Nontuberculous mycobacteria (NTM) are ubiquitous organisms that can cause chronic pulmonary disease (PD), and the burden of the disease is rapidly increasing worldwide [1
]. Mycobacterium avium
complex (MAC), including M. avium
and M. intracellulare
, is the major causative organisms of NTM-PD. The risk factors for the development of MAC-PD are believed to be not only existing structural lung disease such as bronchiectasis or post tuberculosis (TB) fibrosis, but also dysregulated host response to MAC infection [4
]. However, to date, the basis for vulnerability to MAC-PD from an immunologic perspective has yet to be elucidated.
T cells have long been known to play an important role in immune containment of mycobacterial infection such as M. tuberculosis
, proven by increased susceptibility to TB in mice lacking CD4+
T cells [5
T cells differentiate into numerous T cell subpopulation, such as Th1, Th2, Th17, and regulatory T cells (Tregs), a process regulated by specific transcription factors [8
]. Although the IFN-γ/IL-12 axis, representative of Th1, is central to disseminated MAC-PD susceptibility [10
], its role in patients confined to MAC-PD is unclear. In addition to Th1 cells, Th17 cells also play a role in establishing protective immunity against mycobacterial infections by secreting IL-17 which leads to recruitment and activation of neutrophils. Data have also shown that the function of IL-17 can help in controlling MAC-PD, as well as TB or other bacteria. Additionally, data have revealed that Tregs are involved in mycobacterial infection. For example, numbers of CD4+
T cell, subsets of Tregs, are elevated upon TB infection leading to suppression of T-cell mediated IFN-γ production, and IFN-γ knockout mice infected with M. massiliense
showed progressive pulmonary disease and accumulation of Tregs in the lungs. These traits are indicative of T cell dysfunction in NTM infection and raise the possibility of T cell exhaustion in the chronic phase of MAC infection. T cell dysfunction is mediated by several inhibitory pathways including programmed death-1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), and T cell immunoglobulin and mucin domain-containing-3 (TIM-3) pathways, which are widely known targets in cancer immunotherapy [17
]. Several models of chronic viral infections, including chronic human immunodeficiency virus (HIV), hepatitis C virus, and hepatitis B virus, have also been linked to high expression of these inhibitory receptors [18
]. Additionally, these receptors play an important role in T cell dysfunction during chronic mycobacteria infections such as TB [21
]. However, there are only a few reports addressing the role of PD-1 and CTLA-4 pathways, especially in MAC-PD [24
In these contexts, however, there are limited data on the characteristics of circulating CD4+ T cell subsets in MAC-PD patients. Therefore, we aimed to characterize T cell immune phenotype and immune inhibitory receptor in MAC-PD patients compared with healthy controls, by investigating levels of cytokines, proportion of T lymphocytes, and expression of immune checkpoint inhibitors, PD-1, CTLA-4, and TIM-3, on T lymphocytes. Our data may partly help to identify vulnerability to developing MAC-PD and targets of further study.
In the current study, we observed the immunological features of circulating CD4+ T lymphocytes in patients with MAC-PD and found a lower frequency of Th17 cells, but a higher frequency of Th2 cells and Tregs compared to those in control individuals. In addition, our study revealed MAC-induced CD4+ T cell dysfunction and represented significantly higher populations of PD-1+CD4+cells, CTLA-4+CD4+cells, and TIM-3+CD4+ T cells in MAC-PD patients compared with healthy controls. We also showed that the expressions of PD-1, CTLA-4, and TIM-3 were significantly induced upon MAC stimulation in PBMCs of MAC-PD patients compared with those of controls.
IFN-γ plays a pivotal role in immune defense against mycobacteria [29
]. In a recent NTM study, PBMCs of NTM patients cultured with anti-CD3, phytohaemagglutinin (PHA), or MAC showed a decrease in Th1 cytokines compared with that from healthy controls. Vankayalapati et al. found that PBMCs from patients with active pulmonary MAC produced lower IFN-γ, IL-12, and TNF-α than M. avium
sensitive–responsive control individuals [30
]. We, and others, report a marked decrease in IFN-γ secretion in response to stimulation with PHA and diminished production of IFN-γ and TNF-α in MAC bacilli-stimulated PBMCs from patients with MAC-PD [25
]. However, contradictory results have also been reported [11
]. In the present study, IFN-γ production decreased in response to MAC bacilli in PBMCs from MAC-PD patients, but no difference was observed in the frequencies of IFN-γ+
T cells in NTM-PD. Therefore, although Th1 lymphocytes are important in the immune response to mycobacterial infection, IFN-γ alone might be insufficient for complete eradication of the bacteria, suggesting roles for other cytokines in the immune defense response against mycobacteria.
Successful host defense against mycobacteria with clearance/control of mycobacterial infection requires an effective Th1 and, to a lesser extent, functioning Th17 immunological response rather than a Th2-type response [32
]. However, chronic disequilibrium between the different divisions of the adaptive immune system may lead to pathology and susceptibility to infection [34
]. In a previous cohort study of patients infected with M. tuberculosis
, peripheral blood eosinophil count and serum IgE levels in patients with MAC-PD were higher than those in patients with pulmonary TB or other species of NTM [35
]. We observed a similar phenomenon, wherein the frequency of IL-4+
T cells was elevated in MAC-stimulated PBMCs from patients compared with that from control individuals, although there was no change in IL-4 production. The discrepancy between this study and others may be explained by the possibility that MAC may have developed defensive mechanisms to skew immune responses towards a Th2-type response, thereby decreasing the ability of the immune system to clear the mycobacteria.
