nat. immunol 18, 552–562 (2017) gut microbial metabolites ... · data analyzed by one -way anova...

Nat. Immunol. 18, 552–562 (2017)

Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetesEliana Mariño, James L Richards, Keiran H McLeod, Dragana Stanley, Yu Anne Yap, Jacinta Knight, Craig McKenzie, Jan Kranich, Ana Carolina Oliveira, Fernando J Rossello, Balasubramanian Krishnamurthy, Christian M Nefzger, Laurence Macia, Alison Thorburn, Alan G Baxter, Grant Morahan, Lee H Wong, Jose M Polo, Robert J Moore, Trevor J Lockett, Julie M Clarke, David L Topping, Leonard C Harrison & Charles R Mackay

In the supplementary information originally posted online, Supplementary Tables 2 and 3 were missing titles and legends. The error has been corrected in this file as of 20 September 2017.

CORRECT ION NOT ICE

Supplementary Figure 1.

Effect of SCFAs on important parameters for diabetes development.

Nature Immunology: doi:10.1038/ni.3713

(a) T1D incidence in germ-free (GF) NOD.MyD88–/–

mice (n=11) vs. specific pathogen-free (SPF) NOD.MyD88–/–

mice (n=14); ****P<0.0001, Mantel-Cox log rank test. Concentrations of acetate, butyrate and propionate in

feces of (b) 5-10 week-old female SPF and GF NOD and NOD.MyD88–/–

mice, and (c) NP-fed age-matched

female vs male NOD and C57BL/6 mice, n ≥ 4. (d) Body weights of 15 week-old female NOD mice (n=5 per

group) fed with NP, HAMS, HAMSA or HAMSB diets. (e) Energy intake measured from NOD mice fed different

diets. Panels (b,c,e) were analyzed by one-way or (d) two-way ANOVA with Bonferroni’s multiple comparisons

test. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. For all graphs, data represent mean ± SEM; each symbol

represents an individual mouse. All data are representatives of three independent experiments. (f) T1D

incidence in female NOD mice fed for 5 weeks with HAMSA diet starting at age 5 weeks (n=11) or 10 weeks

(n=15). Blue arrows in the graph represent the start and end time point on HAMSA diet. NS= not significant (5

weeks vs 10 weeks). (g) T1D incidence in HAMSA- and HAMSB-fed female NOD mice starting from birth to 30

weeks of age (n=10 per group). Mantel-Cox log rank test.


Supplementary Figure 2

Effect of dietary SCFAs on autoreactive T cells.


(a) Gating strategy used for identification of autoreactive IGRP tetramer+

CD8+ T cells and BDC2.5 tetramer

+ CD4

+ T cells.

TUM and hu CLIP were used as tetramer controls respectively. Data is representative from 10,000 events acquired from the

total viable lymphocyte gate. Representative plots and cumulative data showing frequency and number of PLN and splenic

(b) autoreactive IGRP tetramer+

CD8+ gated from total CD8

+ T cells and (c) BDC2.5 tetramer

+ CD4

+ gated from total CD4

+ T

cells from 15 week-old female NOD mice fed NP, HAMS, HAMSA or HAMSB diet. Data from HAMS and HAMSA for spleen

in b is the same as that is shown in Figure 3a. Numbers shown within the plots are representative of the frequency of

tetramer+

T cells. (d) Representative plots and cumulative data showing frequency and number of IGRP tetramer+

CD8+ T

cells in 6 week-old NOD8.3 mice. All data represent mean ± SEM. ****P<0.0001, **P<0.001, *P<0.05 (NP vs HAMS; HAMSA

vs HAMS; HAMSB vs HAMS). Data analyzed by one-way ANOVA with Bonferroni’s multiple comparisons test. Data is

representative of at least two independent experiments, n ≥ 2.



SCFAs modulate B cell subsets and surface marker expression.

