Mechanism of regu[atiatl; of:A(jtri£ O~tfe S!j1ttfiase -2 f7'{OS2) in Leishmania {ono·van£ infected Macropfiages invo[ves owen sensing transcription factor fi!lPQ~a induciGfe factor 1

Chapter 2 -loG Introduction

Leishmania is among the intracellular microorganisms, which resides in

macrophages. For the clearance of the parasite from the host macrophages Th-l immune

response is required which is being evoked by IFN-y induction by activated T cells.

Parasites are usually sensitive to the macrophage cytotoxic mechanism mediated by nitric

oxide (NO). The NO is produced by the inducible form of nitric oxide synthase (iNOS/

NOS2) enzyme in macrophages (Reiner and Locksley, 1995). The expression ofNOS2 is

stimulated by various cytokines such as TNF-a (Reimann et ai., 1994), IL-6 (Hirohashi

et ai., 1996), IL-18 (Kim et ai., 2000), IL-12 (Kato et ai., 1997) and IFN-y (Munder et

al., 1998). Amongst these cytokines IFN-y has been shown to be critically implicated in

NO induction during LD infection. The physiological relevance ofIFN-y as an inducer of

NOS2 is reported by showing impaired production of NO by macrophages from IFN-y or

IFN-y receptor deficient mice (Daltaon et ai., 1993; Kamijo et ai., 1993). However,

numerous cytokines and microbial products enhance NO induction often acting in

synergistic pairs such as IFN-y and LPS (Xie et ai., 1992)), IFN-y and TNF-a (Deng et

ai., 1993). There are several examples of induced expression of inducible nitric oxide

synthase in macrophages following prior activation by a variety of immunological stimuli

such as interferon y (IFN-y), tumor necrosis factor a (TNF-a), and bacterial

lipopolysaccharide (LPS) and subsequent infection by leishmania (Hibbs et ai., 1988;

Nathan and Xie, 1994). Surprisingly, there are no reports regarding the expression of

NOS2 in the resting macro phages by Leishmania infection, which is a likely scenario

during initial stage of infection in vivo.

The NOS2 synthesis by the cytokines is mainly regulated at the transcriptional

level. The promoter region of the NOS2 gene contains several transcription factor

consensus binding sites (Nathan et al., 1997). The list of participating transcription

factors includes NF-lCB, AP-l, the signal transducer and activator of transcription

(STAT)-1 a, interferon regulatory factor-l (IRF -1), nuclear factor interleukin-6 (NF -IL-6)

and the high-mobility group-I (Y) protein (MacMicking et a/., 1997; Kleinert et a/.,

1998; Dlaska and Weiss, 1999; Pellacani et ai., 2001; Ganster et ai., 2001). Depending

on the cytokine or microbial stimulus and the cell type, different upstream signaling

pathways were shown to promote (for example, Janus kinases Jakl, Jak2 and tyk2; Raf-l

protein kinase; mitogen-activated protein kinases p38, Erk1l2 and JNK; protein kinase C;

52

Chapter 2 -protein phosphatases 1 and 2A) or inhibit (for example, phosphoinositide-3-kinase,

protein tyrosine phosphatases) NOS2 expression (Bogdan, 2000; Karaghiosoff et al.,

2000; Chakravortty et ai.) 2001; Chan et al., 2001; Kristof et ai., 2001). NO itself exerts a

biphasic effect on the transcription ofNOS2. Low concentrations of NO (such as occur at

the onset of macrophage stimulation by cytokines) activate NF-KB and upregulate NOS2

(positive feedback). High concentrations have the opposite effect, which may help

prevent NO overproduction (Umansky et al. 1998; Connelly et al., 2001).

Amongst the several putative trans-acting factor consensus binding sites in 5'­

flanking region of NOS2 gene, hypoxia response element (HRE) is effective by the

binding of the factor hypoxia inducible factor-l (HIF-l) and it has recently implicated as

a critical mechanism in the defence of macrophages by another intracellular pathogen

Salmonella (Kol and Harry, 2005). It has also been shown that HIF-l binds to the HRE

of NOS2 gene in response to hypoxia or iron depletion (Melillo et ai., 1997). HIF -1 is a

heterodimer of two basic-helix-Ioop-helix PAS proteins (HIF-la and HIF-IP). The P

subunit is constitutively expressed while the regulatory alpha subunit (HIF-la) is

degraded under normoxic conditions but stabilized under hypoxic conditions. This

heterodimer is able to bind to the targeted DNA sequence to initiate the gene

transcription (Semenza and Wang, 1992; Wang et ai.) 1995). During hypoxia

stabilization of HIF -1 occurs through the inhibition of 4-prolyl hydroxylase activity, an

enzyme that requires molecular oxygen to be functional (Epstein et ai., 2001). This

enzyme has been proposed to be a critical oxygen sensor and regulator of the HIF-l

transcriptional response to low oxygen conditions. Hydroxylation of HIF -1 a decreases

protein stability under normoxic conditions (Ivan et aI., 2001; Jaakkola et ai., 2001). A

conserved proline residue, proline 564, is hydroxylated under normoxia, allowing the von

Hippel-Lindau (VHL) E3 ubiquitin ligase complex to bind HIF-la (Maxwell et a., 1999;

Ohh et ai., 2000). The VHL complex adds ubiquitin to HIF-Ia, which is then degraded

by the proteasomal pathway. Under hypoxic conditions, it was shown that 4-prolyl

hydroxylase activity decreases because of lack of oxygen, resulting in diminished proline

564 hydroxylation and HIF-la protein stabilization (Maxwell et ai., 1999; Ohh et ai.,

2000). Interestingly, it has been reported that interferon-y-induced NOS2 transcription is

further increased by hypoxia in a macrophages cell line RAW 264.7 (Melillo et ai.,

