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Mucosal Immunology

Kiyoshi Takeda

WPI Immunology Frontier Research CenterGraduate School of Medicine

Osaka University

Mucosal tissues

Esophagus

Mouth

Anus

Small intestine

Stomach

Gastrointestinal tract

Large intestine, colon


Mucosal tissuesTissues that are covered by epithelium

These tissues are inside our body. But they have gateways to the outside.Mucosal tissues are exposed to the external environment.

Mucosal tissues of the human body

UterusBladderVagina

Uro-genitaltract

Sinus

Trachea

Lungs

Respiratorytract

Oral cavity

Esophagus

Stomach

Intestine

Conjunctiva

Gastro-Intestinal

tract

Mammarygland

Kidney

Salivarygland

Lachrymalgland

A target of invasion of pathogenic microorganisms

Mucosal tissues

Microbe Epithelium

Free antigen in tissue

Respiratory tractGastrointestinal tractSkin

Connectivetissue

Antigen thatenters blood steam

Lymphaticvessel

Venule

Dendritic cell-associated

antigen


Routes of infection for pathogensRoutes of infection for pathogens

Route of entry Mode of transmission Pathogen Disease Type of pathogen

Mucosal surfaces

Respiratory tract

Inhalation or ingestion ofinfective material

(e.g.saliva droplets, sputum)

Measles virus Measles Paramyxovirus

Influenza virus Influenza Orthomyxovirus

Varicella-zoster Chickenpox Herpesvirus

Epstein-Barr virus Mononucleosis Herpesvirus

Streptococcus pyogenes Tonsilitis Gram-positive bacterium

Haemophilus influenzae Pneumonia,meningitis Gram-negative bacterium

SporesNeisseria meningitidis Meningococcal meningitis Gram-negative bacterium

Bacillus anthracis Inhalation anthrax Gram-positive bacterium

Gastrointestinal tract Contaminatedwater or food

Rotavirus Diarrhea Rotavirus

Hepatitis A Jaundice Picornavirus

Salmonella enteritidis,S.typhimurium

Food poisoning Gram-negative bacterium

Vibrio cholerae Cholera Gram-negative bacterium

Salmonella typhi Typhoid fever Gram-negative bacterium

Urogenital tract

Sexual transmission/Infected blood

Hepatitis B virus Hepatitis B Hepadnavirus

Human immunodeficiencyvirus(HIV)

Acquired immunodeficiencysyndrome(AIDS)

Retrovirus

Sexual transmissionNeisseria gonorrhoeae Gonorrhea Gram-negative bacterium

Treponema pallidum Syphilis Bacterium(spirochete)

0 1000000 2000000 3000000 4000000

Acute respiratory infections (4 million)

Diarrheal diseases (1.8 million)

Tuberculosis (1.5 million)

HIV (2.9 million)

Measles (600,000)

Hepatitis B (103,000)

Whooping cough (294,000)

Roundworm and hookworm (12,000)

Worldwide deaths annually from mucosal infections


Gastrointestinal tractA tube from mouth to anus

We ingest diets (nonself)Pathogens (nonself) come in to the tract

Surface area corresponds to 1.5 tennis courts

10.97 m2× 1.5

There are the highest numbers of immune cells

in the intestinal mucosa

IgA B cell CD4+ T cell

臨床粘膜免疫学（清野宏：シナジー）

Image is protected by copyright.



Mucosal immune system

Exposed to external environment.

Unlike immune cells residing in lymphoid tissues,immune cells residing in the mucosa are constantly exposed to nonself.

The intestinal mucosa establishes a unique immune system.



Mucosal immune system in the intestine

1. Barrier functions of epithelial cells

2. Intestinal environmental factors

3. Unique immune cells in the intestinal mucosa

Topics

Barrier functions of intestinal epithelia

Prevention of invasion ofpathogenic microbes

Dietarycompounds

Mucus layer(100~300㎛)

Glycocalyx

Microvillus

Viruses Bacteria

Cell adhesion complexes


Epithelial cells





ClaudinOccludin

E-cadherin

Actin Filament

Mechanical barrier:There is no interspace between epithelial cells

Tight junction

Desmosome

Adherens junction






Dietarycompounds

Mucus layer(100~300㎛)

Glycocalyx

Microvillus

Viruses Bacteria



Mechanical barrier

Chemical barrier



Major components of mucus


Mucins

Membrane-bound and secretory forms

“Slimy" material that coats epithelial surfaces

Mucus

There are many mucins (Muc1-21)

