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Keywords:

  • Cytokine;
  • Horse;
  • Inflammation;
  • JAK-STAT;
  • Laminitis

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Background

STAT1 and STAT3 are important signaling molecules in disorders of systemic inflammation and are likely to be involved in laminitis, as laminar and systemic inflammation have been well documented in experimental models of laminitis.

Hypothesis

The STAT1 and STAT3 activation (via phosphorylation of tyrosine and serine moieties) is occurring in the laminar tissue during the developmental and onset of lameness time points in both the black walnut extract (BWE) and carbohydrate overload (CHO) models of laminitis.

Animals

Archived laminar tissue from horses.

Methods

Experimental studies of induced laminitis (BWE and CHO administration) in horses were conducted and laminar tissue samples archived. Western hybridization was performed to determine concentrations of Tyr- and Ser-phosphorylated STAT1 and STAT3 from these archived samples. The RT-qPCR was also performed to assess mRNA concentrations of target genes of STAT1 and STAT3.

Results

Increases (P < .05) in phosphorylation of tyrosine705 and serine727 of STAT3, demonstrated by band intensity ratios, are present in laminar tissue from both the BWE and CHO models at the DEV and OG1 time points. No change in phosphorylation of tyrosine701 or serine727 of STAT1 was present in the laminar tissue from either the BWE or the CHO models. The SOCS3 mRNA concentrations were increased at the onset of lameness in both the CHO and BWE models.

Conclusions and Clinical Relevance

The STAT3 activation likely plays a role in equine laminitis, similar to its reported involvement in organ injury/failure in human sepsis. Regulation of JAK-STAT, through STAT3 inhibitors, might serve as potential therapeutic target for controlling the inflammatory response in the septic horse.

Abbreviations
BWE

black walnut extract

cDNA

complementary DNA

CHO

carbohydrate overload

CIS

cytokine-inducible SH2 protein

CLP

cecal ligation puncture

CON

control

COX 2

cyclooxygenase 2

DEV

developmental

E.CON

early control

ICAM-1

intercellular adhesion molecule 1

ICE-1

caspase-1

IFN

interferon

IL

interleukin

IRF

interferon regulatory factor

JAK

Janus Kinase

L.CON

late control

MMP

matrix metalloproteinase

NF-κB

nuclear factor kappa B

OG1

onset of obel grade 1 lameness

RT-qPCR

real-time quantitative PCR

Ser

serine

SIRS

systemic inflammatory response syndrome

SOCS3

suppressor of cytokine signaling 3

STAT

signal transducing activators of transcription

Tyr

tyrosine

As is well described in organ injury in human sepsis, sepsis-related laminitis in horses is associated with an intense local inflammatory response, most likely occurring because of circulating bacterial products, local blood flow disturbances (ischemia/reperfusion), or both. Dysregulation of inflammatory mediator mRNA expression, including large increases in proinflammatory cytokine expression, is well documented both in experimental models of sepsis-related organ injury[1-3] and models of sepsis-related laminitis.[4-7] One of the major inflammatory signaling pathways believed to be involved in sepsis is the Janus Kinase- Signal Transducing Activators of Transcription (JAK-STAT) pathway,[8, 9] leading to interest in this pathway as a possible target for therapeutic intervention in sepsis.[9-12]

The STATs are latent intracytoplasmic proteins that, once activated via phosphorylation, are translocated to the nucleus and serve as transcription factors that result in the production of various inflammatory genes and those regulating cell growth and development.[13] JAK/STAT signaling is initiated most commonly by cytokine binding via a cell-surface receptor resulting in activation of the Janus kinase (JAK) proteins (associated with the cytoplasmic side of the receptors); STAT proteins associate with the activated JAK/receptor complex and undergo tyrosine phosphorylation resulting in STAT activation.[14] Once phosphorylated at the tyrosine residue, STAT1 and STAT3 associated into dimers and are translocated to the nucleus where they serve as transcription factors for numerous genes related to inflammatory signaling.[15, 16] In addition to tyrosine phosphorylation, serine phosphorylation of STAT1 and 3 by multiple kinases (ie, p38 MAPK, JNK, ERK) can lead to further transcriptional activation of the STAT proteins.[17-20]

