Effect of oral feeding with Clostridium leptum on regulatory T-cell responses and allergic airway inflammation in mice

doi:10.1016/j.anai.2012.06.017

Annals of Allergy, Asthma & Immunology

Volume 109, Issue 3, September 2012, Pages 201-207

https://doi.org/10.1016/j.anai.2012.06.017 Get rights and content

Abstract

Background

Allergic lung inflammation is mediated by allergen-specific T responses, which are negatively regulated by regulatory T cells (Tregs). Previous studies have reported that inoculation of indigenous Clostridium species in the early lives of mice can induce Tregs that colonize the colon. However, whether inoculation of C leptum alone in adult mice could induce systemic Treg responses and inhibit allergic airway inflammation remains unclear.

Objective

To investigate the effect of oral administration of C leptum on systemic Treg responses and allergic airway inflammation in a mouse model of asthma.

Methods

Adult BABL/c mice were injected with ovalbumin to induce asthma and treated orally with C leptum or vehicle daily for 2 weeks. The numbers of Foxp3⁺CD4⁺CD25⁺ Tregs in both the spleen and mediastinal lymph nodes were examined by flow cytometry. After allergen challenge, the airway hyperresponsiveness of individual mice was measured, and the numbers of inflammatory infiltrates and the levels of cytokines in bronchoalveolar lavage fluids ere determined.

Results

Oral feeding with C leptum increased the percentage and total number of Tregs in the spleens and mediastinal lymph nodes at 14 days after inoculation and attenuated allergen-induced airway hyperresponsiveness and inflammation by inhibiting inflammatory cytokine production but enhancing interleukin 10 and transforming growth factor β1 production in the lungs.

Conclusion

Oral treatment with C leptum can attenuate induced allergic airway inflammation in adult mice.

Introduction

Asthma is an inflammatory disease caused by allergen-specific T cells, such as T_H2, T_H1, T_H17, and T_H9, but negatively regulated by regulatory T cells (Tregs) T_H2 cells can secrete interleukin (IL) 4, IL–5, and IL–13 and other cytokines that recruit and activate eosinophils and neutrophils and cause pulmonary inflammation, mucous hypersecretion, and airway hyperresponsiveness.¹ T_H17 with IL–17, T_H1 with interferon γ (IFN-γ), and T_H9 with IL–9 are involved in asthmatic pathogenesis.[2], [3], [4], [5], [6], [7], [8], [9] During the past several decades, the incidence of asthma has been increasing significantly in the world. Notably, Tregs can suppress T_H2, T_H1, T_H17, and T_H9 responses by secreting inhibitory cytokines, such as IL–10 and transforming growth factor β (TGF-β).¹⁰ Previous studies[11], [12] have reported that the number and function of Tregs is impaired or altered in allergic patients compared with that in healthy individuals. Therefore, development of therapeutic strategies to increase the numbers and function of Tregs may be highly promising for the intervention of allergic disease.¹³

Interestingly, previous studies have shown that modulation of the gastrointestinal microbiota composition and oral administration of certain organisms can modulate immune responses in the airway.[14], [15], [16], [17], [18] Treatment with heat-killed nonpathogenic Mycobacterium vaccae can induce Tregs that protect against allergic airway inflammation in vivo.¹⁹ Colonization of mice with human Bacteroides fragilis facilitates Treg cell differentiation and IL-10 production.²⁰ Other selective bacteria have also been shown to induce IL-10–producing Tregs by modulating dendritic cells in vitro.²¹ Clostridium species is one of the most prominent gram-positive and spore-forming bacteria, and increased colonization of Clostridium is associated with the protection against wheezing.²² Recent studies have found that inoculation of indigenous Clostridium species in the early lives of mice can induce Tregs that colonize the colon and subsequently migrate into the lung, liver, spleen, and other tissues, inhibiting toxicant-induced intestinal inflammation and allergen-specific IgE responses.[23], [24], [25] Clostridium leptum and Clostridium coccoides can promote anti-inflammatory immune responses by expanding and activating Tregs, in accord with up-regulating Foxp3 expression, a key transcription factor in programming the differentiation of Tregs.¹¹ C leptum is the highest population in the intestinal tract and accounts for a mean (SD) of approximately 16% (7%) of the total number of intestinal bacteria.²⁶ However, whether oral inoculation with C leptum alone in adult mice could induce systemic Treg response and modulate allergic airway inflammation has not been explored.

