbial Cell Factories volume 24, Article number: 34 (2025) Cite this article

Abstract

Previous studies showed that the female genital tract microbiome plays a crucial role in regulating the host’s immune defense mechanisms. Our previous research has shown that Lactobacillus gasseri LGV03 (L. gasseri LGV03) isolated from cervico-vagina of HPV-cleared women contributes to clearance of HPV infection and beneficially regulate immune response. However, the mechanisms behind the regulation of L. gasseri LGV03 in immune response remain unclear. To better understand the interaction between female genital tract microbiome and immune function, the immunomodulatory activities of L. gasseri LGV03 were investigated in zebrafish models of neutropenia, macrophage and T cells deficiency. L. gasseri LGV03 showed higher potent activities in ameliorating vinorelbine-induced neutropenia, macrophage and T cells deficiency, and significantly enhanced mRNA expressions of cytokines TNF-α, TNF-β and IFN-α. Moreover, the transcriptome sequencing results indicated L. gasseri LGV03 might alleviate vinorelbine-induced immunosuppression in zebrafish. Non-targeted detection and analysis revealed that indole derivatives including phenylacetaldehyde, 3-phenyllactic acid, N-acetylserotonin and indole-3-lactic acid were significantly increased in the lysate and supernatant of L. gasseri LGV03. Meanwhile, L. gasseri LGV03 supernatant and indole-3-lactic acid ameliorated the vinorelbine-induced reduction in abundance of macrophages, neutrophils and T cells. However, the alleviating effects of L. gasseri LGV03 supernatant or indole-3-lactic acid were eliminated by aryl hydrocarbon receptor (AHR) antagonist CH-223,191. Furthermore, L. gasseri LGV03 supernatant and indole-3-lactic acid significantly increased the secretion of IFN-α, IFN-β and chemokines (MIP-1α, MIP-1β) in Ect1/E6E7 cells, meanwhile, these benefits were eliminated by CH-223,191 treatment. In summary, L. gasseri LGV03-derived indole-3-lactic acid can activate AHR-mediated immune response.

Introduction

The host’s innate and adaptive immune systems perform complex interactions with microorganisms and metabolites [1, 2]. Previous studies have shown that microorganisms in reproductive tract play a significant role in the host immune system, serving as key regulators of immune defense [3,4,5]. Microorganisms and their metabolites can interact with host immunity, activate immune responses, and alter the immune status, which is highly associated with persistent genital pathogen infections, the exacerbation of infectious diseases, adverse pregnancy outcomes, and even gynecological cancers [6]. Lactobacillus spp. is the predominant genus in the vagina of healthy women, which produces lactic acid to maintain an acidic microenvironment [7, 8]. It also prevents pathogen growth through competition, adhesion blocking, and the secretion of antibacterial and immunomodulatory substances [9, 10]. Moreover, metabolites of Lactobacillus can recruit and activate innate immune cells in the female reproductive tract, such as neutrophils and monocytes [11]. The disruption of the balance between reproductive tract microbiota, their metabolites, and the host immune system can weaken the host’s immune response to pathogens [6]. Therefore, there is a need to investigate the role of the reproductive tract microbiota and its metabolites in regulating the host immune response during disease pathogenesis, which will help improve diagnosis, treatment, and overall female reproductive health. Our previous study has shown that compared to the patients with persistent HPV infection, Lactobacillus represents the major microbiota in the vagina of women with HPV clearance, where Lactobacillus vaginalis, Lactobacillus jenni, Lactobacillus reuteri, Lactobacillus gasseri and Lactobacillus mucosus were very dominant [12]. Correlation analysis between cytokines and Lactobacillus. demonstrated the positive correlation between L. gasseri and IFN-α and IFN-β expression levels [12]. In addition, L. gasseri LGV03 isolated from the genital tract of HPV-cleared patients was able to regulate the immune response of cervical epithelial cells [12]. Previous studies have shown that the reproductive tract microflora plays an important role in shaping the immune response that responsible for pathogens elimination [6, 13,14,15]. However, very little is known of the specific metabolites and mediating targets involved in the regulation of the immune response by the reproductive tract microbiota.

Many studies have reported the anti-inflammatory, anti-tumor, and host immune regulatory properties of natural metabolites of microorganisms [16,17,18]. Lactobacillus metabolites including indoles and their derivatives, such as indole-3-acrylic acid (IA), indole acetic acid (IAA), indole-3-lactic acid (IL), indole-3-propionic acid (IPA), and indole-3-aldehyde (I3C) have been reported to be ligands for aryl hydrocarbon receptors (AhRs) [19,20,21]. AhRs are widely expressed in different cell types throughout the body and serve an important function in immune regulation in barrier sites such as skin and intestine [22]. Previous studies have shown that microbial metabolites relieve colitis and prevent candida albicans infection by activating AhRs to produce anti-inflammatory cytokine IL-22 in gut [23,24,25]. Additionally, indole-3-formaldehyde, a metabolite released by Lactobacillus reuteri in the tumor microenvironment, can activate the AhRs of CD8+T cells, which promotes the secretion of IFN-γ and kills tumor cells, thereby enhancing the efficacy of immunotherapy [26]. However, whether L. gasseri LGV03 affects AhRs in regulating immune responses remains unclear. Therefore, the present research investigated the regulatory effect of L. gasseri LGV03 on vinorelbine-induced immunosuppression in zebrafish. Next, the metabolites were detected in the fermentation supernatant of L. gasseri LGV03. Finally, the immunomodulatory effects of L. gasseri LGV03 fermentation supernatant and its metabolite indole-3-lactic acid on zebrafish and cervical epithelial cells Ect1/E6E7 via AhR receptor were studied.