(a) Gating strategy used for splenic B cell subsets identification. (b) Cumulative data showing frequency (upper panel) and

numbers (lower panel) of TR B cells, T2-MZ B cells, FO B cells and MZ B cells. ****P<0.0001, **P<0.01, *P<0.05 (NP vs

HAMS; HAMSA vs HAMS; HAMSB vs HAMS). Data shown is from three independent experiments, n ≥ 4. (c) Mean

fluorescence index (MFI) +/- SD of surface protein expression for MHCI and CD86 on a per-cell basis on splenic IgM+B220

+ B

cells (NOD mice fed with diets), n ≥ 5. ***P<0.001, **P<0.01, *P<0.05, NS (HAMSA vs HAMS; HAMSB vs HAMS). (d) Real

time PCR showing expression of CD86 and Il12 on sorted splenic CD21high

CD23low

(MZ B) and CD21mid

CD23high

(FO B)

cells (gated from total IgM+B220

+ B cells), n=3. **P<0.01 (HAMSA vs NP; HAMSB vs NP). (e) Real time PCR showing fold

change in expression of C80/B7.1, C80/B7.2, B2M and PRDM1 relative to -actin in splenic IgM+B220

+ B cells from of 15

week-old HAMS-, HAMSA-, or HAMSB-fed female NOD mice, n ≥ 4. **P<0.01, *P<0.05, NS (HAMSA vs HAMS; HAMSB vs

HAMS). Data in panels b-e are representative of at least three experiments analyzed by one-way ANOVA with Bonferroni’s

multiple comparisons test.



SCFA effect on gene expression and APC function.

(a) Gene expression profile between purified splenic IgM+B220

+ B cells from HAMSA (x-axis) against HAMSB (y-axis),

relative to HAMS contrast. Circles in red represent log2 fold-change expressed genes with FDR < 0.05 for differential

expression test between HAMSA and HAMSB diets. (b) Cumulative data showing frequency of CFSE-labeled NOD.8.3 CD8+

T as CFSE–V8.1/8.3

+CD8

+ T cells in PLN. **P=0.0042 (HAMSA vs HAMS); NS (NP vs HAMS and HAMSB vs HAMS). (c)

Cumulative data showing frequency of CFSE–V8.1/8.3

+CD8

+ T cells in MLN. (d)

3[H]-thymidine uptake by CD4

+CD25

– T

cells cultured with LPS stimulated B cells and DCs from NOD mice in the absence or presence of 100 µM acetate or butyrate

pulsed or not with insulin peptide ± IL-2 in vitro. B cells ##

P=0.0015 (No SCFAs, insulin vs no peptide), **P=0.021, *P=0.001,

(Insulin, acetate vs PBS; butyrate vs PBS) and DCs *P=0.0318 (Insulin, butyrate vs PBS). Panels b and c analyzed by one-

way ANOVA and d by two-way ANOVA with Bonferroni’s multiple comparisons test. Data is representative of three

reproducible experiments.



SCFAs affect Treg cells from colon and peripheral tissue.


(a) Cumulative data showing frequency and numbers of CD4+FoxP3

+CD103

+ T cells from total CD4

+ T cells isolated from the

colon of 15 week-old female NOD mice fed HAMS, HAMSA or HAMSB diet. Data represent mean ± SD, n=5 mice. **P<0.01

(HAMSA vs HAMS; HAMSB vs HAMS). (b) Facs plots showing frequency of splenic, PLN and MLN CD4+FoxP3

+ T cells.

Data represent mean ± SEM, n=5-6 mice. (c) Gating strategy used for identification of CD4+

Foxp3+ T cells gated from total

splenic CD4+ T cells. Data is representative from 10,000 events acquired from the total viable lymphocyte gate. (d) Flow

cytometric analysis of Foxp3 protein expression on a per-cell basis from splenic and PLN Foxp3+

Treg cells from NOD-SCID

recipient mice after 3 weeks post-transfer of CD4+Foxp3

– T cells from NOD.FoxP3-GFP mice, fed with different diets as

indicated. The data are shown as mean fluorescence intensity (MFI) mean ± SEM. Spleen **P=0.0023 (HAMSA vs HAMS);

****P<0.0001 (HAMSB vs HAMS). (e) Gating strategy used for single sorted CD45RBlow

CD25+CD4

+ T cells from PLN

showing expression of Foxp3. (f) Flow cytometric analysis of Foxp3 protein expression on a per-cell basis from PLN Foxp3+

Treg cells from 15 week-old NOD/Lt mice fed with HAMS, HAMSA and HAMSB diets as indicated. *P<0.048 (HAMSB vs

HAMS). Data analyzed by one-way ANOVA with Bonferroni’s multiple comparisons test. Data are representative of at least

three independent experiments.



The role of the metabolite-sensing receptor GPR43 in NOD mice.


