From cells to signaling cascades: manipulation of macrophage defense by Leishmania parasites

Pijush K. Das

Infectious Diseases and Immunology Division

Indian Institute of Chemical Biology Kolkata, India

Alveolar Peritoneal Spleen Kupffer cells Circulating macrophages macrophages macrophages macrophages

Various cells but only Ms are selected

Mannose – Albumin – drug

(Drug conjugate)Parasite multiplication inside Ms

Neoglycoprotein mediated drug delivery for Kala-azar

+Mannose Albumin

Mannose – Albumin (Neoglycoprotein)Drug Doxorubicin

Binding of drug conjugate via specific receptor

Infection

By bitingInfected liver & spleen

Drug entry inside macrophage and parasite killing

Chakraborty, P., Bhaduri, A.N. and Das, P.K. (1990) Biochem. Biophys. Res. commun. 166, 404-410.

Chakraborty, P., Bhaduri, A.N. and Das, P.K. (1990) J. Protozool. 37, 358-364 Chakraborty, P., Basu, N., Sett, R. and Das, P.K. (1991) Indian J. Chem. 30B, 127-132.

Sett, R., Basu, N., Ghosh, A.K. and Das, P.K. (1992) J. Parasitol. 78, 350-354.

Sarkar, K. and Das, P.K. (1997) J. Immunol. 158, 5357-5365. Sett, R., Sarkar, K. and Das, P.K. (1993) J. Infec. Dis. 169, 994-999.

Various cells but only Ms are selected

Parasite multiplication inside Ms

Liposome mediated drug delivery for Kala-azar

+

Mannose

Mannosylated liposomeDrug Doxorubicin

Mannosylated liposome containing drug (Drug conjugate)

Drug entry inside macrophage and parasite killing

Infection

By bitingInfected liver & spleen

Liposome

Binding of drug conjugate via specific receptor

Sett, R., Sarkar, H.S. and Das, P.K. (1993) J. Antimicrob. Chemother. 31, 151-159. Kole, L., Sarkar, K., Mahato, S.B. and Das, P.K. (1994) Biochem. Biophys. Res. Commun. 200, 351-358.Sarkar, K., Sarkar, H.S., Kole, L. and Das, P.K. (1996) Mol. Cell Biochem. 156, 109-116.

Antibody grafted drug containing liposomes for targeting infected macrophage only

Infection by

Leishmania parasites Various cells but only Ms are infected

Parasite antigen

Antigen purified

Ab raised against parasite Ag

Fab part isolated

Fab grafted drug containing liposomes

Specific targeting to macrophages

Drug entry inside macrophage and parasite killing

Immunoliposome

Drug containing liposomes

Basu, N., Kole, L., Ghosh, A. and Das, P.K. (1994) Mol. Cell Biochem. 132, 1-6.Datta, N., Mukherjee, S., Das, L. and Das, P.K. (2003) Eur. J. Immunol. 33, 1508-1518.Mukherjee, S., Das, L., Kole, L., Karmakar, S., Datta, N. and Das, P.K. (2004) J. Infec. Dis. 189, 1024-1034.

Leishmaniasis Immunosuppression

Ideal therapeutic agent Parasite killing agent

+

+ IFN-

+ IFN-

Man

Man

Man

DOX

Man

Man

Man

ManMan

HSA DOXMan

• Highly efficient therapy

• Both in vitro culture model

and in vivo animal model

Immunostimulant

Kole, L., Das, L. and Das, P.K. (1999) J. Infec. Dis. 180, 811-820.Mukherjee, S., Ukil, A. and Das, P.K. (2007) Antimicrob. Agents Chemother. 51, 1700-1707.Kar, S., Ukil, A. and Das, P.K. (2009) Eur. J. Immunol. 39, 741-751.

Cystatin, a natural cysteine protease inhibitor, has tremendous potential as antileishmanial agent. Cystatin could cure experimental visceral leishmaniasis in animal model with upregulation of NO and favourable T cell response.

Das, L., Datta, N., Bandyopadhyay, S. and Das, P.K. (2001). J. Immunol. 166, 4020-4028.Kar, S., Ukil, A. and Das, P.K. (2009) Eur. J. Immunol. 39, 741-751.

Kar, S., Ukil, A. and Das, P.K. (2011) Eur. J. Immunol. 41, 116-127. 

18-Glycyrrhetinic acid, a pentacyclic triterpene of licorice root, has strong immunomodulatory potential for its use in general for macrophage-associated diseases.

Ukil, A., Biswas, A., Das, T. and Das, P.K. (2005) J. Immunol. 175, 1161-1169.

Kar, S., Sharma, G. and Das, P.K. (2011) J. Antimicrob. Chemother. 66, 618-625.

