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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., Competing Interests: EN is member of the advisory board by Gilead, Lilly and Roche and received fees for educational training by Gilead, Lilly and Roche. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships related to this study that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Najafi-Fard, Farroni, Petrone, Altera, Salmi, Vanini, Cuzzi, Alonzi, Nicastri, Gualano, Palmieri, Piacentini and Goletti.)

Objectives: To investigate whether Mycobacterium tuberculosis (Mtb) DNA is detected in peripheral blood mononuclear cells (PBMC) of subjects with tuberculosis (TB) or TB infection (TBI) living in a low-burden country., Methods: We prospectively enrolled 57 patients with TB, 41 subjects with TBI, and 39 controls in Rome, Italy. PBMC were isolated, cluster of differentiation (CD)34 + and CD34 - cells were immunomagnetic separated, DNA was extracted, and digital polymerase chain reaction for IS6110 and rpoB sequences was used to detect Mtb DNA in PBMC subsets and unfractionated PBMC., Results: We detected Mtb DNA at a low copy number in CD34 + cells in 4o f 30 (13%) patients with TB, 2 of 24 (8%) subjects with TBI, and 1 of 24 (4%) controls. Mtb DNA was detected in unfractionated PBMC in 3 of 51 (6%) patients with TB, 2 of 38 (5%) subjects with TBI, and 2 of 36 (6%) controls. In CD34 - cells, only 1 of 31 (3%) subjects with TBI tested positive for Mtb DNA., Conclusions: Mtb DNA was detected at low frequencies and levels in the PBMC of subjects with TBI and donors with TB living in a low-burden country. In particular, Mtb DNA was detected more frequently in CD34 + cells, supporting the hypothesis that these cells may represent a Mtb niche. This finding informs biological understanding of Mtb pathogenesis and may support the development of a microbial blood biomarker for Mtb infection., Competing Interests: Declarations of competing interest DG reported the following competing interest: PBD Biotech. EG reported the competing interest: research grants from Gilead Sciences and Mylan not related to the present work and speaker fees for Gilead Sciences and ViiV not related to this work. The remaining authors have no competing interest to declare., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Tuberculosis (TB) is still responsible for the deaths of >1 million people yearly worldwide, and therefore its correct diagnosis is one of the key components of any TB eradication programme. However, current TB diagnostic tests have many limitations, and improved diagnostic accuracy is urgently needed. To improve the diagnostic performance of traditional serology, a combination of different Mycobacterium tuberculosis (MTB) antigens and different antibody isotypes has been suggested, with some showing promising performance for the diagnosis of active TB. Given the incomplete protection conferred by bacille Calmette-Guérin (BCG) vaccination against adult pulmonary TB, efforts to discover novel TB vaccines are ongoing. Efficacy studies from advanced TB vaccines designed to stimulate cell-mediated immunity failed to show protection, suggesting that they may not be sufficient and warranting the need for other types of immunity. The role of antibodies as tools for TB therapy, TB diagnosis and TB vaccine design is discussed. Finally, we propose that the inclusion of antibody-based TB vaccines in current clinical trials may be advisable to improve protection., Competing Interests: Conflict of interest: S.T. Melkie reports support for the present manuscript: co-funded by the EACEA (Education, Audiovisual and Culture Executive Agency, award 2015–2323) of the European Commission, receives a scholarship from the EACEA. Registered in the EMJMD LIVE (Erasmus+ Mundus Joint Master Degree Leading International Vaccinology Education). Conflict of interest: L. Arias has nothing to disclose. Conflict of interest: C. Farroni has nothing to disclose. Conflict of interest: M. Jankovic Makek reports receiving consulting fees from Insmed incorporated and Biomerieux outside the submitted work. Conflict of interest: D. Goletti has nothing to disclose. Conflict of interest: C. Vilaplana reports support for the present manuscript received from CIBER Enfermedades Respiratorias (CB06/06/0031), payment made to the institution. Grants or contracts received from Spanish Government-FEDER Funds through CPII18/00031, outside the submitted work. Other non-financial interests reported as follows: C. Vilaplana's lab hosts secondments from the EMJMD LIVE MsC students (Erasmus+ Mundus Joint Master Degree Leading International Vaccinology Education), co-funded by the EACEA (Education, Audiovisual and Culture Executive Agency, award 2015–2323)., (Copyright ©The authors 2022.)

Subjects with immune-mediated inflammatory diseases (IMID), such as rheumatoid arthritis (RA), have an intrinsic higher probability to develop active-tuberculosis (TB) compared to the general population. The risk ranges from 2.0 to 8.9 in RA patients not receiving therapies. According to the WHO, the RA prevalence varies between 0.3% and 1% and is more common in women and in developed countries. Therefore, the identification and treatment of TB infection (TBI) in this fragile population is important to propose the TB preventive therapy. We aimed to study the M. tuberculosis (Mtb) specific T-cell response to find immune biomarkers of Mtb burden or Mtb clearance in patients with different TB status and different risk to develop active-TB disease. We enrolled TBI subjects as example of Mtb-containment, the active-TB as example of a replicating Mtb status, and the TBI-IMID as fragile population. To study the Mtb-specific response in a condition of possible Mtb sterilization, we longitudinally enrolled TBI subjects and active-TB patients before and after TB therapy. Peripheral blood mononuclear cells were stimulated overnight with Mtb peptides contained in TB1- and TB2-tubes of the Quantiferon-Plus kit. Then, we characterized by cytometry the Mtb-specific CD4 and CD8 T cells. In TBI-IMID, the TB therapy did not affect the ability of CD4 T cells to produce interferon-γ, tumor necrosis factor-α, and interleukin-2, their functional status, and their phenotype. The TB therapy determined a contraction of the triple functional CD4 T cells of the TBI subjects and active-TB patients. The CD45RA - CD27 + T cells stood out as a main subset of the Mtb-specific response in all groups. Before the TB-preventive therapy, the TBI subjects had higher proportion of Mtb-specific CD45RA - CD27 + CD4 + T cells and the active-TB subjects had higher proportion of Mtb-specific CD45RA - CD27 - CD4 + T cells compared to other groups. The TBI-IMID patients showed a phenotype similar to TBI, suggesting that the type of IMID and the IMID therapy did not affect the activation status of Mtb-specific CD4 T cells. Future studies on a larger and better-stratified TBI-IMID population will help to understand the change of the Mtb-specific immune response over time and to identify possible immune biomarkers of Mtb-containment or active replication., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Petruccioli, Petrone, Chiacchio, Farroni, Cuzzi, Navarra, Vanini, Massafra, Lo Pizzo, Guggino, Caccamo, Cantini, Palmieri and Goletti.)