The gut microbiota is a population of bacteria, viruses, and fungi that inhabits the host intestinal tract and produces physiologically important metabolites.1 Dysbiosis of the microbiota affects many immune-related processes and can be caused by environmental influences, such as antibiotic use, especially early in life.2,3 In order to influence immune responses distal to the intestinal tract, metabolites produced by the gut microbiota are absorbed through the intestinal epithelium and circulate systemically. One class of metabolites, short chain fatty acids (SCFAs), is well known to impact the immune system.4 The most abundant of these SCFAs are acetate, butyrate, and propionate.4 Acetate inhibits the pathologic activation of neutrophils, and butyrate increases Treg.5-8 SCFAs are produced through the fermentation of glycans in mucin and dietary fiber.9 Microbes, such Akkermansia muciniphila, metabolize mucin, and Lactobacillus bacteria are main contributors to the production of SCFAs by the metabolism of dietary fiber.10-12
The gut microbiota and its composition influences the outcome of organ transplantation. This microbial community can be perturbed by transplant-related factors such as antibiotics, immunosuppressive drugs, and surgical stress.13 Clinical studies suggest that the gut microbiota may effect poor outcomes in liver transplantation.14,15 Also, the composition of this microbial community is persistently altered after kidney transplantation and is associated with diarrhea, acute rejection, and diabetes.16-19 In experimental animal studies, the presence of donor and recipient microbiota increases rejection of cardiac and skin allografts through activation of a type I interferon response.20 Also, antibiotics given peritransplant improve cardiac graft survival, possibly by eliminating bacteria that are cross-reactive to allospecific T cells.21 Certain components of the microbiota are also immunoregulatory by inducing development of B cells that produce interleukin-10, affecting Treg and improving the immunosuppressive potency of tacrolimus.22-24
Allograft vascular injury is a main component of acute heart transplant rejection that can be independent of parenchymal immune injury.25 Immunological drivers of allograft vascular injury include neutrophils, donor-reactive antibodies, and T cells.26 Notably, neutrophils directly injure vascular smooth muscle cells in the media of allograft arteries in a NADPH oxidase 2-dependent manner that is independent of adaptive immune responses.27 We previously found that altering the gut microbiota of graft recipients by treatment with broad-spectrum antibiotics early in life (for the first 3 wk only) leads to increased neutrophil-mediated injury of vascular allografts performed later in life at a time point that is comparable to early adulthood.28 Here, we characterize metagenomic changes in the gut microbiota that are caused by treatment with broad-spectrum antibiotics early in life and how they relate to immunological injury of vascular allografts. Dysbiosis of the gut microbiota caused by early life disruption reduces or eliminates a handful of bacterial species and alters its metabolic gene content related to acetate production. Normalizing the gut microbiota of antibiotic-treated mice or increasing their dietary intake of acetate prevents neutrophil-mediated allograft vascular injury. Our findings are one of the first to describe how the metagenome of the gut microbiota is related to immune drivers of transplant rejection and identify specific bacterial species and acetate as influencers of transplant rejection.
MATERIALS AND METHODS
Disruption of the Gut Microbiota
C57Bl/6 (H-2b) and Balb/c (H-2d) mice were purchased from Jackson Laboratories and bred in-house. All study protocols were reviewed and approved by the Simon Fraser University Animal Care Committee based on guidelines by the Canadian Council on Animal Care. The gut microbiota was disrupted in mice using an antibiotic cocktail in the drinking water that contained ampicillin (0.5 g/L), vancomycin (0.25 g/L), neomycin sulfate (0.5 g/L), and metronidazole (0.5 g/L) that was given until 3 wk of age. This treatment leads to the near complete elimination of gut bacteria for the duration of antibiotic exposure in young mice and then to colonization of the intestinal tract by bacterial populations after cessation of antibiotics.28 In some experiments, untreated and antibiotic-treated mice were co-housed starting at 3 wk of age. Acetate was administered by adding 100 mmol/L magnesium acetate in drinking water starting 14 d before transplant and continuing until the end point. The gut microbiota was sampled by collecting fecal pellets at 8 to 12 wk of age.
