Stress in the regulation of the immune response in Leishmaniasis

Felix J. TapiaInstituto de BiomedicinaFacultad de MedicinaUniversidad Central de Venezuela

Reuniäo Cyted 2009Projecto Mecanismo de controle e imunoprofilaxia na LeishmanioseFaculdade de Medicina da Bahia (FAMEB)

Neuroendocrine-immune axis

Diffuse neuroendocrine system

Peripheral Immune system

Bioactive Polypeptides

hormones neuropeptides cytokines

Molecules of communication

Murine models of American cutaneous Leishmaniasis

C57BL/6 BALB/c

Th1IFN-

Th2IL-4

L. mexicanaMHOM/BZ/82/BEL21

Sánchez et al. Acta Micros 1993, 2: 180-187Aguilar-Torrentera et al. Am J Trop Med Hyg 2002, 66: 273–279

Díaz et al. Clin Exp Dermatol 2003, 28: 288-293

Sensory peptides: Calcitonin gene-related peptide (CGRP) Substance (Sub P) mediators of neurogenic inflammation

Epidermal dendritic (Langerhans) cells:Skin take up and antigen processing Peripheral lymphoid organs initiate primary T cell responses

Leishmania infection of BALB/c and C57BL/6 mice:103 amastigotes L. mexicana strain MHOM/BZ/82/BEL21 Course of infection 12 weeks

Sites of evaluation

Background

Dendritic Langerhans cells are involved in the pathogenesis of cutaneous leishmaniasis.

Epidermal cell signaling is essential to determine the type of cytokine-related immune response to be generated against Leishmania parasites.

Dendritic Langerhans cells, SubP and CGRP are affected by acute stress in contact hypersensitivity models.

Phases of Cutaneous Regulation

Activation Inmunostimulation Effector Phase

ChallengingAntigen CaptureMigration

Antigen PresentationClonal Expansion Recruitment

RetentionProliferationSuppression

epidermis

dermis

Mo

ag

Mo

..

.

granuloma

gangliolinfático

endoteliovascular

IL-4

mastocyte

IFN-

NK

MΦ

IL-12

T memory

QCCL

GM-CSF

IL-1TNF-

chemokines

C

T

T

Objective

Analyze the effect of acute stress (immobilization and odorant) on Langerhans cells, Sub P, CGRP, and the natural course of infection in susceptible and resistant mouse models of cutaneous leishmaniasis

Acute stressor (2, 8 hrs):Immobilization by placement in 50 ml polypropylene centrifuge tubes, before inoculation of Leishmania parasites.

Materials and methodsStress procedure

BALB/cC57/BL6

Measurement footpad thickness every week for 12

weeks

Mice stressed for 2 hr and infected with L. mexicana

Mice stressed for 8 hr and infected with L. mexicana

Mice infected with L. mexicanaInfection Control

Mice stressed for 2 hr and non-infected with L. mexicanaStress Control

(n=20)

(n=20)

(n=20)

(n=20)

Footpad biopsies taken at weeks 0, 4 and 8 after

infection

Cryopreservation

Immunodetection of CD205+ Langerhans

cells, CGRP and Sub P

(n=20)

(n=20)

(n=20)

(n=20)

Experimental design

Progression of L. mexicana infection in experimental groups of BALB/c and C57BL/6 mice

0 1 2 3 4 5 6 7 8 9 10 11 12 131

2

3

4

5

6

Weeks of iinfection

Size

of

lesi

on in

mm

0 1 2 3 4 5 6 7 8 9 10 11 12 131

2

3

4

5

6

2hr stress healthy

2hr stress infected8hr stress infectedNon-stressed infected

Langerhans cell density in experimental groups of BALB/c and C57BL/6 at weeks O, 4 and 8

0.0 4.0 8.0 12.00

250

500

750

10002hr stress healthy2hr stress infected8hr stress infecedNon-stressed infected

0

1000

2000

0 sem 4 sem 8 sem

Weeks of infection

cells

/mm

2

Progression of infection with L. mexicana in BALB/c mice subjected to acute stress

Langerhans cells Iad+ in separated epidermis of BALB/c mice infected with L. mexicana

Non stressed Stressed for 2 hr

Substance P in skin lesions of BALB/c mice stressed and infected with L. mexicana

week 4 - stressed for 8 hr

Vascularedotheli

um

infiltrate

Conclusions

Acute immobilization stress affects the numbers and function of Langerhans cells and Substance P and CGRP innervations.

