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Ifnar gene variants influence gut microbial production of palmitoleic acid and host immune responses to tuberculosis

Nature Metabolism volume 4, pages 359–373 (2022)Cite this article

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Abstract

Both host genetics and the gut microbiome have important effects on human health, yet how host genetics regulates gut bacteria and further determines disease susceptibility remains unclear. Here, we find that the gut microbiome pattern of participants with active tuberculosis is characterized by a reduction of core species found across healthy individuals, particularly Akkermansia muciniphila. Oral treatment of A. muciniphila or A. muciniphila-mediated palmitoleic acid strongly inhibits tuberculosis infection through epigenetic inhibition of tumour necrosis factor in mice infected with Mycobacterium tuberculosis. We use three independent cohorts comprising 6,512 individuals and identify that the single-nucleotide polymorphism rs2257167 ‘G’ allele of type I interferon receptor 1 (encoded by IFNAR1 in humans) contributes to stronger type I interferon signalling, impaired colonization and abundance of A. muciniphila, reduced palmitoleic acid production, higher levels of tumour necrosis factor, and more severe tuberculosis disease in humans and transgenic mice. Thus, host genetics are critical in modulating the structure and functions of gut microbiome and gut microbial metabolites, which further determine disease susceptibility.

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Fig. 1: Reduction of A. muciniphila abundance is associated with active tuberculosis infection and higher M. tuberculosis-specific tumour necrosis factor production in humans.
Fig. 2: A. muciniphila may confer anti-tuberculosis protection and reduce tumour necrosis factor expression in wild-type mice.
Fig. 3: A. muciniphila-mediated high levels of palmitoleic acid are associated with anti-TB protection.
Fig. 4: Dietary A. muciniphila-mediated palmitoleic acid reduces tuberculosis pathology, Bacillus burden and tumour necrosis factor production in mice.
Fig. 5: Ifnar1 rs2257167 G allele impairs colonization of A. muciniphila and promotes more severe TB infection.
Fig. 6: Oral gavage of A. muciniphila or palmitoleic acid reduces tuberculosis pathology and Bacillus burdens in M. tuberculosis-infected IFNAR1-p.Val168Val mice.
Fig. 7: Effects of IFNAR1 variants are observed in participants with tuberculosis.

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Data availability

Data of 16S rDNA sequencing are available in a public repository at https://dataview.ncbi.nlm.nih.gov/. The accession number of 16S rDNA sequencing data is PRJNA609532. Figure 1, Extended Data Figs. 1 and 2 have associated raw data. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.

Code availability

Code is available at https://github.com/ZhenhuangGe/Akkermansia-muciniphila/.

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Acknowledgements

This work was supported by grants from the Natural Science Foundation of China (82072250 and 81361120379 to G. Zeng and 81873958 to G. Zhang), the National Science and Technology Major Project (2017ZX10201301, 2017ZX106019 and 2017ZX10103004 to G. Zhang), the Shenzhen Scientific and Technological Foundation (JCYJ20180228162511084 to G. Zhang) and the Sanming Project of Medicine in Shenzhen (SZSM201911009 to G. Zhang).

Author information

Author notes

  1. These authors contributed equally: Lingming Chen, Guoliang Zhang, Guobao Li, Wei Wang, Zhenhuang Ge.

Authors and Affiliations

  1. Department of Microbiology, Zhongshan School of Medicine, Key Laboratory for Tropical Diseases Control of the Ministry of Education, Sun Yat-sen University, Guangzhou, China

    Lingming Chen, Yi Yang, Zhiyi Zhang, Qiongdan Mai, Yiwei Chen, Zixu Chen & Gucheng Zeng

  2. National Clinical Research Center for Infection Diseases, Guangdong Key Laboratory for Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Southern University of Science and Technology, Shenzhen, China