1995). It has also been shown that NOS2 induction by cytokines is modulated by oxygen

tension (Kacimi et al.) 1997; Jung et ai., 2000). Also, transient transfection experiments

53

Chapter 2

revealed that for the murine NOS2 gene, HIF-l is essential for the increased promoter

activity in cardiac myocytes exposed to hypoxia, as mutation or deletion of the HIF-l

binding site abolished hypoxic induction of the NOS2 promoter activity (Jung et al.,

2000). To test that whether there is any IFN-y independent NOS2 induction during

Leishmanial infection we found that there was significant increase in the nitrite level as

well as NOS2 expression without using IFN-y or any prior stimulus in the host

macrophages. In the current study we tested the possibility of IFN-y independent NOS2

induction in macrophages during the infection of Leishmania donovani. We found that

there is significant increase in the NOS2 induction as well as resultant level of nitrite

without IFN-y or any prior stimulus of the host macrophages. To understand the

mechanism we have performed functional studies on the murine NOS2 5'-flanking

region to characterize the cis-acting element(s) responsible for the activation of IFN-y

independent NOS2 transcription by Leishmania donovani. The HIF-l binding to HRE

site in the murine NOS2 gene has been found to be required for LD-infected NOS2

expression in J774A.l macrophage cell line in our study. This study is the first

demonstration of cytokine independent expression of NOS2 expression in host

macrophages during LD infection.

2.0 Materials and Methods

2.1 Reagents and antibodies

Reagents used were obtained from Sigma Chemical Co. (St. Louis, MO) unless

indicated otherwise. Antibody used against NOS2 was from Cayman Chemicals.

Horseradish peroxidase-conjugated anti-rabbit IgG antibodies were obtained from Bio­

Rad (Hercules, CA). L Y 294002 was from Sigma Chemical Co. (St. Louis, MO).

2.2 Parasite culture

Leishmania donovani AG83 (MHOMlINI1983/AG83) promastigotes were

cultured at 22°C in modified M199 medium (Sigma, St. Louis, MO) supplemented with

100 units/ml penicillin (Sigma, St. Louis, MO), 100 Ilg/ml streptomycin (Sigma, St.

Louis, MO), and 10% heat inactivated fetal calf serum (FCS) (InvitrogenlGibco-BRL

Ltd., USA). All the infectivity assays were done using virulent parasites (except where

indicated otherwise). The virulent parasites were maintained in BALB/C mice.

Amastigotes were isolated from spleens (as previously described in materials and

method) and transformed to promastigotes in M199 medium containing 30% fetal calf

serum. Freshly transformed promastigotes were maintained at 22°C in M199 with 10%

54

Chapter 2

fetal calf serum (Hart et ai., 1981). Animals were used in accordance with the

Institutional guidelines. The relevant committee duly approved the use of animals for this

work.

2.3 Macrophage culture

A murine macrophage cell line J774A.1 (American Type Culture Collection,

Rockville, Maryland) and RAW 264.7 were used in this study. The macrophages were

maintained at 37°C in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with

100 units/ml penicillin, 100 Ilg/ml streptomycin and 10% FCS in an atmosphere of 5%

C02 in air. The macrophages were seeded into either in microtitre plates (96 well) at a

density of 5 x 105 cells/well or into tissue culture plates (60 mm) at a density of 1 x 106

cells/plate and incubated for 24 h before being used for the requisite assays.

2.4 Stimulation of macrophages

J774A.I, macrophages were pre-treated with different modulating agents for the

indicated periods of time, followed by the treatment with Leishmania donovani for

required time.

2.5 Nitric oxide assay

J774A.l, macrophage (5 x 105 cells/well) were plated onto 96 well tissue culture

plates and kept in a CO2 incubator (5% C02) for 12 h at 37°C. Cells were preincubated

with specific inhibitors for the indicated period of time at 37°C. Followed by infection

with the parasite for different time points. The samples were then harvested for nitrite

concentration and supernatants were assayed using an automated procedure based on

Griess reagent as described by Stuehr and Nathan (1989). In brief, 100 III of culture

supernatant was mixed with 100 III of Griess reagent (1% sulfanilamide, 0.1% N- [1-

naphthylJ-ethylenediamine dihydrochloride in 2.5% H3P04) and incubated at room

temperature for 30 min. The absorbance was measured at 540 nm (Green et al., 1982).

These results are performed at least three different times in triplicate samples. Nitrite

levels were determined using sodium nitrite (NaN02) as a standard.

2.6 Preparation of celllysates

Stimulated cells (1 x 106/sample) were washed twice with ice-cold TBS (50 mM

Tris-HCI (PH 7.4), 400 mM NaCl and 1 mM sodium orthovanadate) and harvested with a

plastic scraper. The cells were lysed in lysis buffer (50 mM Tris-HCl (PH 7.4), 400 mM

NaCI, 1 mM sodium orthovanadate, 1 % Nonidet P-40, 1 mM EDT A, 1 mM EGTA, 10

mM NaP, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.5 Ilg/ml each

55

Chapter 2

of leupeptin and aprotinin by incubation on ice for 30 min (Feng et al., 1999). Lysates

were then centrifuged at (15,000 x g) at 4°C for 10 min, and supernatants were

transferred to fresh tubes and stored at -80°C till required. Protein concentration of the

lysates was determined using a colorometric assay against a BSA standard (Bradford,

1976).