Materials and methods

Materials

The human cervical squamous epithelial immortalized cell line (Ect1/E6E7, American Type Culture Collection (ATCC): CRL-2614) was purchased from Shanghai Balantek Biotechnology Co., Ltd. The aromatic hydrocarbon receptor (AhR) inhibitor (CH-223191) was purchased from Sigma-Aldrich. Vinorelbine was purchased from medchemexpress. The Tg(mpx-EGFP) and Tg(mpeg-EGFP) transgenic zebrafish were purchased from Nanjing EzeRinka Biotechnology Co., Ltd. The Tg(rag2-DsRed) transgenic zebrafish were purchased from Hangzhou Hunter Biotechnology Co., Ltd. Lactobacillus gasseri LGV03 (L. gasseri LGV03) was collected from cervico-vaginal samples of HPV clearance in women [12].

Preparation of bacterial cells and supernatant

Lactobacillus gasseri LGV03 (CGMCC accession no. 221010; China General Microbiology Culture Collection Center) was incubated in MRS liquid medium at 37 ℃ for 24 h. The bacterial cells and supernatant were collected by centrifugation at 12,000 g for 10 min. The obtained bacterial cells and supernatant could be used for subsequent experimental operations.

Cell culture condition

The Ect1/E6E7 cells were cultured on Dulbecco’s Modified Eagle medium (DMEM, Sangon Biotech, Shanghai, China) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA). The CaSki cells were cultured on Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% FBS. Both cells were kept in an incubator at 37 ℃ in 5% CO2.

Maintenance of zebrafish

Wild-type AB, Tg(mpx-EGFP), Tg(mpeg-EGFP), and Tg(rag2-DsRed) adult zebrafish were maintained in a controlled zebrafish breeding system set at 28.5 °C with a pH of 7.5 and conductivity ranging from 500 to 550 µs/cm, under a 14:10 h light-dark cycle. Following this, zebrafish embryos were obtained through natural mating and subsequently incubated in E3 water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4) at 28 °C for development.

Collection and detection of L. gasseri LGV03 cell lysate and L. gasseri LGV03 strain culture supernatant

The L. gasseri LGV03 bacteria were inoculated in MRS medium (5 mL liquid medium, sterilized at 121 ℃ for 15 min), cultured at 37 ℃ for 24 h then inoculated into corresponding medium at 2% inoculum size for activation (3 parallel portions). After 24 h of culture (the bacterial density was about 109 CFU/mL), the medium underwent centrifugation twice, and the solid and supernatant of bacteria strains were retained. Next, the bacteria were resuspended in 5 mL liquid medium and subjected to ultrasonic cracking, followed by centrifugation at 4 ℃ and 10,000 g for 15 min to obtain bacteria lysate. The cell lysate (LGV03) and the strain culture supernatant (supernatant) were filtered by 0.22 mm microporous filter membrane and then stored in the sample bottle at -20 ℃ for later use.

A 100 µL aliquot of the fermentation supernatant or cell lysate was taken, and 10 µL of internal standard (L-2-chlorophenylalanine, 0.3 mg/mL in methanol) was added. The mixture was vortexed for 10 s followed by an addition of 300 µL of methanol-acetonitrile (2:1, v/v). The mixture was vortexed for 1 min and subjected to ultrasonic extraction in an ice bath for 10 min. It was then left at -20 °C for 30 min. Afterwards, the sample was centrifuged at 13,000 rpm for 15 min at 4 °C, and 200 µL of the supernatant was collected and transferred into a clean glass vial for analysis by liquid chromatography-mass spectrometry (LC-MS). LC-MS was performed with LCMS-8050 triple quadrupole mass spectrometer (Shimadzu) for non-targeted detection of the metabolites of the strains in two groups of solutions. The LCMS-8050 triple quadrupole mass spectrometer was used for primary and secondary mass spectrometry data acquisition in Targeted MS/MS mode, controlled by LabSolutions software. The mass scan range was set from m/z 100 to 1000, and data acquisition was performed in positive ion mode. The ESI ion source parameters were as follows: atomization gas flow, 3 L/min; heating gas flow, 10 L/min; interface temperature, 300 ℃; DL temperature, 250 ℃; heating block temperature, 400 ℃; and dry gas flow, 10 L/min.

Metabolite identification

The disembarking data (.raw) file was imported into CD 3.3 library software for processing, and each metabolite was subjected to a simply screening by the parameters including retention time and mass-charge ratio. Then, the first QC was used for peak area correction to make the identification more accurate. Next, peak extraction was performed by setting the parameters such as mass deviation (5 ppm), signal strength deviation (30%), minimum signal strength, and adduct ion information. Meanwhile, the peak areas were quantified, and then the target ions were integrated. Then, the molecular formula was predicted by molecular ion peak and fragment ions and compared with mzCloud (https://www.mzcloud.org/), mzVault and Masslist databases. The background ions were removed with blank samples. The original quantitative results were standardized according to the formula of original quantitative values of samples/(total quantitative values of metabolites in samples/total quantitative values of metabolites in sample QC1), and the relative peak area was obtained. Then, compounds with relative peak area CV greater than 30% were removed from QC samples, and finally the metabolite identification and relative quantitative results were obtained. During this procedure, data processing part was based on the Linux operating system (CentOS version 6.6) and R and Python software, and the specific program packages and software version are included in the result file readme.

The metabolites identified were annotated using KEGG (https://www.genome.jp/kegg/pathway.html), HMDB (https://hmdb.ca/metabolites) and LIPIDMaps (http://www.lipidmaps.org/) databases.