    Guoliang Zhang, Guobao Li, Xing He & Zhi Liu

  3. Department of Clinical Laboratory, Foshan Fourth People’s Hospital, Foshan, China

    Wei Wang

  4. School of Chemistry, Sun Yat-sen University, Guangzhou, China

    Zhenhuang Ge

  5. Department of Microbiology and Immunology, Center for Primate Biomedical Research, University of Illinois College of Medicine, Chicago, IL, USA

    Jiang Pi, Ling Shen & Zheng W. Chen

  6. Key Laboratory of Gene Function and Regulation of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China

    Shuai Yang & Jun Cui

  7. Shanghai Key Lab of Tuberculosis, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, China

    Haipeng Liu & Baoxue Ge

  8. Clinical Research Center, Department of Medical Records Management, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangzhou, China

    Lingchan Zeng

  9. Guangdong Center for Tuberculosis Control, National Clinical Research Center for Tuberculosis, Guangzhou, China

    Lin Zhou

  10. Department of Pathogen Biology, Shenzhen University School of Medicine, Shenzhen, China

    Xinchun Chen

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  1. Lingming Chen
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Contributions

L.C., Z.G., Y.Y., Z.Z., Y.C., Q.M., Z.C., S.Y., L.C.Z. and G.B.L. performed the experiments and analysed the data. G.L.Z., G.L., Z.L., W.W., X.H. and L. Zhou collected clinical samples and conducted analyses. S.Y. and J.C. helped with signalling analysis experiments. X.H. and G.L.Z. performed SNP and clinical analysis works and X.H., Z.L., J.P., J.C., L.S., L.C.Z., H.L., X.C. and B.G. assisted with the data analysis and manuscript preparation. L.C., G.C.Z., Z.W.C., X.C. and B.G. drafted, discussed and revised the manuscript. G.C.Z. conceived the study. L.S. and Z.W.C. used their overtimes at night to help project design, data evaluation/discussion and writing before 2019. This study addressed scientific questions of the US-China Collaborative Biomedical Research program approved by NIH/UIC/NSFC.

Corresponding author

Correspondence to Gucheng Zeng.

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Competing interests

G.C.Z. is a founder of Revaissant Bioscience. All other authors declare no competing interests.

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Nature Metabolism thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Alfredo Giménez-Cassina.

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Extended data

Extended Data Fig. 1 Healthy controls (HC)and active TB patients (TB) exhibit significant differences in diversity, composition, and abundance of gut bacteria at Shenzhen cohort.

(a) PCoA plot (based on Weighted UniFrac distances). Red points indicated the microbiota enriched in HC (n = 28), blue points indicated the microbiota enriched in TB (n = 26). (b) The relative abundance of gut bacteria at phylum level in the fecal samples from HC (n = 28) and TB (n = 26). (c) The top 10 bacterial species with enriched relative abundance in HC were adjusted P < 0.05 and log2 (HC/TB) > 0, species with enriched relative abundance in TB were adjusted P < 0.05 and log2 (HC/TB) < 0 (HC, n = 28; TB, n = 26). The red box indicates A. muciniphila. (d) The absolute abundance of detected A. muciniphila in the fecal samples from HC (n = 28) and TB (n = 26). (e) Predictive power of top 10 species enriched in HC (that is the top 10 most reduced species in TB) assessed by random forest analysis. Blue boxplots acted as benchmarks. Green boxplots represent confirmed species and red boxplots represent rejected species. Red arrowhead marks A. muciniphila. HC = 28, TB = 26. (f) Histogram of the linear discriminant analysis (LDA) coupled with effect size measurements (LEfSe) identified the species with different abundance between HC and TB. Higher abundant species in TB are shaded with green, higher abundant species in HC are shaded with red. Red arrowhead marks the A. muciniphila, black arrowhead marks B. vulgatus. (g) Circle charts marked the relative abundance of six bacteria with differentiated relative abundance between HC and TB in Shenzhen cohort, and these differentiated six bacteria with differentiated relative abundance were also observed at both Shenzhen and Foshan cohorts. Histogram showed the fold changes of relative abundance of six bacteria, including B. vulgatus, B. uniformis, A. muciniphila, B. caccae, P. merdae, and E. ramosun between HC and TB in Shenzhen cohort (fold changes were calculated as HC/TB). Bacteria are identified by color bars above the chart. Data are presented as mean + /- SD. Pvalues were calculated by PERMANOVA test (a), Mann–Whitney U test [(c) and (d)].

