Global epidemiological studies have clearly shown increased susceptibility of diabetes patients to tuberculosis (TB), which is often called “Diabetes-Tuberculosis Synergy” (Jeon and Murray, 2008; Sen et al., 2009). TB patients with diabetes, present a higher bacillary load in the sputum, delayed sputum conversion, and higher rates of multidrug-resistant (MDR-TB) infection (Jeon and Murray, 2008; Sen et al., 2009). They also present with higher lung cavitary involvement, compared to their non-diabetes counterparts (Jeon and Murray, 2008; Sen et al., 2009). These results imply that patients with TB and diabetes, may be more seriously ill, and may pose a higher risk for spread, in the community (Jeon and Murray, 2008; Sen et al., 2009). Recent epidemiological surveys have clearly shown the possibility of diabetes–tuberculosis (DM-TB) nexus in near future, which needs immediate attention (Jeon and Murray, 2008; Sen et al., 2009). Chronic inflammation has long been identified as a risk factor for diabetes (Martinez and Kornfeld, 2014; Ayelign et al., 2019). Inflammation in diabetes is low-grade, systemic, and non-antigen-specific in nature (Martinez and Kornfeld, 2014; Ayelign et al., 2019). Most importantly, it impairs immunity and makes them more susceptible to TB (Martinez and Kornfeld, 2014; Ayelign et al., 2019). Diabetes subjects also have increased redox stress which fuels inflammation and impairs immunity (Yuan et al., 2019). Diabetes in Asian Indians is characterized by the younger age of onset, lower body mass index, propensity for central obesity, increased fat/muscle ratio, and increased insulin resistance (compared to other ethnic populations), which has been described as the “Asian–Indian Phenotype” (Mohan and Deepa, 2006). Whether the Asian-Indian phenotype (which is not fully characterized) is also a risk factor for TB is not known.

A substantial increase in diabetes is taking place in the world, where approximately one-third of the population is latently infected with Mycobacterium tuberculosis (latent tuberculosis infection-LTBI) (Lee et al., 2016). The increased incidence of TB in patients with diabetes, could either be due to the reactivation of latent tuberculosis (endogenous reactivation) or due to new infection (exogenous re-infection), which is the consequence of the weakening of the immune system (Lee et al., 2016). The Diabetes–Tuberculosis synergy is due to a vicious cycle: The tubercle bacillus induces a strong inflammatory response and redox stress which can worsen insulin resistance (Nishio et al., 1988) and pancreatic beta-cell loss (Park et al., 2014), precipitating diabetes among pre-diabetics, or worsening glucose intolerance in chronic diabetics (Mahat et al., 2019). On the other hand, chronic diabetes weakens the immune system, increasing the chances of TB infection among uninfected (exogenous infection), or augmenting re-activation, in those who are latently infected (endogenous reactivation) (Nematollahi et al., 2020). Compared to active TB in diabetes, LTBI in diabetes is poorly studied (Lee et al., 2016). Further with respect to diabetes, most studies have been carried out only on chronic diabetes subjects and what happens during the pre- and early stages of diabetes is still an enigma. Recently, we reported stage-specific modulation of inflammation, by LTBI, among subjects with various grades of glucose intolerance (Bobhate et al., 2021; Aravindhan et al., 2022). As a logical next step, in the present study, we looked at the prevalence of LTBI, at different stages of glucose intolerance. Diabetes is often associated with obesity, hypertension, and dyslipidemia (Lee et al., 2016). Thus, we took a holistic approach to study the association between metabolic co-morbidities, often seen with diabetes, with LTBI. The immunoendocrine mechanism behind DM–LTB synergy was studied by systemic cytokine and hormone profiling. Systemic redox stress was studied by quantifying circulating levels of lipid peroxidation products (oxidative stress) and nitrites (nitrosative stress). Finally, anti-TB immunity was studied using antigen specific IFN-γ response and T-bet expression in LTB⁺ subjects.

Diabetes patients with tuberculosis suffer from significant morbidity and mortality, which is called as “Diabetes–Tuberculosis Synergy” (Jeon and Murray, 2008; Sen et al., 2009). Hence, we expected a higher prevalence of LTBI among PDM, NDM, and KDM subjects. However, in contrast to our expectations, the study revealed several unexpected findings: (1) The prevalence of LTBI was significantly lower among PDM and NDM groups, compared to NGT and KDM; No difference was seen between NGT and KDM groups, (2) The decreased prevalence of LTBI, in the PDM and NDM groups, was associated with significant immunoendocrine modulation, and (3) In contrast to diabetes, a strong positive association was seen between LTBI and hypertension.

