Investigation of Clostridium butyricum on atopic dermatitis based on gut microbiota and TLR4/MyD88/ NF-κB signaling pathway

Abstract

Background

Probiotics, as common regulators of the gut microbiota, have been used in research to alleviate clinical symptoms of atopic dermatitis (AD).

Objective

Our research team has previously identified a potential relieving effect of Clostridium butyricum on the treatment of AD, but the specific mechanism of how Clostridium butyricum alleviates AD has not yet been confirmed.

Methods

In this study, we explored the relieving effect of Clostridium butyricum on AD through in vivo and in vitro experiments. AD mice induced by 2,4-dinitrofluorobenzene (DNFB) were orally administered with 1 × 10⁸ CFU of Clostridium butyricum for three consecutive weeks.

Results

Oral administration of Clostridium butyricum reduced ear swelling, alleviated back skin lesions, decreased mast cell and inflammatory cell infiltration, and regulated the levels of inflammation-related cytokines. Clostridium butyricum activated the intestinal immune system through the TLR4/MyD88/NF-κB signaling pathway, suppressed the expression of inflammatory factors IL-10 and IL-13, and protected the damaged intestinal mucosa.

Conclusion

Clostridium butyricum administration improved the diversity and abundance of the gut microbiota, enhanced the functionality of the immune system, and protected the epidermal barrier.

Keywords

atopic dermatitis intestinal flora TLR4 MyD88 NF-κB

1 Introduction

Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases, characterized by eczematous skin lesions including erythema, papules, and exudative lesions in specific areas.¹ Toll-like receptors (TLRs) signaling pathway is closely associated with the occurrence and development of AD. In acute exacerbations of AD, research has demonstrated an upregulation of TLR2 and TLR4 within keratinocytes. This is accompanied by a disruption in the Th1/Th2 immune response balance, as well as modifications in the signaling pathways associated with dendritic cell (DC) surface molecules.² These alterations are mediated through TLRs, which trigger downstream signaling cascades leading to the expression of pro-inflammatory factors. Such processes are critical in modulating the skin's immune barrier function. Studies have shown that TLR4 not only plays a role in the regulation of infection, but also may participate in the development of AD as an immune response regulator, affecting related gene expression, cytokine release and immune cell polarization.^3–6 These findings highlighted the crucial role of TLR4 in the development of AD.

Increasing evidence suggests that the composition of gut microbiota, development, and maturation of the immune system are highly correlated with the occurrence of allergic diseases.^7–10 Probiotics may serve as an effective alternative method for immune regulation and restoration of intestinal dysfunction in AD patients. Long-term consumption of probiotics can promote the synthesis of amino acids, vitamins, and other nutrients in the host, as well as increase the content of short-chain fatty acids in the intestines. Particularly, short-chain fatty acids containing acetate, propionate, and butyrate lead to a lower pH in the intestinal environment, thereby inhibiting the growth of pathogens.¹¹ Clostridium butyricum, also known as butyric acid-producing bacteria, is a Gram-positive anaerobic bacterium. In the human intestine, Clostridium butyricum can consume undigested dietary fibers and produce short-chain fatty acids, especially butyrate and acetate salts. The short-chain fatty acids produced by Clostridium butyricum have various important effects on host health, including regulating intestinal immune homeostasis, improving gastrointestinal barrier function, and alleviating inflammation. Therefore, bacteria like Clostridium butyricum that produce butyrate are strong candidates for disease treatment probiotics.¹² In a study on probiotics in mice with normal gut health, administration of Clostridium butyricum significantly increased the thickness of the colonic mucosa,¹³ and significantly reduced epithelial damage in the colon tissue of mice with antibiotic-associated diarrhea induced by clindamycin. There is literature suggesting that Clostridium butyricum administered orally can significantly reduce intestinal inflammation and improve gut microbiota.¹⁴ It has also been reported that Clostridium butyricum can affect the aggregation of intraepithelial lymphocytes (IELs) in the colon, increase the number of Treg cells, thereby inhibiting the expression of inflammatory factors (tumor necrosis factor-alpha, IL-12, I IFN-γ, IL-1β, and IL-6), and upregulating the expression of the inhibitory factor (IL-10).^15,16

We applied animal experiments for the first time to further elucidate the alleviating effect of Clostridium butyricum on AD, as well as explore the role of Clostridium butyricum in AD based on gut microbiota and the TLR4/MyD88/NF-κB signaling pathway.

2 Materials and methods

2.1 Animals

Healthy female SPF-grade BALB/c mice, aged 6–8 weeks, were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All animal experimental procedures were approved and licensed by the Ethics Committee of Tianjin Medical University General Hospital (IRB2022-DW-54). The establishment of the mouse model of atopic dermatitis was performed as previously described^17–19: the fur on the dorsal area of the mice, approximately 2 cm × 3 cm in size, was shaved. On the first day of the experiment, the sensitization was performed by applying a 1.0% DNFB solution evenly on the dorsal and ear skin of the mice. On the 14th day, the mice were challenged with a 0.5% DNFB solution on the dorsal and ear skin twice a week. The modeling was completed after 35 days. At the end of the modeling, typical skin lesions such as erythema, exudation, vesicles, and crusts appeared on the dorsal skin of the mice.