Apical membrane

Lateral membrane

Basal membraneBasement membrane

Release

Secreted mucins


Glycoprotein

High viscosity prevents access of microbes to the epithelial cells

Structure of mucins

Core ProteinTransmembrane Region

Cytoplasmic Region

Sugar chain, glycan


Barrier functions of intestinal epitheliaMuc2 / bacteria / DAPI

Bacterial free space

Epithelia

Intestinallumen

Epithelial cells producing mucins

Goblet cell






In addition to goblet cells,there are other secretory cellsin the intestinal tract

Paneth cells

臨床粘膜免疫学（清野宏：シナジー） Goblet cell



microvilli


Paneth cells

Located in the bottom of cryptsecrete anti-microbial peptides

Microvilli

Crypt

Paneth cells

Anti-microbial peptides

Bacteria





Weak

Electrostatic interaction and

Hydrophobic interaction

Strong

Hydrophobic interaction

Mammalian cell membranes Bacterial cell membranes

Cholesterol Zwitterionic phospholipids

Anti-microbial peptides (AMP)

Electricfield

Electrostatic attraction and thetransmembrane electric field bring the

AMP into the lipid bilayerAMP peptides form a pore

AMP



Prevention of infection for pathogenic bacteria

Intestinallumen

Laminapropria

Mucosalepithelium

Villus

Intraepitheliallymphocytes

Macrophage

Dendritic cell

M cell

T cell

B cell Follicle

Peyer’s patch

lgA

Mast cell

Dendritic cellAfferent

lymphatic

Crypt

Lymphatic drainage

Intestinalepithelia cell

Mucus

Plasma cell


Anti-microbialpeptides

Goblet cell

Paneth cells

Commensal bacteria

Infectious diseases in the gastrointestinal tract

Bacteria

Staphylococus aureus Food poisoningSalmonella enteritidis Food poisoningSalmonella typhi Typhoid feverShigella flexneri ShigelosisVibrio cholera CholeraEnterohemolytic E. coli (O157, O111) Gastroenteritis….

Viruses

Rotavirus GastroenteritisNorovirus Gastroenteritis… Children’s common diarrheal diseases


Intestinallumen

Laminapropria

Mucosalepithelium

Villus


Macrophage

Dendritic cell

M cell

T cell

B cell Follicle

Peyer’s patch

lgA

Mast cell


lymphatic

Crypt

Lymphatic drainage


Mucus

Plasma cell

Paneth cells



Prevention of infection for pathogenic bacteria


Commensal bacteria

Goblet cell

Mucosal tissues in gastrointestinal tract

1.Barrier functions of epithelial cells

2. Intestinal environmental factors (commensal bacteria)

3.Unique immune cells in the intestinal mucosa


Intestinal environmental factors

Commensal bacteriaThere are >1,000 species of bacteria in the intestineIntestinal bacteria outnumber our cells1kg intestinal bacteria in 60kg host

Stomach101~103 CFU/mL

LactobacillusCandida StreptococcusHelicobacter pyloriPeptostreptococcus

Jejunum/Ileum104~107CFU/mL

LactobacillusStreptococcusClostridiumBacteroidesActinomycinaeCorynebacterium

LactobacillusStreptococcus

Colon1011~1012 CFU/mL

Clostridium group Ⅳ andⅩⅣBacteroidesBifidobacteriumEnterobacteriaceae

Duodenum101~103 CFU/mL

Mutual relationship between commensal bacteria and host

Provision of niche for colonization

HostCommensal bacteria


Function of commensal bacteria

Production of metabolitesProduce nutrients（short chain fatty acid, vitamin K, B12 etc）Digest polysaccharides（dietary fiber, cellulose）

Prevention of infection Create biological barrier preventing invasion of pathogenic bacteriaProduce anti-microbial factors (lactic acid)

Development of immune cellsInduction of IgAInduction of Th1/Th17 cellsMaturation of lymphoid tissues

Intestinal environmental factorsCommensal bacteria

Colon lumen Clostridium difficile Pseudomembrane

Infection by Clostridium difficile

The colon is colonized by Large numbers ofcommensal bacteria

Antibiotics kill many of these

commensal bacteria

Clostridium difficilegains a foothold andproduces toxins thatcause mucosal injury

Neutrophils and red blood

cells leak into gut between

Injured epithelial cells

Connective tissuedegradation leads to

colitisand pseudomembrane

formation


Function of commensal bacteria

Production of metabolitesProduce nutrients（short chain fatty acid, vitamin K, B12 etc）Digest polysaccharides（dietary fiber, cellulose）

Prevention of infection Create biological barrier preventing invasion of pathogenic bacteriaProduce anti-microbial factors (lactic acid)

Development of immune cellsInduction of IgAInduction of Th1/Th17 cellsMaturation of lymphoid tissues


Neonatal vitamin K deficiency: bleed easily

Germ-free miceWho have no microorganisms living in or on them.