In the multivariate statistical analysis, the metabolomics data processing software metaX was utilized to transform the data, and then principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) were performed to obtain the VIP value of each metabolite. In univariate analysis, the statistical significance (P value) of each metabolite between the two groups was calculated based on the t test, and the fold change (FC value) of the metabolite between the two groups was calculated. The criteria for differential metabolite screening were VIP > 1, P < 0.05 and FC ≥ 2 or FC ≤ 0.5.

The cluster heatmap was plotted with R-package Pheatmap, and the metabolite data were normalized with z-score. Finally, the identified differential metabolites were inputted into MetaboaAnalyst 3.0 (htttp://www.metaboanalyst.ca/) and KEGG (http://www.Kegg.jp/keg/pathway.html) for the construction of metabolic pathways, completing the analysis of the metabolic pathways of significant differential metabolites in LGV03 strain.

ELISA detection on effect of L. gasseri LGV03 supernatant and indole-3-lactic acid on secretion of cytokines/chemokines by Ect1/E6E7 cells

Ect1/E6E7 cells were inoculated in 6-well plates at a concentration of 1 × 106 cells per well followed by an incubation overnight at 37 ℃ in 5% CO2 in a cell incubator. The cells were then treated with L. gasseri LGV03 supernatant or with indole-3-lactic acid with or without a 30 µM CH-223,191. After incubation at 37 ℃ for 72 h, the supernatant was collected. Following this, ELISA was employed to detect Cytokines/chemokines in the supernatant.

Effects of L. gasseri LGV03 on reduction of macrophages, neutrophils and T cells induced by vinorelbine in zebrafish

The present study investigated the potential effects of L. gasseri LGV03 on the inhibition of macrophages, neutrophils, and T cells induced by vinorelbine in zebrafish. Transgenic zebrafish Tg(mpeg-EGFP) expressing enhanced green fluorescent protein (EGFP) in macrophages, transgenic zebrafish Tg(mpx-EGFP) expressing EGFP in neutrophils, and transgenic zebrafish Tg(rag2-DsRed) expressing red fluorescence in T cells were used as the research objects. 48 h-post fertilization (hpf) Tg(mpx-EGFP), Tg(mpeg-EGFP), or Tg(rag2-DsRed) transgenic zebrafish larvae were randomly divided into five groups with 15 zebrafish larvae in each group. The larvae were then inoculated into 6-well cell culture plates with 15 larvae per well. The five groups were as follows: control group, vinorelbine group (model group), 1 × 105 CFU/mL L. gasseri LGV03 group, 1 × 106 CFU/mL L. gasseri LGV03 group, and 1 × 107 CFU/mL L. gasseri LGV03 group. The control group was added with E3 water. The model group was treated with vinorelbine solution (200 µg/mL). The 1 × 10⁵ CFU/mL L. gasseri LGV03 group was treated with 1 × 10⁵ CFU/mL L. gasseri LGV03 and 200 µg/mL vinorelbine solution. The 1 × 10⁶ CFU/mL L. gasseri LGV03 group received 1 × 10⁶ CFU/mL L. gasseri LGV03 and 200 µg/mL vinorelbine solution. The 1 × 10⁷ CFU/mL L. gasseri LGV03 group received 1 × 10⁷ CFU/mL L. gasseri LGV03 and 200 µg/mL vinorelbine solution. Each well contained 5 mL of solution, and the larvae were incubated at 28 °C. The solution was replaced every 24 h. After 96 h of incubation, the zebrafish larvae were observed under a fluorescence microscope, and images were captured. The number of macrophages and neutrophils and the fluorescence intensity of T cells in the zebrafish were calculated.

Immune-related factor detection by qRT-PCR

48 hpf wild-type AB strain zebrafish larvae were randomly divided five groups with 15 zebrafish larvae in each group then incubated in 6-well cell culture plates with 15 larvae per well. These five groups were descried as follows: control group, vinorelbine group (model group), 1 × 105 CFU/mL L. gasseri LGV03 group, 1 × 106 CFU/mL L. gasseri LGV03 group, and 1 × 107 CFU/mL L. gasseri LGV03 group. The control group was supplemented with E3 water. In contrast, the model group was administered vinorelbine solution at a concentration of 200 µg/mL. The 1 × 105 CFU/mL L. gasseri LGV03 group was treated using a combination of 1 × 10⁵ CFU/mL of L. gasseri LGV03 and 200 µg/mL vinorelbine solution. Similarly, the 1 × 106 CFU/mL L. gasseri LGV03 group was administered 1 × 10⁶ CFU/mL L. gasseri LGV03 along with 200 µg/mL vinorelbine solution. The 1 × 107 CFU/mL L. gasseri LGV03 group received a combination of 1 × 10⁷ CFU/mL L. gasseri LGV03 and 200 µg/mL vinorelbine solution. Triplicates were maintained for each group. Each well was filled with 5 mL of the solution, and the larvae were then incubated at a constant temperature of 28 °C. Every 24 h during the following period, the solution was replaced. After 96 h of intervention, 15 wild type AB strain zebrafish larvae were selected from each group. The RNA Easy Fast Tissue/Cell Kit (TIANGEN, DP451) was employed to extract total RNA from zebrafish and HiScript II Q RT SuperMix was employed to synthesize cDNA. LightCycler®96 fluorescent quantitative PCR was employed to amplify cDNA. The RT-PCR amplification was conducted by following procedure: 95 ℃ for 5 min (denaturation), followed by 95 ℃ for 10 s (denaturation) and 55 ℃ for 30 s (denaturation), which was ran for 45 cycles. β-actin was used as the internal reference gene to calculate gene expression while the folding changes of the measured genes were evaluated by 2−ΔΔCt. The primers are listed in Table 1.

