Role of germ-free animal models in understanding interactions of gut microbiota to host and environmental health: A special reference to zebrafish

Abstract Background Recent accumulating evidence suggests that amino acids have crucial roles in the maintenance of intestinal homeostasis. In inflammatory bowel disease (IBD), amino acid metabolism is changed in both host and the gut microbiota. Among amino acids, L-serine plays a central role in several metabolic processes that are essential for the growth and survival of both mammalian and bacterial cells. However, the role of L-serine in intestinal homeostasis and IBD remains incompletely understood. In this study, we investigated the effect of dietary L-serine on intestinal inflammation in a murine model of colitis. Methods Specific pathogen-free (SPF) mice were fed either a control diet (amino acid-based diet) or an L-serine-deficient diet (SDD). Colitis was induced by the treatment of dextran sodium sulfate (DSS). The gut microbiome was analyzed by 16S rRNA sequencing. We also evaluate the effect of dietary L-serine in germ-free mice and gnotobiotic mice that were colonized by a consortium of non-mucolytic bacterial strains or the consortium plus mucolytic bacterial strains. Results We found that the SDD exacerbated experimental colitis in SPF mice. However, the severity of colitis in SDD-fed mice was comparable to control diet-fed mice in germ-free condition, suggesting that the gut microbiota is required for exacerbation of colitis caused by the restriction of dietary L-serine. The gut microbiome analysis revealed that dietary L-serine restriction fosters the blooms of a mucus-degrading bacterium Akkermansia muciniphila and adherent-invasive Escherichia coli in the inflamed gut. Consistent with the expansion of mucolytic bacteria, SDD-fed mice showed a loss of the intestinal mucus layer. Dysfunction of the mucus barrier resulted in increased intestinal permeability, thereby leading to bacterial translocation to the intestinal mucosa, which subsequently increased the severity of colitis. The increased intestinal permeability and subsequent bacterial translocation were observed in SDD-fed gnotobiotic mice that colonized by mucolytic bacteria. In contrast, dietary L-serine restriction did not alter intestinal barrier integrity in gnotobiotic mice that colonized only by non-mucolytic bacteria. Conclusion Our results suggest that dietary L-serine regulates the integrity of the intestinal mucus barrier during inflammation by limiting the expansion of mucus degrading bacteria.

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A9 DORSAL ROOT GANGLIA NEURONAL RESPONSES AND SUBSTANCE P PRODUCTION ARE HIGHER IN MALE MICE

Journal of the Canadian Association of Gastroenterology ◽

10.1093/jcag/gwab002.008 ◽

2021 ◽

Vol 4 (Supplement_1) ◽

pp. 10-11

Author(s):

J Pujo ◽

G De Palma ◽

J Lu ◽

S M Collins ◽

P Bercik

Keyword(s):

