Bifidobacterium longum RAPO Attenuates Dermal and Pulmonary Fibrosis in a Mouse Model of Systemic Sclerosis through Macrophage Modulation and Growth of Short-Chain Fatty Acid Producers

Abstract

Systemic sclerosis (SSc) is a complex autoimmune disease with an unclear etiology and no effective treatments. Recent research has suggested involvement of the microbiome in SSc pathogenesis. This study aimed to identify specific microbial species associated with SSc and explore their therapeutic potential. Serum Abs against 384 intestinal microbial species revealed a significant depletion in Abs against Bifidobacterium longum in patients with SSc compared to healthy controls. In a bleomycin-induced SSc mouse model, oral administration of B. longum strain RAPO attenuated skin and lung fibrosis, accompanied by reduced infiltration of inflammatory monocytes/macrophages and downregulation of pro-inflammatory cytokines and chemoattractant Ccl2 genes in lymph nodes and fibrotic tissues. B. longum RAPO treatment restored fecal microbial diversity and augmented short-chain fatty acid (SCFA)-producing bacteria in the gut, leading to increased fecal butyrate levels and upregulated SCFA receptor Gpr41 in the mesenteric lymph node. In vitro, B. longum RAPO and its culture supernatant suppressed the expressions of pro-inflammatory cytokine genes in macrophages and inhibited myofibroblast differentiation in fibroblasts. These findings highlight the probiotic potential of B. longum RAPO in preventing tissue fibrosis by modulating macrophage activity and promoting the growth of SCFA-producing bacteria, underscoring the therapeutic potential of microbial modulation in SSc.

Keywords: Bacterial antibodies; Bifidobacterium longum; Fibrosis; Short-chain fatty acid; Systemic sclerosis.

Figure 1. Comparison of serum antimicrobial Ab profile between HC (n=50) and patients with SSc (n=76). (A) Alpha-diversity assessment of antimicrobial Ab abundances (Mann-Whitney test). (B) PCoA plot based on Bray-Curtis dissimilarity of antimicrobial Ab communities (PERMANOVA, p<0.001). Each symbol represents an individual. (C) Cladogram generated using LEfSe analysis. The phylogenetic tree shows taxa enriched in HC and SSc represented in green and red, respectively (pairwise Wilcoxon test, p<0.05, LDA score>3.0). (D) Heatmap illustrating differential antimicrobial Ab abundances between HC and patients with SSc (t- and z-tests, p<0.01, Z-ratio>1.5). Colors represent abundance levels, ranging from blue (low abundance) to red (high abundance). (E) CCA diagram visualizing the relationship between differential antimicrobial Abs (circles) and clinical phenotypes (arrows) in patients with SSc. (F) Comparative levels of significantly different antimicrobial Abs against Bifidobacterium species among HC and patients with SSc, stratified according to clinical phenotypes.

lc, limited cutaneous; Mi, Mild; Mo, Moderate; mRSS, Modified Rodnan skin score. One-way analysis of variance, Sidak post hoc test, ^**p<0.01 vs. HC.

Figure 2. Effects of Bifidobacterium strains on dermal and lung fibrosis in BLM-induced SSc mice. (A) Experimental scheme: SSc was induced in mice through subcutaneous injections of BLM to the dorsal skin, 5 times per week for 2 wk. Oral administration of Bifidobacterium strains commenced 2 wk before BLM injection and continued for the subsequent 4 wk. Skin and lung tissues were then obtained from mice in each experimental group to evaluate fibrosis. (B-D) Representative images of Masson’s trichrome stain illustrating skin and lung fibrosis (B), evaluation of skin fibrosis by dermal thickness (C), and assessment of lung fibrosis using the Ashcroft score and the percentages of collagen and alveolar area (D) in skin and lung tissues obtained from SSc mice treated with PBS (n=6), Bifidobacterium longum BORI (n=7), B. longum RAPO (n=6), or B. bifidum BGN4 (n=4). Arrow length indicates dermal thickness in skin tissues. ^*p<0.05, ^**p<0.01 vs. PBS. (E-G) Representative images of Masson’s trichrome stain (E) and evaluation of skin (F) and lung fibrosis (G) in skin and lung tissues obtained from normal (n=5) and SSc mice treated with B. longum RAPO at doses of 0 (n=13), 1 (n=9), 2 (n=14), or 5×10⁹ CFUs (n=7). ^*p<0.05, ^**p<0.01. Scale bars=100 µm.

Mean ± SD.

Figure 3. Antifibrotic effects of B. longum RAPO on dermal and lung fibrosis in BLM-induced SSc mice. SSc was induced in mice through subcutaneous injections of BLM to the dorsal skin for 2 wk. Oral administration of B. longum RAPO, at a dose of 2×10⁹ CFUs, commenced 2 wk before BLM injection and continued for 4 wk. Skin, lung, and fecal samples were then obtained from mice in each experimental group. (A-C) Representative images of Masson’s trichrome stain showing skin and lung fibrosis (A), accompanied by quantitative analysis of fibrosis in skin (B) and lung tissues (C) obtained from mice of normal (n=6), BLM (n=16), and BLM/RAPO (n=19) groups. Scale bars=100 µm. (D) Quantification of B. longum and its strain RAPO in fecal samples using digital PCR (n=5–6/group). (E) Representative images of immunofluorescence (green) and IHC (brown) staining for α-SMA in skin and lung tissues, respectively, and quantification of α-SMA expression (n=3–5 for normal, 5–8 for BLM, and 5–7 for BLM/RAPO group). Scale bars=50 µm. (F) Expression of α-SMA protein in skin and lung tissues (n=3/group).