2.7 Western blot analysis

Cell lysates were resolved by sodium dodecyl sulfate- polyacrylamide gel

electrophoresis (SDS-PAGE) before transferring to PVDF membrane using a transblot

system. The membrane was then incubated with TBST (50 mM Tris-HCI (PH 7.5), 150

mM NaCl, and 0.05% Tween 20) containing 5% skimmed milk for at least 2 h to block

nonspecific protein binding. Primary antibodies were diluted in TBST and applied to the

filter for at least 2 h at room temperature. The blots were washed with TBST thrice and

incubated with the appropriate horseradish peroxidase-conjugated secondary antibody

(diluted up to 1 :5000 in TBST) for 1 h at room temperature. Immunoreactive bands were

visualized by the enhanced chemiluminescence system. The results shown are from a

single experiment typical of at least three giving identical results.

2.8 Preparation of NucIear extract

Nuclear extracts were prepared from J774A.l munne macrophages cells as

described before (Mukhopadhyay et al., 2000). Briefly, 1 x 106 cells were washed twice

with ice-cold phosphate-buffered saline and once with a solution containing 10 mM Tris­

HCI, pH 7.8, 1.5 mM MgCh, and 10 mM KCI, supplemented with a protease inhibitor

mixture containing 0.5 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, and

2 Jlg/ml each of leupeptin, pepstatin, and aprotinin (Sigma). After incubation on ice for

10 min, the cells were lysed by 10 strokes with a Dounce homogenizer, and the nuclei

were pelleted and resuspended in a solution containing 420 mM KCl, 20 mM Tris-HCl,

pH 7.8, 1.5 mM MgCl2, and 20% glycerol; supplemented with the protease mixture

described above; and incubated at 4 °C with gentle agitation. The nuclear extract was

centrifuged at 1 O,OOO-x g for lO min, and the supernatant was dialyzed twice against a

solution of 20 mM Tris-HCl, pH 7.8, 100 mM KCl, 0.2 mM EDTA, and 20% glycerol.

Protein concentration was determined using the Bio-Rad reagent with bovine serum

albumin as standard.

2.9 Electrophoretic mobility shift assay (EMSA)

56

Chapter 2

Sequences of the sense strands of the oligonucleotide probes used for EMSA

were as follows: 5'_ GTG ACT ACG TGC TGC CTA GGG -3' (mouse NOS2-HIF-EM­

For), and the antisense strands were as follows: 5'- CCC TAG GCA GCA CGT AGT

CAe -3' (mouse NOS2-HIF-EM-Rev). The sense and antisense strands were annealed,

gel-purified, and end-labeled with [Y-32P] ATP (NEN Life Science Products) using T4-

polynucleotide kinase (Promega). Unincorporated nucleotide was removed by gel

filtration using G-25 Sephadex columns (Quick Spin TMTE, Roche Molecular

Biochemicals). To measure DNA-protein interaction, 2-5 x 104 cpm of oligonucleotide

probe was incubated with 4-5 Ilg of nuclear extract and 0.5 Ilg of sonicated, denatured

salmon sperm DNA (Life Technologies, Inc.) in 10 mM Tris-HCI (PH 7.8), 50 mM KCI,

50 mM NaCl, I mM MgCI2, I mM EDTA, 5 mM dithiothreitol, and 5% glycerol, for

20 min at 4 °C in a total volume of 20 Ill. The reaction mixture was subjected to

electrophoresis (200 V in 0.3 x Tris-buffered EDTA, 4°C) using 5% nondenaturing

polyacrylamide gels. Dried gels were subjected to autoradiography for 16-48 h. For

competition experiments, a IOO-fold molar excess of unlabeled, annealed oligonucleotide

was added to the binding reaction just prior to the addition of radio labeled probe. For gel

supershift analysis, 1 III of rabbit monoclonal antibody against HIF-1 a (Novus

biologicals) was added after the initial 20-min incubation, and was further incubated for

1 h at 4 °C before electrophoresis.

2.10 Transient Transfection of Cells and Reporter Gene Assays.

To measure transcriptional efficiency of NOS2 promoter construct, J774A.1

macrophages cells (5 x 105) were plated in 35 mm tissue culture plate (Greiner or

Corning) and kept for adherence. Next day cells were transfected in serum free medium

by 1.5 Ilg of LIPOFECT AMINE TM 2000 (Invitrogen) and 1.0 Ilg of the wild type pGL3-

mouse NOS2 promoter or HIF-I mutant pGL3-mouse NOS2 promoter having a reporter

gene construct. After 6h of transfection the cells were again kept in serum media for at

least 12-16 h for recovery. After that the cells were treated for 16 h under the described

experimental conditions and than the cells were washed twice with PBS and lysed in 1 x

lysis buffer (Promega) on ice. The lysate was centrifuged at 12,000-x g for 15 min and

the supernatant was collected. An aliquote of the supernatant was added with the

substrate (Promega) according to manufacturer's instruction and the reading was taken in

a Lurninometer.

2.11 RNA isolation and RT -PCR.

57

Chapter 2

Macrophages were infected with Leishmania donovani in 1: 10 ratio respectively.

After different time point and treatment, cells were washed twice with PBS and than

lysed in Tripure reagent (Roche applied sciences, Indianapolis). Total RNA was isolated

as per the company's protocol. RNA was estimated and 4 )lg of RNA was used for semi­

quantitative Reverse-Transcriptase PCR reaction using mouse NOS2 gene specific

primers. The RT-PCR condition were as follows: RT at 50°C for 30 min, denaturation at

94°C for 2 min, followed by 25 cycle of denaturation at 94°C for 30 s, rumealing at 55°C

for 30 s and elongation at 68°C for 30 s. Additional elongation step was set at 68°C for 7

min. Electrophoresis of amplified DNA was carried out on 1.7% gel. The primer

sequences for NOS2 used in this study are listed below and were purchased from Sigma

genosys and sequence were as follows: mouse NOS2 forward primer, 5'- AAT GGC

AAC ATC AGG TCG GCC ATC ACT-3', mouse iNOS NOS2 reverse primer, 5'-GCT

GTG TGT CAC AGA AGT CTC GAA CTC-3'. Mouse B-actin forward 5'-GTG GGG

CGC CCC AGG CAC CA-3'; B-actin reverse 5' CTC CTT AA T GTC ACG CAC GAT

TTC-3' were used as loading control. The possible contamination of any PCR component

was excluded by performing a PCR reaction in the absence of Reverse transcriptase in

each set of experiment (negative control).