Ukil, A., Kar, S., Srivastav, S. and Das, P.K. (2011) PLoS ONE, 6, e29062. Sharma, G., Kar, S., Palit, S. and Das, P.K. (2012) J. Cell Physiol. 227, 1923-1931. 

Importance of ECM recognition

• Leishmaniasis transmitted by parasite injection into

blood during blood meal of insect vector.

• Parasites home in macrophages of liver and spleen

- adhere, penetrate, transform and replicate.

• Macrophage lysis - attack of neighbouring cells -

disease process cycle.

• Solid organs liver and spleen contain ECM and BM

for various functions.

• Parasite crossing of ECM and BM needed for

homing in macrophages

• ECM and BM recognizing molecules - absence of

which may result parasite entrapment within ECM

meshwork.

Recognition of Extracellular Matrix

Blood Stream

Laminin Binding Protein (LBP)

Preliminary screening – major laminin binding

Activity - 67 kDa integral membrane glycoprotein which may act as an adhesin.

Ghosh, A., Kole, L., Bandyopadhyay, K., Sarkar, K. and Das, P.K. (1996) Biochem. Biophys. Res. Commun. 226, 101-106.Ghosh, A., Bandyopadhyay, K., Kole, L. and Das, P.K. (1999) Biochem. J. 337, 551-558.Bandyopadhyay, K., Karmakar, S., Ghosh, A. and Das, P.K. (2001) J. Biochem. 130, 141-148. Bandyopadhyay, K., Karmakar, S. and Das, P.K. (2002) Eur. J. Biochem. 269, 1622-1629. Karmakar, S., Bandyopadhyay, K., Biswas, A. and Das, P.K. (2003) Eur. J. Biochem. 270, 1-8.

L. donovani, when attached to the macrophage surface, behaves like any other microbe which are killed by macrophages, but once internalized it somehow switches off the activation signaling for downstream oxidative events.

A soluble nitric oxide synthase (NOS) enzyme was purified from L. donovani. The enzyme kinetics, cofactor requirements, inhibition studies and Western blot analysis with brain anti-NOS antibody suggest its similarity with mammalian NOS isoform I.

Basu, N.K., Kole, L., Ghosh, A. and Das, P.K. FEMS Microbiol. Lett. 156: 43-47, 1997.

Bhunia A.K., Sarkar, D. and Das, P.K. J. Eukaryotic Microbiol. 43: 373-379, 1996.

Ukil, A., Maity, S. and Das, P.K. (2006) Br. J. Pharmacol. 149, 121-131.

Maity, S., Ukil, A., Vedasiromoni, J.R. and Das, P.K. (2006). Int. J. Pharmacol. 2, 240-246.

Ukil, A., Maity, S., Karmakar, S., Datta, N., Vedasiromoni, J.R. and Das, P.K. (2003) Br. J. Pharmacol. 139, 209-218.

Maity, S., Ukil, A., Karmakar, S., Datta, N., Chaudhuri, T., Vedasiromoni, J.R. and Das, P.K. (2003) Eur. J.Pharmacol. 470, 103-112.

During macrophage

invasion:

Oxidative burst

Killing……..

A group of of parasite survives in the hostile environment to establish infection….

Leishmania signaling during infection

1.The macrophage environment with temperature 37C and pH 5.5 provides environmental stress to parasites.

2. The tremendous burst of oxidative species from macrophages

Still, a subset of parasite survives and establish infection in macrophages.

HOW? Who acts as an environmental sensor and initiate a survival signaling cascade for the parasites?

nucleus

mitochondria

mitochondrial ROS

NOsynthaseNOsynthase

L-ARG

NO

NADPH OXIDASENADPH OXIDASE

O2 O-

ROSTOTALROS

How do the parasites survive in the presence of ROS &RNS?

Macrophage environment:Temperature 37 C and pH 5.5

Different aspects of cAMP signaling that we wanted to study

Bhattacharya, A., Biswas, A. and Das, P.K. (2008) Free Radical Biol. Med. 44, 779-794.Bhattacharya, A., Biswas, A. and Das, P.K. (2009) Free Radical Biol. Med. 47, 1494-1506.Biswas, A., Bhattacharya, A., Kar, S. and Das, P.K. (2011) Eur. J. Immunol. 41, 992-1003.Biswas, A., Bhattacharya, A. and Das, P.K. (2011) Mol. Biol. Int. 2011, Article ID 782971, 9 pages.Vassallo, O., …Das, P.K., ..Majumder, H.K. and Desideri, A. (2011) The Open Antimicrob. Agents J. 3, 23-29.Bhattacharya, A., Biswas, A. and Das, P.K. (2012) Mol. Microbiol. 83, 548-564.

IRAK-M

MKPMKP1, MKP3

and PP2A

Validation of pathwaysby

immunomodulators SOCS ??

A20??

