Supplemental Methods RNA sequencing experiment

Transcription

1 Supplemental Methods RNA sequencing experiment Mice were euthanized as described in the Methods and the right lung was removed, placed in a sterile eppendorf tube, and snap frozen in liquid nitrogen. RNA extraction was performed on each sample by TRIZOL extraction (Life Technologies) following by Qiagen RNEasy Mini kit purification with on-column DNAse digestion. The Vanderbilt Technologies for Advanced Genomics Core Facility (VANTAGE, Nashville, TN) used the Illumina TruSeq Stranded mrna Sample Preparation Kit to convert the mrna in 1 ng of total RNA into a library of template molecules suitable for subsequent cluster generation and sequencing on the Illumina HiSeq 5. The first step was to do a quality check of the input total RNA by running an aliquot on the Agilent Bioanalyzer to confirm integrity. The Qubit RNA fluorometry assay was used to measure concentration. The input to library prep was 5 µl of 1 ng of total RNA ( ng/µl). The total RNA underwent enrichment of the poly- A containing mrna molecules using poly- T oligo- attached magnetic beads. Following enrichment, the eluted poly(a) RNA was cleaved into small fragments of 1-1 bp using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cdna using SuperScript II reverse transcriptase and random hexamer primers. This was followed by second strand cdna synthesis using DNA Polymerase I and RNase H. The cdna fragments then went through an end repair process, the addition of a single A base, and then ligation of the Illumina multiplexing adapters. The products were then purified and enriched with PCR to create the final cdna sequencing library. The cdna library underwent quality control on the Agilent Bioanalyzer HS DNA assay to confirm the final library size and on the Agilent Mx35P qpcr machine using the KAPA Illumina library quantification kit to determine concentration. A nm stock was created and samples pooled by molarity for multiplexing. From the pool 1 pm was loaded into each well for the flow cell on the Illumina cbot for cluster generation. The flow cell was then loaded onto the Illumina HiSeq 5 for Paired-end 5 bp sequencing utilizing v3 chemistry and HTA 1.8. The raw sequencing reads in BCL format are processed through CASAVA-1.8. for FASTQ conversion and demultiplexing. The RTA chastity filter was used and only the PF (pass filter) reads are retained for further analysis. Quality control (QC) assessment was performed on raw data using Fastx Toolkit (1) and FastQC. RNA data was aligned to mouse genome build 1 (mm1) by using TopHat..7 () followed by Cufflinks (3,). Gene-level expression measurements were normalized to reads per kilobase per million reads (RPKM) by Scripture and in fragments per kilobase per million reads (FPKM) by Cufflinks. Cuffdiff from Cufflinks package was used to detect differentially expressed genes for all group comparisons, which was based on the test statistics T = E[log(y)]/Var[log(y)], where y is the RPKM/FPKM value, and the ratio approximately follows a normal distribution. Differential expression was considered significant if both of the following criteria were met: (1) false discovery rate (FDR) corrected P <.1 (5), and () log (fold change) > Fastx Toolkit. Available from: Trapnell C, Pachter L, Salzberg SL. 9. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 5: Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MG, Salzberg SL, Wold BJ, Pachter L. 1. Transcript assembly and quantification by

2 RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 8: Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1 Genome Project Data Processing Subgroup. 9. The Sequence Alignment/Map format and SAMtools. Bioinformatics 5: Benjamini Y, and Hochberg Y Controlling the false discovery rate: A practical and powerful approach to multiple testing. Journal of the Royal Statistical Society. Series B (Methodological) 57: 89 3.

3 A Total Airway Cells B Airway Lymphocytes 1 cells/ml cells/ml day 1 day day 1 day C Airway Macrophages D Blood Neutrophils 1 cells/ml Peripheral Blood Neutrophils per µl 3 1 day 1 day day Figure S1. Total airway cells (A), airway lymphocytes (B), and airway macrophages (C) on day 1 and day post-kp infection in mice that underwent the sensitization, challenge, and KP infection protocol described in Figure 1. (D) Absolute peripheral blood neutrophil count from and mice on day post-infection. n=1 mice per group combined from two representative experiments. p<.1, p<.1, p<.5.

4 log 1 cfu/ml Lung KP Burden Right Lung Left Lung 1 hour post-infection Figure S. Pre-existing allergic airway inflammation does not alter lung KP burden 1 hour post-infection. Left and right lungs from KP-infected mice were harvested at 1 hour post-infection and quantitative bacterial culture was performed. Lower limit of detection was 1 cfu/ml (log 1 cfu/ml = ). For KP-infected groups, values below the limit of detection were assigned half the lower limit of detection (5 cfu/ml; log 1 cfu/ml = 1.67).

