Conceived and designed the experiments: RH GP. Performed the experiments: RH ER. Analyzed the data: RH ER GP. Wrote the paper: RH GP.
There were no non-financial competing interests to report. The role of the funding sources, Agria Animal Insurance Co. and the Swedish Kennel Club's Research Foundation, was solely financial, covering parts of the costs for blood collection materials, blood analyses and microarrays. They were not involved in any practical part of the study (collection), nor had they any influence or knowledge of the subsequent data analysis and interpretation, manuscript production or submission decision. The financial support for the study does not in any way prevent that the authors agree to make freely available any materials and information associated with their publication that are reasonably requested by others for the purpose of academic, non-commercial research, according to the PLoS ONE policies on sharing data and materials (as detailed in the guide for authors).
Bacterial infection with the severe complication of sepsis is a frequent and serious condition, being a major cause of death worldwide. To cope with the plethora of occurring bacterial infections there is therefore an urgent need to identify molecular mechanisms operating during the host response, in order both to identify potential targets for therapeutic intervention and to identify biomarkers for disease. Here we addressed this issue by studying global gene expression in uteri from female dogs suffering from spontaneously occurring uterine bacterial infection.
The analysis showed that almost 800 genes were significantly (p<0.05) upregulated (>2-fold) in the uteri of diseased animals. Among these were numerous chemokine and cytokine genes, as well as genes associated with inflammatory cell extravasation, anti-bacterial action, the complement system and innate immune responses, as well as proteoglycan-associated genes. There was also a striking representation of genes associated with proteolysis. Robust upregulation of immunoglobulin components and genes involved in antigen presentation was also evident, indicating elaboration of a strong adaptive immune response. The bacterial infection was also associated with a significant downregulation of almost 700 genes, of which various homeobox and zinc finger transcription factors were highly represented.
Together, these finding outline the molecular patterns involved in bacterial infection of the uterus. The study identified altered expression of numerous genes not previously implicated in bacterial disease, and several of these may be evaluated for potential as biomarkers of disease or as therapeutic targets. Importantly, since humans and dogs show genetic similarity and develop diseases that share many characteristics, the molecular events identified here are likely to reflect the corresponding situation in humans afflicted by similar disease.
Bacterial infection with the severe complication of a systemic inflammatory host response (sepsis) is a serious condition and the most common cause of death in intensive care units at hospitals, with a global incidence that remains rising
Dogs are commonly used in experimental studies of sepsis as well as in safety assessment studies of pharmaceuticals since their inflammatory response is similar to humans
Bacterial uterine infection in dogs has many similarities with severe bacterial infections in humans. For example, infection in both species is associated with induction of local and systemic inflammation, cytokine production, an acute phase reaction, endotoxemia and induction of subsequent sepsis. Therefore, an examination of disease mechanisms involved in pyometra may provide important insights to the mechanisms operating during human bacterial infection and sepsis
This research study was conducted according to national regulations (The Animal Welfare Act and Ordinance, The Swedish Ministry of Agriculture) and international guidelines (the European Convention and the European Commissions Directive 86/609/EEC on protection of animals used for experimental and other scientific purposes). The study was covered by an application approved by the Uppsala Animal Ethics Committee, Uppsala, Sweden. The dogs were privately-owned patients admitted and treated according to the routines at the University Animal Hospital, Swedish University of Agricultural Sciences, Uppsala, Sweden. Written owner consent was obtained before any dog was included.
Fifteen female dogs admitted to the University Animal Hospital, Swedish University of Agricultural Sciences, for diagnosis and subsequent surgical treatment (ovariohysterectomy, OHE) of pyometra were included in the study. The control group consisted of 6 healthy female dogs admitted for elective spay (OHE). Case history and physical examination data were noted by the veterinarian in charge on a specific form at admittance, and continued daily during the hospital stay.
Blood samples for analysis of haematological and serum biochemical parameters were collected from all dogs before surgery from the distal cephalic vein into either non-additive, EDTA-containing or heparinized collection tubes (Becton-Dickinson, Stockholm, Sweden), chilled on ice and centrifuged. Plasma and serum were stored at −80°C until analysis. The removed uterus was cut open and a fibre swab (Culturette; Becton Dickinson AG) was used to sample the uterine contents for bacterial culturing. Tissue biopsies were snap-frozen in liquid nitrogen and stored at −80°C. The remaining uterine tissue and the ovaries were formaldehyde-fixated and used for histopathological examination.
