Upadhyay et al.

BMC Infectious Diseases (2019) 19:52

https://doi.org/10.1186/s12879-018-3601-z

RESEARCH ARTICLE Open Access

Transcription factors STAT-4, STAT-6 and

CREB regulate Th1/Th2 response in leprosy
patients: effect of M. leprae antigens
Rajni Upadhyay1, Bhavyata Dua1, Bhawna Sharma1, Mohan Natrajan2, Ajai Kumar Jain3,
Balaji Kithiganahalli Narayanaswamy4 and Beenu Joshi1*

Abstract
Background: Leprosy is an ideal human disease to study T cell regulation as patients show correlation between
cytokine skewed Th1-Th2 responses and clinical forms of the disease. The Role of transcription factors on the
modulation of Th1 and Th2 responses by M. leprae antigens has not been adequately studied. In the present study,
we studied the effect of M. leprae antigens on transcription factors STAT-4, STAT-6 and CREB and their correlation
with Th1/Th2 cell mediated immune responses in leprosy.
Methods: Leprosy patients of both categories of tuberculoid leprosy (BT/TT) and lepromatous leprosy (BL/LL) were
selected from the OPD of NJ1L & OMD, (ICMR), Agra and healthy individuals (H) were chosen from the staff and
students working in the institute. Peripheral blood mononuclear cells (PBMCs) of the study subjects were stimulated
with M. leprae antigens (WCL, MLSA, and PGL-1). Sandwich ELISA was done in the culture supernatants of healthy
and leprosy patients to detect IL-4, IL-10 and IFN-γ. Further, expression of IFN-γ and IL-4 and activation of STAT4,
STAT6 and CREB transcription factors in CD4+ T cell with or without stimulation of M. leprae antigens was investigated
by flow cytometry.
Results: Lepromatous leprosy patients showed significantly lower IFN-γ and higher IL-4 levels in culture supernatant
and significantly low expression of IFN-γ and higher expression of IL-4 by CD4+ T cells than healthy individuals with
or without antigenic stimulation. Antigenic stimulation significantly increased IL-10 in BL/LL patients but not in
BT/TT patients or healthy individuals. PGL-1 stimulation led to significantly higher activation of STAT-6 in BT/TT
and BL/LL patients in comparison to healthy individuals. All the three antigens led to activation of CREB in healthy and
BT/TT patients but not in BL/LL patients.
Conclusion: Our findings show that M. leprae antigens differentially modulate activation of T cell transcription factors
STAT-4/STAT-6 and CREB. These transcription factors are well known to regulate Th1 and Th2 mediated immune
response which in turn could play vital role in the clinical manifestations of leprosy. These observations may help to
determine how these T cell transcription factors affect the development of immune dysfunction and whether these
new pathways have a role in immunomodulation in intracellular diseases like leprosy and TB.
Keywords: Leprosy, Th1/Th2 cytokine, STAT-4, STAT-6, CREB

* Correspondence: beenuj2002@yahoo.co.in
1
Department of Immunology, National JALMA Institute for Leprosy and
Other Mycobacterial Diseases (ICMR), Tajganj, Agra 282004, India
Full list of author information is available at the end of the article

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Upadhyay et al. BMC Infectious Diseases (2019) 19:52 Page 2 of 11