The most unexpected finding in the present study is the lower prevalence of LTBI among PDM and NDM subjects, compared to NGT and KDM groups. In a recent meta-analysis done on 13 studies (which includes both IGRA and TST based screening) only a very small risk factor was found to be associated with LTBI in diabetes (Lee et al., 2016). Being a cross-sectional study, at this juncture, it is difficult to explain this inverse relationship between diabetes and LTBI. Previously, we and others have reported such an inverse relationship between lymphatic filariasis and diabetes (Aravindhan et al., 2010; Wiria et al., 2012), which paved the way for the formulation of the “metabolic hygiene hypothesis” (Aravindhan and Anand, 2017). A common denominator between filariasis and LTBI is the immunomodulatory effect these infections can have on diabetes. Theoretically, LTBI can confer protection against pre-diabetes through immunomodulation leading to hormonal homeostasis; alternatively, pre-diabetes being a pro-inflammatory state, can confer protection against LTBI, due to high circulating levels of cytokines (Ruotsalainen et al., 2006; Wang et al., 2016). In order to explore this possibility, we carried out systemic cytokine and hormone profiling in the sub-groups of the study population. Among the various cytokines, pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β), and Th1 cytokines (IL-12, IFN-γ, and IL-2) are known to confer significant protection against tuberculosis (Appelberg, 1994), while anti-inflammatory cytokines (IL-10 and TGF-β) (Etna et al., 2014) and type-I interferons (IFN-β) confer susceptibility (Moreira-Teixeira et al., 2020). Interestingly, most of these cytokines were also found to be elevated in pre-diabetes and were responsible for insulin resistance and pancreatic-beta cell destruction (Wei et al., 2008). As expected, serums levels of TNF-α, IL-6, IL-1β, and IFN-β were significantly elevated in PDM. Co-morbidity with LTBI specifically downregulated CRP and TNF-α and up-regulated IL-12 levels. Previously, we have reported increased levels of IL-10 in LTB⁺PDM subjects (Bobhate et al., 2021). The up-regulation of IL-10 and downregulation of TNF-α and CRP can augment insulin sensitivity, while increased levels of IL-12 can augment anti-TB immunity. Further, serum hormonal profiling indicates increased levels of adiponectin and leptin in LTB⁺PDM subjects, which can increase insulin sensitivity (Ahlstrom et al., 2017) and insulin release (Shimizu et al., 1997), respectively. Co-morbidity with LTBI also significantly reduced the nitrite levels in PDM subjects. It is important to note that “nitrosative stress” is an important contributor to the pathogenesis of diabetes (Al-Nimer et al., 2012). These findings indicate downregulation of inflammation, leading to increased insulin sensitivity, augmentation of anti-TB immunity, and hormonal homeostasis as major factors responsible for the inverse relationship between PDM and LTB (Figure 8).

Like PDM, NDM subjects also had a reduced prevalence of LTBI. However, the cytokine profile of PDM, NDM, and KDM did not follow the glycemic profile, indicating stage-specific immunomodulation, as we reported previously (Bobhate et al., 2021; Aravindhan et al., 2022). While serum TNF-α, IL-6, IL-1β, and IFN-β were significantly elevated in NDM, IFN-γ levels were significantly reduced. Co-morbidity with LTBI specifically up-regulated IL-1β, which is known to confer protection against TB (Mayer-Barber et al., 2014). The hormone profiling showed reduced levels of insulin, which was responsible for diabetes precipitation. Co-morbidity with LTBI, specifically upregulated the adiponectin levels, with no effect on insulin and leptin levels. Previously, we have reported increased levels of serum IL-10 in NDM-LTB⁺ compared to NDM-LTB⁻ (Bobhate et al., 2021). Overall, the cytokine and hormone profile again indicate downregulation of inflammation, augmentation of anti-TB immunity, and hormonal homeostatic, as major factors contributing to this inverse relationship, seen during the NDM stage (Figure 8).

Another unexpected finding in this study, is the same prevalence of LTBI in the NGT and KDM groups, while we expected an increase in the latter group, due to severe immune impairment (Masood et al., 2022). In order to understand the mechanism, we compared the IFN-γ levels in the quantiferon culture supernatants in LTB⁺ subjects, belonging to the four diabetic groups. There was no significant difference in the basal level secretion of IFN-γ between the groups (IFN-γ levels in TB antigen NIL tubes -data not shown). However, TB antigen-induced secretion of IFN-γ was drastically reduced in the LTB⁺ KDM subjects, indicating strong defects in adaptive immune responses. This was also accompanied by the lack of IL-1Ra (Bobhate et al., 2021) and IL-2 secretion (data not shown). The poor secretion of IFN-γ might account for the low detection rate of LTBI, among the KDM subjects. Thus, IGRA might increase the false negative cases among chronic diabetes subjects and might not be an ideal test for LTBI screening among these patients, as was reported previously (Shin et al., 2017). The poor secretion of IFN-γ was mainly due to the failure to upregulate T-bet expression, following antigenic stimulation. T-bet is a master regulator of Th1 differentiation and in the absence of which both Th1 differentiation and IFN-γ secretion are drastically altered (Sullivan et al., 2005). The defective adaptive immune response accounts for both the low detection rate of LTB and the possibility of high rates of endogenous reactivation, in this group, accounting for diabetes–tuberculosis synergy (Jeon and Murray, 2008; Sen et al., 2009). Furthermore, the KDM subjects had significantly reduced levels of TBARS (but not nitrites) as was reported previously (Chakraborty et al., 2011). The strong augmentation of adiponectin in LTB⁺ subjects in all the four groups is in accordance with a recent report (Ayyappan et al., 2018).