The following haematological and biochemical parameters were analysed, using routine methods: Packed cell volume (PCV, %), hemoglobin (Hb), total leukocyte count (WBC), differential count and morphological evaluation of blood smears, alanine aminotransferase (ALAT), alkaline phosphatase (AP), creatinine and urea. Serum levels of the acute phase proteins, C-reactive protein (CRP) and serum amyloid A (SAA), were analyzed in one batch with commercially available kits (Tridelta Phase™ range canine CRP ELISA assay, Tridelta™ Phase range SAA ELISA assay (Tridelta, Wicklow, Ireland)). Prostaglandin 15-keto-(13,14)-dihydro-F2α metabolite (PGFM) levels were analyzed as previously described
Pyometra | Control | ||||
Reference value | Mean ± SD (Range) | n | Mean ± SD (Range) | n | |
Hemoglobin (g l−1) | 132–199 | 121±30 (57–175) | 15 | 140±21 (111–162) | 5 |
EVF (1012 l−1) | 0.38–0.57 | 0.34±0.08 (0.17–0.50) | 15 | 0.39±0.06 (0.3–0.5) | 5 |
WBC |
5.2–14.1 | 19.5±15.6 (2.8–59.4) | 15 | 8.1±2.1 (6.3–11.0) | 5 |
BN# (109 l−1) | 0–0.3 | 3.4±5.0 (0–13.7) | 14 | 0.0±0.0 (0.0–0.0) | 5 |
SN |
3.0–11.5 | 13.5±9.7 (2.0–35.6) | 15 | 4.7±1.1 (3.7–6.3) | 4 |
EoN (109 l−1) | 0.1–1.2 | 0.57±0.7 (0.0–2.4) | 15 | 0.4±0.4 (0.2–1.1) | 5 |
BaN (109 l−1) | 0–0.1 | 0.01±0.05 (0–0.2) | 14 | 0.0±0.0 (0.0–0.0) | 4 |
Lymphocytes |
1.4–4.8 | 1.4±0.9 (0.1–3.2) | 15 | 2.2±0.8 (1.6 –3.6) | 5 |
Monocytes |
0.2–1.4 | 1.6±1.9 (0.2–7.7) | 15 | 0.4±0.2 (0.3–0.8) | 5 |
Creatinine (µmol l−1) | 40–130 | 106±177 (22–747) | 15 | 76±14 (52–89) | 6 |
ALAT (µkat l−1) | <1.2 | 0.5±0.3 (0.3–1.3) | 14 | 0.6±0.2 (0.4–0.9) | 6 |
Urea (mmol l−1) | 2.5–8.5 | 7.4±14.0 (1.2–57.5) | 15 | 5.2±1.3 (3.9–7.5) | 6 |
AP |
<5.0 | 5.1±4.3 (0.8–18.2) | 15 | 1.4±0.6 (0.8–2.3) | 6 |
Albumin (g l−1) | 31–43 | 35±40 (16–174) | 14 | 30±2.2 (26–32) | 6 |
Bile acids (g l−1) | <30 | 4.6±5.7 (0.0–20.4) | 15 | 3.1±1.4 (1.2–4.6) | 6 |
PGFM |
3695±3674 (464–13000) | 15 | 477±117 (360–640) | 6 | |
SAA |
61.3±31 (<5–>80) | 15 | All<5 | 6 | |
CRP |
38.5±17.5 (0.7–58.2) | 15 | 1.3±1.2 (0.0–2.9) | 6 |
WBC = total white blood cell count; BN = Band neutrophilic granulocytes; SN = segmented neutrophilic granulocytes; BaN = basophilic granulocytes; EoN = eosiniphilic granulocytes; ALAT = alanine aminotransferase; AP = alkaline phosphatase; PGFM = Prostaglandin F2α metabolite, SAA = Serum amyloid A, CRP = C-reactive protein.
Statistically significant difference between the pyometra group and control group (Student's t-test),
difference not tested.