Background tissue damage, and autoimmune responses, or they could

The prevalence of leprosy at the end of 2017 was be pathogenic in the context of infection and tumor immu-
192,713 cases worldwide and the number of new cases nosurveillance [6].
reported was 210,671. India still contributes the major Role of transcription factors on the modulation of
percentage of leprosy cases due to continued transmission immune responses in leprosy has not been thoroughly
in the community [1]. The disease shows an interesting studied. Kim et al., have shown IL-12 induced STAT-4
immunological phenomenon wherein the host immunity phosphorylation and DNA binding in M. leprae-activated
to Mycobacterium leprae (M. leprae) dictates the clinical T cells in TT but not in LL patients [7]. Regulatory role of
outcome of the disease. Patients with strong cell mediated CREB in production of IFN-γ is well documented in M.
responses are able to restrict the infection and are tuberculosis infection [8]. Therefore, in the present study
grouped into Tuberculoid type (TT) whereas, patients we have studied M. leprae antigens mediated Th1/Th2
with low cell mediated immunity and high antibody specific T cell transcription factors STAT-4, STAT-6,
response harbor numerous organisms and are categorized and CREB activation and cytokine production in leprosy
as Lepromatous type (LL). Leprosy has been an exten- patients and healthy individuals.
sively studied human bacterial infection in terms of Th1/
Th2 immune responses. T helper (Th) cells are classified Methods
into Th1 and Th2 cells based on the cytokines secreted by Study subjects
them [2]. Th1 cells predominantly secrete proinflamma- Leprosy patients of both the categories of tuberculoid
tory cytokines such as IFN-γ whereas IL-4 and IL-10 cyto- leprosy (TT/BT) (N = 15) and lepromatous leprosy (BL/LL)
kines are secreted by Th2 cells. IFN-γ is a crucial cytokine (N = 9) were selected from the OPD of National JALMA
for protection against mycobacterial infections including 1nstitute for Leprosy & OMD (ICMR), Agra (Age range
leprosy. Th1 type of immune response is characteristic of 19-48 yrs). Patients were diagnosed on the basis of clinical
the tuberculoid form of leprosy; conversely, Th2 type and bacteriological criteria and classified according to the
immune response is dominant in the lepromatous form of immunological scale of Ridley-Jopling [9]. Twelve healthy
leprosy. individuals working in the laboratory were included as
A wide range of well defined transcription factors, in- healthy controls. All the healthy individuals were clinically
cluding signal transducer and activator of transcriptions free from infections at the time of sample collection and
(STATs), T-bet, cyclic AMP (cAMP) responsive element had no history of TB and Leprosy. In addition, they were
binding (CREB) are known to shape the Th1/Th2 differen- not contacts of patients, therefore, it is unlikely that they
tiation. Lineage commitment to Th1/Th2 is now better would be harboring the disease. Ten milliliter peripheral
understood in terms of transcription factors. Inappropri- blood was collected in heparinized vials from all study sub-
ate induction of Th1/Th2 cell plays an important role in jects after taking informed written consent and the study
the outcome of the disease. STAT-4 and STAT-6 play was approved by institutional human ethics committee
important roles in regulating the differentiation of Th cell (Human Ethics Committee meeting of National JALMA
subsets. STAT-4 is an essential component of the IL-12 1nstitute for Leprosy & OMD, Agra dated 21.2.2010).
signaling pathway and plays an important role in Thl
differentiation. Although STAT-4 is expressed both in Th1 Antigens
and Th2 cells, STAT-4 can only be phosphorylated by Mycobacterium leprae soluble antigen (MLSA), Whole
IL-12 in Th1 cells as there is marked down-regulation of cell lysate (WCL) and Phenolic glycolipid-1 (PGL-1) were
IL-12Rβ specifically in Th2 cells [3]. However, little is procured from the laboratory of Dr. John T Belisle, Deptt
known about the exact mechanism by which STAT-4 acti- of Microbiology, Immunology and Pathology, Colorado
vation leads to Th1 differentiation. In contrast to STAT-4, State University, (under WHO Contract Number USA
STAT-6 plays a central role in modulating Th2 differenti- NIH-NO1-AI-25469).
ation. Binding of IL-4 to the IL-4 receptor results in the
phosphorylation and dimerization of STAT-6 [4]. Further- Separation of peripheral blood mononuclear cells
more, CREB, a transcription factor which belongs to the (PBMCs) from blood and antigenic stimulation
family of basic leucine zipper (bZIP), binds to cAMP PBMCs were isolated from buffy coats from healthy
responsive element (CRE) and is essential for T cell donors and leprosy patients (both tuberculoid and
function and cytokine production [5]. CREB plays various lepromatous) by density gradient centrifugation using
roles in immune function including its role in promoting Ficoll hypaque. Cells were incubated in RPMI-1640
anti-inflammatory immune responses through inhibition of supplemented with 5% heat inactivated FBS (Hyclone,
NF-κB activity, the induction of IL-10, and the generation USA), 2 mM L- Glutamine, 100 unit penicillin/ml and
of regulatory T cells. These anti-inflammatory responses 100 μg streptomycin/ml (Sigma, USA) at 37 °C and 5%
could be protective by inhibiting unwanted inflammation, CO2 in a humidified incubator. Cells were stimulated
Upadhyay et al. BMC Infectious Diseases (2019) 19:52 Page 3 of 11