Abdominal Pain ◽

Substance P ◽

Gut Microbiota ◽

Sensory Neurons ◽

Dorsal Root ◽

Neuronal Response ◽

Neuronal Responses ◽

Visceral Sensitivity ◽

Germ Free

Abstract Background Abdominal pain is a common complaint in patients with chronic gastrointestinal disorders. Accumulating evidence suggests that gut microbiota is an important determinant of gut function, including visceral sensitivity. Germ-free (GF) mice have been shown to display visceral hypersensitivity, which normalizes after colonization. Sex also appears to play a key role in visceral sensitivity, as women report more abdominal pain than men. Thus, both gut bacteria and sex are important in the regulation of gut nociception, but the underlying mechanisms remain poorly understood. Aims To investigate the role of gut microbiota and sex in abdominal pain. Methods We used primary cultures of sensory neurons from dorsal root ganglia (DRG) of female and male conventionally raised (SPF) or germ-free (GF) mice (7–18 weeks old). To study the visceral afferent activity in vitro, calcium mobilization in DRG sensory neurons was measured by inverted fluorescence microscope using a fluorescent calcium probe Fluo-4 (1mM). Two parameters were considered i) the percentage of responding neurons ii) the intensity of the neuronal response. First, DRG sensory neurons were stimulated by a TRPV1 agonist capsaicin (12.5nM, 125nM and 1.25µM) or by a mixture of G-protein coupled receptors agonist (GPCR: bradykinin, histamine and serotonin; 1µM, 10µM and 100µM). We next measured the neuronal production of substance P and calcitonin gene-related peptide (CGRP), two neuropeptides associated with nociception, in response to capsaicin (1.25µM) or GPCR agonists (100µM) by ELISA and EIA, respectively. Results The percentage of neurons responding to capsaicin and GPCR agonists was similar in male and female SPF and GF mice. However, the intensity of the neuronal response was higher in SPF male compared to SPF female in response to capsaicin (125nM: p=0.0336; 1.25µM: p=0.033) but not to GPCR agonists. Neuronal activation was similar in GF and SPF mice of both sexes after administration of capsaicin or GPCR agonists. Furthermore, substance P and CGRP production by sensory neurons induced by capsaicin or GPCR agonists was similar in SPF and GF mice, regardless of sex. However, while the response to capsaicin was similar, the GPCR agonists-induced production of substance P was higher in SPF male mice compared to SPF females (p=0.003). The GPCR agonists-induced production of CGRP was similar in SPF male and female mice. Conclusions Our data suggest that at the level of DRG neurons, the absence of gut microbiota does not predispose to visceral hypersensitivity. The intensity of DRG neuronal responses to capsaicin and the GPCR agonists-induced production of substance P are higher in male compared to female mice, in contrast to previously published studies in various models of acute and chronic pain. Further studies are thus needed to investigate the role of sex in visceral sensitivity. Funding Agencies CIHR

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Lupus Gut Microbiota Transplants Cause Autoimmunity and Inflammation

10.1101/2021.08.15.456375 ◽

2021 ◽

Author(s):

Yiyangzi Ma ◽

Ruru Guo ◽

Yiduo Sun ◽

Xin Li ◽

Lun He ◽

...

Keyword(s):

Gut Microbiota ◽

Lupus Erythematosus ◽

Fecal Microbiota Transplantation ◽

Fecal Microbiota ◽

Healthy Controls ◽

Germ Free ◽

Autoimmune Antibodies ◽

Expression Of Genes ◽

Rdna Sequencing

Background: The etiology of systemic lupus erythematosus (SLE) is multifactorial. Recently, growing evidence suggests that the microbiota plays a role in SLE, yet whether gut microbiota participates in the development of SLE remains largely unknown. To investigate this issue, we carried out 16s rDNA sequencing analyses in a cohort of 18 female un-treated active SLE patients and 7 female healthy controls, and performed fecal microbiota transplantation from patients and healthy controls to germ-free mice. Results: Compared to the healthy controls, we found no significant different microbial diversity but some significantly different species in SLE patients including Turicibacter genus and other 5 species. Fecal transfer from SLE patients to germ free (GF) C57BL/6 mice caused GF mice to develop a series of lupus-like phenotyptic features, which including an increased serum autoimmune antibodies, and imbalanced cytokines, altered distribution of immune cells in mucosal and peripheral immune response, and upregulated expression of genes related to SLE in recipient mice that received SLE fecal microbiota transplantation (FMT). Moreover, the metabolism of histidine was significantly altered in GF mice treated with SLE patient feces, as compared to those which received healthy fecal transplants. Conclusions: Overall, our results describe a causal role of aberrant gut microbiota in contributing to the pathogenesis of SLE. The interplay of gut microbial and histidine metabolism may be one of the mechanisms intertwined with autoimmune activation in SLE.

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Impact of Nutrition on Pulmonary Arterial Hypertension

Nutrients ◽

10.3390/nu12010169 ◽

2020 ◽

Vol 12 (1) ◽

pp. 169

Author(s):

María Callejo ◽

Joan Albert Barberá ◽

Juan Duarte ◽

Francisco Perez-Vizcaino

Keyword(s):

