REVIEW published: 11 September 2019 doi: 10.3389/fimmu.2019.02139 Regulatory T Cells in Mycobacterium tuberculosis Infection Paula Cardona and Pere-Joan Cardona* Unitat de Tuberculosi Experimental, Fundació Institut Germans Trias i Pujol, CIBER Enfermedades Respiratorias, Universitat Autònoma de Barcelona, Badalona, Spain Anti-inflammatory regulatory T cells have lately attracted attention as part of the immune response to Mycobacterium tuberculosis infection, where they counterbalance the protective but pro-inflammatory immune response mediated by Th17 cells and especially by the better-known Th1 cells. In chronic infectious diseases there is a delicate balance between pro- and anti-inflammatory responses. While Th1 and Th17 are needed in order to control infection by Mycobacterium tuberculosis, the inflammatory onset can ultimately become detrimental for the host. In this review, we assess current information on the controversy over whether counterbalancing regulatory T cells are promoting pathogen growth or protecting the host. Keywords: tuberculosis, Treg cells, Th17 cells, tolerance, inflammation Edited by: Julio Aliberti, National Institute of Allergy and INTRODUCTION Infectious Diseases (NIAID), United States Tuberculosis (TB) is a global disease caused by the bacillus Mycobacterium tuberculosis (Mtb). Reviewed by: According to the World Health Organization, in 2017, 10 million people developed the disease Andre Bafica, and 1.6 million people died because of it (1). Compounding the situation, approximately a quarter Federal University of Santa of the world population is currently infected with Mtb (latent TB infection: LTBI) (2). Catarina, Brazil While progression from Mtb infection to active TB is associated with some known risk factors, Kevin Urdahl, Seattle Biomedical Research Institute, including HIV infection and malnutrition, it is still unclear why some apparently healthy people United States develop the disease. One hypothesis is that poor control of the inflammatory response is the Warwick Britton, culprit. While an effective immune response is essential to control the infection at an early stage, University of Sydney, Australia excessive inflammation may be detrimental later on. The damage-response framework of microbial *Correspondence: pathogenesis (3) suggests that both ends of the scale would lead to the development of the disease, Pere-Joan Cardona where a weak response would benefit the dissemination of the bacilli and a very strong response pj.cardona@gmail.com would favor the lung tissue damage characteristic of active TB. The fact that the majority of cases of Mtb infection develop as LTBI imply that most individuals have intermediate response levels Specialty section: (4). There are diverse studies pointing to the immune balance and the development of TB. The This article was submitted to modulation of the leukotriene A4 hydrolase locus, which is key in the balance between pro- and Microbial Immunology, anti-inflammatory eicosanoids, showed to be important in the regulation of TNF-α levels, and thus a section of the journal in the susceptibility to Mtb (5, 6). Interestingly, these authors showed that among individuals with Frontiers in Immunology meningitis TB, both homozygous forms of a specific genotype affecting this locus were related to a Received: 06 May 2019 decreased survival of patients, supporting the idea that both a low and high inflammatory response Accepted: 27 August 2019 may be detrimental. Published: 11 September 2019 This double-edged nature of the immune response also comes into play in the concept of Citation: disease tolerance, a host defense strategy in which less damage is done by the pathogen or the Cardona P and Cardona P-J (2019) Regulatory T Cells in Mycobacterium immune response it triggers, although the burden of the microorganism itself is not reduced. tuberculosis Infection. In this context, immunosuppressive mechanisms are seen as one of the ways in which the host Front. Immunol. 10:2139. achieves tolerance (7). Regulatory T cells (Tregs), which suppress, and thus counterbalance the doi: 10.3389/fimmu.2019.02139 inflammatory response, are one such mechanism. Frontiers in Immunology | www.frontiersin.org 1 September 2019 | Volume 10 | Article 2139 Cardona and Cardona Tregs and TB There is a recurring debate as to whether Tregs are beneficial IPEX (immune dysregulation, polyendocrinopathy, enteropathy, or detrimental in Mtb infection. Many studies, mainly of blood X-linked) syndrome is an autoimmune disease caused by samples, show higher numbers of Tregs in TB patients than in mutations in FoxP3 that lead to Treg dysfunction (21). This LTBI subjects or healthy controls. Some of these studies also focus mutation has also been described in scurfy mice, which have high on the follow-up of TB patients undergoing treatment. However, lymphocytic infiltration resulting in death after a few weeks (22). it is not yet clear whether high levels of Tregs are a consequence Nevertheless, FoxP3 has also been associated with other cell types of inflammation or a risk factor for development of TB. After (23), and T cells with regulatory properties that do not express it briefly introducing Tregs, the present review will address this have also been described (24), so the case for using it as a marker question by examining the available data from animal models and is considerably weakened. Besides, working with an intracellular human subjects. marker is less appealing and often impractical, since it interferes with functional assays. In the case of CD127, the α chain of the IL-7 receptor, low REGULATORY T to undetectable expression levels have also been used to identify CELL CHARACTERIZATION Tregs among CD4+ CD25+ cells (25). This marker is down- regulated in T cells after activation and re-expressed in all but The immune system has mechanisms for suppressing the FoxP3+ cells. FoxP3 interacts with the promoter CD127 and response to persistent self- or non-self-antigens. Tregs are a represses it (26). lymphocyte subset whose main role is maintaining immune CD39 is an ectonucleotidase that turns ATP or ADP into homeostasis and peripheral tolerance. AMP and is expressed by CD4+ CD25+ and CD4+ FoxP3+ The key cytokines involved in the immunosuppressive cells. This has a dual anti-inflammatory effect: it eliminates function of Tregs are IL-10, TGF-β, and IL-35 (8–10). By down- the inflammatory mediator ATP and increases production of modulating the co-stimulatory molecules CD80 or CD86 in adenosine, which inhibits the NK- and T-cell response (27). a CTLA-4-dependent mechanism, Tregs interfere with T-cell Characterization of CD4+ CD25+ CD127− Tregs from peripheral activation by dendritic cells (11). Tregs also express granzymes blood mononuclear cells (PBMCs) according to CD39 expression and thus induce apoptosis of the target cells (12). Another showed that CD39hi Tregs secrete more IL-10 and have a mechanism of suppression is metabolic disruption, achieved by stronger suppressive activity. Moreover, in the presence of IL- consuming available IL-2 (13). 1β and IL-6, CD39low Tregs differentiated into Th1 or Th17 Tregs might be induced in the thymus during development and down-regulated the expression of FoxP3 as compared to (tTregs) or in peripheral tissue such as mucosa (pTregs). Research CD39hi Tregs (28). Further supporting the use of CD39 to on therapies that modulate these cells, or the administration of in characterize a regulatory T-cell phenotype, it has been described vitro induced Tregs (iTregs), have led to efforts to differentiate that CD4+ CD25hi CD39+ cells produce lower levels of IFN-γ and them (14). tTregs generally target auto-antigens, are more stable, higher of TGF-β than CD4+ CD25hi CD39− cells or CD4+ T cells and have higher TCR affinity. The expression of the transcription with low or no expression of CD25 (29). factor Helios has been linked to tTregs (15) since it is detected in all the CD4+ FoxP3+ thymocytes, but only in around 70% Th17-Counterbalancing Role of CD4+ FoxP3+ from peripheral lymphoid tissues. The high Th17 cells are characterized mainly by the production of IL- expression of the surface marker Neuropilin 1, which is up- 17, which promotes the synthesis of other pro-inflammatory regulated by TGF-β, has also been considered as a tTregs cytokines, and chemokines (30). The transcription factor marker (16). Interestingly, it had been previously shown that required for the development of Th17 cells is RORγt (31). the induction of pTregs, but not tTregs, is dependent on this Tregs and Th17 cells are closely intertwined. TGF-β induces cytokine (17). Neither of these markers clearly identifies the both FoxP3 and RORγt expression; however, in order to induce thymic induced Tregs from pTregs or iTregs. Th17, TGF-β also requires the presence of IL-6, in which case the balance is tipped toward Th17 cells instead of Tregs (32). Treg Phenotype Markers Otherwise, FoxP3 inhibits the function of RORγt (33). It has Identification of an optimal cellular marker for characterizing been shown that CD4+ CD25hi CD39+ Tregs have a role in Tregs in general is also proving difficult. Some markers used, in constraining pathogenic Th17 cells (34). combination with CD3 and CD4, include CD25, FoxP3, CD127, or CD39. Although CD8+ Tregs have also been described, as TREGS AND DISEASE recently reviewed by Yu et al. (18), they have not been considered in the present review. Tregs have been linked to a number of inflammatory diseases, CD25 is highly expressed in Tregs and is in fact related to mostly autoimmune but some of them infectious. Equilibrium one of the suppression mechanisms of these cells. However, this with Th17 cells is widely thought to be the key factor in these extracellular marker, which is the α-chain of the high affinity conditions, but this review will focus on Tregs. receptor for IL-2, is also upregulated in activated T cells (19). Multiple sclerosis, a chronic autoimmune inflammatory Therefore, it is generally used in combination with other markers. disease of the central nervous system, has been related to an FoxP3 is a transcription factor, Forkhead box P3, long used as increase in the number and activity of pro-inflammatory Th17 a marker for Tregs, since it is essential for their development (20). cells and a corresponding decrease in Tregs (35). Rheumatoid Frontiers in Immunology | www.frontiersin.org 2 September 2019 | Volume 10 | Article 2139 Cardona and Cardona Tregs and TB arthritis, also of autoimmune origin, affects synovial tissue, In