Introduction Bordetella pertussis 2006 2003 2005 B. pertussis 2001 B. pertussis B. pertussis B. pertussis 1997 2003 2004 2005 B. pertussis locus 1 2 3 Bps-1 2 3 2005 Tlr4 B. pertussis Tlr4 Tlr4 2006 Bps-1 B. pertussis Bps-2 Bps-3 2005 Bps-1 P P P 2005 B. pertussis B. pertussis Bps-1 2 3 B. pertussis Materials and methods Experimental design 7 B. pertussis 2001 1998 B. pertussis 1949 Animals 1996 Clinical and pathological examinations 2004 Transcription profiling Lungs were incubated in RNAlater (Qiagen) at 4°C for 1 day, after which, the tissues were transferred to a fresh tube and stored at −80°C. RNA was extracted by using the midi-RNA isolation kit (Qiagen). RNA concentrations were measured using a NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE). RNA quality was determined using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and was expressed as the RNA Integrity Number (RIN) as defined by the manufacturer. Only samples with a RIN score of at least eight (on a 1–10 scale) were used for expression profiling. Bps-1 Based on RNA quality and yield, RNA isolated from six mice per group was selected for amplification and labeling. RNA amplification and labeling were carried out using the Amino Allyl MessageAmp II aRNA kit (Ambion, Austin, TX) using 1 μg of total RNA as starting material. RNA samples from individual mice were labeled with Cy3. A common reference containing a RNA pool of all samples isolated was labeled with Cy5. After hybridization and washing, the arrays were scanned using a ScanArray 4000XL microarray scanner (Perkin-Elmer, Wellesley, MA). 2003 2004 2005 1995 2001 1995 B. pertussis http://www.geneontology.org 2003 2003 P http://www.genego.com Results Bacteriologic and pathologic findings B. pertussis 2 5 P B. pertussis- 2 B. pertussis- B. pertussis 3 1 B. pertussis Table 1 B. pertussis Parameter and grade a Mock B. pertussis Day 1 Day 3 Day 5 Day 1 Day 3 Day 5 b Minimal 2  3 4 2 2 Slight    1 2 5 Moderate     3  c (25%) (0%) (38%) (63%) (88%) (88%) Perivasculitis Minimal 2 2 2 2 1  Slight    4 3 3 Moderate     3 5 Marked     1  Total (25%) (25%) (25%) (75%) (100%) (100%) Hypertrophy Minimal 1  1 1 2  Slight      1 Moderate     3 1 Marked     2 4 Strong      1 Total (13%) (0%) (13%) (13%) (88%) (88%) Alveolitis Minimal     2 1 Slight    4  2 Moderate     3 1 Marked     1 3 Strong     3 1 Total (0%) (0%) (0%) (75%) (100%) (88%) Exudate Minimal     2 2 Slight     2 2 Moderate     1 1 Marked     1  Strong     1  Total (0%) (0%) (0%) (0%) (100%) (63%) a B. pertussis b c Expression profiling B. pertussis P 5 1 Fig. 1 B. pertussis a red green b B. pertussis diamonds squares Error bars  B. pertussis 2 Table 2 B. pertussis a   Up b P c d 12 (55%) 0 (0%) 22 −7 Antigen presentation 21 (66%) 0 (0%) 32 −15 Apoptosis 47 (15%) 6 (2%) 324 −5 Cell cycle 43 (8%) 22 (4%) 510 −1 Chemokine activity 21 (58%) 0 (0%) 36 −13 Complement activation 8 (26%) 1 (3%) 31 −3 Cytokine activity 48 (25%) 6 (3%) 192 −13 Cytoskeleton 31 (6%) 40 (8%) 531 −3 Development 96 (7%) 94 (7%) 1,433 −1 Immune response 162 (40%) 3 (1%) 408 −75 Inflammatory response 46 (38%) 2 (2%) 121 −19 Metabolism 353 (7%) 220 (5%) 4,771 −1 Muscle contraction 0 (0%) 12 (24%) 49 −6 Listed in any of the above 576 (9%) 318 (5%) 6,574  Other 328 (6%) 204 (4%) 5,639  Unannotated 278 (3%) 137 (1%) 9,744  Total 1,182 (5%) 659 (3%) 21,957  a b c d 3 Table 3 B. pertussis a Main process P No. of genes in pathway Expressed Total Transcription regulation of granulocyte development Immune response 0.000001925 17 (38%) 45 TLR ligands and common TLR signalling pathway Immune response 0.00001814 16 (34%) 47 ECM remodeling Metabolism 0.00003906 18 (30%) 60 Role of IAP proteins in apoptosis Apoptosis 0.00005458 13 (36%) 36 HETE and HPETE diosynthesis and metabolism Metabolism 0.0003249 13 (31%) 42 TPO signaling via JAK-STAT pathway Cell cycle 0.0003893 9 (39%) 23 Leukotriene 4 biosynthesis and metabolism Metabolism 0.0004655 16 (27%) 60 RhoA regulation pathway (extension, GEFs/GAPs) Immune response 