with MLSA (10 μg /ml), WCL (10 μg /ml) and PGL-1 Results
(15 μg/ml) for 24 h. Antigen doses were standardized M. leprae mediated secretion of Th1/Th2 cytokines in the
earlier in our laboratory for a previous study [10]. In culture supernatant of PBMCs of leprosy patients and
brief, standardization of optimum dose of antigen was healthy individuals
done by both MTT and LTT assay using PBMCs of PBMCs of leprosy patients (BT/TT, BL/LL) and healthy
healthy individuals. The optimum dose corresponds to individuals (H) were stimulated with M. leprae antigens
the log phase of the curve generated by these assays. (MLSA, WCL and PGL-1) and culture supernatants were
collected after 48 h and 5 days. Secreted Th1 cytokine
IFN-γ and Th2 cytokines IL-4 and IL-10 were estimated
Sandwich ELISA in the supernatant.
Cytokine estimation was done in the culture supernatant In absence of antigens significantly higher production
of PBMCs of 15BT/TT, 9BL/LL and 12 healthy donors. of IFN-γ (p = 0.027) was observed in culture supernatant
Supernatants were collected from PBMC culture after of healthy individuals (H) as compared to BL/LL patients
48 h for IL-4 and IL-10 estimation and after 5 days for (Fig. 1(I)A). IFN-γ level was higher in response to MLSA,
IFN-γ estimation. Detection of secreted cytokines was WCL and PGL-1 in culture supernatant of healthy indi-
done by commercially available sandwich ELISA kits viduals as compared to lepromatous patients and tubercu-
from R & D systems, Minneapolis, USA. loid patients. However, no significant difference was noted
among any groups in response to all three antigens
Flow cytometric analysis (Fig. 1(I)B).
Effect of M. leprae antigens (MLSA, WCL, and PGL-1) Significantly higher basal concentration of IL-4 (p =
on phosphorylated status of STAT-4, STAT-6 and CREB 0.026) was noted in lepromatous patients (Fig. 1(II)A)
on CD4+ T cells and on frequency of IFN-γ, IL-4 produ- as compared to healthy individuals. MLSA significantly
cing CD4+T was analysed by flow cytometry. In brief 2 × increased production of IL-4 in lepromatous patients
106 cells/ml were stimulated with standard doses of WCL, and healthy individuals as compared to tuberculoid patients
MLSA and PGL-1, few cells were kept without stimula- (p value =0.048, 0.0364 respectively). On other hand,
tion. Plates were incubated for 24 h at 37 °C in 5% CO2 PGL-1 stimulation did not show any significant difference
with humidified air. Six hours before the termination of in IL-4 levels in healthy individuals and leprosy patients,
incubation, cells were treated with monensin (4 μM, but the lowest level of IL-4 was observed in healthy individ-
Sigma, USA). After incubation cells were stained with uals (Fig. 1(II)B).
antibodies for surface markers - anti- human CD3 PE No difference was noted in IL-10 concentration among
Cy5, anti- human CD4 FITC and were incubated for 30 min patients and healthy individuals in unstimulated PBMCs.
in dark at 4 °C. Cells were then washed and fixed with 4% Significant up regulation of IL-10 was observed post
formaldehyde in phosphate buffer saline (PBS, pH -7.4). stimulation with MLSA and WCL in culture supernatant
Cells were permeabilized and staining was done for intracel- of lepromatous patients in comparison to tuberculoid
lular cytokines and transcription factors. Cells were stained patients (p = 0.014 for MLSA, p value = 0.009 for WCL)
with anti-human IL-4 APC, anti human IFN-γ PE Cy7, anti- and healthy individuals (p = 0.003 for MLSA, p = 0.013
human pSTAT-4 PE, anti- human pSTAT-6 Alexa Flour 647 for WCL) (Fig. 1(III)A). Decreased secretion of IL-10
and anti -human pCREB Alexa Flour 647 and incubated for was observed after PGL-1 stimulation in healthy individuals
30 min in dark at 4 °C. All the antibodies for flowcytometry and tuberculoid patients but the change was not significant
were purchased from BD Biosciences, USA. Stained cells as compared to lepromatous patients (Fig. 1(III)B). No
were acquired in BD FACS Aria (BD, San Hose, USA) and difference in IL-10 production was noted in culture super-
the percentage of cells was calculated using FACS Diva natant of healthy individuals and tuberculoid patients after
Software. MLSA and PGL-1 stimulation (Fig. 1(III)B).

