Fig 1.
Experimental design and course of infection in diet-induced obese C57BL/6 mice.
Animals were fed control (AIN93-G) or hypercaloric (HSB) diets ad libitum for four weeks. On the fourth week mice were infected with 1x106 metacyclic L. major in the ear. Weight gain and food intake were measured weekly. (A) Experimental design. (B) Weekly measurement of C57BL/6 body weight. (C) Weekly measurement of infected ear thickness during eight weeks of infection. (D) Parasite titer in the infected ear, measured by limiting dilution. Data are represented as average ± SD. Statistical analysis was performed by Student´s t test (*p<0.05; **p<0.005). Results are representative of at least 4 independent experiments, n = 4 mice/group.
Fig 2.
HSB diet-induced obesity leads to a more severe inflammatory reaction in the infected ear.
Representative photomicrographs of ear histological sections from C57BL/6 mice submitted to HSB and AIN93-G diets and infected with L. major. (A, B and C) photomicrography from control mice 2, 4 and 8 weeks post infection. The cellular infiltration is predominantly polymorphonuclear (Insert in A, B), focal and discrete to moderate infiltration in the papillary dermis and deep dermis accompanied by mild to moderate thickening of the dermis; erosion and ulceration of the epidermis, predominantly polymorphonuclear inflammatory process with discrete mast cell hyperplasia (Insert in C) and thickening of the dermis, moderate to severe at eight weeks post infection. (D, E and F) photomicrography from HSB-fed mice 2, 4 and 8 weeks post infection. Ulcerated epidermis, predominantly polymorphonuclear and focal inflammatory process in the dermis and hypodermis, with thickening of the dermis, both of intense character, and tissue parasitism (Insert in D) in animals belonging to the obese group, 2 weeks post infection; moderate thickening of the dermis and inflammatory infiltrate also moderate, predominantly polymorphonuclear (Insert in E) and focal, in the papillary dermis, deep dermis and hypodermis 4 weeks post infection; ulcerated epidermis, predominantly polymorphonuclear and focal inflammatory process in the dermis and hypodermis with thickening of the dermis, both moderately characterized with moderate mast cell hyperplasia (Insert in F), 8 weeks after infection. Hematoxylin-Eosin. Bar in the normal image = 25mm.
Fig 3.
Diet-induced obesity increases the concentration of L. major specific immunoglobulins in C57BL/6 serum.
Total IgG (A), IgM (B), IgG1 (C) and IgG2a (D) were measured in sera from mice fed control (AIN93G) and hypercaloric (HSB) diet. ELISA was performed at 2, 4 and 8 weeks of infection. For sensitization, L. major antigen at 20 μg/mL was used. To measure IgG, sera were diluted 1:1000, and 1:100 for IgG1, IgG2a and IgM. Data are represented as average ± SD. Statistical analysis was performed by Student´s t test (*p<0.05; **p<0.005). Results are representative of at least two independently experiments, n = 4 mice/group.
Fig 4.
Cytokine production by lymph node cells in culture.
Cytokine concentrations were measured by ELISA in cell culture supernatants stimulated in vitro with 50 μg/mL of L. major antigen. Cells were collected 2, 4 and 8 weeks after infection and adjusted for 5x106/mL of culture and incubated during 72h. (A) IFN-gamma; (B) TNF-alfa; (C) IL-4; (D) IL-10 and (E) IL-17. Data are represented as average ± SD. Statistical analysis was performed by Student´s t test (* p<0.05). Results are representative of at least two independent experiments, n = 4 mice/group.
Fig 5.
In vitro macrophage infection.
To measure the percentage of infected macrophage (MO) and the amastigote number in infected MO, cells were collected from the peritoneal cavity after thioglycotate stimulation and cultured at 1x105/ml. The infection was performed with 5 total non opsonized promastigotes of L. major per MO 24h after adhesion. Parasite counts were performed at 4 and 72 hours post infection. Arginase and NO were measured 72h after infection, as described in Materials and Methods. (A and D) Percentage of infected MO; (B and E) Amastigote number per infected MO and (C and F) Infection index [(MO*100/infected MO)*(amastigotes/infected MO)]; (G) NO production; and (H) Arginase activity. Data are represented as average ± SD. Statistical analysis was performed by Student´s t test (*) means p<0.05 between MO from control mice versus obese mice; (&) means p<0.05 comparing MO from control group without stimulus and with different culture conditions and ($) means p<0.05 comparing MO from obese group without stimulus and with different culture conditions. n = 6.
Fig 6.
HBS fed IL-17R-/- mice do not show difference in L. major infection profile compared with control AIN93G fed mice.
Wild type and IL17R-/- C57BL/6 mice were fed either control (AIN93G) or hypercaloric (HSB) diets ad libitum for four weeks. On the fifth week mice were infected with 1x106 metacyclic L. major in the ear. (A) Weekly measurement of body weight. (B) Weekly measurement of infected lesion during eight weeks of infection. (C) Parasite titer in the infected ear, measured by limiting dilution. (D) Absolute number of CD45+ cells in the draining lymph node. (E) Representative pictures of ears lesions of IL17R-/- and WT at week 8 post infection. Data are represented as average ± SD. Statistical analysis was performed by Anova test (*p<0.05; **p<0.005). n = 4/ group.