Lactobacillus plantarum Lac16 alleviates dextran sodium sulfate-induced colitis in mice by suppressing NLRP3 inflammasome overactivation through microbiota-derived isobutyric acid

The probiotic strain Lac16 (deposited at the China Center for Type Culture Collection No. M2016259) was cultured in Mann-Rogosa-Sharpe broth at 37°C for 18 h, harvested by centrifugation, resuspended in sterile phosphate-buffered saline (PBS, pH = 7.4), and stored at 4℃ for experimental use.

Male C57BL/6 mice (4 weeks old) were obtained from SLAC Laboratory Animal Co., Ltd. (Shanghai, China), and maintained under specific pathogen-free conditions with ad libitum access to food and water throughout the acclimatization period.

During the final 7 days of the experiment, mice were weighed and scored daily for pathological features as previously described (26). Briefly, the disease activity index (DAI) for evaluating colitis severity was calculated as the sum of scores for weight loss (0, none; 1, 1%–5%; 2, 5%–10%; 3, 10%–15%; and 4, >15%), stool consistency (0, normal; 2, loose; and 4, diarrhea), and hematochezia (0, normal; 2, slight bleeding; and 4, gross bleeding), with all parameters assessed daily in each mouse.

Colon tissue was fixed in 4% (vol/vol) paraformaldehyde (Beyotime, Shanghai, China) and dehydrated, followed by paraffin embedding, sectioning, and staining with hematoxylin and eosin or alcian blue-periodic acid-Schiff. Images were acquired via microscope (Olympus, Tokyo, Japan). Histological scoring was performed according to the established scoring system described by Stillie and Stadnyk (27).

Colon tissue was fixed overnight at 4°C in 2.5% (vol/vol) glutaraldehyde (Sinopharm, Shanghai, China). After that, the tissues were sequentially fixed, dehydrated, embedded, sectioned, stained, and observed using a transmission electron microscopy (Hitachi H-7650; Hitachi, Ibaraki, Japan).

Colon sections were successively dewaxed, rehydrated, antigen repaired, blocked, incubated with primary antibodies (mucin 2, NLRP3, and myeloperoxidase [MPO]; Servicebio, Wuhan, China) and secondary antibody (Servicebio), stained, counterstained, dehydrated, clarified, and mounted for imaging using a microscope (Olympus).

Colon sections were successively dewaxed, rehydrated, antigen repaired, blocked, and incubated with primary antibodies (occludin + zona occludens-1 [zo-1], F4/80 + inducible nitric oxide synthase [iNOS]/arginase 1 [Arg-1]; Servicebio) and secondary antibodies (Servicebio). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (Beyotime), and samples were imaged using laser scanning confocal microscopy (Zeiss, Oberkochen, Germany) with subsequent quantification performed using ImageJ software.

Hematological parameters were analyzed using an automated hematology analyzer (Yuyanbio, Shanghai, China).

MPO activity and cytokine levels (IL-1β, IL-6, TNF-α, IL-18, IL-10, and IFN-γ) in the colon were determined using an assay kit. Serum lipopolysaccharide (LPS) concentrations were quantified by enzyme-linked immunosorbent assay (Nanjing Jiancheng Biology Engineering Institute, Nanjing, China).

Total RNA was extracted from colon tissues using RNAiso Plus (Takara, Dalian, China), followed by reverse transcription. Quantitative real-time PCR was performed on a StepOne Plus system (Applied Biosystems, Carlsbad, CA, USA) with β-actin as the endogenous control. Primer sequences are listed in Table S1. Relative gene expressions were quantified using the 2^−∆∆Ct method (28).

Colon samples were homogenized in lysis buffer, and protein concentrations were determined using a BCA assay kit (Beyotime). Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After blocking, membranes were incubated overnight at 4°C with the following primary antibodies: NLRP3 (HuaBio, Hangzhou, China), apoptosis-associated speck-like protein containing a C-terminal CARD (ASC; Abclonal, Wuhan, China), caspase-1 (AdipoGen, Seoul, Korea), IL-1β (Sigma-Aldrich), and β-actin (Abcam, Cambridge, UK). Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (HuaBio), followed by chemiluminescent detection using an HRP substrate kit (Millipore). Ultimately, protein bands were detected using imaging equipment (Tanon, Shanghai, China) and quantified using ImageJ software.