a murine model, when CD25+ cells were depleted in where an imbalance has been reported between Tregs and Th17 DBA/2 mice prior to Mtb aerosol infection, infection outcomes cells (36). improved: there was a reduction in the bacillary load (BL) of The relevance of the balance between these cell populations lungs and spleen, together with less severe histopathology in the was shown in a Treg phenotyping study carried out in lungs (46). Nevertheless, this effect was seen only at week 2 post- PBMCs in the context of organ transplants. Renal allograft infection, and afterwards there was no difference between groups. rejection was associated with a high ratio of CD4+ CD25− CD39+ There was also no significant effect if CD25+ cell depletion cells, which have a pro-inflammatory memory profile, to was done at the chronic stage of infection. Similar results were CD4+ CD25+ CD39+ Tregs (37). observed using a mixed bone chimera system where FoxP3+ High levels of Tregs have been associated with different types cells were depleted with anti-Thy1.1 antibodies: there was a of cancer, both in peripheral blood samples, and in solid tumors. reduction in BL in the lungs, but not the spleen, at day 23 However, their precise role in cancer is still undefined, with some post-infection (47). researchers linking higher levels to a favorable outcome, and However, depletion of CD25+ cells 3 days prior to infection others to a poorer prognosis. In addition to these differences in in both M. bovis BCG- and Mtb-infected C57BL/6 mice had number, higher Treg suppressive activity has also been described no effect on BL, even though a reduction in CD4+ CD25+ in cancer cases (38). cells was observed for at least 23 days (48). In fact, the Another area of study is the relationship between Tregs and characterization of two mouse strains with the same haplotype different pathogens. In Helicobacter pylori infection, increased but different susceptibility to Mtb infection (C3HeB/FeJ and Treg frequency appears to help limit damage due to inflammatory C3H/HeN) showed that the more susceptible animals had onset, but it also promotes bacterial growth (39). This pattern is lower numbers of Tregs (49). Furthermore, after CD25+ cells quite common and applies to a number of different pathogens, were depleted in the more resistant strain, lung pathology including viruses (40, 41). became more severe. Another study in two mouse strains with The two-edged action of Tregs also plays a role in Mtb different susceptibility, the hyper-susceptible I/StSnEgYCit and infection. The pro-inflammatory response characterized by the the relatively resistant C57BL/6JCit, showed that Treg levels were production of IFN-γ or IL-17 is necessary to control Mtb growth, lower in the mediastinal lymph nodes of the susceptible mice and so Tregs would appear to be detrimental in terms of countering remained practically constant during the follow-up period (until the pathogen. However, if left unchecked, the inflammatory week 17 post-infection), while this cell population increased over response ultimately causes excessive tissue damage to the host. time in the resistant C57 mice. The authors concluded that this is Tables 1, 2 summarize TB-related research with a focus on Tregs. a host protective strategy (50). These studies of animal models and human cohorts are discussed Scott-Browne et al. (47) failed to see a similar percentage below separately. increase, but did find an increase in total numbers of CD4+ FoxP3+ cells in the lungs and pulmonary lymph nodes Tregs in TB Animal Models (pLN) of Mtb-infected C57BL/6 mice. However, they were unable In the guinea pig model, while studying the pathogenicity of to detect production of the characteristic regulatory cytokine different Beijing sublineages of Mtb, it was shown that highly IL-10 by this cell population in vitro. These same researchers virulent strains have increasing levels of FoxP3, IL-10, and TGF- (51) showed that Tregs expand both in lungs and pLN; but this β mRNA in lung tissue as infection progresses (42). In another occurs after Mtb has been transported to pLN, same as effector T study with a similar focus, FoxP3, and TGF-β expression was cells. The adoptive transfer of Mtb-specific Tregs (CD4+ CD25+ ) also found to increase in the lungs with progression of infection, before T cell expansion results in higher BL, but no effects were but pro-inflammatory IL-17 increased as well. With less virulent seen when using non-specific Tregs or specific effector cells. strains, the increase is not as steep (43). In a later study, the authors found that the Mtb-specific Treg Results from a non-human primate (NHP) Mtb infection expansion is higher in pLN, but these cells have a short-life, and model suggest that augmented Treg levels are a response to do not accumulate as infection progresses (52). These FoxP3+ inflammation, rather than the cause of TB disease (44). These cells form pLN have a high expression of the transcription authors found that NHPs that failed to develop TB disease factor Helios and expand from cells already expressing FoxP3+ , (i.e., LTBI cases) had higher levels of Tregs in peripheral blood suggesting they are thymically derived. The reduction in the Treg prior to infection than those that developed