Source data

Extended Data Fig. 2 HC and TB show significantly different diversity, composition and abundance of gut bacteria, and abundance of A. muciniphila and B. vulgatus are significantly higher in HC than TB at Foshan cohort.

(a) PCoAplot (based on Weighted UniFrac distances (HC = 17, TB = 19, Foshan cohort). (b) The relative abundance of gut bacteria in phylum level in the fecal samples from HC (n = 17) and TB (n = 19). (c) A. muciniphila was belonged to top 10 bacterial species in fecal microbiota of HC. Species with enriched relative abundance in HC are adjusted P < 0.05 and log2 (HC/TB) > 0, species with enriched relative abundance in TB are adjusted P < 0.05 and log2 (HC/TB) < 0. The red-boxed area marks the A. muciniphila. (d) The absolute abundance of A. muciniphila in the fecal microbiota from HC (n = 17) and TB (n = 19). (e)Predictive power of top 10 species enriched in HC (that is the top 10 most reduced species in TB) assessed by random forest analysis. Blue boxplots correspond to minimal, average, and maximum Z-score of shadow species, which were shuffled version of real species introduced to random forest classifier and act as benchmarks to detect truly predictive species. Red boxplots represent rejected species, yellow boxplots represent suggestive species, and green boxplots represent confirmed species. The red arrowhead marks the A. muciniphila. HC = 17, TB = 19. (f) Histogram of the Linear discriminant analysis (LDA) coupled with effect size measurements (LEfSe) identified the species with different abundance in HC and TB. Higher abundant species in TB are shaded in green, higher abundant species in HC are shaded in red. Red arrowhead pointed A. muciniphila, black arrowhead pointed B. vulgatus. (g) Circle charts showed the relative abundance of six bacteria with differentiated relative abundance between HC and TB in Foshan cohort, and these six bacteria were also observed at both Shenzhen and Foshan cohorts. Histogram showed the fold change of relative abundance of six bacteria, including B. vulgatus, B. uniformis, A. muciniphila, B. caccae, P. merdae and E. ramosun between HC and TB (calculated as HC/TB) in Foshan cohort. Bacteria are identified by color bars above the chart. Data are presented as mean + /- SD. Pvalue was calculated by PERMANOVA test (a), Mann-Whitney test [(c)and (d)].

Source data

Extended Data Fig. 3 The association of M. tuberculosis-specific cytokinesex vivo produced by PBMCs and B. vulgatus in fecal samples of TB patients.

Spearman correlation analysis showed the association between TNF-α, IFN-γ, or IL-10 and the relative abundance of B. vulgatus. There were no significant correlation between B. vulgatus and M. tuberculosis-specific TNF-α, IFN-γ or IL-10. P-value was calculated by Spearman correlation (two-tailed).

Source data

Extended Data Fig. 4 Antibiotics-treated mice show more severe pulmonary pathology and higher bacillus burdens during M. tuberculosis infection.