Pyometra diagnosis was performed by gross and histopathological examinations of haematoxylin-eosin-stained sections of uteri and ovaries. Diagnostic criteria for pyometra with or without concomitant presence of macro- and/or microscopically visible cystic dilatation and epithelial hyperplasia of endometrial glands were uterine distension of a varying degree with macroscopically visible presence of opaque, yellowish to brownish exudates in the uterine lumen and microscopically visible purulent inflammatory changes in the endometrium and cystic glands.
Total RNA was prepared from cross-sections of frozen uterine tissue samples, using Nucleospin RNA II (Macherey-Nagel, Düren, Germany). Affymetrix gene chip microarray analysis was performed using the Canine Genome 2.0 Array, as described
RNA was prepared using NucleoSpin® (Macherey-Nagel, Germany). First-Strand cDNA was synthesized with SuperScript™ II Reverse Transcriptase (Invitrogen, Inchinnan, UK) according to the manufacturers' instructions with 5 µl RNA and a final concentration of 7.5 ng/µl random hexamers (Invitrogen) in a total volume of 20 µl. The cDNA concentration was determined, and the cDNA was diluted to 800 ng/µl. Real-time PCR (qPCR) was performed on a ABI PRISM 7900 HT using iQ™ SYBR Green Supermix (BioRad, CA, USA) in a total volume of 10 µl, containing 80 ng cDNA and a final primer concentration of 100–300 nM. PCR cycling conditions included a 95°C heating step of 10 min at the beginning of every run. The samples were then cycled 40 times at 95°C for 30 s (denaturation), 58°C for 20 s (annealing) and 72°C for 20 s (extension). A melting curve from 60°C to 90°C was generated at the end of every run. Prior to experiments, the primer efficiency for each primer pair was determined with three different dilutions of the cDNA. The CT-values were plotted against Log concentrations of the dilutions and primer efficiency was calculated according to following formula: efficiency = 10(−1/slope)-1. The results were calculated by the comparative CT method (User Bulletin #2: ABI PRISM 770 Sequence Detection System (P7N 4303859)), using Hypoxanthine guanine phosphoribosyl transferase (Hprt) as housekeeping gene. For primers used and primer efficiency, see
Data from analyses of haematological, biochemical, acute phase protein and inflammatory parameters are displayed in
Pyometra case | Uterine diameter (cm) | General condition | Temp(°C) | HR(/min) | RR(/min) | WBC(109 l−1) | PBN(%) | SIRS | Animal hospital stay (days) |
5.2–14.1 | |||||||||
11 | 3.5 | Mildly depressed | 39.5 | 120 | 46 | 10.6 | 12 | + | 4 |
12 | 4.0 | Moderately depressed | 39.3 | 100 | 20 | 32.7 | 42 | + | 2 |
13 | 5.0 | Mildly depressed | 39.6 | 140 | 20 | 26.8 | 35 | + | 4 |
16 | 2.0 | Mildly depressed | 38.3 | 100 | 20 | 29.3 | 0.1 | − | 2 |
Temp = Core temperature, HR = heart rate, RR = respiratory rate, WBC = total white blood cell count, PBN = percentage band neutrophils, SIRS = systemic inflammatory response syndrome.