Early reports regarded STAT1 activation as a proinflammatory response,[8, 9, 21] and STAT3 activation as primarily an anti-inflammatory response.[8] However, more recent reports have also documented a pro-inflammatory effect of STAT3, particularly in cases of sepsis/SIRS, with some reports stating that STAT3 may promote continued inflammation in septic individuals.[9, 10, 22]

Because laminar injury in experimental models of equine sepsis-related laminitis is associated with a similar marked inflammatory response as that reported in other species with sepsis/SIRS,[4, 7, 23-29] it was the objective of this study to assess laminar STAT1 and STAT3 signaling in the early stages of laminitis.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Laminar Tissue Samples

Frozen (−80°C) archived laminar tissue samples from previous studies were used to assess STAT1 and STAT3 tyrosine and serine phosphorylation and the production of genes associated with the activation of STAT1, STAT3, or both. Laminar samples were collected from horses that received either black walnut extract (BWE) or an overload of carbohydrate (CHO; in the form of cornstarch and wood flour) via nasogastric intubation. Laminar samples were also collected from horses that received only deionized water via nasogastric intubation to serve as controls. Tissue samples used from a previous study at Ohio State University were collected from Standardbred horses (age range 2–15) at 1.5 hours post BWE administration (n = 5) and at the onset of leukopenia (3 hours; range 3–4 hours post BWE) (n = 5); samples from control horses in this animal protocol were collected at 3 hours after administration of deionized water (E. CON, n = 5).[4, 27] Laminar tissue samples from mixed breeds of horses (age range 2–15 years old) collected during a previous study at Auburn University were obtained at the onset of OG1 lameness (shifting weight lameness and lameness observed at trot typically occurring at approximately 12 hours post BWE administration; OG1, n = 5) and from control horses, obtained 12 hours after administration of deionized water (L. CON, n = 5).[4] Laminar samples from the CHO horses (mixed breeds with age range 3–12 years) were collected at the onset of fever (developmental group with median occurrence 17 hours (12–22 hours) post CHO administration, DEV, n = 6) and at the onset of OG1 lameness (OG1 with median occurrence 25 hours (20–48 hours) post CHO administration, n = 6). Laminar tissue was collected from horses at 24 hours post water administration (CON, n = 6) to serve as controls for the CHO treatment. All animal protocols were approved by the Animal Care and Use Committees of The Ohio State University, Auburn University, or the University of Missouri. Induction of laminitis with BWE (6L of extract obtained from soaking black walnut heartwood shavings for 24 hours) and CHO (17.6 g/kg of body weight with 85% corn starch and 15% wood flour) via administration through nasogastric tube and collection/snap freezing of laminar tissue while horses were under general anesthesia was performed as previously described.[4, 7, 27]

Protein Extraction

Protein was extracted from previously snap–frozen laminar tissue. Briefly, laminar tissue was pulverized and homogenized on ice in M-PER lysis buffer1 with the addition of protease cocktail inhibitor,2 PMSF (Pierce, Rockford, IL), and phosphatase inhibitor cocktail.1 After incubation, the supernatant was collected by centrifugation and protein concentrations were determined with Bradford reagent3. After quantification, samples were aliquotted and stored at −80°C until use.

Western Blot Hybridization

Laminar protein from horses receiving BWE and CHO was used to assess total and phosphorylated tyrosine and serine STAT1 and STAT3. Laminar protein samples from each group of horses (CON, DEV, OG1 for the CHO horses and E.CON, L.CON, 1.5H, 3H, and OG1 for the BWE horse) were equally pooled and 30 μg of protein was loaded per group for the CHO treated horses and for the BWE treated horses. Proteins were run on an 8% SDS-PAGE gel and transferred to a PVDF membrane. The membranes were blocked with 5% BSA in TBST for 2 hours at room temperature. Blots were probed with rabbit antiphospho-STAT3 Tyr705,4 antiphospho-STAT Ser727,5 antiphospho-STAT1 Tyr7014 or antiphospho-STAT1 Ser7275 at a 1 : 1000 dilution and incubated overnight at 4°C. After washing, the membranes were probed with an antirabbit IgG secondary antibody coupled to a horseradish peroxidase at a 1 : 20,000 dilution for 1 hour at room temperature. The labeled proteins were detected with west femto developing reagent. Blots were stripped after determination of the presence of STAT1 or STAT3 tyrosine and serine phosphorylation and reprobed with rabbit anti-STAT3 or rabbit anti-STAT1 antibody4 for determination of the total amount of the respective STAT protein. The blots were stripped one more time and reprobed with anti-β actin antibody6 for the confirmation of equal loading among groups. All blots were exposed to film and imaged on the Kodak Imager 2000R.7 The western hybridizations were repeated at least twice to confirm results.

