Le rôle des archées dans l'inflammation et leur impact sur la santé humaine

Les archées sont depuis peu reconnues comme étant des microorganismes ubiquitaires retrouvées dans le système digestif de l’humain et de plusieurs animaux. Les humains peuvent donc être exposés aux archées par leur propre microflore intestinale, mais aussi par le contact avec des fèces animales, dont les composantes peuvent être aérosolisées et inhalées. L’impact de ces archées sur la santé respiratoire et intestinale des humains n’est pas clairement établi. La composition exacte des bioaérosols doit être définie afin de bien comprendre ce à quoi sont exposés les humains. Par ailleurs, l’impact de Methanosphaera stadtmanae (MSS) et Methanobrevibacter smithii (MBS), deux espèces d’archées retrouvées dans le tractus intestinal, est inconnu dans un contexte de maladies inflammatoires de l’intestin. L’étude de la biodiversité des bioaérosols des fermes laitières a démontré la présence de grandes concentrations et de plusieurs espèces de bactéries et d’archées dans l’air de ces environnements. Les mécanismes inflammatoires pulmonaires à la base de la réponse immune causée par les archaea ont été étudiés grâce à un modèle murin d’exposition chronique des voies respiratoires. MSS a démontré un potentiel immunogénique plus fort que MBS, en induisant davantage d’altérations histopathologiques, de cellules inflammatoires et de cellules dendritiques myéloïdes activées que MBS dans les poumons de souris. Le potentiel inflammatoire de MSS et MBS a aussi été confirmé avec un modèle de cellules humaines. Finalement, afin d’étudier le rôle des archées dans l’inflammation intestinale, la présence de MBS et de MSS a été évaluée dans les selles de patients atteints de maladies inflammatoires de l’intestin et de sujets sains. Une prévalence accrue de l’archée inflammatoire MSS a été retrouvée dans les selles des patients. Des anticorps spécifiques à MSS ont aussi été retrouvés en plus grande concentration chez ce groupe de sujets. Ces résultats démontrent que les archées sont présentes à la fois dans les bioaérosols et dans les intestins humains et qu’elles peuvent avoir des impacts sur l’inflammation respiratoire et intestinale. Nous ne commençons qu'à explorer la présence des archées dans l’environnement humain et la réponse à ces agents méconnus. Leur rôle en tant qu'agents protecteurs, pro-inflammatoires ou tolérés mérite d'être approfondi.Archaea have recently been recognized as ubiquitous microorganisms found in the digestive tract of human and several animal species. Humans can thus be exposed to archaea through several routes such as their own intestinal microflora, but also via exposure to animal manure which components can be aerosolized and inhaled. Archaeal impact on respiratory and intestinal human health is not known. Bioaerosol’s exact composition must be defined in order to better understand what humans are exposed to. Moreover, the role of Methanosphaera stadtmanae (MSS) and Methanobrevibacter smithii (MBS), two archaeal species found in human gut, on the health of the intestinal tract and potentially in inflammatory bowel diseases is unknown. Studies on dairy barns’ bioaerosol’s biodiversity revealed the presence of high airborne concentrations and various species of bacteria and archaea. Pulmonary inflammatory mechanisms of immune response to archaea were studied using a chronic airway exposure mouse model. MSS showed a higher immunogenic potential than MBS, with more severe hitopathological alterations and higher numbers of inflammatory cells and activated myeloid dendritic cells in exposed mice lungs than MBS. The inflammatory potential of MSS and MBS was also confirmed with a human cell model. Finally, to study the role of archaea in bowel inflammation, the presence of MBS and MSS was evaluated in stool samples from inflammatory bowel diseases’ patients and control subjects. A higher prevalence of the inflammatory archaea MSS was detected in stool samples from patients. MSS-specific antibodies were also found in higher concentration in this group. These results show that archaea are present in bioaerosols and human gut and that they can have an impact on respiratory and intestinal inflammation. We are just beginning to explore the presence of archaea in human environment and our response to these unheralded agents. Their role as protective, proinflammatory or tolerated agents awaits further studied

Characterization of bioaerosols from dairy barns : reconstructing the puzzle of occupational respiratory diseases by using molecular approaches

