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Mycobacterium tuberculosis is known to modulate the host Pathogenic bacteria employ various host immune evasion
immune responses to facilitate its persistence inside the host strategies to facilitate their survival in the host cells. One such
cells. One of the key mechanisms includes repression of class-II mechanism involves the induction of epigenetic modifications
transactivator (CIITA) and MHC-II expression in infected in the host DNA, histone proteins, and RNA by bacterial pro-
that the observed increased bacterial survival was not due to the cellular signaling pathways that can either support or
differences in the growth kinetics of bacteria. The extracellular inhibit the bacterial growth, depending upon the cytokine
and intracellular expressions of esxL were determined by qRT- milieu (45). Previously, ESAT-6 was shown to induce NO pro-
PCR at the 4-, 12-, and 24-h time points. As shown, esxL was duction in macrophages (46). We found significantly higher
expressed under both in vitro (Fig. 1E) and ex vivo (Fig. 1F) NO production in M. smegmatis esxL-infected RAW 264.7 cells
conditions. In conclusion, the above data showed that M. tuber- as compared with M. smegmatis pSMT3-infected cells at the
culosis esxL has a role in increased bacterial persistence inside indicated time points (p ⱕ 0.05 and p ⱕ 0.01; Fig. 2A). Similarly,
the macrophages. The generation of Mtb⌬esxL is shown in Fig. we observed an ⬃18-fold increase in iNOS at transcriptional
1, G and H. (p ⱕ 0.001; Fig. 2B) and significant increase at translational
levels (Fig. 2C) in M. smegmatis esxL-infected macrophages. Im-
EsxL induces NO production and iNOS expression in munofluorescence studies using iNOS-specific antibody also
macrophages showed significantly increased expression in M. smegmatis
iNOS, which catalyzes formation of NO, has immunomodu- esxL-infected macrophages as compared with uninfected and
latory activities that determine the outcome of M. tuberculosis M. smegmatis pSMT3-infected cells (Fig. 2D). On the contrary,
infection (43). In addition to its antibacterial properties, NO is decreased iNOS expression was observed in Mtb⌬esxL-in-
also known as a key regulator in the initiation and maintenance fected THP-1 cells when compared with M. tuberculosis-in-
of anti-TB protective immunity (44) and is known to modulate fected cells (Fig. 2E). We did not observe any significant differ-
ences in the production of ROS in infected macrophages (data M. smegmatis esxL-infected THP-1 (Fig. 3G) and RAW 264.7
not shown), indicating that M. tuberculosis esxL specifically macrophages (Fig. 3H). In agreement with previous reports, we
induces NO production in macrophages. also observed a time-dependent decrease in MHC-II expres-
sion in M. tuberculosis-infected THP-1 cells (Fig. 3I) when
EsxL down-regulates MHC-II and CIITA in macrophages compared with Mtb⌬esxL-infected cells (Fig. 3J). Altogether,
Pathogenic mycobacteria are known to down-regulate the these data suggest that M. smegmatis esxL-, M. bovis BCG-, and
surface expression of MHC-II molecules in macrophages (47). M. tuberculosis-mediated MHC-II inhibition is due to down-
The MHC-II-dependent antigen presentation is tightly regu- regulation of CIITA.
lated by a key transcription factor, CIITA. Mice deficient for
CIITA showed a marked reduction in MHC-II expression (48). M. smegmatis EsxL infection down-regulates IL-2 and IL-10
It has been shown that M. bovis BCG infection inhibited and up-regulates IL-6 and TNF-␣ production in macrophages
MHC-II expression by inducing NO production in macro- It is known that inhibition of antigen presentation prevents
phages (29). In view of these reports, we checked the expression T-cell activation (49). As mentioned above, IL-2 is a key cyto-
of MHC-II and CIITA in M. tuberculosis, Mtb⌬esxL, M. smeg- kine involved in T-cell activation (50, 51). Therefore, we
matis pSMT3, and M. smegmatis esxL-infected macrophages. checked IL-2 levels using the Bioplex cytokine analysis kit. For
As shown, M. smegmatis esxL infection abrogated CIITA this, macrophages were first infected with M. smegmatis
expression at both transcriptional (p ⱕ 0.01; Fig. 3A) and trans- pSMT3 and M. smegmatis esxL strains, followed by co-culture
lational levels (Fig. 3B). M. tuberculosis-infected THP-1 cells with BALB/c mouse splenocytes. We found significant down-
also showed a time-dependent decrease in CIITA expression regulation of IL-2 (p ⱕ 0.05; Fig. 4A) and IL-10 (p ⱕ 0.05; Fig.