Once the pooled samples were evaluated, western hybridizations were performed as described above with laminar protein samples from each horse (n = 5 per group for BWE and n = 6 per group for CHO) from the CON, 3H (developmental time point used for BWE), DEV (for CHO), and OG1 groups to evaluate signaling events that appeared differentially regulated on the pooled blots (phosphorylated STAT3 tyrosine 705 and serine 727). Band intensities values were determined by Image J8 software and ratios of pSTAT/tSTAT were calculated for each sample. Data were tested for normality and subsequently analyzed by a one-way ANOVA and tukey's multiple comparison post hoc tests with statistical significance set at P < .05 to maintain type I error for all tests.

Immunohistochemistry

Laminar sections from control horses and from horses at the onset of lameness (OG1) of both the BWE and CHO models were used to immunolocalize the presence of phospho-tyrosine 705-STAT3. The paraffin-embedded samples were sectioned at 5-μm thickness, deparaffinized, and treated by boiling with EDTA for antigen retrieval. After blocking, sections were incubated at 4°C overnight with a rabbit mAb phospho-tyrosine 705 STAT3 antibody4 at a 1 : 50 dilution. A biotinylated antirabbit antibody9 was then applied at a 1 : 100 dilution at room temperature for 30 minutes. The immunoreactivity was observed by a peroxidase10 following the instructions of the manufacturer and Harris hematoxylin11 was used as the counterstain. Whole slide digital images (WSIs) were obtained from stained sections with an automated scanning robot12 with a 40× magnification at a spatial sampling period of 0.2 μm per pixel. The WSIs were assessed using a software program13 to capture images from each section, which were then transformed into jpg files. Comparisons between the OG1 and CON groups were used for immunolocalization only.

RNA Isolation and cDNA Synthesis

Total RNA was extracted from previous snap frozen laminar tissues with a kit14 which includes a DNase treatment to remove genomic DNA contamination. PolyA mRNA was then isolated15 and used to make complementary DNA (cDNA) for each sample via reverse transcription16 with a total of 400 ng of mRNA. The cDNA was frozen at −20°C and stored until used for real-time quantitative PCR (RT-qPCR) analysis.

Real-Time qPCR Procedure

Real-time quantitative PCR was performed with a thermocycler17 and quantified with external standards with the fluorescent format for SYBR Green I dye as previously described.[7, 28, 29] Primers were designed from equine-specific sequences. The housekeeping genes, β-actin, β-2 microglobulin, glyceraldehyde-3 phosphate dehydrogenase, and TATA-box binding protein, have been previously reported (Waguespack et al 2004a and Waguespack et al 2004b). The primers for ICE-1, IRF-1, CIS, and SOCS-3 were as follows: ICE-1 f- 5′-TGGTCGAAAGGATATGGAAAGAAAACTCAG-3′ and r- 5′-TGGGCGGGCAGCAAATC-3′; IRF-1f- 5′-GTGAAGGACCAGAGCAGGAACA-3′ and r- 5′-TCTTGGTGAGGGGTGGGAGCAT-3′; CIS f- 5′-CCAGCGAGGCCCGGCAACAC-3′ and r- 5′-TGCTGCACAAGGCTGACCACATC-3′; and SOCS3 f- 5′-CCCGCCGGCACCTTTCT-3′ and r- 5′-GCACCAGCTTGAGCACGCAGTC-3′. All primers were screened by gel electrophoresis and melt curve analysis17 to confirm amplification of a single cDNA fragment of the correct melting temperature and size.[29] Amplified cDNA fragments of each gene were ligated into a vector18 and the vectors linearized with HindIII restriction enzyme5 for use as templates to generate a standard curve for the RT-qPCR reaction.[28, 29] Amplified cDNA fragments were sequenced after cloning to confirm correct DNA sequence for the products of each primer.[29]

Each cDNA sample was diluted 1 : 5 and 1 : 500 with 1× TE buffer to be used in the cytokine and housekeeping PCR reactions, respectively. The PCR reactions were performed in glass capillaries containing 5 μL of diluted sample and 15 μL of PCR master mixture. Master mix included the following: 1 unit of Taq polymerase5, 0.2 units of uracil-N-glycosylase2, 1 : 10,000 dilution of SYBR Green stock solution, forward and reverse primers, PCR nucleotide plus2 and PCR buffer. The PCR buffer (20 mmol/L Tris-HCL) contained 0.05% each of Tween 20 and nonionic detergent. Primers were used at a concentration of 5 μmol/L.