The V3–V4 hypervariable regions of bacterial 16S rRNA genes were amplified from cecal content DNA using primers 338F (5′--3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The PCR amplicons were sequenced on the Illumina MiSeq platform. Raw sequences were quality-filtered and clustered into operational taxonomic units at 97% similarity using QIIME software. Microbial α-diversity (Ace, Chao1, Shannon, and Simpson indices) and β-diversity were analyzed using the QIIME software. β-Diversity was visualized through principal coordinate analysis (PCoA) plots. The relative abundance of microbiota was analyzed across several taxonomic levels. ACTCCTACGGGAGGCAGCA

The method for determining SCFA concentrations was adapted from Gong et al. (29) with modifications. Fecal samples were homogenized and centrifuged. The supernatant was mixed with 25% (wt/vol) metaphosphoric acid, incubated at −20°C for 24 h, then centrifuged and filtered. SCFAs were detected using the NEXIS GC-2030 ATF (Shimadzu Group Company, Kyoto, Japan).

Fecal samples were collected aseptically, weighed, homogenized, serially diluted, and plated on brain heart infusion agar. Colony growth was documented photographically after 48 h of incubation.

HT-29 cells were cultured in Dulbecco’s modified Eagle’s F12 ham medium (DMEM/F12) augmented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were maintained at an atmosphere of 37°C and 5% CO₂.

Bone marrow-derived macrophages (BMDMs) were isolated from femurs and tibias of mice. Both ends of the bones were cut, and the bone marrow was flushed with sterile PBS. The cell suspension was collected, and red blood cells were lysed. After centrifugation, the cells were resuspended in complete DMEM supplemented with 10 ng/mL macrophage colony-stimulating factor (M-CSF). After 72 h of culture, fresh complete DMEM with 10 ng/mL M-CSF was added for another 72 h. On day 7, cells were trypsinized, seeded, and cultured in M-CSF-free complete DMEM.

Cytotoxicity assay was evaluated using a CCK-8 assay (Beyotime). When HT-29 cells reached 80% confluence, they were treated with isobutyric acid (0.1, 1.0, 10.0, and 100.0 µM and 1.0 and 10.0 mM) for 24 h. Subsequently, cells were incubated in serum-free DMEM/F12 containing 10% (vol/vol) CCK-8 solution, and absorbance was measured at 450 nm. Cell viability was assessed according to the manufacturer’s protocol.

To evaluate the protective effect of isobutyric acid, HT-29 cells were co-treated with increasing concentrations of isobutyric acid (0.001, 0.01, 0.1, 1.0, 10.0, and 100 µM and 1 mM) and 10 µg/mL LPS (Sigma-Aldrich) for 24 h. Cell viability was then determined as described previously.

To assess the anti-inflammatory effects of isobutyric acid, BMDMs were co-treated with 1 mM isobutyric acid and 1 µg/mL LPS for 24 h, followed by cell viability analysis as previously described.

Cell apoptosis was analyzed using an Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime). HT-29 cells treated with 1 mM isobutyric acid and 10 µg/mL LPS for 24 h, then harvested, washed, stained, and examined using flow cytometry (Becton Dickinson, Parsippany, NJ, USA).

Upon reaching 90% confluence, a wound was scratched in HT-29 cells, followed by PBS washing to remove debris. Cells were then treated with 1 mM isobutyric acid and 10 µg/mL LPS for 24 h. Wound healing progression was quantified by measuring scratch width at 0 and 24 h using an EVOS M7000 Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).