A total of 36 female BALB/c mice were randomly divided into 4 groups, namely (1) the blank control group (Control), (2) the AD model group (Model), (3) the Clostridium butyricum group (C.b), (4) the positive drug dexamethasone group (Dex.). Except for the blank control group, all other groups were subjected to modeling. During the modeling period, the mice in the blank control group and AD model group were administered physiological saline by oral gavage. The mice in the dexamethasone group were administered a 60 µg/mL dexamethasone solution by oral gavage. The mice in the Clostridium butyricum group were administered live bacteria at a dose of 5 × 10⁸ CFU/mL of Clostridium butyricum liquid by oral gavage. Each mouse was orally gavaged once a day with a volume of 200 μL for a total of 3 weeks, until the end of the experiment.

2.2 Ad mouse ear swelling degree detection

DNFB was used to sensitize the right ear of the mice. At the end of the experiment, an electronic vernier caliper was used to measure the skin thickness at the same location on the right ear of all mice, and the values were recorded. The measurer took an average after three readings.

2.3 Assessment of damaged skin on the back of ad mice

The skin in the hair loss area on the back of each group of mice was observed and scored based on the severity and improvement of skin damage.²⁰ This scoring was based on four symptoms: dryness/scaling, bleeding/redness, ulceration/epidermal detachment, and edema. Each symptom was graded as none, mild, moderate, or severe, represented by scores of 0, 1, 2, or 3, respectively. The final score for the severity of skin damage in mice was the sum of the scores for the four symptoms, ranging from 0 to 12. The scoring process was performed by two independent observers who were blinded to the grouping of the mice. The observers did not need to be members of the experimental team, but they needed to be familiar with the scoring criteria. After modeling, the two observers measured the scores once a week and the average of their recorded scores was taken as the final result until the end of the experiment.

2.4 Collection of dorsal skin tissue samples

Experimental mice were euthanized by cervical dislocation. The mice were placed with their abdomen facing down and their limbs fixed on a pre-prepared foam board. Using ophthalmic scissors and forceps, a 1.0 cm × 1.0 cm piece of dorsal skin tissue was removed from the back of the mice. Half of the tissue was stored at −80°C for ELISA, WB, and qPCR tests, while the other half was placed in a 4% polyformaldehyde solution for pathological and immunohistochemical tests. The dorsal skin tissue stored in the −80°C freezer was retrieved, and the weight of the skin tissue was accurately measured. It was then mixed with PBS in a 1:9 ratio and homogenized using a tissue grinder until a homogeneous solution was obtained. The solution was then placed in a low-temperature high-speed centrifuge at 4°C and 3000 rpm for 30 min. After centrifugation, the supernatant was collected for subsequent testing.

2.5 Colon tissue sample collection

After euthanizing the experimental mice using cervical dislocation method, the mice were placed with their abdomen facing up and their limbs fixed on a foam board. The abdominal skin was disinfected with alcohol, and then the chest and abdomen were opened using ophthalmic scissors to fully expose the abdominal area. The colon was then separated, and the cecum was located. The entire colon tissue from the anus to the cecum was collected, and the length of the colon was measured. Physiological saline was used to rinse the intestinal tissue using a syringe. Half of the intestinal tissue was placed in a low-temperature storage tube and frozen at −80°C for future use. The other half was fixed in a 4% paraformaldehyde solution for intestinal pathology and immunohistochemistry testing.

2.6 Measurement of total IgE

Blood samples were collected from BALB/c experimental mice using the retro-orbital bleeding method. The collected blood was placed in EP tubes and centrifuged at low temperature and high speed: 4°C, 3000 ×g for 10 min. After centrifugation, the upper serum layer was aspirated using a pipette and transferred into another EP tube, which was then stored at −80°C. The final separated serum of each group of mice will be used for the detection of serum immune factors using the ELISA method. According to the manufacturer's instructions, the serum total IgE concentration was measured using the IgE ELISA kit (Beijing Dingguo Changsheng Biotechnology, China).

2.7 Analysis of intestinal Microbiota

Frozen mouse fecal samples were taken out from the −80°C freezer. Approximately 0.2–0.5 g of the samples were added to a centrifuge tube containing extraction lysis buffer and subjected to grinding treatment. For the ground samples, genomic DNA was extracted using a DNA extraction kit. After extraction, the concentration and purity of the sample DNA were quantified using a NanoDrop instrument. Additionally, agarose gel electrophoresis was performed to analyze the integrity of the sample DNA and detect any contamination. Based on the concentration of each DNA sample, an appropriate amount of DNA sample was taken and diluted to 1 ng/μl using sterile water. Subsequently, PCR amplification, identification, purification, and quantification of PCR products were carried out. The NEB Next^® Ultra™ DNA Library Prep Kit was used to construct the libraries. The constructed libraries were evaluated using the Qubit and Agilent Bioanalyzer 2100 systems. After passing library quality control, the libraries were sequenced on the Illumina NovaSeq 6000 sequencer, generating 250 bp paired-end reads. The resulting raw data was subjected to quality control analysis, including paired-end sequence assembly, filtering, and removal of chimeras, to obtain valid data. Based on the obtained valid data, operational taxonomic units (OTUs) were clustered, species were annotated, and abundance analysis was performed. Furthermore, the above analysis can also be used to explore the differences between samples and select differential microbial communities.