Finally, the most unexpected finding of this study was the strong association seen between LTBI and hypertension. In all the four groups, LTB⁺ subjects had higher SBP and DBP compared to LTB⁻ subjects. The prevalence of LTBI was much higher in the hypertensive group compared to the non-hypertensive group. Few studies have shown an association between extra pulmonary tuberculosis and pulmonary hypertension (Bhalla et al., 2010; Mojtahedzadeh et al., 2012). Recently, the importance of hypertension in active TB disease was reviewed with a limited number of studies (Seegert et al., 2017). Since the primary objective of our study was the diabetes status, a detailed history of hypertension (newly diagnosed vs. chronic, disease duration, treatment, etc) could not be collected. However, to the best of our knowledge, this is the first report to show a strong positive correlation between LTBI and hypertension and this obviously needs further exploration.

Recently, Mycobacterium has been shown to confer significant protection against heterologous diseases, by a mechanism called “trained immunity” (Gopalaswamy et al., 2020). In trained immunity, the innate immune cells, such as macrophages, DCs and NK cells, can be trained by mycobacterium to develop a pro-inflammatory phenotype, which can confer protection against infection by non-mycobacterial, fungal, or viral pathogens (Singh et al., 2020). However, the contribution of such a non-specific immune response, elicited by LTBI, in the setting of diabetes, is not fully understood. It is important to note that the immune status during active TB and LTB are quite different: while active TB is associated with a strong inflammatory reaction, LTB is associated with immunomodulation (Madan et al., 2021). Recently, in a small study carried out in India, LTBI was shown to confer significant protection against the severe form of the covid-19 disease (Madan et al., 2021). Similarly, the metabolic status of diabetes and pre-diabetes are quite different: while frank diabetes is associated with insulin resistance and deficiency, pre-diabetes is associated with insulin resistance and hyperinsulinemia. In contrast to diabetes and metabolic syndrome, isolated insulin resistance, under certain conditions, can confer some evolutionary advantage, at the organism level (Soeters and Soeters, 2012), and in this case anti-TB immunity. India has long been identified as an endemic zone for tuberculosis (Pathak et al., 2021) and is competing with China in becoming the diabetes capital of the world (Joshi and Parikh, 2007). The active implementation of the national end TB (NETB) program in India over the past several years might have reduced the LTBI burden (Dolla et al., 2019; Bhargava et al., 2021), which could have gradually contributed to the increase in diabetes, over the years (“metabolic hygiene hypothesis”) (Aravindhan and Anand, 2017). Further studies are thus needed to substantiate this inverse relationship between diabetes and LTBI.

The most interesting finding in this study is that the relationship between diabetes-latent tuberculosis is complex and biphasic: At the early stages of diabetes, it is reciprocal; while at a late stage it is synergistic—this important phenomenon obviously needs further research (Figure 8). Cytokine and hormone profiling indicated that IL-10 mediated secretion of adiponectin could be the most important mechanism by which LTBI can confer protection against insulin resistance. Insulin resistance which was associated with high circulating levels of IL-12, leptin, and IL-1β could be the most possible reason behind anti-TB immunity. During chronic diabetes, a synergistic interaction was seen with LTBI. The reduced expression of T-Bet and defective antigen-specific IFN-γ response, as seen in the KDM group, could account for both the low detection rate of LTBI and the high probability of endogenous reactivation. Thus, the interaction between diabetes and LTBI is complex involving a multifaceted immune-endocrine mechanism.

Diabetes patients have impaired immunity which makes them more susceptible to tuberculosis. In general, diabetes patients have higher sputum bacillary load, delayed sputum clearance, high rates of treatment failure, higher incidence of multi-drug resistance, increased cavitary lung lesion, etc. This is called as diabetes-tuberculosis nexus, which could either be due to an increased infection rate or increased reactivation of latent tuberculosis infection (LTBI). In the present study, we looked at the prevalence of LTBI among subjects with various stages of glucose intolerance. In contrast to our hypothesis, the prevalence of LTBI was the same between control subjects and chronic diabetes patients, while it was significantly reduced in pre-diabetes and newly diagnosed diabetes patients. Further, hypertension which is a common co-morbidity seen with diabetes was found to be a major risk factor for LTBI, which hasn't been reported to date. Apart from prevalence, we also studied the disease mechanism by quantifying circulating levels of cytokines, hormones, and redox markers. Our study indicated modulation of the immunoendocrine system, as a probable mechanism behind the biphasic relationship between LTBI and diabetes.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by MV Hospital Institutional Human Ethical Committee. The patients/participants provided their written informed consent to participate in this study.

VA and VV conceived and designed the experiment, drafted the manuscript, and garantors of the data. AB and KS performed the experiment. VA, SK, and AP analyzed the data. VA, AB, AP, and VV contributed to the discussion and reviewed the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by Department of Biotechnology, New Delhi (BT/PR5693/MED/29/585/2012). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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.

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