Cell Biology of Leishmania

Dr. Anuradha Dube

Division of Parasitology
Leishmaniasis
An Overview
 Major Tropical Diseases

Most important diseases of World Health Organization

/Tropical Disease Research (WHO/TDR)
 Malaria1st
 Schistosomiasis
 Filariasis
 Chagas disease
 African Trypanosomiasis
 Leishmaniasis2nd
 Leprosy
 Tuberculosis
Introduction

Leishmaniasis
Protozoal disease of mammals
Largely zoonotic
23+ pathogenic species

Cutaneous leishmaniasis
Visceral leishmaniasis
The Parasite (Classification)

Phylum Sarcomastigophora

Order Kinetoplastida

Family Trypanosomatidae

Genus Leishmania
 Types of Leishmaniases
Leishmania species cause three clinical syndromes depending on the
spread of the infection in the body

Cutaneous Mucocutaneous Visceral

Self limiting but chronic Initially causes similar Very severe

Skin ulcer dev at the site skin ulcers that heal but systemic disease
Which may takes months subsequently lesions with the organisms
To heal (scar) reappear primarily in homing to liver,
mucous tissue of nose spleen & B.M.
& mouth often accom- (usually fatal if
panied by sec. infection not treated)
L.major & massive tissue
L.tropica destruction L.donovani
L.mexicana complex L.infantum
L. aethiopica L.braziliensis L.chagasi
complex
Species Pathogenic in Humans

Leishmania aethiopica

Leishmania brazilieinsis (complex)

Leishmania mexicana (complex)

Leishmania tropica major

Leishmania tropica (minor)

Leishmania donovani (complex)

 Leishmaniasis in Sudan, India, Afghanistan, and the USA

Kala azar clinic in Duar, Sudan,

Ward at Kala azar

Medical Research
Centre in Muzaffarpur,
Bihar , India
Cutaneous infection in Cutaneous infection
Kabul, Afghanistan; school (both legs) in a
children with facial lesions contrasting setting
(private clinic in New
York USA) in a traveller
returned from Israel.
 Global distribution of Leishmaniasis
 12 million people infected
in 88 countries

 350 million at risk (WHO

report)

 3-5 million clinical cases

Cutaneous every year
Leishmaniasis

Visceral
Leishmaniasis

 Endemic in 62 countries
 200 million people at risk
 500 000 new cases each year
worldwide
 41 000 recorded deaths in the
year 2000
 Endemic areas of Kala-azar (VL) in India

Bihar: the state most badly

affected by visceral leishmaniasis
Accounts for >90%of the cases

A new wave of epidemic VL is

sweeping through

Reactivation of sub-clinical
infection into disease
in AIDS patients
General
habitat
where VL
cases
occurs
 Visceral leishmaniasis ( Kala-azar/Black fever
/Black sickness /Burdwan fever/Dumdum fever/sarkari bimari)

1903 William Leishman

1920 Pentavalent antimony
1931 Experimental transmission

Leishmania donovani (complex) Leishman and

L.d. archibaldi (Sudan/Ethiopia) Donovan discovered
the Leishmania
L.d. infantum (Mediterranean) parasite

L.d. donovani (Indian subcont., Sudan, Kenya)

L.d. chagasi (S.America)
Kala Azar - Visceral Leishmaniasis
 Caused by the L. donovani complex

 General infection of macrophages in

the entire RES

 Weeks to months (3-100 weeks)

incubation period

 Abdominal swelling (hepato- and

splenomegaly

 Often but not always fever occurs in

two daily peaks (Prolonged Low-
grade fever)