2.8 Statistical analysis

GraphPad Prism 8 software (La Jolla, CA, USA) was used to perform statistical analysis on the experimental data. The experimental data were expressed as mean ± standard deviation. For the comparison of means between multiple groups, one-way ANOVA was used, followed by Tukey's post-hoc test to identify specific group differences. For comparisons between two groups, an unpaired t-test was employed. A p-value < 0.05 was considered statistically significant. And a value of p < 0.05 was considered statistically significant.

3 Results

3.1 Probiotic treatment improves Ad-like symptoms induced by 2,4-dinitrofluorobenzene (DNFB)

To investigate the effects of different probiotics on AD, we established a mouse model of AD-like skin lesions by treating mice with DNFB. As shown in Figure 1A and B, compared to the control group, the AD model mice exhibited significantly thickened skin lesions, erythema, obvious desquamation, accompanied by inflammatory exudates, and some areas formed scabs. In addition, histopathological results showed abundant infiltration of inflammatory cells in the dermis of the AD model group, severe intercellular and intracellular edema at the site of skin lesions, severe epidermal keratinization, and extensive cortical loss (Figure 1C). Compared with the model group, mice in the B.f group showed a slight decrease in ear thickness, while the ear thickness of the C.b, B.I, and Dex groups decreased more significantly, with the C.b group showing the most significant difference compared to the model group (P < 0.001) (Figure 1D). Mice treated with C.b, B.I, and Dex by gavage showed significant improvement in thickened skin lesions, erythema, desquamation, inflammatory exudates, and the appearance of blisters and scabs in some areas, with C.b showing more significant effects (Figure 1E). The back skin of mice in the B.I group showed improvement in erythema, scabs, and ulcer-like injuries compared to the AD model group; the back skin of mice in the C.b and Dex groups appeared smooth and almost no erythema, scabs, or ulcer-like injuries were observed (Figure 1F). Therefore, C.b exhibited the most prominent effect among several probiotics for specific dermatitis.

Figure 1.

Probiotic treatment improves AD-like symptoms induced by 2,4-dinitrofluorobenzene (DNFB). Comparison images of the ears (A), back skin (B), and HE-stained histopathology (C) between the control group mice and the AD model group mice. (HE-stained histopathology images are at 100× magnification). (D) Effects of different probiotics on the degree of ear swelling in mice with atopic dermatitis. (E) Effects of different probiotics on skin lesions in the ear area of mice with atopic dermatitis. (F) Effects of different probiotics on skin lesions in the back area of mice with atopic dermatitis. Control: blank control group; Model: AD model group; B.f: Bacteroides fragilis group; C.b: Clostridium butyricum group; B.I: Bifidobacterium infantis group; Dex: Dexamethasone group. Statistical analysis was performed by Mann-Whitney U test comparing with the model group mice (n = 9). Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

3.2 Clostridium butyricum treatment relieves DNFB-induced Ad-like symptoms

To investigate the alleviating effect of Clostridium butyricum on AD in mice, we examined the changes in ear thickness. In weeks 3 to 5 of the experiment, the ear skin thickness of mice in the Clostridium butyricum group and dexamethasone group showed a significant decrease compared to the model group, with a significant difference (P < 0.01). At week 5 of the experiment, the average ear thickness of mice in the Clostridium butyricum group was lower than that of the dexamethasone group (Figure 2A). In addition, we observed and scored the skin lesions on the back of the mice to determine the degree of improvement. The results showed that the mice in the Clostridium butyricum group and dexamethasone group had only a small amount of crust and ulcer-like damage on the back skin, which was significantly improved compared to the skin damage in the AD model group. Furthermore, the symptom score of back skin lesions in the Clostridium butyricum group was significantly lower than that in the AD model group (Figure 2B, C). HE results showed that in the Clostridium butyricum group, there were few infiltrated neutrophils and lymphocytes in the dermis, with no obvious intracellular edema, and the stratum corneum was basically normal, while the spinous layer was significantly reduced compared to the thickening in the AD model group (Figure 2D). Compared with the AD model, after Clostridium butyricum treatment, there were slightly enlarged spindle-shaped mast cells in the mouse skin tissue, with relatively intact cell membranes (Figure 2E). In addition, after Clostridium butyricum treatment, the serum IgE levels, and IL-4 in the back skin of mice were significantly decreased (Figure 2F).

Figure 2.

Clostridium butyricum treatment alleviates DNFB-induced AD-like symptoms. (A) Trend chart of ear thickness in each group of mice (n = 9); (B) Back skin images of mice in each group after 4 weeks of treatment (n = 9); (C) Trend chart of back skin lesion score in each group of mice (n = 9); (D) Histopathological section of mouse back skin stained with HE (100×); (E) Histopathological section of mouse back skin stained with toluidine blue (400×); (F) Serum IgE levels, IL-4 levels in back skin tissue of mice.

