Corso di laurea:

Biotecnologie mediche - A.A. 2014/2015

Immunologia e immunopatologia
Prof.sse Angela Santoni, Cristina Cerboni, Helena Stabile

Lezione 7. Immunit Mucosale

Il materiale presente in questo documento viene distribuito solamente per uso interno ed esclusivamente a scopo didattico.

The Organization of the mucosal immune system

- The mucosal immune system:

GI tract
Respiratory tract
Urogenital tract
Exocrine glands associated with these organ

The mucosal immune system protects the internal

surfaces of the body

Mucosal tissue of human body

Immunobiology, Janeway 2008

Mucosal Immune System

Mucosa-Associated Lymphoid Tissues (MALT)

Worldwide deaths annually from mucosal infections

 The mucosal surfaces are thin and permeable barriers to

the interior of the body
effective defense mechanism
vulnerability to infection
The vast majority of infectious agents invade the human
body by these routes
The mucosal surfaces are also portals of entry for a vast
array of foreign antigens that are not pathogenic (e.g.
food antigens)
The healthy large intestine is colonized by at least 1000
species of microorganisms : commensal microorganisms
contain many foreign antigens

 The mucosal immune system may be the original vertebrate

immune system
- The mucosal immune system forms the largest
part of the bodys immune tissues
3/4 of all lymphocytes
producing the majority of immunoglobulins!!

- The mucosal immune system has many unique

and unusual features
- It has been proposed that the mucosal immune
system represents the original vertebrate immune
system and that the spleen and lymph nodes are
later specialization

MALT: mucosal associated lymphoid tissue

Soluble components: IgA, antimicrobial peptides, mucous layer, etc
Intraepithelial lymphocytes
Isolated lymphoid follicles (ILF)
Lymphoid organs (such as tonsils, adenoids, Peyers patches, appendix)
Regional lymph nodes (mesenteric, cervical, bronchial/mediastinal, iliac,)
BALT (bronchus), GALT (gut), and others

organized tissues=
INDUCTION sites

scattered lymphoid
cells=
EFFECTOR sites

Control of development of GALT compared with

systemic lymphoid tissues

Stromal cells

LTi
lymphoid tissue-inducer

VCAM-1
CCL19-21
CXCL13

Lymphoid-tissue inducer (LTi) cells are required for initiation of

lymph-node and Peyer's-patch development in the fetus

Eberl G., Nat Rev Immunol. 2005

Gut-Associated Lymphoid Tissue Anatomy

Abbas A.K, Lichtman A.H and Pillai A., Immunologia cellulare e molecolare, Elsevier (VII edizione 2012)

Peyers patches (Gut-Associated Lymphoid Tissue)

Mucosal epith
elium
Lamina propria
Lymphoid
follicle

Peyers patch

M cell

- Peyers patches have a distinctive appearance,

forming dome-like aggregates of lymphoid cells
- Microfold cells (M cells):
the route by which Ag enters the Peyers patch from lumen
- isolated lymphoid follicles in the small and large
intestine contain mainly B cells

- Similar isolated follicles are found in other sites

bronchus-associated lymphoid issues (BALT)
nasal-associated lymphoid tissues (NALT)

The intestine has distinctive routes and mechanisms

of antigen uptake
- The M cells in the follicle-associated epithelium are
continually taking up molecules and particles from the
gut lumen by endocytosis or phagocytosis

released into the extracellular space

transcytosis
DC take up the transported material

Presentation to T lymphocytes

transcytosis

Salmonella typhimurium infection

Chemokines released by the epithelial cells:

CCL20 (MIP-3a) and CCL9 (MIP-1g)

recruitment of DC
- DCs are abundant in the wall of the intestine mainly in lamina
propria:
acquire antigens across an intact epithelial barrier
(without the need for M cells)
transport antigen to the T cell areas of mesenteric
lymph nodes

Lymphocytes.

