... cytolysins (CDC) produced by Gram-positive bacteria such as. Clostridium and Streptococcus are structurally related to the MACPF family proteins recently.
Supplementary Information for
The oral and gut microbiome are perturbed in rheumatoid arthritis and partly normalized following treatment
Zhang et al.
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Nature Medicine: doi:10.1038/nm.3914
List of contents Supplementary Note........................................................................................................ 3 Supplementary Figures ................................................................................................... 7 Supplementary Figure 1 ........................................................................................... 7 Supplementary Figure 2 ........................................................................................... 8 Supplementary Figure 3 ........................................................................................... 9 Supplementary Figure 4 ......................................................................................... 10 Supplementary Figure 5 ......................................................................................... 11 Supplementary Figure 6 ......................................................................................... 12 Supplementary Figure 7 ......................................................................................... 14 Supplementary Figure 8 ......................................................................................... 16 Supplementary Figure 9 ......................................................................................... 17 Supplementary Figure 10 ....................................................................................... 19 Supplementary Figure 11 ....................................................................................... 20 Supplementary Figure 12 ....................................................................................... 22 Supplementary Figure 13 ....................................................................................... 23 Supplementary Figure 14 ....................................................................................... 24 Supplementary Figure 15 ....................................................................................... 25 Supplementary Tables..................................................................................................... 26 Supplementary References ............................................................................................. 29
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Nature Medicine: doi:10.1038/nm.3914
Supplementary Note Gram-negative bacteria including Escherichia and Bacteroides have been reported to suppress arthritis in animal models, possibly through LPS or other virulence-related factors, while gram-positive bacteria such as Bifidobacterium and Lactobacillus aggravate the disease17,49,50. In colitis-associated CRC mouse models, enterotoxigenic Bacteroides fragilis induces colitis and colonic tumors through a T helper type 17 (Th17) inflammatory response51–53. B. ovatus and B. vulgatus have been reported to be higher in human cases of Crohn’s disease54. Thus, the Gram-negative bacteria relatively depleted in RA patients might provide healthy stimulations for the immune system. These bacteria also serve important functions in nitrogen and sulfur metabolism, which usually take place in the periplasm and could modulate the redox environment for neighboring microbes. The sulphogly-solysis pathway recently discovered in Enterobacteriaceae sustains their growth on sulphoquinovose, a compound abundant in plants and photosynthetic bacteria, and the degradation product of sulphoqunovose, 2,3dihydroxypropane-1-sulphonate, could then be mineralized by other bacteria55.
The increase in Lactobacillus salivarius might also be due to the fact that they do not require iron for growth and could tolerate high concentrations of ROS generated at inflammatory sites. Lactobacillus and Bifidobacterium have actually been found to stimulate NADPH oxidase 1 (Nox1)-dependent ROS generation and intestinal stem cell proliferation56. Lactic acidosis has been observed in RA synovial tissues, a phenomenon attributed to hypoxia, increased glycolysis and nitric oxide production57,58. Moreover, Lactobacillus has been reported to be enriched in the oral cavity of patients with hyposalivation (xerostomia), a common complication of RA and the key symptom of Sjögren's syndrome59–62. L. salivarius was also higher in a mouse model of the autoinflammatory disease osteo-myelitis, in contrast to L. vaginalis and L. reuter enriched in wildtype63.
Due to hepcidin secretion, chronic RA patients show impaired iron absorption at the gut mucosa and increased sequestration of iron by macrophages1. Heme, a metabolite with altered transport and
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Nature Medicine: doi:10.1038/nm.3914
synthesis in the RA gut and oral microbiome, has recently been found to promote development of red pulp macrophages (RPM), cells that degrade senescent erythrocytes and recycle hemeassociated iron64. Hemin (iron(III)-bound heme), on the other hand, could regulate the nuclear export and degradation of the tumor suppressor protein p5365. Hemolysin-producing bacteria could obtain iron from red or white blood cells, while the others express iron transporters, especially iron(III) transporters in RA saliva, to draw on the limited iron resource.
In the patients’ saliva, the sulfate adenylyltransferase was more often the Sat genes, seen in bacteria such as Lactobacillus and Staphylococcus, instead of the CysND genes found in bacteria such as Escherichia and Yersinia, which were accompanied by other Cys genes that further reduce adeno-sine 5’-phosphosulfate (APS) to sulfide (Supplementary Fig. 9b, Supplementary Tables 15,16). In our oral samples, KOs responsible for hydrogen sulfide production correlated with control MLGs such as Aggregatibacter sp., Haemophilus sp. and Kingella dinitrificans, in contrast to RA-enriched Bac-teroides sp. in the gut (Supplementary Tables 17,18). Hydrogen sulfide is an important gaseous messenger molecule implicated in processes such as vasorelaxation, inflammation and bone homeostasis66–68 and has recently been reported to react with nitric oxide to form the TRPA1 agonist nitroxyl (HNO)69.