2.12 Cloning of Mouse NOS2 5'-flanking region

Mouse NOS2 promoter was designed from the mouse NOS2 gene (GenBank@

accession no NM_OI0927 XM_OOlO015) in such a way that it contains both the HIFI

binding site (TACGTGCT) as well as IFN-gamma binding site (CACTGTCAATAT

ITCACTTTCATAAT). This promoter was cloned into the pGL3-Luciferase reporter

vector (promega) by PCR using primers designed to contain restriction sites for Xho I

and Hind III in forward and reverse primers respectively. The forward primer was 5'­

ATA CAT C·TCGA .. G GTC CIT GTA CAT GCA AGG-3' and the reverse primer was

5'-ATA CAT A"'AGCT .. T GGA GTG AAC AAG ACe CAA-3'. The amplified PCR

product was purified from the gel by using gel elution kit from Qiagen and was digested

with Xho I and Hind III, and ligated into pGL3-control Luciferase reporter vector,

previously digested with the same restriction enzyme. The resultant DNA construct was

amplified, purified and etimated (Quigen kit) and designated as pGL3-mouse NOS2

promoter. Site-directed mutagenesis of the HIFI in NOS2 promoter was done by the

megaprimer method (Mukhopadhyay et al., 2000). All deletion and mutation constructs

were verified by sequencing.

58

Chapter 2

2.13 Gene silencing by siRNA for HIF-1a. in J774A.1 murine macrophages.

To knock down HIF-Ia gene silencing was performed by siRNA from Qiagen

and was done as per manufacturer's protocol. After silencing the gene macrophage cells

were infected with Leishmanai donovani and its surface molecule LPG for given time

point. The effect of gene silecing was monitored for HIF -1 a by western analysis and for

NOS2 at mRNA level by RT-PCR. The target sequence was AAC ACA CAG CGG AGC

TTT TTT, this sequence was designed for mouse HIF-I by using QIAGEN online siRNA

Design Tool.

3.0 Results

3.1 Effect of Leishmania donovani on Nitric oxide generation in J774A.1

macrophages.

Nitric oxide (NO) is known to play a critical role in host immune system.

Generation of NO by activated macrophages has been correlated directly with the

leishmanialcidal capacity of the cells (Proudfoot et ai., 1996). Previously, L. major LPG

has been reported to activate as well as inhibit NO synthesis by the murine macrophages,

thereby, playing an important role in host - parasite relationship. Surprisingly, no direct

report has been shown on IFNy or LPS independent NO generation in host macrophages

during infection by the Leishmania donovani. To address this issue in the present study,

we infected murine macrophages J774A.1 by Leishmania donovani and checked NO

generation in J774A.l cells. After 16 h of infection the nitrite level was increased about 5

fold in infected J774A.1 cells compared to uninfected control (Fig. 1A). The specificity

of NO generation was checked by using NOS specific inhibitor L-NAME (300 nM).

About 80% of nitrite generation was blocked by L-NAME. The generation of nitric oxide

was further estimated by different multiplicity of infection in macrophages. When

infected with 1:5 MOl or 1:10 (J774A.1: LD), the NO generation was maximum in

murine macrophage J774A.1 (Fig. IB). Nitric oxide generation was also checked with

and without IFN gamma treatment (Fig. 1 C). It was found that the cells only infected

with Leishmania donovani shows lesser NO generation as compared with the IFN-y

treated cells.

3.2 Effect of Leishmania donovan; on the protein level of NOS2 in murine

macrophages.

59

A

B

Fig. 1:

8

6

~ '-'

'" 4 0 Z

2

0 -. L. donovani + + L-NAME + +

8

6

'-' 4 '" o z

2

o o 1:2.5 1:5 1:10

J774A.l : L. donovani

Leishmania donovani induced nitrite levels In J774A.l

macrophages. J774A.1 macrophages (lx105) cells/well were seeded

onto 96 well tissue culture plates and kept in a CO2 incubator at

37°C. (A) Cells were then infected with Leishmania donovani and

treated with Nitric oxide inhibitor (B) Cells were treated with

different doses of parasite. Both the set were again incubated at

37°C for 16 h. Nitrite levels present in the supernatants were

quantified by Griess reagent. Data are mean ± standard deviation

(S.D.) of triplicate values.

5

c: .~ 4 .-

C.I ::I

"0 c: 3 -"0 "0 ~ 2 Q,j

OIl eo: J. Q,j

1 .. -<

0 Control L. donovani IFN-y

Fig. Ie: Leishmania donovani and IFN-y induced nitrite levels in J774A.1

macrophages. Macrophages (l x 1 05) cells/well were seeded onto 96

well tissue culture plates and kept in a CO2 incubator at 37°C. Cells

were then infected /treated with Leishmania donovani / IFN-y

respectively. The plate were again incubated at 37°C for 16 h.

Nitrite levels present in the supernatants were quantified by Griess

reagent. Data are mean ± standard deviation (S.D.) of triplicate

values.