• TLRs present on macrophages, which upon countering a pathogen, result in the activation of NF-κB and production of pro-inflammatory cytokines leading parasite clearance.

• A20 inhibits NF-κB activation at an upstream level, by directly modulating the ubiquitination state of TRAF6

A20, an ubiquitin editing enzyme, inhibits NF-κB activation at an upstream level, by directly modulating the ubiquitination state of TRAF6

Administration of A20 siRNA prior to infection results in enhanced proinflammatory cytokine response and decreased parasite survival.

Srivastav, S., Kar, S., Chande, A.G., Mukhopadhyaya, R. and Das, P.K. (2012) J. Immunol. 189, 924-934.

TLR 2MyD88

IRAK4 IRAK1

TRAF6

TAB2TAK

TAB

IKK

IκB

A20

Proteasome

NF-κB

Pro-inflammatory cytokines

L. donovani

Lys 63 linked ub chain

P

P

P

P

Srivastav, S., Kar, S., Chande, A.G., Mukhopadhyaya, R. and Das, P.K. (2012) J. Immunol. 189, 924-934.

• Activation of MAPK occurs primarily through phosphorylation of the TxY motif.

• Because MAPK have to be phosphorylated on both threonine and tyrosine residues for kinase activity, inactivation can be brought about by members of different phosphatase families.

• MAP Kinase directed Phosphatases include serine-threonine phosphatases (PP2A and PP2C), the tyrosine phosphatases (PTPs) and members of the DUSP family.

MAP Kinase directed Phosphatases

• Gene expression profiling revealed that L. donovani infection markedly upregulated the expression of 3 phosphatases, MAP kinase phosphatase (MKP)1, MKP3 and protein phosphatase 2A (PP2A).

• Inhibition of these phosphatases prior to infection points towards preferential induction of Th2 response through deactivation of p38 by MKP1.

• On the other hand, MKP3 and PP2A might play significant roles in the inhibition of inducible nitric oxide synthase (iNOS) expression through deactivation of ERK1/2.

Kar, S., Ukil, A., Sharma, G. and Das, P. K. (2010) J Leukocyte Biol. 86: 1-12

Another strategy deployed by Leishmania could be to suppress the oxidative burst of the host macrophages, the first line of defense against any invading pathogen.

Mitochondria is the major cellular organelle related to generation of reactive oxygen species, thereby contributing significantly to cellular oxidative burst.

Our aim-

Is Leishmania capable of suppressing mitochondrial ROS generation?

If so, what are the factors responsible for that? Elucidation of the functional significance of the factor(s).

Leishmania infection upregulates UCP2, knock-down of which results in mitochondrial ROS generation, decreased spleen parasite burden, reduced PTP activity and induction of

leishmanicidal Th1 cytokines ,in vivo

Basu Ball, W., Kar, S., Mukherjee, M., Chande, A.G., Mukhopadhyaya, R. and Das, P.K. (2011) J. Immunol. 187, 1322-1332.

M

UCP2 knocked-down macrophages

PTPMAPK

P

Th1 cytokines M

Normal infected macrophages

PTPMAPK

P

Killed Ld

Th1 cytokines

Basu Ball, W., Kar, S., Mukherjee, M., Chande, A.G., Mukhopadhyaya, R. and Das, P.K. (2011) J. Immunol. 187, 1322-1332.

IRAK-M

MKPMKP1, MKP3

and PP2A

Validation of pathwaysby

immunomodulators SOCS ??

A20??

Lab Alumni

Dr. Prasanta Chakraborty Dr. Lopamudra Das

Dr. Rupnarayan Sett Dr. Neeta Datta

Dr. Nilanjana Basu Dr. Sudipan Karmakar

Dr. Kakali Sarkar Dr. Tapasi Das

Dr. Labanyamoy Kole Dr. Snigdha Mukherjee

Dr. Abhijit Ghosh Dr. Anindita Bhattacharya

Dr. Anil Bhunia Dr. Aruna Biswas

Dr. Chaiti Ganguly Dr. Arijit Bhattacharya

Dr. Keya Bandyopadhyay Dr. Susanta Kar

Dr. Nikhil K. Basu Dr. Arunima Biswas

Technical Staff

Ms. Arti Kheterpaul Mr. Dipak K Guin

Mr. Biswajit Mandal

Graduate Students

Mr. Gunjan Sharma Mr. Kuntal GhoshMr. Writoban Basu Ball Ms. Purnima GuptaMs. Supriya Srivastava Ms. Jayeeta GiriMs. Madhuchanda Mukherjee Ms. Amrita SahaMs. Shreyasi Palit

Financial Aid

 

1. United Nations Development Programme (UNDP)

2. Department of Biotechnology (DBT)

3. Indian Council of Medical Research (ICMR)

4. Department of Science & Technology (DST)

Thanks for your patience