In addition, cholesterol-dependent cytolysins (CDC) produced by Gram-positive bacteria such as Clostridium and Streptococcus are structurally related to the MACPF family proteins recently identified as being responsible for inducing cellular hypercitrullination, through pore formation and thus calcium influx sufficient to activate endogenous PAD enzymes70. Besides extranuclear targets such as collagen and enolase, PAD enzymes have been reported to citrullinate histones and thus regulate chromatin decondensation and pluripotency71,72, an epigenetic mechanism that might operate in the RA synovium in addition to DNA hypomethylation.
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Nature Medicine: doi:10.1038/nm.3914
Rheumatoid arthritis synovial fibroblasts (RASFs) are known to over-express spermidine/spermine N1-acetyltransferase (SSAT1) which recycles the polyamines spermine and spermidine back to putrescine, leading to S-adenosyl methionine (SAM) depletion and global DNA hypomethylation in RASFs73. Enrichment of the methionine salvage pathway in the patients’ oral samples but the control gut samples might suggest differences in the concentration of homocysteine (Fig. 1, Supplementary Tables 5,15,16), a metabolite implicated in various conditions including cardiovascular diseases.
E. faecalis was known to bind platelets, cells with emerging antimicrobial and immune-modulating functions in addition to their traditional role in coagulation26,27. L. salivarius was reported to be able to aggregate platelets74. RA oral MLGs might even be used to predict platelet counts (R2 = 0.1676 for dental, 0.2451 for salivary, Supplementary Tables 13,14).
Among the gut MLGs, the RA-enriched Eggerthella lenta and Bacteroides sp. (20_3) and the controlenriched Haemophillus (parainfluenzae) were also identified from a Chinese study on type 2 diabetes20 as enriched in patients and controls, respectively, suggesting common features in dysbiosis and inflammation. Interestingly, Eggerthella lenta produces ATP through the arginine dihydrolase pathway, could inactive the cardiac drug digoxin75, and is responsible for synthesizing the non-steroidal estrogen equol76,77. Eggerthella and Bacteroides proteins also contained motifs similar to RA-related human antigens (Supplementary Table 19). Further investigations would be necessary to explore their potential role in the metabolism of hormones and drugs, and immune homeostasis.
DMARD treatment led to promising changes in the gut, dental and saliva microbiome. Increases in control-enriched bacteria at least serve as markers for, if not directly contributing to reduction in the RA autoantibodies, anti-CCP and RF. No decrease, however, could be detected for Lactobacillus salivarius, whose presence correlated with very active RA (Fig. 3, Fig. 6), indicating that other thera5
Nature Medicine: doi:10.1038/nm.3914
pies might be needed to target this bacterium. Our data suggest that patient stratification based on the gut and oral microbiome before DMARD treatment might facilitate selection of the optimal treatment strategy, which could be further adjusted according to the gut and oral microbiome during or after treatment. Dietary interventions and traditional Chinese medicine in addition to standard drugs would possibly facilitate recovery of the microbiome. Similar strategies might also apply to the treatment of other autoimmune diseases such as systemic lupus erythematosus (SLE) and ankylosing spondylitis78,79.
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Nature Medicine: doi:10.1038/nm.3914
Supplementary Figures
a
Shannon index 12.5
All (n=157) RA (n=77) Control (n=80)
1.0
12.0
Density
Shannon index
Shannon index distribution
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11.5 11.0
0.8 0.6 0.4
10.5
0.2
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RA Control
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Shannon index
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Gene counts distribution 0.020
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Density
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80 60
All (n=157) RA (n=77) Control (n=80)
0.010 0.005
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Supplementary Figure 1
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RA Control
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Diversity of the gut microbiome in RA. Box plot and distribution of
Shannon index at gene level (a) and gene count (b) in RA cases and controls.
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Nature Medicine: doi:10.1038/nm.3914
120
Gene counts( 10 ) x
Gene counts(×105 )
Gene counts
a
b
10 Yr
Dental −4
RA−2767
−6
MLG abundance(lg)
MLG abundance (lg)
PC2(20.5%)
−4
PC1(28.1%)
c
Gut
−8
Control 10 Yr
−10
−12
d
n=80 n=10 n=5 n=11 n=3 n=19 n=10 n=4 n=15
Con−4759
Centipeda periodontii
Con−2374
−6
−8
−10
−12
Control 10 Yr
n=50 n=6 n=3 n=8 n=1 n=11 n=12 n=2 n=7
Saliva −4
Con−536
Con−4088
Oribacterium sp.
Con−993
RA−7843
RA−7834
Streptococcus sp.
RA−7823
RA−11401
Shuttleworthia satelles
MLG abundance(lg)
−6
−8
−10 Control