Chapter 2

To check whether the increased generation of NO is due to the increase in the

protein level J774A.1 cells (1 x 1 06 cells/ml) were treated with Leishmania donovani (1:5

MOl) for 16 hours and the cell lysates were made. The activation of NOS2 was

investigated by western blotting analysis using NOS2 antibody. It was found that the

nitric oxide synthase was stimulated about 3 fold after 16 hours of infection when

compared with the uninfected macrophages, (Fig 2A). Cobalt chloride (l00 J.lM) was

used as a positive control. Like the NO generation maximal induction of NOS2 was

found when macrophages were treated by 5 times more of parasite. To check that

activation of NOS2 by LD infection is not limited to the J774A.l murine macrophages

only, another macrophage cell line RAW 264.7 was also tested. When RAW 264.7

macrophage cell line was infected with the LD (l :5, MOl) for 16 h and the NOS2 protein

level was detected by Western blot analysis more then 2 fold increase of NOS2 protein

was found (Fig. 2B). Incidentally, the induction of NOS2 was in a lesser degree than

J774A.l macrophages probably due to different origin of these two macrophages (Fig.

2B). Further the nitric oxide generation was also checked in J774A.l macro phages after

infection with virulent and non-virulent parasites. It was found that more nitric oxide was

being generated by the infection with virulent parasite (Fig 3A). This was further

supported by western blot analysis. It was found that virulent parasite infection in

J774A.l increased greater NOS2 induction as compared to the non-virulent parasite

infection (Fig 3B).

3.3 Leishmania donovani induces NOS2 mRNA expression in murine

macrophages.

To determine whether Leishmania donovani induced expression of NOS2 is due

to increase in mRNA, J774A.l macrophages were infected with the parasite and the total

RNA was harvested after different time of infection. Semi-quantitative reverse

transcriptase-PCR was performed by using murine NOS2 specific primers to detect

NOS2 mRNA expression. Infection with Leishmania donovani caused a significant

increase in NOS2 mRNA expression and was detectable as early as 8 h of infection (Fig.

4A). The maximal induction was found about 16 h and remained up-regulated even at 24

h.

To confirm that the increased NOS2 mRNA expreSSIOn due to Leishmania

donovani was not restricted to only J774A.1 murine macrophages, we also checked the

expression of NOS2 mRNA in RAW 264.7 macrophages after infecting it with

60

1774A.1 : L. donovani

o 1:2.5 1:5 1:10 Cobalt

.......,.-75_.;: \",~i_iii iN$ I!I._II r- NOS2 Immunuhlut using anti-NOS2 antihody

Fig.2A: Immunoblot analysis of NOS2 in J774A.l macrophages following L.

donovani infection. Macrophages (l x 101i) were infected with the L.

donovani for 16 hours. Whole cell lysates were resolved on SDS-PAGE

gel followed by Western blot analysis of the separated proteins using anti­

NOS2 antibodies. The data shown is from one of the three independent

experiments that yielded similar results.

-LD +LD

Immunoblot using anti-NOS2 antihody

+- alpha-Actin

Immunoblot with a-actin antihody

Fig.2B: Immunoblot analysis of NOS2 in RAW 264.7 cells following L. donovani

infection. Macrophages (1 x 106) were infected with the L. donovani (1 x

107) for 16 hours. Whole celllysates were resolved on 7.5 % SDS-PAGE

followed by Western blot analysis of the separated proteins using mouse

NOS2 antibody. The data shown is from one of the three independent

experiments of similar results.

Fig.3:

A 4 _._------.•. --c .: t: 3 == "0 C -"0 2 '0 "-OJ OL 1 Ol ... OJ 0-

-< 0 Cont. nVLD vLD

B Cont. nVLD vLD

I U_ ,~ NOS2

Immunohlot using anti-NOS2 antibody

II!Z!\W!5lc"". 'I ... a-AClin Immunohlot with a-actin antihody

(A) Nitric oxide generation by virulent and non-virulent LD in J774A.l

macrophages. 1774A.1 macrophages (lxI05) cells/well were seeded onto 96

well tissue culture plates and kept in a CO2 incubator at 37°C. Cells were

then infected with both virulent and non virulent Leishmania dOllovalli and

again incubated at 37°C for 16 h. Nitrite levels present in the supernatants

were quantified by Griess reagent. Data are mean ± standard deviation (S.D.)

of triplicate values.

(B) Induction of NOS2 by non-virulent and virulent LD. Macrophages (1

x 106) were infected with the virulent and non virulent L. dOllovani for 16

hours. Whole cell lysates were resolved on SDS-PAGE gel followed by

Western blot analysis of the separated proteins using anti-NOS2 antibodies.

The data shown is from one of the three independent experiments that

yielded similar results

Time (h)

o 8 16 24 M --NOS2 -+ ---' ..... -.

(bp)

- 600 -500 - 400

RT-PCR using mouse NOS2 transcript specific primers

Beta-actin -+

RT-PCR using mouse ~-actin transcript specific primers

Fig.4A: RT-PCR analysis of NOS2 transcript levels in L.donovani infected and

uninfected 1774A.l cells. J774A.l cells were infected with Leishmania

donovani at a parasite to host ratio 10: 1 for indicated time points. Total

RNA was isolated and NOS2 transcript were reverse transcribed and

amplified by using one-step RT-PCR protocol. Amplified mRNA were

separated by agarose gel electrophoresis. The data shown is from one of

the three independent experiments that yielded similar results.

Chapter 2

Leishmania donovani (Fig. 4B) by RT-PCR. The result demonstrated that Leishmania

donovani also induce NOS2 mRNA in RAW 264.7 macrophage cell line.

3.4 Functional requirement of the NOS2-HRE for the induction of mouse NOS2

promoter activity in macro phages by Leishmania donovani infection

To understand the mechanism involved in Leishmania donovani mediated NOS2

mRNA expression in macro phages the 1376 nt upstream of the 5'-flanking region of

mouse NOS2 from the transcription start site was cloned from genomic DNA in to

promoter less vector pGL3 containing luciferase reporter gene (Fig SA). The cloned

chimera pGL3-NOS2-prom was transiently transfected into J774A.l murine macrophage

cells and was infected with the Leishmania donovani. Upon infection the luciferase

activity was increased 2.1 fold over the uninfected control. Cobalt chloride was used as a

positive control (Fig. 5B). This result indicates that L. donovani infection induced a

transcription factor, which binds within the 1376 nt upstream S' -flanking region ofNOS2

gene for its increased expression. This fragment contained the regulatory sequences like

hypoxia responsive element, which binds the oxygen sensitive transcription factor HIF-!

