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SUPPLEMENTARY INFORMATION

ZAP-70 genotype disrupts the relationship between microbiota and host, leading to

spondyloarthritis and ileitis in SKG mice

Linda M REHAUME PhD1, Stanislas MONDOT PhD2, Daniel AGUIRRE DE CÁRCER

PhD2, Jared VELASCO BSc (Hons)1, Helen BENHAM MBBS1, Sumaira Z HASNAIN

PhD3, Jaclyn BOWMAN BSc1, Merja RUUTU PhD1, Philip M HANSBRO PhD4, Michael A

MCGUCKIN PhD3, Mark MORRISON PhD2 and Ranjeny THOMAS MD1

1 The University of Queensland Diamantina Institute, Brisbane, QLD, Australia

2 Animal, Food and Health Sciences, CSIRO, Queensland Bioscience Precinct, Brisbane,

QLD, Australia

3 Mater Research Institute – The University of Queensland, Translational Research Institute,

Brisbane, QLD, Australia

4 Centre for Asthma and Respiratory Disease, Hunter Medical Research Institute and

University of Newcastle, Newcastle, Australia.

DOI: 10.1002/art.38773

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Supplementary Figure 1

Supplementary Figure 1. Microbiota increase enthesitis incidence but the composition does

not affect severity. Representative photomicrographs of naive specific pathogen-free (SPF)

SKG (A), and SPF SKG (B), germ-free (GF) SKG (C) and Charles River altered Schaedler

flora (ASF)-recolonized GF SKG (E) ankle joints 8, 14 and 8 weeks post-curdlan,

respectively. The entheseal site of attachment of the Achilles tendon to the calcaneus is

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indicated (arrows). Scale bars equal 1000 m at 4X magnification. Histological scores of

enthesitis for GF SKG (D) and ASF-recolonized GF SKG (F) ankle joints 14 and 8 weeks

following curdlan treatment, respectively, relative to 8 week curdlan-treated SPF SKG ankle

joints. In D, F each dot represents an individual mouse.

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Supplementary Figure 2

Supplementary Figure 2. The incidence of arthritis and ileitis triggered by curdlan is

reduced in germ-free conditions. SKG mice were housed under specific pathogen-free (SPF)

conditions or rederived to germ-free (GF) facilities, treated with curdlan and then monitored

for 8 weeks (SPF) or 14 weeks (GF). BALB/c mice were housed under SPF or GF conditions,

treated with curdlan, and then monitored for 8 weeks. GF SKG mice were recolonized with

altered Shaedler flora (ASF) for three weeks followed by curdlan treatment and analysis 8

weeks later. The incidence of ankle arthritis, vertebral spondylitis, and ileitis of SKG and

BALB/c mice under SPF and GF conditions, or after ASF recolonization, is shown.

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Supplementary Figure 3

Supplementary Figure 3. The effect on microbiota composition caused by curdlan is similar

in cecal and fecal samples in SKG and BALB/c mice. Double principal coordinate analyses

based on the bacterial community profiles of the cecal (A,C) and fecal (B,D) samples from

the BALB/c (A,B) and SKG (C,D) mice prior to curdlan (day 0), and 3, 7, and 14 days after

curdlan. This analysis groups the samples based on the phylogenetic relationships and

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abundances of the bacterial species present in their resident microbiota. The labels and

colours represent the different sampling times. Corresponding cecal and fecal samples from a

total of 12 BALB/c and 25 SKG mice were analyzed.

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Supplementary Figure 4

Supplementary Figure 4. A significant cage effect was determined for the bacterial

composition in co-housed SKG and BALB/c mice. The dataset for each sample was subjected

to a Principal Component Analysis, and the results were then constrained for cage (G1 to

G4), using Between Class Analysis (A). A Monte Carlo test was computed to assess the

robustness of the observed clustering result (p value 0.001). OTUs specific to each cage were

removed and the samples re-clustered (B). No taxonomic specificity was found related to a

cage, therefore the overall diversity was not skewed by the cage effect.

