OCTOPUS

A sound microbiota in a sound body through apolipoprotein A-I and HDL: from mouse models to humans

The main objectives of the OCTOPUS project were:

to determine, in mouse models, to what extent apoA-I/HDL modulate gut microbiota composition, intestinal homeostasis/immunity, plasma metabolome and atherosclerosis development.
to determine whether apoA-I/HDL levels modulate gut microbiota composition and plasma metabolome in human cohorts, and to identify possible correlations between microbiota composition and metabolic parameters.
to conduct SNPs analyses to highlight polymorphisms in genes involved in the microbiota-host cross-talk which could influence apoA-I/HDL levels.
to disseminate to the general public, scientific community, and stakeholders the scientific questions, aims, initiatives and results of the project.

The results obtained in mouse models confirm the protective role of apoA-I/HDL against atherosclerosis. However, HDL seems to be mandatory for the TMAO-mediated deleterious effects on atherosclerosis. Those effects do not seem to involve an increased activation of the immune system, nor macroscopic changes in the morphology of lymphoid organs, liver, kidney, intestine, and lungs.

Metabolomic analyses on plasma and hepatic gene expression indicated that increased dietary choline besides elevating TMAO levels also modulates other metabolic pathways, including the methionine cycle leading to altered homocysteine concentration.

Data obtained in germfree mice that received the cecal microbiota from donor mice with varying levels of HDL and fed diets with different choline content showed that the microbiota per se is not able to modulate atherosclerosis development.

The evaluation of fecal microbiota composition showed significant differences between subjects with low or high HDL, but plasma levels of TMAO did not associate with plasma HDL levels. Evaluation of SNPs in candidate genes with a pivotal role in HDL metabolism and gut mucosa-microbiota interaction did not detect the presence of major variants of these genes in the LURIC population. Epigenome wide association studies were performed and significant associations with ApoA-1 were identified, whose most significant was a CpG at the DHCR24 locus.

The dissemination has been pursued throughout the entire project and will continue beyond the project closure. Both general public and peers were approached with focused preparation of social media posts, scientific articles, and sharing of results at scientific conferences.

Expected impacts. Although no individual bacterial species have been identified/isolated that may impact on atherosclerosis development through a modulation of dietary choline metabolization, this project demonstrates with some consistency that varying levels of HDL, both in humans and mice, do not make the gut microbiota more or less pro-atherogenic. At the same time, the project shows that HDL are essential for the pro-atherogenic effects of TMAO to occur, thus a stratification of patients combining HDL levels with TMAO levels can be hypothesized.

Consortium

Partner Organization	Partner Country
Université Paris-Saclay (INRA)	France
Heidelberg University	Germany
Berlin Institute of Health (BIH)	Germany

Highlights

The OCTOPUS project was designed and realized with the aim of shedding light on an aspect of apoA-I/HDL pathophysiology that was never properly investigated before: does apoA-I/HDL affect atherosclerosis development, intestinal inflammation and the immunoinflammatory status by modulating the presence of TMAO-producing (or other) bacteria in the gut?
The most relevant results obtained in OCTOPUS project are:

ApoA-I/HDL are confirmed to be protective against atherosclerosis development in animal models: mice expressing apoA-I have smaller lesion size than mice not expressing apoA-I.
A high intake of dietary choline results into an increased plasma concentration of TMAO and worsens atherosclerosis development, but only if apoA-I is expressed. In other words, HDL is a prerequisite for this to happen.
As a consequence, although HDL is per se protective against the development of atherosclerotic lesions, its presence seems to be necessary for TMAO to exhibit its pro-atherogenic effect: mice expressing apoA-I fed low-choline diet develop smaller lesions than mice fed high-choline diet. That either suggests that TMAO may impair the athero-protective activity of HDL, or that HDL may work as TMAO carrier throughout the bloodstream, contributing to the exploitation of its effects.
The microbiota composition of mice expressing or not apoA-I showed little, yet sizeable, differences. Differences are also found in the gut microbiota of subjects with low or elevated HDL plasma levels. These differences, however, do not make the microbiota any pro- or anti- atherogenic per se, as fecal transplants into atherosclerosis-prone mice failed to show any difference in plaque formation.