(a) Two representative lungs derived from M. tuberculosis-infected mice with antibiotics or water control treatment. Red circles mark the cystic changes, hemorrhage or necrosis on the lungs of infected mice. (b-c) Hematoxylin and eosin (H&E) staining of two representative lungs (b) and the histological scores (c) of M. tuberculosis-infected mice with antibiotics or water control treatment at 5 weeks post infection. Scale bars, 500 μm (top: original magnification), 200 μm (middle: 2.5×) and 50 μm (bottom: 10×). The red-boxed areas at the top are enlarged below. As marked by yellow arrowheads, antibiotics-treated mice showed more severe lesions and more infiltration of inflammatory cells than untreated mice. (d) Quantification analysis of M. tuberculosis CFU in the lung homogenates of M. tuberculosis-infected mice at 5 weeks post infection. (e) The acid-fast staining of M. tuberculosis in lung section of mice at 5 weeks post infection. Note that more acid-fast staining-positive bacilli in lung sections derived from antibiotics-treated mice. Scale bars, 5 μm (top: 100× of original magnification). The red-boxed areas at the top are enlarged below. N = 6 mice per group. Data are representative at least two biological replicates.Data are presented as mean + /- SD. P values were calculated by Student’s two-tailed unpaired t-test [(c) and (d)].

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Extended Data Fig. 5 A. muciniphila-mediated effects of reduced TB pathology and bacillus burdens were impaired in the absence of TNF-α signaling.

(a) Experimental diagram for determining whether the effect of A. muciniphila depends on TNF-α signaling during M. tuberculosis infection. Antibiotics pre-treated mice were gavaged with A. muciniphila (2 × 108 CFU) or saline for 3 times per week followed by aerosol M. tuberculosis infection. Mice were injected with 500 μg anti-TNF-α MAb or IgG antibody control at the third day after M. tuberculosis infection and injections were performed every third day for a total of six injections. (b) Gross pathology shows the hemorrhage and necrosis in lungs of M. tuberculosis-infected mice at 5 weeks post infection. Red circles mark the severe, unresolved hemorrhage, massive disruption or caseous necrosis on the lungs of M. tuberculosis-infected mice. (c-d) Hematoxylin and eosin (H&E) staining of four representative lungs (c) and the histological scores (d) of M. tuberculosis-infected mice at 5 weeks post infection. Scale bars, 500 μm (top: original magnification), 200 μm (middle: 2.5×) and 50 μm (bottom: 10×). The red-boxed areas at the top are enlarged below. Yellow arrowheads mark lesions and infiltration of inflammatory cells. (e) Quantification analysis of M. tuberculosis CFU in the lung homogenates of M. tuberculosis-infected mice at 5 weeks post infection. N = 6 mice per group. Data are representative at least two biological replicates.Data are presented as mean + /- SD. Pvalues were calculated by Student’s two-tailed unpaired t-test [(d) and (e)].

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Extended Data Fig. 6 Intestine-colonized A. muciniphila and anA. muciniphila-mediated metabolite palmitoleic acid,inhibit M. tuberculosis-specific TNF-α expression via an epigenetic mechanism.

(a) The experimental diagram of transwell assay to mimic the effects of epithelium-colonized gut bacteria on cytokine production. (b) Representative CBA analysis of culture supernatants of CD3 + T cells in the lower chamber for transwell assay. Red and black-boxed areas mark the fluorescent clusters of TNF-α and IFN-γ, respectively, and dashed line mark the shift of fluorescent clusters of TNF-α and IFN-γ, respectively. (c) Pooled bar graphic data show the expression of TNF-α/IFN-γ/IL-2/IL-4/IL-6/IL-10 in culture supernatants of CD3 + T cells in the lower chamber. N = 7 mice per group. One-way ANOVA, P = 0.0064. (d) Representative CBA analysis of culture supernatants of CD3 + T cells of mice. CD3 + T cells were co-cultured with A. muciniphila lysates (10 μg/ml) or BHI in presence of M. tuberculosis lysates (10 μg/ml). Red and black-boxed areas mark the fluorescent clusters of TNF-α and IFN-γ, respectively, and dashed line mark the shift of fluorescent clusters of TNF-α and IFN-γ, respectively. (e) Pooled bar graphic data show the in vitro cytokines expression in culture supernatants of CD3 + T cells. N = 7 mice per group. (f) CHIP-qPCR analysis of IgG, H3K4Me3, H3K9Me1, H3K9Me3, H3K27Me2 and H3K27Me3, and control antibodies at the promoter of tnfα. N = 7 biologically independent samples. (g) Immunoblot analysis of H3K4Me3, H3K9Me1, H3K9Me3, H3K27Me2 and H3K27Me3. (h) Quantitative immunoblot analysis of expression of H3K4Me3, H3K9Me1, H3K9Me3, H3K27Me2 and H3K27Me3. ImageJ was used for quantitative analysis of immunoblot. N = 3 biologically independent samples. Data in (d) to (h) are from one experiment. Box and whisker plots depict the following: line, median; box limits, first and third quartiles; whiskers, Min to Max and Show all points. Data are representative at least two biological replicates.Data are presented as mean + /- SD. P values were calculated by one-way ANOVA with Tukey’s multiple comparison test (c) and Student’s two-tailed unpaired t-test [(e), (f) and (h)].