(HRE- 5'-TACGTGCT-3') and interferon-y sensitive responsive element (ISRE- 5'­

CACTGTC AATATTTCACTT TCATAAT-3'), those are known to regulate NOS2

expression in different conditions. To fmd the contribution of HRE and ISRE for the

induction ofNOS2 expression a mutagenesis strategy was used. We mutated the HRE on

the NOS2 promoter from wild type 5'-TACGTG CT -3' to mutated type 5'- TAAAAG

CT -3' and ISRE from wild type 5'- CACTGTCAATATTTCACTTTCATAAT-3' to

mutated type S'-CCATTATAGGCATGAAATATTGAC-3'. To find out the contribution

of the each of the sites, they were mutated individually. All the fragments were ligated

upstream of luciferase in the promoterless reporter vector pGL3basic. The murine

macrophages J774A.l cells were transfectedwith these constructs and infected for 16 h

with Leishmania donovani. The chimera with HRE mutation lost more than 85%

inducibility of luciferase activity upon infection indicating the contribution of HRE in the

process (Fig SC). Interestingly, ISRE mutant showed greater induction of promoter

activity by both LD infection and cobalt treatment, indicating an interplay of these two

transcription factors may exist during stressed conditions like LD infection or hypoxic

stimuli as indicated before (Melillo et ai., 1995).

3.4 Leishmania donovani induces DNA binding activity to the NOS2-HRE

61

M L. donovani

+

RT-PCR using mouse NOS2 transcript specific primers

LD +

NOS2

+- Beta -actin

RT-PCR using mouse ~-actin transcript specific primers

Fig.4B: RT-PCR analysis of NOS2 transcript levels in L.donovani infected and

uninfected RAW 264.7 cells. After 16 hours of infection of cells at a

parasite to host ratio 10: 1 total RNA was isolated and NOS2 transcript

were reverse transcribed and amplitied by using one-step RT-PCR

protocol. Amplified mRNA were separated by agarose gel

electrophoresis. The data shown is from one of the three independent

experiments that yielded similar results.

-1376 -1077 -388 -1

.. / .......... . ~ ~

•••• • •••• ...... . ..... . .. .,.

·tACTGTCAATATTTCACTTTCATAAT

• · . • • • • · . • • • • • • • • • • -TACGTGCT-

Fig. SA: Schemetic diagram of mouse NOS2 promoter (1.376 kb ) containing HIF-I (hypoxia inducible factor I) and ISRE (lFN-stimulated response element) site was cloned in pOL3 basic vector

;>-. ...... 4 ~------~-----~-

. ->-.-...... u C\l <l.)

T (/j 3 C\l ;..,

~ .-u j T ...... 2 0 c:: 0 .-...... u = _0 "0 c:: .-

"0 -& 0 -------~- ----

Control L donovani Cobalt

Fig.5B: 1774A.l macrophage cells were transfected with 1.376 kb long mouse

NOS2 promoter region in pGL3 vector. After the recovery, cells were

either infected with LD or treated with cobalt (1 00 ~M) or remain

untreated. Luciferase activity was checked after 16 h from cell extracts.

All the cells were cotransfected with ~-galactosidase under the control of

CMV promoter. Final results were depicted after normalization with ~­

galactosidase activity as a transfection control and at least three

independent experiments performed in duplicates

8,-----------------------------

7

6

5

4

3

2

1

o

Fig. 5C: Wild type (WT) and sized matched HIF-l mutant (HRE-mut) as well as

Interferron-y response element mutant (lSRE-mut) were transfected into

J774A.1 cells. After recovery cells were either infected with 10 times

more LD compared to J774A.l cells or treated with HIF-inducer cobalt

chloride (100 J.lM) for 16 h. Luciferase activity was checked in cell

extracts and normalized with ~-galactosidase activity as previously

described. Results are depicted from three independent experiments

performed in duplicates.

Chapter 2 -To confinn whether Leishmania donovani induces DNA binding activity to the

NOS2-HRE, EMSA was performed. Nuclear extract were prepared from J774A.l

macrophages infected with Leishmania donovani, and incubated with a radiolabeled

probe of 21-base pair (bp) oligonucleotide containing the HIF -1 binding site of the mouse

NOS2 promoter. In response to Leishmania donovani, specific formation of a

radio labeled complex with increased intensity was observed in compare to uninfected

cells (Fig.6). Competition experiments were performed to show the specificity of binding

of the complex to the NOS2-HRE probe. The binding of radiolabeled NOS2-HRE probe

to the putative HIF -1 complex in nuclear extracts from Leishmania donovani infected

murine macrophages cells was effectively competed by a lOO-fold molar excess of

unlabeled probe. To confinn the contribution ofHIF-l in DNA protein binding complex

supershift analysis was performed (Fig.6). When the radio labeled probe was incubated

with a mixture of nuclear extract with HIF-la antibody, the HIF-l binding shifted to a

slower mobility complex (Fig. 6, lane 5). This experiment strongly suggests that HIF-l is

activated and bind to the NOS2 promoter region for its increased expression in host

macrophages during Leishmania donovani infection.

3.5 The involvement of HIF-l in Leishmania donovani induced NOS2 expression

was confirmed by knocking down HIF-l from J774A.l macrophages.

