Discussion
The association between LTBi and DM-2 has implications on global TB
control: a mathematical model predicts that elimination of TB by the
year 2035 might be hampered if the incidence of DM-2 continues to
increase worldwide (29). An elevated glucose or HbA1c levels has been
shown to be related with an increased risk of developing LTBi (30).
The aim of our study is to contribute to understanding the impact of
diabetes on host immunity during mycobacterial infection and disease.
Diabetes has been shown to be associated with altered cellular and
humoral immune responses. It has been suggested that when DM co-occurs
with TB, host protective immune function is affected (31). This leads to
more un-favourable outcomes of both TB and DM (31), with a slower
recovery and increased chance of TB relapse (32). Less is known about
the alterations of host immune responses in DM in LTBi individuals.
Limited reports indicate that there is an IL-10 and TGF-beta driven
decrease in host protective CD4 T cell responses (10).
We studied PPD-triggered cytokine responses in healthy subjects with and
without LTBi, diabetics with and without LTBi and also patients with TB.
We observed increased PPD-induced IL-2, TNF-α and IL-6 protein levels in
DM-LTBi as compared with EC, LTBi and DM. PPD- stimulated GM-CSF levels
were also raised in DM-LTBi as compared with EC and LTBi groups.
IL-5 protein titers were higher in DM-LTBi compared with EC cases and
IL-13 levels were raised in DM-LTBi as compared with DM as well,
suggesting heightened levels of some Th2 cytokines in diabetics with
latent TB. As the latter are markers of alternate macrophage activation
(11), this suggests a possible shift to macrophage dysfunction in
diabetics who are latently infected with MTB.
The increased susceptibility to M. tuberculosis infection is
regulated by alterations in of IFN-γ, TNF-α, IL1-β, IL-6 and IL-10
levels (33, 34). Studies in diabetic mice have shown reduced levels of
IFN-γ in lungs after infection with M. tuberculosis and increased
bacterial load as compared with the non-diabetic ones (35).
An impaired trafficking of immune cells, resulting in delayed T cell
responses was observed to the site of infection in diabetic mouse model
resulting in increased susceptibility to infection with M.
tuberculosis (36, 37). TNF-α is a major regulator of granuloma
formation and hence containment of M. tuberculosis infection.
However, dysregulation in TNF-α levels also leads to the rupture of
granulomas resulting in dissemination of granulomas (38).
We observed increased TNF-α gene expression at the protein and mRNA
levels in DM-LTB as compared with EC and LTBi. Elevated levels of TNF-α
have been reported in patients with chronic diabetes (39). Raised TNFα
levels have also observed in patients with raised HbA1c levels (39).
IL-2 and GM-CSF were shown to be raised in DM-LTB as compared with EC.
These findings are in line with previous studies (40, 41). Patients with
diabetes are known to exhibit a persistent pro-inflammatory state linked
with the development of insulin resistance and pathology linked with
diabetes (42).
We found IL-6 protein levels to be significantly raised in DM-LTB cases
as compared with both EC and LTBi subjects. Similarly, we found
PPD-induced IL-6 mRNA expression to be raised in DM-LTB subjects as
compared with EC. Raised levels of IL-6 in plasma has been shown to be
associated with metabolic disorders (43). Raised IL-6 mRNA levels have
been reported in insulin resistant humans (43). In another study, a
positive association has been reported between elevated levels of IL-6
and poorly controlled diabetes (39). In TB, IL-6 has been shown to play
a vital role in pro-inflammatory responses including promoting T cell
and B cell responses (44). IL-6 deficient mice have been shown to be
susceptible to TB infection (45).
We also observed IL-5 levels to be raised in DM-LTB as compared with
healthy controls. Previously, TB with diabetes has been associated with
an increase in circulating IL-5 levels(46) . Further, PPD-induced IL-13
secretion was raised in DM-LTB cases as compared with those with DM
only. IL-13 has previously been shown to be raised in TB patients with
pre-DM (47). IL-13 regulation is required for effective glucogenesis
(48).
Our observation that individuals with PPD-induced SOCS1 was upregulated
the most in TB patients is concordant with previous reports (49).
Previously, it has been shown that M. bovis BCG (50) and M.
tuberculosis infection upregulates SOCS1 expression (51). We also
observed an increase in SOCS1 upregulation in diabetics. Previous
reports showed that SOCS1 inhibit insulin receptor substrate (IRS)
proteins thereby causing insulin resistance (52).
After PPD-stimulation of PBMCs, SOCS3 expression was found to be the
highest in LTBi cases and reduced in EC, DM, DM-LTBi and TB cases.
Upregulation of SOCS3 in response to M. tuberculosis infection
has been shown previously (23). SOCS3 is required for protection against
MTB infection and mice deficient in SOCS3 are susceptible to
mycobacterial infections (53). Upregulation of SOCS3 in macrophages has
been shown to provide protection against infection by intracellular
pathogens (23). We have previously found reduced SOCS3 gene expression
in patients with far advanced PTB (54). In this case, the association of
downregulated SOCS3 together with raised inflammatory cytokines in
DM-LTBi may be suggestive of less protective immune state in the host,
making it more prone to disease progression.
This work further points to the importance of early identification of
diabetes and of latent TB both as important independent predisposing
factors for TB progression. It emphasizes the need to identify health
interventions and treatments which may aid management of the individual
prior to progression towards active TB disease.