Aurora Salageanu

Laborator Imunitate Antiinfectioasa

Institutul Cantacuzino
 SISTEM IMUN

Imunitate Imunitate
innascuta dobindita
(prima linie de aparare) (a II-a linie de aparare)

Componente Componente Componente Componente

celulare umorale celulare umorale

adaptat dupa
“Microbiology and Immunology on-line”, University of South California, School of Medicine
http://pathmicro.med.sc.edu/book/immunol-sta.htm
 Celulele sistemului imun

preluat din
“Microbiology and Immunology on-line”, University of South California, School of Medicine
http://pathmicro.med.sc.edu/book/immunol-sta.htm
preluat din
“Microbiology and Immunology on-line”, University of South California, School of Medicine
http://pathmicro.med.sc.edu/book/immunol-sta.htm
 Tesutul limfoid
Distributia organelor limfoide

preluat din
“Microbiology and Immunology on-line”, University of South California, School of Medicine
http://pathmicro.med.sc.edu/book/immunol-sta.htm
 Circulatia limfocitelor in organele limfoide periferice

preluat din
“Microbiology and Immunology on-line”, University of South California, School of Medicine
http://pathmicro.med.sc.edu/book/immunol-sta.htm
preluat din
“Microbiology and Immunology on-line”, University of South California, School of Medicine
http://pathmicro.med.sc.edu/book/immunol-sta.htm
Raspunsul imun fata de patogeni
Raspunsul imun fata de patogeni
 IMUNITATE INNASCUTA

Raspuns initial pentru:

• eliminare microbi
• prevenire infectii

Intra in actiune imediat (in citeva ore) dupa

expunere la antigen

Disponibila in orice moment

(innascuta)
 IMUNITATE INNASCUTA
 Mecanism universal de aparare fata de infectii
 Prima linie de aparare a organismului
 Precede raspunsul imun adaptativ
 prezent la toate organismele pluricelulare
 receptori si efectori ancestrali
 raspunde la o mare varietate de patogeni
 Distinctie perfecta self-nonself
 Defecte foarte rare si in general letale
 IMUNITATE INNASCUTA

preluat din Janeway, CA, Travers, P, Walport M, Shlomchik M “Immunobiology“, New York and
London: Garland Science; c20
http://www.ncbi.nlm.nih.gov/sites/entrez?db=books
 IMUNITATE
INNASCUTA / nespecifica DOBINDITA / specifica
(innate immunity) (adaptive immunity)

 raspuns independent de antigen  raspuns dependent de antigen

 receptori pt. patogeni:
 codificati in genom
 specificitate joasa
 raspuns imediat
 lipsa memorie
 prezenta la toate organismele
pluricelulare
 receptori pt. patogeni:
 generati (rearanjare genica)
 specificitate inalta
 raspuns lent
 memorie imunologica
(expansiune clonala ly)
 prezenta doar la vertebrate

SISTEM INTEGRAT
 Specificitatea raspunsului imun adaptativ
Teoria selectiei clonale

 Fiecare limfocit: un singur receptor (specificitate unica)

 Interactia receptor-ligand  activarea limfocitului

 Celulele efectoare diferentiate dint-un limfocit activat:

acelasi receptor

 Limfocitele cu receptori self sunt eliminate (toleranta)

Innate immunity

ROL SI FUNCTII
originea si contextul atg
Recunoastere
Functii efectoare
microorganisme
• prevenire intrare patogene
• eliminare

Rol “instructiv” asupra imunitatii specifice

initierea si tipul raspunsului

Innate immunity

ELEMENTE CONSTITUTIVE

1. Bariere anatomice
2. Componente moleculare
3. Componente celulare
Innate immunity
1. Bariere anatomice
- piele
- mucoase

• Functia de prevenire a intrarii

microorganismelor patogene
• motilitate: muco-ciliara, peristaltica
Factori fizici / mecanici • mucus
• fluxul fluidelor prin organism

• pH
Factori chimici • molecule antimicrobiene

• limfocite T intraepiteliale ()

Factori biologici • celule B-1
Innate immunity
2. Componente moleculare
- receptori
- molecule secretate
 Functia de recunoastere
 Functii efectorii
Mol. anorganice: HCl, NO, H2O2
Peptide antibacteriene: defensine, cathelicidine, histatine
Proteine antibacteriene: lizozim, lactoferina, transferina
Lectine: colectine, ficoline, receptori manoza
Complement
Citokine: IFN-/, IL-1, TNF-, CSF
Chemokine: IL-8, MIP, MCP
Receptori TLR
Innate immunity