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Supplementary Figure 5

Supplementary Figure 5. Low abundance of segmented filamentous bacteria (SFB) in local

UQBRF and UQAIBN animal houses does not correlate with disease severity. DNA was

extracted from the cecal contents of SKG and BALB/c mice housed at UQAIBN, SKG mice

housed at UQBRF, and BALB/c mice from ARC, and quantitative real-time PCR was

performed using SFB-specific (A) and total bacteria (TB) primers (B). Quantification was

performed using 10-fold serial dilutions of an amplicon generated from a mix of SKG and

BALB/c DNA. The relative SFB gene copy number expressed as a percentage of the total

bacterial gene copy number (C). Histological score of ankle joint (D), vertebrae (E), and

ileum (F) of SKG mice housed at UQBRF or UQAIBN animal facilities 8 weeks following

curdlan treatment. The p values of significantly different means measured using one-way

ANOVA with Tukey's multiple comparisons test (A-C) or the Mann-Whitney t test (D-F) are

indicated as * p<0.05. Error bars represent SEM. Each dot represents an individual mouse. In

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A-C each group contained samples from 5 mice, and in D-F the UQBRF SKG group had 12-

16 mice, depending on tissue type, and the UQAIBN SKG group had 8 mice. For comparison

the scores for the curdlan-treated UQAIBN SKG mice are from the same mice as the curdlan-

treated SPF SKG mice in Figure 1A-C.

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Supplementary Figure 6

Supplementary Figure 6. SKG mice housed under SPF (white bars) or GF (red bars)

conditions were treated i.p. with curdlan and ileum was collected 3 or 7 days later, or from

naive mice (day 0) as controls. sXbp1 mRNA expression relative to Gapdh mRNA expression

was determined by quantitative real-time PCR. The p value of significantly different means

between SPF and GF conditions at each time point, using two-way ANOVA with Tukey's

multiple comparisons is indicated as * p<0.05. The numbers of mice range from 3-5.

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Supplementary Figure 7

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Supplementary Figure 7. SKG mice housed under SPF or GF conditions were treated i.p.

with curdlan and tissues collected 3 or 7 days later or from naive mice (day 0) as controls. Il6

(A,B), Il1b (C,D), and Tgfb1 (E,F) mRNA expression in the ileum, mesenteric lymph nodes

(MLN), spleen, inguinal lymph nodes (ILN) and liver, relative to Hprt mRNA expression,

was determined by quantitative real-time PCR. The p values of significantly different means,

measured using one-way ANOVA with Tukey's multiple comparisons test are indicated as *

p<0.05, ** p<0.01. The box plot whiskers represent the minimum and maximum values. For

Il6 and Il1b, the numbers of mice range from 1-7 with a median of 6. For Tgfb1, the numbers

of mice range from 2-5 with a median of 3.

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Supplementary Table 1. The OTU identifiers (ID) and corresponding bacterial species,

listed in the same order, from the heatmap in Figure 3F.

OTU ID SPECIES 16923 Prevotella shahii 15657 Clostridium xylanolyticum14415 Barnesiella intestinihominis 12636 Prevotella loescheii 5378 Roseburia intestinalis 3202 Barnesiella intestinihominis 2489 Butyricimonas virosa 1804 Butyricimonas virosa 1240 Alistipes shahii 261 Prevotella dentasini 249 Howardella ureilytica 192 Clostridium proteolyticum 189 Roseburia inulinivorans150 Barnesiella intestinihominis103 Alistipes finegoldii 72 Butyricimonas virosa 150 Barnesiella intestinihominis17426 Odoribacter laneus 13425 Butyricimonas virosa 12636 Prevotella loescheii 2129 Clostridium amygdalinum 1240 Alistipes shahii 192 Clostridium proteolyticum189 Roseburia inulinivorans 150 Barnesiella intestinihominis72 Butyricimonas virosa 18323 Barnesiella intestinihominis 17247 Butyricimonas virosa 17240 Odoribacter laneus 15905 Barnesiella intestinihominis 15686 Barnesiella intestinihominis15597 Barnesiella intestinihominis15294 Barnesiella intestinihominis 14004 Barnesiella intestinihominis11432 Lactococcus plantarum 4494 Bacteroides acidifaciens 1164 Barnesiella intestinihominis685 Butyricimonas virosa 639 Tannerella forsythia 200 Bacteroides acidifaciens120 Barnesiella intestinihominis90 Ruminococcus flavefaciens 65 Barnesiella intestinihominis

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4 Barnesiella intestinihominis 862 Butyricimonas virosa 417 Barnesiella intestinihominis32 Barnesiella intestinihominis 18387 Ruminococcus gauvreauii15384 Barnesiella intestinihominis14618 Barnesiella intestinihominis 14391 Marvinbryantia formatexigens11651 Barnesiella viscericoli 11445 Barnesiella intestinihominis 11061 Barnesiella intestinihominis10927 Butyricimonas virosa 10284 Eubacterium ramulus 2457 Barnesiella viscericoli 923 Eubacterium ramulus 321 Barnesiella intestinihominis 215 Barnesiella viscericoli 122 Barnesiella intestinihominis69 Clostridium leptum 63 Butyricimonas virosa 44 Meniscus glaucopis 12500 Bacteroides acidifaciens 1638 Bacteroides acidifaciens1304 Barnesiella intestinihominis215 Barnesiella viscericoli