Aortic Interposition Grafting
Aortic transplantation was performed as described previously in mice that were 8 to 12 wk of age.28,29 Briefly, a section of infrarenal aorta from Balb/c donors was interposed into the infrarenal aorta of sex-matched C57Bl/6 recipient mice. C57Bl/6 donors were used as syngraft controls.
Histology and Immunohistochemistry
Artery grafts were perfusion fixed with 4% paraformaldehyde. For histological examination of medial injury, 2 cross-sections per artery that were at least 100 μm apart were H&E stained and the average thickness of the media measured in Image J (National Institutes of Health). The medial thickness was normalized to arterial radius.28
Immunohistochemistry was performed using antibodies toward myeloperoxidase (Abcam, Cambridge, United Kingdom) to visualize neutrophils, CD4 (4SM95; eBioscience, Waltham, Massachusetts), CD8 (4SM15, eBioscience), Foxp3 (FJK-16S, eBioscience), and Mac-3 (BD Biosciences, Franklin Lakes, New Jersey) to visualize macrophages as described previously.29 Staining was visualized by aminoethyl carbazole chromagen (red; Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin (blue). For quantification, the total number of cells in each cross-section was manually counted in a blinded manner and reported per mm2 of media or adventitia. Two cross-sections that were at least 100 μm apart were evaluated for each artery and an average value from the cross-sections calculated and used as a single data point.
Microbiome 16S rRNA Sequencing and Analysis
DNA from fecal samples was extracted, amplified, and 16S rRNA gene sequenced as described previously.28 Analysis of sequences was performed using QIIME2, version 2019.2.30 Reads were filtered for quality and denoised using divisive amplicon denoising algorithm.31 Phylogenetic trees for rarefaction were constructed using multiple alignment using fast Fourier transform and randomized axelerated maximum likelihood.32,33 Samples were each rarefied to 1.2 × 104 reads, and the resulting amplicon sequence variants were classified using a Bayesian classifier trained on reads extracted from the SILVA 132 release.34 Genetic content was inferred using PICRUSt2 and grouped into MetaCyc pathways and enzyme commission (EC) reactions.35-37
Metagenomic Shotgun Sequencing and Analysis
Whole genome shotgun sequencing was performed at the Michael Smith Genome Sciences Center (Vancouver, British Columbia, Canada) on the Illumina HiSeq platform using V2.5 chemistry. Before analysis, raw reads were filtered for quality and presence of mouse sequences using KneadData.38 Resulting data were analyzed using MetaPhlAn3 using the included ChocoPhlAn3 pipeline based on UniProt and NCBI repositories.38,39 HUMAnN3 was used to determine genetic content; genes were detected using the UniRef90 database and regrouped under MetaCyc pathways and EC reactions.38,40 Gene abundance data were normalized to reads per kilobase. Pathways and EC reactions were filtered for those that appeared in at least 3 mice across all treatment groups.
Splenocytes and blood cells were stained with antibodies recognizing CD4 (RM4.5, BDBiosciences), CD8 (53–6.7, BD Biosciences), Foxp3 (MF23, BD Biosciences), Ly6G (1A8, BD Biosciences), and CD11b (M1/70, eBioscience). Data were acquired on a BD LSRFortessa X-20 (BD Biosciences) and analyzed using FlowJo.
Differences between groups were determined using a Mann-Whitney U-test. Volcano plots display results from Kruskal-Wallis rank-sum tests with Benjamin Hochberg correction. Ordination plots were generated using the Vegan package in R. Bray-Curtis or weighted-Unifrac distances were calculated, and these matrices were used to produce a 2-dimensional nonmetric multidimensional scaling (NMDS) plot. Covariance ellipses were calculated using Vegan. Significant differences were defined as having a P < 0.05.