Statistical analyses were performed using SPSS software. Data are presented as mean ± SD. Between-group comparisons were analyzed by two-tailed Student’s t-test, while multigroup comparisons used one-way analysis of variance with Tukey’s post hoc test. Differences were deemed statistically significant when P < 0.05. Graphical representations were created using GraphPad Prism 9 (San Diego, CA, USA).

To investigate the effects of Lac16 on DSS-induced colitis, mice were administered 2 × 10⁸ CFU of Lac16 for 4 weeks, with colitis induction occurring during the final 7 days (Fig. 1A). DSS administration induced significant weight loss, colon shortening, and elevated DAI scores in mice, all of which were markedly attenuated by Lac16 (Fig. 1B through D). Pretreatment with Lac16 significantly ameliorated multiple DSS-induced colonic pathologies, including ulcer formation, inflammatory infiltration, crypt loss, microvillus damage, goblet cell depletion, and downregulation of mucin 2 expression (Fig. 1E through G; Fig. S1A). Furthermore, Lac16 pretreatment significantly decreased serum LPS levels (Fig. 1H), a well-established biomarker of intestinal barrier dysfunction (30). Consistent with the observed barrier dysfunction, DSS challenge markedly reduced colonic levels of key tight junction proteins (occludin, zo-1, and claudin-1), while Lac16 pretreatment largely preserved their expression (Fig. 1I through K; Fig. S1B through D).

To assess the impact of Lac16 on systemic and colonic inflammation, we evaluated hematological parameters, MPO activity (as a marker of neutrophil infiltration), inflammatory cytokine levels, and intestinal macrophage polarization. Notably, the DSS challenge significantly increased circulating leukocyte populations (white blood cells, monocytes, granulocytes, and lymphocytes), effects that were substantially attenuated by Lac16 pretreatment (Fig. S2A). Furthermore, DSS treatment significantly elevated both colonic MPO expression and activity, indicating severe inflammation, while Lac16 intervention markedly attenuated these effects (Fig. 2A; Fig. S2B). Consistently, Lac16 significantly decreased colonic levels of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6, and IL-18) while concurrently elevating the anti-inflammatory cytokine IL-10 (Fig. 2B). DSS treatment significantly increased the proportion of pro-inflammatory M1 macrophages (F4/80⁺iNOS⁺) while also elevating anti-inflammatory M2 macrophages (F4/80⁺Arg-1⁺), with Lac16 intervention both attenuating the M1 response and further augmenting the M2 population (Fig. 2C and D). Both M1-associated genes (iNOS, IL-1β, TNF-α, and IL-12/p40) and M2-associated markers (Arg1, MR, Ym1, and Fizz1) showed concordant expression patterns at the transcriptional level, mirroring the protein-level observations of macrophage polarization (Fig. S2C).

Consistent with established links between macrophage polarization and NLRP3 inflammasome activation (7), our analysis demonstrated that DSS challenge significantly upregulated both NLRP3 protein expression and gene transcription in colonic tissue, effects that were markedly attenuated by probiotic pretreatment (Fig. 3A and B). Concurrently, the same trend was obtained in ASC, another crucial protein in the NLRP3 inflammasome pathway (Fig. 3B). Western blot analysis demonstrated significant upregulation of NLRP3 inflammasome pathway components in DSS-treated mice, including NLRP3, ASC, cleaved caspase-1 (p20), and mature IL-1β (p17), indicating robust inflammasome activation (Fig. 3C and D). Notably, Lac16 pretreatment substantially attenuated the expression of all these proteins.

