In Vitro Model of the Human Blood–Brain Barrier to Explore HTLV-1 Immunopathogenesis
Abstract
1. Introduction
2. Materials and Methods
2.1. Study Population
2.2. Ethical Considerations
2.3. Sample Processing
2.4. Cell Culture
2.5. Indirect Co-Cultivation Standard Procedure
2.6. Mimetic-Human BBB
2.7. Assessing Barrier Integrity
2.8. Quantification of Inflammatory Cytokines
2.9. Cell Death Assay
2.10. Statistical Analysis
3. Results
3.1. Innate Immunity-Mediated Inflammatory Response Regulates the BBB Cells
3.1.1. Pro- and Anti-Inflammatory Cytokines Modulate the In Vitro BBB Endothelium by Cellular Stimuli
3.1.2. Innate Immune Response from BBB Model Contributes to Neuroinflammatory Progression in HAM/TSP
3.2. HTLV-1-Infected PBMCs Trigger Neuronal Apoptosis in a BBB Mimic System
4. Discussion
4.1. Cytokines and BBB Permeability
4.2. Mechanisms of Neuronal Damage
4.3. Comparison with Previous In Vitro Studies
4.4. Implications for Future Research
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
HTLV | Human T-lymphotropic virus |
ATL | Adult T-Cell Leukemia |
HAM/TSP | HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis |
CNS | Central nervous system |
BBB | Blood–brain barrier |
TNF | Tumor necrosis factor |
IFN-γ | Interferon-gamma |
IL | Interleukin |
PBMCs | Peripheral blood mononuclear cells |
PBS | Phosphate-buffered saline |
hBMEC | Human brain microvascular endothelial cells |
SH-SY5Y | Neuroblastoma cells |
MT-2 | HTLV-1 immortalized T cells |
DMEM/F12 | Dulbecco’s Modified Eagle Medium with HAM/F12 |
M199 | Medium 199 |
FBS | Fetal Bovine Serum |
Pen/Strep | Penicillin/Streptomycin |
NEAA | Non-essential amino acids |
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Cell Compartment | Time | Cytokines | Mean of Quantification (pg/µL) | |||
---|---|---|---|---|---|---|
Negative Control (n = 4) | Positive Control (MT-2 Cells) | HTLV+ HAM/TSP− (n = 4) | HTLV+ HAM/TSP+ (n = 4) | |||
hBMEC line | ||||||
24 h | IL-12p70 | 0 | 0.06 | 17.25 | 7.60 | |
TNF | 721.45 | 57.76 | 88.61 | 2250.08 | ||
IL-10 | 13.41 | 0 | 9.45 | 63.75 | ||
IL-6 | 12,350.11 | 11,848.32 | 2,622,369.02 | 11,281,468.06 | ||
IL-1β | 996.48 | 19.82 | 6292.02 | 14,425.55 | ||
IL-8 | 23,773.84 | 12,396.19 | 28,920.41 | 34,775.30 | ||
48 h | IL-12p70 | 0 | 0.17 | 6.90 | 10.14 | |
TNF | 135.08 | 22.10 | 242.58 | 782.57 | ||
IL-10 | 3.49 | 0 | 10.34 | 30.89 | ||
IL-6 | 12,290.31 | 11,906.03 | 10,167,982.96 | 10,410,740.41 | ||
IL-1β | 990.17 | 19.82 | 9881.23 | 19,174.38 | ||
IL-8 | 14,711.46 | 15,370.40 | 48,510.48 | 50,203.04 | ||
72 h | IL-12p70 | 0 | 1.25 | 5.21 | 9.50 | |
TNF | 32.78 | 12.01 | 79.76 | 209.30 | ||
IL-10 | 0.28 | 0 | 6.81 | 17.78 | ||
IL-6 | 12,291.21 | 11,539.45 | 7,249,746.83 | 10,596,709.93 | ||