Uracil-N-glycosylase activation, to prevent PCR product carryover, was conducted at 50°C for 2 minutes and was followed by denaturation at 95°C for 2 minutes. Amplification occurred for 40–45 cycles, with the annealing temperature set at 1–5°C below the melting temperature for each specific set of primers, extension was set at 72°C for 5 seconds, and fluorescence acquisition for 10 seconds in the SYBR Green format. Single fluorescence acquisition in each cycle occurred at a temperature determined from the melting temperature of the cDNA product of interest as previously described.[28, 29] After amplification cycling, melting curves of the PCR product were acquired through a stepwise increase in temperature from 65 to 95°C. Standard and target samples were prepared in separate capillaries. Standard curves and water for negative control were performed for each gene of interest and amplified with each series of reactions. Standards were made up of 10-fold serial dilutions of linearized plasmids containing the different gene-specific cDNA inserts. All samples were run in duplicate.

qRT-PCR Data Analysis

Average copy number from each sample was determined for each gene (housekeeping and inflammatory marker). As previously reported, RT-qPCR data from the 4 housekeeping genes for each sample was evaluated by the computer software program geNorm to determine which 2 genes received the best acceptable score to be used for normalization.[23] The β-actin and glyceraldehyde-3 phosphate dehydrogenase were selected and then used by the geNorm19 software to create a normalization factor for each sample. To determine the corrected copy number value for each sample the amplification data obtained by RT-qPCR for each gene was divided by the normalization factor of the selected housekeeping gene for the same sample. After normalization, the fold change from the average control value was calculated for each sample. The qRT-PCR data from the CHO group were analyzed nonparametrically by the Kruskal-Wallis with Dunn's multiple comparisons test to make comparisons among the CON, DEV, and OG1 horses. The qRT-PCR data from the BWE horses were analyzed by a Mann-Whitney unpaired t-test to compare the control horses to 1.5H, 3H, and the OG1 horses. Statistical significance was set at P < .05 to maintain type I error for all tests.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Western blot hybridization yielded no apparent differences in tyrosine 701 or serine 727 phosphorylation of STAT1 between groups in the lamina of the 1.5H, 3H, and OG1 groups administered BWE when compared with their respective controls (Figs 1, 2). There was also no apparent difference in phosphorylation of the tyrosine 701 or serine 727 moieties of STAT1 between groups of horses administered a carbohydrate overload (CHO) (Figs 3, 4). In contrast, increased tyrosine 705 and serine 727 phosphorylation of STAT3 in lamina was present in the 1.5H, 3H, and the OG1 group of horses administered BWE when compared with their respective controls (Figs 5, 6); however, there was no apparent difference in signal intensity between the early (1.5H and 3H) horses and the OG1 horses. Increased (P < .05) tyrosine 705 and serine 727 phosphorylation in the 3H (both tyrosine and serine) and the OG1 (tyrosine only) time points compared with controls was confirmed through western hybridization of each individual horse's laminar sample and calculation of pSTAT/tSTAT band intensity ratios (Fig 7). Increased tyrosine 705 and serine 727 phosphorylation of STAT3 in the lamina was also present in the DEV and OG1 group of horses administered CHO when compared with the CON group (Figs 8, 9). Greater signal was present in the OG1 group compared with DEV for both tyrosine 705 and serine 727 STAT3 phosphorylation. Increased (P < .05) tyrosine 705 and serine 727 phosphorylation of STAT3 in the DEV and OG1 horses administered CHO versus controls was also confirmed through western hybridization of each individual horse's laminar sample and calculation of pSTAT/tSTAT band intensity ratios (Fig 10).

image

Figure 1. STAT1 tyrosine responses in laminar tissue from horses receiving black walnut extract (BWE). Western blot analysis of STAT1 tyrosine 701 phosphorylation is shown in the top image from control horses (E. CON = early control; L. CON = late control), horses during the developmental phases (1.5H post BWE and 3H post BWE), and from horses at the onset of lameness (OG1) that received BWE. Note there is no difference between groups. Total STAT1 tyrosine 701 and β-actin images were acquired after stripping membrane probed for phosphor-tyrosine and used to demonstrate equal loading of protein on the gel. + lane represents a positive control for STAT1 tyrosine 701 phosphorylation.