0.002412 10 (29%) 34 MIF in innate immunity response Immune response 0.002435 14 (25%) 57 Retinol metabolism Metabolism 0.002842 12 (26%) 46 Classic complement pathway Complement activation 0.002842 12 (26%) 46 Putative erythropoietin signaling pathway (part 1) Immune response 0.002905 13 (25%) 52 MAPK cascade Cell cycle 0.004975 12 (24%) 49 Ceramide-dependent NO antiapoptotic action Apoptosis 0.006738 13 (23%) 57 IL9 signaling pathway Cytokine activity 0.006809 9 (27%) 33 Ephrins signaling Other 0.007847 13 (22%) 58 Polyamine metabolism Metabolism 0.008731 7 (30%) 23 Integrin outside-in signaling Cell cycle 0.009444 16 (20%) 79 Chemokines and adhesion Chemokine activity 0.009657 34 (16%) 209 P P B. pertussis ECM GAP GEF HPETE HETE IAP IL9 JAK MIF MAPK NO RhoA STAT a Bps-1 B. pertussis 4 Bps-1 Wars Tnfaip2 Zfyve21 Kif26a Table 4 Bps-1 B. pertussis GenBank accession Gene symbol a Fold ratio change P Description b Day 1 Day 3 Day 5 NM_011705 Vrk1 106 1.29 1.52 1.41 0.00003 Vaccinia-related kinase 1 Protein kinase activity NM_011710 Wars 109 1.95 3.58 4.01 0.00000 Tryptophanyl-tRNA synthetase Aminoacyl-tRNA ligase activity NM_010480 Hspca 111 −1.14 1.40 1.27 0.00064 Heat shock protein 1, alpha Protein folding NM_009396 Tnfaip2 112 1.43 4.48 5.40 0.00000 Tumor necrosis factor, alpha-induced protein 2 Angiogenesis XM_138272 Gm266 112 −1.08 −1.25 −1.35 0.00110 Gene model 266, (NCBI) G-protein coupled receptor activity NM_026752 Zfyve21 112 −1.33 −1.41 −1.50 0.00001 Zinc finger, FYVE domain containing 21 Zinc ion binding XM_138275 Kif26a 113 −1.32 −1.68 −1.73 0.00030 Kinesin family member 26A Microtubule motor activity NM_178911 Pld4 113 1.21 1.82 2.52 0.00000 Phospholipase D family, member 4 Catalytic activity XM_619046 LOC434166 113 1.13 1.42 1.42 0.00141 Similar to hypothetical protein MGC37588 Unknown a Bps-1 b Bps-2 -3 B. pertussis 6 Bps-2 Bps-3 Discussion B. pertussis 2005 2006 B. pertussis B. pertussis B B. pertussis Bps-1 2 3 B. pertussis B. pertussis Francisella tularensis 2006 F. tularensis 2006 As expected, most upregulated genes are involved in immune and inflammation-related processes, such as antigen presentation, complement, cytokine, and chemokine activity. Other processes involved include the acute phase response and metabolism. Most downregulated genes are involved in nonimmune processes, such as metabolism, development, cell cycle, or muscle contraction. We speculate that by downregulating these genes, the energy balance is shifted in favor of the immune response. Evidently, cellular influx in the lungs is likely to influence the gene-expression profile. B. pertussis B. pertussis 2005 2001 2005 B. pertussis 2005a B. pertussis 2005 B. pertussis 2006 2005a b B. pertussis B. pertussis Muc Muc4 Muc5b 2000 Muc4 B. pertussis Muc5b 2000 B. pertussis Ipr1 Ipr1 mycobacterium tuberculosis 2005 2005 Ipr1 Ipr1 B. pertussis B. pertussis 1999 2001 B. pertussis 1999 2005 B. pertussis B. pertussis B. pertussis Tlr4 Tnf-α B. pertussis 2006 Tlr4 Tnfaip 2006 B. pertussis B. pertussis B. pertussis 2005 B. pertussis B. pertussis B. pertussis 2001 2005 2003 2004 B. pertussis Bps-1 B. pertussis B. pertussis Tnfaip2 1992 Wars 2000 B. pertussis Bps-1 B. pertussis Bps-1 Bps-2 -3 B. pertussis B. pertussis Bps-2 2004 Bps-3 B. pertussis B. pertussis B. pertussis B. pertussis Electronic supplementary material Raw data as well as the detailed description of the experiment will be uploaded to the freely accessible online database ArrayExpress ( http://www.ebi.ac.uk/arrayexpress/ ) upon acceptance of the manuscript. Expression levels of all 1,841 genes as well as comparisons to other studies are presented in the supplementary data Table  5 .
 Supplementary data Fig. 2 B. pertussis Boxes B. pertussis diamonds High resolution image file (EPS 493 kb) Supplementary data Fig. 3 B. pertussis Solid bars B. pertussis grey bars High resolution image file (EPS 680 kb). Supplementary data Table 5 B. pertussis a B. pertussis b 2005 c d Supplementary data Table 6 Bps-1 -2 -3 B. pertussis