3. Componente celulare

• Functia de eliminare a
microorganismelor patogene

neutrofile
monocite/macrofage Fagocitoza
celule dendritice

bazofile
mastocite Inflamatie
eozinofile

celule NK Citotoxicitate
 Recunoastere imuna non-
self self

origine antigen / context biologic

Ipoteze
 Receptori (self vs. nonself microbian)
PRR Pattern Recognition Receptor

PAMP Pathogen Associated Molecular Pattern

(Medzhitov & Janeway, 1997)
Recunoastere imuna
RECEPTORI

INNASCUTA DOBINDITA
(innate immunity) (adaptive immunity)
 codificati in genom  generati prin rearanjare
genica

 distributie non-clonala  distributie clonala

(expansiune clonala ly)

 recunosc structuri  recunosc detalii de

conservate structura moleculara
(epitopi)
PAMP •lipopolizaharid
•peptidoglican
 invariabile •acid lipoteichoic
 inalt conservate •lipoproteine
 specifice microbilor •manoza
(patogeni + nonpatogeni) •ADN
 comune pentru o clasa de microbi •ARN dc
 vitale pentru microorganisme •flagelina
•pilina
•zimozan

Gram-negative Gram-positive
Pattern Recognition Receptors - PRR

• glycosylphosphatidylinositol
 LPS
(gpi)-linked receptor
• Scavenger receptors (SR)
Endocytic PRRs  oxLDL; microbes
• C-type lectin receptors (CLR)
 Microbes; microbial moieties

• Toll-like receptors (TLR)

 Microbial moieties
• NOD-like receptors (NLR)
 Bacterial lipoproteins
Signaling PRRs • RIG1-like helicases (RLH)
 Nucleic acids

 Sugars
• Collectins
Bridging  Glycoproteins
• Ficolins
molecules
(adaptat dupa Jeannin et al. “Pattern recognition receptors in the immune response against
 Microbes; microbial moieties
dying cells”, Current Opinion• Pentraxins
in Immunology 2008, 20:1–8)
Pattern Recognition Receptors - PRR

a) ENDOCITOZA Endocytic PRRs

Bacterie

glicoproteina proteina bact.

bact. LPS, LTA
man C3b

receptori manoza receptori scavenger receptori opsonine

(lectina C) (CD36, CD68, SRB-1) (CR1)

FAGOCIT
Pattern Recognition Receptors - PRR

b) TRANSMITERE SEMNAL Signaling PRRs

TLR
Toll-like receptors

NOD
nucleotide-binding
oligomerization domain

RIG-1
retinoic acid-inducible gene-1
Toll-like receptors (TLR)
 proteine transmembranare tip I
 conservate filogenetic (Drosophila  om)
 domeniu extracelular bogat in leucina
 domeniu intracelular TIR (similar IL-1R)
 TLR mamifere (11-om, 13-soarece):
 recunoastere PAMPs
 asociere cu alti TLR sau alte proteine (MD2,CD14)

TLR2 TLR4 TLR5 TLR9

+(TLR1/TLR6)
Lipoproteins, lipopeptides LPS Flagellin CpG DNA
Peptidoglycan Taxol
Zymosan
HSP60
LPS Leptospira interrogans
LPS P.gingivalis Fibronectin
GPI (T.cruzi) F Protein (RSV)
Lipoarabinomannan
Phosphatidylinositol dimannoside
(M. tuberculosis)
TLR
 TLR
LPS
MD-2 LPS
LPS
LBP LBP
TLR4 LBP
mCD14
sCD14 TLR4
LBP
sCD14 RP105
(CD180)
MD-1

Celule CD14+
(mo/M, PMN)
Celule CD14-
(endoteliu, epiteliu)
Celule CD14-
(ly B)

(adaptat dupa Donata Vercelli: Innate Immunity in Diseases of Lung, Heart and Blood)
TLR Infectie

PAMP

Spatzle PAMP
Toll Toll
Insecte Mamifere
dorsal NFkB

Peptide antimicrobiene • Peptide antimicrobiene

• iNOS (soarece)
Rol “instructiv” • Molecule costimulatoare
asupra imunitatii specifice • Citokine proinflamatorii
TLR: semnalizare intracelulara
 TLR & cai semnalizare intracelulara

