Periodic Reporting for period 1 - I-AM-GUT (Impact of maternal Adrenomedullin on the Microbiome and Gut health: insights into preventing chronic intestinal disorders)
Periodo di rendicontazione: 2019-11-01 al 2021-10-31
The I-AM-GUT Project will investigate the role of maternal adrenomedullin (AM), a hormone naturally produced in the body, in bacterial colonisation of the gut in early-life, thus affecting gut development, health, and future susceptibility to suffer from intestinal disorders. This project also aims to test the efficacy of AM as a biomarker for inflammatory bowel disease (IBD).
IBD is a chronic disease characterised by relapsing episodes of gastrointestinal inflammation. It has become a global disease with over 3 million patients only in Europe. The financial costs of the disease are, in general, high with an estimated €4.6-5.6 billion per year in Europe. However, these estimates do not factor the "real" price of IBD, which can impede career aspirations, instil social stigma and impair quality of life in patients (with 20-25% of European patients experiencing continuous symptoms, such as diarrhoea, rectal bleeding, abdominal pain, joint pain, weight loss, without permanent relief). Studies addressing IBD have increased exponentially in the last decades, but its causes and pathogenesis remains largely unkown, especially at the molecular level. These highlight the need for research aimed to predict the evolution of IBD and for the discovery of new biomarkers for diagnosis, which will result in the development of innovative prevention strategies and treatments to manage this complex disease.
Several scientific studies have indicated that an adequate establishment of bacteria composition (i.e. microbiota) in our intestines during early life and its maintenance throughout childhood is crucial for developing a healthy gut and to reduce the risk of developing certain disease conditions later in life. As previously mentioned, AM is a hormone that is beneficial for the gut and is involved in the promotion of a healthy microbiota by being a potent killer agent of harmful bacteria and promoter of beneficial bacteria in the gut. AM is also detected at high concentrations in all body fluids and secretions, including the mammal's milk, but the role of AM found in the milk for intestinal health and disease is currently unknown.
IBD is a chronic disease characterised by relapsing episodes of gastrointestinal inflammation. It has become a global disease with over 3 million patients only in Europe. The financial costs of the disease are, in general, high with an estimated €4.6-5.6 billion per year in Europe. However, these estimates do not factor the "real" price of IBD, which can impede career aspirations, instil social stigma and impair quality of life in patients (with 20-25% of European patients experiencing continuous symptoms, such as diarrhoea, rectal bleeding, abdominal pain, joint pain, weight loss, without permanent relief). Studies addressing IBD have increased exponentially in the last decades, but its causes and pathogenesis remains largely unkown, especially at the molecular level. These highlight the need for research aimed to predict the evolution of IBD and for the discovery of new biomarkers for diagnosis, which will result in the development of innovative prevention strategies and treatments to manage this complex disease.
Several scientific studies have indicated that an adequate establishment of bacteria composition (i.e. microbiota) in our intestines during early life and its maintenance throughout childhood is crucial for developing a healthy gut and to reduce the risk of developing certain disease conditions later in life. As previously mentioned, AM is a hormone that is beneficial for the gut and is involved in the promotion of a healthy microbiota by being a potent killer agent of harmful bacteria and promoter of beneficial bacteria in the gut. AM is also detected at high concentrations in all body fluids and secretions, including the mammal's milk, but the role of AM found in the milk for intestinal health and disease is currently unknown.
The I-AM-GUT Project aimed to investigate the role of maternal adrenomedullin (AM), a hormone naturally produced in the body, in bacterial colonisation of the gut in early-life, thus affecting gut development, health, and future susceptibility to suffer from intestinal disorders. It also aimed to test the efficacy of AM as a biomarker for inflammatory bowel disease (IBD) in patients suffering from this condition.
Generation of mammary gland conditional Knock-out for AM
In order to do achieve the aims of this project, we needed to create a mammary gland conditional knock-out (cKO) model for AM to identify the role of maternal AM on breast and faecal microbiome, gut physiology including epithelial and immune cell regulation by AM, and susceptibility to colitis. A cKO model was needed because of the lethal phenotype of the total KO model for this hormone. To generate the mammary gland cKO for AM, mice with the AM gene (Adm) flanked by the loxP sequences ("floxed" mice) previously generated in the fellow’s laboratory (rederived at Charles River, France) were crossed with transgenic mice expressing Cre recombinase under the control of whey acidic protein promoter (WAP-CRE, imported from Jackson Labs, USA). Cre-mediated recombination resulted in the deletion of the floxed Adm only in the mammary gland epithelium during pregnancy and lactation.
Evaluation of the AM anti-inflammatory action using an in vitro cell model
One of the hypothesized mechanism by which AM may be exerting a protective action in in vivo colitis studies is by regulating the immune system response, thereby preventing the over expression of pro-inflammatory cytokines during colitis development.
Evaluation of the AM system in colonic biopsies from healthy donors and IBD patients
The I-AM-GUT Project also aimed to identify the role of endogenous AM in IBD pathogenesis exploring human samples (WP3). Using samples previously collected by the groups of Dr Melgar and Nally, the expression of the AM system (peptide and receptors) were studied in colonic biopsies from females and males with IBD (active and remission phase), and from healthy controls (HL) by using quantitative (q)RT-PCR.