M. leprae antigens mediated expression of Th1 and Th2

Statistical analysis cytokines, STAT and CREB transcription factors in CD4+ T
Statistical analysis was done using Prism 3 software cells in leprosy patients and healthy individuals
(Graph pad version 3, LA Jolla, USA). Data was presented Interaction of T cell receptor with MHC/antigen complex
as mean ± SEM. Variation between the groups was cal- leads to activation of naïve T cells and its differentiation to
culated by non parametric Mann Whitney test. P value Th1 or Th2 cell type expressing IFN-γ and IL-4 cytokines
less than 0.05 was considered as significant. Values of respectively. Role of transcription factor STAT-4 in the
stimulated PBMCs were normalised by subtracting the induction of IFN-γ by IL-12 has been suggested whereas
unstimulated values for comparison among different differentiation of Th2 cells is mediated by STAT6.
study subjects. Transcription factor CREB plays diverse role in immune
Upadhyay et al. BMC Infectious Diseases (2019) 19:52 Page 4 of 11

Fig. 1 Cytokine levels in culture supernatant of healthy and leprosy patients. Cytokine levels in culture supernatant of unstimulated (A) M. leprae
antigens stimulated (B) PBMCs of healthy individuals (H), TT/BT and BL/LL patients. PBMCs from the study subjects were cultured with or without
antigens for 5 days in RPMI at 37 °C in 5% humidified atmosphere. Supernatants were collected after 48 h and 5 days of culture and cytokine was
estimated. Concentration of cytokine was normalized after subtracting values of unstimulated supernatant from stimulated supernatant. I = IFNγ,
II = IL-4, III = IL-10. * = p < 0.05, ** = p < 0.001. MLSA- M. leprae soluble antigen, WCL- whole cell lysate, PGL-1- Phenolic glycolipid-1. BT/TT
(Borderline tuberculoid/Tuberculoid), BL/LL (Borderline lepromatous/Lepromatous)

response. It regulates Th1, Th2 and Th17 type of immune tuberculoid leprosy patients also showed significantly
response differentially. Therefore, expression of cytokines higher frequencies of these cells in response to antigenic
IFN-γ, IL-4 and transcription factors STAT-4, STAT-6 and stimulation (p = 0.003 for MLSA, p = 0.0001 for WCL,
CREB on CD4+ T cells with and without stimulation of p < 0.033 for PGL-1) as compared to lepromatous patients
MLSA, WCL and PGL-1 antigen was studied. PBMCs (Fig. 2(II)B).
from leprosy patients and healthy individuals were stimu- Basal mean percentage of IL-4 expressing CD4+ T cells
lated with antigens and stained with fluorochrome conju- was significantly higher (p = 0.0008) in PBMCs of leproma-
gated anti-CD4 antibody to intracellular cytokines. Cells tous leprosy patients as compared to healthy individuals.
were acquired by flow cytometer. Analyzed results were Significantly higher IL-4 expressing CD4+ T cells were
presented as mean percentage of cytokines expressing noted in LL patients after stimulation with MLSA in com-
CD4+ T cells in blood of healthy individuals and leprosy parison to healthy and BT/TT patients whereas significant
patients. difference was observed in the expression of these cells in
only BL/LL and BT/TT patients after stimulation with
Expression of IFN-γ and IL-4 in CD4+T cells WCL and PGL-1(p = 0.0057 for MLSA, p = 0.0133 for
Leprosy patients showed significantly reduced IFN-γ ex- WCL, p = 0.0041 for PGL-1) (Fig. 2(III)A & B).
pressing CD4+ T cells (p = 0.005 for BT/TT, p = 0.0006
for BL/LL) as compared to healthy individuals at basal Expression of phosphorylated STAT-4 and STAT-6 in CD4+T
level (Fig. 2(II)A). These cells were also significantly cells
higher in healthy individuals than lepromatous patients Significantly higher pSTAT-4 expression by CD4+ T cells
in response to antigenic stimulation (p value = 0.002 for was observed in healthy individuals (p = 0.046) and tu-
MLSA, p = 0.0002 for WCL, p < 0.0001 for PGL-1) and berculoid patients (p = 0.042) as compared to lepromatous
Upadhyay et al. BMC Infectious Diseases (2019) 19:52 Page 5 of 11