Disruption of the Gut Microbiota With Antibiotics Early in Life Exacerbates Neutrophil Accumulation in Female Recipients of Vascular Transplants
The effects of early life disruption of the gut microbiota were examined by administering a cocktail of antibiotics in the drinking water of recipient mice for the first 3 wk of life and then maintaining them on normal water thereafter. Aortic transplants were then performed into these mice at 8 to 12 wk of age and collected at day 7 posttransplantation.28 When stratified by sex, antibiotic treatment early in life markedly and significantly exacerbated neutrophil accumulation in both the media and adventitia of allograft artery transplants in female recipients, whereas there was minimal effect in males (Figure 1A). There was no effect on accumulation of CD4 T cells, CD8 T cells, Treg, or macrophages in either females or males (Figure 1B–E). Also, immune cell accumulation was minimal in syngrafts (Figure 1 and Figure S1A, SDC, https://links.lww.com/TP/C423). When the systemic levels of leukocytes were examined in female mice, there was no effect of antibiotic treatment on CD4 T cells, CD8 T cells, Treg, monocytes, or neutrophils (Figure S1B–D, SDC, https://links.lww.com/TP/C423). All subsequent studies were performed in female mice.
Antibiotic Treatment Early in Life Changes the Inferred Metagenome of the Gut Microbiota
We examined how antibiotic treatment early in life alters the taxonomic and metagenomic landscape of the gut microbiota of adult mice. Fecal samples were collected from mice at 8 to 12 wk of age, DNA was isolated, and bacterial composition was determined by 16S rRNA sequencing. Antibiotic treatment early in life altered the gut microbiota of adult mice. This was characterized by an increase of the bacterial classes Clostridia and Erysipelotrichia, a decrease of Bacteroidia, and an absence of Verrucomicrobiae (Figure 2A). NMDS analysis of weighted-UniFrac distances showed a clear separation between untreated and antibiotic-treated mice (Figure 2B). There was also a slight reduction in microbial diversity caused by antibiotic treatment early in life (Figure 2C).
Next, genomic composition of potential metabolic pathways was inferred from 16S data based on a reference set of genomes using PICRUSt2.35 After grouping metabolic genes under MetaCyc pathways, NMDS ordination of Bray-Curtis distances showed that the total inferred metabolome of antibiotic-treated mice was significantly different than untreated counterparts (Figure 3A). Genes encoding enzymes in metabolically relevant reactions were then examined by regrouping the PICRUSt2 data under EC reactions. Inferred enzymatic reactions clustered separately between untreated and antibiotic-treated mice (Figure 3B). This analysis suggested that antibiotic treatment early in life changes the resultant gut microbiota in adults and alters its metabolic capacity. Direct comparison of pathways and EC reactions between groups showed that there may be alterations in those related to nucleotide and amino acid biosynthesis, but these did not reach statistical significance.
Because of the role of acetate in regulating neutrophils and the production of this metabolite as a by-product of mucin metabolism, we also examined the inferred metagenomic landscape for genes involved in reactions that metabolize mucin and could potentially affect the production of SCFAs. Mucin degradation pathways have yet to be curated in the databases we queried, but studies have established key enzymatic reactions in this process (Figure 3C).41 Six such reactions were inferred from the 16S rRNA, data and of these, 4 were significantly reduced in antibiotic-treated mice, suggesting a potentially reduced capacity to metabolize mucin and produce acetate (Figure 3D). In addition to mucin, we also queried our data for inferred genes involved in the production of SCFAs from fermentation of fiber but were unable to assign genes specifically to this process.