To understand the etiology of exposure-related diseases and to establish standards for reducing the risks associated with working in contaminated environments, the exact nature of the bioaerosol components must be defined. Molecular biology tools were used to evaluate airborne bacterial and, for the first time, archaeal content of dairy barns. Three air samplers were tested in each of the 13 barns sampled. Up to 106 archaeal and 108 bacterial 16S rRNA genes per m3 of air were detected. Archaeal methanogens, mainly Methanobrevibacter species, were represented. Saccharopolyspora rectivirgula, the causative agent of farmer’s lung, was quantified to up to 107 16S rRNA genes per m3 of air. In addition, a wide variety of bacterial agents were present in our air samples within the high airborne bioaerosol concentration range. Despite recommendations regarding hay preservation and baling conditions, farmers still develop an S. rectivirgula-specific humoral immune response, suggesting intense and continuous exposure. Our results demonstrate the complexity of bioaerosol components in dairy barns which could play a role in occupational respiratory diseas

Microbial contents of vacuum cleaner bag dust and emitted bioaerosols and their implications for human exposure indoors

Vacuum cleaners can release large concentrations of particles, both in their exhaust air and from resuspension of settled dust. However, the size, variability, and microbial diversity of these emissions are unknown, despite evidence to suggest they may contribute to allergic responses and infection transmission indoors. This study aimed to evaluate bioaerosol emission from various vacuum cleaners. We sampled the air in an experimental flow tunnel where vacuum cleaners were run, and their airborne emissions were sampled with closed-face cassettes. Dust samples were also collected from the dust bag. Total bacteria, total archaea, Penicillium/Aspergillus, and total Clostridium cluster 1 were quantified with specific quantitative PCR protocols, and emission rates were calculated. Clostridium botulinum and antibiotic resistance genes were detected in each sample using endpoint PCR. Bacterial diversity was also analyzed using denaturing gradient gel electrophoresis (DGGE), image analysis, and band sequencing. We demonstrated that emission of bacteria and molds (Penicillium/Aspergillus) can reach values as high as 1E5 cell equivalents/min and that those emissions are not related to each other. The bag dust bacterial and mold content was also consistent across the vacuums we assessed, reaching up to 1E7 bacterial or mold cell equivalents/g. Antibiotic resistance genes were detected in several samples. No archaea or C. botulinum was detected in any air samples. Diversity analyses showed that most bacteria are from human sources, in keeping with other recent results. These results highlight the potential capability of vacuum cleaners to disseminate appreciable quantities of molds and human-associated bacteria indoors and their role as a source of exposure to bioaerosols

Immunogenic properties of archaeal species found in bioaerosols.

The etiology of bioaerosol-related pulmonary diseases remains poorly understood. Recently, archaea emerged as prominent airborne components of agricultural environments, but the consequences of airway exposure to archaea remain unknown. Since subcomponents of archaea can be immunogenic, we used a murine model to study the pulmonary immune responses to two archaeal species found in agricultural facilities: Methanobrevibacter smithii (MBS) and Methanosphaera stadtmanae (MSS). Mice were administered intranasally with 6.25, 25 or 100 µg of MBS or MSS, once daily, 3 days a week, for 3 weeks. MSS induced more severe histopathological alterations than MBS with perivascular accumulation of granulocytes, pronounced thickening of the alveolar septa, alveolar macrophages accumulation and increased perivascular mononucleated cell accumulation. Analyses of bronchoalveolar lavage fluids revealed up to 3 times greater leukocyte accumulation with MSS compared to MBS. Instillation of 100 µg of MBS or MSS caused predominant accumulation of monocyte/macrophages (4.5×10(5) and 4.8×10(5) cells/ml respectively) followed by CD4(+) T cells (1.38×10(5) and 1.94×10(5) cells/ml respectively), B cells (0.73×10(5) and 1.28×10(5) cells/ml respectively), and CD8(+) T cells (0.20×10(5) and 0.31×10(5) cells/ml respectively) in the airways. Both archaeal species induced similar titers of antigen-specific IgGs in plasma. MSS but not MBS caused an accumulation of eosinophils and neutrophils in the lungs, which surprisingly, correlated inversely with the size of the inoculum. Stronger immunogenicity of MSS was confirmed by a 3 fold higher accumulation of myeloid dendritic cells in the airways, compared to MBS. Thus, the dose and species of archaea determine the magnitude and nature of the pulmonary immune response. This is the first report of an immunomodulatory role of archaeal species found in bioaerosols

Increased Prevalence of <i>Methanosphaera stadtmanae</i> in Inflammatory Bowel Diseases