5 H&E 1X H&E X Lung bacteria (-) Sirius Red 1X 3 Lung neutrophils (-) 3 Lung granulocytes (-) Lung eosinophils (-) D α-neutrophil X A B C E 1 1

6 Figure S3. Allergic airway inflammation decreases lung neutrophils and bacteria following acute KP infection. Day post-infection lung sections were obtained. (A) H&E X. (B) H&E 1X, arrow indicates bacteria. (C) Sirius Red 1X demonstrating eosinophils (arrows) and neutrophils (arrowheads). (D) α-neutrophil elastase. (E) Lung granulocyte, bacteria, eosinophil, and neutrophil scores. p<.1, p<.1, p<.5., n=5;, n=6;, n=7;, n=1. Combined from two representative experiments.

7 1 Lung KP Burden 8 log 1 cfu/ml 6 No aerosol PBS aerosol OVA aerosol day Figure S. Aerosol exposure to PBS or OVA does not decrease lung KP burden. WT mice were exposed to PBS aerosol or 1% OVA (in PBS) aerosol for consecutive days for minutes per day or were not aerosol exposed. Twenty-four hours following the final aerosol exposure, mice were infected with KP and harvested on day post-infection. Left lung was removed and quantitative bacterial culture was performed. n=9-1 mice per group, combined results from two representative experiments.

8 1 cells/ml A 3 1 Airway Eosinophils pg/ml B 5 3 day 1 day IL-5 1 pg/ml C 8 6 day 1 day IL-13 day 1 day Figure S5. Acute KP lung infection alters post-infection kinetics of allergic airway inflammation. (A) Total airway eosinophils. Lung IL-5 expression (B) and lung IL-13 expression (C) were quantified by cytokine bead array. p<.1, p<.5. n=1 mice per group combined from two representative experiments.

9 A WT- WT- B Mucus Score (-3) 3 1 Airway Mucus WT- WT- WT- WT- WT- WT- day IL-13KO- IL-13KO- IL-13KO- IL-13KO- Figure S6. Decreased airway mucus in the absence of IL-13. (A) Representative PAS stained lung sections from WT and IL-13KO mice that underwent OVA sensitization and challenge or mock sensitization followed by PBS or KP infection. Lungs were harvested on day post-infection. (B) Airway mucus score based on PAS positivity. n=-3 mice per group. p<.1, p<.5.

10 A hour 6 hour WT- ALUM WT- OVA WT- ALUM WT- OVA WT- ALUM WT- OVA STAT6KO- ALUM STAT6KO- OVA WT- ALUM WT- OVA STAT6KO- ALUM STAT6KO- OVA

11 B WT hour STAT6KO WT 6 hour STAT6KO Transcripts Increased in OVA vs. ALUM Transcripts Decreased in OVA vs. ALUM Figure S7. (A) Heatmap showing normalized log-transformed intensities for transcripts with significant different expression levels between ALUM and OVA experimental groups. Significant different expression was defined as q.1 and a log-fold expression difference 1.5. The locus ID is shown when a gene name has not been assigned. n= mice per group for hour timepoint and n=-5 mice per group for the 6 hour timepoint. (B) Venn diagrams demonstrating the number of transcripts for which expression was significantly different in OVA mice compared to ALUM mice. Areas of overlap denote the number of transcripts for which OVA mice had significantly different expression compared to ALUM mice for both WT and STAT6KO genotypes.

12 hours (pre- infection) Transcripts Increased in OVA vs. ALUM lungs in both WT and STAT6KO mice Transcript Name RPKM WT ALUM RPKM WT OVA Log fold change q value RPKM STAT6KO- ALUM RPKM STAT6KO- OVA Log fold change q value Slc6a Ccl Mmp Cxcl E- 7 Cxcl E E- 1 Saa Ccl E- 5 Cxcl E Reg3g E- 13 Bpifb E- 9 Birc E Rgs E Ctsk E- 5 Pigr E- 8 Kifc E Cxcl Ubec Gm E Mki E- 5 Lcn Slfn E Kif E Cenpe Tpx Iqgap Ckap E Ckapl E Adamts Mcm E E- 6 Nusap E

13 hours (pre- infection) Transcripts Decreased in OVA vs. ALUM in both WT and STAT6KO lungs Zbtb E- 5 6 hours Transcripts Increased in OVA vs. ALUM in both WT and STAT6KO lungs Transcript Name RPKM WT ALUM RPKM WT OVA Log fold change q value RPKM STAT6KO- ALUM RPKM STAT6KO- OVA Log fold change q value Ccl Retnla Agr Tff E- 6 Kntc Reg3g Shcbp E Birc E Ckapl E Ndc E Dtl Tpx Rrm E E- 5 Kif18b Ccno Cxcl E- 5 Ubec E Gp AC E31N8Rik hours Transcripts Decreased in OVA vs. ALUM in both WT and STAT6KO lungs None Table S1: Lung transcripts with significantly different expression between OVA and ALUM mice for both WT and STAT6KO genotypes. Transcript name, reads per kilobase per million reads (RPKM), logfold expression change between OVA and ALUM groups, and q value are shown.