 The mucosal immune system contains large numbers of effector

lymphocytes (even in the absence of disease)
- In the intestine, effector cells are found in two main
compartments: the epithelium and the lamina propria
- The epithelium contains mainly lymphocytes, the vast majority
of which are CD8 T cells
- The lamina propria: CD4, CD8 T cell, plasma cells,
macrophages, DC, eosinophils, mast cells
- The total number of lymphocytes in the epithelium and lamina
propria probably exceeds that of most other parts of the body
- The healthy intestinal mucosa displays many characteristics of
chronic inflammatory response
Local responses to the myriad of innocuous Ags
Overt disease is rare due to regulatory mechanisms

Signals guiding the migration of lymphocytes into

mucosal sites

CXCR7:

CD62L

CCL19

CCR9:

CCL21

CCR10
CCL28 (colon, ghiandole
mammarie, salivari)

a4b7

CCL25 (tenue)

 Priming of lymphocytes in one mucosal tissue can

induce protective immunity at other mucosal surface!
- MAdCAM-1 is not restricted entirely to the blood
vesseles of the intestine
also found on the vasculature in the other
mucosal surfaces!!
lymphocytes primed in GALT,
migrate to other common mucosal
immune system

Oral vaccination

Signals guiding the migration of activated T cells into

mucosal sites
MALT

aEb7-E-cadherin
CCR9-CCL25
CCR10-CCL28
MALT

a4b7-MAdCAM
CCR9-CCL25

Vitamin A and the imprinting of small intestine

trafficking

alcohol dehydrogenases
(ADH)
Retinol

retinal dehydrogenases
(RALDH)
Retinal

(Epithelium, intestinal DC)

Retinoic acid, RA (active metabolite)

RAR, retinoic acid receptor; RXR, retinoid X receptor

DC
effettrici

DC
regolatorie

IL-10

The mucosal immune system contains

unusual T lymphocytes

In the intestine, scattered T cells are found in two distinct locations:

- lamina propria
- epithelium

Lamina propria
- Rapporto CD4:CD8 di 3:1
- Hanno marker associati a funzioni effettrici o memoria,
come CD45RO
- Esprimono markes di localizzazione intestinale CCR9, a4b7 recettori
per chemochine pro-infiammatorie come CCL5 (RANTES)
- Le cellule T CD4 effettrici della lamina propria proliferano poco se
stimolate da mitogeni o Ags
- Secernono grandi quantit di citochine come IFNg, IL-5,
IL-10 anche in assenza di infiammazione
- Sono le principali cellule T effettrici in caso di Celiachia o malattie
infiammatorie dellintestino (IBDs)
- Possono favorire la produzione di IgA o essere cellule regolatrici
coinvolte nella prevenzione reazioni di ipersensibilit

Epitelio
- I linfociti intraepiteliali (IEL) hanno caratteristiche ben definite
- Ci sono 10~15 linfociti ogni 100 cellule epiteliali

- Pi del 90% degli IEL sono linfociti T e l80% esprime il CD8

- Hanno aspetto attivato con granuli contenenti granzimi e perforina
- Mostrano un uso limitato dei segmenti genici VDJ si espandono quindi
in risposta ad un numero limitato di peptidi antigenici

- Esprimono CCR9, aEb7 integrin (ligando E caderina sullepitelio)

IEL

B Lymphocytes.

Immunita umorale nel tratto gastrointestinale

IgA costituiscono lisotipo anticorpale pi rappresentato
nel tratto gastrointestinale: IgA (40): IgM (3): IgG (1)

IgA secretorie prodotte nel GALT e trasportate attraverso

lepitelio mucoso nel lume intestinale
La prevalenza di plasmacellule producenti IgA dovuta:
- Scambio isotipico verso IgA che avviene nel GALT e linfonodi
mesenterici
- Preferenziale homing allintestino di cellule producenti IgA
via CCR10-CCL28