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Extended Data Fig. 7 A. muciniphila played a dominant role in contributing to increased levels of palmitoleic acid in mice.

(a) Representative total ion chromatograms (TICs) of metabolites profiles in culture supernatants of A. muciniphila by GC-TOF-MS-based metabolic analysis. Black arrowhead marks the identification of palmitoleic acid (CAS: 373-49-9). (b) Comparative KEGG pathway analyses of palmitoleic acid synthesis and degradation between A. muciniphila (marked with oval black dashed line) and B. vulgatus (marked with green solid line). The green box mark the presence of long-chain-fatty-acid-CoA ligase for degrading/processing palmitoleic acid in B. vulgatus, and the gray box point the lack of presence (or at least lower level) of long-chain-fatty-acid-CoA ligase in A. muciniphila. The solid green arrow mark the downstream metabolic pathways for degrading/processing palmitoleic acid in B. vulgatus, and the black dashed arrow point that no (or at least less) long-chain-fatty-acid-CoA ligase to degrade/process palmitoleic acid in A. muciniphila. (c) Schematic diagram for palmitoleic acid biosynthesis pathway in A. muciniphila. (d) Genes involved in palmitoleic acid biosynthesis pathway in A. muciniphila and B. vulgatus, respectively. (e-f) The existences of genes involved in palmitoleic acid biosynthesis pathway in A. muciniphila and B. vulgatus were confirmed from the bacterial genome DNA and bacterial mRNA. (g) The qPCR-based analysis of fatty acyl-ACP thioesterase (FAT) mRNA expression in fecal samples from M. tuberculosis-infected mice. (h) Genes involved in palmitoleic acid metabolism pathway in gut bacteria, which showed significantly different abundance between HC and TB. These bacteria includeAcinetobacter johnsonii (AJ), Lactobacillus mucosae (LM), Lactobacillus salivarius (LS), Clostridium innocuum (CI), Plesiomonas shigelloides (PS), Comamonas kerstersii (CK), Alistipes onderdonkii (AO), Lactobacillus ruminis (LR), Streptococcus anginosus (SA), Lactobacillus fermentum (LF), Faecalibacterium prausnitzii (FP), Bifidobacterium adolescentis (BA), Bacteroides uniformis (BU) and Bacteroides caccae (BC). (i-j)The relative abundance of A. muciniphila, A. johnsonii (AJ) and L. salivarius (LS) in stool samples, and palmitoleic acid concentrations in plasmaand fecal samples fromantibiotics-treated mice at the third day after bacteria gavage. N = 6 mice per group. Data are representative at least two biological replicates.Data are presented as mean + /- SD. P values were calculated by Student’s two-tailed unpaired t-test [(g) and (i)] and one-way ANOVA with Tukey’s multiple comparison test (j).

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Extended Data Fig. 8 Live but not inactive A. muciniphila reduces TB pathology and bacillus burdens.