Communication & Dissemination Activities

Target group	Authors	Means of communication	Hyperlink
Scientists	Graciela Delgado, Winfried März, Marcus E. Kleber, Marco Busnelli, Philippe Gérard, Olivia Gräbner, Giulia Chiesa, A sound microbiota in a sound body through apolipoprotein A-I and HDL: from mouse models to humans (The OCTOPUS Consortium), Biomarkermeeting, Mannheim, 2019	Poster
Poster	Marco Busnelli, Giulia Chiesa, Stefano Manzini*, HDL, aterosclerosi e… microrganismi intestinali: parte uno studio per studiarne lo stretto rapporto, University of Milan RicercaMix Blog, 2019	Blog post
Scientists, General Public	Marco Busnelli, Giulia Chiesa, Stefano Manzini*, A sound microbiota in a sound body through apolipoprotein A-I and HDL, Hot Nut 2: Hot Topics in Nutrition, Università degli Studi di Milano, Milan, 2019	Talk	Link
General Public	Marco Busnelli*, Interview about Hot Nut 2 talk, by https://microbioma.it/, 2019	Talk/Interview	Link
Scientists	Stefano Manzini*, Genetic HDL deficiency: insights from a double knock-out mouse model, Rare Disorders of Lipid Metabolism: from Phenotype to Precision Medicine, Università degli Studi di Milano, Milan, 2019	Talk
General public	Consortium, Upkeeping of Project Website. Migration of the site to a long-term domain, URL: https://piermatteobaramban.wixsite.com/octopus	Website	Link
General public	Consortium, OCTOPUS Facebook social page. The page hosts multilingual (Italian and English) content related to the Project’s activities, with topics ranging from everyday lab work and routine, to the interesting news in the field. Further, Projects meetings and activities are advertised. Date of activity: 12/02/2019 – current Total posts: 88 Followers: 220 Coverage (mean ± SD): 311 ± 342 Clicks (mean ± SD): 42.0 ± 43.2 Highest coverage: 1,753 Highest clicks: 250	Social media	Link
General public	Consortium, OCTOPUS Twitter social account. The account hosts multilingual (Italian and English) content related to the Project’s activities, with topics ranging from everyday lab work and routine, to the interesting news in the field. Further, Projects meetings and activities are advertised. Date of activity: 12/02/2019 – current Total tweets: 132 Followers: 20	Social media	Link
Scientists	Marco Busnelli, Stefano Manzini, Maria Conti, Giulia Chiesa* Impact Of Dietary Choline On Atherosclerosis Development In Conventionally Raised Apoe-KO Mice Expressing Different Levels Of Apoa-I, 87th EAS Congress, Maastricht, 2019	Talk	Link
Scientists	Graciela Delgado, Marcus Kleber, Influence of apoA-I levels on gene expression and DNA methylation in mice and humans: the OCTOPUS Consortium, Maikammer meeting, Klinischer Lipidstoffwechsel, Maikammer, 2019	Talk
University students	Marco Busnelli*: “The importance of gut microbiota”. Academic courses in Toxicology, Biotechnology and Pharmacy. School of Pharmacy – University of Milan, May2020	Lesson
Scientists	Stefano Manzini, Marco Busnelli, Amin Jablaoui, Aurélia Bruneau, Aïcha Kriaa, Catherine Philippe, Francesca Arnaboldi, Alice Colombo, Benedetta Ferrari, Emmanuelle Maguin, Moez Rhimi, Giulia Chiesa, Philippe Gérard: Fat-Shaped Microbiota Affects Lipid Metabolism, Liver Steatosis And Intestinal Homeostasis In Mice Fed A Low-Protein Diet, Fifth SISA Spring Meeting, Virtual, June/July 2020	Poster