The skew of the skin immune response after stress and infection may be diverted by epidermal accessory signals to distinct Th1 or Th2 responses in resistant or susceptible mice, respectively.

The genetic background of the mouse strain determined the response to acute stress in murine leishmaniasis.

Odorant inhalation stress in resistant and susceptible mouse models of cutaneous leishmaniasis

Acute stressor (48 hrs):Inhalation of citral for 48 hours.

At 24 hrs of stress, mice were infected Leishmania mexicana

Materials and methodsStress procedure

Odorant inhalation stress alters the immune response in resistant and susceptible mice infected with L. mexicana

Odorant inhalation stress alters the immune response in resistant and susceptible mice infected with L. mexicana

Odorant inhalation stress alters the immune response in resistant and susceptible mice infected with L. mexicana

Conclusions

Citral inhalation stress induces clinical and immunological alterations in the natural course of infection in mice inoculated with L. mexicana.

These alterations include decreased numbers of dendritic Langerhans cells and their morphological, and downregulation of Th1 and Th2 cytokines in both resistant and susceptible mice. Only IL-12 and iNOS remained in normal values.

The results indicate that citral inhalation suppress the immune response against the Leishmania parasite regardless the genetic background of the host.

Macrophages treated by sera from mice sressed and infected with L. mexicana24 hours, Giemsa

1 3 5 9 110

100

200

BALB/c stressed & infect

BALB/c infect

Weeks of infection

cells

/mm

2

Density of DC-SIGN positive cells in lesions of stressed and L.mexicana-infected BALB/c mice

1 3 5 9 110

1000

2000

3000

BALB/c infectado yestresado BALB/c infectado

* *

*

*

Density of macrophages MOMA-2 positive cells in lesions of stressed and L.mexicana-infected BALB/c mice

* p 0.05

cells

/mm

2

Weeks of infection

TLR-2 positive cells in BALB/c mice stressed and infected with L. mexicana5 weeks

1 3 5 9 110

100

200

300

BALB/c stressed & infect

BALB/c infect

Weeks of infection

cells

/mm

2

1 3 5 9 110

100

200

BALB/c stressed & infect

BALB/c infect

*

Weeks of infection

cells

/mm

2

TLR-4 positive cells in BALB/c mice stressed and infected with L. mexicana5 weeks

* p 0.05

CD14 positive cells in BALB/c mice stressed and infected with L. mexicana5 weeks

1 3 5 9 110

100

200

300

BALB/c stressed & infect

BALB/c infect

Weeks of infectionce

lls/m

m2

1 3 5 9 110

100

200

BALB/c stressed & infect

BALB/c infect

TLR-9 positive cells in BALB/c mice stressed and infected with L. mexicana5 weeks

cells

/mm

2

Weeks of infection

Laboratorio de Biología MolecularInstituto de Biomedicina

Estudiantes:Ysamar Chirinos

Alba QuiñonesMaría del Rosario Ruiz

Fabiola CabreraMaría Alejandra Sarabia

Eliana FigueiraMónica Fernández

Adriana ArbeláezCelsy Hernández

Alejandra Da AlmeidaPedro SalazarMaría García

Izaskun Urdanibia

Laboratorio:Nilka L. Díaz

Iraima B. MonsalveMónica SuárezMiguel MarzalLuis González

Colaboradores:Martín A. Sánchez

Zelandia FermínLadys Sarmiento

ObrigadoThanksGracias

American cutaneous leishmaniasis

C57BL/6IFN-

BALB/cIL-4

LCL

Intermediate forms

MCL CCL

DCL

CBAIFN / IL-4