TINTE FARMACOLOGICE

Adjuvanti vaccinare Boli inflamatorii (sepsis, astm, artrita

reumatoida, SLE, etc)
• antagonisti
• agonisti
• inhibitori/supresori
TLR & cai semnalizare intracelulara
 imbunatatire eficienta vaccinuri existente
Vaccinare
 noi vaccinuri profilactice (boli infectioase)
 vaccinuri terapeutice (cancer, Alzheimer)

 Vaccinuri ce contin liganzi TLR

• antigen: BCG [TLR2 & TLR4]
• adjuvant: HiB – OMBC conjugate [TLR2]
(outer membrane complex Neisseria)

 Adjuvanti liganzi TLR (agonisti)

 Pam3Cys [TLR1 + TLR2]
 MPL A, CFA, BCG, analogi LPS [TLR4]
 Imiquimod, Resiquimod [TLR7]
 CpG ODN [TLR9]
 Immune Response to Toll-Like Receptor 9-Agonist Adjuvanted
Pneumococcal Vaccination in HIV Infected Adults

This study has been completed.

First Received: November 23, 2007 Last Updated: January 20, 2009
Purpose
Pneumococcal disease is a major source of morbidity and mortality
in HIV-patients. HIV-patients are vaccine hyporesponders.A good
immune response to pneumococcal vaccination enhances vaccine
effectiveness, thereby preventing the morbidity and mortality
caused by pneumococcal disease. Even when an optimized
regimen containing both conjugated and polysaccharide
pneumococcal vaccine is used, only 13% of the immunized HIV
patients are high responders at week 96. Recent data indicate that
TLR9-agonists have excellent vaccine adjuvant potential and
are safe to use in immunocompetent as well as
immunocompromised individuals. The aim of this study is to
evaluate the qualitative and quantitive immune response to
pneumococcal vaccination with or without TLR9-agonist in HIV-
infected adults
Imiquimod for Breast Cancer Patients With Chest Wall Recurrence or Skin
Metastases

This study is currently recruiting participants.

Verified by New York University School of Medicine, August 2009

Purpose
TLR agonists are novel agents for cancer therapy which
modify the immune response.

Imiquimod, a synthetic TLR7 agonist has proven

immunomodulatory activity when applied topically,
leading to clearance of HPV-induced genital warts and
primary skin malignancies. Its effects will now be
examined in breast cancer metastatic to the skin. If
effective, it will add a relatively non-toxic approach to
the treatment armamentarium for this patient population
frequently resistant to conventional therapies.
 Blocking of Responses to Endotoxin by E5564
in Healthy Volunteers with Experimental
Endotoxemia
The Journal of Infectious Diseases 187:631–9, 2003

These results demonstrate that E5564 blocks the effects of LPS in

a human model of clinical sepsis and indicate its potential in the
treatment and/or prevention of clinical sepsis.
A Phase II, Double-Blind, Placebo-Controlled, Ascending-Dose Study of
Eritoran (E5564), a Lipid A Antagonist, in Patients Undergoing Cardiac
Surgery with Cardiopulmonary Bypass
Anesth Analg 104:378-383, 2007

CONCLUSIONS:
This Phase II safety study suggests that the
administration of the novel lipid A antagonist, eritoran, is
not associated with overt toxicity in cardiac surgical
patients.

Blocking lipid A with eritoran does not appear to confer

any clear benefit to elective cardiac surgical patients.
Recunoastere imuna

IPOTEZE

 Receptori (self vs. nonself microbian)

(Medzhitov & Janeway, 1997)

 Semnale de pericol “Danger hypothesis”

(self vs. self alterat)

(Polly Matzinger, 1994)
Danger hypothesis

Naive
T cells
Signal 1 Signal 2
(costimulation)

APC

Danger
signal

- infection Damaged Normal

- tissue damage cell cell
- stress cells
- hypoxia
- temperature shifts
- hsp
Recunoastere imuna
IPOTEZE

- Receptori
(Medzhitov & Janeway, 1997)
- Semnale de pericol (“danger hypothesis”)
(Polly Matzinger, 1994)

- Recunoastere “prin lipsa”(„„missing self”)

Missing self

MHC
NKR cls I
Celula  Celula Absenta
NK  tinta citotoxicitatii
NCR Ligand
activator

NKR
Celula  Celula Citotoxicitate
NK  tinta
NCR Ligand
activator
Innate immunity

ROL SI FUNCTII

Recunoastere
Functii efectoare
•prevenire intrare
microorganisme
•eliminare patogene
Functiile imunitatii innascute