In order to investigate the molecular events associated with the infection, total RNA extracted from uterine tissue of 4 diseased and 4 healthy control animals (blood parameters specified in
Gene Title | Gene symbol | ID | Fold change | adj. |
secretory leukocyte peptidase inhibitor | SLPI | CfaAffx.15167.1.S1_s_at | 344,8 | 0,0006 |
interleukin 8 | IL8 | Cfa.3510.1.S2_at | 242,9 | 0,0003 |
sphingomyelin phosphodiesterase, acid-like 3A | SMPDL3A | CfaAffx.2399.1.S1_s_at | 194,4 | 0,0002 |
S100 calcium binding protein A9 | S100A9 | CfaAffx.26854.1.S1_at | 160,7 | 0,0002 |
interferon induced transmembrane protein 2 | IFITM2 | CfaAffx.10684.1.S1_s_at | 137,3 | 0,0002 |
similar to Ig lambda chain V region 4A precursor | LOC612066 | CfaAffx.345.1.S1_s_at | 129,5 | 0,0018 |
similar to Ig kappa chain C region, B allele | LOC475754 | Cfa.12195.14.S1_s_at | 124,8 | 0,0053 |
insulin-like growth factor binding protein 1 | IGFBP1 | CfaAffx.19068.1.S1_s_at | 104,4 | 0,0004 |
similar to Ig heavy chain V-III region VH26 precursor | LOC490894 | Cfa.4556.3.A1_a_at | 94,5 | 0,0010 |
prostaglandin-endoperoxide synthase 2 | PTGS2 | Cfa.3449.1.S1_s_at | 88,7 | 0,0017 |
serum amyloid A protein /// serum amyloid A1 | SAA1 | CfaAffx.14443.1.S1_at | 65,9 | 0,0031 |
S100 calcium binding protein A8 | S100A8 | CfaAffx.26852.1.S1_at | 56,6 | 0,0007 |
haptoglobin-related protein | HPR | Cfa.12245.2.A1_a_at | 56,1 | 0,0025 |
similar to Immunoglobulin lambda-like polypeptide 1 precursor | LOC607558 | Cfa.4465.2.S1_at | 53,3 | 0,0010 |
serglycin | SRGN | Cfa.20785.1.S1_s_at | 53,2 | 0,0002 |
similar to immunoglobulin iota chain preproprotein | LOC486411 | Cfa.4465.2.S1_s_at | 50,7 | 0,0010 |
similar to Ig lambda chain V-I region BL2 precursor | LOC607020 | CfaAffx.265.1.S1_s_at | 49,8 | 0,0162 |
complement component 6 | C6 | CfaAffx.28425.1.S1_s_at | 48,5 | 0,0014 |
tissue factor pathway inhibitor 2 | TFPI2 | CfaAffx.3983.1.S1_at | 48,1 | 0,0016 |
chemokine (C-X-C motif) ligand 14 | CXCL14 | CfaAffx.2498.1.S1_s_at | 47,0 | 0,0010 |
chemokine (C-C motif) ligand 2 | CCL2 | Cfa.3851.1.S1_s_at | 44,8 | 0,0008 |
matrix metallopeptidase 1 (interstitial collagenase) | MMP1 | CfaAffx.23166.1.S1_s_at | 44,7 | 0,0139 |
complement component 5a receptor 1 | C5AR1 | Cfa.3834.1.S1_at | 43,4 | 0,0002 |
CD5 molecule-like | CD5L | Cfa.5955.1.S1_at | 42,6 | 0,0068 |
Fc fragment of IgG, high affinity Ia, receptor (CD64) | FCGR1A | Cfa.173.1.A1_s_at | 42,5 | 0,0004 |
macrophage receptor with collagenous structure | MARCO | Cfa.15713.1.A1_s_at | 40,7 | 0,0002 |
similar to Small inducible cytokine A23 precursor (CCL23) | LOC480602 | Cfa.12237.1.A1_at | 40,7 | 0,0007 |
neutrophil cytosolic factor 2 | NCF2 | Cfa.2804.1.S1_at | 39,7 | 0,0001 |
CD48 molecule | CD48 | Cfa.14560.1.S1_at | 38,9 | 0,0001 |
acyloxyacyl hydrolase (neutrophil) | AOAH | CfaAffx.5812.1.S1_at | 37,0 | 0,0001 |
thrombospondin 4 | THBS4 | CfaAffx.14209.1.S1_s_at | 31,2 | 0,0033 |
chemokine (C-X-C motif) ligand 10 | CXCL10 | Cfa.16590.1.S2_at | 31,1 | 0,0264 |
similar to Small inducible cytokine A4 precursor (CCL4) | LOC480601 | Cfa.5334.1.A1_s_at | 30,8 | 0,0020 |
