L.Donovani influenced cytokines and TLRs expression
among Sudanese visceral leishmaniasis patients
By: Dr.DinaTag Elsir
Symposium on: Advances in Parasitology “Education and Research in
Parasitology in the service of Mankind”
Background
 Leishmaniasis
remains
a
serious
health
problem. The outcome of Leishmania infection
depends on the early innate response that
directs the subsequent adaptive immune
response to the infecting Leishmania parasite.
 The early innate immune response by the host,
including macrophage - parasite interaction,
are crucial in determining the persistence or
clearance of the parasites (Bosque, et al 2000).
 Susceptibility to visceral leishmaniasis
correlates with the presence of a Th2 response
(Nyl´en S. and Sacks D. 2007).
 Toll-like receptors (TLRs) are hallmarks of cellular
receptors that recognize pathogen-associated
molecules and participate in innate responses to
infections (Kaye and Aebischer 2011).
 We use in this study a whole blood model of
stimulation to mimic the real infection scene
and also a THP-1 cells model to detect the
influence of the macrophage cell alone
without the presence of the rest of other
immune cells.
 We compare some of the outcome of these experiments in
each model to detect if there is a similarity in stimulation and
infection of these cells of both models which can give us a
guide for forthcoming experiments and can help in
understanding of the interplay between Leishmania and its
hosts that leads to resistance or susceptibility.
 We measure the expressions of TLR 2, TLR4,
and TLR9 and we measure the production of
some cytokines in whole blood samples of
visceral leishmaniasis patients and
in the
human macrophage cell line THP1 following
stimulation
promastigotes.
with
live
L.
donovani
• Early events during Leishmania infections are
a crucial phase that determines the
persistence of the parasite or successful cure
by the host immune response. The fact that
the host cells of Leishmania parasites are the
macrophage lineages cells suggest an
important role of these cells in the
pathogenesis of leishmaniasis.
• Macrophages are known to recognize
microbes via their toll like receptors which are
currently the focus of the analysis of the
innate immune responses.
Materials and Methods
 The study was approved by the ethics
committee of the Institute of Endemic
Diseases, University of Khartoum.
 A consent forms were obtained from all
participants before their enrolment.
THP1 cell
line
Patients and
Controls whole
Blood
TLRs
L.donovani Live
Real Time
PCR
cDNA
Synthesis
Cytokines
RNA
Extraction
β Actin, TLR2, TLR4 and TLR9 oligonucleotides primers:
Actin β
TLR2
TLR4
TLR9
FW
5’-CTG TGG CAT CCA CGA AAC TA-3’
RV
5’-AGT ACT TGC GCT CAG GAG GA-3’
FW
5’-CGA TAT GCT AAA CAC AAT GAC -3’
RV
5’- CAA ATG ACG GTA CAT CCA CGT -3’
FW
5’CAGACATCAAGGCGCAT-3’
RV
5’TTCTTCACCTGCTCCACG-3’
FW
RV
5’- GGG TTG GAA GAT GCT AGA AGA -3’
5’- CGA GCA GGG GAG GGT CAG ACC -3’
Cytokines:
1. IFN-γ
6. IL-1β
11. IL-6
16.IL-12p70
2. IFN-α2
7. IL-2
12. IL-7
17. IL-13
22. CCL2
3. TNF-α
8. IL-3
13. IL-8
18. IL-15
23. CCL3
4. TNF-β
9. IL-4
14. IL-10
19. IL-17
24. CCL4
20. G-CSF
25. CCL11
5. IL-1α
10. IL-5 15.IL-12p40
21.GM-CSF 26. CXCL10
Results & Disscusion
Cytokines
• G-CSF stimulates the survival, proliferation, differentiation, and
function of neutrophil precursors and mature neutrophils. This
cytokine action could highlight the role of neutrophil in first hours
post infection possibly the protection against Leishmania is
granulocyte mediated with other several mediators. G-CSF can be
used as an antileishmanial treatment since its correlation with
the protection mode found in LST -ve and LST+ve groups. These
intracellular parasites can use granulocytes as “Trojan horses” to
invade their definitive host cells, the macrophages (van
Zandbergen et al 2004).
• IL-1 production was altered in VL patients. It has been
shown that IL-1α administration in the susceptible BALB/c
mice resulted in increased Th1 and strikingly decreased
Th2 cytokine production. (Von Stebut . et al, 2003), IL-1
has been shown to affect the pathogenicity of
leishmaniasis by generating an inflammatory response in
afflicted tissues and by modulating adaptive T cellmediated immune responses, which act to limit parasite
dissemination (Von Stebut . et al, 2003, Kostka. et al
2006).
• Increased GM-CSF production following infection allows
enhanced support of myelopoiesis. It was reported previously
the addition of GM-CSF to human monocytes invitro increases
their leishmanicidal effects (Dumas, et al 2003). Also when mice
infected with L. donovani were treated with murine GM-CSF,
they showed increased leishmanicidal activity, as well as
leukocytosis and myelomonocytic accumulation in infected
viscera (Singal, and Singh. 2005).
• GM-CSF can activate macrophages to produce high
levels
of
proinflammatory
cytokines
such
as
interleukin-1β (IL-1β), IL-6, and IL-18 and various
chemokines including CCL5, CCL4, CCL3, CCL2, which
enhance parasite killing. In this study, they were
found to be very high and non discriminating
between the study groups which in accordance with
production of GM-CSF in all the three clinical groups.