Table 1 Primers for qRT-PCR
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Total RNA extraction and transcriptome sequencing

48 hpf wild-type AB strain zebrafish larvae were randomly sorted into three groups with 15 larvae in each group. Afterwards, the zebrafish larvae were placed in 6-well cell culture plates for incubation with 15 larvae per well. The three groups were as follows: control group, vinorelbine group (model group), and 1 × 10⁷ CFU/mL L. gasseri LGV03 group. The control group was supplemented with E3 water, while the model group received vinorelbine solution at a concentration of 200 µg/mL. The 1 × 10⁷ CFU/mL L. gasseri LGV03 group was treated with 1 × 10⁷ CFU/mL L. gasseri LGV03 along with 200 µg/mL vinorelbine solution. Triplicates were maintained for each group. Each well contained 5 mL of solution, and the larvae were incubated at 28 °C. Every 24 h during the following period, the solution was replaced. After 96 h of intervention, 15 wild-type AB strain zebrafish larvae were selected from each group for total RNA extraction using the RNA Easy Fast Tissue/Cell Kit (TIANGEN, DP451).

Each group of zebrafish was placed into a 2 mL centrifuge tube. After one wash of nuclease-free water, 350 µL of lysis buffer RLA was added to each tube, and approximately 10 beads. The tubes were then placed in TGrinder H24 Tissue Grinding Homogenizer (OSE-TH-01). The homogenizer was set to run at a speed of 6 m/s for 30 s, cooled for 30 s, and then run a second time to thoroughly homogenize the tissue. Following this, 10 µL of proteinase K was added, and the mixture was pipetted gently to mix well. The samples were kept at room temperature for 5 min centrifuged at 12,000 rpm for 5 min. The supernatant was taken and added to a genomic DNA removal column and centrifugated (12,000 rpm) for 30 s to collect filtrate. An equal volume of 70% ethanol was then slowly added to the filtrate then mixed using gentle inversion and transferred into an RNase-Free adsorption column CR4 (with the adsorption column placed in a collection tube). After centrifugation at 12,000 rpm for 30 s, the waste liquid in the collection tube was then discarded followed by a collection of adsorption column back into the collection tube. Afterwards, 700 µL of protein removal buffer RW3 was added to the RNase-Free adsorption column CR4 and centrifuged at 12,000 rpm for 30 s, then the adsorption column was put back into the collection tube after the discard of waste liquid. Following this, 500 µL of wash buffer RW was added to the RNase-Free adsorption column CR4 and kept at room temperature for 2 min, then centrifuged at 12,000 rpm for 30 s, discard the waste liquid, and put the adsorption column back into the collection tube. The RNase-Free adsorption column CR4 was kept at room temperature for 2 min to completely dry the residual wash buffer in the adsorbent material. The RNase-Free adsorption column CR4 was then transferred into a new RNase-Free centrifuge tube, and 35 µL of RNase-Free ddH2O was slowly dripped onto the center of the adsorption membrane. RNase-Free adsorption column CR4 was kept at room temperature for 2 min, and centrifuged at 12,000 rpm for 2 min to obtain the RNA solution. RNA concentration was then determined by NanoDrop 2000. RNA quality was analyzed with by Agilent 2100 Bioanalyzer (RIN ≥ 7 and 28 S/18S ≥ 0.7)., The total RNA was then digested by DNase, the mRNA was enriched by magnetic beads with Oligo(dT), which was broken into short fragments by adding interrupting reagents. ThemRNA fragments was taken as template, afterwards, one strand of cDNA was synthesized, and then the other strand of cDNA was synthesized based on the first strand, followed by purification of the double-strand cDNA using a kit. Next, purified double-strand cDNA underwent end repair, dA-tailing, and adaptor ligation. Subsequently, fragments were selected according to their size, followed by PCR amplification in final stage. The constructed library was then subjected to quality testing by Agilent 2100 Bioanalyzer, and the qualified library was then sequenced by Illumina HiSeq X Ten sequencing platform to generate paired-end data of 150 bp.

Transcriptome data analysis

A large quantity of paired-end sequencing data of samples was attained through Illumina platform along with massive generation of raw reads during sequencing, which were needed to be filtered to obtain high quality reads for further analysis. First, Trimmomatic software was employed to test the quality of the data and remove adaptors to filter out the low-quality bases, thereby obtaining high-quality reads. Second, Hisat 2 software was employed to compare the clean reads with the reference genome of zebrafish to determine genome comparison rate to assess the condition of sample. The expression value of gene FPKM was quantified by using Cufflinks software. The number of gene reads in each sample was obtained by Htseqcount software when calculating the difference in gene expression, and the data were standardized using DESeq(2012)R Package. NbinomTest function was applied to calculate the P value and fold change value of the differences, and DESeq(2012)R software was used for differential expression analysis. |log2(fold change)|≥2 (or 1.5) and P < 0.05 were set as the threshold of significant differential expression. Stratified cluster analysis of differentially expressed genes (DEGs) was performed to demonstrate the expression patterns of genes in different groups of samples. The Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of DEGs were conducted based on the hypergeometric distribution to analyze the biological functions or pathways affected by DEGs.