Scambio isotipico
T-dipendente

1) DC cattura Ag batterici e migra verso

la zona interfollicolare
2) Presentazione Ag CD4+T naive
3) T helper interagisce cellule B IgM+IgD+
-CD40L
-TGF-b
- NO
SCAMBIO ISOTIPICO

IgA alta affinit

vs patogeni e tossine

Abbas A.K, Lichtman A.H and Pillai A., Im

munologia cellulare e molecolare, Elsevier
(VII edizione 2012)

DC attivate dai ligandi TLR:

Scambio isotipico
T-indipendente

-APRIL
-TGF-b
- IL-6
- Acido retinoico
SCAMBIO ISOTIPICO

IgA bassa affinit

batteri intestinali

Abbas A.K, Lichtman A.H and Pillai A., Im

munologia cellulare e molecolare, Elsevier
(VII edizione 2012)

 Secretory Ig A
- In human, two isotypic forms IgA1, IgA2
In blood, IgA1 : IgA2 10:1 : mainly monomer
In mucosal, IgA1 : IgA2 3:2 : mainly dimer
- nave B cell

TGFb

IgA secreting plasma cells

- B lymphoblasts express the mucosal homing integrin a4b7

- In the lamina propria, plasma cell:

J chain linked IgA dimers

polymeric Ig receptors
transcytosis to the lumen
proteolytic cleavage of the
extracellular domain of the
poly-Ig receptor
IgA dimer + secretory
component (Secretory IgA)

- In some animals, there is a second route of IgA secretion into the

intestine : hepatobiliary route
- In the liver, small veins (sinusoids) are lined by hepatocytes that
express the poly-Ig receptor
transcytosis of IgA into the adjoining bile ducts
deliveryed directly into the upper small intestine via
the common bile duct

- Secretory IgA has several functions in epithelial surface

IgA deficiency is common in humans but may be overcome by
secretory IgM

IgA Functions

The mucosal response to infection

and regulation of mucosal immune responses
Enteric pathogen cause a local inflammatory response
and the development of protective immunity
- The gut is the most frequent site of infection by
pathogenic microorganisms

- Innate mechanisms eliminate most intestinal

infections rapidly and without significant spread
beyond the intestine

EPITHELIAL CELLS HAVE A CRUCIAL ROLE IN

INNATE DEFENSE AGAINST PATHOGENS
-

Epithelial cells do bear TLR-5 on their basal surface flagellin on

the basal surface

Epithelial cells carry TLRs in intracellular vacuoles that can detect

pathogens and their products that have been internalized by endocytosis

Epithelial cells also have intracellular sensors

interact with microorganism or their products
in the cytoplasm
e.g. NOD1 muramyl tripeptide in G(-) bacteria
NOD2 muramyl dipeptide in most bacteria
Epithelial cells express NLRP3
Injury and stress to the enterocytes stimulated the expression of
MIC-A and MIC-B proteins.

TLR, NOD1 e NOD2 attivano la via di NFkB,

inducendo le cellule epiteliali ad esprimere
numerose citochine infiammatorie, chemochine
ed altri mediatori che attivano:
neutrofili, macrofagi e DC.

The outcome of infection by intestinal pathogens is

determined by a complex interplay between the
microorganism and the host immune response

- moreover, many enteric pathogens need to exploit

host mechanisms of antigen uptake via M cells and
inflammation as part of their invasive strategy.
e.g. Salmonella typhimurium, Shigella flexneri

- The host inflammatory response is an additional and often essential

part of this invasive process
- Bacteria transcytosed through M cells are free to interact with
TLRs on inflammatory cells and the epithelial cells
- After being ingested by phagocytes, many of these microbes induce
caspase-dependent apoptosis of the phagocyte
- The production of a cascade of inflammatory mediators e.g.
(IL-1b, TNFa)

loosen the tight junctions between epithelial cells

allow microorganisms to flood in the intestinal tissue

Salmonella Typhimurim

Shigella flexneri

- The principal role of the mediators and cells induced by the

innate immune response:
initiate the adaptive immune response
- IL-12, IL-18 produced by macrophages:
IFNg production by antigen specific T cells
enhances the ability of the macrophage to kill
the bacteria it has infected
- The host-pathogen interaction is further complicated by the
ability of many enteric microbes to modulate the inflammatory
response.
essential to bacteria ability to cause disease