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image

Figure 2. STAT1 serine responses in laminar tissue from horses receiving black walnut extract (BWE). Western blot analysis of STAT1 serine 727 phosphorylation is shown in the top image from control horses (E. CON = early control; L. CON = late control), horses during the developmental phases (1.5H post BWE and 3H post BWE), and from horses at the onset of lameness (OG1) that received BWE. Note there is no difference between groups. Total STAT1 serine 727 and β-actin images were acquired after stripping membrane probed for phosphor-tyrosine and demonstrate even loading of the gel.

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image

Figure 3. STAT1 tyrosine responses in laminar tissue from horses receiving carbohydrate overload. Western blot analysis of STAT1 tyrosine 701 phosphorylation is shown in the top image from Control (CON), onset of fever (DEV), and onset of lameness (OG1) horses that received an overload of carbohydrate. Note there appears to be no real difference between the groups. β-actin was used to demonstrate equal loading of the gel. + lane represents a positive control for STAT1 tyrosine 701 phosphorylation.

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image

Figure 4. STAT1 serine responses in laminar tissue from horses receiving carbohydrate overload. Western blot analysis of STAT1 serine 727 phosphorylation is shown in the top image from control (CON), onset of fever (DEV), and onset of lameness (OG1) horses that have received an overload of carbohydrate. Note there is no difference between groups. Total STAT1 serine 727 and β-actin images were acquired after stripping membrane probed for phosphor-serine and demonstrate even loading of the gel.

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image

Figure 5. STAT3 tyrosine responses in laminar tissue from horses receiving black walnut extract (BWE). Western blot analysis of STAT3 tyrosine 705 phosphorylation is shown in the top image from control horses (E. CON = early control; L. CON = late control), horses during the developmental phases (1.5H post BWE and 3H post BWE), and from horses at the onset of lameness (OG1) after receiving BWE. Note increased signal in the 1.5H, 3H, and OG1 horses compared with the 2 CON groups. Double bands may be seen at 79 and 86 kDa for both phospho-STAT3 (Tyr705) and for total STAT3. 3 Total STAT3 tyrosine 705 and β-actin images were acquired after stripping membrane probed for phosphor-tyrosine and demonstrate even loading of the gel.

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image

Figure 6. STAT3 serine responses in laminar tissue from horses receiving black walnut extract (BWE). Western blot analysis of STAT3 serine 727 phosphorylation is shown in the top image from control horses (E. CON = early control; L. CON = late control), horses during the developmental phases (1.5H post BWE and 3H post BWE), and from horses at the onset of lameness (OG1) after receiving BWE. Note increased signal in the 1.5H, 3H, and OG1 horses compared with the 2 CON groups. Total STAT3 serine 727 and β-actin images were acquired after stripping membrane probed for phosphor-tyrosine and demonstrate even loading of the gel. Double bands may be seen at 79 and 86 kDa for total STAT3. 3

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image

Figure 7. Band intensity ratios (pSTAT/tSTAT) calculated from CON, 3H, and OG1 horses after black walnut extract administration. Significant increases (P < .05) in the phosphorylation of tyrosine 705 were present in the 3H and OG1 horses when compared with CON. Increases (*P < .05) in the phosphorylation of serine 727 were also present in the 3H horses when compared with controls.

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image

Figure 8. STAT3 tyrosine responses in laminar tissue from horses receiving carbohydrate overload. Western blot analysis of STAT3 tyrosine 705 phosphorylation is shown in the top image from control horses (CON), horses at the onset of fever (DEV), and from horses at the onset of lameness (OG1) that received an overload of carbohydrate. Note there an increase in tyrosine phosphorylation in the DEV and OG1 horses compared with controls, with horses developing lameness (OG1) having the greatest response. Total STAT3 tyrosine 705 and β-actin images were acquired after stripping membrane probed for phosphor-tyrosine and demonstrate even loading of the gel.

