Outline of the Proposed Topic of Research

Name of Candidate : ***********

ID No. : ***********

Place of Research Work and Organisation : Institute of ********, New Delhi

Proposed Supervisor’s Details

Name : ***************

Qualification : M.Sc., Ph.D

Designation : ***************

Organization : Institute of *********, New Delhi

Proposed Topic of Research

Role of Antigen Presenting Cells (APCs) and Toll like Receptors in Providing a Protective

Immune Response during Chlamydia trachomatis Infection

Objective of the Proposed Research

1. In vitro study of processing and presentation of chlamydial antigens by Dendritic cells

(DC’s) and monocytes/macrophages to CD8+ and CD4+ T lymphocytes.

2. Differential regulation of cytokine production by CD8+ and CD4+ T lymphocytes.

3. Role of Toll like receptors 2 and 4 in recognition of Chlamydial antigens and regulation

of cytokine production.

4. Regulation of nitric oxide production by chlamydial antigens.

Background of the Proposed Research

Introduction

Worldwide, an estimated 90 million sexually transmitted Chlamydia trachomatis infections

occur each year. Sexually transmitted C. trachomatis infection is an important public health

concern because of its adverse effects on reproduction [1]. In India alone a high chlamydial

prevalence rate (28%) was found in symptomatic patients [2]. In women, infection with C.

trachomatis causes pelvic inflammatory disease (PID) and has long term consequences – such as

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infertility, ectopic pregnancy and chronic pelvic pain- that are secondary to scarring of the

fallopian tubes (caused by salpingitis) and ovaries. In addition, infection with C. trachomatis

felicitates the transmission of HIV [3] and might be a co factor in human papilloma virus (HPV)-

induced cervical neoplasia [4-5]. The pathological mechanism by which C. trachomatis induces

scarring is not well understood. In all cases the pathology seems to be related to a chronic

inflammation caused by a persistent chlamydial infection or by repeated infections with the

bacterium.

After initial infection of the host with C. trachomatis, Dendritic cells (DC) are the first

professional antigen presenting cells (APC’s) encountering the bacteria. DC’s are present in the

epithelium of cervix and vagina [6] and they prime the T cells to modulate the type of T-cell

responses (Th1/Th2), contributing to the inflammatory response which largely depends on the

up-regulation of co-stimulatory and adhesion molecules and on secretion of inflammatory

cytokines [7-9]. Protection against chlamydial infection has been shown to be primarily mediated

by IFN- producing T-cells [10,11] and it has been shown that DC can process and present

chlamydial antigens to T cells [12-14]. Little is known, however, about the interaction between

human DC and Chlamydia species.

Review

Chlamydia trachomatis is an obligate intracellular gram –ve bacterium which causes a wide

spectrum of human diseases. C. trachomatis infection of genital tract is now recognized as one of

the most common major cause of sexually transmitted diseases in developed countries [1].

Without treatment the chlamydial genital infections can cause pelvic inflammatory disease (PID)

and its sequelae of ectopic pregnancy and infertility.

The chlamydial developmental cycle involves a metabolically inactive non replicating infectious

form called elementary body (EB), that, after entry into the host cells differentiate into

metabolically active Reticulate Body (RB). The organism infects the epithelial cells often

inducing an acute inflammatory response, caused by persistent chlamydial infections, or by

repeated infections with the bacterium, however, protective immunity is limited.

C. trachomatis infection remains sub clinical in a high proportion of infected individuals (70-

90% of women and 30-50% of men). Clinical symptoms if present include dysuria, abnormal

vaginal discharge and lower abdominal pain. Infection ascends the endometrial epithelium to the

fallopian tubes, where C. trachomatis can establish persistent infection and can cause PID.

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Overall, 11% of women with PID develop tubal factor infertility and 9% develop ectopic

pregnancies.

Both humoral and cellular responses can be readily detected in patients suffering from C.

trachomatis infection. Since infection is intracellular, neutralizing antibodies have little

relevance in resolving infection [10]. An early epidemiological observation suggested an inverse

correlation between the amount of IgA in cervical secretions and the amount of C. trachomatis

recovered from the cervix of infected women [15]. In vitro, antibodies specific for C.

trachomatis can neutralize infection in tissue culture [16], however, in humans high titers of C.

trachomatis specific antibodies do not correlate with resolution of infection and , in fact, more

strongly correlated with increased severity of sequelae of infection, such as tubal infertility [17].

In contrast, there is good evidence that T-cell mediated immune (CMI) responses play a major

role in clearance and resolution of chlamydial infections and T-cell responses are critical in host

resistance to C. trachomatis. Transfer of T lymphocytes into naïve mice have been shown to

protect the mice against C. trachomatis infection [18].