Evaluation of effects of L. gasseri LGV03 supernatant and indole-3-lactic acid on reduction of macrophages, neutrophils and T cells induced by vinorelbine in zebrafish

Normally developed Tg(mpx-EGFP), Tg(mpeg-EGFP), or Tg(rag2-DsRed) transgenic zebrafish of 48 h-post fertilization (hpf) were randomly selected and inoculated in 6-well cell culture plates, with 15 fish per well. The zebrafish were divided into six groups including control group, vinorelbine group (model group), L. gasseri LGV03 supernatant group, L. gasseri LGV03 supernatant combined with AhR antagonist (CH-223191) group, indole-3-lactic acid group, and indole-3-lactic acid combined with CH-223,191 group. The control group was added with 4 mL zebrafish E3 water, and the model group was added with 4 mL vinorelbine solution (200 µg/mL). L. gasseri LGV03 supernatant group was added with L. gasseri LGV03 supernatant (40%, V/V) plus vinorelbine solution (200 µg/mL). L. gasseri LGV03 supernatant combined with AhR antagonist (CH-223191) group was added with L. gasseri LGV03 supernatant (40%, V/V), vinorelbine solution (200 µg/mL), and 50 µM CH-223,191 solution. Indole-3-lactic acid group was added with indole-3-lactic acid (12 µM) plus vinorelbine solution (200 µg/mL). Indole-3-lactic acid combined with AhR antagonist (CH-223191) group was added with indole-3-lactic acid (12 µM), vinorelbine solution (200 µg/mL), and 50 µM CH-223,191 solution. The 6-well cell culture plates were incubated in a biochemical incubator at 28 ℃ for 96 h, and the solutions were updated every 24 h with newly prepared solutions. After 96 h, 15 zebrafish were taken from each group followed by observation and photograph under a fluorescence microscope. The number of macrophages and neutrophils were counted and the fluorescence intensity of T cells in zebrafish was recorded.

Data statistical analysis

All data were statistically analyzed using GraphPad Prism 8, and experimental data were represented by mean ± SEM. For the Student t test, comparison between the normal group and the model group: #P < 0.05, ##P < 0.01, and ###P < 0.001. For one-way analysis of variance, comparison between the L. gasseri LGV03 group and the model group: *P < 0.05, **P < 0.01, and ***P < 0.001.

Results

L. Gasseri LGV03 alleviates vinorelbine-induced immunosuppression

Our previous studies have shown that L. gasseri LGV03 isolated from the cervico-vagina of HPV-cleared women contributes to the clearance of HPV infection and beneficially regulate immune response [12]. However, the detailed underlying mechanisms by which L. gasseri LGV03 regulates the immune response remain unclear. Therefore, this study first investigated the effect of L. gasseri LGV03 on the immunosuppression induced by vinorelbine in zebrafish. As the results shown, vinorelbine (model control group) significantly inhibited the formation of macrophages and neutrophils in zebrafish (Fig. 1A and B). However, the number of macrophages and neutrophils in L. gasseri LGV03 group increased with the rise of L. gasseri LGV03 concentration, indicating that L. gasseri LGV03 inhibited the reduction of vinorelbine-induced macrophages and neutrophils in zebrafish. In addition, the potential effect of L. gasseri LGV03 on vinorelbine-induced T cell inhibition was examined in zebrafish. A production of red fluorescence was observed in T cells of transgenic zebrafish Tg(rag2-DsRed) while strong fluorescence signals were generated in thymus where T cells gather. Compared to those zebrafish in blank control group, the fluorescence signal of T cells was reduced by vinorelbine in thymus. L. gasseri LGV03 combined with vinorelbine treatment restored the fluorescence signal of T cells in the thymus to normal level, that is, L. gasseri LGV03 inhibited the vinorelbine-induced T cell inhibition (Fig. 1C). Finally, we examined the expression levels of some immune-related factors at transcriptional level. Compared to those zebrafish in control group, decreased mRNA expressions of immune-related genes (such as TNF-α, TNF-β, IFN-α) were observed in the vinorelbine group. Interestingly, mRNA expression of TNF-α, TNF-β and IFN-α were significantly increased when L. gasseri LGV03 was used in combination with vinorelbine (Fig. 1D). These results suggested that L. gasseri LGV03 alleviate the immunosuppression induced by vinorelbine.

Fig. 1
figure 1

Biomarkers of the immune function were significantly increased in larval zebrafish following L. gasseri LGV03 exposure. (A) L. gasseri LGV03 inhibits vinorelbine-induced macrophage reduction in zebrafish model Tg (mpeg-EGFP) (right). Number of macrophages in zebrafish after treatment of vinorelbine and L. gasseri LGV03 (left). (B) L. gasseri LGV03 inhibits vinorelbine-induced neutrophil reduction in zebrafish model Tg (mpx-EGFP) (right). Number of neutrophils in zebrafish after treatment of vinorelbine and L. gasseri LGV03 (left). (C) L. gasseri LGV03 suppresses vinorelbine-induced T cell decrease in zebrafish model Tg(rag2-DsRed). The dotted box indicates the thymus where T cells gather (right). Fluorescence intensity of T cells in zebrafish after treatment of vinorelbine and L. gasseri LGV03 (left). (D) mRNA gene expression of cytokines TNF-α, TNF-β and IFN-α in zebrafish

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L. Gasseri LGV03 inhibits the expression of vinorelbine-mediated immune-related genes in zebrafish