Briefly, total RNA was isolated from colonic biopsies using The mirVana™ miRNA Isolation Kit (Ambion), according to the manufacturer’s instructions. Total RNA (1µg) of each sample was reverse transcribed using the Transcriptor Reverse Transcriptase kit (Sigma Aldrich) according to manufacturer’s instructions. The synthesized cDNA was amplified by qRT-PCR in a LightCycler® 480 Real-Time PCR System (Roche Life Sciences). Relative gene expression was calculated by the 2-△△CT method and ACTB gene was used as reference for normalization of samples. Target genes, primers and probes (Roche) are described in Table 2.
Generation of mammary gland conditional Knock-out for AM
In order to do achieve the aims of this project, we needed to create a mammary gland conditional knock-out (cKO) model for AM to identify the role of maternal AM on breast and faecal microbiome, gut physiology including epithelial and immune cell regulation by AM, and susceptibility to colitis. A cKO model was needed because of the lethal phenotype of the total KO model for this hormone. To generate the mammary gland cKO for AM, mice with the AM gene (Adm) flanked by the loxP sequences ("floxed" mice) previously generated in the fellow’s laboratory (rederived at Charles River, France) were crossed with transgenic mice expressing Cre recombinase under the control of whey acidic protein promoter (WAP-CRE, imported from Jackson Labs, USA). Cre-mediated recombination resulted in the deletion of the floxed Adm only in the mammary gland epithelium during pregnancy and lactation.
Evaluation of the AM anti-inflammatory action using an in vitro cell model
One of the hypothesized mechanism by which AM may be exerting a protective action in in vivo colitis studies is by regulating the immune system response, thereby preventing the over expression of pro-inflammatory cytokines during colitis development.
Evaluation of the AM system in colonic biopsies from healthy donors and IBD patients
The I-AM-GUT Project also aimed to identify the role of endogenous AM in IBD pathogenesis exploring human samples (WP3). Using samples previously collected by the groups of Dr Melgar and Nally, the expression of the AM system (peptide and receptors) were studied in colonic biopsies from females and males with IBD (active and remission phase), and from healthy controls (HL) by using quantitative (q)RT-PCR.
Briefly, total RNA was isolated from colonic biopsies using The mirVana™ miRNA Isolation Kit (Ambion), according to the manufacturer’s instructions. Total RNA (1µg) of each sample was reverse transcribed using the Transcriptor Reverse Transcriptase kit (Sigma Aldrich) according to manufacturer’s instructions. The synthesized cDNA was amplified by qRT-PCR in a LightCycler® 480 Real-Time PCR System (Roche Life Sciences). Relative gene expression was calculated by the 2-△△CT method and ACTB gene was used as reference for normalization of samples. Target genes, primers and probes (Roche) are described in Table 2.
To date, the impact of AM on gut development, IBD susceptibility and breast-gut microbiome interaction is largely unknown.
To our knowledge this is the first time that a mammary gland cKO mice for AM has been created. This totally new transgenic model can be used to fundamentally reveal a deeper understanding on how AM, secreted through breast milk, during lactation can support the development of a healthy gut and microbiota in the infant. Furthermore, this new cKO can also shed light in breast cancer. AM is an angiogenic peptide, directly involved in tumor metabolism and growth. Our cKO offers a new opportunity to study the role of this hormone in breast cancer and to test new AM-based therapies in this type of cancer, which every year causes more than 600 000 dead all over the world.
The I-AM-GUT project also aimed to correlate the genetic and molecular causes of alterations in AM biology with IBD aetiology; thus being the first study to determine the suitability of AM as a prognostic biomarker for patients with IBD. Our research has demonstrated for the first time that AM system is deeply altered in the colon of IBD patients, with a reduction in the synthesis of AM and its normal receptor RAMP2, and an overexpression of RAMP3 (a receptor that decreases AM binding affinity) in UC patients. These findings can have significant implications for basic research on IBD and they can be used to open a new line of research for detection and prevention of development of IBD, as well as new treatments based on AM application.
To our knowledge this is the first time that a mammary gland cKO mice for AM has been created. This totally new transgenic model can be used to fundamentally reveal a deeper understanding on how AM, secreted through breast milk, during lactation can support the development of a healthy gut and microbiota in the infant. Furthermore, this new cKO can also shed light in breast cancer. AM is an angiogenic peptide, directly involved in tumor metabolism and growth. Our cKO offers a new opportunity to study the role of this hormone in breast cancer and to test new AM-based therapies in this type of cancer, which every year causes more than 600 000 dead all over the world.
The I-AM-GUT project also aimed to correlate the genetic and molecular causes of alterations in AM biology with IBD aetiology; thus being the first study to determine the suitability of AM as a prognostic biomarker for patients with IBD. Our research has demonstrated for the first time that AM system is deeply altered in the colon of IBD patients, with a reduction in the synthesis of AM and its normal receptor RAMP2, and an overexpression of RAMP3 (a receptor that decreases AM binding affinity) in UC patients. These findings can have significant implications for basic research on IBD and they can be used to open a new line of research for detection and prevention of development of IBD, as well as new treatments based on AM application.