Genetic and Functional Role of TNF-alpha in the Development Trypanosoma cruzi Infection

TNF-alpha plays an important role in trypanocidal mechanisms and is related to tissue injury. This cytokine has been detected in the heart of human chagasic patients where it is associated with tissue damage. This study investigated whether TNF-alpha levels and the presence of genetic polymorphisms are associated with the presence of T. cruzi infection and/or with the development of the cardiac form in chronic chagasic patients. Genomic DNA of 300 subjects from an endemic area was extracted and analyzed by PCR using specific primers. TNF-alpha was assayed in culture supernatants by ELISA. An association was observed between the absence of the TNF-238A allele and negative serology. Furthermore, seropositive individuals carrying the TNF-238A allele produced significantly higher TNF-alpha levels without stimulation (p = 0.04) and after stimulation with LPS (p = 0.007) and T. cruzi antigens (p = 0.004). The present results suggest that the polymorphism at position -238 influences susceptibility to infection and that this allele is associated with higher TNF-alpha production in seropositive individuals

Levels of TNF-alpha in culture supernatants.

<p>TNF-alpha was assayed by ELISA in supernatants of PBMC cultured for 48 h in the absence of any stimulus and in the presence of <i>T. cruzi</i> antigen, LPS and PHA. <b>A.</b> Subjects were grouped according to positive or negative serology; *p = 0.0003 (Mann-Whitney test). <b>B.</b> Subjects were grouped according to the cardiac or indeterminate form and negative serology: Cardiac x negative to medium: p = 0.0002; cardiac x negative to LPS stimulation: p = 0.005; indeterminate x negative to PHA: p = 0.03; cardiac x indeterminate to medium: p = 0.01; to LPS: p = 0.005; to <i>T. cruzi</i> antigens: p = 0.01 (Mann-Whitney test). Results are expressed as pg/mL. The horizontal line indicates the median, bars the 25% and 75% percentiles, and vertical lines the 10% and 90% percentiles.</p

Levels of TNF-alpha in culture supernatants.

<p>TNF-alpha was assayed by ELISA in supernatants of PBMC cultured for 48 h in the absence of any stimulus and in the presence of <i>T. cruzi</i> antigen, LPS and PHA. Subjects were grouped according to TNF-238A genotypes (TNF-238AA, TNF-238GA and TNF-238GG). Individuals with positive and negative serology were analyzed. Results are expressed as pg/mL. The horizontal line indicates the median, bars the 25% and 75% percentiles, and vertical lines the 10% and 90% percentiles.</p

TNF-alpha genotypes found in subjects from an endemic area according to clinical form.

<p>TNF-alpha genotypes found in subjects from an endemic area according to clinical form.</p

TNF-alpha genotypes found in subjects from an endemic area according to the presence or absence of infection.

<p>TNF-alpha genotypes found in subjects from an endemic area according to the presence or absence of infection.</p

Levels of TNF-alpha in culture supernatants.

<p>TNF-alpha was assayed by ELISA in supernatants of PBMC cultured for 48 h in the absence of any stimulus and in the presence of <i>T. cruzi</i> antigen, LPS and PHA. <b>A.</b> Subjects with positive serology and negative serology were grouped according to the presence or absence of the TNF-238A allele; *p = 0.045 (Mann-Whitney test). <b>B.</b> Only subjects with positive serology were grouped according to the presence or absence of the TNF-238A allele; * p = 0.04 compared to medium, p = 0.004 compared to <i>T. cruzi</i> antigen, and p = 0.007 compared to LPS (Mann-Whitney test). Results are expressed as pg/mL. The horizontal line indicates the median, bars the 25% and 75% percentiles, and vertical lines the 10% and 90% percentiles.</p

Presence and absence of the TNF-238A allele in seropositive and seronegative individuals.

<p>Presence and absence of the TNF-238A allele in seropositive and seronegative individuals.</p

TNF-alpha expression on CD8⁺ and CD4⁺ lymphocytes.

<p><b>A.</b> Percentage of TNF-alpha-producing cells among the CD8⁺ and CD4⁺ population of patients with the cardiac (gray bars) and indeterminate form (white bars) after 48 h of culture in the presence of <i>T. cruzi</i> antigens. <b>B.</b> Percentage of TNF-alpha-producing cells among the CD14⁺ population of patients with the cardiac (gray bars) and indeterminate form (white bars) after 48 h of culture in the presence of <i>T. cruzi</i> antigens. For analysis, a gate was established using FSC and surface marker flowed by analysis of intracellular TNF-alpha expression.</p