 Progressive weight loss

 Darkening of the skin

 Mortality of untreated disease 75-

95%
Visceral leishmaniasis (L donovani) in Bihar State, India
Clinical Symptoms of VL
•Hepatosplenomegaly
•Bone marrow hyperplasia
•Leucopenia & cachexia
•High titres of IgG
(Hypergammaglobulins-
mainly IgG from polyclonal
B cell activation)
•Depressed CMI
•Complicated by secondary
opportunistic infections
•Giemsa-stained splenic
aspirate smear, showing
clumped mononuclear cells
numerous amastigotes.
• Serodiagnosis of KA by
anti-K39 ab detection in
Immuno-chromatographic
strip test.
Right-hand strip shows second pink
band Indicating presence of anti K39.
Left- hand strip shows a negative
result.
 Post Kala Azar Dermal
Leishmanoid
(non-ulcerative cutaneous lesion)

Normally develops< 2 years

Recrudescence
Restricted to skin
Rare but varies geographically
 CL- Cutaneous Leishmaniasis
(Oriental Sore)

Old World New World

 Leishmania  Leishmania braziliensis

aethiopica  Leishmania mexicana
 Leishmania major
 Leishmania tropica
Spectrum (clinical features)
LCL Self healing (0.5 –2 years) –MCL Self healing (2-3
years)-DCL Life long non-ulcerative infection
Cutaneous Leishmaniasis is usually
self-limiting
 Old world oriental sore is caused by
parasites of the L. tropica complex.
(similar disease in the new world is
caused by L. mexicana)
 A chronic but self-limiting dry
ulceration at the site of the bite
 Ulceration start months after infection
 Parasites are not found outside the
lesion
 Nearly absolute resistance to re-
infection (however, there is long term
persistence and persistence is
required for resistance)
Localized Cutaneous
Leishmaniasis

Single or multiple lesions

Usually on head and /or neck
Generally self-healing
Variable few week to many
months
Ulceration followed by healing and scar
Secondary infection & tissue erosion
(lesion enlarges remaining red
but without pain; leads to necrosis;
formation of healing granuloma)
 Mucocutaneous Leishmaniasis (Espundia)

Direct inoculation or extension

L.aethiopica & others
Low cell mediated immunity (CMI)

Metastatic spread
Caused by L. braziliensis
~20% of infected patients develop ulcers of the
oral & nasal mucosa
Progression of ulceration is slow but steady,
ultimately destroying all soft parts of nose,
lips, & soft palate
High CMI & extensive tissue destruction
Extensive destruction of naso-oral &
pharyngeal cavities with hideous disfiguring
lesions; Mutilation of face & great suffering for life)
Death occurs usually through secondary
bacterial infection
Diffuse Cutaneous Leishmanasis

Multiple diffuse spreading

lesions
Usually face & limbs rarely
trunk

No ulceration
Non-healing-life long
infection
No cell mediated immunity
Good antibody response
L. aethiopica (Old world)
L. mexicana mexicana (New World)
 Vectors
Phlebotomine Sandflies
6 genera world wide distribution
Phlebotomus & Lutzomia
500 species
Females Haematophagus
Males sap feeders
Size of sand flies very small (< 3.5 mm)
Hard to see
Most active from dusk to dawn
Less active during hottest time of day
Female lays eggs - burrows of certain
rodents, bark of old trees, ruined buildings,
cracks in house walls, animal shelters & in
household rubbish (WHO website).
 Morphological types of Leishmania

Leishmania exist as pro- and amastigotes

Promastigotes: (Size: 2x 2-20 m) Nucleus
flagellated, motile (Insect midgut)
Kinetoplast
Procyclic (immature log phase)
metacyclic (mature, infective, stationary
phase)

Amastigotes: (size: 2-5  m)

non-flagellated, non-motile
(Mammalian macrophages)
 The Leishmania life cycle.

The Leishmania parasite shuttles between the sandfly vector (a,b) and the human host (c,d).
 Initial Infection

Leishmania invades
Similar in all species macrophages by receptor-
mediated phagocytosis

Inoculation of promastigotes

Inflammation & chemotaxis

Receptor mediated phagocytosis

Promastigote Amastigote

Transformation
 Parasite Spread

Macrophage lysis & parasite release

Lymphatic spread

Blood spread

Target organs

Skin/lymph nodes/spleen/liver/bone
marrow
Leishmania amastigotes
replicate in acidic vacuoles
containing lysosomal
enzymes and membrane
proteins
 Infection

Sub-clinical or inapparent infection

Immune
system of
the host

Recovery Death

Immune to reinfection concurrent infection

PKDL
Leishmania infects and thrives in
macrophages

How do they
get in and
how to they
avoid being
killed?
 Host cell for Leishmania
 Leishmania parasite do not force their entry, but rely completely
on the phagocytic capacity of host cells for uptake.