3.3 Clostridium butyricum regulates intestinal immunity and protects damaged intestinal mucosa through the TLR4/MyD88/Nf-κB signaling pathway

The potential mechanisms of probiotics in disease treatment mainly involve regulating the intestinal microbiota, improving intestinal mucosal barrier function, and modulating intestinal immune function, thereby affecting the body's immune system.^21,22 We explored the effects of Clostridium butyricum on the intestinal immune system of AD mice. RT-qPCR and WB analysis were used to investigate the impact of Clostridium butyricum on the TLR4/MyD88/NF-κB inflammatory signaling pathway in the colonic tissues of different groups of mice. The results showed that administration of Clostridium butyricum acidophilus down-regulated the expression of TLR4, MyD88, and NF-κB P65 at the transcriptional and translational levels, thereby exerting an effect on the intestinal immunity of mice (Figure 3A, B). The down-regulation of the TLR4/MyD88/NF-κB pathway contributes to the reduction of inflammatory responses, thereby reducing the impact of dermatitis. Our results showed that the levels of IL-10 and IL-13 in the colonic tissues of mice in the Clostridium butyricum group were significantly lower than those in the AD model group (Figure 3C). The intestinal barrier is an epithelial tissue structure that supports intestinal nutrient absorption and waste excretion while preventing potential leakage of harmful luminal substances. Immunohistochemical staining of ZO-1 and Occludin in intestinal tissues indicated that AD modeling may lead to damage and disruption of the intestinal barrier epithelial tissue in mice, while Clostridium butyricum effectively enhances the protection of the intestinal barrier (Figure 3D, E).

Figure 3.

Clostridium butyricum regulates intestinal immunity and protects damaged intestinal mucosa through the TLR4/MyD88/NF-κB signaling pathway. (A) mRNA expression of TLR4, MyD88, and NF-κB P65 in mouse colon tissues. (B) Protein levels of TLR4, MyD88, and NF-κB P65 in mouse colon tissues. (C) Levels of IL-10 and IL-13, two inflammation-related factors, in mouse colon tissues. (D) Immunohistochemical staining of ZO-1 protein in mouse colon tissues (400×). (E) Immunohistochemical staining of Occludin protein in mouse colon tissues (400×).

3.4 Clostridium butyricum alleviates inflammation in the back skin of mice

The back skin tissue, as the site where we induced DNFB administration, is the ultimate destination for evaluating the effectiveness of our model. Clostridium butyricum can downregulate the TLR4/MyD88/NF-κB P65 pathway, but whether the alleviating effect of Clostridium butyricum on the dermatitis site is mediated through this pathway is the key for us to explore the effectiveness of the pathway. Next, we focused on the back skin tissue and investigated the effects of Clostridium butyricum on mRNA, protein, and ELISA levels in these tissues, as well as whether the mechanism of action is consistent with that in the intestinal tissues. Our results showed that administration of Clostridium butyricum downregulated the expression of TLR4, MyD88, and NF-κB P65 in mouse back skin tissues at the transcriptional and translational levels (Figure 4A, B). Furthermore, Clostridium butyricum administration significantly reduced the levels of IL-10 and IL-13, two inflammation-related factors, in mouse back skin tissues (Figure 4C).

Figure 4.

Clostridium butyricum alleviates inflammation in the back skin of mice through the TLR4/MyD88/NF-κB signaling pathway. (A) mRNA expression of TLR4, MyD88, and NF-κB P65 in mouse back skin tissues. (B) Protein levels of TLR4, MyD88, and NF-κB P65 in mouse back skin tissues. (C) Levels of IL-10 and IL-13, two inflammation-related factors, in mouse back skin tissues.

3.5 Clostridium butyricum significantly improves the diversity and abundance of gut microbiota in mice

Metabolomic sequencing analysis of mouse feces showed differences at the OTU level among different treatment groups (Figure 5A). There were 600 shared OTUs between the control group, AD model group, Clostridium butyricum group, and the positive drug dexamethasone group, referred to as core microbiota. Furthermore, our sequencing depth has covered almost all species in the samples, with an adequate number of samples to reflect the majority of microbial information (Figure 5B). Clostridium butyricum administration can to some extent enhance the microbial diversity and richness of intestinal microbiota in atopic dermatitis mice (Table 1). Additionally, the species composition of metabolites significantly differentiated between the Clostridium butyricum group and the control group and AD group (Figure 5C). At the phylum level, Clostridium butyricum administration significantly increased the abundance of Firmicutes, Bacteroidetes, and Actinobacteria (Figure 5D). Further taxonomic analysis showed that uncultured genera were dominant in the control group and AD model group, with higher abundance in the control group than the AD model group. In the Clostridium butyricum group, genera such as Parabacteroides, Mucispirillum, and Bacteroides were dominant, with higher abundance compared to other groups. The genus Helicobacter was dominant in the dexamethasone group, with significantly higher abundance than the other groups (Figure 5E). Additionally, linear discriminant analysis showed enrichment of Bacteroidetes and Muribaculaceae in the control group, enrichment of Eubacterium_xylanophilum_group and uncultured Bacteroidales bacterium in the AD model group, enrichment of Aerococcus and Erysipelotrichaceae_UGG_003 in the Clostridium butyricum group, and enrichment of Helicobacter and campylobacteria in the dexamethasone group (Figure 5F). KEGG functional prediction analysis showed that the Clostridium butyricum group was mainly enriched in membrane transport, carbohydrate metabolism, amino acid metabolism, replication and repair, translation, energy metabolism, nucleotide metabolism, vitamin metabolism, as well as cellular processes and signal transduction (Figure 5G). Further LEfSe analysis indicated that the functional items of the Clostridium butyricum group were mainly responsible for regulating the endocrine system and immune system processes (Figure 5H).