Pulmonary Arterial Hypertension ◽

Animal Models ◽

Gut Microbiota ◽

Arterial Hypertension ◽

Case Reports ◽

Host Immune System ◽

Dietary Polyphenols ◽

Pulmonary Arterial ◽

Near Future

Pulmonary arterial hypertension (PAH) is characterized by sustained vasoconstriction, vascular remodeling, inflammation, and in situ thrombosis. Although there have been important advances in the knowledge of the pathophysiology of PAH, it remains a debilitating, limiting, and rapidly progressive disease. Vitamin D and iron deficiency are worldwide health problems of pandemic proportions. Notably, these nutritional alterations are largely more prevalent in PAH patients than in the general population and there are several pieces of evidence suggesting that they may trigger or aggravate disease progression. There are also several case reports associating scurvy, due to severe vitamin C deficiency, with PAH. Flavonoids such as quercetin, isoflavonoids such as genistein, and other dietary polyphenols including resveratrol slow the progression of the disease in animal models of PAH. Finally, the role of the gut microbiota and its interplay with the diet, host immune system, and energy metabolism is emerging in multiple cardiovascular diseases. The alteration of the gut microbiota has also been reported in animal models of PAH. It is thus possible that in the near future interventions targeting the nutritional status and the gut dysbiosis will improve the outcome of these patients.

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Putative Pathobionts in HLA-B27-Associated Spondyloarthropathy

Frontiers in Immunology ◽

10.3389/fimmu.2020.586494 ◽

2021 ◽

Vol 11 ◽

Author(s):

Tejpal Gill ◽

James T. Rosenbaum

Keyword(s):

Animal Models ◽

Microbial Communities ◽

Underlying Disease ◽

Disease Development ◽

Hla B27 ◽

Disease Pathogenesis ◽

Fecal Microbiota Transplant ◽

Germ Free ◽

Associated Diseases

Spondyloarthritis (SpA) is a group of immune mediated inflammatory diseases with a strong association to the major histocompatibility (MHC) class I molecule, HLA-B27. Although the association between HLA-B27 and AS has been known for almost 50 years, the mechanisms underlying disease pathogenesis are elusive. Over the years, three hypotheses have been proposed to explain HLA-B27 and disease association: 1) HLA B27 presents arthritogenic peptides and thus creates a pathological immune response; 2) HLA-B27 misfolding causes endoplasmic reticulum (ER) stress which activates the unfolded protein response (UPR); 3) HLA-B27 dimerizes on the cell surface and acts as a target for natural killer (NK) cells. None of these hypotheses explains SpA pathogenesis completely. Evidence supports the hypothesis that HLA-B27-related diseases have a microbial pathogenesis. In animal models of various SpAs, a germ-free environment abrogates disease development and colonizing these animals with gut commensal microbes can restore disease manifestations. The depth of microbial influence on SpA development has been realized due to our ability to characterize microbial communities in the gut using next-generation sequencing approaches. In this review, we will discuss various putative pathobionts in the pathogenesis of HLA-B27-associated diseases. We pursue whether a single pathobiont or a disruption of microbial community and function is associated with HLA-B27-related diseases. Furthermore, rather than a specific pathobiont, metabolic functions of various disease-associated microbes might be key. While the use of germ-free models of SpA have facilitated understanding the role of microbes in disease development, future studies with animal models that mimic diverse microbial communities instead of mono-colonization are indispensable. We discuss the causal mechanisms underlying disease pathogenesis including the role of these pathobionts on mucin degradation, mucosal adherence, and gut epithelial barrier disruption and inflammation. Finally, we review the various uses of microbes as therapeutic modalities including pre/probiotics, diet, microbial metabolites and fecal microbiota transplant. Unravelling these complex host-microbe interactions will lead to the development of new targets/therapies for alleviation of SpA and other HLA-B27 associated diseases.

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A122 THE INFLUENCE OF SEX AND THE GUT MICROBIOTA ON VISCERAL PAIN SENSITIVITY IN MICE

Journal of the Canadian Association of Gastroenterology ◽

10.1093/jcag/gwz047.121 ◽

2020 ◽

Vol 3 (Supplement_1) ◽

pp. 142-143

Author(s):

J Pujo ◽

G De Palma ◽

J Lu ◽

S M Collins ◽

P Bercik

Keyword(s):