 No. of host cells investigated for their susceptibility to Leish

parasites in vitro or in vivo: such as: fibroblasts, kidney cells,
dendritic cells, mast cells, turtle heart cells etc.

 Not restricted to ‘Professional’ phagocytes (neutrophils,

monocytes & MQ).
 Every mammalian cell type can constitutively or facultatively
engulf particulate matter including microbes, provided that
phagocytosis promoting receptors complementary to the
respective particle ligands are expressed on the cell surface.
 Parasite invasion

o Promastigote Invasion and Establishment

of Infection

o Promastigote to amastigote differentiation

in macrophages

o Mechanism of Parasite persistence

1. Promastigote invasion & establishment of infection
1.1 Promastigote entry into the mammalian host
Sand fly secrete saliva during blood feeding & Prom. are deposited by
this process
 Prom. taken up by mononuclear phagocytes from the site of bite of
infected sandflies
Prom. escape complement pathways

Uptake of L. amazonensis metacyclic

promastigote by a mouse
macrophage. The parasite is
phagocytosed with the cell body
entering first and through the
formation of a long tubular
pseudopod. Images were captured
every 0.5 seconds over the course of
367 seconds.
 Evasion of the host complement system
Resistance to complement is due to:
 +nce of dense phosphoglycan coat on its surface so membrane attack
complex (MAC) unable to penetrate
Leishmanolysin or gp63 (membrane
protease)
 Cleaves C3b to a form which cannot
fix MAC
 Avoid activation of the lytic
MAC (C5b-C9)
 Shedding of lytic membrane
attack complex (C5b-C9)
Interaction of
 Gp63 converts C3b to C3bi on Leishmania
parasite surface, C3bi facilitates with
macrophages
binding to CR3 of macrophages
- interplay of
receptors
 Parasite protein kinases (LPK-1,
c-LPK2) phosphorylate C3,C5 & C9
of complement system thus inhibit
their further steps
Evasion of complement system by Leishmania-at a glance

 Leishmania stimulates this process by

binding elements of the complement
system to its surface
 Binding of complement can destroy
pathogens but also tags them for
phagocytosis (opsonization: pathogen
bound 3Cb is a potent ‘eat me’ signal for
macrophages & neutrophils)
 However, the parasite prevents the
formation of the fully functional
membrane attack complex
 A molecule on the surface of the
parasite seems to be responsible both
for complement activation and
prevention of the final attack
 1.2 Phagocytosis of promastigotes by macrophages
 Attachment and internalization:
parasites attached to MQ in a random non-oriented manner
 Prom binding & phagocytosis are receptor mediated events
 Bind to complement receptors CR1 & CR3

Two ways exists:

 Zipper type
phagocytosis

 Coiling type
phagocytosis

Uptake occurs via

circumferential engulfment
by pseudopods, resulting
in a strictly phagolysosomal
localization of Leishmania
 Phagocytosis of parasites by macrophages

Phagocytosis of a L. amazonensis metacyclic promastigote by a mouse

macrophage. The parasite binds to the macrophage plasma membrane by the tip
of the flagellum. It then turns around and is finally ingested via the cell body.
 1.3 Parasite Surface and secreted molecules
(Promastigote Ligands) for parasite- macrophages

 In addition to being distinguished by morphology & location,

various developmental stages of Leish parasites can be
distinguished by their surface molecule composition.

 Procyclic prom are covered by a 7-nm-thick glycocalyx. The

glycocalyx of metacyclic prom is even thicker, at least 17 nm, but
it is almost completely absent from amast.