Figure 5.

Clostridium butyricum significantly improves the diversity and abundance of gut microbiota in mice. (A) Venn diagram showing the distribution of OTUs in each sample. (B) Dilution curve of the samples. (C) Principal coordinates analysis plot. (D) Bar chart showing the relative abundance of species at the phylum level in each sample. (E) Bar chart showing the relative abundance of species at the genus level in each sample. (F) Analysis of microbial abundance among samples. (G) KEGG enrichment analysis among samples. (H) Abundance analysis of functional items among samples.

Table 1.

Alpha diversity index of each sample.

Sample ID	Shannon	Simpson	ACE	Coverage	Chao1	Observed_species	PD_whole_tree
C3	5.037964	0.912139	621.5084	0.998015	596.6282	502	78.74042
C4	6.164708	0.972353	763.1633	0.99754	746.1299	601	113.6642
C5	5.676597	0.95014	755.3797	0.997909	717.6333	640	109.3505
C6	4.677867	0.874385	664.5151	0.9986	631.5618	554	67.07261
C7	5.549293	0.953045	719.5822	0.997955	704.6145	584	82.12461
C8	5.959949	0.958636	745.6816	0.997711	719.2059	637	52.89918
Average	5.511063	0.936783	711.6384	0.997955	685.9623	586.3333	83.97524
CB1	5.357288	0.931696	790.6199	0.997828	751.8529	624	112.7244
CB2	6.168563	0.967599	795.3159	0.997536	778.619	642	109.8207
CB3	5.427817	0.954253	647.5292	0.998432	619.2614	534	99.95986
CB4	5.880467	0.950349	812.9502	0.997923	770.5224	701	83.20996
CB5	5.419975	0.947735	722.4509	0.9983	688.875	607	106.6304
CB6	5.314067	0.943517	600.2225	0.998349	577.6667	505	107.9438
Average	5.594696	0.949192	728.1814	0.998061	697.7996	602.1667	103.3815
D1	5.738814	0.95111	688.9153	0.99835	648.7292	568	80.71893
D2	6.187359	0.964447	843.8056	0.997962	814.0263	716	140.9635
D3	4.254746	0.83657	568.881	0.99856	557.016	436	98.99227
Sample ID	Shannon	Simpson	ACE	Coverage	Chao1	Observed_species	PD_whole_tree
D7	5.152831	0.943068	565.9624	0.998337	528.05	413	85.37038
D8	5.318497	0.940157	674.7552	0.998482	682.0938	561	72.79661
D9	4.824601	0.875439	672.3346	0.998464	654.7692	557	93.69881
Average	5.246141	0.918465	669.109	0.998359	647.4474	541.8333	95.42341
M2	6.154906	0.968232	784.8651	0.997562	761.0095	656	77.539
M4	5.224257	0.907348	755.418	0.998075	752.679	611	113.7948
M6	5.603846	0.934226	722.5845	0.99829	701.8947	619	72.23412
M7	5.184174	0.94087	567.3665	0.998602	548.9048	452	98.4335
M8	5.676164	0.945345	763.2477	0.998122	728.9231	622	87.54955
M9	4.628194	0.892432	677.2817	0.998143	659.625	538	46.56784
Average	5.411924	0.931409	711.7939	0.998132	692.1727	583	82.68647

4. Discussion

AD is a chronic disorder, carrying significant burden for children and adults and impacting their life quality. Since the underlying mechanism of AD has not been fully understood, further investigating its pathogenesis is of great importance. Previous studies have highlighted the vital role of TLR4-related signaling in AD. Belderbos ME et al. demonstrated that the production of interleukin-10 mediated by toll-like receptor 4 in neonates is associated with secondary AD.³ Hüls A et al. conducted a survey study on the symptoms of AD and found that GSTP1, TNF, TLR2, and TLR4 genes are closely related to the occurrence and development of AD.⁴ Yoon J et al. found that after scratching, IL-23 is released in the skin of AD mice, leading to polarization of skin dendritic cells to drive the immune response of IL-22 in the skin.⁵ Lin L's team investigated the immune regulatory function of TLR4 in the AD model induced by 2,4-dinitrochlorobenzene (DNCB), and the results showed that TLR4-deficient mice exhibited more severe AD symptoms, higher levels of inflammatory cytokine expression, and stronger Th2 response compared to control mice after DNCB application. In addition, compared to control mice, TLR4-deficient mice showed significantly increased expression of thymic stromal lymphopoietin (TSLP) in the skin, which is an important potential factor for allergic inflammation.⁶ In consistence with these researches, we found that Clostridium butyricum alleviates AD by regulating the TLR4/MyD88/NF-κB signaling pathway in the intestinal immune system, thereby modulating the immune balance and relieving atopic dermatitis. Additionally, administration of Bifidobacterium infantis could reduce skin swelling, inflammatory cell infiltration, and mast cell infiltration, while decreasing the levels of inflammatory factors in the serum, skin, and intestinal tissues of AD mice. Furthermore, it increased the diversity of gut microbiota and enhances the protective effect of intestinal mucosa, thus alleviating atopic dermatitis.