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image

Figure 9. STAT3 serine responses in laminar tissue from horses receiving carbohydrate overload. Western blot analysis of STAT3 serine phosphorylation is shown in the top image from control horses (CON), horses at the onset of fever (DEV), and from horses at the onset of lameness (OG1) that received an overload of carbohydrate. Note there an increase in serine phosphorylation in the DEV and OG1 horses compared with controls, with horses developing lameness (OG1) having the greatest response. Total STAT3 tyrosine 705 and β-actin images were acquired after stripping membrane probed for phosphor-tyrosine and demonstrate even loading of the gel.

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image

Figure 10. Band intensity ratios (pSTAT/tSTAT) calculated from individual CON, DEV, and OG1 horses after carbohydrate overload administration. Increases (*P < .05) in STAT3 phosphorylation of tyrosine 705 and serine 727 were present in the 3H and OG1 horses when compared with CON. The OG1 horses also had increases (*P < .05) in STAT3 phosphorylation of tyrosine 705 and serine 727 when compared with DEV horses.

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Immunohistochemistry of paraffin-embedded laminar sections from CON and OG1 horses was used to determine the cell types within the laminae that were positive for phosphorylated STAT3 tyrosine 705. The P-Tyr705 STAT3 staining was dramatically increased in the OG1 laminar sections in BWE and CHO models (compared to CON samples) and was characterized primarily by immunopositive nuclei present in the laminar epithelial cells (Fig 11); positive cells were also present within the laminar dermis (of which many of the positive cells are suspected to be neutrophils or macrophages).

image

Figure 11. Immunohistochemical staining of STAT3 tyrosine705 phosphorylation. Images demonstrate increased signal (brown staining primarily localized to the nucleus) in the epithelial (closed arrows) but also in the dermal (open arrows) cells of the laminar tissue at the onset of lameness (OG1) from both the black walnut extract (BWE) and carbohydrate overload (CHO) models of laminitis when compared to minimal signal in the respective control (CON) horses. Note that majority of cells (counterstained with hematoxylin) are negative for P-Tyr705 STAT3 tyrosine in sections from CON animals.

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The STAT1-responsive (CIS, ICE-1, IRF-1) and STAT3-responsive (CIS, SOCS3) genes were evaluated in both the BWE and CHO models of laminitis (Tables 1 and 2). Increases in expression of both STAT1 and STAT3 responsive genes were present in horses administered BWE (Table 1) as evidenced by increased (P < .05) mRNA concentrations of IRF-1, ICE-1, and CIS in the 1.5-hour horses and ICE-1, CIS, and SOCS3 in the 3-hour horses when compared with the respective CON horses. Only SOCS3 mRNA concentrations were increased in the OG1 group when compared with CON horses. In the CHO model, only increases in SOCS3 mRNA concentrations were found in the OG1 horses when compared with controls (Table 2). There were no differences in expression of STAT1-responsive genes between groups in the CHO model.

Table 1. Laminar STAT responsive genes after black walnut extract administration
Gene CON1.5HCON3HCONLAM
  1. a

    mRNA values expressed as median cDNA copies per normalization factor.

  2. b

    Fold increase expressed in medians from control. NS = nonsignificant fold change from control.

ICE-1mRNA concentrationa771411111534491040
(25–75% percentile)(51–101)(98–249)(82–123)(71–341)(385–556)(441–1740)
Fold increaseb 2.5 0.7 2.2
P-value P < .05 P < .05 NS
IRF-1mRNA concentrationa4765070220187013501230
(25–75% percentile)(298–592)(1,412–9,200)(118–259)(1,221–5,940)(1,129–2,160)(687–3,295)
Fold increaseb 11.2 14 0.85
P-value P < .01 NS NS
CISmRNA concentrationa1,6803,4406784,7202,8102,400
(25–75% percentile)(1,186–2,245)(2,595–4,335)(518–1,076)(1,455–9,710)(1,357–4,110)(1,940–7,890)
Fold increaseb 2 6.1 0.8
P-value P < .01 P < .01 NS
SOCS3mRNA concentrationa0.150.260.093.120.133.77
(25–75% percentile)(0.1–0.45)(0.24–0.75)(0–0.49)(1.2–5.9)(0.08–0.36)(1.9–4.9)
Fold increaseb 1.2 14.5 18.7
P-value NS P < .05 P < .01
Table 2. Laminar STAT responsive genes after carbohydrate overload administration
Gene CONDEVLAM
  1. a

    mRNA values expressed as median cDNA copies per normalization factor.