Fig. 2 IFN-γ and IL-4 expressing CD4+T cells in healthy individuals and leprosy patients. I Representative histogram showing CD4+T cells
expressing IFN-γ and IL-4 in healthy individuals and leprosy patients. II Mean Percentage of IFN-γ and III IL-4 expressing CD4+T cells in healthy
individuals (H) and leprosy patients with or without stimulation with M. leprae antigens. PBMCs of different study subjects were incubated with
medium only (A) or stimulated with MLSA, WCL and PGL-1antigens (B) and incubated for 24 h. After incubation cells were washed and stained with
fluorescent labeled anti-CD3, anti-CD4, anti IFN-γ and anti-IL-4 antibodies and acquired in flow cytometer. *p < 0.05 and **p < 0.001 *** = p < 0.0001.
BT/TT (Borderline tuberculoid/Tuberculoid), BL/LL (Borderline lepromatous/Lepromatous)

patients when no antigen was added. However, no differ- when PBMCs were stimulated with MLSA however,
ence was noted among various groups after antigenic higher pSTAT-6 expression was observed in BT/TT and
stimulation (Fig. 3(II)A & B). BL/LL patients than healthy individuals. After WCL
Mean percentage of pSTAT-6 expressing CD4+ T stimulation, significantly higher expression of pSTAT-6 by
cells was compared among leprosy patients and healthy CD4+ T cells was noted in BT/TT patients (p = 0.0487) as
individuals. Basal pSTAT-6 expression by CD4+ T cells compared to healthy individuals. BL/LL (p = 0.007) and
was significantly higher in BL/LL and BT/TT patients BT/TT (p = 0.0487) patients showed significantly higher
both in comparison to healthy individuals (p = 0.0175, expression of STAT-6 after PGL-1 stimulation when com-
0.035 respectively) (Fig. 3(III)A). Further, no significant pared to healthy individuals (Fig. 3(III)B).
difference in pSTAT-6 expression by CD4+ T cells was Ratio of mean percentages of basal and antigen mediated
noted in healthy individuals, BL/LL and BT/TT patients IFN-γ and IL-4 expressing CD4+ T cells was significantly
Upadhyay et al. BMC Infectious Diseases (2019) 19:52 Page 6 of 11

Fig. 3 Phosphorylated STAT-4 and STAT-6 expressing CD4+T cells in healthy individuals and leprosy patients. I Representative histogram showing
CD4 + T cells expressing STAT-4 and STAT-6 in healthy individuals and leprosy patients. II Mean Percentage of pSTAT-4 and III pSTAT-6 expressing
CD4+T cells in healthy individuals (H) and leprosy patients with or without stimulation with M. leprae antigens. PBMCs of different study subjects
were incubated with medium only (A) or stimulated with MLSA, WCL and PGL-1antigens (B) and incubated for 24 h. After incubation cells were
washed and stained with fluorescent labeled anti-CD3, anti-CD4, anti pSTAT-4 and anti pSTAT-6 antibodies and acquired in flow cytometer. * = p < 0.05.
BT/TT (Borderline tuberculoid/Tuberculoid), BL/LL (Borderline lepromatous/Lepromatous)

higher in healthy and BT/TT patients than BL/LL patients also analyzed after antigenic stimulation. Basal expression
(Fig. 4(I)A & B). However, significantly higher ratio of of phosphorylated CREB by CD4+ T cell was significantly
STAT4/STAT6 expressing CD4+ T cells was noted in un- higher (p = 0.0039) in PBMCs of healthy individuals
stimulated PBMCs in healthy individuals only as compared and BT/TT patients as compared to BL/LL patients
to BL/LL patients and to both BT/TT and BL/LL patients (Fig. 5(II)A). Higher expression of pCREB by CD4+ T cells
in response to MLSA (Fig. 4(II)A & B) was also noted in healthy individuals and BT/TT patients
as compared to lepromatous patients after MLSA, WCL
Activation of CREB in CD4+T cells and PGL-1 stimulation. However, significant difference
Expression of activated transcription factor CREB which was noted in healthy individuals and BL/LL patients only
regulates proliferation and differentiation of T cells was with PGL-1 (p = 0.046) stimulation (Fig. 5(II)B).
Upadhyay et al. BMC Infectious Diseases (2019) 19:52 Page 7 of 11

Fig. 4 Ratio of IFN-γ/IL-4, pSTAT4/pSTAT6 expressing CD4+T cells in healthy individuals and leprosy patients. Ratio of IFN-γ/IL-4 (I), pSTAT4/pSTAT6
(II) expressing CD4+T cells in healthy individuals (H) and leprosy patients with or without stimulation with M. leprae antigens. * = p < 0.05 and ** = p < 0.001.
BT/TT (Borderline tuberculoid/Tuberculoid), BL/LL (Borderline lepromatous/Lepromatous)

Discussion patients than healthy individuals and tuberculoid patients.