Antibiotic Treatment Early in Life Reduces Bacterial Taxa Involved in Production of SCFAs
The bacterial composition of the gut microbiota was then examined in greater detail by whole genome shotgun sequencing, and the effect of co-housing was also determined. Fecal samples were collected at 8 to 12 wk of age. The data showed similar trends to 16S analysis. NMDS analysis of Bray-Curtis distances showed a clear separation between the composition of the gut microbiota in untreated and antibiotic-treated mice (Figure 4A). There was an increase in the bacterial class Clostridia and decrease in Bacteroidia in antibiotic-treated mice compared with untreated counterparts (Figure 4B). There was also a reduction of Verrucomicrobiae, Bacilli, and Actinomycetia in antibiotic-treated mice. Notably, co-housing mice normalized the gut microbiota, resulting in a bacterial population that was comparable to untreated mice (Figure 4A and B). Of the bacterial species detected in our samples, 8 were significantly different between the untreated and antibiotic-treated groups (Figure 4C). A. muciniphila was the most abundant species that was completely eliminated by antibiotics and completely restored to normal levels after co-housing. Reduction of A. muciniphila in the gut microbiota of antibiotic-treated mice was also observed by 16S PCR (Figure 4D). A. muciniphila is well known to degrade mucin, which results in production of acetate as a by-product.14 The second- and third most abundant bacterial species that were reduced by antibiotics and completely rescued by co-housing were Lactobacillus murinus and Lactobacillus johnsonii (Figure 4C). Notably, these 2 Lactobacillus species can also contribute to the production of acetate by the gut microbiota.42
Genes that are related to metabolic pathways were then examined using HUMAnN3 and clustered into MetaCyc pathways. Of these, 103 were significantly different between untreated and antibiotic-treated mice (Figure S1E, SDC, https://links.lww.com/TP/C423). The NMDS plot of Bray-Curtis distances showed that mice treated with antibiotics have distinct genomic composition of metabolic genes as compared with untreated mice. This was normalized by co-housing (Figure 5A). Significantly different pathways were related to vitamin biosynthesis, amino acid biosynthesis, and nucleotide biosynthesis (Table S1, SDC, https://links.lww.com/TP/C423). Under current classifications, there are no pathways that specifically examine the metabolism of mucin and production of SCFAs from fiber.
The abundance of genes related to metabolically relevant enzymatic pathways was then assessed. Antibiotic-treated mice clustered separately from untreated and co-housed mice in the NDMS plot, with the latter groups clustering together (Figure 5B). There were 558 enzymatic reactions that were significantly different between untreated and antibiotic-treated mice that were related to enzyme cofactor biosynthesis, secondary metabolite degradation, and carbohydrate degradation (Figure S1F and Table S2, SDC, https://links.lww.com/TP/C423). When enzymes related to mucin degradation were specifically examined, genes related to 4 mucin-degrading enzyme reactions were detected. Similar to findings from inferred gene content using 16S analysis, β-N-acetylhexosaminidase was significantly decreased in antibiotic-treated mice as compared with untreated counterparts, and co-housing completely restored its abundance (Figure 5C). There was no detectable difference in abundance of the other 3 enzyme genes. β-N-acetylhexosaminidase catalyzes the cleavage of N-acetylglucosamine and N-acetylhexoseamine and is an initial step of mucin degradation.14 In untreated mice, a consistent proportion of identified reads from this gene in the microbiome was associated with the genome of A. muciniphila (Figure 5D). In antibiotic-treated mice, which lack A. muciniphila, the lower abundance of β-N-acetylhexosaminidase was disproportionally present from the genome of F. plautii. Co-housing mice restored the abundance of this gene from A. muciniphila. These findings indicate that the deficiency in A. muciniphila caused by antibiotic treatment reduces the metagenomic content of a mucin-degrading enzyme gene that may hamper mucin metabolism, and potentially the production of downstream SCFAs as a by-product. We were unable to identify enzymatic reactions that produce SCFAs specifically from degradation of resistant starches/fiber in the databases queried.