similar to Ig kappa chain V-II region RPMI 6410 precursor | LOC491492 | CfaAffx.23613.1.S1_x_at | 30,0 | 0,0114 |
matrix metallopeptidase 9 (gelatinase B) | MMP9 | Cfa.3470.1.S1_s_at | 29,4 | 0,0001 |
selectin L | SELL | CfaAffx.23335.1.S1_s_at | 28,3 | 0,0004 |
similar to normal mucosa of esophagus specific 1 | LOC478287 | CfaAffx.25306.1.S1_x_at | 28,2 | 0,0036 |
caspase 4, apoptosis-related cysteine peptidase | CASP4 | Cfa.3589.1.S1_s_at | 28,1 | 0,0002 |
similar to immunoglobulin J chain | LOC475166 | CfaAffx.5291.1.S1_s_at | 27,7 | 0,0022 |
secreted phosphoprotein 1 | SPP1 | Cfa.9240.1.S1_at | 27,7 | 0,0038 |
CD163 molecule | CD163 | Cfa.9647.1.A1_at | 27,5 | 0,0010 |
chemokine (C-X-C motif) ligand 14 | CXCL14 | Cfa.21149.1.S1_at | 27,5 | 0,0005 |
plasminogen activator inhibitor type 1 | SERPINE2 | CfaAffx.24902.1.S1_at | 25,6 | 0,0004 |
peptidase inhibitor 3, skin-derived (SKALP) | PI3 | CfaAffx.15155.1.S1_s_at | 24,1 | 0,0044 |
similar to normal mucosa of esophagus specific 1 | LOC478287 | Cfa.11815.1.A1_at | 23,8 | 0,0067 |
lymphocyte cytosolic protein 2 | LCP2 | Cfa.18362.1.S1_at | 23,4 | 0,0002 |
regenerating islet-derived 3 gamma | REG3G | Cfa.16734.1.S1_s_at | 23,2 | 0,0474 |
Fc fragment of IgG, low affinity IIIa, receptor (CD16a) | FCGR3A | Cfa.21258.1.S1_at | 23,2 | 0,0010 |
membrane-spanning 4-domains, subfamily A, member 7 | MS4A7 | CfaAffx.16226.1.S1_at | 22,7 | 0,0015 |
Gene title | Gene symbol | ID | Fold change | adj. |
sulfotransferase | SULT1D1 | Cfa.3502.1.S1_at | −30,6 | 0,018 |
EPH receptor A7 | EPHA7 | CfaAffx.6057.1.S1_s_at | −26,0 | 0,003 |
transcription factor CP2-like 1 | TFCP2L1 | Cfa.15666.1.A1_at | −15,2 | 0,002 |
synuclein, alpha interacting protein | SNCAIP | Cfa.13718.1.S1_s_at | −10,9 | 0,001 |
fasciculation and elongation protein zeta 1 (zygin I) | FEZ1 | Cfa.18202.2.S1_a_at | −10,7 | 0,042 |
similar to phosphatidylethanolamine-binding protein 4 | LOC608950 | Cfa.17117.1.S1_at | −10,6 | 0,004 |
similar to esophageal cancer related gene 4 protein | LOC611190 | CfaAffx.4062.1.S1_at | −10,5 | 0,001 |
trefoil factor 2 | TFF2 | Cfa.201.1.S1_at | −9,2 | 0,048 |
regulator of G-protein signaling 22 | RGS22 | CfaAffx.1736.1.S1_at | −8,9 | 0,002 |
epoxide hydrolase 2, cytoplasmic | EPHX2 | Cfa.574.1.A1_at | −8,3 | 0,001 |
Norrie disease (pseudoglioma) | NDP | Cfa.4725.1.S1_at | −8,3 | 0,007 |
ankyrin 3, node of Ranvier (ankyrin G) | ANK3 | CfaAffx.19834.1.S1_s_at | −8,1 | 0,003 |
rhophilin, Rho GTPase binding protein 2 | RHPN2 | Cfa.15.1.S1_at | −8,0 | 0,006 |
hydroxypyruvate isomerase homolog (E. coli) | HYI | CfaAffx.8731.1.S1_s_at | −7,9 | 0,004 |
ectonucleotide pyrophosphatase/phosphodiesterase 6 | ENPP6 | CfaAffx.12520.1.S1_at | −7,9 | 0,012 |
msh homeobox 2 | MSX2 | Cfa.3529.1.S1_at | −7,9 | 0,014 |
forkhead box A2 | FOXA2 | Cfa.13382.1.A1_at | −7,8 | 0,007 |
similar to Homeobox protein DLX-6 | LOC482312 | CfaAffx.4169.1.S1_at | −7,5 | 0,001 |
distal-less homeobox 5 | DLX5 | CfaAffx.4173.1.S1_at | −7,4 | 0,006 |
solute carrier family 30 (zinc transporter), member 2 | SLC30A2 | Cfa.5561.1.A1_at | −7,4 | 0,014 |