In this study, we used a well-known asthmatic mouse model to examine the effect of oral administration of C leptum on inducing Treg response and allergic inflammation and to determine the potential mechanisms by which this therapeutic strategy modulates airway inflammation. We found that oral administration of C leptum alone in adult mice induced vigorous Treg responses and inhibited airway hyperresponsiveness.

Section snippets

C leptum Preparation

The C leptum were purchased from Jilin Baoxin Biological Technology, Changchun, China. The strain was isolated from a human feces sample and stored at −80°C. A single colony of C leptum was grown in chopped meat broth for 24 hours and harvested by centrifugation, followed by washing with phosphate-buffered saline (PBS). The freshly prepared bacteria in PBS were used for oral feeding.

Animals

Female BALB/c mice at 6 to 8 weeks of age and 18 to 22 g were obtained from the Animal Research Center of Jilin

Oral feeding with C leptum and Treg responses in mice

To investigate whether oral treatment with C leptum alone could induce systemic Treg responses, adult female BALB/c mice were fed C leptum or vehicle PBS for 14 days and the numbers of splenic mononuclear cells, CD3⁺ T cells, and CD4⁺CD25⁺Foxp3⁺ Tregs were evaluated at successive time points. No significant difference was found in the numbers of splenic mononuclear cells and CD3⁺ T cells between the PBS and PBS-CL groups of mice at 1, 7, and 14 days after inoculation (data not shown). Although

Discussion

Allergic airway inflammation results from allergen-specific T-cell responses. T_H2, T_H9, and T_H17 cells secrete IL-4, IL-17A, IL-9, and other cytokines, which contribute to pulmonary inflammation and airway hyperresponsiveness. [4], [5], [6], [7], [8], [9] T_H1 cells secrete IFN-γ and TNF-α, which suppress T_H2 responses and cytokine secretion, and induce epithelial and smooth muscle cells apoptosis.[2], [3] Tregs are important for the maintenance of peripheral immune tolerance and can inhibit

Acknowledgment

We thank Professor Feng Fang, for her technical assistance.