In order to further explore the immune regulation caused by L. gasseri LGV03, this study used transcriptome sequencing technology, GO biological annotation database, and KEGG database to screen for significantly altered signaling pathways. As shown in Fig. 2A, there were significant differences in the expression levels of 114 genes between the control group and the L. gasseri LGV03 intervention group, among which 46 genes were up-regulated (red) and 68 genes were down-regulated (blue) (Fig. 2A). Heatmaps of differentially expressed genes (DEGs) (Fig. 2B) showed that the expression levels of DEGs were separated from the model group after the intervention of L. gasseri LGV03, with CLCA1 and PLAC8.1 significantly up-regulated and ZNF1162 significantly down-regulated (Fig. 2C). CLCA1 can activate macrophages and increase the expression of cytokines. Consistent with this, decreased expressions of CLCA1 and PLAC8.1 mRNA was detectedin the vinorelbine group, as compared with the control group (Fig. 2D). Interestingly, when L. gasseri LGV03 was used in combination with vinorelbine, the expression of CLCA1 and PLAC8.1 mRNA was significantly up-regulated. PLAC8.1 can regulate the expression of Th cell differentiation inducers, promote the expression of IL-6, IL-4, IL-17 A and TGF-β, and make Th cell differentiation shift from Th1 to Th2, Th17 and Treg. In addition, GO analysis was used to analyze biological processes, cell components, and molecular functions and predict the relationship between biological function and DEGs (Fig. 2E). To further predict the pathways involved, we performed a KEGG analysis. Compared with the model group, L. gasseri LGV03 intervention group showed DEG enrichment in immunoinflammatory pathways (cytokine-cytokine receptor interaction, MAPK signaling), biological process and molecular function related pathways (amino sugar and nucleotide sugar metabolism, spliceosome, protein processing in endoplasmic reticulum, phagosome, cellular senescence, endocytosis, and neuroactive ligand-receptor interaction) (Fig. 2F). Therefore, the above results indicate that L. gasseri LGV03 can affect the immune response of zebrafish.

Fig. 2
figure 2

L. gasseri LGV03 altered gene expression and pathways in zebrafish. (A) The volcano plot in comparison of two groups of zebrafish. (B) Heat map of the differentially expressed genes (DEGs) compared between group B and M. (C) mRNA expression of CLCA1, PLAC8.1 and ZNF1162 in zebrafish by RNA-Seq. (D) mRNA gene expression of CLCA1, PLAC8.1 in zebrafish after treatment of vinorelbine and L. gasseri LGV03. (E) Go analysis comparison of group B and M. (F) Analysis of DEGs KEGG pathway enrichment in groups B and M. B: L. gasseri LGV03 plus Vinorelbine group, M: Vinorelbine group

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L. Gasseri LGV03 can secrete indole derivatives

An increasing number of studies indicate that microorganisms play an important role in mediating host immune responses primarily through their metabolites [22, 26]. Non-targeted detection was carried out for the metabolites in L. gasseri LGV03 strain lysate (Group A) and L. gasseri LGV03 strain culture supernatant (Group B), and it was found that compared with MRS Medium, there were 142 differential metabolites in the lysate of L. gasseri LGV03 strain lysate and 216 differential metabolites in the culture supernatant of L. gasseri LGV03 strain (Figure S1 and Figure S2), among which 70 and 100 were upregulated, respectively (Fig. 3A and B). The intersection of the two groups of upregulated differential metabolites included 26 metabolites (Fig. 3C). The top 4 upregulated metabolites were phenylacetaldehyde, 3-phenyllactic acid, N-acetylserotonin and indole-3-lactic acid. Among them, phenylacetaldehyde and 3-phenyllactic acid may be derived from phenylalanine metabolism, and N-acetylserotonin and indole-3-lactic acid may be derived from tryptophan metabolism. Tryptophan metabolites N-acetylserotonin and indole-3-lactic acid are both AhR agonists, which can induce T cell differentiation and promote the expression of inflammatory factors IL-1β and TNF-α. KEGG enrichment analysis of metabolic pathways (Fig. 4A and B) verified that L. gasseri LGV03 mainly engaged in purine metabolism, phenylalanine metabolism and tryptophan metabolism. Among them, tryptophan can produce N-acetylserotonin, iindole-3-lactic acid and 5-hydroxytryptophan, while phenylacetaldehyde and 3-phenyllactic acid are produced by phenylalanine metabolism.

Fig. 3
figure 3

(A) Heat map of up-regulated differential metabolites of group L. gasseri LGV03 compared with MRS broth. (B) Heat map of up-regulated differential metabolites of group L. gasseri LGV03 supernatant compared with MRS broth. (C) Shared up-regulated differential metabolites between L. gasseri LGV03 vs. broth and L. gasseri LGV03 supernatant vs. broth

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Fig. 4
figure 4

(A) Enriched bubble map of metabolic pathway analysis of the differential metabolites between group L. gasseri LGV03 and MRS broth. (B) Enriched bubble map of metabolic pathway analysis of the differential metabolites between group L. gasseri LGV03 supernatant and MRS broth

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L. gasseri LGV03 regulates the immune response in zebrafish through its activation of AhR via its metabolite indole-3-lactic acid

Lactobacillus spp. has been shown to release several immunomodulatory metabolites, including indole derivatives such as indole-3-formaldehyde, which exhibit T cell immunomodulatory properties by activating AhRs, a type of universally expressed transcription factor [19]. More recently, study has shown that indole-3-formaldehyde, a metabolite released by Lactobacillus reuteri in the tumor microenvironment, can activate AhR of CD8+T cells, promote the secretion of IFN-γ and kill tumor cells, thus enhancing the efficacy of immunotherapy [26]. Therefore, we further studied the effect of L. gasseri LGV03 metabolite on the immune response in zebrafish. As the results reveal, vinorelbine (model group) significantly inhibited the formation of macrophages and neutrophils in zebrafish (Fig. 5A and B). In addition, compared to the model group, the number of macrophages and neutrophils increased significantly in the L. gasseri LGV03 fermentation supernatant plus vinorelbine group and indole-3-lactic acid plus vinorelbine group. AhR inhibitor CH-223,191 can significantly inhibit the promoting effect of L. gasseri LGV03 fermentation supernatant or indole-3-lactic acid on macrophages and neutrophils production in zebrafish. Meanwhile, a similar phenomenon was also found in transgenic zebrafish Tg(rag2-DsRed). Compared with the model group, the fluorescence intensity of T cells in the zebrafish thymus in the L. gasseri LGV03 fermentation supernatant plus vinorelbine group and indole-3-lactic acid plus vinorelbine group was significantly enhanced and comparable with that in the control group. CH-223,191 could significantly inhibit the inducing of fluorescence signal recover of T cells in the thymus by L. gasseri LGV03 fermentation supernatant or indole-3-lactic acid (Fig. 5C). These results indicate that L. gasseri LGV03 regulated the immune response in zebrafish through activating AhR by its metabolite indole-3-lactic acid.