lymphoid enhancer-binding factor 1 | LEF1 | CfaAffx.17535.1.S1_s_at | −7,4 | 0,019 |
F-box and WD repeat domain containing 10 | FBXW10 | Cfa.11549.1.A1_at | −7,3 | 0,026 |
cholecystokinin | CCK | CfaAffx.8825.1.S1_s_at | −7,1 | 0,027 |
aldehyde dehydrogenase 1 family, member A1 | ALDH1A1 | Cfa.1715.1.S1_at | −7,1 | 0,018 |
epoxide hydrolase 2, cytoplasmic | EPHX2 | CfaAffx.13394.1.S1_s_at | −7,0 | 0,001 |
glutamate-cysteine ligase, catalytic subunit | GCLC | CfaAffx.4309.1.S1_s_at | −7,0 | 0,014 |
phosphatidic acid phosphatase type 2 domain containing 1A | PPAPDC1A | Cfa.5652.1.A1_at | −6,9 | 0,023 |
similar to RIKEN cDNA 5133401N09 | LOC484150 | CfaAffx.3054.1.S1_at | −6,6 | 0,042 |
protein phosphatase 1, regulatory (inhibitor) subunit 1B | PPP1R1B | Cfa.20636.1.S1_at | −6,6 | 0,027 |
similar to Epithelial-cadherin precursor (E-cadherin) | LOC489647 | CfaAffx.30291.1.S1_at | −6,6 | 0,009 |
DEP domain containing 7 | DEPDC7 | CfaAffx.11821.1.S1_at | −6,6 | 0,044 |
similar to EGFR-coamplified and overexpressed protein | LOC608562 | Cfa.20305.1.S1_at | −6,6 | 0,009 |
betaine-homocysteine methyltransferase | BHMT | Cfa.11111.1.A1_at | −6,5 | 0,002 |
N-acetylated alpha-linked acidic dipeptidase 2 | NAALAD2 | CfaAffx.7397.1.S1_s_at | −6,4 | 0,038 |
similar to dachshund homolog 1 isoform a | LOC485489 | CfaAffx.8384.1.S1_at | −6,4 | 0,016 |
carboxylesterase 2 (intestine, liver) | CES2 | Cfa.19114.1.S1_at | −6,3 | 0,005 |
msh homeobox 1 | MSX1 | CfaAffx.24056.1.S1_at | −6,3 | 0,010 |
spermatid perinuclear RNA binding protein | STRBP | Cfa.19369.1.S1_at | −6,2 | 0,002 |
EF-hand domain (C-terminal) containing 2 | EFHC2 | Cfa.9584.1.A1_s_at | −6,1 | 0,001 |
cystathionase (cystathionine gamma-lyase) | CTH | Cfa.359.1.S1_at | −6,1 | 0,005 |
SH3 domain binding glutamic acid-rich protein like 2 | SH3BGRL2 | CfaAffx.5151.1.S1_s_at | −6,0 | 0,033 |
glutamate receptor interacting protein 1 | GRIP1 | Cfa.8622.1.A1_s_at | −5,9 | 0,006 |
CKLF-like MARVEL transmembrane domain containing 8 | CMTM8 | Cfa.8530.1.A1_s_at | −5,7 | 0,017 |
tumor-associated calcium signal transducer 1 | TACSTD1 | CfaAffx.4844.1.S1_at | −5,7 | 0,004 |
similar to myosin 18A isoform b | LOC475308 | Cfa.1186.1.A1_s_at | −5,6 | 0,003 |
similar to ankyrin repeat domain 26 | LOC610965 | CfaAffx.12169.1.S1_at | −5,6 | 0,009 |
WNT inhibitory factor 1 | WIF1 | Cfa.4881.1.A1_at | −5,5 | 0,018 |
similar to Arg/Abl-interacting protein 2 isoform 1 | LOC482906 | CfaAffx.12091.1.S1_s_at | −5,4 | 0,012 |
RAS guanyl releasing protein 1 (calcium and DAG-regulated) | RASGRP1 | Cfa.7809.1.A1_at | −5,4 | 0,040 |
similar to membrane-associated guanylate kinase-related (MAGI-3) | LOC479761 | CfaAffx.24689.1.S1_at | −5,4 | 0,008 |
A large number of chemokine genes were found among the most upregulated genes (
The normalized 2 log intensity values were centered to the median value of each probe set and colored on a range of −3 to +3. Red denotes upregulated expression levels, and green denotes downregulated expression levels as compared with the median value.