  2. b

    Fold increase expressed in medians from control. NS = nonsignificant fold change from control.

ICE-1mRNA concentrationa5366811,345
(25–75% percentile)(373–810)(299–1,720)(902–2,560)
Fold increaseb 1.172.3
P-value NSNS
IRF-1mRNA concentrationa53,600110,45076,850
(25–75% percentile)(29,825–61,025)(50,625–313,250)(53,375–194,500)
Fold increaseb 2.341.62
P-value NSNS
CISmRNA concentrationa2,9453,3353,395
(25–75% percentile)(2,295–4,130)(2,715–4,785)(2,150–8,280)
Fold increaseb 0.991.01
P-value NSNS
SOCS3mRNA concentrationa33.4178.5372
(25–75% percentile)(29–59)(103–652)(311–1,565)
Fold increaseb 3.737.78
P-value NSP < .01

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Because the presence of laminar inflammation has been well documented early in the developmental phases and onset of lameness in both the BWE and CHO models of laminitis,[4, 7, 23, 26, 30, 31] it is now essential that signaling pathways involved in laminar inflammation be evaluated in these models to discover novel targets for pharmaceutical intervention. This study evaluated 2 STAT signal transduction molecules, STAT1 and 3, which have been reported to play a role in sepsis/SIRS in humans and rodent models[8-10, 32, 33] and were suspected to play a role in laminar inflammation and subsequent failure. Because STAT1 is primarily activated by type I and type II interferons[34] and because there have been no significant increases in mRNA concentrations of IFNα, IFNβ (unpublished data, Belknap), or IFNγ documented at any stage in laminar tissue from horses administered BWE or the traditional CHO model (combined wood/corn starch) used in the present study,[4, 7] the absence of a significant STAT1 response is not completely unexpected. The lack of STAT1 activation in the CHO model, represented by no difference in phosphorylation of tyrosine 701 or serine 727 between groups, was consistent with the lack of increased laminar mRNA concentrations of STAT-1 responsive genes IRF-1 and ICE-1. In the BWE model, increases in laminar mRNA concentrations of the STAT1-responsive genes IRF-1 and ICE-1 in the developmental stages with no evidence of STAT1 activation indicate that other signaling mechanisms (ie, TLR9/Myd88 induction of IRF-1[35] and flagellin/NOD-like receptor Ipaf induction of ICE-1[36]) are responsible for activating the increased gene expression of IRF-1 and ICE-1 in this model.

Laminar STAT3 activation was indicated in both BWE and CHO models with the increased phosphorylation of tyrosine 705 moiety of STAT3 in the DEV and OG1 time points in both models and increased phosphorylation of serine 727 moieties of STAT3 in both DEV and OG1 horses after CHO administration and in the DEV (3H) horses after BWE administration. Because Il-6, one of the major activators of STAT3,[37, 38] is greatly increased in the laminar tissue after BWE and CHO administration[7, 29] laminar STAT3 activation fits well with the previously published works on laminar inflammatory signaling in laminitis. However, the increase in laminar phospho-Tyr705 concentration at the DEV time point in the CHO model (before reported increases in laminar IL-6 mRNA concentration) indicates that this early activation of STAT3 may be because of either increased circulating IL-6 concentrations[39] or STAT3 activation by other signaling mechanisms (ie, growth factor signaling through either epidermal growth factor or platelet derived growth factor).[40-42] Although IL-10 can also induce STAT3 activation,[40] laminar IL-10 is not increased in either model at any time point.

Phosphorylation of STAT3 tyrosine and serine moieties results in up-regulation of MMP-9, MMP-2, ICAM1, and COX-2, whereas IL-1β, IL-6, CXCL-8, and MCP-1 have been reported to be either up-regulated or down-regulated in response to STAT3 activation depending on various conditions.[42-45] Interestingly, all of these mediators have been reported to be increased in laminar tissue of horses either at the developmental period, the onset of lameness in the BWE or both and CHO models of laminitis. In the CHO models (corn starch/wood flour and oligofructose models), circulating endotoxin has been documented[46, 47] and likely plays a role in induction of the expression of these inflammatory proteins. Although NF-κB signaling has been more frequently described as pivotal to the induction of a wide array of inflammatory mediators in response to LPS-induced TLR signaling, STAT3 tyrosine phosphorylation has also been reported to be critical for gene expression of the central cytokines IL-1β and IL-6 in LPS-activated murine macrophages.[48] In addition, administration of a STAT3 inhibitor blocks the expression of IL-1β and IL-6 in LPS-stimulated macrophages, but has no effect on TNFα production.[48] Thus, the consistent pattern of increased laminar IL-1β and IL-6 mRNA concentrations in the face of no increase in TNFα mRNA concentrations in the different models of laminitis may be explained by the inability of STAT3 to stimulate TNFα gene expression, while still stimulating IL-1β and IL-6 expression.[4, 7, 27] It is also possible that STAT3 activation works synergistically with NF-κB to promote a prolongation of the inflammatory response as has been reported in other studies.[43, 45, 49] Furthermore, although the production of some inflammatory genes results from either STAT3 or NF-κB stimulation, some genes may only be expressed via one transcription factor or may be dependent on the presence of both transcription factors.[45]

