Microbial Transplantation With Human Gut Commensals Containing CutC Is Sufficient to Transmit Enhanced Platelet Reactivity and Thrombosis Potential - PubMed (original) (raw)

Clinical Trial

. 2018 Oct 26;123(10):1164-1176.

doi: 10.1161/CIRCRESAHA.118.313142.

Weifei Zhu 1 2, Kymberleigh A Romano 1 2 3, Chun-Jun Guo 4, Zeneng Wang 1 2, Xun Jia 1, Jennifer Kirsop 1 2, Bridget Haag 1, Jennifer M Lang 5 6, Joseph A DiDonato 1 2, W H Wilson Tang 1 2 7, Aldons J Lusis 5 6, Federico E Rey 3, Michael A Fischbach 1 4, Stanley L Hazen 2 7

Affiliations

Clinical Trial

Microbial Transplantation With Human Gut Commensals Containing CutC Is Sufficient to Transmit Enhanced Platelet Reactivity and Thrombosis Potential

Sarah M Skye et al. Circ Res. 2018.

Abstract

Rationale: Gut microbes influence cardiovascular disease and thrombosis risks through the production of trimethylamine N-oxide (TMAO). Microbiota-dependent generation of trimethylamine (TMA)-the precursor to TMAO-is rate limiting in the metaorganismal TMAO pathway in most humans and is catalyzed by several distinct microbial choline TMA-lyases, including the proteins encoded by the cutC/D (choline utilization C/D) genes in multiple human commensals.

Objective: Direct demonstration that the gut microbial cutC gene is sufficient to transmit enhanced platelet reactivity and thrombosis potential in a host via TMA/TMAO generation has not yet been reported.

Methods and results: Herein, we use gnotobiotic mice and a series of microbial colonization studies to show that microbial cutC-dependent TMA/TMAO production is sufficient to transmit heightened platelet reactivity and thrombosis potential in a host. Specifically, we examine in vivo thrombosis potential employing germ-free mice colonized with either high TMA-producing stable human fecal polymcrobial communities or a defined CutC-deficient background microbial community coupled with a CutC-expressing human commensal±genetic disruption of its cutC gene (ie, Clostridium sporogenes Δ cutC).

Conclusions: Collectively, these studies point to the microbial choline TMA-lyase pathway as a rational molecular target for the treatment of atherothrombotic heart disease.

Keywords: cardiovascular diseases; gastrointestinal microbiome; humans; metabolism; thrombosis.

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Conflict of interest statement

CONFLICT OF INTEREST

Z.W. and S.L.H. are named as co-inventors on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics and therapeutics and have the right to receive royalty payment for inventions or discoveries related to cardiovascular diagnostics or therapeutics from Cleveland Heart Lab, Quest Diagnostics and Proctor & Gamble. S.L.H. also reports having been paid as a consultant from Proctor & Gamble and having received research funds from Proctor & Gamble and Roche.

Figures

Figure 1.

Figure 1.. Transmission of thrombotic risk phenotypes through human fecal transplant to a germ-free recipient.

A) Scheme illustrating fecal microbial transplant study design. Germ-free C57Bl/6J mice were colonized by oral gavage with a fecal microbial community from B) human subjects with either high or low plasma TMAO levels (donors shown with dark symbols along with corresponding TMAO levels). Recipient mice were maintained on a choline diet. Five days post-transplant, blood was collected, tissues harvested, and C) Cecal choline TMA-lyase activity was measured; D) Hepatic FMO activity was measured; E) plasma TMAO was measured; and F) platelet function was assessed by ex vivo monitoring of aggregation response to sub-maximal ADP (1μM). P-values were determined by two-tailed unpaired t-test with Welch’s correction.

Figure 2.

Figure 2.. Relationship between plasma TMAO levels and platelet aggregation.

Relationship between A) plasma TMAO levels and in vitro aggregometry in platelet rich plasma recovered from recipient groups of mice of human fecal microbiota from the human TMAO high or low donors and B) plasma TMAO levels and time to cessation of blood flow in the carotid artery injury model. Spearman rank correlation A) R = 0.75, p = 0.003 and B) R = −0.75, p=0.0009.

Figure 3.

Figure 3.. Microbial composition of human fecal transplantation.