active TB. Although levels would be mediated by the transcription factor T-bet in an all subjects experienced an initial decrease in Treg frequencies, IL-12 dependent manner. animals that remained latently infected remained at higher levels Contrary to these observations, a reduction in the frequency during the period of decline and eventually returned to pre- of Tregs in lungs after infection was described by McBride infection levels as infection progressed, while Tregs continued et al. (53) in both toll-like receptor 2 (TLR2) knockout mice to increase in the NHPs with TB. In a separate study, IL-2 (C57BL/6 strain) and their wild-type counterparts, although less treatment of NHP in early-stage infection expanded Foxp3+ in the former, which had a higher BL. In this study, the authors Tregs, and CD4+ /CD8+ /γδ T effector cells (45). The authors demonstrated that TLR2 activation on myeloid cells is necessary linked ensuing resistance to severe TB lesions to an optimal to induce the accumulation of CD4+ FoxP3+ in the lungs, and cooperation between IL-2-expanded CD4+ T cells and Tregs, by that these accumulated Tregs prevent immunopathology and preventing overreacting inflammatory responses. associated tissue damage. This suggested that Tregs may function Frontiers in Immunology | www.frontiersin.org 3 September 2019 | Volume 10 | Article 2139 Cardona and Cardona Tregs and TB TABLE 1 | Summary of data on Tregs and TB from animal models. Animal model Sample Treg phenotype Relationship between Tregs and TB References Guinea pig, aerosol infection with Mtb H37Rv Lungs FoxP3 mRNA Progression of infection increases Treg levels (42) and Erdman laboratory strains, and 3 clinical isolates of Mtb Guinea pig, aerosol infection with clinical Lungs FoxP3 mRNA Progression of infection increases Treg levels (43) isolates of Mtb Beijing strains Cynomolgus Macaque, bronchoscopic Lungs CD3+ CD4+ FoxP3+ Higher levels of Tregs in Mtb-positive lungs and (44) instillation infection with Mtb Erdman strain and LNs draining LNs PBMCs Higher levels of Tregs found prior to infection in animals that did not develop the disease Cynomolgus Macaque, bronchoscopic Blood CD4+ CD25+ FoxP3+ Higher levels of Tregs correlates with less (45) instillation infection with Mtb Erdman strain severe lung pathology after IL-2 treatment BAL DBA/2 mice, aerosol infection with Mtb Kurono Systemic CD25+ Treg depletion decreases spleen and lung BL (46) or Erdman strains and improves lung pathology (at early stages of infection) C57BL/6 & bone marrow chimeric mice, Lungs CD4+ FoxP3+ Progression of infection increases Treg levels (47) aerosol infection with Mtb H37Rv strain and LNs (WT) Systemic FoxP3+ Treg depletion decreases lung BL C57BL/6, aerosol infection with Mtb Erdman Systemic CD25+ Treg depletion does not affect BL or lung (48) strain pathology C57BL/6, intranasal infection with M. bovis BCG Pasteur strain 1173P2 C3HeB/FeJ & C3HeN, intravenous infection Spleen CD4+ CD25+ FoxP3+ Higher levels of Tregs in strain less susceptible (49) with Mtb H37Rv Pasteur strain to infection (C3HeN) Systemic CD25+ Treg depletion exacerbates lung pathology (C3HeN) I/StSnEgYCit and C57BL/6JCit mice, aerosol Mediastinal CD4+ CD25+ FoxP3+ Higher levels of Tregs found in strain less (50) infection with Mtb H37Rv Pasteur strain LNs susceptible to infection (C57BL/6JCit) Progression of infection increases Treg levels (C57BL/6JCit) C57BL/6 & P25 TCR transgenic mice, aerosol Systemic CD4+ CD25+ Mtb-specific Treg adoptive transfer increases (51) infection with Mtb H37Rv strain BL C57BL/6, FoxP3-GFP reporter & TCR KO Pulmonary CD4+ FoxP3+ Early expansion of Mtb-specific thymically (52) mice, aerosol infection with Mtb H37Rv strain LN derived Tregs C57BL/6 & IL-12p35 or IL12p40 KO mice, Progression of infection decreases Mtb-specific aerosol infection with Mtb H37Rv strain Tregs in an IL-12 dependent manner C57BL/6 & TLR2 KO mice, aerosol infection Lungs CD4+ FoxP3+ Progression of infection decreases Treg levels (53) with Mtb Erdman strain KO have fewer Tregs than WT after infection, and higher BL C57BL/6, BALB/c, DBA/2 mice, aerosol Lungs CD4+ FoxP3+ Higher levels of Tregs found in strains less (54) infection with clinical isolate of Mtb Harlingen susceptible to infection (C57BL/6 and BALB/c) strain C57BL/6, aerosol infection with Mtb H37Rv Systemic CD4+ CD25+ FoxP3+ Treg depletion does not interfere with BCG (55) strain effect C57BL/6, aerosol infection with Mtb Erdman Systemic CD25+ Treg depletion has no effect on BCG-mediated (56) strain or M. bovis Wag201 strain reduction of BL and improves lung pathology BALB/c, intratracheal infection with Mtb H37Rv Lungs CD4+ FoxP3+ Lower number of Tregs correlate with better (57) strain vaccine protection Systemic CD25+ Treg depletion does not interfere with vaccine effect BAL, bronchoalveolar lavage; BL, bacillary load; KO, knock-out; LN, lymph node; PBMCs, peripheral blood mononuclear cells; WT, wild-type. by restraining the influx of monocytes and neutrophils, serving BL was caused by the absence of TLR2-mediated antimicrobial to limit the availability of niches for Mtb to replicate. In this activity in the granuloma or by enhanced inflammation. The study authors were not able to discern whether the increased latter hypothesis