In spite of several investigations, the mechanisms of M. After MLSA and WCL stimulation, levels of IL-4 were
leprae specific T cell anergy in LL patients are not well lower in tuberculoid patients than healthy individuals and
understood. This study has been done to correlate BL/LL patients which is intriguing. However, it could sug-
differential Th1/Th2 cell mediated immune responses gest higher Th1 response in tuberculoid type of patients
observed in leprosy with known T cell transcription fac- which may be due to the presence of well developed cell
tors (STAT-4, STAT-6 and CREB) ex vivo along with the mediated immunity in tuberculoid patients that is directly
effect of M. leprae antigens (MLSA, WCL and PGL-1) related with T cell activation and differentiation. Highest
on the modulation of these transcription factors. production in IL-4 after PGL-1 stimulation is due to Th2
Detection of Th1 cytokine IFN-γ and Th2 cytokines response exhibited by lepromatous patients as well as
IL-4 and IL-10 in the PBMCs confirmed Th1/Th2 immunosuppressive nature of PGL-1. These findings show
polarization of immune response in leprosy patients. that PGL-1 might be responsible in inducing Th2 re-
Significantly lower basal concentration of IFN-γ was sponse which lowers Th1 response and help in bacterial
observed in culture supernatant of BL/LL patients than growth which is seen in LL patients.
healthy individuals. When IFN-γ production to different Expression of IFN-γ, IL-4 by CD4+T cells with and
antigens was compared, no difference in the cytokine level without stimulation of M. leprae antigen was also studied
was noted among all subject categories. This could be due by flow cytometry in order to confirm observations noted
to different cell populations namely, CD4+T cells, NK cells by ELISA. Similar findings were noted in the case of both
and CD8+T cells secreting the cytokines in the medium. It IFN-γ and IL-4 secreting CD4+T cells. Even more signifi-
was also noticed that very low level of IFN-γ was pro- cance is found in flow cytometry data as compared to
duced in response to PGL-1 in healthy individuals and in ELISA results because of its higher sensitivity and T cell
both types of leprosy patients. These observations confirm specific cytokine secretion. Basal IFN-γ level was signifi-
low immunogenicity of PGL-1. It also shows that IFN-γ cantly higher in healthy individuals than leprosy patients.
might be helping tuberculoid patients to restrict the M. However, all the three antigens induced increase in IFN-γ
leprae growth as IFN-γ was significantly higher in BT/TT expressing CD4+T cells in healthy individuals and tuber-
than BL/LL patients after stimulation with M. leprae culoid leprosy patients than in LL patients which is in
antigens. Earlier similar observations were reported in contrast to findings observed by ELISA. In the culture
studies done by Misra et al. [11]; Dockrell et al. [12] supernatant several cells like CD4+T cells, NK cells and
and Weir et al. [13]. CD8+T cells are the source for IFNγ production whereas
Significantly higher basal level of Th2 cytokines IL-4 in flow cytometry we have targeted specific cells, namely
was observed in culture supernatant of lepromatous CD4+T cells hence significant antigen specific response
Upadhyay et al. BMC Infectious Diseases (2019) 19:52 Page 8 of 11