<div><p>Background</p><p>The gut microbiota is associated with the modulation of mucosal immunity and the etiology of inflammatory bowel diseases (IBD). Previous studies focused on the impact of bacterial species on IBD but seldom suspected archaea, which can be a major constituent of intestinal microbiota, to be implicated in the diseases. Recent evidence supports that two main archaeal species found in the digestive system of humans, <i>Methanobrevibacter smithii</i> (MBS) and <i>Methanosphaera stadtmanae</i> (MSS) can have differential immunogenic properties in lungs of mice; with MSS but not MBS being a strong inducer of the inflammatory response. We thus aimed at documenting the immunogenic potential of MBS and MSS in humans and to explore their association with IBD.</p><p>Methods</p><p>To validate the immunogenicity of MBS and MSS in humans, peripheral blood mononuclear cells from healthy subjects were stimulated with these two microorganisms and the production of inflammatory cytokine TNF was measured by ELISA. To verify MBS and MSS prevalence in IBD, stool samples from 29 healthy control subjects and 29 patients suffering from IBD were collected for DNA extraction. Plasma was also collected from these subjects to measure antigen-specific IgGs by ELISA. Quantitative PCR was used for bacteria, methanogens, MBS and MSS quantification.</p><p>Results</p><p>Mononuclear cells stimulated with MSS produced higher concentrations of TNF (39.5 ng/ml) compared to MBS stimulation (9.1 ng/ml). Bacterial concentrations and frequency of MBS-containing stools were similar in both groups. However, the number of stool samples positive for the inflammatory archaea MSS was higher in patients than in controls (47% vs 20%). Importantly, only IBD patients developed a significant anti-MSS IgG response.</p><p>Conclusion</p><p>The prevalence of MSS is increased in IBD patients and is associated with an antigen-specific IgG response.</p></div

Metabolic Adaptation of Airway Smooth Muscle Cells to an SPHK2 Substrate Precedes Cytostasis

Thickening of the airway smooth muscle is central to bronchial hyperreactivity. We have shown that the sphingosine analog (R)-2-amino-4-(4-heptyloxyphenyl)-2-methylbutanol (AAL-R) can reverse preestablished airway hyperreactivity in a chronic asthma model. Because sphingosine analogs can be metabolized by SPHK2 (sphingosine kinase 2), we investigated whether this enzyme was required for AAL-R to perturb mechanisms sustaining airway smooth muscle cell proliferation. We found that AAL-R pretreatment reduced the capacity of live airway smooth muscle cells to use oxygen for oxidative phosphorylation and increased lactate dehydrogenase activity. We also determined that SPHK2 was upregulated in airway smooth muscle cells bearing the proliferation marker Ki67 relative to their Ki67-negative counterpart. Comparing different stromal cell subsets of the lung, we found that high SPHK2 concentrations were associated with the ability of AAL-R to inhibit metabolic activity assessed by conversion of the tetrazolium dye MTT. Knockdown or pharmacological inhibition of SPHK2 reversed the effect of AAL-R on MTT conversion, indicating the essential role for this kinase in the metabolic perturbations induced by sphingosine analogs. Our results support the hypothesis that increased SPHK2 levels in proliferating airway smooth muscle cells could be exploited to counteract airway smooth muscle thickening with synthetic substrates

Dominance of CD4⁺ T lymphocytes and CD19⁺ B lymphocytes in BALF of mice instilled with archaeal species.

<p>The numbers of CD4⁺ T cells, CD8⁺ T cells and CD19⁺ B cells were analyzed in BALF of mice exposed for 3 weeks to 6.25, 25 and 100 µg of A) MBS or B) MSS. Compared to MBS, MSS induced strong CD4⁺ helper T cell and CD19⁺ B cell responses in the airways, which plateaued from the lowest dose instilled. Results are expressed as median ± interquartile intervals. Letters a and b represent statistical differences between treatment regimens within a same cell type. Six mice per group were analyzed. p<0.05.</p

Quantification of leukocyte subsets in the BALF of mice instilled with increasing doses of two archaeal species.

<p>A) Total immune cell numbers in BALF of mice instilled with 6.25, 25 or 100 µg of MBS or MSS for 3 weeks. B) Leukocyte subset numbers in BALF of mice exposed to 6.25, 25 and 100 µg of MBS for 3 weeks. C) Leukocyte subset numbers in BALF of mice exposed to 6.25, 25 and 100 µg of MSS for 3 weeks. Results expressed as average ± SEM. For both archaeal species, BALF cell subtypes were mainly composed of macrophages and lymphocytes. MSS also induced a significant accumulation of eosinophils and of neutrophils. Letters a and b represent statistical differences between treatment regimens within a same cell type. Results from two pooled experiments obtained with similar results are presented and 14 to 22 mice per group were analyzed. p<0.05.</p

Histopathological alterations of mice lungs exposed to MBS or MSS.

<p>Results are expressed as median scores for each group, graded on a scale from 0 (no alteration) to 5 (severe alterations). Letters represent statistical differences between each dose of MBS and MSS within a same criteria. Six mice per group were analyzed. p<0.05.</p