After infection of host with C. trachomatis, dendritic cells (DC) and macrophages/monocytes are

the main antigen presenting cells (APC’s) encountering the bacteria. DC’s are key players in

immunity that dictate the type of immune response generated to a particular antigen. DC’s are

professional antigen presenting cells that have extraordinary capacity to stimulate naive T-cell

and initiate primary immune response. Immature DC present in the epithelium of cervix and

vagina capture microbial antigens, process them and present them to T lymphocytes. Mature

DC’s are highly effective at presenting antigen and priming protective adaptive immune

responses.

TLR’s comprise a family of cell surface receptors that recognize pathogen associated molecular

patterns (PAMP’s), including lipopolysaccharide (LPS) and hypomethylated CpG- rich DNA as

well as double stranded and single stranded RNA. Toll like receptors detect microbial infection

and have an essential role in the induction innate and adaptive immune responses [19]. Of the 10

cloned mammalian TLRs, TLR2 and TLR4 are the best characterized with respect to innate

responses to bacteria. TLR4, in association with accessory molecules MD-2 and CD 14, is the

signal transduction receptor for gram- negative bacterial lipopolysaccharide and heat shock

proteins. A broader range of microbial products activate immune responses through engagement

of TLR2, including peptidoglycan from gram-positive bacteria, bacterial lipopeptides, and

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zymosan. A recent hypothesis states that differential expression and engagement of TLR family

members at the surface of dendritic cells and macrophages influences the type of immune

response that is induced by a microbial pathogen. Infection with Chlamydia muridarum has been

shown to stimulate DC’s to produce IL-12 (TH1 type response) [20]. It is not confirmed which

particular TLR’s expressed by dendritic cells are engaged by Chlamydia spp., TLR2 might have

an important role in the activation of DC’s by C. pneumoniae [21]. Furthermore, signaling

through TLR2 , but not TLR4, is associated with increase fallopian tube pathology in C.

muridarum infected mice [22], indicating that engagement of TLR2 is a potential common

pathway in both the immunity and immunopathology induced by Chlamydia spp. Given the high

level of expression of TLR’s by DC’s and there ability to polarize immune responses, the

identification of the role of DC’s in Chlamydia specific immune responses is crucial for

understanding the type of immune response that is elicited and therefore also for designing a

vaccine against infection with Chlamydia trachomatis [23].

Studies in the animal models have clearly established that T cells have a crucial role in the

resolution of infection with Chlamydia spp [24]. Nude mice cannot control infection and

adoptive transfer of Chlamydia specific CD4+ or CD8+ cells allow these mice to successfully

control infections. Specifically protection seems to be mediated by CD4+ T cells that produce

IFN- [25]. The role and effector mechanisms of Chlamydia positive CD8+ T cells are less clear.

MHC class I peptide presentation to CD8+ T cells is not essential for clearance of infection with

Chlamydia spp. In some situation CD8+ T cells might be important in elimination of cells

infected with Chlamydia spp. [26]. Also adoptive transfer of CD8+ T cell lines specific for

serovar L2 of C. trachomatis protected mice against infection with C. trachomatis through a

mechanism involving the production of IFN- [27]. Thus to establish the real role played by

CD8+ T cells in C. trachomatis infection further studies are required.

Stimulated T cells produce a variety of cytokines for clearance of Chlamydial infection including

IFN- and IL-12. Most of the cytokines secreted by T cells and macrophages are T helper1 (TH1)

cytokines, which have a role in polarizing the immune response to Chlamydia spp towards a

protective TH1 type response. By contrast, cytokines such as TNF, IL-1 and IL-10 might be

involved in the pathology associated with infection with Chlamydia spp. IFN-, the main TH1

type cytokine is essential for the clearance of Chlamydial infections from genital tract. It controls

the in vitro growth of C. trachomatis through inducing production of enzyme indoleamine-2,3-

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dioxygenase (IDO) [28]. Activation of IDO by IFN- leads to degradation of tryptophan and

lack of this essential amino acid causes the death of C. trachomatis through tryptophan starvation

[28]. Additional immune effector mechanisms induced by IFN- include induction of nitric

oxide production, which inhibits growth of C. muridarum [29] and the promotion of TH1 type

protective immune response, which downregulate non protective TH2 type responses, thereby,

promoting persistent infection [30]. Persistent infection might induce the secretion of

proinflammatory cytokines, leading to chronic inflammatory cellular response and tissue damage

[31, 32]. Overall, these data show that Chlamydia specific CD4+ TH1 cells and to a more limited

extent CD8+ T cells are required to control C. muridarum infection in genital tract of mice [33].

Observations from humans infected with C. trachomatis indicate that similar immune effector

mechanisms occur in humans [33].