As displayed in
The normalized 2 log intensity values were centered to the median value of each probe set and colored on a range of −3 to +3. Red denotes upregulated expression levels, and green denotes downregulated expression levels as compared with the median value.
The uterine infection caused a marked upregulation of a number of genes related to the complement system (
The normalized 2 log intensity values were centered to the median value of each probe set and colored on a range of −3 to +3. Red denotes upregulated expression levels, and green denotes downregulated expression levels as compared with the median value.
An examination of the list of upregulated genes reveals a striking representation of genes related to proteolysis. Notably, the gene that showed the highest extent of upregulation among all genes was a protease inhibitor, secretory leukocyte peptidase inhibitor (SLPI;
The normalized 2 log intensity values for 50 genes (out of total 172 genes filtered as proteases/protease inhibitors) were centered to the median value of each probe set and colored on a range of -3 to +3. Red indicates upregulated expression, and green indicates downregulated expression as compared with the median value.
(A) SLPI; (B) MMP1; (C) MMP9; (D) SRGN; (E) MSX2; (F) HOXA6. All values are relative to one of the control samples. n = 7 (control); n = 5 (pyometra). p<0.05 *; p<0.01 **; p<0.001 ***; Student's t-test
One of the genes that showed the highest extent of upregulation was the gene coding for the core protein of serglycin proteoglycan (SRGN), as shown both by Affymetrix gene chip analysis (
The normalized 2 log intensity values for 30 genes (out of total 72 filtered genes) were centered to the median value of each probe set and colored on a range of -3 to +3. Red denotes upregulated expression levels, and green denotes downregulated expression levels as compared with the median value.
The uterine infection caused a massive (∼90-fold) upregulation of the prostaglandin-endoperoxide synthase 2/cyclooxygenase-2 gene (PTGS2)(
Various immunoglobulin (Ig)-related genes were highly represented (
A natural consequence of the uterine infection would be an upregulated expression of various anti-bacterial proteins. Indeed, strong upregulated expression of lysozyme was apparent and there was also a marked upregulation of acyloxyacyl hydrolase, a lipase that partially deacylates bacterial lipopolysaccharide (LPS)(
Pyometra and other bacterial infections are associated with elevated plasma levels of various acute phase reactants
The onset of innate immunity reactions is typically triggered through the various pattern recognition receptors, including the toll-like receptors (TLRs). Out of these, we detected significant upregulation of, in particular, TLR2 (∼9-fold), but also TLR1 (∼5-fold) and, to a somewhat lesser extent, TLR4 (∼2.4-fold)(
Cell adhesion to the endothelium is a crucial step in the inflammatory process. Accordingly, several endothelial adhesion molecules were significantly upregulated in infected animals, in particular selectin L (28-fold), VCAM-1 (18-fold), selectin P (8.5-fold), ICAM-1 (7.6-fold) and selectin E (4-fold)(
Among the remaining, upregulated genes, the gene coding for Sphingomyelin phosphodiesterase, acid-like 3A (SMPDL3A) showed the most dramatic extent of induction, being increased almost 200-fold in uteri from diseased animals (
The genes that were significantly downregulated in diseased animals included a number of genes with functions associated with signaling pathways (
The normalized 2 log intensity values for 50 homeobox genes (out of 176) were centered to the median value of each probe set and colored on a range of −3 to +3. Red denotes upregulated expression levels, and green denotes downregulated expression levels as compared with the median value.
The normalized 2 log intensity values for 50 zinc finger genes (out of 489) were centered to the median value of each probe set and colored on a range of −3 to +3. Red denotes upregulated expression levels, and green denotes downregulated expression levels as compared with the median value.