A) Unweighted Unifrac distances plotted in principle component analysis comparing the fecal (donors) or cecal (mouse) microbiota of donors and recipients. Each data point represents a sample from a distinct human donor or mouse recipient projected onto the first three principal coordinates (representing 89% of the total variance). B) Heirarchical clustering using unweighted pair group method with arithmetic mean (UPGMA) of human donor and recipient mice reveals a relationship among recipient mice in which the mice colonized with the high TMAO donor cluster together and distinctly from the mice colonized with the low TMAO donor. Branch lengths indicate distance of separation based on unweighted unifrac distance matrices. C) Linear discriminant analysis (LDA) effect size (LEfSe) identified cecal taxa from mouse characteristic in low TMAO recipient (empty bars) and high TMAO recipients (filled bars). D-F) The relationship of specific indicated genera abundance to plasma TMAO levels or aggregation values in low TMAO recipient mice (empty dots, n=6) and high TMAO recipient mice (filled dots, n=7). P-values were determined by unpaired t test with Welch’s correction.

Figure 4.

Figure 4.. Microbial colonization of a cutC containing human commensal transmits TMA/TMAO generation and heightened platelet responsiveness.

A) Capability to produce d9-TMA from d9-choline substrate in individual microbes or in combination was determined in vitro. B) C57Bl/6 germ-free mice colonized by gastric gavage with a core community +/− C. sporogenes (TMA producer). The non-TMA producing core microorganisms consist of B. caccae, B. ovatus, B. thetaiotaomicron, C aerofaciencs, E. rectale. Core + TMAO mice received TMAO in drinking water (0.2% w/w TMAO). All mice received a choline diet. Fourteen days post-transplant blood was collected, platelet rich plasma recovered and ex vivo platelet aggregation response to ADP (1μM) was measured. C) Cecal choline TMA-lyase activity; and D) Hepatic FMO enzyme activity were assessed as described under Methods. E) Cecal microbial community composition was determined sequencing of mouse cecum, as described in Methods. Two mice from the Core + C. sporogenes group and one mouse from the Core + TMAO group failed to sequence. All p-values shown were determined by One-Way ANOVA with Tukey’s multiple comparison post-test (B-E).

Figure 5.

Figure 5.. Microbial colonization of a cutC containing human commensal transmits TMA/TMAO generation and heightens thrombosis potential.

Germ-free C57Bl/6 mice were colonized by oral gavage. Core mice received non-TMA producing B. caccae, B.ovatus, B. thetaiotaomicron, C. aerofaciens, E. rectale. Core + C. sporogenes mice received the additional TMA producing microorganism. All mice maintained on a choline diet. A) Fourteen days after colonization, thrombosis potential (FeCl3 carotid injury model) and plasma TMAO levels were assessed. Example clot formation images are shown with average plasma TMAO ± SD values. B) Time to cessation of blood flow was determined as described in Methods. C) Cecal choline TMA-lyase activity; and D) Hepatic FMO enzyme activity were assessed as described under Methods. E) Cecal microbial community composition was determined by sequencing of mouse cecum, as described in Methods. One animal in the Core group died before terminal measurements could be completed was still used in sequencing analysis. P-values were determined by unpaired t test with Welch’s correction.

Figure 6.

Figure 6.. Deletion of the cutC gene from C. sporogenes blocks generation of TMAO in the host reducing thrombosis potential.

A) Growth curves of C. sporogenes WT or C. sporogenes ΔcutC strains in mega media supplemented with d9-Choline (1mM). LC/MS/MS quantification of d9-TMA produced by C. sporogenes WT or C. sporogenes Δ_cutC_ strains in mega media containing d9-choline (1mM). All values are averages of three independent replicates ± SD. B) Germ-free C57Bl/6 mice were colonized by oral gavage with core (B. caccae, B. ovatus, B. thetaiotaomicron, C. aerofaciens, E. rectale) +/− TMA producing C. sporogenes or C. sporogenes Δ_cutC_. Core + TMAO mice also harbored the non-TMA producing Core and received TMAO in drinking water (0.2% w/w). All mice were maintained on a choline diet. After two weeks of colonization the FeCl3 carotid artery injury model was performed. Representative images are shown. C) Time to cessation of blood flow was determined during the FeCl3 carotid injury model as described under Methods. Respective plasma TMAO values are indicated as measured by LC/MS/MS. D) Cecal choline TMA-lyase activity; and E) Hepatic FMO enzyme activity were assessed as described under Methods. F) Microbial community composition was assessed via sequencing of mouse cecal material as described under Methods. One animal in the Core + C. sporogenes group died before terminal measurements could be completed was still used in sequencing analysis. One animal in the Core + TMAO group failed to sequence. All p-values shown were determined by One-Way ANOVA followed by Tukey’s multiple comparison post-test.

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