would support the findings in the NHP model Frontiers in Immunology | www.frontiersin.org 4 September 2019 | Volume 10 | Article 2139 Cardona and Cardona Tregs and TB TABLE 2 | Summary of data on Tregs and TB from human cohorts. Population of study Sample Treg phenotype Relationship between Tregs and TB References PTB (n = 13) vs. HC (n = 12) PBMCs stimulated with CD4+ CD25hi FoxP3+ or Higher levels of Tregs in PTB (29) Mtb-RD1 proteins for 1 CD4+ CD25hi CD39+ or 6 d HC (n = 9) vs. LTBI (n = 8) vs. PTB Cryopreserved PBMCs CD4+ CD25+ or LTBI have higher Tregs than HC, but lower (58) (n = 13) CD4+ CD25hi than PTB HC (n = 28) vs. LTBI (n = 20) vs. TB Cryopreserved PBMCs CD4+ CD25+ CD127− Lower levels of Tregs in HC, no differences (59) (n = 20) between LTBI and PTB LTBI pre and post ATT (n = 20) CD4+ CD25+ FoxP3+ Increase in Tregs after prophylactic treatment LTBI (n = 18) vs. PTB (n = 33) vs. cured PBMCs CD4+ CD25+ FoxP3+ Higher levels of Tregs in PTB, no (60) TB (n = 20) differences between LTBI and cured TB Follicular hyperplasia LN tissue FoxP3+ Expression of FoxP3 in LNTB (n = 6) vs. LNTB (n = 20) HC (n = 23) vs. TB (n = 27) PBMCs CD4+ CD25hi Higher levels of Tregs in TB (61) FoxP3 mRNA Healthy TB case contacts (n = 134) vs. Total RNA from whole FoxP3 mRNA LTBI have higher Tregs than HC, but lower (62) LTBI (n = 151) vs. TB (n = 126) blood than active TB PTB (n = 19 in PBMCs and n = 6 in BAL) PBMCs and BAL CD4+ CD25+ FoxP3+ Higher levels of Tregs in PTB (both in (63) vs. LTBI (n = 10 in PBMCs and n = 6 in PBMCs and BAL) BAL) Higher levels of Tregs in BAL than in PBMCs in PTB LNTB (n = 18) Cryopreserved LNMCs CD4+ CD25+ FoxP3+ CD127− Higher levels of Tregs in LNMCs than in (64) and PBMCs PBMCs HC (n = 66) vs. LTBI (n = 27) vs. TB Whole blood CD3+ CD4+ FoxP3+ Higher levels of Tregs in TB than HC or (65) (n = 62) LTBI, no differences between these two groups PTB (n = 35) vs. STB (n = 13) Higher levels of Tregs in PTB Pleural TB (n = 14) Whole blood and Higher levels of Tregs in PFMCs PFMCs LTBI (n = 20) vs. PTB (n = 20) vs. pleural PBMCs CD4+ CD25+ FoxP3+ Higher levels of Tregs in PTB or pleural TB (66) TB (n = 15) Pleural TB (n = 15) PBMCs and PFMCs Higher levels of Tregs in PFMCs TB (n = 31) vs. LTBI (n = 30) Cryopreserved PBMCs CD4+ CD134+ CD25+ CD39+ Higher levels of Tregs in TB (67) stimulated with PPD, ESAT-6 or CFP-10 for 24 h PTB (n = 48) and/or EPTB (n = 28) (before Whole blood stimulated CD3+ CD4+ CD25+ CD39+ Higher levels of Tregs in TB, but no (68) and after ATT) vs. HC (n = 83) with BCG and FoxP3+ differences between PTB and EPTB cryopreserved afterwards Increase in Tregs after ATT in PTB PTB during ATT (n = 20, AFB+ vs. AFB-) PBMCs CD4+ CD25+ CD127− Tregs increase with TB treatment (69) LTBI (n = 19) vs. PTB (n = 21) PBMCs CD4+ FoxP3+ , Higher levels of Tregs in PTB (70) CD4+ CD25+ or CD4+ CD25+ FoxP3+ PTB (n = 21) CD4+ CD25+ FoxP3+ Higher levels of Tregs with higher BL in sputum DS-PTB (n = 16) and MDR-PTB (n = 4) Decrease of Tregs with ATT only if DS-PTB during ATT PTB (n = 41) vs. HC (n = 24) PBMCs CD4+ CD25+ FoxP3+ Higher levels of Tregs in PTB (71) PTB during ATT (n = 20) Tregs increase transiently with ATT and decrease to HC levels HC (n = 15) vs. PTB (n = 10) PBMCs stimulated with CD3+ CD4+ CD25+ FoxP3+ Higher levels of Tregs in PTB (72) rESAT6-CFP10 during 6 days (Continued) Frontiers in Immunology | www.frontiersin.org 5 September 2019 | Volume 10 | Article 2139 Cardona and Cardona Tregs and TB TABLE 1 | Continued Population of study Sample Treg phenotype Relationship between Tregs and TB References PTB during ATT (n = 10) Treg decrease with ATT PTB (n = 15) vs. HC (n = 17, 41% QTF+) PBMCs cultured CD4+ CD25+ CD39+ or Higher levels of Tregs in PTB, no (73) overnight CD4+ CD25+ FoxP3+ differences between QTF+ and QTF- PTB during ATT (n = 12) Treg levels decrease with ATT HC (n = 15) vs. PTB (n = 13) vs. PBMCs cultured CD4+ CD25+ CD39+ or Higher levels of Tregs in PTB than HC and (74) completed ATT (n = 21) overnight CD4+ CD25+ FoxP3+ cured TB. Treatment failure: increase in Tregs LTBI (n = 10) vs. PTB (n = 10) vs. cured PBMCs stimulated with CD4+ CD25hi FoxP3+ Lower levels of Tregs in LTBI (75) TB (n = 10) M. bovis antigens for 48 h HC (n = 80) vs. LTBI (n = 80) vs. PTB PBMCs CD4+ CD25+ C127− LTBI have higher Tregs than HC, but lower (76) (n = 90) than PTB PTB (n = 30) vs. cured TB (n = 10) or HC Whole blood CD4+ CD25+ or Higher levels of Tregs in PTB (77) (n = 30) CD4+ CD25+ FoxP3+ AFB + (n = 21) vs. AFB – (n = 9) Higher levels of Tregs in AFB + Cavitary TB (n = 19) vs. non-cavitary TB Higher levels of Tregs in cavitary PTB (n = 11) HC (n = 14) vs. DS-PTB (n = 33) vs. PBMCs CD4+ FoxP3+ Higher levels of Tregs in active PTB (DS or (78) MDR-PTB (n = 7) vs. stable MDR-PTB MDR), no differences between stable (n = 16) MDR-PTB and HC HC/LTBI (n = 20) vs. DS-PTB (n = 20) vs. PBMCs CD4+ CD25+ FoxP3+ Higher levels of Tregs in DS-PTB than (79) MDR-PTB (n = 18) HC/LTBI, but lower than MDR-PTB HC (n = 30) vs. DS-TB (n = 30) vs. PBMCs CD4+ CD25+ Higher levels of Tregs in DS-TB than HC, (80) MDR-TB (n = 30) but lower than MDR-TB AFB, Acid-fast bacilli; ATT, antituberculous treatment; BAL, bronchoalveolar lavage; EPTB, extrapulmonary tuberculosis; HC, healthy control; LNTB, lymph node