Gene expression of adipokines and inflammatory cytokines in peripheral blood mononuclear cells of obese dogs

Abstract Background Peripheral blood mononuclear cells (PBMCs) have been identified as a possible marker of inflammation in obesity. Understanding the expression of pro‐ and anti‐inflammatory cytokines in PBMCs in obese dogs will help control obesity‐related inflammatory diseases. Objectives The aim of this study was to evaluate the role of PBMCs in obesity‐associated chronic inflammation by analyzing the expression of adipokines and inflammatory cytokines. Methods Blood samples were obtained from 25 subjects and real‐time quantitative polymerase chain reaction determinations were performed to quantify the gene expression levels of adipokines and inflammatory cytokines, including TNF‐α, IL‐17, leptin, MCP‐1, and adiponectin, in the PBMCs. Results The results showed that the gene expression levels of TNF‐α (p < 0.001), IL‐17 (p < 0.0001), and leptin (p < 0.0001) were strongly upregulated in the PBMCs of obese dogs compared to that in non‐obese dogs. Conclusions The changes in gene expression levels of inflammation‐related adipokines and pro‐inflammatory cytokines occur in PBMCs, which may contribute to the low‐grade chronic inflammation that is present in obesity.

respiratory disease (Piantedosi et al., 2016). However, limited studies have investigated the difference in gene expression between obese and normal dogs.
Peripheral blood mononuclear cells (PBMCs) are conventional and universal blood samples that are used to analyse biomarkers at a transcriptional level. PBMCs are mainly composed of lymphocytes, monocytes and natural killer cells, which are all essential components of the immune system. They are generally useful in identifying changes in cytokine activity during inflammation since direct measurement of blood concentration level is limited due to the short half-life of cytokines and the presence of many inhibitors in the blood serum (Friberg et al., 1994). In addition, a more suitable amount of sample can be obtained when using PBMCs to confirm gene expression levels, in contrast to other test samples such as fat and muscles (de Mello et al., 2008). Several studies have compared adipokine and cytokine gene expressions in circulating PBMCs to discuss the correlation between obesity and inflammation in humans. However, in veterinary medicine, most studies have evaluated the correlation between obesity and inflammation by comparing cytokine concentration in serum samples, and only one study utilised gene expression (Vendramini et al., 2020).
However, this study used whole blood samples instead of PBMCs; hence, there is a need for further research in this area.
In this study, we aimed to evaluate how obesity affects the expression of inflammation-related adipokines in circulating PBMCs. We analyzed the messenger RNA (mRNA) expression of not only adipokines and chemokines such as leptin, adiponectin and monocyte chemoattractant protein-1 (MCP-1), but also pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α), and interleukin-17 (IL-17).
The haematological effects of obesity were also considered and analysed by comparing the results of general blood tests on obese and nonobese dogs.

Animals
All animals included in this study were client-owned, young, healthy dogs with no known systemic disease and were brought in for blood donation. Informed consent was obtained from the owners before each procedure. Using a thorough physical examination and the BCS 9 score system, dogs with BCS 7-9 were defined as obese dogs, while dogs with BCS 4-6 were classified as the normal group (Laflamme, 1997). The blood samples taken for analysis were part of their health screening, which is also evaluated and discussed in this study. Furthermore, circulating PBMC samples were taken from whole blood and used to analyse gene expression.

Sample collection
All dogs included in this study underwent over 12 hours of fasting. A physical examination was done, after which, blood samples were obtained by venipuncture from either the jugular or cephalic vein.

Complete blood count and serum biochemistry
Blood samples treated with EDTA were used for complete blood count (CBC) while samples taken in SST tubes were centrifuged and used for the serum chemistry panel including serum triglycerides (TG) and total cholesterol (T-chol) level measurements.

PBMC isolation, RNA extraction and real-time quantitative PCR
PBMCs were isolated from the EDTA-treated blood samples by  Table 1.

Statistical analysis
All data are presented as mean values and standard error of the mean (SEM). Statistical comparisons between the two groups were performed using an unpaired two-tailed Student's t test. pvalues < 0.05 were considered statistically significant. All statistical analyses were performed using GraphPad Prism v 6.01 (GraphPad Software Inc., La Jolla, CA, USA).

