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”
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.
Background
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 with live L. donovani
promastigotes.
• 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
Real Time PCR Cytokine
s
TLRs
cDNA Synthesi
s
Patients and
Controls whole Blood
L.donovani Live
RNA Extractio
n
Actin β FW 5’-CTG TGG CAT CCA CGA AAC TA-3’
RV 5’-AGT ACT TGC GCT CAG GAG GA-3’
TLR2 FW 5’-CGA TAT GCT AAA CAC AAT GAC -3’
RV 5’- CAA ATG ACG GTA CAT CCA CGT -3’
TLR4 FW 5’CAGACATCAAGGCGCAT-3’
RV 5’TTCTTCACCTGCTCCACG-3’
TLR9 FW 5’- GGG TTG GAA GAT GCT AGA AGA -3’
RV 5’- CGA GCA GGG GAG GGT CAG ACC -3’
β Actin, TLR2, TLR4 and TLR9 oligonucleotides primers:
1. IFN-γ 6. IL-1β 11. IL-6 16.IL-12p7021.GM-
CSF
26.
CXCL10
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
5. IL-1α 10. IL-515.IL-
12p4020. G-CSF 25. CCL11
Cytokines:
Results & Disscusion
CytokinCytokineses
• 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 cell-mediated 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 IL-13 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 co-
production 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 between
macrophages and the clinical groups 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 Real Time PCRTime 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 (55,56).
Interestingly, LST-ve controls 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.