Normalizing the Gut Microbiota by Co-housing Prevents Neutrophil-mediated Allograft Vascular Injury in Antibiotic-treated Mice
We next examined whether dysbiosis of the gut microbiota caused by its early life disruption with antibiotics affects neutrophil-mediated allograft vascular injury by performing co-housing experiments. Aortic interposition allografts were performed into recipients at 8 to 12 wk of age. Antibiotic treatment markedly and significantly exacerbated neutrophil accumulation in both the arterial media and adventitia of allograft arteries (Figure 6A). This was related to increased medial injury as determined by a reduction in medial thickness that was accompanied by loss of cellularity and elastic laminae in the media of allograft arteries at day 30 posttransplantation (Figure 6B). Accumulation of other immune cells was not affected (Figure S2, SDC, https://links.lww.com/TP/C423). Notably, normalizing the microbiota of antibiotic-treated mice to resemble that of untreated counterparts via co-housing completely prevented neutrophil accumulation (Figure 6A). These findings directly establish that changes in the gut microbiota of adult graft recipients caused by early life disruption with antibiotics lead to dysregulation of neutrophils and increase medial injury after transplantation.
Acetate Prevents Neutrophil Accumulation and Related Injury in Vascular Allografts Caused by Early Life Disruption of the Gut Microbiota
We observed that early life disruption of the gut microbiota with antibiotics results in metagenomic changes, that relate to reduced acetate production, such as diminished abundance of the mucin-degrader A. muciniphila, reduction of genes encoding enzymes that metabolize mucin, and diminished abundance of bacterial species (L. murinus and L. johnsonii) that contribute to the production of acetate from dietary components. As such, the effect of acetate on regulating neutrophil responses in vascular allografts was examined by administering magnesium acetate (100 mmol/L) in the drinking water of some antibiotic-treated mice for 14 d before transplantation until end point. Administration of acetate in this manner has been shown to increase acetate levels and inhibit pathological neutrophil responses.43 Aortic interposition allografts were performed and artery segments collected at day 7 and 30 posttransplantation. Administration of acetate significantly and completely prevented the exacerbation of neutrophil accumulation caused by early life disruption of the gut microbiota by antibiotics (Figure 6A). Moreover, acetate also prevented medial injury assessed at day 30 posttransplantation, as determined by a preservation of medial thickness, cellularity, and elastic laminae integrity in the media of allograft arteries (Figure 6B). Acetate did not effect the accumulation of CD4 T cells, CD8 T cells, Tregs or macrophages (Figure S2, SDC, https://links.lww.com/TP/C423). This establishes that neutrophil-mediated allograft vascular injury caused by early life disruption of the gut microbiota with antibiotics is inhibited by acetate.
In this study, we provide new information on how early life disruption of the gut microbiota affects its metagenome and related immunoregulatory properties and how the resulting changes influence immune injury of allograft blood vessels. Specifically, dysbiosis of the gut microbiota that exacerbates neutrophil-mediated vascular injury is characterized by a reduction in metabolic genes related to the production of SCFAs, and acetate prevents neutrophil-mediated allograft vascular injury. These findings advance our knowledge of how the gut microbiota controls immune responses that injure organ transplants by establishing the role of acetate as an immune regulator.