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Clostridium leptum induces the generation of interleukin-10<sup>+</sup> regulatory B cells to alleviate airway inflammation in asthma
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Therefore, the development of new drugs for clinical use is urgently needed. In recent years, it has been found that Clostridium leptum (CL), a bacterial strain of the human gut microbiota, can significantly interfere with the function of immune cells and provide beneficial roles in the process of immune-related diseases like asthma (Li et al., 2012; Sghir et al., 2000). For example, Atarashi et al. have found that CL can reduce airway inflammation and airway hyperresponsiveness in HDM-induced mice(Atarashi et al., 2011).
Asthma is one of the most common chronic inflammatory diseases of the respiratory tract. Previous studies have shown that the reduction of regulatory B cells (Bregs) can increase inflammation of the body and promote the formation of chronic airway inflammation in asthma, but the detailed mechanisms have not been fully elucidated. The intestinal flora Clostridium leptum (CL) has been reported to modulate immune regulatory cells in the body, but the specific mechanisms are not clear. This study aimed to investigate the effects of CL on the differentiation of interleukin (IL)− 10⁺ Bregs and the regulation of the asthmatic inflammation-associated immune network.
The abundances of CL and the frequencies of blood Bregs from asthmatic patients and healthy controls were compared. The house dust mite (HDM)-induced asthma model was established in mice. The effects of CL exposure and B cell infusion on Breg differentiation, T cell cytokine production, and inflammatory cell infiltration in mouse lungs were examined. Bregs were cocultured with regulatory T cells (Tregs) and CD4⁺ non-Tregs to evaluate their roles on Foxp3 expression and T cell differentiation, respectively.
Compared with healthy controls, asthmatic patients had significantly reduced frequencies of blood Bregs and abundances of fecal CL, and these two parameters were positively correlated. In the asthma model, the frequencies of Bregs in lungs were significantly reduced; while the infusion of Bregs isolated from CL- supplemented mice significantly reduced airway inflammation and hyperresponsiveness. In addition, Bregs inhibited the differentiation of cocultured non-Tregs into multiple effector cells and enhanced Foxp3 expression in cocultured Tregs.
Bregs contribute to the alleviation of airway inflammation, which provides insight on implementing CL-based microbial induction of Bregs in asthma therapy.
Immunologic Effects of the Microbiota in Organ Transplantation
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This seems to be a result of antigen-stimulation of Tregs because dendritic cells cocultured with heat-inactivated Clostridia are sufficient to elicit this effect.61 These Tregs are capable of regulating immunopathologies in vivo as indicated by decreased eosinophil infiltration in mice fed with Clostridium leptum in a mouse model of airway allergy.62 The microbiota exerts a considerable portion of its influence on immune function via by-products of fiber fermentation.
The protective effect of Lactobacillus and Bifidobacterium as the gut microbiota members against chronic urticaria
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Chronic Urticaria is a common disorder which is defined by recurrent occurrence of wheals and sometimes angioedema. It has a notable influence on the patients' quality of life. Regulation of the immune system is one of the important roles of the gut microbiota. The effect of dysbiosis considering some members of gut microbiota in patients with chronic urticaria has been demonstrated in our previous study.
Comparing the frequency and bacterial load of Lactobacillus, Bifidobacterium, and Bacteroides between patients with chronic urticaria and healthy controls.
20 patients with chronic urticaria and 20 age and sex matched healthy individuals were included in the present study. Stool samples were analyzed for determining the frequency and bacterial load of Lactobacillus, Bifidobacterium, and Bacteroides genera.
There were no significant differences among the frequencies of detectable Lactobacillus, Bifidobacterium, or Bacteroides in stool samples of patients with chronic urticaria and healthy controls. The relative amounts of Lactobacillus and Bifidobacterium were significantly higher in fecal samples from controls compared to patients with chronic urticaria (P = 0.038 and 0.039, respectively).
It is the first study on the implication of Lactobacillus, Bifidobacterium, and Bacteroides genera as gut microbiota members in patients with chronic urticaria.
Lactobacillus johnsonii supplementation attenuates respiratory viral infection via metabolic reprogramming and immune cell modulation
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Regulation of respiratory mucosal immunity by microbial-derived metabolites has been a proposed mechanism that may provide airway protection. Here we examine the effect of oral Lactobacillus johnsonii supplementation on metabolic and immune response dynamics during respiratory syncytial virus (RSV) infection. L. johnsonii supplementation reduced airway T helper type 2 cytokines and dendritic cell (DC) function, increased regulatory T cells, and was associated with a reprogrammed circulating metabolic environment, including docosahexanoic acid (DHA) enrichment. RSV-infected bone marrow–derived DCs (BMDCs) from L. johnsonii–supplemented mice had altered cytokine secretion, reduced expression of co-stimulatory molecules, and modified CD4+ T-cell cytokines. This was replicated upon co-incubation of wild-type BMDCs with either plasma from L. johnsonii–supplemented mice or DHA. Finally, airway transfer of BMDCs from L. johnsonii–supplemented mice or with wild-type derived BMDCs pretreated with plasma from L. johnsonii–supplemented mice reduced airway pathological responses to infection in recipient animals. Thus L. johnsonii supplementation mediates airway mucosal protection via immunomodulatory metabolites and altered immune function.