To investigate the role of gut microbiota in Lac16-mediated amelioration of colitis, mice received Abx pretreatment via drinking water to deplete intestinal microbiota prior to Lac16 administration and DSS challenge (Fig. 4A). After 1 week of Abx administration, fecal microorganisms in the Abx + Lac16 + DSS group showed a dramatic reduction, indicating effective gut microbiota elimination (Fig. 4B). Following microbiota depletion, the Abx + Lac16 + DSS group exhibited significant reductions in body weight and colon length, while the DAI score remained unchanged (Fig. 4C through E). In the absence of gut microbiota, the protective effect of Lac16 pretreatment against DSS-induced colonic epithelial damage was partially attenuated (Fig. 4F). Concurrently, levels of IL-1β and TNF-α, as well as NLRP3 and ASC protein expression, were significantly elevated compared to the Lac16 + DSS group (Fig. 4G and H). Conversely, the gene expression of occludin, claudin-1, and zo-1 of the Abx + Lac16 + DSS group was significantly reduced compared to the Lac16 + DSS group (Fig. 4I). These phenomena suggested that Lac16 had reduced relieving effects on colitis after the removal of the gut microbiota.

To investigate the role of Lac16-modulated microbiota in colitis amelioration, we performed FMT from donor mice (with or without Lac16 treatment) into recipient mice with DSS-induced colitis (Fig. 5A). FMT experiments revealed that recipient mice receiving microbiota from Lac16-treated donors (Lac16-FMT and Lac16 + DSS FMT groups) showed significantly attenuated colitis symptoms compared to DSS-FMT controls (Fig. 5B through D). The FMT-Lac16 and FMT-Lac16 + DSS groups exhibited significantly attenuated colonic epithelial damage, accompanied by reduced levels of the pro-inflammatory cytokines IL-1β and TNF-α, as well as downregulated gene expression of the inflammasome components NLRP3 and ASC (Fig. 5E through G). The colonic gene expression of occludin, claudin-1, and zo-1 was significantly elevated in recipients of microbiota from Lac16-treated or Lac16 + DSS-treated donors compared to those receiving microbiota from DSS-treated donors (Fig. 5H). Collectively, these findings demonstrate that FMT from Lac16-treated donors recapitulates the probiotic’s protective effects against colitis, further confirming the crucial contribution of microbiota in the anticolitis benefits of Lac16.

To investigate the microbiota-dependent mechanisms underlying Lac16-mediated colitis amelioration, we systematically analyzed gut microbial composition in DSS-treated mice with or without Lac16 pretreatment. The current analysis revealed no significant alteration in α-diversity between the Lac16 + DSS and the DSS group (Fig. S3). β-Diversity analysis revealed significant compositional differences among the three groups, as demonstrated by distinct clustering in PCoA plots using the unweighted_unifrac method. Notably, the Lac16 + DSS group showed clear separation from the DSS group, indicating distinct microbial community structures resulting from probiotic intervention (Fig. 6A). Phylum-level analysis demonstrated that DSS treatment significantly decreased the relative abundance of Firmicutes while increasing the abundances of Desulfobacterota, Campylobacterota, and Proteobacteria compared to the control group (Fig. 6B). Compared to the DSS group, the Lac16 + DSS group showed a significantly increased relative abundance of Firmicutes and decreased proportions of Desulfobacterota, Campylobacterota, Proteobacteria, and Deferribacterota. At the genus level, DSS treatment significantly increased the relative abundances of Bacteroides and Helicobacter while decreasing Lactobacillus and Alloprevotella. In contrast, Lac16 supplementation in DSS-treated mice substantially enriched Dubosiella and Alloprevotella and reduced Bacteroides and Helicobacter compared to DSS controls (Fig. 6C and D).

SCFAs, key microbial metabolites produced by intestinal commensal bacteria, play an essential role in maintaining and restoring intestinal epithelial barrier integrity following mucosal damage (31). Our analysis revealed that DSS treatment significantly decreased concentrations of acetic acid, butyric acid, and total SCFAs. Lac16 pretreatment attenuated these reductions, maintaining higher levels of acetic acid and total SCFAs compared to DSS controls. Notably, isobutyric acid levels were uniquely elevated in the Lac16 + DSS group relative to both control and DSS-treated mice (Fig. 6E). Spearman correlation analysis of combined data from the DSS and Lac16 + DSS groups (Fig. 6F) demonstrated that acetic acid and isobutyric acid levels were positively correlated with body weight, colon length, IL-10 concentration, and expression of tight junction genes (occludin, claudin-1, and zo-1). Conversely, acetic acid and isobutyric acid showed significant negative correlations with pro-inflammatory cytokines (acetic acid: IL-1β, IL-6, IL-18, and IFN-γ; isobutyric acid: IL-1β, TNF-α, and IL-18), disease activity index (isobutyric acid only), and NLRP3 inflammasome components (NLRP3 and ASC gene expression). Butyric acid showed significant positive correlations with occludin and zo-1 mRNA expression while exhibiting strong negative correlations with IL-6 concentration and ASC gene expression. Propionic acid demonstrated a specific positive correlation with IL-10 concentration. Collectively, these findings indicate that Lac16’s therapeutic effects on colitis are mediated through microbiota-derived SCFAs, which collectively modulate epithelial barrier integrity and inflammatory responses.