 The mucosal immune system must maintain a balance between

protective immunity and homeostasis to a large number of different
foreign antigens
- The majority of antigens encountered by the normal intestinal
immune system are not derived from pathogen but come from food
and commensal bacteria
- The mucosal immune system has developed sophisticated means of
discriminating between pathogens and innocuous antigens
- The default response to oral administration of a protein antigen:
specific peripheral unresponsiveness (oral tolerance)

- A similar phenomenon in the respiratory tract

mucosal tolerance

The default response to oral administration

of a protein antigen
specific peripheral unresponsiveness
(oral tolerance)

- A breakdown in oral tolerance ??

celiac disease :
IFNg producing CD4 T cell
inflammation in the upper
small intestine

- The mechanisms of the oral tolerance:

anergy or clonal deletion
the generation of regulatory T cell of different
types : Foxp3+ TReg, TH3
- Commensal bacterial also do not provoke a systemic primary
immune response, but there is no active tolerance to these
antigens
Stimulate local IgA antibody production and
..active suppression of local effector T cell response

 The healthy intestine contains large quantities of bacteria but

does not generate productive immunity against them

- The protective role of the commensal flora is dramatically

illustrated by the adverse effects of broad spectrum antibiotics

 The healthy intestine contains large quantities of bacteria but

does not generate productive immunity against them

- The protective effect of TLR seems to involve the epithelial cells

being made more resistant to inflammation-induced damage

Basal TLR stimulation by commensal bacterial products enhances

the ability of epithelial cells to repair damage

Examples of microbiota influence on the innate immune responses

Reduced in patients with IBD

Examples of microbiotal influence on the adaptive immune responses

SFB = Segmented filamentous bacter; SAA= serum amyloid A; PSA = polysaccharide A; SCFA = short-chain fatty acids

Microbes and obesity?

The absence of TLR-5 in mice alt
ers the gut microbiota, causing
increased food intake, insulin
resistance, and obesity.

Possible factors controlling the gut microflora

Possible routes of immunization for human vaccines

Possible alternative (needle-free) routes for vaccination

NO
l

requirement of professiona
skill and syringe/needle for
administration

NO

requirement for cold-chain

(or refrigeration storage)

HIGHER

stability

physico-chemical

Different oral vaccine delivery strategies

Davitt et al., Adv. Drug. Deliv. Rev. 2015

The Ideal oral vaccine delivery strategy

Davitt et al., Adv. Drug. Deliv. Rev. 2015

Overview of rice-based mucosal vaccine development

Why oral vaccines?

serum Abs do not efficien

tly protect against mucos
al infections!

Celiac disease

- Commensal bacteria actively inhibit proinflammatory NF-kB

mediated signaling responses induced in epithelial cells by
pathogenic bacteria

PPARg

recettore g attivato del prolif

eratore del perossisoma

 Intestinal helminths provoke strong TH2-mediated immune responses

- Intestinal helminth infection
- In virtually all cases, the dominant protective immune response
is generated by TH2 cells whereas a TH1 response does not
clear the pathogen and tends to produce an inflammatory
reaction

DCs at mucosal surface favor the induction of tolerance under

physiological conditions and maintain the presence of physiological
inflammation

Intestinal helminth infection

Many factors contribute to the tolerance against

commensal bacteria

(defensins)

