Benznidazole affects expression of Th1, Th17 and Treg cytokines during acute experimental Trypanosoma cruzi infection

<div><p>Abstract Background The present study evaluated the effect of treatment with benznidazole on mRNA expression of IFN-γ, IL-17, IL-10, TGF-β and FoxP3 in spleen and heart tissue of BALB/c mice in the acute phase of an experimental infection with Trypanosoma cruzi, strains JLP or Y. Methods The mRNA expression of cytokines and parasite load were assessed by q-PCR. Dependent groups were compared using Student's paired t-test and independent groups were compared using Student's unpaired t-test. Results Infection with the JLP or Y strains increased expression of IFN-γ in the heart and of IL-10 and IL-17 in the spleen and heart compared to uninfected animals. Treatment increased the expression of IFN-γ and decreased the expression of IL-17, IL-10, TGF- β and Foxp3 in spleen and heart tissue compared to untreated infected animals. Conclusion Benznidazole can induce Th1 profile in the initial of the acute phase. The treatment decreased the parasite load in both organs, although the number of parasites in Y-strain-infected mice remained high. The data suggest that benznidazole may modulate cytokine expression in infection and can be dependent of the strain. However, treatment was not fully effective in the infection provoked by Y strain, probably due to the characteristics of the strain itself.</p></div

The Involvement of TLR2 and TLR4 in Cytokine and Nitric Oxide Production in Visceral Leishmaniasis Patients before and after Treatment with Anti-Leishmanial Drugs - Table 1

<p>* <i>Glucantime (M-metyl-glucamine)scheme</i>: <i>20 mg/Kg/day for 20 days</i></p><p>** <i>Deoxycholate Amphotericin B scheme</i>: <i>1 mg/Kg/day for 20 days</i></p><p>*** <i>Liposomal Amphotericin B scheme</i>: <i>5 mg/Kg/day for 5 days</i></p><p>The Involvement of TLR2 and TLR4 in Cytokine and Nitric Oxide Production in Visceral Leishmaniasis Patients before and after Treatment with Anti-Leishmanial Drugs - Table 1 </p

Cytokine production after stimulation with LPS and PGN agonists.

<p>Levels of TNF-α (A), IFN-γ (B), IL-17 (C), IL-10 (D) and TGF-β (E) in supernatants were analyzed after 24 hours of PBMCs cultured (1x10⁶ cells/ml), obtained from patients with VL pre-treatment, post-treatment and control subjects, and stimulated or not with LPS ultra-purified (1 μg/ml), PGN (5 μg/ml) and LPS+PGN. Lowercase letters represent significant differences among agonists in the same group: p<0.05 <b>a</b>—PBMC <i>vs</i>. LPS, PGN, LPS+PGN; <b>b</b>—PBMC, PGN <i>vs</i>. LPS, LPS+PGN; <b>c</b>—PBMC, LPS <i>vs</i>. PGN, LPS+PGN; <b>d</b>—LPS <i>vs</i>. LPS+PGN. Capital letters represent significant differences in the same agonists among groups: p<0.05 <b>A</b>—pre-treatment <i>vs</i>. post-treatment; <b>B</b>—pre-treatment <i>vs</i>. control individuals; <b>C</b>—post-treatment <i>vs</i>. control individuals. Cytokine levels were measured by CBA. Each dot represent a different patient and each bar represents the median.</p

NO production after stimulation with LPS and PGN agonists.

<p>Levels of NO in supernatants were analyzed after 24 hours of PBMCs cultured (1x10⁶ cells/ml), obtained from patients with VL pre-treatment, post-treatment and control subjects, and stimulated or not with LPS ultra-purified (1 μg/ml), PGN (5 μg/ml) and LPS+PGN. Lowercase letters represent significant differences among agonists in the same group: p<0.05 <b>a</b>—PBMC <i>vs</i>. LPS, PGN, LPS+PGN. Capital letters represent significant differences in the same agonists among groups: p<0.05 <b>A</b>—pre-treatment <i>vs</i>. post-treatment; <b>B</b>—pre-treatment <i>vs</i>. control individuals. Each dot represent a different patient and each bar represents the median. NO levels were measured by the levels of nitrite/nitrate and the data are representative of triplicates.</p

Expression of surface TLR2 and TLR4.

<p>Frequency of CD3⁺ and CD14⁺ cells expressing TLR2, TLR4 and co-expressing TLR2 and TLR4 (A) and mean of fluorescence intensity (B) of TLR2 and TLR4 in CD3⁺ and CD14⁺ cells in whole blood from patients with visceral leishmaniasis pre-treatment, post-treatment and control subjects. Gating strategies to distinguish between cell populations analyzed by flow cytometry. FSC-SSC profile was used to distinguish total lymphocytes and monocytes and these subtype cells were gated according to light scatter profile and the expression of CD3 or CD14. Representative histograms plots of TLR2 and TLR4 expression in CD3⁺ (C,D) and CD14⁺ (E,F) cells. The results are expressed as the mean and standard deviation. *p<0.05 compared pre-treatment <i>vs</i>. post-treatment and control subjects; ▪ p<0.05 compared pre-treatment <i>vs</i>. control subjects and • p<0.05 compared post-treatment <i>vs</i>. control subjects.</p

Gene expression and cell surface of TLR2 and TLR4.

<p>mRNA expression of TLR2 (A) and TLR4 (B) and mean percentages of CD3⁺ (C) and CD14⁺ (D) cells expressing TLR2, co-expressing TLR2 and TLR4 and expressing TLR4 among PBMCs from control individuals (G1) and pulmonary tuberculosis patients (G2) during anti-tuberculosis treatment at M1 (up to one month of treatment), M2 (three months of treatment) and M3 (end of treatment). Gating strategies to distinguish between PBMC populations analyzed by flow cytometry. FSC-SSC profile was used to distinguish total lymphocytes and monocytes (E). T lymphocytes and monocytes were gated according to light scatter profile and the expression of CD3 (F) and CD14 (G), respectively. Representative histograms plots of TLR2 and TLR4 expression are shown in figures H–K, in which blue histograms represent isotype controls and red histograms indicate TLR-specific antibodies with the respective expression of TLR2⁺ and TLR4⁺ in monocytes (H, I) and T lymphocytes (J, K). The results are expressed as mean and standard deviation and are representative of three independent replicates. *p<0.05 compared with G1; #p<0.05 compared with M2 and M3; ^○p<0.05 compared with M1 and M2; Δp<0.05 compared with M1.</p

Expression and production cytokines inflammatory.

<p>mRNA expression and production of IL-12 (A, B), IFN-γ (C, D), TNF-α (E, F) and IL-17 (G, H) in the control group (G1) and in the patient group during anti-tuberculosis treatment (G2) and at M1 (up to one month of treatment), M2 (three months of treatment) and M3 (end of treatment). The results are expressed as the mean and standard deviation and are representative of three independent replicates. *p<0.05 compared with G1; #p<0.05 compared with M1.</p

Expression and production cytokines IL-10 and TGF-β.

<p>mRNA expression and production of IL-10 (A, B) and TGF-β (C, D) in the control group (G1) and in the patient group during anti-tuberculosis treatment (G2) and at M1 (up to one month of treatment), M2 (three months of treatment) and M3 (end of treatment). The results are expressed as the mean and standard deviation and are representative of three independent replicates. *p<0.05 compared with G1.</p