 Glycocalyx comprises of glycoproteins & other glycosylated sp.,

which are anchored to surface membrane by a distinctive
glycosylphosphatidylinositol (GPI) linkage.
1.Lipophosphoglycan (LPG)
2. Glycoprotein (gp63)
3. Glycosylinositol phospholipid (GIPL)
4. Cysteine peptidases
5. Proteophosphoglycans
 Lipophosphoglycans play important
roles in Leishmania pathogenesis
• Lipophosphoglycan or LPG – a glycolipid, is the
dominant molecule on the surface of Leishmania
• The mole is anchored in the membrane by a lipid
to which a long chain of highly hydrophilic sugar-
phosphate repeats are attached
• LPG is structurally modified during the develop-
mental process of metacylogenesis (parasites
decide to stop replicating and begin to pre-adjust
to the mammalian host) to adapt to various
functions.
• LPG in metacylics has 2-3 times the no. of repeat
units
• Sidechains with terminal galactose are down-
regulated & remaining galactose residues are
capped with another terminal sugar
• LPG is a pathogenesis factor in the mammalian
host and important for the life cycle within the
sandfly
• Are these chemical changes responsible for the
pathogens attachment phenotype?
Only LPG from procyclics attaches to the midgut

• Phosphoglycans were isolated

from procyclics (those that
attach) and metacyclics (those
that detach and infect) and were
labeled with a dye (resulting in
fluorescence seen as ‘white in
the lower panels to the right)

• Opened sandfly midguts were

incubated with PG from
procyclics (A/B) and metacyclics
(C/D) and detected with an
antibody

• Only procyclic phosphoglycan

binds to the midgut
 1.3 Parasite Surface and secreted molecules
(Promastigotes Ligands) for host macrophages-contd

2. Glycoprotein (gp63)- prom surface protein, a

Zn-metalloprotease with a wide range of
substrates, including casein, gelatin,
albumin, hemoglobin, and fibrinogen

 10-fold less abundant than LPG, it is still

found throughout the prom surface, its
shorter length means that it is essentially
buried under a sea of LPG.

 Like LPG, gp63 is down-regulated in the

amast form. This reduced expression may
be counteracted by the absence of LPG
on the amastigote surface, meaning that
gp63 is no longer masked and may
therefore play an important role in amast
survival and modulation of the host
response.
 1.3 Parasite Surface and secreted molecules (Promastigotes
Ligands) for host macrophages-contd
3. Glycosylinositol phospholipid (GIPL)- a class of GPI-linked glycolipids,
most abundant prom surface mole
 10 times more abundant than LPG, but their small size keeps them
close to the parasite membrane, the role in interaction with the host
is unclear
 Unlike LPG, which is continually shed, GIPL has a long half-life and
hence play a protective role at the prom. surface.

4. Cysteine peptidases: some of these have roles in promoting parasite

survival & disease progression,
 A significant no. of CPs are released following amast death as a
result of their unusual intracellular trafficking pathway;
 these released proteases can modulate MQ activity by acting
directly on the host cell surface or following entry into the MQ
endoplasmic reticulum from the phagosome.

5. Proteophosphoglycans: Mucin like secretory and membranous

glycoproteins, its role in survival and pathogenesis.
 2. Conversion of promastigotes to amastigotes in
macrophages
2.1 Parasitophorous Vacuole (PV) formation & microbicidal
mechanism
• Uptake of promastigote in Phagosome; Phagosome fuses with sec.
Lysosome forming phagolysosome or PV
• PV is acidic, rich in hydrolytic
enzymes, microbicidal peptides
produced by membrane
proton pumps
• Conversion by loss of flagellum,
reduction in size, change in
gene expression
•Transformation into amast., which
are very resistant to low pH, NO,
lysosomal enzymes

Transition mechanism is unclear.