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Supplementary Methods and Materials

DNA extraction and pyrosequencing

DNA was extracted from the samples using the rapid bead beating plus column method (1,

2). The quality and quantity of DNA was determined by agarose gel electrophoresis, and

spectrophotometry (ND-1000, Nanodrop Technologies) or fluorometry (Qubit, Life

Technologies). The V1-V3 region of the rrs-genes from each DNA sample was PCR-

amplified using a barcoded pyrotagging approach with fusion primers containing the Roche-

454 sequencing adapter followed by a small linker segment and the 5’ end of either primer 8f

or 515r. The reverse primer carried a short barcode sequence after the linker sequence. The

resulting products were quality checked by agarose gel electrophoresis. Equal amounts of

each product were pooled and cleaned by gel extraction using the QIAquick Gel Extraction

Kit (QIAGEN). The resulting product, checked again by agarose gel electrophoresis, and

spectrophotometry or fluorometry, was then sequenced using a Roche 454 GS FLX

sequencer with titanium chemistry.

Sequence processing and data collection

The sequences obtained were initially processed using QIIME (3) (Figure 3), or QIIME and

MOTHUR (4) (Supplementary Figure 3). The sequences were first binned using their

respective barcodes, filtered for correct length and quality values (length = 250-600 bp Figure

3 or 350-550 bp Supplemental Figure 3, mean quality score = >25, maximum homopolymer

length = 10 Figure 3 or 6 Supplemental Figure 3, and maximum mismatches in primer = 0),

and grouped into operational taxonomic units (OTUs) at 97% sequence similarity distance. A

representative sequence from each OTU was used to obtain a proxy for taxonomic affiliation

of each OTU using the Ribosomal Database Project algorithm (5, 6). OTUs with a sequence

abundance lower than 0.05% (Figure 3), not present in at least two samples (Supplemental

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Figure 3), and/or not identified as Bacteria were removed from downstream analyses. After

filtering, the dataset was comprised of 2928±276 (SD) (Figure 3) or 3772±2786 (SD)

(Supplemental Figure 3) sequences per sample.

Sequencing data statistical analysis

Statistical analyses for Figure 3 were performed using R and the ADE4 package. Differences

between SKG and BALB/c microbiota across sampling times were assessed using analysis of

variance, and the other comparisons were made using the Wilcoxon test.

Statistical analysis for Supplemental Figure 3 were performed as follows. The samples-by-

OTUs matrix was sub-sampled 100 times to a common depth and the averaged matrix was

retained, and then values less than 1.01 were recoded as 0 (7). The resulting normalized

matrix was used, in conjunction with the distance matrix describing the phylogenetic

relationships between all OTU representative sequences, to perform double principal

coordinate analyses (dpcoa) (8), which ordinates the samples based on the phylogenetic

relationships and abundances of the species present in their resident community. For the

exploration of the data by dpcoa, the dataset was divided into adequate groups to be able to

observe possible effects due to time after curdlan treatment without confounding factors.

Possible differences between groups of samples were tested by between group analysis (9).

Then, the relative abundances of chosen phylogenetic groups across the samples were

recovered, and observed differences between the sample classes were analyzed.

qPCR detection of bacteria in mouse cecal and fecal samples

Primers for total bacteria (TB) and segmented filamentous bacteria (SFB) are those described

by Denman et al. and Snel et al., respectively (10, 11). PCR was performed on mixed DNA

extracted from SKG and BALB/c cecal contents using Taq DNA Polymerase (Invitrogen)

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and TB or SFB primer sets individually. Amplicons were purified using the MinElute PCR

purification kit (QIAGEN), and DNA concentration was determined using fluorometry and

the dsDNA BR Assay (Qubit, Life Technologies). Serial 10-fold amplicon dilutions were

performed to generate a standard curve for qPCR.

qPCR was performed using an ABI 7900HT Sequence Detection System (Applied-

Biosystems). Amplification and detection were carried out using Platinum® SYBR® Green

qPCR SuperMix-UDG (Invitrogen). Each reaction was run in quadruplicate in a final volume

of 5 µL with 0.2 µM final concentration of each primer and 1 µL of the appropriate dilutions

of DNA samples. Amplification was performed using the following cycling conditions: 1

cycle at 50oC for 2 min, 1 cycle at 95oC for 2 min followed by 40 cycles of 95oC for 15 s and

60oC for 1 min. Melt curve analysis ensured amplification specificity.

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