Gap in Existing research

More than two-third of the Chlamydial infections cases occur in the developing countries, where

diagnostic and treatment services are almost absent. An estimated 15 million new cases are

occurring in Africa and 45 million new cases in Southern Asia every year [34]. Because of its

effect on reproduction, programmes to control C. trachomatis have been implemented in many

developed countries but many regions are now showing an increase in the number of infected

individuals [35]. Since current programmes for the control of C. trachomatis are not affordable

for much of the developing countries, vaccine development have been identified as an essential

to controlling infection with C. trachomatis. Mouse models of chlamydial infections with C.

muridarum have provided information on the immune mechanisms of clearance of infection and

resistance to reinfection, but there are several important differences between C. muridarum and

C. trachomatis that might effect the immunobiology of infection. Firstly, C. trachomatis

infection in humans is much more prolonged than C. muridarum infection in mice [36].

Secondly, immune-evasion mechanisms also differ. These differences limit the direct

extrapolation of findings from C. muridarum infection to C. trachomatis infection. Thus, a better

definition of human immune response correlates with C. trachomatis protective immunity and

disease pathogenesis needs to remain an important research priority if we are to develop a

vaccine against C. trachomatis infection that has protective and not deleterious effects.

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Methodology

For carrying out the proposed research work facilities such as tissue culture room equipped with

bio safety hood and incubator, thermocyclers for Polymerase Chain reaction, cell sorter, flow

cytometer, confocal microscope and computers for analysis and storage of data are required

which are available at the Institute of Pathology.

Phase 1:

This phase will comprise of Literature survey.

Phase 2:

(A) Enrollment of patients:

Symptomatic female patients attending the Gynecology Out Patient Department of Safdarjung

Hospital, New Delhi, and having complaints of cervical/ vaginal discharge, abdominal pain,

dysuria or infertility will be enrolled. Cervical lavage samples and peripheral blood will be

collected from these women. Diagnosis for Chlamydia trachomatis and other STD pathogen will

be done using standard methodology.

(B) Flow Cytometry:

Quantification of different T-cell subsets, monocytes, dendritic cells and expression of TLR’s on

the surface of dendritic cells and monocytes will be performed by standard flow cytometry

methodology.

Phase 3: Culture of Monocyte derived Dendritic Cells (MDDC)

Peripheral blood mononuclear cells (PBMC’s) will be purified on Ficoll gradients and will be

washed three times with RPMI 1640. They will be then stained with CD14 microbeads and will

be separated using a cell sorter. These CD14 positive cells will be inoculated (1-2 x 10 6/ml) into

each well of a six well plate and will be cultured in RPMI 1640 supplemented with 10% heat

inactivated Fetal Calf Serum, 2mM L-glutamine, 25mM HEPES, 0.02M 2-mercaptoethanol,

10g/ml Gentamycin and 2g/ml Amphotericin B at 370c in a 5% CO2 incubator, in the

presence of 50 ng/ml Granulocyte Macrophage Colony Stimulating factor and 20 ng/ml IL-4 for

6-7 days. After 6-7 days immature MDDC’s will be washed and analyzed for CD14 and CD1a

expression (marker for immature DC’s)

Phase 4: Coculture of Dendritic cells with CD8 and CD4 T lymphocytes

Immature Dendritic cells and Monocytes will be infected with live C. trachomatis EB’s and

cultured in RPMI 1640 for 4 days at 370C. Autologous CD4+ and CD8+ T cells will be separated

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using a cell sorter and will be cocultured with the EB pulsed dendritic cells and monocytes for

further 4 days in the presence of recombinant IL-2 for generation of T-cell clones. The culture

supernatants will be then analyzed for production of various cytokines.

Phase 5:

(A) Blocking of Toll like receptors on dendritic cells and monocytes

Toll like receptors 2 and 4 present on the surface of Dendritic cells and monocytes will be

blocked with the help of blocking peptides before pulsing them with chlamydial EB’s and will

then be cocultured with CD8+ and CD4+ T cells. The culture supernatants will be again analyzed

for production of various cytokines. Changes in the gene expression patterns of Toll like

receptors, upon infection with Chlamydial EB’s will be done with RT-PCR (reverse transcriptase

Polymerase chain reaction).

(B) Regulation of nitric oxide production by chlamydial antigens

Nitric oxide production by antigen presenting cells and T cells will be analyzed in the culture

supernatants and its regulation by IFN- and Toll like receptors will be studied.

Phase 6: Conclusion and Thesis writing

Results will be concluded with the help of the data, which we obtained throughout our research

work and will be compiled in thesis.

Work Plan :

0 6 12 18 24 30 36 42 48

Duration in Months

7

PHASE 6

PHASE 5

PHASE 4

PHASE 3

PHASE 2

PHASE 1

PHASE 4

PHASE 1

ACTIVITY

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