tuberculosis; LTBI, latent tuberculosis infection; MDR-TB, multidrug-resistant tuberculosis; PBMCs, peripheral blood mononuclear cells; PFMCs, pleural fluid mononuclear cells; PTB, pulmonary tuberculosis; QTF, quantiferon; STB, severe tuberculosis (PTB with meningitis). (44) and the idea that Tregs and T effector cells act together to A similar finding had been noted before by Wergeland et al. control inflammation without increasing Mtb replication (45). In (59), who found lower levels of CD4+ CD25+ CD127− Tregs, but another study, the lungs of Mtb-infected resistant mouse strains not of CD4+ CD25+ FoxP3+ Tregs, in healthy controls compared (C57BL/6 and BALB/c), compared to the DBA/2 susceptible to subjects with either active TB or LTBI. The specific parent strain, had higher numbers of Tregs as well as of CD103+ population in the hierarchy of the analysis is also relevant. For dendritic cells, which showed an anti-inflammatory profile (54). example, in one study the percentage of CD4+ CD25hi cells The concern that Tregs might interfere with the protective expressing FoxP3 was the same in both study groups, but the effect of BCG vaccination has been addressed. C57BL/6 mice percentage of CD4+ CD25hi FoxP3+ relative to total CD4+ cells were administered an adenoviral vector that expresses murine was significantly different between healthy donors and patients IL-28B, which is thought to down-regulate Tregs. When this with pulmonary TB (PTB) (60). was given together with BCG and a subunit vaccine booster, The first association between Tregs and TB patients was Tregs were depleted in the spleen but this had no influence on shown by Guyot-Revol et al. (61). Before starting treatment, the effects of vaccination (55). These results support what was TB patients had higher frequencies of CD4+ CD25hi Tregs and previously observed by Quinn et al. (56), who inactivated Tregs a higher expression of FoxP3 in PBMCs compared to healthy with anti-CD25 mAb prior to vaccination and found that this did controls. This difference has been shown in various cohorts not affect BCG effectiveness. Nonetheless, another study of BCG summarized below. booster vaccine candidates in Mtb-infected BALB/c mice showed Burl et al. (62) performed a study of FoxP3 gene expression in an association between lower BL and a higher ratio of CD4+ to TB contacts. They found that non-infected contacts (ELISPOT- CD4+ FoxP3+ in the lungs (57). and tuberculin skin test (TST)-negative) had higher levels of FoxP3 mRNA in blood than Mtb-infected contacts (ELISPOT- Tregs in Human TB and TST-positive), but lower levels than patients with active The importance of the phenotypic markers used to define TB. They hypothesize that this may be due to the sequestering Tregs, discussed at the beginning of this review, is highlighted of Tregs in the lungs at early stages of infection and their in the work carried out by Zewdie et al. (58), who found subsequent reappearance in the periphery. However, it has that while CD4+ CD25+/hi T-cell frequencies were higher in been shown that in patients with PTB, but not in LTBI patients with active TB than individuals with LTBI, there subjects, Treg levels are higher in bronchoalveolar lavage (BAL) were no differences in the frequency of CD4+ CD25+ CD127lo , than in blood (63). FoxP3 expression was detected in the CD4+ CD25+ FoxP3+ , or CD4+ CD25+ FoxP3+ CD127lo T cells. immunohistochemical analysis of Mtb-infected lymph nodes Frontiers in Immunology | www.frontiersin.org 6 September 2019 | Volume 10 | Article 2139 Cardona and Cardona Tregs and TB (LN) (60). In patients with LN TB, there were higher levels were Treg percentages higher in patients with active TB than in of Tregs and activated CD4+ T cells among lymph node those with LTBI or controls, but so were Th17 cell percentages mononuclear cells than among PBMCs (64). Higher levels of (and their corresponding cytokines in plasma). Furthermore, Tregs have also been described in pleural effusion fluid compared LTBI patients presented higher levels of Tregs and IL-10 than to PBMCs (65, 66). healthy controls. Conversely, Chen et al. (65) found that the Chiacchio et al. found no differences in Treg levels blood of TB patients had higher levels of FoxP3+ CD4+ cells unless they did an Mtb-specific in vitro stimulation. After but lower levels of IL-17-producing CD4+ cells compared to that 1- or 6-day stimulation with Mtb-specific RD1 proteins, TB patients had higher levels of both CD4+ CD25hi FoxP3+ and CD4+ CD25hi CD39+ Tregs than healthy controls (29). In PBMCs stimulated with the mycobacterial antigens PPD, ESAT-6, or CFP-10, the characterization of activated CD4+ T cells showed a higher percentage of CD4+ CD25+ CD134+ -expressing CD39 in active TB than in LTBI. In line with the high expression of this immunosuppressive molecule, the ratio of IL-10 to IFN-γ production was increased in cell cultures from active TB cases compared to LTBI (67). The characterization of BCG-stimulated whole blood in a pediatric TB population supports these results: Tregs were higher in TB than in healthy controls (68). Moreover, the analysis of cytokines in the supernatants of these cultures showed higher levels of pro-inflammatory mediators in healthy controls. When comparing PTB to extrapulmonary TB, the