The intestine is not only an important organ for digestion and absorption of nutrients, but also plays a crucial role in the interaction between the body and the external environment, and it is a vital site in the immune system of organisms. Increasing evidence suggests that maintaining the integrity of the intestinal morphology and function can prevent various systemic diseases.^23–25 With the development of sequencing technology, many studies have revealed the important role of the gut microbiota in allergic diseases.^26–28 Probiotics, as common regulators of the gut microbiota, can improve the host's gut microbiota, promote digestion and absorption of nutrients, and regulate gut microbiota disorders caused by antibiotics or other factors, thus helping to restore the beneficial active microorganisms in the body.^29,30 Studies on animals and humans have shown that probiotics of different strains play important roles in various disease models. Bifidobacterium is a human gut symbiont that produces butyrate, and it has been safely used as a probiotic for decades. It has been reported that the combination of Bifidobacterium Sx-01 and Lactobacillus C-1–3 can significantly improve intestinal health and reduce serum lipid levels in mice, and these benefits may be related to the regulation of gut microbiota.³¹ Zhang HQ et al. demonstrated that Bifidobacterium treats experimental colitis in rats by reducing the levels of interleukin-23 (IL-23) and tumor necrosis factor-α (TNF-α) in the serum.³² In addition, increasing evidence suggests that Bifidobacterium plays a protective role in intestinal injury, irritable bowel syndrome, inflammatory bowel disease, metabolic diseases, and colitis by regulating the composition of the host's gut microbiota and increasing certain beneficial bacterial groups, such as Lactobacillus and Bifidobacterium, thereby modulating the immune system.^33–35 Bacteroidetes is a commonly occurring symbiotic bacteria, accounting for about 30% of the human gut microbiota and playing an essential role in maintaining the host's health. According to reports, Bacteroidetes can enhance intestinal mucosal barrier function, degrade high molecular weight organic compounds, and regulate innate immune responses.^36–38 Bacteroides fragilis is a member of the Bacteroidetes phylum, Bacteroidaceae family, and Bacteroides genus, commonly found in the lower gastrointestinal tract of mammals.³⁹ As research advances, scholars have discovered that Bacteroides fragilis, which colonizes the intestine, has established a mutually beneficial relationship with the host through long-term evolution, showing promising prospects for the treatment of obesity, glucose metabolism, and immunodeficiency diseases.^38,40–43 Research by Nagalingam et al. has shown that a decrease in the abundance of Bacteroides fragilis is associated with the occurrence of inflammatory bowel disease, indicating a link between the occurrence of inflammatory bowel disease and an imbalance in the gut microbiota.⁴⁴ Sha S and their team used a sequence-based method to investigate and characterize the composition of the major fecal microbiota in patients with inflammatory bowel disease and healthy controls. The results showed that the abundance of Bacteroides fragilis was lower in patients with ulcerative colitis and Crohn's disease compared to healthy controls. This suggests that Bacteroides fragilis is crucial for maintaining human health.⁴⁵ In probiotic research, Bifidobacterium is a dominant probiotic that exists in the human body and plays a crucial role in human health and various diseases. Schwiertz et al. evaluated the differences in gut microbiota and short-chain fatty acid concentrations in the feces of lean and obese subjects, indicating that some Bifidobacterium and their metabolites in the gut microbiota have immunomodulatory effects.¹⁴ Rabe et al. studied the colonization patterns of the gut using 16S rRNA next-generation sequencing and culture-based techniques, and found that Bifidobacterium plays an important role in inducing infant immune maturation.¹⁵ Furthermore, clinical trials have confirmed that probiotics containing Bifidobacterium can significantly alleviate antibiotic-associated diarrhea,¹⁶ regulate systemic inflammation and immune dysfunction,⁴⁶ and reduce the levels of systemic pro-inflammatory biomarkers in gastrointestinal and non-gastrointestinal diseases.⁴⁷ The Bifidobacterium strains covered in these studies mainly include Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium breve, and Bifidobacterium animalis. In this study, significant improvements were observed in the groups treated with Bacteroides fragilis, Clostridium butyricum, Bifidobacterium infantis, and dexamethasone compared to the AD model group in mice, with the most significant effect observed with Clostridium butyricum, and the statistical difference between the improvement of Clostridium butyricum and AD model mice was the most significant. Towards the end of the gavage administration, a large number of deaths occurred among the mice in the Bacteroides fragilis group. We speculate that this may be due to the dual effects of DNFB dermatitis modeling and Bifidobacterium fragilis, which leads to more aggressive behavior in the mice, thus causing mutual death. Therefore, Clostridium butyricum and Bifidobacterium infantis may be more suitable probiotics for our selection. Compared to the control group, the mice in the Clostridium butyricum group, Bifidobacterium infantis group, and dexamethasone group all showed significant improvements in ear and back skin lesions, with Clostridium butyricum showing greater improvement than Bifidobacterium infantis, and its significant effect is comparable to that of the positive drug.