Maintained in an isolator


Immunological defects in germ-free mice

Commensal bacteria are required for development of adaptive immunity

Development of T cellsTh1 cells: fewerTh17 cells: fewerTreg cells: fewer

Development of plasma cells IgA-secreting cells: fewer

Good bacteria Bad bacteria

“Next generation sequencer” enables analysis of commensal bacteria

Many commensal bacteria are obligate anaerobic, and hard to culture

Precise pictures of commensal bacteria remained long unknown

VS



Inflammatory Bowel Diseases (IBD) in humans

Crohn’s diseases

Ulcerative colitis

Intractable diseases with unknown etiology

Photo: T. Kanai (Keio Univ)

Cell Host & Microbes 3, 417, 2008

Percentage of bacteria in gut habitat

Normal IBD


Altered composition of commensal bacteria (dysbiosis)

Correlates with development of several diseases

Altered commensal bacterial composition

Allergy Autoimmune diseasesMultiple sclerosis

Arthritis

Inflammatory bowel disease

Crohn’s diseaseUlcerative colitis

Metabolic diseaseObesity

Diabetes mellitus

CancerColon cancerLiver cancer




2. Intestinal environmental factors (dietary compounds)



Intestinal environmental factors

Dietary compounds

Oral tolerance

Protective immunity Mucosal tolerance

Non-selfPathogenic bacteria,

viruses, toxinsDietary

compoundsCommensal

bacteria

Immune response

Yes No No



2. Intestinal environmental factors



Intestinallumen

Laminapropria

Mucosalepithelium

Villus

Dendritic cell

M cell

Follicle

Peyer’s patch

lgA

Afferentlymphatic

Crypt

Lymphatic drainage


Mucus

Mucosal tissues in the gastrointestinal tract


Goblet cell

Paneth cells

Commensal bacteria

Lamina propria: tissues existing just beneath the epithelia of mucosa

T cell

Mast cellMacrophage

Plasma cell

Dendritic cell


B cell

Gastrointestinal tractA tube from mouth to anus

We ingest diets (nonself)Pathogens (nonself) come in to the tract

Surface area corresponds to 1.5 tennis courts

There are the highest numbers of immune cells

in the intestinal mucosa

IgA B cell CD4+ T cell






Intestinallumen

Laminapropria

Mucosalepithelium

VillusIntraepitheliallymphocytes

Macrophage

Dendritic cell

M cell

T cell

B cell Follicle

Peyer’s patch

lgA

Mast cell


lymphatic

Crypt

Lymphatic drainage


Mucus

Plasma cell


Goblet cell

Paneth cells

Commensal bacteria

Mucosal tissues in the gastrointestinal tractGut-associated lymphoid tissues (GALT)

Lymphoid tissue, Lymphoid organs

TonsilAdenoid

Right subclavian vein

Lymph node

Kidney

Appendix

Lymphatics

Left subclavian vein

Thymus

Heart

Thoracic duct

Spleen

Peyer’s patch in small intestine

Large intestine

Bone marrow

Primary lymphoid organs

Secondary lymphoid organs

Tissues where lymphocytes are generated (naïve lymphocytes)(Bone marrow, thymus)

Tissues where naïve lymphocytes (they are not exposed to antigens) are activated(Lymph nodes, spleen, gut-associated lymphoid tissues)


Structure of lymph node

Secondarylymphoid follicle(with germinal center)

Afferentlymphatic vessel

Paracortical area(mostly T cells)

Germinal center

Primarylymphoid follicle(mostly B cells)

Medullary cords(macrophagesand plasma cells)

Artery

Vein

Efferentlymphatic vessel

Secondary lymphoid organs

Cortical sinus

Senescent germinal center

Marginal sinus

Medullary sinus

Gut-associated lymphoid tissues (GALT)