PAMP

Stimulare PRR
 stimuleaza fagocitoza
 induce activitate microbicida
 induce citokine inflamatorii:
 IL-1, IL-6, TNF- (NF-kB)
 activeaza imunitatea dobindita
 expresia mol. costim.
(MHC cls.II, CD80/CD86)
 Functii efectorii: Fagocitoza

celule fagocitare Chemotaxia microorganisme patogene

neutrofile
eosinofile
monocite
macrofage
celule dendritice
limfocite B
Atasarea

Ingerarea

Distrugerea
Fagocitoza

Mecanism
dependent de oxigen independent de oxigen

O2 – - defensine
H 2 O2 - cathepsina B
1O - lizozim
2

OCl - lactoferina
OH - enzime proteolitice

• eliminare a microorganismelor patogene

Rol
• prelucrare atg pt. prezentare
 Inflamatia

- citokine pro-
inflamatorii (TNF-,
IL-1) PAMP / PRR
- vasodilatatoare
- complement
- bradikinine

 permeabilitate
capilara
 expresia mol.
adeziune cel.
endoteliale

diapedeza
 Citokine Macrofag activat

IL-1 TNF- IL-6 IL-12

EFECTE LOCALE
- activeaza - activeaza - activeaza ly - activeaza NK
endoteliul vascular endoteliul vascular -  producere atc. - induce dif. Th1
- activeaza ly - creste
-  acces cel. efect. permeab. vasculara
-  acces cel. efect.

EFECTE SISTEMICE
- febra - febra - febra
- producere de IL-6 - mobilizare metaboliti - inducere proteine
- soc faza acuta
Chemokine
 proteine masa moleculara mica (8-12 kDa)
 rol: inflamatie, reglare raspuns imun, angiogeneza, hematopoieza,
interactie cu SNC
 4 Cys pozitie conservata (punti disulfidice)
 structura tertiara caracteristica (1- 3, )
 redundanta (receptori si celule)

neutrofile monocite limfocite limfocite

CXC CC C CXXXC

IL-8 MCP-1
-mo/m
-fibroblaste MIP-1,  LIMFOTAKTINA FRAKTALKINA
-cel.endoteliale RANTES
NAP-2
-plachete MDC
IP-10 - mo/m
-fibroblaste - fibroblaste
- cel.endoteliale
-endoteliu - cel.epiteliale etc
SDF-1
-celule stromale
 Functii efectorii: Rolul interferon

Celula infectata viral Celula neinfectata

IFN receptor
IFN-/

2-5 (A) Sintetaza PKR

degradare mRNA  translatie mRNA

Inhibare sinteza proteica

Innate immunity

ROL SI FUNCTII

Recunoastere
Functii efectoare
• prevenire intrare
• eliminare

Rol “instructiv” asupra imunitatii specifice

initierea si tipul raspunsului

Co-stimulare: initierea raspunsului imun specific

PAMP
Infectie
TLR

APC

costimulator
MHC / peptide

CD28
TCR

T cell Activare
 PAMP
ADAPTIVE IMMUNITY
pathogen cellular immune response
TLR
Endocytic IL-12
PRR

Th1
IFN-
CD80/86 CD28
DC
TCR Naive Th1
MHC-II
T cells IFN-

INNATE IMMUNITY

(adapted after Medzhitov R, Nature Reviews Immunology, 1, 2001, 135-145)

Celule dendritice umane diferentiate din monocite izolate din singele periferic
Laborator Imunitate Antiinfectioasa, INCDMI “Cantacuzino”, 2006
Citokine imunoreglatoare: orientarea raspunsului imun specific

NIPC = Natural Interferon-Producing Cell

(Plasmacytoid precursor dendritic cells)
INFECTIE
VIRALA
 1/500 – 1/200 din celulele
mononucleare din singele priferic
 Produc de 1000x mai mult IFN decit
alte celule

IFN-/

Immature DC Mature DC
Imunitate innascuta

 Nu este doar un sistem de aparare simplu,

menit sa tina in loc infectia pina la interventia
imunitatii dobindite
 “Instruieste” sistemul imunitatii dobindite
pentru a raspunde la infectii

Decizia majora de a raspunde sau

nu unui antigen este luata de
imunitatea innascuta, prin
receptorii codificati in genom !
 BIBLIOGRAFIE

 http://microvet.arizona.edu/Courses/MIC419/tutorials.html

 http://pathmicro.med.sc.edu/book/immunol-sta.htm

 http://immunology.ucsf.edu/immuno/courses/micro204/index
1.html