Immunomodulatory effects of cysteamine and its potential use as a host-directed therapy for tuberculosis

Abstract

Objective: Cysteamine, a drug approved to treat cystinosis, has been proposed as a host-directed therapy for M. tuberculosis (Mtb) and SARS-CoV-2. The impact of cysteamine on the immune responses has not been fully investigated. We aimed to in vitro evaluate the immunomodulatory effects of cysteamine on peripheral blood mononuclear cells (PBMCs) using the purified protein derivative (PPD) as a recall antigen, and an unspecific stimulus as staphylococcal enterotoxin B (SEB).

Methods: PBMCs isolated from subjects with tuberculosis infection (TBI), those with tuberculosis disease (TB), and healthy controls (HC) were in vitro stimulated with PPD or SEB and treated or not with cysteamine at different concentrations (50 µM-400 µM) for 6 hours (h) and 24 h. We evaluated the T helper1 (Th1) and T cytotoxic1 (Tc1) cell cytokine production by flow cytometry and immune-enzymatic assays. In HC, we also evaluated apoptosis and/or necrosis by flow cytometry.

Results: We observed an immunomodulatory effect of cysteamine at 400 µM in PBMCs from TB and TBI subjects. It significantly reduced PPD-specific Th1 responses at 24 h and at 6 h (p=0.0004 and p=0.0009, respectively), and a similar non-significant trend was observed with cysteamine at 200 µM (p=0.06 at 24 h and p=0.14 at 6 h). Moreover, cysteamine at both 400 µM (p<0.0001 and p=0.0187 at 24 h, respectively, and p<0.0001 at 6 h for both) and 200 µM (p=0.0119 and p=0.0028 at 24 h and p=0.0028 and p=0.0003 at 6 h, respectively) significantly reduced SEB-induced Th1 and Tc1 responses. Furthermore, we found that cysteamine induced morphological lymphocyte changes and significantly reduced the lymphocyte percentage in a dose- and time-dependent manner. Cysteamine at 400 µM induced 8% late apoptosis and 1.6% necrosis (p<0.05) at 24 h. In contrast, despite significant differences from untreated conditions (p<0.05), cysteamine at 400 µM for 6 h induced approximately 1% late apoptosis and 0.1% necrosis in the cells.

Conclusions: High doses of cysteamine in vitro reduce the percentages of PPD- and SEB-induced Th1 and Tc1 cells and induce late apoptosis and necrosis. Differently, cysteamine at lower doses retains the immunomodulatory effect without affecting cell viability. These findings suggest cysteamine as a potential adjunct to antimicrobial regimens as in the TB or COVID-19 field, for its ability to reduce the inflammatory status.

Keywords: Ag-specific response; PPD-specific response; apoptosis; cysteamine; host-directed therapy; inflammation; necrosis; tuberculosis.

Figure 1

The effect of cysteamine on the specific Th1 and Tc1 responses in TB and TBI subjects. PBMCs from TB (n=12), TBI (n=10), and HC (n=9) were stimulated in vitro for 24 h and treated with cysteamine at 400 µM (A–C) and, in a subgroup of subjects, also with 200 µM (D–F). (A, D) By flow cytometry, the CD4⁺ Th1-specific response was evaluated in PPD- and (B, C, E, F) the CD8⁺ Tc1-specific response in SEB-stimulated cells. The total number of each group are reported as evaluable totals. Statistical analysis was performed using Wilcoxon matched-pairs rank test. Significant p values are indicated in bold. Data are reported as median, and each dot represents a different individual.

Figure 2

Evaluation of Th1- and Tc1-specific responses after treatment with cysteamine in HC BCG-vaccinated individuals. PBMCs from BCG-vaccinated HC (n=10) were stimulated with (A–D) PPD and (B, C, E, F) SEB for 6 h or 24 h, as indicated in each graph. Cells were treated or not with cysteamine at different concentrations (50 µM–400 µM), and the percentages of Th1- and Tc1-specific response were evaluated by flow cytometry. Each dot represents a single subject. Friedman test followed by Dunn’s multiple comparisons was performed, and significant p values are indicated in bold. Data are reported as median.

Figure 3

Treatment with cysteamine modulates the cytokine production. Cytokine production was evaluated in supernatants of PBMCs (BCG-vaccinated HC, n=10) stimulated in vitro with PPD for 6 h (A–C) and 24 h (D–F) and treated or not with cysteamine (50 µM–400 µM). Levels of (A, D) IFN-γ, (B, E) TNF, and (C, F) IL-2 were measured using an automated ELISA assay (ELLA). Statistical analysis was performed using Friedman test followed by Dunn’s multiple comparisons. Values from stimulated samples were subtracted from the respective unstimulated control. Red lines indicated the median, and each dot represents a different individual.

Figure 4

Cysteamine affects the percentage of lymphocytes in a dose- and time-dependent manner. Unstimulated PBMCs from 10 BCG-vaccinated HC were treated with cysteamine at different concentrations (50 µM–400 µM) for 6 hours (h) and 24 h. (A) The percentage of total lymphocytes was analyzed according to the morphological parameters (SSC-A and FSC-A) after 6 h (top panels) and 24 h (bottom panels). (B) Percentage of lymphocytes after cysteamine treatment for 6 h (left graph) or 24 h (right graph). (C) Comparison of the percentage of lymphocytes, 6 h red square and 24 h black triangle. Statistical analysis was performed using (B) Friedman test followed by Dunn’s multiple comparisons and (C) Wilcoxon matched-pair rank test. Data are expressed as median, and each dot represents a different subject.

Figure 5

Analysis of apoptosis and necrosis in PBMCs treated with cysteamine at different concentrations and different time points by flow cytometry. Unstimulated PBMCs (BCG-vaccinated HC, n=10) were treated or not with cysteamine (50 µM–400 µM) for 6 h and 24 h. (A) All events acquired were gated, as schematically described at the top, after 6 h (middle) or 24 h (bottom) of incubation of unstimulated and untreated PBMCs as a representative gating strategy, based on the single or double positivity of Annexin V and/or PI. The apoptosis and necrosis were evaluated after (B) 6 h or (C) 24 h by flow cytometry. Statistical analysis was performed using Friedman test followed by Dunn’s multiple comparisons. Data are expressed as median, and each dot represents a single subject. PI, propidium iodide; w/o, without.