• The development of cell-mediated immune responses capable
of controlling Leishmania infection and resolving disease is
critically dependent on interferon gamma (IFNG), secreted
primarily by activated T cells and NK cells in response to IL-12
(Ghosh et al 2008). Since the protective role is provided to LST ve group as well as LST+ve controls, the non discriminating
production of IL-2 and IFN-γ between the VL patients and LST ve group suggesting factors other than TH1 may be genetic
factors and it has been demonstrated previously the differences
in genetic susceptibility to VL in the Sudanese population
(Mohamed et al 2003).
• IL-10 is known to play a regulatory role in Leishmaniasis.
A previous report showed that few hours following
exposure to L. amazonensis, PBMCs from naive
volunteers presented a predominant Th2 response.
Such response is supported by a high production of IL13 and IL-10 production. However, despite of this
cytokine milieu, IFNG was produced (Rogers and Titus,
2004).
• Dendritic cells can induce in vivo development
of Th2 cells in the absence of IL-4 is in
agreement with our result suggesting that IL-4
is not required for the initiation of Th2
response and other cytokines such as IL-13
that influence the development of Th2
response (Sacks and Anderson, 2004).
• Moreover our data showed an increase in coproduction of IL-10 and IFNG levels in patients and
controls, which reflected the mixed T cell subsets
pattern in our study groups, and this is in consistent
with many previous reports (Louzir
et al 1998,
Bosque et al 2000, Belkaid et al 2001, Khalil et al
2005,Costa et al 2012).
• A number of earlier studies have implicated IL-10 in
the immune suppression associated with Leishmania
infection, arguing either for Th2 response (Ghalib et
al 1995) or regulatory T cells (Belkaid et al 2002) as
the main source of immune-suppressive IL-10.
• CXCL10 chemokine is capable of recruiting and
activating NK cells, which are IFNG producers, (Vester
et al 1999). In this study the production of CXCL10 is
clearly associated with the pathogenicity of VL and
can be a biomarker for disease progression.
• The correlation of this chemokine with other tropical
diseases had been reported, CXCL10 were found tightly
associated with fatal cerebral malaria (Wilson. et al,
2011) and Tuberculosis (Hassan. et al, 2011). CXCL10
and other proinflammatory factors in patients with
active skin and mucosal lesions suggest a possible
involvement of these molecules in disease outcome.
• Measurement of cytokines concentration in THP1
human macrophages produced IFNG concentration
more than all other clinical groups, it has been
reported
previously
the
auto
activation
of
macrophages (Schindler, et al 2001).
• A few studies reported production of IFNG by human
and murine macrophage after Leishmania infection
(Schindler, et al 2001, Bogdan and Schleicher. 2006).
• TNF
has
no
significant
difference
macrophages and the clinical groups
between
while
IL-10,
IL-1β and IL-6 were less than other groups. These
findings suggest that the presence of other cells in
the blood modulates the chemokine and cytokine
responses of macrophages to Leishmania infections.
Real Time
PCR
• A significant increase in expression of TLR4, a receptor
known to bind to the proteoglycolipid complex ligand on
Leishmania parasite that, favoring a Th1 response and
increasing the production of inducible nitric oxide
synthase (Kropf et al 2004,Chandra et al 2008).
• Similarly, the same VL samples showed a significant
increase in the expression of TLR9, a known Th1 inducer
leading to protective immunity. TLR9 activation in NK
cells is known to induce IFNG production and enhance
the clearance of the parasite (Abou Fakher et al 2009)
• TLR2 is known to bind to lipophosphoglycan (LPG) on
Leishmania parasites leading to production of IFNG and
TNF proinflammatory cytokines production
Interestingly, LST-ve controls
(55,56).
showed similar TLR
expression profiles as the VL patients. The fact that
those individuals tested negative with Leishmanin skin
test (LST-ve) suggests their susceptibility to clinical VL.
• Despite the high production of IL-10 and IFNG
in LST+ve controls expression of these TLRs
were
negatively
correlated
with
the
production of these cytokines which reported
also by Srivastava et al 2013.
• The significant expression of TLR4 and TLR9 in
whole blood samples of VL patients compared
with stimulated THP1 cell line suggests the
participation of other cells eg: NK, DC, and
neutrophils in VL patients responses.
Conclusions:
• Live
Leishmania
promastigotes
increased
the
expression of TLR4 and TLR9 in peripheral blood
samples of VL patients leading to the induction of
aTh1 and Th2 or T reg mixed cytokine response. TLR9
seems to be recognising L.donovani DNA.
• On the other hand, our data support the hypothesis
that the TLRs activation seems to be the major
mechanism associated with active disease.
• In this context also the protective function of IFNG
was demonstrated among the LST+ve controls.
• Macrophage infection reveals a varied immune response
distinct of that of whole blood infection scenario.
• The increasing expression of TLR2 in THP1 cell line
induced IFNG-γ production more than in whole blood
samples, which suggesting the presence of other factors
in whole blood samples that affect the IFNG production.
• Despite infection and stimulation with one parasite
strain within these study groups, different immune
responses were elicited.