Interaction between peripheral blood mononuclear cells and Trypanosoma cruzi-infected adipocytes: implications for treatment failure and induction of immunomodulatory mechanisms in adipose tissue

Abstract

Background/introduction: Adipose tissue (AT) has been highlighted as a promising reservoir of infection for viruses, bacteria and parasites. Among them is Trypanosoma cruzi, which causes Chagas disease. The recommended treatment for the disease in Brazil is Benznidazole (BZ). However, its efficacy may vary according to the stage of the disease, geographical origin, age, immune background of the host and sensitivity of the strains to the drug. In this context, AT may act as an ally for the parasite survival and persistence in the host and a barrier for BZ action. Therefore, we investigated the immunomodulation of T. cruzi-infected human AT in the presence of peripheral blood mononuclear cells (PBMC) where BZ treatment was added.

Methods: We performed indirect cultivation between T. cruzi-infected adipocytes, PBMC and the addition of BZ. After 72h of treatment, the supernatant was collected for cytokine, chemokine and adipokine assay. Infected adipocytes were removed to quantify T. cruzi DNA, and PBMC were removed for immunophenotyping.

Results: Our findings showed elevated secretion of interleukin (IL)-6, IL-2 and monocyte chemoattractant protein-1 (MCP-1/CCL2) in the AT+PBMC condition compared to the other controls. In contrast, there was a decrease in tumor necrosis factor (TNF) and IL-8/CXCL-8 in the groups with AT. We also found high adipsin secretion in PBMC+AT+T compared to the treated condition (PBMC+AT+T+BZ). Likewise, the expression of CD80+ and HLA-DR+ in CD14+ cells decreased in the presence of T. cruzi.

Discussion: Thus, our findings indicate that AT promotes up-regulation of inflammatory products such as IL-6, IL-2, and MCP-1/CCL2. However, adipogenic inducers may have triggered the downregulation of TNF and IL-8/CXCL8 through the peroxisome proliferator agonist gamma (PPAR-g) or receptor expression. On the other hand, the administration of BZ only managed to reduce inflammation in the microenvironment by decreasing adipsin in the infected culture conditions. Therefore, given the findings, we can see that AT is an ally of the parasite in evading the host's immune response and the pharmacological action of BZ.

Keywords: Trypanosoma cruzi; adipokine; adipose tissue; benznidazole; chemokine; cytokine; immunomodulation.

Figure 1

Schematic representation of indirect culture between Trypanosoma cruzi-infected AT, PBMC and benznidazole treatment. PBMC, Peripheral blood mononuclear cells (PBMC); AT, Adipose tissue from adipogenic differentiation of ADSC (AT); Trypanosoma cruzi (T); Benz nidazole (BZ). Design created with the aid of Biorender ^®.

Figure 2

Adipogenic differentiation of human adipose-derived stem cells (ADSC). Legend: Human adipose tissue-derived stem cells (ADSC) after adipogenic differentiation. Lipid vesicles were highlighted by green color after staining with AdipoRed™ and visualized in confocal microscopy. (A) Adipogenic differentiation of ADSC (phase-contrast); (B) Adipogenic differentiation of ADSC (phase-contrast + fluorescence); (C) Adipogenic differentiation of ADSC (fluorescence).

Figure 3

Quantification of parasite load of Trypanosoma cruzi infected adipocytes in indirect culture between adipose tissue, peripheral blood mononuclear cells and Benznidazole treatment. PBMC, Peripheral blood mononuclear cells (PBMC); AT, Adipose tissue from adipogenic differentiation of ADSC (AT); Trypanosoma cruzi (T); Benznidazole (BZ). Horizontal bars represent the median and vertical bars the lower and upper limit. Symbols (*) above the bars indicate statistical difference with p ≤ 0.05.