Amastigote Survival in Macrophage

What are the biochemical and

immunological changes that make the
amastigotes so well adapted to the
hostile intracellular environment of the
PV with its acidic pH & abundance of
hydrolases?
 Essential nutrients, cations &
Life in the macrophage. carbon sources are delivered to
phagolysosome via endocytic
pathway or directly from
macrophage cytosol.
Amast. might internalize low
mol wt nutrients (e.g. hexose,
amino acids, polyamines,
purines & vitamins) & cations
(Fe2+, Ms2+) via plasma membr.
transporters, often in
competition with phagolyso-
some membr. transporters.
Also internalize large
macromolecules (e.g. proteins,
carbohydrates, DNA & RNA by
endocytosis.
Heme can be obtained by
endocytosis of host proteins or
through uptake of free heme by
Flagellar Pocket receptors.
A tight junction might form
between posterior membr. of the
amast. & phagolysosome
membr. & be involved in
scavenging host lipids.
Evasion strategies of Leishmania within macrophage
Inhibition of macrophage functions

Sequestering itself insides cells of host allows

Leishmania to escape many of immune responses that
would otherwise be directed against it. However, its also
necessary to inhibit numerous MQ functions,
particularly those involved in immune surveillance and
macrophage activation, at protein or gene expression level.

1. Protection against antileishmanial products of macrophages or

suppression of synthesis of antileishmanial molecules
2. Antigen processing, presentation and T cell stimulation
3. Modulation of cytokine production
4. Alteration of T cell differentiation
5. Modulation of macrophage apoptosis
1. Protection against antileishmanial products of
macrophages or suppression of synthesis of
antileishmanial molecules
Invasion of host cells lacking leishmaniacidal effector mechanisms “safe
targets”- Immature or stromal MQS, Langerhans cells, dermal or
lymph node fibroblasts
“Pregnant pause” strategy for survival:
Inhibition of phagosome-lysosomal fusion (by LPG) and of degrading
phagolysosomal enzymes (by gp63)
Scavenging of ROI (by LPG & leishmanial superoxide dismutase)
Transformation into Amast. (Enhanced resistance against low pH, H2O2,
NO, lysosomal enzymes)
Inhibition of oxidative burst and drastic decrease in H2O2, NO, or O2-.
Production ( LPG,Gp63)
Inhibition of iNOS expression or activity (L. major parasites, GIPLs,
phosphoglycan, KMP-11)
Suppression of protein kinase C activity by LPG
 3. Inhibition of antigen processing, presentation
and T cell stimulation
Activation of Th1 cells by APCs require

 expression of MHC class II antigens

 Interaction of co stimulatory receptor-ligand pairs like
B7/CD28, MHC class II/CD4
 Peptide presentation by MHC class II

But during Leishmania infection, there is

 Suppression of MHC class II expression

 Internalization & degradation of MHC class II antigens
Sequestration of amastigote antigens from presentation in an
endocytic compartment
 Reduced expression of co-stimulatory molecules by
macrophages
 Inhibition of peptide loading of MHC molecules
Reduced adhesion of T cells to infected macrophages
4. Modulation of cytokine production and T-cell
response of the host
Suppression of protein kinase C
activity by LPG & GIPLs
Inhibition of iNOS expression or
activity
Intact parasites or their products
inhibits IL-1, TNF-alpha,
IL-12 production

Parasites enhance TGF-beta &

IL-10 production

Induction of macrophage deactivating cytokines e.g. TGF-, IL-10

TGF- suppress NK cell, macrophage effector functions (e.g. NO,

superoxide production)

Suppression of macrophage activating cytokines IL-1 and IL-12

 5. Alteration of T cell differentiation

Rapid production of
IL-4 at early stage of
infection

Parasite antigens
promote develop
ment of counter-
protective T helper
lymphocytes

Results in disease
exacerbating Th2
response
 MECHANISM OF PARASITE PERSISTENCE

 Modulation of macrophage apoptosis

TNF- and GM-CSF induced in macrophages after infection protect
infected macrophages from apoptosis
Survival of infected macrophages followed by their necrosis helps
in spreading of infection

Invasion of New Macrophages and dendritic cells by

amastigote
Invasion of host cells lacking leishmanicidal effector mechanisms
e.g. immature or stromal macrophages

Invasion of dendritic cells which carry amastigotes from site of

infection in skin to draining lymph nodes, where the antigen +ntation
to the naïve T cell occurs & where parasites persist indefinitely.