(a) Three representative lungs of M. tuberculosis-infected mice treated with live A. muciniphila, inactive A. muciniphila or saline. Red circles mark the severe, unresolved hemorrhage, massive disruption or caseous necrosis on the lungs of M. tuberculosis-infected mice. (b-c) Hematoxylin and eosin (H&E) staining of three representative lungs (b) and histological score (c) of M. tuberculosis-infected mice treated with live A. muciniphila, inactive A. muciniphila and saline at 5 weeks post infection. Scale bars, 500 μm (top: original magnification), 200 μm (middle: 2.5×) and 50 μm (bottom: 10×). The red-boxed areas at the top are enlarged below. Yellow arrowheads mark lesions and infiltration of inflammatory cells. (d) Quantitative analysis of bacillus CFU in lung homogenates of M. tuberculosis-infected mice treated with live A. muciniphila, inactive A. muciniphila and saline at 5 weeks post infection. N = 6 mice per group. Data are representative at least two biological replicates.Data are presented as mean + /- SD. P values were calculated by one-way ANOVA with Tukey’s multiple comparison test [(c) and (d)].

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Extended Data Fig. 9 Knock-out of an IFN-I receptor (Ifnar1) increases the intestinal colonization and abundance of bulk anaerobic bacteria and A. muciniphila.

(a) Experimental design for determining the effect of IFN-I signalingon A. muciniphilaabundance. (b) The proportion analysis of anaerobic bacteria in jejunum, ileum, cecum or stool of Ifnar1-/- mice and wild-type miceat day 18. (c) Pooled data of relative amounts of anaerobic bacteria recovered from jejunum, ileum or cecum of Ifnar1-/- mice (red bar) and wild-type mice (black bar), respectively. Mice were administrated with fecal samples of HC (Left panel) or TB (Right panel) as described in (a), and then tissues of jejunum, ileum or cecum and fecal samples of mice were subjected to anaerobic culture, followed by quantitative analysis of anaerobic bacteria CFU. Fold changes were calculated as CFU of anaerobic bacteria recovered from jejunum, ileum or cecum and fecal samples of mice administrated with fecal samples of HC or TB normalized to those of saline-treated Ifnar1-/- mice and wild-type mice. (d) The abundance analysis of A. muciniphila in jejunum, ileum, or cecum of Ifnar1-/- mice and wild-type mice, respectively. Tissues of jejunum, ileum and cecum of mice were collected at day 18 to analyze the copies of A. muciniphila per gram feces. (e) Pooled data of relative gene expression levels of A. muciniphila recovered from jejunum, ileum, or cecum of Ifnar1-/- mice (red bar) and wild-type mice (black bar), respectively. Mice were administrated with fecal samples of HC (Left panel) or TB (Right panel) as described in (a), and then tissues of jejunum, ileum, or cecum were subjected to qPCR analysis of DNA extracted from jejunum, ileum, and cecum of mice. Fold changes were calculated as gene expression levels of A. muciniphila recovered from jejunum, ileum, or cecum of mice administrated with fecal samples of HC or TB normalized to those of same genotype mice with oral saline administration. N = 6 mice per group. Data are representative at least two biological replicates.Data are presented as mean + /- SD.P values were calculated byStudent’s two-tailed unpaired t-test (d).

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Extended Data Fig. 10 Pooled bar graphic data shows the expression of IL-10, IL-6, IL-2, and IL-4 in culture supernatants of PBMCs derived from TB patients carrying genotype GG, GC, CC.

sPBMCs derived from TB patients carrying genotype GG (n = 185), GC (n = 206) or CC (n = 62) were ex vivo stimulated with M. tuberculosis lysates and analyzed as in Fig. 7. Data are presented as mean + /- SD. P values were calculated by one-way ANOVA with Tukey’s multiple comparison test.

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Chen, L., Zhang, G., Li, G. et al. Ifnar gene variants influence gut microbial production of palmitoleic acid and host immune responses to tuberculosis. Nat Metab 4, 359–373 (2022). https://doi.org/10.1038/s42255-022-00547-3

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