Abdominal Pain ◽

Gut Microbiota ◽

Sensory Neurons ◽

Neuronal Response ◽

Calcium Mobilization ◽

Visceral Sensitivity ◽

Drg Neurons ◽

E Coli ◽

Germ Free

Abstract Background Abdominal pain is a common complaint in patients with chronic gastrointestinal disorders. Its treatment is of limited efficacy as the pathophysiology is largely unknown. Accumulating evidence suggests that gut microbiota is an important determinant of gut function, including visceral sensitivity. Germ-free (GF) mice have been shown to have altered pain signaling, which normalizes after colonization. Sex also appears to play a key role in visceral sensitivity, as abdominal pain is diagnosed predominantly in female patients. Thus, both gut bacteria and sex are important in the regulation of gut nociception, but the underlying mechanisms remain poorly understood. Aims To investigate the role of gut microbiota and sex in abdominal pain. Methods We used primary cultures of sensory neurons from dorsal root ganglia (DRG) of female and male conventional mice (SPF) or germ-free (GF) mice, age 7–18 weeks. To study the visceral afferent activity in vitro, calcium mobilization in DRG sensory neurons was measured by inverted fluorescence microscope using a fluorescent calcium probe Fluo-4 (1mM). Two parameters were considered: i) percentage of responding neurons and ii) intensity of neuronal response. First, DRG sensory neurons were stimulated by a TRPV1 agonist capsaicin (12.5nM, 125nM and 1.25µM) or by an agonist mix of G-protein coupled receptors (GPCR: bradykinin, histamine and serotonin; 1µM, 10µM and 100µM). We next cultured Escherichia coli JM83 (E. coli) and Enterobacter aerogenes (E. aer) overnight in LB and LDMIIG medium, respectively. Bacterial supernatant of 1010 CFU/mL was diluted to 1% in Krebs Ringer solution to treat DRG neurons from GF/SPF mice and calcium mobilization was measured. Results The percentage of neurons responding to capsaicin and GPCR agonist was similar in SPF male and SPF female. In contrast, the intensity of the neuronal response was higher in SPF male compared to SPF female mice in response to capsaicin (125nM p<0.035 and 1.25µM p<0.038) but not in response to GPCR. The same trend was observed in GF mice. Neuronal activation induced by capsaicin or GPCR agonist was similar in SPF and GF mice. While bacterial supernatant from E. coli did not affect the activity of sensory neurons, the bacterial supernatant from E. aer induced changes in calcium mobilization in DRG neurons. Conclusions Our data suggest that at the level of DRG neurons from healthy mice, female sex and the absence of gut microbiota do not predispose to visceral hypersensitivity. In fact, the intensity of neuronal responses to capsaicin appear to be higher in DRGs from male mice. Furthermore, we show that metabolites from certain bacteria can activate sensory neurons. Thus, further studies are needed to investigate the role of gut microbiota and sex in visceral sensitivity Funding Agencies CIHR

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Managing the Microbiome: How the Gut Influences Development and Disease

Nutrients ◽

10.3390/nu13010074 ◽

2020 ◽

Vol 13 (1) ◽

pp. 74

Author(s):

Noah Weinstein ◽

Brandon Garten ◽

Jonathan Vainer ◽

Dulce Minaya ◽

Krzysztof Czaja

Keyword(s):

Clinical Applications ◽

Great Promise ◽

Germ Free ◽

Gut Dysbiosis ◽

Prevention And Treatment ◽

Microbiome Research ◽

The Common ◽

Physiological Systems ◽

Free Animal

The microbiome lies at the forefront of scientific research, as researchers work to uncover its mysterious influence on human development and disease. This paper reviews how the microbiome is studied, how researchers can improve its study, and what clinical applications microbiome research might yield. For this review, we analyzed studies concerning the role of the microbiome in disease and early development, the common methodologies by which the microbiome is researched in the lab, and modern clinical treatments for dysbiosis and their possible future applications. We found that the gut microbiome is essential for proper development of various physiological systems and that gut dysbiosis is a clear factor in the etiology of various diseases. Furthermore, we found that germ-free animal models and microbiome manipulation techniques are inadequate, reducing the efficacy of microbiome research. Nonetheless, research continues to show the significance of microbiome manipulation in the clinical treatment of disease, having shown great promise in the prevention and treatment of dysbiosis. Though the clinical applications of microbiome manipulation are currently limited, the significance of dysbiosis in the etiology of a wide array of diseases indicates the significance of this research and highlights the need for more effective research methods concerning the microbiome.