In established infection mechanism of interaction of obligatory

intracellular amastigotes & its host cells are not well known
Exit of amastigotes from infected macrophages
Mechanism of exit of amastigotes is unclear

The accepted view :

infected macrophage
bursts, discharging
parasites in the
immediate vicinity of the
macrophage ghost.
However, data obtained
by video-microscopy
suggests that
amastigote-containing
vacuoles accumulate
at the periphery of the
infected cell, and the
amastigotes are released
over several hrs in a
process reminiscent of
exocytosis
A TH1 response is required for parasite control
and healing
• Stimulation with different cytokines leads to
the development of two types of T-cells
specialized for different immune responses

• Th1 & Th2 strongly downregulate each other

• This polarization has important consequen-

ces for the downstream response and can
spell life or death

• Non healing Leishmania infections are

characterized by a strong TH2 response

• Healing infections are characterized by TH1

• The parasites seems to manipulate this

balance in his favor, we don’t understand
yet how that is done
 Summary of host parasite interaction
 Evasion from complement cascade
 Synchronization with phagocytosis phenomenon
 Prom & amast interact with only those MQ which have poor
triggering of oxidative burst
 Parasite’s survival in MQ (Phagolysosome - a hostile
environment)
 Amastigote are provided with strong antioxidant enzymes (catalase,
SOD, glutathione peroxidase) to down regulate the O2
dependant killing of MQ
 Inhibition of antigen presentation, Suppression of T-cell function
and Modulation of host cytokine response
 Passive protection of parasite against antileishmanial products
 Induction & expansion of counter production of T helper cells
 Presence of promastigote ligands- LPG & Gp63 which helps at
various stages of interaction.
 Conclusions
Macrophages acts as ‘safe target cells’ for Leishmania
Multiple evasion & survival mechanisms exists
Alone they are not sufficient for survival but together
all of them help in persistence & survival of
Leishmania
Understanding of complex interactions of host cells
with Leishmania will influence the development of
novel strategies of immunoprophylaxis and
therapy
Further studies on mechanisms of parasite evasion will
enrich our understanding of anti-parasite immune
defense
 Evasion strategies of Leishmania - A summary

1. Evasion of the host complement system by

Promastigotes
2. Protection against antileishmanial products of
macrophage or Suppression of synthesis of
antileishmanial molecules
3. Modulation of cytokine and T-cell response of the
host
4. Modulation of Macrophage apoptosis
5. Inhibition of antigen processing, presentation in
macrophage and T-cell stimulation
6. Alteration of T cell differentiation/function
Induction of VL
In asymptomatic individuals
(left), a Th1 type immune
response is induced.
Dendritic cells produce IL-12
and prime antigen-specific T
cells to production of
effector cytokines (IFN-g,
TNF-a). The parasite is
controlled within functional
granulomas (based on
experimental models and
the examination of
asymptomatic dogs, which
are natural reservoirs. There
is a balance between
effector responses, which
control the parasites, and
regulatory cytokines and
cells (e.g. IL-10, natural
regulatory Foxp3 T cells),
which limit collateral tissue
damage. As a result of
combinations of genetic and
environmental factors
(orange arrow),

VL ensues when innate and/or acquired immune responses are inadequate to clear or control the infection
(right). Persistent stimulation with parasite antigen induce high levels of pro-inflammatory cytokines, which in
turn triggers anti-inflammatory control mechanisms, including differentiation and expansion of IL-10-producing T
cells. The elevated levels of IL-10 promote progressive disease by the activities as depicted in Figure .
Macrophages infected in vitro with L donovani

(A) (A) Ingestion of two flagellated

promastigotes (arrow) by
human monocyte derived
macrophages. Cells were fixed
to show macrophage
morphology.