Fig. 5
figure 5

Biomarkers of the immune function were significantly increased in larval zebrafish following L. gasseri LGV03 supernatant exposure. (A) L. gasseri LGV03 supernatant and indole-3-lactic acid inhibited vinorelbine-induced macrophage reduction in zebrafish model Tg (mpeg-EGFP) (right). Number of macrophages in zebrafish after treatment of vinorelbine and L. gasseri LGV03 supernatant or indole-3-lactic acid (left). (B) L. gasseri LGV03 supernatant and indole-3-lactic acid inhibited vinorelbine-induced neutrophil reduction in zebrafish model Tg (mpx-EGFP) (right). Number of neutrophils in zebrafish after treatment of vinorelbine and L. gasseri LGV03 supernatant or indole-3-lactic acid (left). (C) L. gasseri LGV03 supernatant and indole-3-lactic acid inhibited suppressed vinorelbine-induced T cell decrease in zebrafish model Tg(rag2-DsRed) (right). The dotted box indicates the thymus where T cells gather. Fluorescence intensity of T cells in zebrafish after treatment of vinorelbine and L. gasseri LGV03 supernatant or indole-3-lactic acid (left)

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L. gasseri LGV03 regulates the immune response of cervical epithelial cells Ect1/E6E7 by activating AhR through its metabolite indole-3-lactic acid

To explore the regulation of the immune effect of cervical epithelial cells by L. gasseri LGV03 metabolites, we detected the regulatory role of L. gasseri LGV03 supernatant and indole-3-lactic acid on the cytokine secretion of Ect1/E6E7 cells by using ELISA. As shown in Fig. 6, L. gasseri LGV03 fermentation supernatant and indole-3-lactic acid can induce Ect1/E6E7 cells to significantly secrete IFN-α, IFN-β and chemokines (MIP-1α, MIP-1β). However, AhR inhibitor CH-223,191 could significantly inhibit the inducing role of L. gasseri LGV03 fermentation supernatant or indole-3-lactic acid on the secretion of these cytokines from Ect1/E6E7 cells. Therefore, L. gasseri LGV03-derived indole-3-lactic acid promoted the secretion of cytokines by activating AhR receptor of Ect1/E6E7 cells.

Fig. 6
figure 6

L. gasseri LGV03 supernatant and indole-3-lactic acid stimulated End1/E6E7 cells to secreta IFN-α, IFN-β and CC chemokine (MIP-1α, MIP-1β)

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Discussion

The microenvironment of the female reproductive tract, including microorganisms, metabolites, and immune components, relies on the balance of their interactions to maintain female reproductive tract homeostasis and health [1, 6, 27]. Vaginal mucosa serves as a protective barrier to against invading pathogens through the interaction between its epithelial cells, immune system, and symbiotic microorganisms [27, 28]. The microbiota residing in the vaginal environment are active and critical components of this defense system against infections [29]. Moreover, alterations in the cervicovaginal microbial community not only induce changes in the cervicovaginal metabolome but also further affect immunity, thereby contributing to the onset and progression of diseases as well as the manifestation of corresponding symptoms and physical signs [6, 30]. In particular, the dominance of Lactobacillus in the genital tract is crucial due to its ability to inhibit pathogens and maintain immune balance [8, 11, 29]. Our previous studies have shown that the Lactobacillus-dominated vaginal microbiota, specifically L. gasseri LGV03, regulates immune homeostasis in vulva and cervix and inhibits viral escape [12]. However, how L. gasseri LGV03 regulates immunity remains unclear.

Zebrafish share up to 85% genetic homology with humans and have immune cell types and morphologies similar to humans [31]. Here, we used transgenic zebrafish with fluorescent protein-labelled neutrophils, macrophages and T cells to observe the immunoregulatory effects of L. gasseri LGV03 on vinorelbine treated immunocompromised zebrafish [32,33,34]. Studies have found that L. gasseri LGV03 can significantly increase the number of macrophages and neutrophils and fluorescence intensity of T cells in immunocompromised zebrafish. Macrophages, as an important type of immune cells in immune system, can initiate congenital immune response, thereby helping the body to resist infection and inflammation. Meanwhile, L. gasseri LGV03 can significantly increase the mRNA expression levels of immune factors TNF-α, TNF-β and IFN-β in vinorelbine-induced immunocompromised zebrafish. In other words, L. gasseri LGV03 can enhance the immune function of vinorelbine-induced immunocompromised zebrafish.

To further explore the immune regulation caused by L. gasseri LGV03, this study adopted transcriptome sequencing technology, GO biological annotation database and KEGG database to screen the signaling pathways with significant changes. It can be inferred that L. gasseri LGV03 might regulate the immunity of vinorelbine-induced immunocompromised zebrafish models. Significant differences were observed in the expression levels of 114 genes after L. gasseri LGV03 intervention, among which 46 genes were up-regulated and 68 genes were down-regulated. The relationship between biological functions and DEGs was predicted by GO analysis. Moreover, KEGG enrichment found that DEGs were enriched in immunoinflammatory pathways such as cytokine-cytokine receptor interaction and MAPK signaling pathways.

