Trypanosoma cruzi infection of cultured adipocytes results in an inflammatory phenotype

Abstract

Infection with Trypanosoma cruzi, the etiologic agent of Chagas disease is accompanied by an intense inflammatory reaction. Our laboratory group has identified adipose tissue as one of the major sites of inflammation during disease progression. Because adipose tissue is composed of many cell types, we were interested in investigating whether the adipocyte per se was a source of inflammatory mediators in this infection. Cultured adipocytes were infected with the Tulahuen strain of T. cruzi for 48-96 h. Immunoblot and quantitative PCR (qPCR) analyses demonstrated an increase in the expression of proinflammatory cytokines and chemokines, including interleukin (IL)-1 beta, interferon-gamma, tumor necrosis factor-alpha, CCL2, CCL5, and CXCL10 as well as an increase in the expression of Toll-like receptors-2 and 9 and activation of the notch pathway. Interestingly, caveolin-1 expression was reduced while cyclin D1 and extracellular signal-regulated kinase (ERK) expression was increased. The expression of PI3kinase and the activation of AKT (phosphorylated AKT) were increased suggesting that infection may induce components of the insulin/IGF-1 receptor cascade. There was an infection-associated decrease in adiponectin and peroxisome proliferator-activated receptor-gamma (PPAR-gamma). These data provide a mechanism for the increase in the inflammatory phenotype that occurs in T. cruzi-infected adipocytes. Overall, these data implicate the adipocyte as an important target of T. cruzi, and one which contributes significantly to the inflammatory response observed in Chagas disease.

Figure 1

Representative electron micrograph of a Trypanosoma cruzi–infected cultured adipocyte. Asterisks indicate lipid droplet. Arrows indicate parasites. Nu indicates adipocyte nucleus. Bar = 1 μm.

Figure 2

Representative immunoblot/quantitative PCR analyses of the effect of Trypanosoma cruzi infection on expression of mitogen activated protein kinases, cell cycle regulatory proteins, and proteins of the notch signaling pathway in cultured adipocytes. (a) Immunoblot analysis of pERK (ERK42/44), p38 MAPK, and pJNK 48–96 h postinfection (PI). (b) Quantitative analysis of immunoblot (a) normalized to guanine nucleotide dissociation inhibitor (GDI). (c) Immunoblot analysis of pCaveolin-1 96 h PI. (d) Immunoblot analysis of cyclin D1 48, 72, and 96 h PI. (e) Quantitative analysis of immunoblots (d) demonstrating a significant increase in cyclin D1 protein levels normalized to GDI. (f) Immunoblot analysis of cyclin A1, cyclin D1, and cyclin E1 in infected adipocytes 96 h PI. (g) Fold-increase in the expression of cyclin D1 mRNA level as analyzed by quantitative reverse transcriptase–PCR 48–96 h PI. (h) Representative immunoblot analysis of levels of ICN, Hey-1, and Hes-5 in infected adipocytes. (i) Quantitative representation of data shown in h. C, control; I, infected. (n = 3, asterisk denotes significance, P < 0.05).

Figure 3

Effects of Trypanosoma cruzi infection on expression of cytokines, chemokines, and Toll-like receptors (TLRs) in adipocytes. (a) Quantitative PCR (qPCR) analysis of CCL2, CCL3, CCL5, and CXCL10 represented as fold-increase in mRNA levels 72 and 96 h postinfection (PI). (b) qPCR analyses demonstrate fold-increases in mRNA expression levels of interleukin (IL)-1β, IL-4, IL-10, and IFN-γ 72 and 96 h PI. (c) Fold-increase in the mRNA levels of TNF-α determined by qPCR 72 and 96 h PI. (C, control; I, infected). (n = 3). (d) qPCR analysis of TLR-2, TLR-4, and TLR-9 in fat cells 96 h PI. The data are shown as fold change normalized to glyceraldehyde-3-phosphate dehydrogenase. The increases in TLR-2 and TLR-9 are significant (n = 3, asterisk denotes significance, P < 0.05).

Figure 4

Effect of Trypanosoma cruzi infection on expression of pSTAT3, PPAR-γ, adipocyte-specific proteins and insulin/IGF-1 receptor signaling proteins in adipocytes. (a) Representative immunoblot analysis of pSTAT3 expression 48, 72, and 96 h postinfection (PI). (b) Immunoblot analysis of PPAR-γ abundance 48, 72, and 96 h PI. (c) Quantitative analysis of protein abundance presented as fold-increase. PPAR-γ is significantly reduced and pSTAT3 is significantly increased at all time-points. (d) Representative immunoblot analysis of PPAR-γ, resistin, and adiponectin in fat cells 96 h postinfection. (e) Quantitative PCR showing fold decrease in the expression of PPAR-γ 48, 72, and 96 h PI (n = 3). (f) Representative immunoblot analysis of adiponectin (MW = 30 kDa) abundance 48, 72, and 96 h PI. There is a significant reduction in abundance 72 and 96 h PI (asterisk denotes significance, P < 0.05). (g) Quantitative representation of data shown in f normalized to GDI. (h) Representative immunoblot analysis of pAKT/IRS-1 and PI3K abundance in control (C) and infected (I) adipocytes 96 h PI. (i) Quantitative representation of data shown in h. (n = 3). All changes in the infected cells are significant (P < 0.05).