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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