Clostridium butyricum is a Gram-positive anaerobic bacterium. Butyrate is one of the main fermentation end products produced by Clostridium butyricum through the butyrate kinase (buk) pathway. Short-chain fatty acids produced by Clostridium butyricum have multiple important roles in host health, including regulating intestinal immune homeostasis, improving gastrointestinal barrier function, and reducing inflammation.^48,49 Therefore, microorganisms like Clostridium butyricum that produce butyrate are strong candidates for disease treatment probiotics. In a study on probiotics in healthy mice, administration of Clostridium butyricum significantly increased colonic mucosal thickness and significantly reduced epithelial damage in the colon tissue of mice with antibiotic-associated diarrhea induced by clindamycin. The impact of Clostridium butyricum on mucosal health can be explained by its ability to produce butyrate. In this study, administration of Clostridium butyricum significantly reduced ear thickening caused by atopic dermatitis, improved the recovery of skin in the back dermatitis area, optimized the mouse back skin lesion score, reduced skin swelling, hyperplasia, and inflammatory cell infiltration, reduced mast cell infiltration, and decreased the levels of serum IgE, as well as inflammatory cytokines such as IL-4 in skin tissue.

The immune system of the gastrointestinal tract can be divided into the innate immune system and the adaptive immune system. The gastrointestinal innate immune system mainly includes the intestinal mucosal barrier, immune cells associated with innate immunity, and the cytokines secreted by these cells.^50,51 TLRs are expressed on various cells in the intestines and mainly mediate interactions between microorganisms and cells. They play an important role in the recognition of pathogenic microorganisms and the transmission of inflammatory signals in both the innate and adaptive immune systems in the gastrointestinal tract.^52,53 Studies have shown that in patients with AD during acute flare-ups, there is an excessive expression of TLR2 and TLR4 in the keratinocytes of their skin lesions. The imbalance of Th1/Th2 immune response in AD patients is associated with changes in the signaling pathway of surface molecules of dendritic cells (DCs), which are specifically activated by Toll-like receptors, leading to the activation of downstream signaling and induction of expression of inflammatory factors. This highlights their important role in the skin immune barrier.² TLR4/MyD88/NF-κB is a critical signaling pathway for inflammation, closely involved in cell differentiation, proliferation, apoptosis, and pro-inflammatory responses.^54–57 In recent years, the role of TLRs/MyD88/NF-κB signaling pathway in various types of inflammation has been increasingly studied. Research has reported that butyrate-producing bacteria can regulate Th17 cells in the treatment of colitis through the TLRs/MyD88/NF-κB signaling pathway, with TLR4 downstream signaling pathway playing an important role.⁵⁸ In this study, we have demonstrated the effect of butyrate-producing bacteria on atopic dermatitis from the initial site of action (colonic tissue) to the final site of action (dorsal skin tissue) through the inflammatory pathway TLR4/MyD88/NF-κB. Administration of butyrate-producing bacteria can downregulate the TLR4/MyD88/NF-κB signaling pathway associated with colonic tissue and inflammatory responses, reduce the levels of inflammatory cytokines, and enhance the protective function of the intestinal barrier. Moreover, the immunomodulatory effects of butyrate-producing bacteria in the gastrointestinal tract can truly respond to the site of dermatitis, downregulate the TLR4/MyD88/NF-κB signaling pathway and IL10, IL13 cytokines in dermatitis tissue, and provide relief for atopic dermatitis.

Research has shown that the occurrence of allergic diseases such as AD is closely related to the diversity and composition of intestinal microorganisms.^7–10 Liang et al. and Wang et al. have also found that the addition of a certain amount of Bifidobacterium longum in feed promotes the abundance and diversity of intestinal microbial flora in weaned piglets and elderly laying hens.^59,60 In addition, relevant clinical studies have found that AD patients generally have microbial imbalances and low diversity, indirectly proving that Bifidobacterium longum can improve the abundance of intestinal flora in AD mice. In this study, after gavage with Bifidobacterium longum, the diversity and abundance of intestinal microorganisms in AD mice were significantly increased. Multiple studies have shown that probiotic intervention can change the structure of intestinal flora by regulating the ratio of Firmicutes/Bacteroidetes⁶¹ and Proteobacteria/Bacteroidetes.⁶² Our research results also support this view. In this experiment, the sequencing results of the intestinal flora of each group of mice showed that at the phylum level, the dominant intestinal flora were Firmicutes, Bacteroidetes, and Actinobacteria. Compared with the control group, the abundance of Firmicutes and Bacteroidetes in the Bifidobacterium longum group increased, while the abundance of Actinobacteria (Proteobacteria) was lower than that in the dexamethasone group. This indicates that Bifidobacterium longum has the ability to regulate the abundance of intestinal flora in mice and protect the intestinal health of animals with atopic dermatitis.

