Ubiquitous transgenic overexpression of C-C chemokine ligand 2: a model to assess the combined effect of high energy intake and continuous low-grade inflammation

Abstract

Excessive energy management leads to low-grade, chronic inflammation, which is a significant factor predicting noncommunicable diseases. In turn, inflammation, oxidation, and metabolism are associated with the course of these diseases; mitochondrial dysfunction seems to be at the crossroads of mutual relationships. The migration of immune cells during inflammation is governed by the interaction between chemokines and chemokine receptors. Chemokines, especially C-C-chemokine ligand 2 (CCL2), have a variety of additional functions that are involved in the maintenance of normal metabolism. It is our hypothesis that a ubiquitous and continuous secretion of CCL2 may represent an animal model of low-grade chronic inflammation that, in the presence of an energy surplus, could help to ascertain the afore-mentioned relationships and/or to search for specific therapeutic approaches. Here, we present preliminary data on a mouse model created by using targeted gene knock-in technology to integrate an additional copy of the CCl2 gene in the Gt(ROSA)26Sor locus of the mouse genome via homologous recombination in embryonic stem cells. Short-term dietary manipulations were assessed and the findings include metabolic disturbances, premature death, and the manipulation of macrophage plasticity and autophagy. These results raise a number of mechanistic questions for future study.

Figure 1

A STOP sequence flanked by loxP sites was inserted between the Ubiquitin promoter and the mouse Ccl2 gene (a). The sequences of both the STOP cassette (bold) and the loxP sites (underlined) are shown later (b). The wild-type allele for Ccl2 gene is located in the region 11 C-E1 of chromosome 11 and the transgenic vector (bottom) is inserted in the ROSA26 locus of chromosome 6 (c). The procedure is designed to avoid chromosomal instabilities.

Figure 2

Simplified strategy for genotyping that includes the sequence of each primer (a), the reaction proposed for each primer (b), and the expected PCR products for each strain (c). The method is designed for the concomitant use of all primers and a representative gel is shown in (d).

Figure 3

Overexpression of CCL2 with respect to wild type and knockout was observed in all selected tissues (extracts) in the transgenic mice as measured by ELISA. Differences were also observed in plasma and serum and there was cross-reactivity with similar chemokines that could explain the detection of CCL2 in KO mice (a). CCL2 was also detected by immunochemistry in different types of cells (b). *P < 0.005; Micrographs in the left column are representative for liver, pancreas, and kidney. Those in the right column were for brain, intestine, and stomach.

Figure 4

The effect of high-fat diet in body weight increase was evident in transgenic and wild-type mice ((a), (b)), but the different increase was immediate after dietary manipulation in transgenic. This effect was negligible in knockout mice (c). The combination of these effects with high-fat diet (d) shows similar results to facilitate comparison. These findings are not due to differences in the cumulative food intake ((e), (f)) indicating that CCL2 probably has no effect on appetite. *P < 0.05.

Figure 5

The size of the adipocytes was significantly higher in transgenic mice than in wild-type and knockout mice and the effect was observed with both dietary interventions regardless of the duration of the dietary treatment (6 or 14 weeks) (a) but it was more intense when mice were fed a high-fat diet. For clarity, values are indicated only for adipocytes in epididymal white adipose tissue. Representative micrographs are shown for transgenic, wild-type, and knockout animals ((b), (c) and (d), resp.) when fed a chow diet and for the corresponding animals fed a high-fat diet ((e), (f), (g)) at 16 and 24 weeks' old.

Figure 6

The effect of CCL2 expression in the weight of adipose tissue ((a)–(d)) of animals fed either chow (left column) or high-fat diet (right column). Of note, differences among strains were more evident during energy surplus and no change was observed in brown adipose tissue.

Figure 7

We found no significant differences among strains in the appearance of liver tissue when mice were fed a chow diet (left column (a), (c), (e); transgenic, wild-type, and knockout mice resp.). Representative micrographs show in the right column that a high fat diet produces steatosis in transgenic mice (b), dispersed lipid droplets in the liver of wild type mice (d), and no change in knockout mice (f).

Figure 8

Dietary fat (right column) and CCL2 expression modify the size, number, and morphology of liver macrophages with respect to those fed a chow diet (left column) as assessed with F4/80 staining. Values for stained area and length of macrophages ((a)–(d)) are illustrated with representative microphotographs from transgenic ((e), (f)), WT ((g), (h)) and KO mice ((i), (j)).

Figure 9

The appearance of mitochondria was affected by the dietary manipulation and the expression of CCL2 as shown in representative microphotographs (a) and these changes were accompanied by a significant effect in fusion-fission balance (b). The number of autophagosomes per cell was counted and was significantly higher in transgenic mice. Further, these were rare in both WT and KO and independent of diet (c). The heterogeneous nature of autophagic elements is illustrated in (d) (photographs obtained in transgenic mice).

Figure 10

Relative mRNA expression in transgenic mice with respect to WT mice of selected markers for M1 and M2 macrophages in cells treated in vitro with either GC-MSF or M-CSF. Acronyms used were C-C chemokine receptor type 2 (CCR2), TNFα, Inhibin, beta A (INHBA), inducible nitric oxide synthase (iNOS), C-C chemokine receptor type 7 (CCR7), Egl nine homolog 3 (EGLN3), Arginase (ARG), EGF module-containing mucin-like hormone receptor EMR1 (F4/80), insulin growth factor-1 (IGF1), IL-10, the mannose receptor CD206, and Growth arrest-specific 6 (GAS6).

Figure 11

The relative CCL2 mRNA expression (a) and the secretion in supernatants of selected cytokines in bone marrow-derived macrophages of transgenic and WT cells treated in vitro with either GC-MSF (b) or M-CSF (c).