Several genes induced by STAT proteins can act as negative feedback because of their inhibitory action on JAK-STAT signaling; these STAT inhibitors include suppressor of cytokine signaling (SOCS), cytokine-inducible SH2-continuing protein (CIS), and protein inhibitor of activated STAT (PIAS).[15, 50] In this study, we assessed SOCS-3 and CIS. Significant increases in laminar SOCS-3 mRNA concentrations in both the BWE and CHO models most likely occurred secondary to STAT3 activation in these horses as a negative feedback mechanism to limit the STAT3 transcriptional response in the laminar tissue. SOCS3 has been reported to negatively regulate the IL-6 activated gp130-STAT3 response in keratinocytes and immune cells, thus preventing excessive neutrophil accumulation and chemokine production,[50, 51] and is reported to be important in controlling cytokine expression secondary to mild to moderate ischemia-reperfusion injury in the liver.[52] The lack of increased laminar CIS mRNA concentrations in the CHO model (increases were only present at the DEV stages of the BWE model) may reflect the fact that this protein is more commonly associated with induction by growth factors (ie, EPO, growth hormone, prolactin) than by cytokines such as IL-6.[53] Although activation of both STAT1 and STAT3 has been reported in models of human sepsis/SIRS,[8, 9, 54] only STAT3 activation was present in the laminar tissue from the CHO and BWE models of laminitis. With the development of STAT inhibitors to be used in the treatment of many cancers, determination of the effect STAT3 activation has on sepsis/SIRS is important to establish whether these therapies can be used in septic equine patients to improve outcome. STAT3 inhibition has had mixed effects in the rodent cecal ligation puncture models of sepsis, with administration of STAT inhibitors reported to decrease organ injury and mortality in one report,[10] whereas liver-specific STAT3 knockout mice had increased mortality compared with wild-type mice (suggesting that STAT3 activation could be important as a protective hepatic response to sepsis) in another report.[33] Therefore, although the current results and several compelling studies in other species suggest that STAT3 inhibition has the potential to decrease laminar inflammation and injury in sepsis-related laminitis, further studies are needed to determine if STAT3 inhibition results in a beneficial or detrimental effect in the early stages of laminitis.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Supported by U.S. Equestrian Federation Grant #EHRF 2009-1.

Footnotes
  1. 1

    Pierce, Rockford, IL

  2. 2

    Roche, Indianapolis, IN

  3. 3

    Bio-Rad, Hercules, CA

  4. 4

    Cell Signaling Technology Inc, Danvers, MA

  5. 5

    Invitrogen, Carlsbad, CA

  6. 6

    Santa Cruz Biotechnology Inc, Santa Cruz, CA

  7. 7

    Eastman Kodak, Windsor, CO

  8. 8

    Image J, National Institute of Health, Bethesda, MD

  9. 9

    VECTASTAIN ABC kit; Vector Laboratories, Burlingame, CA

  10. 10

    DAB kit; Vector Laboratories

  11. 11

    Fisher Scientific, Kalamazoo, MI

  12. 12

    T2 Scan Scope; Aperio, Vista, CA

  13. 13

    Aperio Image Scope

  14. 14

    Absolutely RNA Miniprep; Stratagene, LaJolla, CA

  15. 15

    mRNA extraction kit; Roche

  16. 16

    Retroscript; Ambion Inc, Austin, TX

  17. 17

    LightCyler; Roche Molecular Biochemical

  18. 18

    TOPO 010 E.Coli; Invitrogen

  19. 19

    Ghent University, Ghent, Belgium

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References