(a) C57.Gpr43–/–

mice were backcrossed 13 generations onto the NOD strain (NOD.Gpr43–/–

). Once fully

backcrossed, NOD.Gpr43–/–

mice were genotyped at over 70,000 SNPs genome-wide. DNA from liver was purified and

genotyped using the Mega-MUGA array (Geneseek, NB). Genotypes were compared to the reference (C57BL/6) alleles and

to NOD alleles determined by the NOD genome sequence. The knockout was produced on the FVB background, so

haplotypes are depicted as coming from the NOD genome (blue) or non-NOD genomes (red); grey indicates non-informative

regions in which C57BL/6 and NOD have the same genotypes. This figure shows the strain of origin of haplotypes on each

mouse chromosome, with the physical size of each chromosome shown on the X axis. This analysis demonstrated that all

chromosomes were derived from the NOD strain, except for a region around the Gpr43 locus on chromosome 7. Thus, these

mice harbor all NOD T1D susceptibility loci including the Idd7 and Idd27 loci mapped to ~19Mb and ~80-120 MB

on chromosome 7, respectively. This enabled us to confirm that there have been no T1D susceptibility genes reported in the

non-NOD interval. (b, c) T1D incidence in female NOD.Gpr43+/+

and NOD.Gpr43–/–

littermates fed NP, HAMSA diet.

*P=0.0392 (NOD.Gpr43+/+

mice vs NOD.Gpr43–/–

fed HAMSA diet); *P=0.0067 (NOD.Gpr43-/-

mice, HAMSB vs NP). Mantel-

Cox log-rank test. Data shown is from two independent experiments (NOD.Gpr43–/–

on HAMSA in b were from a different

experiment to those shown in c). (d) Cumulative data showing absolute numbers of splenic and PLN CD4+FoxP3

+ T cells and

(e) IgM+B220

+ B cells, n ≥ 5. Data were analyzed by one-way ANOVA with Bonferroni’s multiple comparisons test. Data

shown is from three independent experiments. (f) Frequency of PLN autoreactive IGRP tetramer+

CD8+ and IA

g7/BDC2.5

tetramer+

CD4+ T cells from 15 week-old female NOD.Gpr43

+/+ and NOD.Gpr43

–/– mice fed NP or HAMSA diet. Data were

analyzed by one-way ANOVA with Bonferroni’s multiple comparisons test. IGRP+CD8

+ T cells ***P<0.001 (NOD.Gpr43

–/– vs

NOD.Gpr43+/+

NP-fed; NOD.Gpr43–/–

vs NOD.Gpr43+/+

HAMSA-fed; NS, NOD.Gpr43+/+

fed HAMSA vs NP); BDC2.5 CD4+ T

cells (NOD.Gpr43+/+

, HAMSA vs NP; NS, NOD.Gpr43–/–

vs NOD.Gpr43+/+

NP-fed; NOD.Gpr43–/–

vs NOD.Gpr43+/+

HAMSA-

fed). Data represent mean ± SEM. Data shown is from three independent experiments.



Acetate is associated with beneficial changes in gut microbial ecology.

(a) Microbial profile analysis of feces from NOD donors and GF re-colonized NOD mice fed HAMS, HAMSA and HAMSB diet

by PCoA diagrams based on unweighted (left panel) and weighted (right panel) Unifrac distance metrics. The bacterial

communities of different diets were significantly different based on both weighted and unweighted (P<1E-7

) Unifrac, 1E7

permutations and Adonis permutational multivariate statistics. The legend represents the microbiota from donors NOD mice

(circles) and GF NOD mice re-colonized by fecal transfer (FT) with HAMS, HAMSA and HAMSB modified microbiota in the

same colour (squares). (b) Relative abundance of selected bacterial populations at (genus level) in donor NOD mice fed NP,

HAMS, HAMSA or HAMSB diet and in GF NOD mice after fecal transfer. Data were analyzed by one-way ANOVA with

Bonferroni’s multiple comparisons test. *P<0.05 (HAMSA vs HAMS; HAMSB vs HAMS; HAMSA.FT vs HAMS.FT); **P<0.01

(HAMS vs NP). Data represent mean ± SEM; each symbol represents individual mice. All data are representatives of two

independent experiments. (c) Bar chart showing distribution of genera detected in feces from SPF donor NOD mice and GF

NOD mice after fecal transfer (FT) for different diets, n=5-6 per group. Each genus is represented by a different colour and is

proportional to the relative abundance in each sample. The legend shows the genera with relative abundance higher than

1%.