General characteristics of patients
The general characteristics of each patient are shown in Table 2. A total of 25 dogs participated; five dogs were classified in the obese group (BCS above 7) and the other 20 were classified in the non-obese control group. All patients are large breed dogs, mainly Labradors and Golden Retrievers. The mean BCS of the obese dogs was at 7.2 ± 0.2, while the TA B L E 1 Primer sequences used to detect gene expression of adipokines, chemokines, cytokines and housekeeping gene

Hematochemical parameters
The results of the blood analysis are shown in Table 4. Among the hematologic tests and chemistry panels performed, only the blood TG concentration level was significantly different between the obese and non-obese dogs (p < 0.0001).

Gene expression of pro-inflammatory cytokines and adipokines
In comparing the gene expression levels between the obese and nonobese dogs, TNF-α, IL-17 and leptin showed a statistically significant increase in obese dogs (p < 0.001) while MCP-1 (p = 0.7068) and adiponectin (p = 0.3800) showed a tendency to increase and decrease in obese dogs, respectively. Diagrams comparing the expression of mRNAs are given in Figure 1.

DISCUSSION
The aim of this study was to compare the expression levels of pro-   (Ghanim et al., 2004). Since PBMCs significantly reflect the expression of obesity-related systemic cytokines and adipokines as well as represent a more convenient sample extraction process in contrast to the use of adipose, liver and muscle tissues, many studies on human obesity and inflammation have been conducted on PBMCs. In this study, PBMCs were used as a novel sample to identify in vivo cytokine gene expression to characterize the effects of dog obesity on adipokine expression and systemic inflammation.
As pro-inflammatory cytokines TNF-α and IL-17 significantly increased in the circulating PBMCs of obese dogs, it can be established that obese dogs are in a low-grade chronic inflammatory status.
Studies done in the early 1990s in obese mice showed an increase in the expression of TNF-α. TNF-α is expressed and secreted in adipose tissue; therefore, its expression increases as the degree of obesity increases, thereby causing clinical symptoms such as insulin resistance (Hotamisligil et al., 1993). Additionally, TNF-α also has tumour-promoting activity; hence, the correlation between obesity and cancer in humans, which is also a major factor discussed in recent studies. IL-17, a cytokine derived mainly from T cells and secreted by adipose tissues, plays a major role in the initiation of inflammation. It is also known to be involved in glucose homeostasis and adipogenesis (Zúñiga et al., 2010). Studies also reported that IL-17 promotes inflammatory response in obese mice  and is associated with autoimmune diseases and chronic inflammation in humans with obesity (Chehimi et al., 2017).
Upon comparison of the expression of adipokines and chemokines such as leptin, adiponectin and MCP-1 in circulating PBMCs, a difference between obese and non-obese dogs was found. Among them, leptin is an adipokine produced in adipocytes and is known to be greatly affected by adipose tissue (Münzberg & Morrison, 2015). Obesity can F I G U R E 1 mRNA expression of pro-inflammatory cytokines and Adipokines in PBMC of obese dogs. Expression of pro-inflammatory cytokines such as TNF-α and IL-17 and Adipokine such as Leptin, MCP-1, adiponectin were analyzed using RT-qPCR. Results are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant result in an increase in blood leptin levels that can lead to various clinical symptoms due to its effects on glucose and lipid metabolism, angiogenesis, cell-mediated immunity, blood pressure homeostasis, body temperature regulation and appetite regulation (Francisco et al., 2018 The neutrophil-lymphocyte ratio (N/L ratio) has been used as a marker of systemic inflammation and as an indicator of some inflammatory diseases in dogs (Benvenuti et al., 2020;Pierini et al., 2019). In this experiment, we were able to confirm if the N/L ratio increased in obese dogs. Several studies conducted on human obesity have reported an increase in the N/L ratio (Furuncuoglu et al., 2016;Rodríguez-Rodríguez et al., 2020); however, based on the author's knowledge, no previous studies regarding changes in the N/L ratio have been conducted on dogs. Therefore, we recommend further discussion on the use of the N/L ratio as an inflammatory marker in future studies on dog obesity.
Another limitation of this study is the imbalance in the number of dogs included in the obese group (n = 5) and the control group (n = 20).
Analysis of small breeds was also limited, and advanced tests such as blood insulin concentration and lymphocyte differentiation were not performed. Nevertheless, the results of this experiment confirmed that the expression of inflammatory cytokines and adipokines was significantly high in obese dogs, it could serve as the basis for further research on larger population sizes, more diverse breeds and more comprehensive laboratory blood test items.

CONCLUSIONS
This is the first study to confirm changes in the expression levels of inflammatory cytokines and adipokines in PBMCs of obese dogs. Our findings could serve as a basis for further research on the association of obesity with chronic inflammation as well as its correlation with the incidence of systemic diseases associated with adipokines in dogs.