The genus Fusobacterium is an anaerobic bacterium that primarily exists in the microbiota of the human gastrointestinal tract.⁶³ Research has found that Fusobacterium may have a protective effect on certain diseases in terms of pathogenicity, including liver fibrosis,^64,65 cancer immunotherapy,^66,67 and cardiovascular diseases.^68,69 It is considered by researchers to be a potential genus for regulating inflammation and emotions. Metabolites secreted by different members of the Bacteroides genus can help maintain the stability of the immune system and are key participants in immune regulation.⁷⁰ The genus Ruminococcus belongs to the Firmicutes phylum and is commonly found in the human intestines. It participates in the metabolism of various carbohydrates. One study found that Ruminococcus is a marker of the anti-histamine effect in chronic spontaneous urticaria.⁷¹ These pieces of evidence support our results. At the genus level, we found that the abundance of Fusobacterium, Bacteroides, and Ruminococcus in the Bacteroides group of Clostridium butyricum was higher than in the control group. This suggests that Clostridium butyricum can increase the levels of Fusobacterium, Bacteroides, and Ruminococcus, enhance immune function, and promote the epidermal recovery of mice with atopic dermatitis. It has been reported that probiotics and symbiotic microbial communities ferment carbohydrates in the gastrointestinal tract, resulting in the production of metabolic products such as acetic acid, formic acid, succinic acid, and lactic acid, which acidify the intestinal environment and inhibit the growth of pathogenic bacteria.¹¹ Therefore, administering Clostridium butyricum orally to AD model mice can improve carbohydrate metabolism, maintain the intestinal environment, and inhibit the infiltration of bacterial pathogens. Previous studies have shown that the intake of probiotics can affect the host's digestive tract (motility, secretion) and feeding behavior, ensuring optimal supply and delivery of required nutrients.^72,73 More importantly, probiotics have immunoregulatory properties. They can enhance non-specific cellular immune responses, characterized by the activation of macrophages, natural killer cells, and cytotoxic T lymphocytes, and improve the intestinal mucosal immune system by releasing various cytokines in a strain-specific and dose-dependent manner.^74,75 This study indicates that the intake of Clostridium butyricum can enhance the abilities of the immune system and endocrine system, thereby reducing the levels of pathogenic bacteria and exerting a protective effect on mice with atopic dermatitis. These results are completely consistent with our previous experimental conclusions, and the addition of Clostridium butyricum precisely alleviates and treats atopic dermatitis by regulating intestinal immune function, providing further supplementation and support for our research conclusions.

While this study provides compelling evidence for the therapeutic potential of Clostridium butyricum in alleviating AD symptoms and modulating immune responses, several limitations should be acknowledged. Firstly, the use of a mouse model, although valuable for initial investigations, may not fully capture the complexity and variability of human AD, including differences in immune response, skin barrier function, and genetic factors. Secondly, the study primarily focuses on short-term effects, and the long-term safety and efficacy of C. butyricum treatment in humans remain to be determined. Additionally, the mechanisms by which C. butyricum exerts its beneficial effects, while partially elucidated, require further investigation to fully understand the intricate interactions with the host's immune system and gut microbiota. Lastly, the study does not explore the potential side effects or interactions with other treatments, which are important considerations for clinical application. Further clinical trials and mechanistic studies are necessary to validate these findings and to establish the optimal dosing, duration, and safety profile of C. butyricum in the treatment of AD.

5. Conclusion

The results of this study demonstrate that Clostridium butyricum, as a probiotic, exhibits significant therapeutic effects in a DNFB-induced AD mouse model. C. butyricum not only alleviates skin inflammatory symptoms such as erythema, desquamation, and thickening, but also shows superior efficacy in reducing serum IgE levels and IL-4 expression compared to dexamethasone. Furthermore, by downregulating the TLR4/MyD88/NF-κB signaling pathway, C. butyricum improves intestinal immune function and protects the damaged intestinal mucosa, while also modulating the gut microbiota, increasing microbial diversity and abundance. These findings suggest that C. butyricum may alleviate AD symptoms through the modulation of host immune responses and the gut microbiome, providing a novel approach for the clinical treatment of AD.

Footnotes

Ethics approval and consent to participate

This study was approved by the ethics committee of Tianjin Medical University General Hospital.

Consent for publications

The author read and approved the final manuscript for publication.

Informed consent

Signed written informed consents were obtained from the patients and/or guardians.

Authors’ contributions

Xiaojing Yang and Huiping Wang designed the study and performed the experiments, Qian Zhao and Xing Wang collected the data, Yiming Zhang, Jingyue Ma and Yuanjun Liu analyzed the data, Xiaojing Yang and Huiping Wang prepared the manuscript. All authors read and approved the final manuscript.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was funded by Tianjin Key Medical Discipline (Specialty) Construction Project (No:TJYXZDXK-057B).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Availability of data and material

The data that support the findings of this study are available from the corresponding author upon reasonable request

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