Scientists	Marco Busnelli, Stefano Manzini, Alice Colombo, Giulia Chiesa*: Impact of dietary choline on atherosclerosis development in conventionally raised ApoE-KO mice expressing different levels of ApoA-I Fifth SISA Spring Meeting, Virtual, June/July 2020	Poster
Scientists	Mariel A. García-Rivera, Graciela Delgado, Winfried März, Marcus E. Kleber, Marco Busnelli, Philippe Gérard, Jennifer Kirwan, Giulia Chiesa* A sound microbiota in a sound body through apolipoprotein A-I and HDL: from mouse models to humans: the OCTOPUS Consortium. Metabolomics 2020 Online, October 27-29 2020	Poster
Scientists	Mariel A. García-Rivera* OCTOPUS: a sound microbiota in a sound body through apolipoprotein A-I and HDL: from mouse models to humans. Postdoc Day 2020, MDC-Berlin, November 13th 2020	Virtual poster
General public	Coordinator, HDL e microbiota: un legame per curare l’aterosclerosi Journal: Platinum (Italian version), edited by PubliScoop Più, Il Sole 24 Ore. No. 070; Pub date: 22/03/2021	Press/Magazine	Link
General public	Coordinator, HDL and microbiota: a possible link to treat atherosclerosis Journal: Platinum (English version), edited by PubliScoop Più, Il Sole 24 Ore. No. 070; Pub date: 22/03/2021	Press/Magazine	Link
Scientists	Gérard P. Role of the gut microbiota in health and disease. How to prove causality ? International Interdisciplinary Colloquium : new challenges induced by microbiomes, Paris, France, 23 March 2021	Talk
Scientists	Busnelli M, Manzini S, Colombo A, Fontana F, Oleari R, Potì F, Castiglioni S, Bellosta S, Chiesa G; Gene Expression Profile Reveals That Apoa-I Levels Modulate Inflammation And Lysosomal Activity In Murine Advanced Atherosclerosis, European Atherosclerosis Society (EAS) Congress May 30-June 2, 2021, Helsinki, Finland	Poster
Scientists	Manzini S, Busnelli M, Colombo A, Kiamehr M, & Chiesa G; Hands-Down Extraction Of Individual Lipid Moieties From Lipidomics Datasets: Liputils, A Python Module, European Atherosclerosis Society (EAS) Congress May 30-June 2, 2021, Helsinki, Finland	Poster
Scientists	Zhang X, Busnelli M, Manzini S, Etienne C, Monnoye M, Dubard A, Tenorio Na, Chiesa G, Gerard P. Emerging Role of the Gut Microbiota in Choline-induced Atherosclerosis through the Modulation of Apolipoprotein A-I/HDL levels , 12th International Symposium On Gut Microbiology, Clermont-Ferrand, France, 23-25 October 2021	Poster
Scientists	Zhang X, Busnelli M, Manzini S, Etienne C, Monnoye M, Dubard A, Tenorio Na, Chiesa G, Gerard P. Effet protecteur de l’apolipoprotéine A-I vis-à-vis de l’athérosclérose : rôle du microbiote intestinal ? Journées Francophones de Nutrition, Lille, France, 9-11 November 2021	Poster
Scientists	Busnelli M, Franchi E, Manzini S, Colombo A, García-Rivera MA, Kirwan J, Zhang X, Gérard P, G. Chiesa. Dietary choline content and HDL levels contribute to a pro-atherogenic plasma metabolomic profile in mice, 35° Congresso Nazionale Società Italiana per lo Studio dell’Aterosclerosi, virtual, 28–30 novembre 2021	Talk
Scientists + General Public	Gérard P. Contribution du microbiote dans le lien Nutrition – Santé : Exemple de deux projets impliquant des transferts de microbiote. Webinar of the Paris Center for Microbiome Medicine (PaCeMM), 13 December 2021	Talk