Nonvirulent Salmonella species inhibit NFB activation

The commensal bacterial species Bacteroides thetaiotaomicron
es proinflammatory gene expression by activating the antiscription factor PPARg

attenuat
inflammatory tran

Intestinal bacteria in mammalian

health and disease

A model for the coevolution of adaptive immunity with the microbiota

 Mucosa-Associated Lymphoid Tissues (MALT). is located in

anatomically defined compartments in the gut.
- Gut-Associated Lymphoid Tissues (GALT)
Peyers patches
solitary lymphoid follicles of the intestine
appendix
tonsils
adenoids
mesenteric lymph nodes
- The tonsils and adenoids form a ring of lymphoid
tissue, Waldeyers ring around the entrance of the
gut and airway

CARATTERISTICHE ANATOMICHE DEL SISTEMA

IMMUNITARIO MUCOSALE:
-Barriera epiteliale esterna
-Connettivo sottostante detto lamina propria con cellule
dellimmunit innata e adattativa

-Tessuti linfoidi secondari organizzati ma non incapsulati posti

immediatamente al disotto della barriera epiteliale
- Linfonodi drenanti nel mesentere sottostante

Major GALT components

T cell and B cell
area in a Peyer
s patch

T cells and B cel

ls close to M cell
s

IgG and IgA plasma cells

Intraepithelial CD
4+ and CD8+ ly
mphocytes

Different pathways for the uptake of microbes across mucosal s

urfaces
M cell
lumen
intestinal epi
thelium

DC

Lamina propria

GALT
Peyers p
atches
appendix
colon folic
les

Mesenteric lymph node

Transcytosed
bacteria are tr
ansferred to i
mmature DC (i
DC)

DCs undergo
maturation (m
DC), and migra
te to the T-cellrich region for
Ag presentatio
n

 Vasta surperficie

- 400m2

Stimolazione antigenica continua

Batteri patogeni, virus, parassiti

Batteri Commensali - >1014
>1000 specie

Proteine del cibo - 25-30kg/anno

The circulation of lymphocytes within the mucosal immune system is controlled

by tissue-specific adhesion molecules and chemokine receptors:

Role of DC

Abbas A.K et all., Im

munologia cellulare e
molecolare, Elsevier
(VII edizione 2012)

Funzioni delle IgA

Prevengono ladesione dei microrganismi
Neutralizzano batteri, tossine, virus
Scarsa capacit di attivare la via classica del complemento e
opsonizzare Ag

no infiammazione

Ruolo nella relazione simbiontica tra individuo e batteri commensali

che vengono mantenuti nel lume dellintestino

3-4 g di IgA prodotte giornalmente >> di altre Ig

 The healthy intestine contains large quantities of bacteria but

does not generate productive immunity against them
- The protective role of the commensal flora is dramatically
illustrated by the adverse effects of broad spectrum antibiotics
- Triggering of TLRs by commensal bacteria is also important in
protecting against dangerous inflammation in the intestine
e.g. TLR-2, TLR-9, MyD88 deficient mice
more susceptible to the induction of experimental
inflammatory bowel diseases (IBDs)

- The protective effect of TLR seems to involve the epithelial cells

being made more resistant to inflammation-induced damage

- Commensal bacteria: their products are recognized by the

adaptive immune system
e.g. germ-free animals :

marked reduction in the

size of all peripheral
lymphoid organ, serum
Ig and immune response!!

- Commensal do not posses the virulence factors necessary for

penetrating the epithelium and cannot disseminate throughout
the body

The presence of intestinal bacteria has a large impact on bot

h gut and systemic lymphoid structures
splenic CD4
(inset:CD8)

Germ-free mouse

Mouse colonized
with intestinal ba
cteria

intestinal CD4
(inset:CD8)

intestinal IgA
(inset: Peyers patch
IgA)

In the presence of commensal bacteria:

Gut epithelial cell and mesenchymal cells product:

TGF-b, TSLP and PGE2

maintain local DC in a quiescent state with low

levels of co-stimulatory molecules

TReg induction

Conditions in which commensal bacteria penetrate in large numbers:

Full activation of local DCs

co-stimulatory molecules: CD40, CD28

Pro-inflammatory cytokines: IL-12

strong inflammatory T cell response

leading to severe intestinal damage