IL-1β | 738.29 | 40.80 | 6757.40 | 14,618.64 | ||
IL-8 | 22,237.97 | 13,970.70 | 49,066.70 | 46,673.98 | ||
SH-SY5Y cells | ||||||
24 h | IL-12p70 | 0 | 0 | 5.40 | 4.72 | |
TNF | 162.71 | 15.62 | 131.70 | 117.86 | ||
IL-10 | 2.74 | 0 | 6.94 | 10.71 | ||
IL-6 | 12,504.31 | 8759.12 | 420,576.35 | 7,827,617.52 | ||
IL-1β | 246.24 | 0 | 4499.44 | 2674.93 | ||
IL-8 | 22,758.84 | 8944.92 | 25,013.93 | 31,979.71 | ||
48 h | IL-12p70 | 0 | 0 | 4.86 | 18.25 | |
TNF | 45.64 | 10.44 | 65.24 | 95.71 | ||
IL-10 | 1.81 | 0 | 6.99 | 13.07 | ||
IL-6 | 9190.90 | 10,571.36 | 3,400,817.07 | 10,602,510.19 | ||
IL-1β | 460.01 | 9.98 | 3825.55 | 7974.24 | ||
IL-8 | 8944.92 | 12,485.92 | 42,245.11 | 45,132.37 | ||
72 h | IL-12p70 | 0 | 8.02 | 5.48 | 6.86 | |
TNF | 20.45 | 3.61 | 50.17 | 58.77 | ||
IL-10 | 0.74 | 0 | 6.26 | 12.00 | ||
IL-6 | 13,062.54 | 10,760.72 | 7,297,915.70 | 12,406,516.19 | ||
IL-1β | 617.44 | 14.88 | 5135.01 | 8782.95 | ||
IL-8 | 24,822.20 | 12,165.40 | 46,664.63 | 49,356.05 |
Comparisons | Mean Difference in Cell Death (%) | p-Value |
---|---|---|
HTLV+ HAM/TSP− 24 h and HTLV+ HAM- 72 h | −13.5 | 0.0017 |
HTLV+ HAM/TSP− 24 h and HTLV+ HAM+ 72 h | −17.0 | <0.0005 |
HTLV+ HAM/TSP− 24 h and Positive control 48 h | 21.0 | <0.0005 |
HTLV+ HAM/TSP− 24 h and Positive control 72 h | 18.88 | <0.0005 |
HTLV+ HAM/TSP− 24 h and Negative control 48 h | 20.13 | <0.0005 |
HTLV+ HAM/TSP− 24 h and Negative control 72 h | 20.0 | <0.0005 |
HTLV+ HAM/TSP− 48 h and Positive control 24 h | 16.5 | 0.0001 |
HTLV+ HAM/TSP− 48 h and Positive control 48 h | 29.0 | <0.0005 |
HTLV+ HAM/TSP− 48 h and Positive control 72 h | 26.88 | <0.0005 |
HTLV+ HAM/TSP− 48 h and Negative control 24 h | 15.25 | 0.0003 |
HTLV+ HAM/TSP− 48 h and Negative control 48 h | 28.13 | <0.0005 |
HTLV+ HAM/TSP− 48 h and Negative control 72 h | 28.0 | <0.0005 |
HTLV+ HAM/TSP− 72 h and HTLV+ HAM+ 24 h | 13.75 | 0.0013 |
HTLV+ HAM/TSP− 72 h and Positive control 24 h | 22.0 | <0.0005 |
HTLV+ HAM/TSP− 72 h and Positive control 48 h | 34.5 | <0.0005 |
HTLV+ HAM/TSP− 72 h and Positive control 72 h | 32.38 | <0.0005 |
HTLV+ HAM/TSP− 72 h and Negative control 24 h | 20.75 | <0.0005 |
HTLV+ HAM/TSP− 72 h and Negative control 48 h | 33.63 | <0.0005 |
HTLV+ HAM/TSP− 72 h and Negative control 72 h | 33.5 | <0.0005 |
HTLV+ HAM/TSP+ 24 h and HTLV+ HAM+ 72 h | −17.25 | <0.0005 |
HTLV+ HAM/TSP+ 24 h and Positive control 48 h | 20.75 | <0.0005 |
HTLV+ HAM/TSP+ 24 h and Positive control 72 h | 18.63 | <0.0005 |
HTLV+ HAM/TSP+ 24 h and Negative control 48 h | 19.88 | <0.0005 |
HTLV+ HAM/TSP+ 24 h and Negative control 72 h | 19.75 | <0.0005 |
HTLV+ HAM/TSP+ 48 h and Positive control 24 h | 18.0 | <0.0005 |
HTLV+ HAM/TSP+ 48 h and Positive control 48 h | 30.5 | <0.0005 |