We observe a sex-dependent effect on the influence of the gut microbiota on neutrophil responses, which is consistent with the differential susceptibility of females to some immunopathologies through effects of the gut microbiota.44,45 This differential susceptibility may relate specifically to neutrophils because females are more susceptible than males to systemic lupus erythamatosus and the etiology of this autoimmune disease involves the development of immunopathological responses toward autoantigens from neutrophils.46 Recently, RNA sequencing has identified an enhanced responsiveness of female neutrophils to type I interferon stimulation as compared with male counterparts. This is related to altered bioenergetics of female and male neutrophils that are driven by estradiol exposure.47,48 With regard to vascular effects, eliminating the gut microbiota increases outward hypertrophic remodeling of resistance arteries in female mice to a greater extent than males, so the gut microbiota may contribute to sex differences in certain vascular conditions.49
In our studies, co-housing mice that were treated with antibiotics early in life with untreated counterparts prevented neutrophil-mediated allograft vascular injury, indicating that changes in the composition of the gut microbiota were responsible for exacerbating neutrophil responses. Treatment of mice with antibiotics in our experiments reduces the amount of bacteria to nearly undetectable levels, so dysbiosis of the gut microbiota in adults is likely to be a result of differential population of the intestinal tract by bacterial species caused by the delay in exposure to bacteria until after weaning.28 Indeed, altering the timing of exposure of animals to gut colonizing bacteria around the time of weaning leads to differences in the establishment of gut microbiota populations later in life.50 This may be a result of the differential availability of intestinal niches available for population by bacteria at different times during development.51
The anaerobic bacterium A. muciniphila contributes to the production of acetate as a by-product of mucin degradation and was the most abundant bacterial species reduced by early life disruption of the gut microbiota with antibiotics. In humans, A. muciniphila is highly abundant in the colonic mucosa, and it is being investigated as a probiotic for several immune-mediated conditions.14,52-55 The reduction in A. muciniphila caused by antibiotics early in life was related to a reduced abundance of genes that encode mucin-degrading enzymes, such as β-N-acetylhexosaminidase, which catalyzes the cleavage of terminal β-D-N-acetylglucosamine and β-D-GalNAc residues of oligosaccharides that are important for bacterial taxa to derive resources from mucin.56,57 The second- and thirdmost abundant bacterial species that were reduced by disruption of the gut microbiota with antibiotics were L. johnsonii and L. murinus. Lactobacillus bacteria, which are implicated in many aspects of health and disease.58 One of the defining features of bacteria within the Lactobacillus genus is the production of lactic acid through the fermentation of indigestible carbohydrates such as dietary fiber. Lactic acid is then converted to SCFAs, including acetate, by other bacteria in the community.42L. johnsonii may also be capable of directly producing acetate.59,60 In patients infected with HIV, reduction in Lactobacillus species in the gut microbiota increases neutrophil accumulation in the intestinal mucosa, but the roles of L. murinus and L. johnsonii were not specifically examined.61
Our observation that directly providing acetate to mice reduces neutrophil-mediated injury of allografts indicates that this SCFA is immunoregulatory. Although the mechanism by which neutrophils exacerbate medial injury in our experiments was not specifically examined, these innate immune cells induce vascular smooth muscle apoptosis in allograft arteries through a reactive oxygen species-mediated pathway.27 Neutrophils can also produce high levels of nitric oxide, and inhibition of inducible nitric oxide synthase protects against early ischemic injury of liver allografts.62 Acetate inhibits neutrophil responses by activating the G-protein coupled receptor 43.43 The cellular mechanisms by which this SCFA inhibits neutrophils are poorly understood. Neutrophils can migrate toward a chemotactic gradient of acetate in vitro.63 Although this suggests a proinflammatory effect of this SCFA, elevated levels of acetate in the blood could inhibit the extravasation of neutrophils to sites of inflammation that are outside the blood vessel lumen. Acetate also induces neutrophil apoptosis in vitro.43 Administration of acetate in the drinking water or a high fiber diet is efficacious for attenuating neutrophil pathologies such as gout.10 In transplantation, Wu et al64 recently demonstrated that acetate from the gut microbiota reduces rejection of renal transplants through an effect on Treg. We were not able to quantify circulating levels of acetate because of technical challenges, and this is a limitation of our studies.
In summary, we show that early life disruption of the gut microbiota can enhance immune responses that injure transplant blood vessels through effects on the immunoregulatory SCFA acetate. These findings provide a basis for further study of immunoregulatory effects of the gut microbiota in transplantation and of immune regulatory properties of A. muciniphila, Lactobacillus spp., and acetate.
We are grateful to the SFU Big Data Hub, the Cedar supercluster, and Compute Canada for maintenance of bioinformatics data and software. Expert animal care and technical assistance were provided by the Animal Research Center at SFU.
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