Scientists	Busnelli M, Franchi E, Zhang X, Manzini S, Colombo A, García-Rivera MA, Kirwan J, Gérard P. Chiesa G. Dietary choline content and HDL levels modulate gut microbiota composition and contribute to a pro-atherogenic plasma metabolomic profile in mice. World of Microbiome, 28-30 aprile 2022, Wien, Austria	Poster
Scientists	Zhang X, Busnelli M, Manzini S, Etienne C, Monnoye M, Dubard A, Tenorio Na, Chiesa G, Gerard P. Protective effect of Apolipoprotein A-I/HDL levels in Choline-induced Atherosclerosis : a role for the gut microbiota ? World of Microbiome, Vienna, Austria, 28-30 April 2022	Poster
Scientists	Chiesa G, Manzini S, Colombo A, Franchi E, Garcia-Rivera MA, Kirwan J, Busnelli M; Dietary Choline Supplementation Increases The Plasma Concentration Of Several Amino Acids During Atherosclerosis Development, European Atherosclerosis Society (EAS) Congress May 22-25, 2022, Milan, Italy	Poster
Scientists	Franchi E, Busnelli M, Manzini S, Colombo A, Chiesa G; Histological assessment of lipid deposition in peripheral tissues from genetically modified mice with deficiency or overexpression of apoA-I, European Atherosclerosis Society (EAS) Congress, May 22-25, 2022, Milan, Italy	Poster
Scientists	Colombo A, Manzini S, Busnelli M, Franchi E, Chiesa G; HDL levels do not impact on the expression of genes playing a pivotal role in intestinal lipid metabolism in apolipoprotein E- knockout mice, European Atherosclerosis Society (EAS) Congress, May 22-25, 2022, Milan, Italy	Poster
Scientists	Manzini S, Busnelli M, Colombo A, Franchi E, Grossano P, Chiesa G; Restring: a software to manage functional enrichment of complex experimental designs, European Atherosclerosis Society (EAS) Congress, May 22-25, 2022, Milan, Italy	Poster
Scientists	Busnelli M, Franchi E, Colombo A, Manzini, S, Garcia-Rivera MA, Kirwan J, Chiesa G; Dietary choline increases plasma concentration of gut-derived uremic toxins and metabolites associated with chronic kidney disease progression in apoE- knockout mice with elevated HDL; European Atherosclerosis Society (EAS) Congress, May 22-25, 2022, Milan, Italy	Poster
Scientists	Colombo A, Busnelli M, Manzini S, Franchi E, Garcia-Rivera MA, Kirwan J, Chiesa G; Impact Of HDL Levels And Dietary Choline Content On Lipid Metabolism And Atherosclerosis Development In Mice, 7th Spring Meeting Giovani Ricercatori (SIIA, SIMI, SIPREC, SISA), June 5-7, 2022, Rimini, Italy	Poster
Scientists	Franchi E, Busnelli M, Colombo A, Manzini S, García-Rivera MA, Kirwan J, Zhang X, Gérard P, Chiesa G; Increased Dietary Choline Alters The Plasma Metabolome, Worsens Atherosclerosis Development, But Does Not Make Gut Microbiota Pro-Atherogenic Per Se, 7th Spring Meeting Giovani Ricercatori (SIIA, SIMI, SIPREC, SISA), June 5-7, 2022, Rimini, Italy	Talk
Scientists	Franchi E, Busnelli M, Colombo A, Manzini S, García-Rivera MA, Kirwan J, Zhang X, Gérard P, Chiesa G; Increased Dietary Choline Alters The Plasma Metabolome, Worsens Atherosclerosis Development, But Does Not Make Gut Microbiota Pro-Atherogenic Per Se, V Giornata della Ricerca del Centro Enrica Grossi Paoletti, June 17, 2022, Milan, Italy	Talk