Introduction
Interleukin (IL)-10, also known as cytokine synthesis inhibitory factor is a class-2 low molecular-weight protein encoded by the IL-10 gene. It is an anti-inflammatory cytokine with other members of the class 2 cytokine family comprising of IL-19, IL-20, IL-22, IL-24 (Mda-7), IL-26, interferon and interferon-like molecules such as limitin [1]. While monocytes are primarily the producers of IL-10 with lymphocytes, namely; type 2 T-helper cells (TH2), mastocytes, CD4+CD25+Foxp3+ regulatory T cells, and a certain subset of activated T cells and B cells also contribute significantly to the overall IL-10 circulatory levels [2–4].
IL-10 has been shown to interact with IL-10 receptor-alpha subunit with the receptor complex for IL-10 requiring the IL-10R2 chain to initiate signaling [5]. Induction of IL-10 involves ERK1/2 (extracellular signal-regulated kinases 1 and 2), p38 (mitogen-activated protein kinases), and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling. IL-10 expression is tightly regulated at the transcriptional and post-transcriptional (involving the control of mRNA stability via AU-rich elements (adenylate-uridylate-rich elements) and by microRNAs such as let-7 and miR-106) levels with extensive IL-10 locus remodeling observed in monocytes upon stimulation of its receptors (TLR or Fc receptor pathways) [3,6,7]. IL-10 blockade of NF-κB activity is involved in regulating the JAK-STAT signaling pathway. IL-10 binding induces signal transducers and activators of transcription (STATs) 3 signaling via the phosphorylation of the cytoplasmic tails of IL-10 receptor 1 and 2 by Janus kinase 1 (JAK 1) and tyrosine kinase 2 (Tyk2), respectively [8–10].
This study aims to investigate the time course of IL-10 in dogs with cutaneous wounds and their relationship with some hematologic parameters.
Material and Methods
Ethical approval
Ethical clearance for this study was secured from the Ahmadu Bello University Committee on Animal Use and Care with reference number ABUCAUC/2016/026.
Study subjects and location
Twelve (12) apparently healthy dogs aged between 9–13 months with a live weight between 8 and 12 kg were enrolled for this study, housed and maintained throughout the research period in the Small Animal Clinic Kennels of the Ahmadu Bello University Veterinary Teaching Hospital.
Groupings and surgical considerations
The experimental and control groups each comprised of six dogs that were randomly assigned. Pre-surgical considerations [11] included aseptic surgical preparations of patients. Anaesthesia was achieved by intravenous (cephalic veins) administration of atropine sulfate (Atropine® – Shanxi Shuguang Pharmaceutical Co., Ltd, Qixian, China) at 0.05 mg/kg, pentazocine (Pentalab® – Laborate Pharmaceuticals Ltd, India) at 1 mg/kg and midazolam (Roche Pharmaceuticals, Switzerland) at 0.25 mg/kg as pre-anesthetics, while thiopental sodium 1 g (Pauco Pharmaceuticals, Nigeria) at 12 mg/kg was the anesthetic agent employed. Four-centimeter squared cutaneous excisions were created on the mid-lateral aspect of the fully draped right antebrachium of the dogs. The dogs were subsequently monitored until complete recovery from anesthesia.
Hematology, sample acquisition, and storage
Five milliliters (5 ml) of whole blood was collected from each dog via cephalic venipuncture at 12 hours, 36 hours, 60 hours, 156 hours, and 324 hours post-operation. The blood sample (3 ml) was dispensed into a sodium ethylenediamintetraacetate-containing sample bottle for full blood cell count. Mindray automated hematologic analyzer (Mindray® BC-3,600, Shenzhen, China) was used for processing the anticoagulated whole blood. Serum was obtained from centrifuged whole blood (2 ml in plain sample bottle) at 4,500 g for 15 minutes after storage at 21°C without anticoagulant for 1 hour [12,13]. The wound washout method was used to collect wound fluids sequentially over 156 hours post-operation [14]. The extracted serum and wound fluid were stored at −20°C for IL-10 determination.
Table 1.