authors found that Tregs could not be used to distinguish the two populations. Interestingly, however, when they looked at Treg levels after 6 months of treatment, they found that children with PTB had a significant increase but those with extrapulmonary TB did not. Xu et al. (69) studied PTB patients at the time of diagnosis (sputum smear positive) and the same subjects after 3 weeks of treatment (sputum smear negative). Like Whittaker et al. (68), they found that with the course of treatment, both Treg frequency in PBMCs and plasma levels of IL-10 increased, and at the same time correlated negatively with plasma levels of IL-17. In contrast, Singh et al. (70) described a positive correlation between Tregs and BL in sputum. A follow-up study performed over 9 months, comprising the 6 months of treatment, showed that Tregs continued to increase until month 4, but later decreased until levels matched those of healthy controls (71). Two similar studies with 2- or 6-month follow-up periods during treatment showed that Treg levels were higher in pulmonary TB patients than healthy controls, and decreased with chemotherapy, with no transient increase (72, 73). Agrawal et al. (74) suggest that this could be a way to monitor treatment success or failure, since the one case of failure they had in a new cohort had had an increase in Treg levels. In fact, the lack of Treg decrease with treatment has been linked to multidrug-resistant TB (MDR-TB) and has been proposed for monitoring purposes (70). Nevertheless, the PBMCs stimulus with BCG antigens showed higher levels of Tregs in TB patients but also in cured individuals when comparing with LTBI (75). Prophylactic treatment of LTBI subjects resulted in augmented FIGURE 1 | Hypothesis on the role of the balance between Tregs and Th17 levels of Tregs with the phenotype CD4+ CD25+ FoxP3+ , but cells in the development of TB. In a context of immunosuppression, where no differences were seen in levels of CD4+ CD25+ CD127− (59). Tregs are predominant over Th17, Mtb disseminates more easily (A). The However, the latter phenotype was the one that differentiated immune balance between these populations gives place to the encapsulation between control subjects and those with TB or LTBI. and control of lung lesions (B). If there is a high Th17 response, inflammation and neutrophils recruitment fuel the growth of the granuloma and development Regarding the importance of the balance between pro- and of TB (C). anti-inflammatory responses, Luo et al. (76) found that not only Frontiers in Immunology | www.frontiersin.org 7 September 2019 | Volume 10 | Article 2139 Cardona and Cardona Tregs and TB of both healthy donors and LTBI subjects. Nevertheless, IL-6 There have been many studies carried out in patient cohorts, and TGF-β plasma levels were higher, a fact typically linked to with the main limitation being that they are mostly based on induction of the Th17 population. The authors suggest a possible PBMCs. It has been shown that in TB patients Treg levels are explanation in terms of reduced expression of IL-6R CD4+ cells higher at the site of infection, so the study of PBMCs may caused by Mtb. seem somewhat inaccurate. Furthermore, even when contact Pang et al. (77) also found a higher frequency of Tregs in blood trace studies may give an approximate time of exposure to Mtb, in active TB than in LTBI or healthy controls. When they took a the exact steps of infection are hard to elucidate in human TB. closer look at this group of subjects, they observed that Treg levels Longitudinal studies, the ones that could really shed more light were higher in patients with a positive sputum smear and in those on whether Tregs have a beneficial or detrimental role in TB, are with cavitary PTB. A different approach was taken by Lim et al. rare. Potentially, monitoring of Tregs as an exploratory endpoint (78), who looked into the differences between drug-sensitive TB in future prospective cohort study addressing progression from and MDR-TB. They did not find significant differences between LTBI toward active TB could be of interest to solve this question. these groups, but once again Treg levels were higher in active Many studies suggest the potential use of Tregs as a diagnostic TB than in healthy controls. Treg frequency was higher in method for active TB. In general, Tregs are higher in active smear-positive patients and did not correlate with radiological TB than in LTBI or healthy controls. However, the difference extent or cavitation. MDR-TB patients whose sputum results had between LTBI and non-infected persons is less clear. So far remained negative for at least 6 months had Treg levels similar this seems unrealistic and much work remains to be done in to those of healthy controls. More recently, research with the this context, not only because of the everlasting issue of Treg same target population showed that MDR-TB patients had higher phenotyping, but also because it is difficult to set a cut-off value Treg frequencies than those with drug-sensitive TB, and this for this parameter. Tregs have also been suggested as a way of was supported by blood levels of regulatory cytokines IL-10, and monitoring the effectiveness of treatment. This option is even TGF-β (79). The same differences