The aforementioned three investigations demonstrated that Lac16-mediated modulation of gut microbiota is essential for colitis remission, with a particularly notable elevation in fecal isobutyric acid levels observed in Lac16-pretreated mice. As a microbiota-derived metabolite, isobutyric acid exhibited distinct correlations with multiple colitis-associated phenotypic and inflammatory cytokines. These results suggest that isobutyric acid may serve as a key mediator of Lac16’s therapeutic effects against colitis. To directly assess isobutyric acid’s anticolitis effects, mice received 150 mM isobutyric acid in drinking water for 3 weeks prior to and during DSS challenge (Fig. 7A). Isobutyric acid pretreatment significantly ameliorated DSS-induced colitis, as evidenced by attenuated body weight loss, preserved colon length, reduced disease activity index scores, and improved histopathological damage compared to the DSS group (Fig. 7B through E). Isobutyric acid pretreatment significantly downregulated pro-inflammatory gene expression (IL-1β, TNF-α, NLRP3, and ASC) while upregulating tight junction components (occludin, claudin-1, and zo-1), demonstrating its protective role against DSS-induced colitis (Fig. 7G and H).

To investigate isobutyric acid’s direct effects on intestinal epithelial cells and macrophages, we established LPS-stimulated HT-29 cells and BMDM models in vitro. Isobutyric acid (≤1 mM) showed no cytotoxicity in HT-29 cells (Fig. 8A) but dose-dependently protected against LPS-induced damage (10 µM–1 mM, Fig. 8B). Isobutyric acid (1 mM) significantly reduced both early apoptosis (Annexin V-FITC⁺/PI⁻) and late apoptosis/necrosis (Annexin V-FITC⁺/PI⁺) in LPS-stimulated HT-29 cells (Fig. 8C). The scratch wound assay demonstrated that isobutyric acid significantly accelerated wound closure in LPS-stimulated HT-29 cells (Fig. 8D). Consistent with in vivo observations, isobutyric acid upregulated mRNA expression of tight junction components (occludin, claudin-1, and zo-1) in vitro (Fig. 8E). In LPS-activated BMDMs, 1 mM isobutyrate significantly attenuated LPS-induced cytotoxicity while maintaining baseline cell viability (Fig. 8F). Furthermore, it markedly downregulated expression of pro-inflammatory cytokines (TNF-α and IL-6, Fig. 8G) and inflammasome components (NLRP3 and ASC, Fig. 8H). Collectively, these findings further validated that isobutyric acid attenuates inflammatory responses and restores intestinal epithelial homeostasis through its dual actions on immune modulation and barrier reinforcement.

In summary, our findings demonstrate that Lac16 alleviates DSS-induced colitis by preserving intestinal barrier integrity, reducing inflammation, regulating macrophage polarization, modulating NLRP3 inflammasome activation, and maintaining gut microbiota homeostasis. Crucially, Lac16’s suppression of NLRP3 inflammasome overactivation depends on gut microbiota, with isobutyric acid serving as a key mediator of this protective effect (Fig. 9). This study elucidates novel mechanisms underlying Lac16’s dual modulation of host immunity and gut microbiota, positioning it as a potential therapeutic strategy for IBD management.