(B) Replication of intracellular

amastigotes within mouse
peritoneal macrophages.
(B) Methanol fixation shows
characteristic amastigote
features (round nucleus, rod-
shaped kinetoplast; arrows)
seen in clinical specimens.
Original magnification, 500.
 Dynamic relation in the clinical spectrum of human leishmaniasis
between effective CMI, DTH and parasite burden
(1) asymptomatic infection (vigorous
but not pathologic CMI/DTH; low
parasite burden) and moving
counterclockwise, CMI/DTH and
parasite burden are inversely related.
At end of spectrum (4), however,
nonhealing chronic disease is
produced differently, by either: (a)
uncontrolled infection because of
absent (diffuse cutaneous
leishmaniasis) or ineffective CMI/DTH
(visceral leishmaniasis, PKDL in India),
or (b) unrestrained CMI/DTH and
pathologic inflammation despite low
parasite burden (mucosal
leishmaniasis, chronic cutaneous
leishmaniasis). Self-healing disease,
associated with cutaneous (2) or
visceral infection (3), is shown at
In this scheme, reactivation (not shown) of separate points since clinical disease is
subclinical (asymptomatic), self-healing, or different and initially CMI/DTH is less
successfully-treated infection would be associated well-developed and parasite burden
with impaired CMI/DTH and high parasite burden. higher in (3).
 The actions of IL-10 might contribute to
IL-10 and VL pathology VL pathology in multiple ways:
a) IL-10 renders MQs unresponsive to
activation signals (i.e. IFN-g) and
inhibits the induction of RNI and ROI.
(b) In Ag-+nting cells (DCs & MQs ), IL-
10 downregulates expression of MHC
class II, co-stimulatory molecules & IL-
12, which will inhibit the effective
generation and/or maintenance of ag-
specific Th1 cells. Moreover, IL-10
inhibits DC maturation and migration
and can induce tolerogenic DCs, which
produce IL-10 and promote the
generation of IL-10-producing Tr1 cells.
(c) IL-10 might also contribute to
enhanced activation-induced T-cell
death and
(d) promote B-cell survival and plasma-
cell differentiation. As disease
progresses, B cells, which might also
be a source of IL-10, and abs could be
imp.contributors to VL pathology
because self-reactive antibodies and
immune-complex deposition might
cause tissue damage.
(e) Moreover, immune complexes can
stimulate the MQs and monocytes to
produce IL-10 as well as proinflamma-
tory cytokines (e.g. IL-6, TNF-a), a loop
that will promote the generation of more
immune complexes and more IL-10.
Black arrows indicate sources of IL-10; the red
lines indicate blocking/ downmodulating
activities of IL-10; the green arrows indicate
differentiation/apoptosis promoted by IL-10.
 Cutaneous and mucosal disease
A. Old world infection (L major) acquired in Iraq;
note five papular and nodular lesions on
neck .
B. New world infection (L panamensis) in
Colombia; purely ulcerative lesion is
characteristic of new world disease .
C. Healed infection in patient shown in (B) 70
days after 20 days of meglumine antimoniate
treatment; note paper-thin scar tissue over
flat re-epithelialised skin.
D. Old world (L tropica) infection in Afghanistan
with extensive hyperkeratotic plaque .
E. Destructive mucosal disease (presumed L
braziliensis) in Peru .
F. Diffuse cutaneous disease (L panamensis) in
Venezuela with mutliple nodular lesions.
G. Post kala azar dermal leishmaniasis in India
A. (L donovani) with papular-nodular lesions
over face, chest, and arms .
3.2. Evasion and subversion of the host immune response
 Leish (chronic disease) – slow in turning on the host protective MQ -
activating immune responses.
 Evolved various ways to interfere with host immune responses:

-Modulating cytokine production

L. mexicana, L. major, L. braziliensis – trigger production of TGF- &
IL-10 which inhibit killing of amastigote.
Recovery from infection is critically dependent on MQ- activating
cytokine & infection with prom inhibits production of IL-12 & TNF-.