Association of altered gut microbiota composition with chronic urticaria
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Some mechanisms can be suggested for the protective effects of these bacteria against CU, including induction of regulatory T (Treg) cells. This mechanism is supported by other studies that revealed that these bacteria could induce Treg cells.50–54 Treg cells can reduce inflammation through secretion of anti-inflammatory mediators,55 and an anti-inflammatory milieu is protective against CU.
An altered gut microbiota composition has recently been linked to some types of allergies.
To compare the relative amounts of Akkermansia muciniphila, Clostridium leptum, Faecalibacterium prausnitzii, and Enterobacteriaceae as members of gut microbiota among patients with chronic urticaria (CU) and healthy controls.
A total of 20 patients with CU and 20 healthy individuals matched by age and sex participated in the study. Fresh fecal samples were collected, and DNA extracted from stool samples was analyzed by real-time polymerase chain reaction for the qualitative and quantitative assays of the so-called bacteria.
The frequencies of A muciniphila, C leptum, and F prausnitzii in healthy controls' stool samples were significantly more than those of patients with CU (P < .001, P < .01, and P < .05, respectively), whereas the Enterobacteriaceae family was detected in all patients and healthy controls' stool samples. The relative amounts of A muciniphila in healthy control positive samples were significantly higher than those of samples from patients with CU (P < .001). Furthermore, there was a corresponding increase of relative amounts of C leptum and F prausnitzii in healthy control positive samples compared with those of patients with CU (P = .09 and P = .08, respectively). The mean of the relative amounts of Enterobacteriaceae family in the stool samples from patients with CU was more than that of healthy controls; however, the difference was nearly significant (P = .12).
The results reveal a change of frequency and relative amounts of A muciniphila, C leptum, and F prausnitzii in patients with CU compared with healthy controls. This is the first study, to our knowledge, to show the change of microbiota composition in patients with CU.
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Both asthma16 and obesity17–19 often begin in early childhood, when the gut microbiota is primarily developed. Recent studies in animal models and in human subjects have found that an altered or less diverse gut microbiota composition has been associated with asthma and allergic disease diseases (Table 1).20–26 Moreover, certain gut microbial strains have been shown to inhibit or attenuate immune responses associated with chronic inflammation in experimental models.
Asthma and atopy, classically associated with hyper-activation of the T helper 2 (Th2) arm of adaptive immunity, are among the most common chronic illnesses worldwide. Emerging evidence relates atopy and asthma to the composition and function of gut microbiota composition. Moreover, certain gut microbial strains have been shown to inhibit or attenuate immune responses associated with chronic inflammation in experimental models. Although still a relatively nascent field of research, evidence to date suggests that the gut microbiome may represent fertile targets for prevention or management of allergic asthma and other diseases in which adaptive immune dysfunction is a prominent feature. The oral probiotics/prebiotic represents a possible therapeutic for improving asthma and allergic disease. Especially, recent technological developments that permit identification of microbes and their products using culture-independent molecular detection techniques. In this review, we literaturely summarise the aggravation or improvement of metabolic diseases by role of gut microbiota, probiotics/prebiotic treatment.

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: Disclosures: Authors have nothing to disclose.

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Original articleMechanisms of allergic and immune diseasesEffect of oral feeding with Clostridium leptum on regulatory T-cell responses and allergic airway inflammation in mice

Abstract

Background

Objective

Methods

Results

Conclusion

Introduction

Section snippets

C leptum Preparation

Animals

Oral feeding with C leptum and Treg responses in mice

Discussion

Acknowledgment

Mucosal Immunol

Immunity

J Allergy Clin Immunol

Exp Toxicol Pathol

J Immunol Methods

J Allergy Clin Immunol

Curr Opin Pharmacol

J Allergy Clin Immunol

Am J Clin Nutr

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Original article
Mechanisms of allergic and immune diseases
Effect of oral feeding with Clostridium leptum on regulatory T-cell responses and allergic airway inflammation in mice

The levels of CD4⁺CD25⁺ regulatory T cells in paediatric patients with allergic rhinitis and bronchial asthma