(Fig. 3C), whereas increased CIITA expression was observed in 4B) cytokines in supernatant obtained from M. smegmatis
Mtb⌬esxL-infected cells (Fig. 3D). We further confirmed the esxL-infected cells as compared with M. smegmatis pSMT3
effect of CIITA on MHC-II expression. A significant decrease infection after 24 h. However, the presence of G9a inhibitor
in MHC-II expression was observed at both transcriptional UNC0638 increased production of both of the cytokines, sug-
(p ⱕ 0.01; Fig. 3E) and translational (Fig. 3F) levels in M. smeg- gesting that M. smegmatis esxL infection suppressed T-cell
matis esxL-infected RAW 264.7 macrophages when compared activation. In contrast, TNF-␣ (p ⱕ 0.01; Fig. 4C) and IL-6 (p ⱕ
with M. smegmatis pSMT3-infected cells. Flow cytometry anal- 0.01; Fig. 4D) were up-regulated in M. smegmatis esxL-infected
ysis also showed a significant decrease in MHC-II expression in cells.
M. smegmatis EsxL induces histone modification (H3K9 H3K4me3 and total H3 in M. smegmatis pSMT3- and M. smeg-
hypermethylation) in macrophages matis esxL-infected macrophages (Fig. 5C), indicating that esxL
A few studies have shown that pathogenic mycobacteria and induces H3K9me2/3 in macrophages.
its antigens induce epigenetic changes to evade host immune
responses (16, 18, 51). We hypothesized that EsxL might render M. bovis BCG and M. tuberculosis infection induces
repressive epigenetic modifications at the CIITA promoter that H3K9me2/3 in macrophages
subsequently inhibit MHC-II-dependent antigen presentation. To further confirm the role of EsxL in inducing repressive his-
H3K9me2/3 is involved in transcriptional repression (52). tone modification, we analyzed the expression of H3K9me2/3 in
Therefore, we analyzed the status of H3K9me2/3 in infected M. bovis BCG-, M. tuberculosis-, and Mtb⌬esxL-infected macro-
macrophages. Indeed, immunoblotting (Fig. 5A) and immuno- phages. Concordantly, Western-blotting analysis showed in-
fluorescence (Fig. 5B) analysis showed significantly elevated creased levels of H3K9me2/3 in M. bovis BCG (Fig. 5D)- and
levels of H3K9me2/3 in M. smegmatis esxL-infected macro- M. tuberculosis (Fig. 5E)-infected macrophages, suggesting that
phages as compared with control cells. A significant increase in M. tuberculosis and BCG may down-regulate CIITA expression
H3K9me2/3 puncta was observed in M. smegmatis esxL-in- by inducing H3K9me2/3 in macrophages. In contrast, de-
fected cells. We did not observe any significant differences in creased H3K9me2/3 expression was observed in Mtb⌬esxL-in-
fected THP-1 cells when compared with M. tuberculosis-in- To assess whether CIITA down-regulation during M.
fected cells (Fig. 5F). smegmatis esxL infection is dependent on G9a-mediated
H3K9me2/3, we checked the expression of CIITA in untreated
M. smegmatis esxL and M. bovis BCG induce H3K9me2/3 and G9a inhibitor-treated macrophages. Immunoblotting anal-
modification by up-regulating EHMT2 methyltransferase ysis showed that pretreatment with G9a inhibitor severely
activity reduced the capacity of M. smegmatis esxL to inhibit CIITA
To further investigate the mechanism of H3K9me2/3 induc- expression in macrophages (Fig. 5I), indicating that observed
tion, we studied the activity of methyltransferases in infected CIITA down-regulation was due to G9a-mediated induction of
macrophages. Several H3K9-specific lysine methyltransferases, H3K9me2/3 in infected macrophages.
such as Eset, KMT1E, and G9a, are involved in H3K9 methyla-
tion (53). Among them, G9a, also known as EHMT2, is a dom- ChIP analysis shows that H3K9 hypermethylation occurs at the
inant histone methyl transferase responsible for methylation promoter IV region of CIITA
of H3K9 (54). Transcriptional analysis showed a significant A ChIP assay was performed to check H3K9me2/3 in the
increase in G9a level in M. smegmatis esxL (p ⱕ 0.01; Fig. 5G)- CIITA promoter. Sequence analysis revealed the presence of
and M. tuberculosis (p ⱕ 0.001; Fig. 5H)-infected THP-1 cells, three promoter regions (CIITApI, CIITApIII, and CIITApIV)
whereas Mtb⌬esxL infection down-regulated G9a expression in CIITA (16). As shown, M. smegmatis esxL down-regulated
(p ⱕ 0.001; Fig. 5H). Otherwise, treatment with G9a inhibi- CIITA expression by inducing H3K9me2/3 at the promoter IV
tor (UNC0638) subdued the expression of H3K9me2/3 in region of CIITA (p ⱕ 0.001; Fig. 5J), whereas no such modifica-
M. smegmatis esxL-infected macrophages when compared tion was observed at CIITApI and CIITApIII promoters (data
with untreated cells (Fig. 5I). Similar results were obtained not shown). Moreover, we did not observe any H3K9me2/3
with M. bovis BCG infection, where BCG infection increased enrichment in M. smegmatis pSMT3-infected cells. Impor-
the level of H3K9me2/3, whereas treatment with G9a inhib- tantly, inhibition of G9a significantly decreased H3K9 hyper-
itor reduced H3K9me2/3 level (Fig. 5D). Collectively, these methylation at CIITA promoter IV in M. smegmatis esxL-in-
results indicate that M. smegmatis esxL, M. tuberculosis, and fected macrophages (p ⱕ 0.01; Fig. 5K). These results clearly
M. bovis BCG induce H3K9me2/3 via G9a methyltransferase. indicate that M. smegmatis esxL down-regulates G9a-depen-
dent CIITA expression by promoting H3K9me2/3 in the pro- Increased MAPK leads to recruitment of histone deacetylases
moter IV region of CIITA. (HDACs), leading to gene repression (58). Mycobacteria, in
addition to selective antigens, are known to induce MAPK sig-
M. smegmatis esxL triggers H3K9me2/3-mediated CIITA naling in macrophages (59). In this context, we addressed the
inhibition by inducing the MAPK-signaling pathway role of the MAPK-signaling pathway in the regulation of
NO acts as a key intermediate in regulation of cell-fate deci- H3K9me2/3 and CIITA expression. We found that M. smeg-
sions by modulating several signaling pathways in the host cells matis esxL infection triggered the activation of pp38 (Fig. 6A)
(55, 56). Hence, we postulated that signaling cascades that reg- and pERK (Fig. 6B) when compared with control conditions.