Amygdala

Tonsil

Adenoid

Peyer’s patch

Isolated lymphoid follicle

Cecal patch

Mesenteric lymph node

Peculiar lymphoid tissues present in the gastrointestinal tract


Pharyngeal tonsil (adenoid)

Tubal tonsil

Lingual tonsils

Waldeyer's ring

Palatine tonsilPalatine tonsil



Pharyngeal tonsil (adenoid)

An anatomical term forthe annular arrangement oflymphoid tissues in the pharynx



AmygdalaTonsil Adenoid Peyer’s patch Isolated lymphoid follicle Cecal patchMesenteric lymph node

Waldeyer’s ring in the pharynx


Peculiar lymphoid tissues present in the gastrointestinal tract


A typical gut-associated lymphoid tissuePeyer’s patch

B

TT

A typical gut-associated lymphoid tissue: Peyer’s patchScattered lymphoid cells

Epithelium

Lamina propria

Lamina propria lymphocyte

Intraepithelial lymphocyte

Crypt

Peyer’s patchIsolatedlymphoidfollicle

Afferent lymphaticTo mesenteric lymph node


Peyer’s patch Lymph node

Structural difference between lymph node and Peyer’s patch

Antigens are delivered by dendritic cells, which capture them in the peripheryand migrate via afferent lymphatic vessels

Antigens are directly captured from the intestinal lumen

Antigen uptake

Primarylymphoid follicle

Paracorticalarea

Lymphatic vessel

Lymphaticfollicle

VillusAntigen

High endothelialvenules

M cell




M cell (microfold cell)

Peyer’s patches are covered by an epithelial layer containing specialized cells called M cells, which have characteristic membrane ruffles

Figure 11‐6 Immunobiology, 7th ed（©Garland Science 2008)


Cellular and Molecular Immunology (A. Abbas: Elsevier)

Intestinallumen

Laminapropria

Mucosalepithelium

Villus


Macrophage

Dendritic cell

M cell

T cell

B cell Follicle

Peyer’s patch

lgA

Mast cell


lymphatic

Crypt

Lymphatic drainage


Mucus

Plasma cell

A unique mechanism for antigen uptake in the intestine

Anti-mircobialpeptides

Goblet cell

Paneth cells

Commensal bacteria

Direct antigen uptake by1. M cells on Peyer’s patch2. CX3CR1+ dendritic cells in the lamina propria

M cell

M cells take up antigen by endocytosis

and phagocytosis

M cells take up intestinal antigens and deliver them to dendritic cells in GALT

A unique mechanism for antigen uptake in GALT

Antigen is transported across the M cells in vesicles and released at the basal surface

Antigen is bound by dendritic cells, which

activate T cells

M cell



A subset of dendritic cells extend their dendrites across the epithelial layerto capture antigen from the lumen of the gut

Unique subsets of dendritic cells in the intestine

Epithelial cells

Antigen Antigen

CD11b+

CX3CR1+

dendritic cell

CD103+

CX3CR1-

dendritic cell

TGF-β

T cell

Foxp3+

Treg cell

RA

INF-γTGF-β

IL-6

IL-17

FOXP3

A B


Intestinallumen

Laminapropria

Mucosalepithelium

Villus


Macrophage

Dendritic cell

M cell

T cell

B cell Follicle

Peyer’s patch

lgA

Mast cell


lymphatic

Crypt

Lymphatic drainage


Mucus

Plasma cell



Goblet cell

Paneth cells

Commensal bacteria

Direct antigen uptake by1. M cells on Peyer’s patch2. CX3CR1+ dendritic cells in the lamina propria

Intestinallumen

Laminapropria

Mucosalepithelium

Villus


Macrophage

Dendritic cell

M cell

T cell

B cell Follicle

Peyer’s patch

lgA

Mast cell


lymphatic

Crypt

Lymphatic drainage


Mucus

Plasma cell

Lymphocytes residing in the intestine


Goblet cell

Paneth cells

Commensal bacteria

T cells： Th1, Th17, Treg cellsIntraepithelial lymphocytes (IEL)