Figure 4

Measurement of cytokines in the culture supernatant of indirect culture between Trypanosoma cruzi-infected adipose tissue, peripheral blood mononuclear cells and benznidazole treatment. PBMC, Peripheral blood mononuclear cells (PBMC); AT, Adipose tissue from adipogenic differentiation of ADSC (AT); Trypanosoma cruzi (T); Benznidazole (BZ). (A) IL-2 cytokine measurement; (B) IL-4 cytokine measurement; (C) IL-10 cytokine measurement; (D) IL-6 cytokine measurement; (E) TNF cytokine measurement; (F) IFN-gamma cytokine measurement. Horizontal bars represent the median and vertical bars the lower and upper limit. Symbols (*) above the bars indicate statistical difference with p ≤ 0.05.

Figure 5

Measurement of chemokines in the culture supernatant of indirect culture between adipose tissue infected by Trypanosoma cruzi, peripheral blood mononuclear cells and treatment with benznidazole. PBMC, Peripheral blood mononuclear cells (PBMC); AT, Adipose tissue from adipogenic differentiation of ADSC (AT); Trypanosoma cruzi (T); Benznidazole (BZ). (A) IP-10/CXCL10 chemokine measurement; (B) MIG/CXCL9 chemokine measurement; (C) MCP-1/CCL2 chemokine measurement; (D) RANTES/CCL5 chemokine measurement; (E) IL-8/CXCL8 chemokine measurement. Horizontal bars represent the median and vertical bars the lower and upper limit. Symbols (*) above the bars indicate statistical difference with p ≤ 0.05.

Figure 6

Measurement of adipokines in the culture supernatant of indirect culture between adipose tissue infected by Trypanosoma cruzi, peripheral blood mononuclear cells and treatment with benznidazole. PBMC, Peripheral blood mononuclear cells (PBMC); AT, Adipose tissue from adipogenic differentiation of ADSC (AT); Trypanosoma cruzi (T); Benznidazole (BZ). (A) Adiponectin adipokine measurement; (B) Adipsin adipokine measurement; (C) Leptin adipokine measurement; (D) Resistin adipokine measurement. Horizontal bars represent the median and vertical bars the lower and upper limit. Symbols (*) above the bars indicate statistical difference with p ≤ 0.05.

Figure 7

Evaluation of the expression of activation/inhibition molecules in CD4+ and CD8+ T lymphocytes from indirect culture between Trypanosoma cruzi-infected adipose tissue, peripheral blood mononuclear cells and benznidazole treatment. PBMC, Peripheral blood mononuclear cells (PBMC); AT, Adipose tissue from adipogenic differentiation of ADSC (AT); Trypanosoma cruzi (T); Benznidazole (BZ). (A) CD4+ expression on lymphocytes; (B) CD8+ expression on lymphocytes; (C) CD28+ expression on CD4+ T lymphocytes; (D) CD28+ expression on CD8+ T lymphocytes; (E) CTLA-4+ expression in CD4+ T lymphocytes; (F) CTLA-4+ expression in CD8+ T lymphocytes. Horizontal bars represent the median and vertical bars the lower and upper limit. Symbols (*) above the bars indicate statistical difference with p ≤ 0.05.

Figure 8

Evaluation of the expression of activation/inhibition molecules in CD14⁺ cells from indirect culture between Trypanosoma cruzi-infected adipose tissue, peripheral blood mononuclear cells and benznidazole treatment. PBMC, Peripheral blood mononuclear cells (PBMC); AT, Adipose tissue from adipogenic differentiation of ADSC (AT); Trypanosoma cruzi (T); Benznidazole (BZ). (A) CD80+ expression on CD14+ monocytes; (B) HLA-DR+ expression on CD14+ monocytes Horizontal bars represent the median and vertical bars the lower and upper limit. Symbols (*) above the bars indicate statistical difference with p ≤ 0.05.

Figure 9

Schematic representation of the study’s main findings. PBMC, Peripheral blood mononuclear cells (PBMC); AT, Adipose tissue from adipogenic differentiation of ADSC (AT); Trypanosoma cruzi (T); Benznidazole (BZ). Design created with the aid of Biorender ^®.