Mean ± SD, ^*p<0.05, ^**p<0.01.

Figure 4. Anti-inflammatory effects of B. longum RAPO on dermal and lung fibrosis in BLM-induced SSc mice. SSc was induced in mice through subcutaneous injections of BLM to the dorsal skin for 2 wk. Oral administration of B. longum RAPO commenced 2 wk before BLM injection and continued for 4 wk. Skin, lung, and BAL fluid were then obtained from mice of normal (n=3–4), BLM (n=4–7), and BLM/RAPO (n=5–6) groups. (A) Immunofluorescent images and quantification showing expression of CD11b⁺CX3CR1⁺ macrophages in skin tissues stained for DAPI (blue), CD11b (green), and CX3CR1 (red). Scale bars=50 µm. (B) Differential cell counts of BAL cells stained with Diff-Quik. Scale bars=25 µm. (C) Flow cytometric analysis of macrophage populations in BAL cells. Total macrophages (MPs, CD45⁺CD64⁺) were assessed, and macrophage subsets were further divided into alveolar macrophages (CD11c⁺CD11b⁻) and exudate macrophages (exMac, CD11c⁺CD11b⁺). (D, E) Quantification of total protein (D) and pro-inflammatory cytokine concentrations in BAL fluid (E). (F) Expression of genes for profibrotic and pro-inflammatory cytokines and chemokine in the skin and lung tissues.

Mean ± SD, ^*p<0.05, ^**p<0.01.

Figure 5. Immunomodulatory impacts of B. longum RAPO on the MLNs and spleen in BLM-induced SSc mice. Mice were subjected to subcutaneous injections of BLM to the dorsal skin for 2 wk and treated with or without B. longum RAPO 2 wk before BLM injection. Cells from MLN and spleen were isolated from normal (n=3–4), BLM-treated (n=4–5), and BLM/RAPO-treated (n=4–6) mice. (A) Flow cytometric analysis characterized macrophage populations in MLN and spleen cells. The numbers and percentages of Ly6C⁺ inflammatory monocytes, including newly recruited (Ly6C⁺MHCII⁻) and maturing monocytes (Ly6C⁺MHCII⁺), were assessed among total macrophages (CD45⁺CD64⁺CD11b⁺CX3CR1⁺). (B) Expression levels of pro-inflammatory cytokines and chemokine genes in MLN and spleen cells. (C) Immune responses of MLN and spleen cells. Cytokine levels secreted upon stimulation with LPS were measured in CSs from MLN and spleen cells.

Mean ± SD, ^*p<0.05, ^**p<0.01.

Figure 6. Influences of B. longum RAPO on gut microbiota in BLM-induced SSc mice. Mice were subjected to subcutaneous injections of BLM to the dorsal skin for 2 wk and treated with or without B. longum RAPO 2 wk before BLM injection. Fecal samples were collected from normal (n=5), BLM-treated (n=5), and BLM/RAPO-treated (n=6) mice, and 16S rRNA gene sequencing was conducted. (A) Alpha-diversity analysis of gut microbiota. (B) Beta-diversity assessed using PCoA based on Weighted UniFrac distance matrix. (C) Relative abundances of gut microbiota at the phylum level. (D) Heatmap illustrating significantly differential genera between groups (p<0.05). Colors represent abundance levels, ranging from blue (low abundance) to red (high abundance). (E) Measurement of SCFA levels in fecal samples from normal, BLM-treated, and BLM/RAPO-treated mice (n=4/group). (F) Expression levels of GPCR genes in MLN cells isolated from normal (n=3), BLM-treated (n=5), and BLM/RAPO-treated (n=6) mice, as determined using quantitative PCR.

Mean ± SD, ^*p<0.05, ^**p<0.01.

Figure 7. Modulating ability of B. longum RAPO and its derived components on macrophages and fibroblasts activation in vitro. (A) Pro-inflammatory gene expression of RAW 264.7 macrophage cells stimulated with LPS in the absence or presence of HK-B. longum RAPO or its CS (n=3–4/group). (B, C) α-SMA protein expression (B) and profibrotic and pro-inflammatory gene expression (C) of primary NHDFs stimulated with hTGF-β1 in the absence or presence of B. longum RAPO CS. (D, E) α-SMA protein expression (D) and profibrotic and pro-inflammatory gene expression (E) of normal-LFs (LL24) and IPF-LFs (LL29) stimulated with hTGF-β1 in the absence or presence of B. longum RAPO CS (n=3/group).

Mean ± SEM, ^*p<0.05, ^**p<0.01.