Finally, we aimed at elucidating the importance of HIF-la in the Leishmania

donovani induced expression of NOS2 gene. In the first step we established a specific

SiRNA against HIF-la. Murine J774A.l macrophages cells were transfected with HIF-

1a SiRNA and then the cells were infected with Leishmania donovani for 16 h. Initially,

the HIF-la expression was checked by western analysis, which shows specific SiRNA to

the mouse HIF-la decreased HIF-la synthesis during LD infection, whereas non­

specific SiRNA shows no effect (Fig. 7 A). Then total RNA was extracted from SiRNA

transfected and LD-infected J774A.l cells and RT-PCR was performed for NOS2

expression. A significant decrease in the NOS2 mRNA expression was seen in the HIF­

la knocked down sample (Fig. 7B). Specificity of the selected sequence was confirmed

by transfecting a control non-specific SiRNA. This data demonstrate that the HIFla is

critically involved in Leishmania donovani induced expression of NOS2 in murine

macrophages.

3.6 Leishmania donovani induces NOS2 expression in murine macrophages

through PI3 Kinase pathway

62

F'ig.6:

::'2 C U

X >. 0 C 0

0 -v +

.D Cl Cl Cl 0 .....l .....l .....l .... Q... + +

.D < i:;

u.. -:r: + Cl , +

+- Super-shift +- HIF-l +- NS

+- Free Probe EMSA with /JUJU.lf NOS2 liRE

EMSA was performed using 12p labeled mouse NOS2 HRE. Nuclear

extracts (5 ~g) were prepared from J774A.1 cells after 4 h of infection by

virulent LD (MOI- 1: I 0) and incubated with labeled probe for 20 min at

4()C, run into 5% acrylamide geL Then gel was dried and kept for

autoradiography for over night. lOOX cold probe was added just before

the addition of radiolabeled probe. HIF-I C1 monoclonal antobody (2 ~g)

was incubated with nuclear extract for 1 h before the addition of the

radiolabeled probe.

Fig. 7:

A.

B.

600 -500 -400 -

ScRNA SiRNA

+

Immunoblot analysis of HIF-J ex

ScRNA SiRNA

(LO)

HIF-J ex

(LO)

+- NOS2

RT-PCR using mouse NOS2 transcript specific primers

A. HIF-J ex expression by Western analysis in .J774A.1 macrophages by

LO infection after transfection with specific SiRNA and Scrambled Si

RNA (ScRNA). B. NOS2 expression in LD infected .J774A.!

macrophages after HlF-J ex SiRNA transfection in by semi-quantitative

reverse transcriptase PCR usmg mOllse transcript specific primers.

Chapter 2 -There are several reports of involvement of PI3-kinase in HIF-l activation during

normoxic condition (Biswas et ai., 2007). To find out the whether a similar signalling

pathway involved in NOS2 expression in Leishmania donovani infected macrophages

J774A.l cells were pretreated with PI3-Kinase inhibitor L Y 294002 (25 J.lM) followed by

infection with the parasite. After infection for 16 h RT -PCR was performed to check

NOS2 expression. The increase in NOS2 mRNA expression by LD infection was almost

completely blocked by PI3-kinase inhibitor L Y 294002, confirming the role of PI3-

kinase in the process (Fig. SA). In a complimentary experiment we also checked PI-3

kinase activity by LD infection. AKT -kinase is one of the major substrate of PI3kinase as

it is phosphorylated by PI3-kinase when it is activated. We tested the AKT

phosphorylation in LD-infected J774A.l cells by using phosphor-serine specific

antibody. The AKT phosphorylation was increased within 5 minutes of infection

substantiating the earlier observation of involvement of PI3K in the process (Fig. SB).

4.0 Discussion Protozoan organisms of the genus Leishmania are obligate intracellular parasites

of macrophages that are responsible for severe morbidity and mortality in infected people

in many parts of the world. A principal function of macrophage is to destroy intracellular

pathogens. Hence, the manner in which Leishmania and other intracellular pathogens are

able to survive and replicate within this hostile intracellular milieu is an important

question in cell biology and immunology. Leishmania and other intracellular pathogens

have evolved mechanisms to modulate gene expression in order to facilitate invasion and

survival. Diverse lines of evidence also indicate that Leishmania interferes with signal

transduction in macrophages (Reiner, 1994). Earlier reports show that in addition to

parasite products and virulence factors that facilitate survival and entry of metacyclic

promastigotes into the host cell, sandfly saliva suppresses macrophage leislunanicidal

activity by inhibiting nitric oxide (NO) production (Hall and Titus, 1995) and accelerates

lesion development (Hall and Titus, 1995). This activity has been attributed to the

sandfly salivary peptide maxadiIan, a selective agonist of the pituitary adenylate-cyclase­

activating polypeptide type 1 receptor, which inhibits tumour necrosis factor-a (TNF-a)

production by lipopolysaccharide (LPS)-stimulated macrophages (Bozza et ai., 1998) and

diminishes their ability to produce NO and kill Leishmania in vitro (David et ai., 1997).

Consequently, administration of maxadilan together with L. major promastigotes

63

A

L. donovani LY (50 ~M)

+ + + + M

Fig. SA: RT-PCR analysis of NOS2 transcript levels in L.donovani infected and

uninfected J774A.l cells. J774A.1 cells were pretreated with specific

inhibitor of PI3 kinas, L Y 294002 (25 JlM) followed by infection with the

parasite to host ratio 10:1 for 16 h and total RNA was isolated and NOS2

transcript were reverse transcribed and amplified by using one-step RT­

PCR protocol. Amplified mRNA were separated by agarose gel

electrophoresis. The data shown is from one of the three independent

experiments that yielded similar results.