HTLV+ HAM/TSP+ 48 h and Positive control 72 h | 28.38 | <0.0005 |
HTLV+ HAM/TSP+ 48 h and Negative control 24 h | 16.75 | <0.0005 |
HTLV+ HAM/TSP+ 48 h and Negative control 48 h | 29.63 | <0.0005 |
HTLV+ HAM/TSP+ 48 h and Negative control 72 h | 29.5 | <0.0005 |
HTLV+ HAM/TSP+ 72 h and Positive control 24 h | 25.5 | <0.0005 |
HTLV+ HAM/TSP+ 72 h and Positive control 48 h | 38.0 | <0.0005 |
HTLV+ HAM/TSP+ 72 h and Positive control 72 h | 35.88 | <0.0005 |
HTLV+ HAM/TSP+ 72 h and Negative control 24 h | 24.25 | <0.0005 |
HTLV+ HAM/TSP+ 72 h and Negative control 48 h | 37.13 | <0.0005 |
HTLV+ HAM/TSP+ 72 h and Negative control 72 h | 37.0 | <0.0005 |
Positive control 24 h and Positive control 48 h | 12.5 | 0.0051 |
Positive control 24 h and Positive control 72 h | 10.38 | 0.0472 |
Positive control 24 h and Negative control 48 h | 11.63 | 0.0129 |
Positive control 24 h and Negative control 72 h | 11.5 | 0.0148 |
Positive control 48 h and Negative control 24 h | −13.75 | 0.0013 |
Positive control 72 h and Negative control 24 h | −11.63 | 0.0129 |
Negative control 24 h and Negative control 48 h | 12.88 | 0.0034 |
Negative control 24 h and Negative control 72 h | 12.75 | 0.0039 |
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Guimarães, A.B.; Bernardo-Menezes, L.; Azevedo, E.; Agrelli, A.; Silva, P.; Sena, M.; Araújo Júnior, W.; Diniz, G.; Gaião, W.D.; Rodrigues, C.; et al. In Vitro Model of the Human Blood–Brain Barrier to Explore HTLV-1 Immunopathogenesis. Curr. Issues Mol. Biol. 2025, 47, 818. https://doi.org/10.3390/cimb47100818
Guimarães AB, Bernardo-Menezes L, Azevedo E, Agrelli A, Silva P, Sena M, Araújo Júnior W, Diniz G, Gaião WD, Rodrigues C, et al. In Vitro Model of the Human Blood–Brain Barrier to Explore HTLV-1 Immunopathogenesis. Current Issues in Molecular Biology. 2025; 47(10):818. https://doi.org/10.3390/cimb47100818
Chicago/Turabian StyleGuimarães, Ana Beatriz, Lucas Bernardo-Menezes, Elisa Azevedo, Almerinda Agrelli, Poliana Silva, Marília Sena, Waldecir Araújo Júnior, George Diniz, Wyndly Daniel Gaião, Claudio Rodrigues, and et al. 2025. "In Vitro Model of the Human Blood–Brain Barrier to Explore HTLV-1 Immunopathogenesis" Current Issues in Molecular Biology 47, no. 10: 818. https://doi.org/10.3390/cimb47100818
APA StyleGuimarães, A. B., Bernardo-Menezes, L., Azevedo, E., Agrelli, A., Silva, P., Sena, M., Araújo Júnior, W., Diniz, G., Gaião, W. D., Rodrigues, C., Cavalcante, M., Castellano, L. R., Souza, J., Magalhães, P., Vallinoto, A. C., & Morais, C. (2025). In Vitro Model of the Human Blood–Brain Barrier to Explore HTLV-1 Immunopathogenesis. Current Issues in Molecular Biology, 47(10), 818. https://doi.org/10.3390/cimb47100818