Fig. 5 Phosphorylated CREB expressing CD4+T cells in healthy individuals and leprosy patients. I Representative histogram showing CD4 + T cells
expressing CREB in healthy individuals and leprosy patients. II Mean Percentage of pCREB expressing CD4+T cells in healthy individuals (H) and
leprosy patients with or without stimulation with M. leprae antigens. PBMCs of different study subjects were incubated with medium only (A) or
stimulated with MLSA, WCL and PGL-1antigens (B) and incubated for 24 h. After incubation cells were washed and stained with fluorescent
labeled anti-CD3, anti-CD4, anti-pCREB and acquired in flow cytometer. * = p < 0.05 and ** = p < 0.001. BT/TT (Borderline tuberculoid/Tuberculoid),
BL/LL (Borderline lepromatous/Lepromatous)

was observed. On the contrary, IL-4 expressing CD4+T programme in macrophages but not triggering anti-
cells showed significantly higher mean percentage of these microbial pathway in leprosy has been earlier reported.
cells in BL/LL patients than healthy individuals and This leads to survival of mycobacteria leading to extensive
BT/TT patients. The study confirms Th2 type of disease in LL [17, 18]. Inhibition of M. leprae specific T cell
response in the lepromatous pole and intact CMI showing proliferation by IL-10 has been reported [19]. Boussiotis et
Th1 response (high IFN-γ and low IL-4) in TT/BT to M. al., 2000 also reported that increased susceptibility to
leprae antigens. All of these results are in concordance mycobacterial infection is linked with secretion of IL-10 by
with the findings of Misra et al. [11]; Dockrell et al. [12] T cells [20].
and Weir et al. [13] which have shown strong Th1 T cell transcription factors regulate specific immune
response in tuberculoid patients and Th2 response and T response against pathogen after infection, hence we
cell unresponsiveness in lepromatous patients to M. leprae further studied the activation of different transcription
antigens. Dockrell et al., 1996 showed strong lymphopro- factors STAT-4, STAT-6 after stimulation with M. leprae
liferative response and IFN-γ secretion in response to antigens. Role of these factors have not been studied
fractionated cell wall or cytosol and membrane proteins of widely in leprosy. Activation of STAT-4 is critical for
M. leprae in tuberculoid leprosy patients [12]. Our results Th1 differentiation and plays an important role in IL-12
are also in favor of other two studies done by our group signaling pathway. IL-12 receptor is composed of two
earlier which states suppressive nature of M. leprae β-like chains, IL-12Rβ1 and IL-12Rβ2. IL-12Rβ2 is
antigens MLSA, WCL and PGL-1 respectively in Jurkat selectively expressed on Th1 but not Th2 cells. IL-12Rβ2
T cells [10, 14]. production and expression is limited to activated T-cells
As IL-10 can directly inhibit Th1 and Th2 cytokine which respond to IL-12 by secreting IFN- γ and is
production at the level of T cell [15, 16], this cytokine down-regulated by IL-4 and IL-10 [21]. In case of infection
was also quantified in culture supernatants. All of three with intracellular pathogen development toward the Th1
antigens induced an increase in IL-10 in BL/LL patients subset is initiated by stimulation with IL-12 and IFN-γ,
which was significantly higher than healthy individuals which are secreted by dendritic cells and macrophages.
and BT/TT patients especially after MLSA and PGL-1 This Th1 differentiation is thus linked to activation of the
stimulation. Role of IL-10 in induction of phagocytic transcription factors STAT-1 and STAT-4 downstream of
Upadhyay et al. BMC Infectious Diseases (2019) 19:52 Page 9 of 11