Maize starch - 3401C 379.5 379.5 379.5 379.5 229.5HAMS 150 0 0 0 150HAMSA 0 150 0 0 0HAMSP 0 0 150 0 0HAMSB 0 0 0 150 150Casein 200 200 200 200 200Sucrose 100 100 100 100 100Sunflower Seed Oil 70 70 70 70 70alpha cellulose 50 50 50 50 50Mineral Mix AIN 93G 35 35 35 35 35Vitamin Mix AIN 93VX 10 10 10 10 10L-Cystine 3 3 3 3 3Choline bitartrate 2.5 2.5 2.5 2.5 2.5Total 1000 1000 1000 1000 1000

15% HAMSA/B

Ingredient 15% HAMS 15% HAMSA 15% HAMSP 15% HAMSB

Supplementary Table 1. Calculated nutritional parameters from diets used in this study.

Approximate ingredients from the non purified diet used in this study.Ingredient Non-purifiedMin. Crude Protein 14%Max. Crude Fat 4%Max crude Fibre 6%Metabolizable energy 11mj/kgLysine 4.6 g/kgMethionine & cystine 3.5 g/kgThreonine 4.0 g/kgHistidine 3.1 g/kgLeucine 10.0 g/kgArginine 6.7 g/kgValine 5.6 g/kgIsoleucine 3.8 g/kgPhenylaline & Tyrosine 9.9 g/kgTryptophan 2.0 g/kg


Marino et al. Supplementary Table 2

Putative gene functionMouse gene P-value Fold Change P-value FDR Fold Change P-value FDR Fold Change P-value FDR Fold Change P-value FDR

Hspa1b 2.38x10-11 3.752129344 8.05x10-7 0.000110908 -0.89623749 0.19 0.62422829 -4.70185541 3.72x10-9 2.02x10-6 3.805617929 6.97x10-7 0.00045324 Protein folding and stability

Hspa1a 3.05x10-10 3.809996495 8.05x10-7 0.000115422 -0.73735795 0.29 0.70519504 -4.24136796 8.14x10-8 2.77x10-5 3.504010014 4.95x10-6 0.00211719 Protein folding and stability, protein ubiquitination

Scand1 4.27x10-12 -0.04190582 0.86 0.959720438 1.038765285 2.56x10-5 0.00968193 -0.7619719 1.94x10-3 0.07324618 1.800737183 8.54x10-13 4.63x10-9 Antigen presentation and BCR signalling, transcriptional activity (?)

Sik1 4.42x10-21 1.622604662 6.44x10-9 2.27593E-06 -1.20630541 1.3x10-5 0.00595178 -2.68899185 2.07x10-20 1.68x10-16 1.482686447 1.06x10-7 0.00013211 Antigen presentation, HDAC inhibition

Bpgm 9.02x10-8 -0.565455182 0.09 0.399442055 1.736110063 3.13x10-7 0.00063576 0.266462255 0.42 0.83982193 1.469647808 1.26x10-5 0.00371354 NFKB and antigen presentation

Pex11a 4.09x10-4 0.666211038 0.07 0.357378131 -0.00621757 0.99 0.99804971 -1.43338041 1.53x10-4 0.01247286 1.427162836 2.63x10-4 0.02780121 Dysfunction in gene linked to T1D

Ddit4 4.41x10-20 0.775371036 5.95x10-4 0.020289264 -1.03054346 5.68x10-6 0.00346103 -2.23944571 1.62x10-21 2.64x10-17 1.208902249 1.19x10-7 0.00013775 Cell growth, proliforation and survival

Bcl7c 2.15x10-9 0.072874061 0.67 0.881557363 0.534614945 1.76x10-3 0.10661562 -0.58956646 5.64x10-4 0.03263404 1.12418141 7.66x10-11 3.11x10-7 Unknown function, possible cell survival

nudc 2.6x10-7 0.368644876 0.02 0.172017871 0.025389948 0.87 0.96487045 -0.75800898 8.03x10-7 0.00018647 0.783398932 3.72x10-7 0.00029179 Cytokinesis and cell proliforation