Time (min)

5 15 30

C I C I C I .• ' . _.~I pAKT

I ....... ----~ IAKT

C - Control I - Infected

Fig.8B: Kinetics of pAKT in 1774A.l macrophages following L. donovani

infection. Macrophages were infected with L. donovani for the indicated

times. Whole cell lysates were resolved on SDS-PAGE gel followed by

Western blot analysis of the separated proteins using anti-pAKT and anti­

AKT antibodies. The data shown is from one of the three independent

experiments that yielded similar results.

Chapter 2

significantly exacerbated disease in resistant mice, which is associated with diminished

NO production in draining lymph nodes (David et ai., 1997).

It has been reported earlier that in epidermal Langerhan cells, L. major parasites

survive due to the lack of cytokine-inducible nitric oxide synthase (iNOS, NOS-2) (Blank

et ai., 1996). Thus these Langerhan cells form a safe habitat for parasite and transporting

the parasites from the infected skin to the draining lymph node (Blank et al., 1996). In

contrast, cytokine-activated macrophages express NOS2 and synthesize high levels of

NO from L-arginine. Proudfoot et ai., (1995) demonstrated that infection of a

macrophage cell line with L. major promastigotes prior to stimulation with IFN-y and

lipopolysaccharide partially inhibited the release of NO. This effect was mimicked by

addition of glycoinositolphospholipids from L. major, which is abundantly expressed on

the surface. of both the promastigote and amastigote parasite form. Another parasite

molecule, which downregulate NOS2 activity in infected macrophages, is the LPG­

associated kinetoplastid membrane protein-ll. At amino acid position 45, this protein

contains NG-monomethyl-L-arginine, a structural analogue of L-arginine and well-known

inhibitor of NOS2 (1 ardim et al., 1995). Persistence of the parasites in macrophages,

dentritic cells was paralleled by a sustained, lifelong expression of NOS2 mRNA and

protein, which was dependent on CD4+ and not on CD8+ T cells. Furthermore, 30-40% of

the parasites detected co-localized with NOS2-positive cells (macrophages and dendritic

cells), whereas 60-70% were found in areas negative for NOS2.

The most relevant and novel finding of the present study was the involvement of

the active HIF-l transcription factor in the promoter enhancer region of the mouse

NOS2, which result in the increased NOS2 expression in murine macrophages during

Leishmania donovani infection. One mechanism by which hypoxia is known to increase

NOS2 gene expression is through the induction of transcription factors such as HIF-l. In

this in vitro studies presented, the 5'-flanking region of the murine NOS2 gene was

shown to contain a DNA sequence that was functionally essential for hypoxia induced

transcriptional activation in murine macrophages. This 8-bp sequence, 5'-TACGTGCT-

3', was identical to the binding site for HIF -1 identified in the human erythropoietin gene

(Melillo et ai., 1997). It has been shown previously that NOS2 gene expression is

induced by hypoxia in a macrophage cell line only when cells were costimulated with

interferon-y (Melillo et ai., 1997). Our results demonstrate that Leishmania donovani

induced NOS2 expression through HIF-l in murine macrophages even in the absence of

64

Chapter 2 .

interferon-yo The Leishmania donovani dependent induction ofHIF-l binding and NOS2

promoter activity was associated with increased expression of the NOS2 gene in murine

macrophages. Leishmania donovani induced NOS2 mRNA expression and increased the

rate of transcription of the NOS2 gene without IFN-y. The induction of NOS2 rnRNA

was time-dependent and required PI3 kinase signalling intermediates, as demonstrated by

inhibition of Leishmania donovani induced NOS2 mRNA expression in the presence of

specific inhibitor of PI3 kinase. It was observed from the present study that Leishmania

donovani resulted in a significant dose-dependent and time-dependent increase in nitrite

levels.

The role HIF-l plays in the transcriptional regulation of gene expression in

response to hypoxia may be both cell type and gene specific. For instance, in the human

hepatoblastoma cell line Hep 3B, transcriptional activation mediated by HIF-l requires

the binding of a second unidentified factor at site 2 of the erythropoietin gene enhancer

(Semenza et al., 1992). In the murine macrophage line ANA-I, the effects of hypoxia on

NOS2 transcription that require the HIF-l binding site are augmented by interferon-y

treatment (Melillo et a., 1995). Regulation of the lactate dehydrogenase A gene by

hypoxia in the human cervical carcinoma cell line HeLa is augmented by forskolin and is

dependent on the HIF-I binding site and an adenosine 3', 5'-cyclic monophosphate

response element (Firth et a!., 1995). So far, the requirement for additional factors for

transcriptional activation of the NOS2 gene through HIF-I in Leishmania infected

macrophages is not known.

Comparison of sequences around the HIF -1 site present in the 5' -flanking region of

the NOS2 gene and the 3'enhancer of the erythropoietin gene shows a region of

similarity 10 bp downstream from the HIF-l site. This 5-bp sequence, 5'- CACTG-3', is

similar to site 5'-CACAG-3', of the erythropoietin gene enhancer. Mutations of the 5'­

CACAG-3' sequence eliminated the ability of the erythropoietin enhancer to activate

transcription in response to hypoxia (Semenza et al., 1992). Thus it is possible that the

5'-CACTG-3' sequence in the NOS gene may also be involved in the LD induced HIF-I

activation

It has been reported that activating transcription factor (ATF)-1 and adenosine 3',

5'-cyclic monophosphate response element binding protein (CREB)-1 constitutively bind

to the HIF-l consensus sequence (Kvietikova et al., 1995). This may indicate the

presence of other transcription factors, the binding sites of which are close to or overlap

65

Chapter 2

with the HIF-1 consensus site and are involved in regulating basal NOS2 gene expression

in murine macrophages. We have also found one ISRE binding site that is located 681 bp

upstream ofHIF-1 binding site but whether it is helping HIF-1 for NOS2 expression is

not clear so far.

65A