IFN-γ and IL-12 signaling, respectively. Together with the of STAT-6 by CD4+T cells in BT/TT patients as compared
transcription factors such as nuclear factor of activated T to healthy individuals after WCL stimulation and in both
cell (NFAT), adaptor related protein complex 1 (AP-1) and BL/LL and BT/TT patients as compared to healthy indi-
nuclear factor κB (NFκB)] that are activated by TCR en- viduals in response to PGL-1 also pinpoints role of
gagement, STAT1 induces the expression of the master STAT-6 in favoring of Th2 response and hence establish-
transcriptional factor of the Th1 subset, T-bet. Subse- ing infection. These findings also confirm role of PGL-1 in
quently, STAT-4 and T-bet act coordinately to produce the immunosuppression seen in leprosy by evoking Th2
large amounts of IFN-γ production in Th1 cells [22]. response through STAT-6 pathway.
Higher basal levels of STAT-4 in healthy and BT/TT CREB is a transcription factor that regulates diverse
individuals in our study correlated with significant Th1 cellular responses, including proliferation and differenti-
response in these individuals and may be responsible for ation of T cells [29]. Liu et al., 2010 showed that CREB
their protective immunity which ultimately prevents the could promote the transcription and production of IFN-γ
infection in healthy and restricts the same in BT/TT through binding with the IFN-γ proximal promoter [8].
patients. Antigen specific activation of STAT-4 was not The proximal IFN-γ promoter contains CRE-like sequences
noted in our study which suggests that pathway other than (ACGT) where CREB binds and regulates IFN-γ transcrip-
STAT-4 could be involved in IFNγ secretion in response to tion. Although some studies on Jurkat T cells and trans-
M. leprae antigens. genic mice suggest that CREB proteins inhibit the
Expression and up-regulation of IL-12Rβ2 in leprosy transcription of IFN-γ [30, 31], positive regulation by
patients which is correlated with cell mediated immunity CREB of IFN-γ production by M. tuberculosis respon-
has been reported earlier by Kim et al. [7]. They reported sive human T cells has been shown [32]. Reduced
IL-12 induced STAT-4 phosphorylation in tuberculoid but amounts of CREB binding to the IFN-γ proximal
not in lepromatous patients after M. leprae stimulation. promoter, and absence or diminished expression of
This is due to inability of the lepromatous patients to phosphorylated CREB was reported in tuberculosis
mount an appropriate Th response to M. leprae, which patients which in turn was responsible for reduced
was also noted in our study showing low basal level of IFN-γ production in TB patients [8, 32]. We also
STAT4 activation. Watford et al., 2008 has identified one observed similar finding in case of leprosy patients
new STAT-4 target Map3K8 that has a rather different showing significantly higher basal expression of CREB
function [23]. Map3K8 is an upstream activator of ERK, in healthy individuals and BT/TT patients than BL/LL
which is inducible by IL-12 and T cell receptor-dependent patients. It is inferred from this observation that cross
signals. Chromatin immunoprecipitation assays have regulatory pathways may be involved in the expression
revealed that STAT-4 directly binds the Map3k8 gene. of IFN-γ in leprosy patients as BT patients are able to
Deficiency of Map3k8 in T cells interferes with IFN-γ restrict the M. leprae infection. CREB activation may
production. Our group had earlier shown the inhibitory be directly linked to Th 1 cells differentiation. Induc-
effects of M. leprae antigens on phosphorylation of tion of CREB after M. leprae antigens stimulation also
MAPKs [10, 24]. Hence it is possible that M. leprae ac- supports this fact.
tivates STAT-4 which leads to phosphorylation of
MAPKs which is responsible for cytokine expression Conclusion
thereby killing of M. leprae. Deficiency of activation of Our study is an effort to investigate the correlation of T
STAT-4 or MAPKs may lead to low IFN-γ which we have cell polarization observed in leprosy with the expression
also shown, thereby leading to bacterial survival in LL. of transcription factors in a few leprosy patients. This
STAT-6 is another important Th2 specific transcrip- study shows differential expression of T cell transcription
tion factor. Binding of IL-4 receptor to IL-4 induces the factor STAT-4, STAT-6 and CREB in leprosy patients and
phosphorylation of STAT-6 which further activates healthy individuals correlating with Th1 and Th2 cytokine
GATA-3. GATA-3 in return enhances the expression of expression. Regulation of cell mediated immune response
Th2 cytokines IL-4, IL-5, IL-10 and IL-13 [25]. STAT-6 through transcription factors could play an important role
is not only implicated in the initiation of Th2 differenti- in the clinical manifestation of leprosy. More detailed
ation, but it also contributes to maintenance of the Th2 studies of expression profile of different transcription fac-
phenotype [4].GATA-3 also inhibits Stat4 transcription tor on large number of patients are needed to decipher
[26, 27] and directly represses Ifn-γ [28]. In our study we the in vivo regulatory mechanism of T cell differentiation.
noted higher STAT-6 expression in unstimulated PBMCs These observations may provide new tools to study and
in patients in comparison to healthy individuals which monitor patients, to determine how these T cell transcrip-
correlated with higher Th1 response in healthy individuals tion factors affect the development of immune dysfunc-
and suggests role of this transcription factor in the devel- tion, and to study new pathways to block suppressor
opment of disease. Further, significantly higher expression mechanisms. The findings may also help in better
Upadhyay et al. BMC Infectious Diseases (2019) 19:52 Page 10 of 11

understanding of defects in cell mediated immunity in Received: 13 April 2018 Accepted: 7 December 2018
leprosy as well as other intracellular infections such as tu-
berculosis and leishmaniasis.
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