Other examples of markedly downregulated genes included SULT1D1 (∼30-fold; coding for sulfotransferase), EPHA7 (∼26-fold; coding for EPH receptor A7), TFCP2L1 (∼15-fold; coding for transcription factor CP2-like 1) and SNCAIP (∼11-fold; coding for synuclein, alpha interacting protein)(
To the best of our knowledge, this is the first study in which the global gene expression pattern in the uterus is studied following a naturally occurring bacterial infection. Importantly, since a spontaneous disease rather than experimentally induced infection was used, the findings reflect a clinically relevant situation. Notably, previous attempts to extrapolate findings derived from experimental models of sepsis into a clinical setting have often encountered serious problems
The pattern of upregulated genes clearly reflects an ongoing inflammatory response, as shown by the upregulated expression of several endothelial adhesion molecules, chemotactic proteins and cytokines. Among the cytokines, IL-6 and IL-1 were upregulated to the largest extent, and this was also reflected by a strong induction of the IL-1 receptor. Marked upregulation of IL-18 and IL-33 was also evident. Notably, IL-1, IL-18 and IL-33 are closely related cytokines, sharing structural and functional properties and, in addition, they are all activated by a caspase-1/inflammasome-dependent pathway
As judged from the present study, a major feature of uterine infection is the upregulated expression of a large panel of proteases. In particular, there was a profound upregulation of various MMP members implicated in extracellular matrix (ECM) and chemokine
Uterine infection also caused a strong induction of several cysteine cathepsins. Traditionally, cystein cathepsins are mostly known as lysosomal enzymes involved in intracellular degradation processes. However, more recent data have revealed a much wider repertoire of functions, extending from roles in apoptosis to roles in cancer progression, wound healing and also in inflammatory disorders
Since uncontrolled activation of proteolytic pathways may be harmful, it is critical that proteolytic activities are in balance with corresponding inhibitors. Indeed, a major finding in this study was the strong upregulation of various protease inhibitors. Strikingly, out of all upregulated genes, the gene coding for SLPI showed the highest extent of upregulation (∼340-fold). SLPI is an inhibitor of neutrophil elastase and its dramatic upregulation thus indicates that control of elastase activity is an important feature of the uterine infection. This notion is also supported by the strong upregulation of another elastase inhibitor, SKALP (24-fold). There was also a robust upregulation of MMP inhibitors, TIMP-1 and -2. In addition, a number of serine protease inhibitors of serpin type were induced. Out of these, plasminogen activator inhibitor 1 (PAI-1) showed the largest degree of upregulation and, notably, this was matched by a strong upregulation of the corresponding target, i.e. urokinase-type plasminogen activator.
SRGN, i.e. the gene coding for the core protein of serglycin proteoglycan, was one of the genes showing the largest extent of upregulation in diseased uteri. Serglycin has previously been shown to be critical for maintaining storage of secretory granule proteases in such cells
The massive downregulation of a number of homeobox and zinc finger genes during uterine infection is intriguing. Homeobox transcription factors have been widely implicated mainly in embryonal development and in cancer
An obvious extension of the present work will be to evaluate whether any of the identified upregulated genes can be utilized either as biomarkers for disease or as therapeutic targets. Moreover, it will be important to address whether the respective identified gene product is specifically associated with uterine bacterial infection or if its upregulation is a general consequence of bacterial insult. We believe that the results presented here may provide a basis for numerous future investigations where the usefulness of the candidate genes/gene products identified are evaluated in both canine and corresponding human disease.
Primers used in qPCR. Primers were designed for qPCR using the software Primer Express Version 1.0 O or Primer-BLAST, NCBI.
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Haematological-, biochemical-, acute phase protein- and inflammatory parameters in 4 female dogs with bacterial uterine infection (pyometra) and 4 healthy control dogs subjected to Affymetrix gene chip analysis.
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All genes showing significant (adj p<0.05) and more than 2-fold upregulation, in comparison with healthy controls, in uteri from animals diagnosed with uterine bacterial infection.
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All genes showing significant (adj p<0.05) and more than 2-fold downregulation, in comparison with healthy controls, in uteri from animals diagnosed with uterine bacterial infection
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Histology of uteri from control (A) dogs and dogs diagnosed with uterine bacterial infection (pyometra; B). Note the extensive infiltration of mononuclear cells in infected animals, as well as the extensive loss of tissue organization.
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Unsupervised clustering of gene expression in uteri from control animals and animal diagnosed with uterine bacterial infection (pyometra), data derived from the Affymetrix Canine Genome 2.0 Array.
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We are grateful to Hanna Göransson (Uppsala Array Platform) for helpful discussions and excellent support throughout this investigation, and to Erika Karlstam (National Veterinary Institute) for performing histopathological examinations.