Time-course of IL-10 (ng/ml) levels in male and female dogs with cutaneous wounds.
| Time (hour) | WF. level (ng/ml) | Exp. serum level (ng/ml) | Cont. serum level (ng/ml) | |||
|---|---|---|---|---|---|---|
| Males (n=3) |
Females (n=3) |
Males (n=3) |
Females (n=3) |
Males (n=3) |
Females (n=3) |
|
| Pre-operative (Start) | - | - | 0.90 ± 0.02 | 0.81 ± 0.00 | 0.89 ± 0.02 | 0.81 ± 0.0 |
| - | - | (0.85–0.93) | (0.81–0.81) | (0.85–0.93) | (0.81–0.81) | |
| Post-operative 12 |
1.02 ± 0.16 | 1.05 ± 0.04 | 0.92 ± 0.08 | 0.88 ± 0.02 | 0.86 ± 0.03 | 0.86 ± 0.19 |
| (0.74–1.29) | (0.82–1.42) | (0.82–1.07) | (0.85–0.91) | (0.81–0.91) | (0.79–0.91) | |
| 36 | 0.86 ± 0.02 | 0.91 ± 0.05 | 0.96 ± 0.08 | 1.01 ± 0.08 | 0.79 ± 0.18 | 0.82 ± 0.02 |
| (0.83–0.90) | (0.81–0.99) | (0.87–1.11) | (0.80–1.36) | (0.62–0.88) | (0.78–0.87) | |
| 60 | 0.82 ± 0.02 | 0.82 ± 0.05 | 0.93 ± 0.08 | 0.88 ± 0.03 | 0.88 ± 0.02 | 0.84 ± 0.03 |
| (0.81–0.86) | (0.74–0.90) | (0.82–1.10) | (0.81–0.91) | (0.86–0.91) | (0.81–0.90) | |
| 156 | 0.85 ± 0.53a,d | 0.90 ± 0.50a,d | 1.73 ± 0.03b | 1.62 ± 0.04c | 1.16 ± 0.31a | 0.72 ± 0.03d |
| (0.82–0.91) | (0.85–0.98) | (0.85–2.68) | (0.86–2.55) | (0.83–1.78) | (0.68–0.77) | |
| 324 | - | - | 0.86 ± 0.05 | 0.82 ± 0.05 | 0.87 ± 0.02 | 0.86 ± 0.03 |
| - | - | (0.81–0.95) | (0.72–0.89) | (0.83–0.91) | (0.81–0.90) | |
| *Mean ± SEM | 0.89 ± 0.04 | 0.92 ± 0.05 | 1.05 ± 0.14 | 1.00 ± 0.13 | 0.91 ± 0.05 | 0.82 ± 0.02 |
a,b,c,d=Mean (Mean ± Standard Error of Mean) with different superscript letters are significantly (p < 0.05) different. Sources of variation (Interaction: p < 0.001; Time: p < 0.001; Groups: p < 0.001). Values in parenthesis are the ranges (minimum – maximum) from the values. Exp.=Experimental, WF.=wound fluids, Cont.=control, ng/ml=nano-gram per millilitre, h.=hour, n=total number of individuals in group and (*)=Overrall values.
Enzyme-linked immunosorbent assay
IL-10 Canine ELISA (Enzyme-linked Immunosorbent Assay) Kit was supplied by Abcam Ltd, United Kingdom, with reference number ab193685, Lot: GR252141-2. The assays were carried out in strict adherence to the manufacturer’s protocols and the absorbances at 450 nm were read spectrophotometrically using a Thermo Multiskan Ascent Photometer (Thermo Scientific, USA).
Data analysis
The numerical data were expressed as means and standard error of the means (means ± SEM). The significance of data was evaluated by analysis of variance with Tukey’s multiple comparison test. The relationships between the parameters were assessed by Person’s correlation test. A p-value of less than 0.05 was accepted as significant. The analysis was performed using GraphPad Prism Version 5.03 (San Diego, California, USA) for Windows [15].
Results
Clinical examination of dogs in this study revealed rectal temperature, pulse, and respiratory rates of 38.02°C ± 0.02°C, 72.13 ± ٠.٢٧°C beats/minute, and 22.47 ± 0.07 cycles/minute, respectively. Table 1 summarizes the IL-10 protein levels obtained. Varying levels of minor fluctuations were observed in the complete leucocyte count (Figs. 1–5). Figure 6 shows some significant associations between IL-10 and the leucocyte count.