ulate NO production could act as a focal point in M. smegmatis Similarly, M. tuberculosis infection also induced pp38 expres-
esxL infection-triggered histone modification that subse- sion when compared with Mtb⌬esxL-infected THP-1 cells (Fig.
quently leads to inhibition of MHC-II or CIITA. MAPK path- 6C). Importantly, pretreatment with p38 inhibitor (SB203580)
ways are known to regulate eukaryotic gene expression by mod- abrogated the M. smegmatis esxL-induced inhibition of CIITA
ulating the chromatin structure of regulatory elements (57). (Fig. 6D, top). Similarly, treatment with SB203580 down-regu-
lated the expression of H3K9me2/3 in M. smegmatis esxL-in- CFP-10, lipoproteins, and PE/PPE (proline-glutamic acid/
fected macrophages (Fig. 6D, middle). On the other hand, inhi- proline-proline-glutamic acid) proteins, are known to be in-
bition of pERK by the pharmacological inhibitor U0126 did volved in the establishment of the infection process (60 – 67).
not show any effect on the expression of either CIITA or Herein, we reported that M. tuberculosis esxL represses
H3K9me2/3 during M. smegmatis esxL infection when com- CIITA/MHC-II expression by inducing H3K9me2/3 in the
pared with untreated macrophages (Fig. 6E). These results CIITA promoter. Previously, we and several others have
clearly suggest that the M. smegmatis esxL-triggered p38 MAPK- used M. smegmatis as a surrogate model to elucidate the
signaling pathway holds the capacity to modulate H3K9me2/3 function of M. tuberculosis proteins in pathogenesis. For
expression to regulate CIITA/MHC-II expression. example, expression of M. tuberculosis Mce4A protein in
M. smegmatis esxL-induced NO production regulates non-pathogenic Escherichia coli increased invasion in HeLa
induction of H3K9me2/3 and inhibition of CIITA expression cells (68), whereas expression of M. tuberculosis PE proteins in
M. smegmatis increased its virulence properties (69). Based on
Next, we assessed the role of iNOS/NO during M. smegmatis
this evidence, we expressed M. tuberculosis esxL in an M. smeg-
esxL infection in modulating the expression of H3K9me3 and
matis strain and also deleted esxL from the M. tuberculosis
CIITA. For this, macrophages were infected with M. smegmatis
genome (Mtb⌬esxL) and proved its function using a macro-
pSMT3 and M. smegmatis esxL strains and then treated with an
phage infection model.
iNOS inhibitor, 1400W. Treatment with the 1400W inhibitor
The M. smegmatis genome does not contain esxL ortho-
severely down-regulated the expression of H3K9me2/3 in
M. smegmatis esxL-infected macrophages (Fig. 6F). On the logue; therefore, the observed phenotypes can be attributed to
other hand, inhibition of iNOS led to increased expression of the ectopic expression of esxL in M. smegmatis. Using human
CIITA when compared with untreated conditions (Fig. 6F). and mice macrophage infection models, we showed that
These results confirm the crucial role of NO in repression of recombinant M. smegmatis esxL strain survives more as com-
CIITA by increasing hypermethylation of H3K9. pared with control strains, indicating that EsxL is involved in
bacillary persistence in macrophages. It is well established that
Discussion pathogenic M. tuberculosis facilitates its survival by modulating
M. tuberculosis adopts various strategies to evade the host ROS and NO production (70 –73). We observed that M. smeg-
defense mechanisms to facilitate its survival. One such mecha- matis esxL strain increased NO and iNOS production in
nism involves epigenetic modifications in the host proteins to infected macrophages, whereas inhibition of iNOS decreased
dampen antibacterial effector functions of host cells. In this the intracellular survival of M. smegmatis esxL. On the con-
context, various M. tuberculosis proteins, including ESAT-6, trary, Mtb⌬esxL reduced iNOS expression. The role of NO as
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