Plaur 2.94x10-23 1.109035669 8.74x10-16 4.73x10-12 -0.67759214 7.79x10-7 0.00115079 -1.25604865 1.09x10-19 5.91E-16 0.57845651 2.49x10-5 0.00587923 Antigen presentation

Pde2a 4.9x10-4 -0.28229256 0.05 0.297190851 -0.02392869 0.87 0.96422568 0.480204947 6.66x10-4 0.03643162 -0.50413364 3.69x10-4 0.03403973 Antigen processing and presentation

slfn8 4.5x10-4 0.088903806 1.11x10-4 0.825353554 -0.26487679 0.08 0.48609691 0.343844109 0.02 0.28424977 -0.6087209 5.21x10-5 0.00976277 Modulates T cell development

Pydc3 1.56x10-3 -0.00691397 1.11x10-4 0.994152665 -0.59087823 0.02 0.28957283 0.368394563 0.13 0.59158602 -0.95927279 9.22x10-5 0.01318228 Transcriptional repressor, linked to increased IL-10 production in T cells

Gnb3 6.05X10-4 0.440355251 0.58 0.83800049 -2.55018401 0.03 0.37498951 1.425253743 0.04 0.3977084 -3.97543775 1.79x10-4 0.02207143 Promotes HDAC3 expression

HAMSA relative to HAMS HAMSB relative to HAMS HAMSA relative to HAMSBHAMS relative to NPEdgeR all 4 groups

Columns left to right: 15 week-old HAMS- vs NP-fed female NOD mice; HAMSA- vs HAMS-fed female NOD mice; HAMSB- vs HAMS-fed female NOD mice and HAMSA- vs HAMSB-fed female NOD mice. Data is expressed as mean fold change in gene expression, P-values were obtained for all four groups together and between paired groups using EgdeR by carrying out a likelihood ratio test after fitting genewise dispersions using its own algorithm (prior counts setting = 0.125). A false discovery rate (FDR) cut-off of q<0.05 was used to define differentially expressed genes between groups. HAMSA up-regulated 10 of 14 transcript genes, Hspa1b, Hspa1a, Scand1, Sik1, Bpgm, Pex11a, Ddit4, Bcl7c, nudc and Plaur, compared to HAMSB (left to right, fourth column). Meanwhile, HAMSB down-regulated 4 of 14 transcript genes, Pde2a, slfn8, Pydc3 and Gnb3, (left to right, forth column). Down-regulated genes highlighted in blue and up-regulated genes highlighted in orange. Less expression shown in lighter colors and higher expression shown in darker colors.

Differentially expressed transcripts in splenic IgM+B220+ B cells from NOD mice fed with different diets.

Nature Immunology doi:10.1038/ni.3713

Marino et al. Supplementary Table 3

Primer name Forward sequence Reverse sequence

5’-ACCAGAGGCATACAGGGACA-3’ 5’-CTAAGGCCAACCGTGAAAAG-3’

5’-TCCGTGAGGCCTTTTGAA-3’ 5’-GGTGCATAATGATTGGGTTTG-3’

5’-GCCAGATATCTATCAGATTGCAAA A-3’ 5’-GATGCCAGAGCTACGATGG-3’

5’-ACCGGA AGTGACTCGAAATGATGT-3’ 5’-CTTCAGAACCACTGCCCTCGTAAT-3’

5’-TCCTGACCAAGAGCGAACAC-3’ 5’-ACAGCACGACAGTCTTCAGG-3’

Actb

Ocln

Tjp1

Cdh1

Muc2B7-1

B7-2

Prdm1

B2m

5’-CACGAGCTTTGACAGGAACA-3’ 5’-TTAGGTTTCGGGTGACCTTG-3’

5’-CAG AGAGCA GAG CAC CTC AG-3’ 5’-ACA TGT AGC AAGGCC ATC AA-3’

5’-CCT TCA GCAAGGACTGGTCT-3’ 5’-TGT CTC GAT CCC AGT AGA CG-3’

5’-TTCCCAGCAATGACAGACAG-3’ 5’-CCATGTCCAAGGCTCATTCT-3’

List of oligo(dT)18 primers used to amplify mRNA for RT-PCR expression analysis.

Nature Immunology doi:10.1038/ni.3713

nat. immunol 18, 552–562 (2017) gut microbial metabolites ... · data analyzed by one -way anova...

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