Discussion
The study subjects were ascertained to be apparently healthy by pre-operative clinical examination with all values within the normal reference range limit [11]. IL-10 proteins were detected at similar levels to those observed in different exotic dog breeds [16] and the findings by Risnes et al. [17], where IL-10 levels increased very early upon sustaining an injury. IL-10 being a pleiotropic cytokine plays a fundament role in modulating inflammation via its anti-inflammatory function while maintaining cellular homeostasis. Therefore, the early local and systemic IL-10 release with observed peak levels in wound fluids by 12 hours could be attributed to the body’s protective mechanism against cytokine storm which is a result of an uncontrolled immune response, mostly mediated through the Jak1/Tyk2 and STAT3 signaling pathway during the acute phase reaction [18,19]. The significantly (p < 0.05) and (p < 0.001) high peaks of Serum IL-10 attained at 156 hours for males and females, respectively, in comparison with the wound fluid levels is likely due to immuno-stimulatory function of IL-10 given its pivotal role in immune modulation in the migratory/proliferative phase of wound healing which is characterized by a transient hyper-inflammatory state [19]. It has been postulated that the programmed death (PD-1) pathway which is a co-receptor that is predominantly expressed on T-cells binds to the ligands PD-L1 or PD-L2 triggering monocytes to produce IL-10 with the minimal expression of IL-10 in unstimulated tissues, regulating the immune system by the inhibition of macrophages and self-reactive T-cells’ synthesis of pro-inflammatory cytokines such as IFN-γ, IL-2, IL-3, IL-6, IL-8, TNFα, and GM-CSF which serve to protect against autoimmune diseases [4,20].
Figure 1.
Changes in total white blood cells count in male and female dogs with cutaneous wounds.
All values are maintained within normal range limits for the NIDs. Mean (Means ± SEM), n=total number of individuals in group.
Figure 2.
Changes in neutrophils count in male and female dogs with cutaneous wounds.
All values are maintained within normal range limits for the NIDs. Mean (Means ± SEM), n=total number of individuals in group.
Figure 3.
Changes in lymphocytes count in male and female dogs with cutaneous wounds.
All values are maintained within normal range limits for the NIDs. Mean (Means ± SEM), n=total number of individuals in group.
Figure 4.
Changes in monocytes count in male and female dogs with cutaneous wounds.
All values are maintained within normal range limits for the NIDs. Mean (Means ± SEM), n=total number of individuals in group.
Figure 5.
Changes in eosinophils count in male and female dogs with cutaneous wounds.
All values are maintained within normal range limits for the NIDs. Mean (Means ± SEM), n=total number of individuals in group.
The slight elevation (p > 0.05) in total WBC count following injury is probably due to efficient regulation of the immune cells under the combined signaling of IL-10 and other inflammatory cytokines produced by the circulatory leucocytes [21]. The sharp spike in neutrophil counts in females at 60 hours. is indicative of an increase in wound debridement [22]. Lymphocyte count rose gradually in injured NIDs with significant (p < 0.01) variation over time, especially at its peak value at 156 hours for males and females, while the control values remained within the reference range [11]. This finding corroborated the reports by Park and Barbul, [21] that T-lymphocytes migrate into wounds immediately after neutrophils and macrophages about the fifth day during the proliferative phase, and peak about 48 hours later. There was a significant (p < 0.05) rise in monocyte count 12 hours post injury which was sustained through to 36 hours and 60 hours followed by a decline to values within control ranges at 60 hours and 156 hours, respectively, for females and males. This could be attributed to the migration of macrophages into wounds 2–4 days after sustaining an injury, predominating, with the secretion of cytokines and growth factors, enhancing the inflammatory and debridement processes of healing [23,24]. The cytokines balancing activities in cell recruitment may be responsible for the minimal fluctuations in eosinophils count within reference ranges during the period of the experiment.
The significant (p < 0.05) associations in this study show the balancing role of IL-10 in inflammatory responses, which could serve as a prospective predictive tool of outcomes and further influence decisions regarding the adoption of specific wound management modalities.
Figure 6.
Significant associations between the levels of IL-10 with some hematologic parameters in the experimental group. (A) Serum IL-10 with lymphocytes irrespective of sex, (B)HGB and serum IL-10 in female NIDs, (C) Lymphocytes and serum IL-10 in females, and (D) Eosinophils and serum IL-10 levels in females. (E) Lymphocytes and serum IL-10 in male NIDs.
HGB: hemoglobin concentration; r=Pearson’s correlation coefficient, p < 0.05 are considered significant.
Conclusion
The study demonstrated that IL-10 levels were significantly (p < 0.05) elevated following cutaneous injury with varying degrees of modulatory effects on circulatory lymphocytes. It provided evidence that further investigations into the expression patterns of IL-10 in cutaneous wounds may improve the quality of wound management.
Acknowledgment
The authors thank the technical support rendered by Mallam Sanni, Mr. Mohammed I., Dr. Yunusa Y, Mr. Suleiman I., Bimi B., and Mrs Daniel C.
Funding
No funding was declared.
Conflict of interest
The authors have no conflict of interest disclosed.
Author contributions
Avazi DO, Awasum CA, and Hassan AZ. contributed to the study conceptualization and design, material preparation, data collection, analysis, manuscript write-up, and proofreading.