were found when looking at more challenging as direct correlation between bacillary load and CD4+ CD25+ cells expressing the co-stimulatory receptors PD-1 Treg has never been done in the context of chemotherapy. or CTLA-4. Similarly, patients with MDR-TB had higher (though All in all, the research carried out so far on Tregs and not significantly higher) percentages of CD4+ CD25+ Tregs in TB suggests it is not a black-and-white issue. In a setting of blood and higher IL-10 levels in serum than those with drug- excessive inflammation, they would appear to be beneficial for sensitive TB (80). We note that neither of these studies provided the host. However, their immunosuppressive role could also be any data on the extent of pulmonary disease, which usually is advantageous for the pathogen. It is most probably a matter worse in MDR-TB (81). of balance between both responses (Figure 1). Mtb is a slow growing pathogen that generates a delayed immune response. CONCLUDING REMARKS The scale tipped toward Tregs at this instance would play in favor of the bacillus, allowing its dissemination, and intracellular It is clear that the balance between pro- and anti-inflammatory growth. Also, the relatively low Th17 response would delay the responses to Mtb infection is important in the development of recruitment of neutrophils to the site of infection. However, in the disease and should be further studied. the opposite scenario, a low Treg response would benefit the Research performed on animal models is a useful way of onset of an excessive inflammatory response, characteristic of focusing on different aspects in the progression of Mtb infection pulmonary TB. Ideally, a balanced situation in which the immune and their relationship with Tregs. One of the main advantages system controls Mtb growth without generating tissue damage, of these studies is the possibility of easily looking into the would allow the encapsulation of the small granulomas and limit site of infection and lymphoid tissues. They also provide the Mtb spread. More efforts should be done on analyzing not just possibility of modulating many of the involved actors to further the regulatory response against Mtb, but rather its equilibrium understand the mechanisms behind Mtb infection. In this sense, with the pro-inflammatory response, especially Th17. the development of knock outs and monoclonal antibodies has been of great help. However, using animal models has obvious AUTHOR CONTRIBUTIONS limitations when the time comes to translate findings to human TB. It is crucial to set appropriate objectives in each model and to PC and P-JC conceptualized the review. PC provided an initial contextualize results. draft. P-JC performed the final edits. Most work carried out on animal models describes an increase in Treg levels as Mtb infection progresses. Experimental FUNDING depletion of CD25+ T cells as a proxy of Treg deletion, even being a rough approach to the issue, resulted in worse This work was supported by the Plan Nacional I + D + I pathology or higher bacillary load, arguing they have in fact a co-financed by ISCIII-Subdirección General de Evaluación and detrimental role for the host. Nonetheless, higher frequencies Fondo-EU de Desarrollo Regional (FEDER) through PC contract of Tregs have been related to less susceptible mouse strains IFI14/00015 and project PI14/01038. The project leading to and better lung pathology or non-progression toward disease these results has received funding from la Caixa Foundation (ID in NHP. 100010434), under agreement LCF/PR/GN16/10290002. Frontiers in Immunology | www.frontiersin.org 8 September 2019 | Volume 10 | Article 2139 Cardona and Cardona Tregs and TB REFERENCES 21. Bacchetta R, Passerini L, Gambineri E, Dai M, Allan SE, Perroni L, et al. Defective regulatory and effector T cell functions in patients with FOXP3 1. World Health Organization. Global Tuberculosis Report 2018. Geneva. (2018). mutations. J Clin Invest. (2006) 116:1713–22. doi: 10.1172/JCI25112 2. Houben RMGJ, Dodd PJ. The global burden of latent tuberculosis 22. 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(2013) 8:4– 10. doi: 10.1371/journal.pone.0065496 P-JC is co-founder of two spin-offs related to tuberculosis: Archivel Farma 78. Lim HJ, Park JS, Cho YJ, Yoon H Il, Park KU, Lee CT, et al. and Manremyc. CD4+FoxP3+T regulatory cells in drug-susceptible and multidrug- resistant tuberculosis. Tuberculosis. (2013) 93:523–8. doi: 10.1016/j.tube.2013. Copyright © 2019 Cardona and Cardona. This is an open-access article distributed 06.001 under the terms of the Creative Commons Attribution License (CC BY). The use, 79. Li N, Xie WP, Kong H, Min R, Hu CM, Zhou XB, et al. Enrichment distribution or reproduction in other forums is permitted, provided the original of regulatory T-cells in blood of patients with multidrug-resistant author(s) and the copyright owner(s) are credited and that the original publication tuberculosis. Int J Tuberc Lung Dis. (2015) 19:1230–8. doi: 10.5588/ijtld. in this journal is cited, in accordance with accepted academic practice. No use, 15.0148 distribution or reproduction is permitted which does not comply with these terms. Frontiers in Immunology | www.frontiersin.org 11 September 2019 | Volume 10 | Article 2139