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.