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The Role of Host Glycobiology and Gut Microbiota in Rotavirus and Norovirus Infection, an Update

10.20944/preprints202112.0045.v1 ◽

2021 ◽

Author(s):

Nazaret Peña-Gil ◽

Cristina Santiso-Bellón ◽

Roberto Gozalbo-Rovira ◽

Javier Buesa ◽

Vicente Monedero ◽

...

Keyword(s):

Gut Microbiota ◽

Animal Studies ◽

Viral Infections ◽

Host Cells ◽

Blood Group Antigens ◽

Virus Attachment ◽

Germ Free ◽

Complex Relationships

Rotavirus (RV) and norovirus (NoV) are the leading cause of acute gastroenteritis (AGE) worldwide. Several studies have demonstrated that histo-blood group antigens (HBGAs) have a role in NoV and RV infections, since their presence on the gut epithelial surfaces is essential for the susceptibility to many NoV and RV genotypes. Polymorphisms in genes that code for enzymes required for HBGAs synthesis lead to secretor or non-secretor and Lewis positive and Lewis negative individuals. While secretor individuals appear to be more susceptible to RV infections, regarding NoVs infections there are too many discrepancies that prevent drawing conclusions. A second factor that influences enteric viral infections is the gut microbiota of the host. In vitro and animal studies have determined that the gut microbiota limits, but in some cases enhances, enteric viral infection. The ways microbiota can enhance NoV or RV infection include virion stabilization and promotion of virus attachment to host cells, whereas experiments with microbiota-depleted and germ-free animals point to immunoregulation as the mechanism by which the microbiota restricts infection. Human trials with live, attenuated RV vaccines and analysis of the microbiota in responders and non-responders individuals also allowed the identification of bacterial taxa linked to vaccine efficacy. As more information is gained on the complex relationships that are established between the host (glycobiology and immune system), the gut microbiota and the intestinal viruses, new avenues will be open for the development of novel anti-NoV and anti-RV therapies.

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The Role of Host Glycobiology and Gut Microbiota in Rotavirus and Norovirus Infection, an Update

International Journal of Molecular Sciences ◽

10.3390/ijms222413473 ◽

2021 ◽

Vol 22 (24) ◽

pp. 13473

Author(s):

Nazaret Peña-Gil ◽

Cristina Santiso-Bellón ◽

Roberto Gozalbo-Rovira ◽

Javier Buesa ◽

Vicente Monedero ◽

...

Keyword(s):

Gut Microbiota ◽

Animal Studies ◽

Viral Infections ◽

Host Cells ◽

Blood Group Antigens ◽

Virus Attachment ◽

Germ Free ◽

Complex Relationships

Rotavirus (RV) and norovirus (NoV) are the leading causes of acute gastroenteritis (AGE) worldwide. Several studies have demonstrated that histo-blood group antigens (HBGAs) have a role in NoV and RV infections since their presence on the gut epithelial surfaces is essential for the susceptibility to many NoV and RV genotypes. Polymorphisms in genes that code for enzymes required for HBGAs synthesis lead to secretor or non-secretor and Lewis positive or Lewis negative individuals. While secretor individuals appear to be more susceptible to RV infections, regarding NoVs infections, there are too many discrepancies that prevent the ability to draw conclusions. A second factor that influences enteric viral infections is the gut microbiota of the host. In vitro and animal studies have determined that the gut microbiota limits, but in some cases enhances enteric viral infection. The ways that microbiota can enhance NoV or RV infection include virion stabilization and promotion of virus attachment to host cells, whereas experiments with microbiota-depleted and germ-free animals point to immunoregulation as the mechanism by which the microbiota restrict infection. Human trials with live, attenuated RV vaccines and analysis of the microbiota in responder and non-responder individuals also allowed the identification of bacterial taxa linked to vaccine efficacy. As more information is gained on the complex relationships that are established between the host (glycobiology and immune system), the gut microbiota and intestinal viruses, new avenues will open for the development of novel anti-NoV and anti-RV therapies.

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