-Inhibiting antigen presentation

Parasite causes reduction of host MHC class II moles available for
binding to parasite antigens which presumably helps prevent its
detection in MQ by T-cells.
In L. amazonensis & L. major – achieved by sequestration and
degradation of class II antigens by amastigote. In L. amazonensis, the
MHC class II molecules seem to accumulate in amastigote specific
organelles = as megasomes and amastigote specific proteases are
involved in hydrolysis of MHC moles.
IL-10 and VL pathology The actions of IL-10 might
contribute to VL pathology in
multiple ways.

(a) IL-10 renders macrophages

unresponsive to activation
signals (i.e. IFN-g) and inhibits
the induction of RNI and ROI.

b) In ag-+ing cells (DCs &Mqs),

IL-10 downregulates the
expression of MHC class II, co-
stimulatory moles & IL-12, which
will inhibit the effective
generation and/or maintenance
of ag-specific Th1 cells.
Moreover, IL-10 inhibits DC
maturation and migration and
can induce tolerogenic DCs,
which produce IL-10 and
promote the generation of IL-10-
producing Tr1 cells.

(c) IL-10 might also contribute to

enhanced activation-induced T-
cell death and

(d) promote B-cell survival &

plasma-cell differentiation.

As disease progresses, B cells, which might also be a source of IL-10, and antibodies could be important contributors to VL
pathology because self-reactive antibodies and immune-complex deposition might cause tissue damage. (e) Moreover, immune
complexes can stimulate the macrophages and monocytes to produce IL-10 as well as proinflammatory cytokines (e.g. IL-6,
TNF-a), a loop that will promote the generation of more immune complexes and more IL-10. Black arrows indicate sources of IL-
10; the red lines indicate blocking/downmodulating activities of IL-10; the green arrows indicate differentiation/apoptosis
promoted by IL-10. Abbreviations: ROI, reactive oxygen intermediates; RNI, reactive nitrogen intermediates.
2. Microbial Free Radical Production
Two types of microbicidal molecules are recognized for their efficacy
against Leishmania:
(1) Nitric Oxide (NO)-
 Critical for parasite clearance since host lacking inducible nitric oxide
synthase (iNOS) are unable to control infection and MQs derived from
these hosts are incapable of eliminating promastigotes in culture.
 IFN-g & LPG can synergize to generate NO when administered
simultaneously to naive macrophages.
 Infected MQs or MQs incubated with purified LPG or GIPL Leishmania
surface molecules lose their ability to induce iNOS or to generate NO
in response to IFN-g and/or LPS.
 This suggests that contact between the parasite and the macrophage
prevents the macrophage from responding to subsequent exposure to
IFN-g produced by lymphoid cells.
 Inhibition of NO production may result from the production of IL-10
and/or TGF-b, inactivation of the JAK/STAT pathway, activation of
phosphotyrosine phosphatases, and/or ceramide production,
2. Microbial Free Radical Production-Contd
(2) Reactive Oxygen Intermediates(ROI)

 Play a less important role in parasite clearance since in contrast to

mice deficient for NO production, mice deficient for the generation of
ROI can ultimately control the infection after an initial period of
increased susceptibility

 ROI generation is also inhibited by L.donovani infection. Inhibition

appears to be dependent on the surface molecules LPG and gp63
and has been shown to involve abnormal PKC activity
 DENDRITIC CELLS
 Abundant evidence: host cell carries amastigote from
initial site of infection in skin to draining lymph
nodes, where the antigen +ntation to the naïve T cell
occurs & where parasites persist indefinitely.
 Cell which ferries amastigotes from the skin to lymph
nodes - a dendritic cell (Langerhans cell). The receptor
responsible for interaction with amastigote, as well
as ligand on the amastigote, are unknown.
 Dendritic cells are required to initiate primary T-cell
responses where MQ can only +nt antigen to T-cells
that are already primed.
 In contrast to infected MQs which seems to be
impaired in antigen +ntation, the infected
dendritic cells are competent to +nt antigen and
initiate T-cell immune responses to the parasite.