1 National Veterinary Institute, Technical University of Denmark2 Section for Immunology and Vaccinology, National Veterinary Institute, Technical University of Denmark3 Aarhus University
Innate immune gene expression and circulating biomarkers in obesity-induced inflammation
Overweight and obesity are increasing at an alarming rate globally, and are quickly becoming one of the largest medical problems in the world. Overweight and obesity are risk factors of metabolic diseases such as diabetes, cardiovascular diseases and cancer. Obesity is associated with a chronic state of low-grade inflammation in the adipose tissues, which involves several factors of the innate immune response having a range of systemic effects and which has been implicated in the development of the metabolic syndrome. To investigate the impact of obesity and obesity-related diseases good translational animal models are needed, and as such pigs have been proposed as relevant models for human obesity-induced inflammation as pigs share many genetic, anatomical and physiological features with humans. In this project the up- and downregulation of genes and proteins involved in the innate immune response to obesity-induced inflammation were examined to define the response and to identify a pig model that resembles the human response. Regulation was examined in the liver, two adipose tissues from the abdomen (subcutaneous adipose tissue (SAT) from the abdomen or retroperitoneal adipose tissue (RPAT) and visceral adipose tissue (VAT)) and one from the neck (neck SAT), as well as in the blood. Three pig breeds, all previously used as obesity models, have been examined, namely domestic pigs, Göttingen minipigs and Ossabaw minipigs, as well as cloned domestic pigs in an attempt to reduce variation, and thus the number of animals to be used in a trial to obtain statistical power. For the gene regulation analysis, two platforms for quantitative real-time PCR (qPCR) were employed: The Rotor-Gene Q instrument and the microfluidics-based high-throughput Bio-Mark. For the serum protein concentrations analysis several enzyme-linked immunosorbent assays (ELISAs) were used on the blood. In the clones, both cloning and obesity changed the response of the innate immune genes in the tissues and in the blood, as fewer genes were differentially regulated in the clones and in the obese, than in the controls and lean pigs. These effects were additive, so that obese, cloned pigs showed the least differential gene expression. However, the obese clones showed up-regulation of two serum proteins, namely haptoglobin and orosomucoid (ORM), as the only group of domestic pigs. Furthermore, cloning did not reduce the inter-individual phenotypic variation between the pigs. This changed expression of the innate immune response seen in cloned pigs may have phenotypic effects over time, and should therefore be taken into consideration when using cloned pigs for nutritional studies. In the Göttingen minipigs, obesity induced extensive changes in gene expression in the tissues as nine genes in the liver (out of 35), 12 genes in the VAT, 11 in the RPAT and eight genes in the neck SAT (out of 33) were significantly differentially regulated in the obese Göttingen minipigs compared to lean. Three genes were differentially expressed in all three adipose tissues, namely the proinflammatory CC Chemokine ligand 3-like 1 (CCL3L1), the anti-inflammatory CD200 and the anti-inflammatory IL1RN, indicating that several factors in the adipose tissues are suppressing the effects of obesity. This response is like the response observed in obese humans. However, the obese Göttingen minipigs do not show elevated serum protein concentrations, which is observed in obese humans. The obese Ossabaw minipigs did not show significant differential expression in the adipose tissues, although this could be an artifact due to small group sizes. However, a number of genes in the liver (six out of 26 genes) were down-regulated, which is not like the human response to obesity. When comparing the three breeds of porcine obesity-models with regards to the acute phase protein ORM, none of the three breeds showed any difference in the gene regulation of ORM between the lean and obese pigs. ORM has been found to have anti-inflammatory and immunomodulatory effects and has been associated with maintaining the metabolic homeostasis in obesity. Furthermore, no expression differences were found between the adipose tissues, which is similar what has been observed in obese humans. In obese humans, raised plasma levels of ORM have been found, as was the case for obese Ossabaw minipigs. The plasma levels between lean and obese domestic pigs and Göttingen minipigs were unchanged. Therefore, with regard to ORM, the obese Ossabaw minipigs respond similarly to obesity as humans. In conclusion, none of the investigated breeds of porcine obesity models showed a complete human-like response to obesity, under the investigated conditions, though the obese Göttingen minipigs showed a response similar to obese humans with regards to gene expression in the adipose tissues, and the obese Ossabaw minipigs showed a similar response with regard to serum ORM concentrations.