B cells： IgA-secreting cells


Naïve lymphocytes (B, T cells)Peyer’s patch Activation by Ag presentationMesenteric lymph nodesBlood circulation via thoracic ductRecruitment to

the intestinal lamina propria

Recruitment of lymphocytes to the intestine

Lamina propria T cellor plasma cell

Lamina propria venuleBlood

Effector T or B cell

α4β7Naive T or B cell

RALDHPeyer’s patchor mesentericlymph node

Dendritic cell

TSLP, other factors

Retinoic acid

Vitamin A

Gut epithelial barrier

CCL25

CCR9

MadCAM

Recruitment of lymphocytes to the intestine

Blood vessel

CCR10

CCR9

α4:β7

MAdCAM-1L-selectin

Lamina propria

E-cadherin

αE:β7

CCL25

EpitheliumSmall intestine

Large intestine

CCL28

Endothelium

Gut-homing effector T cells bind MAdCAM-1 on endothelium

Gut epithelial cells express chemokines specific for

gut-homing T cells


Intestinallumen

Laminapropria

Mucosalepithelium

Villus


Macrophage

Dendritic cell

M cell

T cell

B cell Follicle

Peyer’s patch

lgA

Mast cell


lymphatic

Crypt

Lymphatic drainage


Mucus

Plasma cell



Goblet cell

Paneth cells

Commensal bacteria




90% of IEL are T cells, among them 80% are CD8-positiveIntestinal

epithelial cell

Intraepithelial lymphocytes (IEL)

Intraepithelial lymphocytes

Lymphocytes called intraepithelial lymphocytes(IELs) lie within the epithelial lining of the gut

Mucus


Intraepithelial lymphocytes (IEL)Kill viral-infected epithelial cells Kill stressed epithelial cells

Gliadin peptide

MIC-A,BNKG2D

CD8α:β

CD8α:α

TL

Perforin Fasligand

Fas

Granzyme

Virus infects mucosal

epithelium cell

Infected cell displays viral

peptide to CD8 IEL via MHC

class I

Activated IEL kills infected epithelial

cell by perforin/granzyme

and Fas-dependent

pathways

Epithelial cells undergo stress as

a result of infection, damage, or toxic peptides,

and express MIC-A and MIC-B

NKG2D on IELs binds to MIC-A,B and activate the

IEL. CD8α:α

homodimers also bind to TL

Activated IEL kills the stressed cell

via the perforin/granzyme

pathway

Intestinallumen

Laminapropria

Mucosalepithelium

Villus


Macrophage

Dendritic cell

M cell

T cell

B cell Follicle

Peyer’s patch

lgA

Mast cell


lymphatic

Crypt

Lymphatic drainage


Mucus

Plasma cell

Lymphocytes residing in the intestine


Goblet cell

Paneth cells

Commensal bacteria




Early stage protection

Major Ig againstinvading pathogens

Found in mucosa and prevents colonization by pathogens

IgG

IgD

IgE

IgA

Antibody（Immunoglobulin)

IgM

Recruitment of IgA-secreting cells to the intestine

Plasmablast

IgA secreting plasma cell

Lamina propria

Luminal antigen

Mucus

M cell

IgA

IgA

TGF-β Dendritic cell

NO

B cell

CD40

Peyer’s patch

CD40 ligand

Activated helper T cell

Naive CD4+

T cell Via blood


IgA production in the gutPolymeric immunoglobulin receptor (poly-Ig receptor)

Lamina propria Mucosal epithelial cell Lumen

Secreted IgA

Proteolytic cleavage

Endocytosedcomplex of IgA and poly-Ig receptor

Dimeric IgA

IgA-producing plasma cell

J chainPoly-Ig receptor with bound IgA

Function of secreted IgA

IgG, IgM: complement activationOpsonization (Induction of phagocytosis)

Induction of inflammation

IgA does not induce inflammation

IgA binds intestinal bacteria (commensal and pathogenic) and inhibits invasion of these bacteria into epithelial cells

Prevention of intestinal infectious diseasesMaintenance of commensal bacteria

Lamina propria

Toxin

Epithelial cell

Bacteria


Intestinallumen

Laminapropria

Mucosalepithelium

Villus


Macrophage

Dendritic cell

M cell

T cell

B cell Follicle

Peyer’s patch

lgA

Mast cell


lymphatic

Crypt

Lymphatic drainage


Mucus

Plasma cell

anti-mircobialpeptides

Goblet cell

Paneth cells

Commensal bacteria

Mechanical barrier: Cell adhesion complexChemical barrier: Mucus layer


Commensal bacteriaDietary compounds

Gut-associated lymphoid tissues (GALT)Unique antigen uptakeLamina propria lymphocytes (IEL & IgA)

Summary

Mucosal Immunology

mucosal immunology - d37djvu3ytnwxt.cloudfront.net · mucosal immunology kiyoshi takeda ......

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