In the present study, we observed elevated levels of IL-17 in MAC-PD patients. In addition, The IL-17 level from lymphocytes was higher after MAC bacilli stimulation both of MAC-PD patients and healthy controls, but the IL-17 level expressed from lymphocytes activated in MAC-stimulated PBMCs was lower in patients with MAC-PD than that in healthy controls. This result is consistent with a previous study conducted on M. avium
in macrophages [25
]. A recent study showed increased IL-17 and IL-23 gene expression in the lungs of patients with active TB [36
], while another reported that MAC lung disease was associated with defects or biases in Th1 and Th17 immunity [11
]. Thus, the attenuated IL-17 response might contribute to host vulnerability or pathogen evasion in MAC-PD via impairment of neutrophil recruitment and granulopoiesis.
Tregs play a role in the immunosuppression observed in chronic infectious diseases [38
], suggesting that Tregs might contribute to impaired specific MAC-induced T cell responses. Antigen-stimulated blood cells from NTM-PD patients showed an elevated Treg population compared with that from control individuals [39
]. Furthermore, Tregs were increased in the peripheral blood of cystic fibrosis NTM patients compared with that of controls [24
]. Our study demonstrates that Tregs were increased in MAC-stimulated PBMCs of MAC-PD patients compared with that of control individuals, suggesting that the elevated levels of Tregs might suppress T-cell responses, thereby protecting MAC and playing a pathogenic role in MAC-PD.
Recent studies demonstrated that PD-1 regulates T cell activation, peripheral tolerance and autoimmunity, principally as an inhibitory molecule [40
]. Several studies have demonstrated that the PD-1 signaling pathway is activated during persistent infection with various microorganisms and contributes to impairment of protective immunity [22
]. Few studies have addressed the effects of PD-1 on T lymphocytes in NTM infection. It was recently shown that in vitro blockade of PD-1 signaling enhanced MAC-specific IFN-γ production by T cells [25
] and NK T cells of PTB patients [42
], indicating that this inhibitory pathway also affects T cell functions during mycobacterial infection. Elevated CTLA-4 was also observed on CD4+
T cells in NTM infection [24
]. Consistent with these reports, we found that PD-1+
T cells and CTLA-4+
T cells were more prevalent in patients with MAC-PD compared with that in control participants. In addition to these molecules, there is a variety of other immune checkpoints with unknown function in relation to mycobacteria. Tim-3 is a membrane protein expressed at late stages of interferon-gamma secreting CD4+
T helper type 1 (Th1) cell differentiation [43
]. T-bet binds directly to the Tim-3 promoter in CD4+
Th1 cells [44
]. Among inhibitory factors, TIM-3 has not been studied with respect to MAC-PD. Our data indicate, for the first time, that TIM-3 is elevated in CD4+
T cells after MAC infection, further indicating the degree of immunosuppression in MAC-PD.
T cell exhaustion is common during chronic infections. Exhausted CD4+
T cells display poor production of effector cytokines (TNF and IFN-γ) and express high levels of PD-1 [45
]. It has been suggested that CD4+
T cells increased expression of mRNA encoding several transcription factors including PR domain zinc-finger protein 1, nuclear factor of activated T cell, and T-bet have been implicated in the development of different T cell subsets in chronic infection [46
]. We observed a decrease in effector cytokine production and the high populations of transcription factors related T cells subset in MAC-stimulated PBMCs in MAC-PD. The low IFN-γ and IL-17 production by MAC stimulated- CD4+
T cells probably explained by exhausted CD4+
Th1 cells to induce the binding of T-bet to the Tim-3 promoter. T-bet has been reported to be a transcription factor for regulating Tim-3 promoter during chronic infection [44
]. T-bet enhances Tim-3 expression via a c-Jun N-terminal kinases pathway, leading to dampened monocyte/macrophages function during hepatitis C virus infection [47
This study had several limitations. First, the patients and controls were not age-matched. The average age of the patients with MAC-PD was higher than that of the controls, possibly leading to some bias, although we found similar results in age-matched subgroups. Second, an MOI of 100 is high, warranting caution; thus, further validation studies using viable bacilli are needed. Third, we could not identify the intracellular mechanism by which MAC induces the expression of PD-1, CTLA-4, and TIM-3. Finally, we did not perform lymphocyte proliferation assays or intracellular cytokine staining to evaluate lymphocyte function.
In summary, our study provides evidence for CD4+ T cell dysfunction in patients with MAC-PD. The population of CD4+ and CD4+IL-17+ T cells was decreased in MAC-induced PBMCs from patients with MAC-PD, while that of CD4+IL-4+ and CD4+CD25+Foxp3+T cells was increased in MAC-induced PBMCs from patients with MAC-PD. An increasing population of PD-1, CTLA-4, and TIM-3 might be responsible for Th1, Th2 and Th17 cells in MAC-PD. Our findings suggest a complex immune response in MAC-PD patients and targeted interventions against the inhibitor pathways may help restore local and systemic immunity in these patients.