Accumulating studies have shown that microorganisms exert their influence on the local and even the whole body mainly through their metabolites. Tryptophan can be decomposed into indole and various indole derivatives by microorganisms, playing an important role in mediating the immune response of hosts [22, 26]. Non-targeted detection and analysis of metabolites of L. gasseri LGV03 strain revealed that phenylacetaldehyde, 3-phenyllactic acid, N-acetylserotonin and indole-3-lactic acid were significantly increased in the lysate and supernatant of L. gasseri LGV03. KEGG enrichment analysis of metabolic pathways showed that L. gasseri LGV03 was mainly involved in purine metabolism, phenylalanine metabolism and tryptophan metabolism. The metabolism of tryptophan can produce N-acetylserotonin and indole-3-lactic acid, both of which are AhR agonists, can induce T cell differentiation, promote the expression of inflammatory cytokines IL-1 and TNF-α, and regulate immune response. Further study showed that L. gasseri LGV03 fermentation supernatant and indole-3-lactic acid could significantly enhance the number and fluorescence intensity of immune cells (macrophages, neutrophils, and T cells) in immunocompromised zebrafish. However, AhR inhibitor CH-223,191 can significantly inhibit L. gasseri LGV03 fermentation supernatant or indole-3-lactic acid from promoting the increase of macrophages and neutrophils and the fluorescence intensity of T cells in the thymus of zebrafish. Thus, L. gasseri LGV03 can regulate the immune response in zebrafish by activating AhR through the indole-3-lactic acid from its metabolism.

As immune sentinels, cervical epithelial cells play a key role in the maintenance of local immune response of cervical mucosa [35]. These immune sentinels can recruit and activate immune cells by expressing soluble factors such as pro-inflammatory cytokines and chemokines, thereby inducing mucosal immune responses to pathogens. Of those antiviral factors produced by infected cells, IFN-α and IFN-β are key cytokines involved in the protection to viral infection, that is the reason for the improvement of these antiviral cytokines in cervical epithelial cells, which has been used as a biomarker to find beneficial microorganisms capable of protecting against viral infection [36,37,38,39]. Thus, whether L. gasseri LGV03 fermentation supernatant and indole-3-lactic acid induced the expression of IFN-α and IFN-β in Ect1/E6E7 cells was examined. The results showed that L. gasseri LGV03 fermentation supernatant and indole-3-lactic acid actually activated the immune response of Ect1/E6E7 cells and significantly increase the secretion of IFN-α, IFN-β and chemokines (MIP-1α, MIP-1β) from Ect1/E6E7 cells, but this activated immune response can be inhibited by the AhR antagonist CH-223,191. This suggested that L. gasseri LGV03-derived indole-3-lactic acid can activate AHR-mediated immune response.

Conclusion

In summary, the results of this study demonstrated that L. gasseri LGV03 isolated from the cervico-vagina of HPV-cleared women exhibited potent activities in alleviating vinorelbine-induced reduction in the abundance of macrophages, neutrophils and T cells, and significantly enhanced mRNA expressions of cytokines TNF-α, TNF-β and IFN-β. Meanwhile, indole-3-lactic acid was significantly increased in the lysate and supernatant of L. gasseri LGV03. In addition, L. gasseri LGV03 fermentation supernatant and indole-3-lactic acid alleviated vinorelbine-induced macrophages, neutrophils and T cells deficiency. However, the alleviating effects of L. gasseri LGV03 supernatant and indole-3-lactic acid were eliminated by AHR antagonist CH-223,191. Consistently, both L. gasseri LGV03 fermentation supernatant and indole-3-lactic acid significantly increased the secretion of IFN-α, IFN-β and chemokines (MIP-1α, MIP-1β) in Ect1/E6E7 cells, while these benefits were eliminated by CH-223,191 treatment. Our findings suggested that L. gasseri LGV03 produced indole-3-lactic acid to activate AHR-mediated immune response.

Data availability

The raw data generated with transcriptome sequencing in this study are available in the NCBI SRA database under BioProject Accession Numbers PRJNA1073976. Available online: (https://dataview.ncbi.nlm.nih.gov/object/48550801).

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Acknowledgements

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Funding

This work was supported by Natural Science Foundation of Guangdong Province (Grant NO. 2023A1515011439), Basic and Applied Basic Research Foundation of Guangdong Province (Grant NO. 2021A1515220025).

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  1. Zikang Zhang and Kangdi Zheng contributed equally to this work.

Authors and Affiliations

  1. Department of Laboratory Medicine, The Seventh Affiliated Hospital of Southern Medical University, Foshan, Guangdong, 528244, China

    Zikang Zhang, Longbin Cao, Weimin Sun & Feng Qiu

  2. Research and Development Department, Guangdong Longseek Testing Co., Ltd., Guangzhou, Guangdong, 510700, China

    Kangdi Zheng & Zhao Zhang

  3. Department of General Practice Center, The Seventh Affiliated Hospital of Southern Medical University, Foshan, Guangdong, 528244, China

    Lizhu Lin

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Contributions

Zikang Zhang and Kangdi Zheng conceived and designed the study. Longbin Cao and Lizhu Lin performed all the experiments. Zhao Zhang analyzed the data. All authors discussed the results. Weimin Sun wrote the main manuscript text. All the authors have read and approved the final manuscript. Feng Qiu supervised all aspects of this study.

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Arrive: All the procedure of the study is followed by the ARRIVE guidelines.

The animal study protocol was approved by Animal Welfare and Ethics Committee of Guangdong Human Microecology Engineering Technology Research Center’s Laboratory (approval number: IACUC MC 0106-03-2023; date of approval: 10 March 2023).

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Zhang, Z., Zheng, K., Zhang, Z. et al. Lactobacillus gasseri LGV03-derived indole-3-lactic acid ameliorates immune response by activating aryl hydrocarbon receptor. Microb Cell Fact 24, 34 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12934-025-02662-8

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Microbial Cell Factories

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