Helminth infection protects against high fat diet-induced obesity via induction of alternatively activated macrophages

Abstract

Epidemiological studies indicate an inverse correlation between the prevalence of the so-called western diseases, such as obesity and metabolic syndrome, and the exposure to helminths. Obesity, a key risk factor for many chronic health problems, is rising globally and is accompanied by low-grade inflammation in adipose tissues. The precise mechanism by which helminths modulate metabolic syndrome and obesity is not fully understood. We infected high fat diet (HFD)-induced obese mice with the intestinal nematode parasite Heligmosomoides polygyrus and observed that helminth infection resulted in significantly attenuated obesity. Attenuated obesity corresponded with marked upregulation of uncoupling protein 1 (UCP1), a key protein involved in energy expenditure, in adipose tissue, suppression of glucose and triglyceride levels, and alteration in the expression of key genes involved in lipid metabolism. Moreover, the attenuated obesity in infected mice was associated with enhanced helminth-induced Th2/Treg responses and M2 macrophage polarization. Adoptive transfer of helminth-stimulated M2 cells to mice that were not infected with H. polygyrus resulted in a significant amelioration of HFD-induced obesity and increased adipose tissue browning. Thus, our results provide evidence that the helminth-dependent protection against obesity involves the induction of M2 macrophages.

Figure 1

Experimental models. Schematic diagram illustrates H. polygyrus infection on HFD-fed mice. Schematic diagram illustrates H. polygyrus infection on long-term HFD experiment. The schematic diagram (3) illustrates the adoptive transfer of macrophage experiment in HFD-induced obesity; here the objective was to observe the potential regulatory role of helminth-induced M2 cells in HFD-obesity.

Figure 2

H. polygyrus infection attenuates HFD-induced obesity in mice. C57BL/6 mice fed with the HFD showed steady gain in body weight than those HFD infected with H. polygyrus through the course of experiment (a). Blood glucose levels were attenuated by H. polygyrus infection (b). Mice on the HFD accumulated more gonadal and subcutaneous fat than HFD fed infected mice (c). In long term experiments, C57BL/6 mice on HFD gradually gained more body weight (red line) than those HFD but infected with H. polygyrus (blue line) along a 104-day course (d). No differences in food intakes of HFD obese mice with (blue line) or without (red line) H. polygyrus infection (e). Blood glucose levels showed significant differences at 15, 30 and 45 minutes, between the two groups of mice (f). Triglyceride and cholesterol levels significantly dropped in H. polygyrus infected mice (g and h). The physical appearance (body size) in HFD-fed mice with (Hp) and without infection (Un-inf) (i) and the histological analysis of liver section of HFD induced mice showing severe steatosis, with fat deposits shown in un-infected mice using ORO staining (j). (n = 5–6; *p < 0.05; **p < 0.01. Two-tailed Student’s t-test).

Figure 3

H. polygyrus infection influences gene expression of key enzymes/mediators of lipid metabolism. H. polygyrus infection results in decreased gene expression of leptin enzymes compared with normal mice in gonadal fat tissues: (a) leptin; (b) C/EBPα; and (c) PPARγ. (n = 5–6; *p < 0.05; **p < 0.01; two-tailed Student’s t-test).

Figure 4

H. polygyrus infection results in upregulation of UCP1 expression and adipose tissue browning. qPCR analysis revealed that H. polygyrus infection results in a significant upregulation of UCP-1 expression in gonadal fat tissue at mRNA level (a). Immunoblot analysis showed increased UCP1 levels in adipose and liver tissue (b). qPCR results revealed significant upregulation of mitochondrial mass and UPC-1 expression (c). (n = 5–6; *p < 0.05; **p < 0.01; two-tailed Student’s t-test). (d and e) The adipose tissues showed marked reduction in the adipocyte size and increased expression of UCP1 in helminth-infected HFD fed mice (HFD-HP) compared to non-infected (HFD) mice. Adipose (including brown adipose, subcutaneous and gonadal adipose tissues) from HFD-fed mice with and without helminth infection collected. Ten-μm sections were cut on a Leica CM1850 Cryostat (Leica Biosystem) and were stained with hematoxylin and eosin (d) or anti-UCP1 antibody (e). (d) H&E staining: arrows indicate the presence of multilocular lipid droplets in adipocytes in the white adipose tissues of infected mice absent in uninfected obese mice. (e) Immunohistochemical staining of the brown (upper panel) and white (low panel) adipose tissues revealed a markedly intensified UCP1 expression in the adipose tissue of helminth-infected mice compared to non-infected HFD-fed mice.

Figure 5

H. polygyrus infection promotes a strong type 2 immune response. Real time qPCR analysis shows a clear suppression in the T-bet and RORγt mRNAs expression (a and b) and increase in the expression of GATA3 and Foxp3 mRNAs (c and d) in the MLN cells from HFD-fed H. polygyrus-infected mice. H. polygyrus-infected obese mice showed decreases in IFN-γ and IL-17 secretions in the supernatant of cultured MLN cells (e and f). H. polygyrus-infected obese mice had higher production of cytokines IL-4 and IL-10 in MLN (g and h). H. polygyrus-infected obese mice had increased levels of serum IgE (i) and IgG1 (j) and a decreased IgG2a level (k). (n = 5–6; *p < 0.05; two-tailed Student’s t-test).

Figure 6

H. polygyrus-infection influences M1/M2 macrophage polarization in gonadal fat of obese mice towards anti-inflammatory M2 phenotype. (a) H. polygyrus infection results in reduced HFD-induced inducible oxide synthase (iNOS) expression, indicating inhibition M1 macrophages. (b and c) H. polygyrus infection results in increased expression of YM1 and Arg1, promoting the development of M2 cells in HFD-fed mice. (d and e) H. polygyrus infection results in increased IL-10 expression and decreased TNF-α expression. (n = 5–6; *p < 0.05; two-tailed Student’s t-test). (f and g) Peritoneal F4/80⁺ macrophages from normal control (g, mostly F4/80⁺EGR2⁻ M1) and H. polygyrus-infected mice (h: most F4/80⁺EGR2⁺ M2 cells).

Figure 7

Adoptive transfer of M2 cells results in attenuated obesity in HFD-fed mice. (a) Body weigh change in mice received macrophages from normal and helminth-infected donor mice. (b) AUC of OGTT, Blood glucose level; (c) fat tissue and liver weight; (d) blood chemistry test; (e) M2 cell transfer alters gene expression of leptin and TNF-α; (f) M2 transfer displays M2 phenotype of increased IL-10 expression. (n = 5–6; *p < 0.05; **p < 0.01; two-tailed Student’s t-test).

Figure 8

Adoptive transferring of H. polygyrus-induced M2 cells promotes adipose tissue browning. Purified F4/80⁺ macrophages from normal or H. polygyrus-infected donors were transferred into HFD-fed recipient mice. Adipose tissues were collected. (a) qPCR data showed significant increase in UCP1 expression and mitochondrial levels in adipose tissues from mice that received helminth-stimulated M2 cells. (n = 5–6; *p < 0.05; two-tailed Student’s t-test). (b) Representative of immunohistochemical stained sections showed marked reduction of the adipocyte size and increased intensity of UCP1 expression in the adipose tissue of the recipient mice transferred with helminth-induced M2 cells compared to mice harboring normal macrophages.