Dr.T.V.Rao. MD

INNATE IMMUNITY NOBEL PRIZE WINNING TOPIC - 2011

DR.T.V.RAO MD 1

2011 NOBEL PRIZE IN PHYSIOLOGY OR

MEDICINE

• The 2011 Nobel Prize in Physiology or Medicine was

awarded to Bruce Beutler at the Scripps Research

Institute in California, Jules Hoffmann at the French

National Center for Scientific Research and Ralph

Steinman at The Rockefeller University in New York

City. Beutler and Hoffman helped to elucidate innate

immunity, the non-specific array of initial responses by

the body’s immune system that can recognize invading

microorganisms as being foreign and try to destroy

them.

DR.T.V.RAO MD 2

• The Nobel Prize in

Physiology or Medicine

2011 was divided, one half

jointly to Bruce A. Beutler

and Jules A. Hoffmann "for

their discoveries

concerning the activation of

innate immunity" and the

other half to Ralph M.

Steinman "for his discovery

of the dendritic cell and its

role in adaptive immunity".

THE NOBEL PRIZE IN PHYSIOLOGY OR

MEDICINE 2011

DR.T.V.RAO MD 3

Infection of the human body by pathogenic microorganisms such as bacteria, viruses, parasites or fungi triggers the immune response. It occurs in a two-step process: innate immunity halts the infection, and adaptive immunity subsequently clears it.

THE IMMUNE SYSTEM

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• We are constantly being

exposed to infectious

agents and yet, in most

cases, we are able to resist

these infections. It is our

immune system that

enables us to resist

infections. The immune

system is composed of two

major subdivisions, the innate

or non-specific immune

system and the adaptive or

specific immune system

OVERVIEW OF THE IMMUNE SYSTEM

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DR.T.V.RAO MD 6

• no need for prolonged induction

• no clonal expansion of Ag

specificity

• act quickly

• immediate direct response 0-

4 hrs

• rapid induced 4-96 hrs

• failure ==> adaptive immune

response

• dependence on germ line encoded

receptors

• high discrimination of host and

pathogen

INNATE IMMUNITY :DEFINITION

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What happens when the physical and chemical barriers are breached?

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Characteristics: - rapid - does not generate immunologic memory - dependent upon germ line encoded receptors recognizing

structures common to many pathogens

Innate Immunity- First Line of Defense

Innate Immunity

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Leukocyte Players of Innate Immune Responses

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• The elements of the innate (non-

specific) immune system include

anatomical barriers, secretory

molecules and cellular

components. Among the

mechanical anatomical barriers

are the skin and internal

epithelial layers, the movement

of the intestines and the

oscillation of broncho-pulmonary

cilia. Associated with these

protective surfaces are chemical

and biological agents.

INNATE (NON-SPECIFIC) IMMUNITY

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The Innate Immune System composed of ?

- includes physical, chemical, and cellular barriers

- physical barriers include skin and mucus membranes

- chemical barriers include stomach acidity, secreted anti-microbial peptides

- cellular barriers include macrophages, neutrophils

- innate immune response activation occurs within minutes of pathogen recognition

DR.T.V.RAO MD 12

HOW INNATE IMMUNITY PROTECTS • 1. Provides a barrier to prevent the spread of infection

• Mechanical (tight junctions, movement)

• Chemical (fatty acids, enzymes, pH, antimicrobial peptides)

• Microbiological (normal flora)

• Mucosal surfaces

• Nasopharyngeal, Oral, Respiratory, Intestinal tract

Urogenital tract

• Skin (epithelial cells)

• Wounds, burns, insect bites

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• Identifies and eliminates pathogens

• Non-adaptive recognition systems

• Activates molecules that target the microbe and aid in it’s identification.

• These factors may be expressed at the surface or within cells, released from immune cells or are secreted and present within circulatory system

HOW INNATE IMMUNITY ELIMINATES

PATHOGENS

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INNATE IMMUNITY • 3. Initiates an inflammatory response

• Reaction to injury or infection

• Trauma to tissues or cells

• Presence of foreign matter (self vs. non-self)

• Infectious agents (viruses, bacteria, fungi)

• Delivers effector molecules & immune cells to the site of infection

• Components

• Leukocytes & secreted factors

• Blood vessels

• Plasma proteins

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Once the PRRs are activated by the PAMPs, phagocytosis is initiated

Phagocytosis is active process:

- Internalization of pathogen into phagosome

- Acidification of phagosome

- Fusion of phagosome with lysosomes that contain anti-microbial compounds (phagolysosome)

- This may be sufficient to kill the pathogen

- If not, reactive oxygen and nitrogen species may need to be generated

Macrophage Microbial Killing

INNATE IMMUNITY • Provides signals to activate and regulate the type of

adaptive immune response generated

• Stimulation of co-stimulatory molecules • B7 family (CD80/86, PD-L, ICOSL)

• TNFR family (OX40L)

• Induction of a cytokine/chemokine response • Cytokines: IL-12, IL-23, IL-4

• Chemokine's: CXCR1, CXCR2, CCL20

• a variety and depends on stimulus

DR.T.V.RAO MD 17

FIRST LINE OF DEFENSE -- EPITHELIA

• Mechanical

• tight junctions, air/fluid flow, ciliary rejection

• Chemical

• lysozyme, pH, defensins, surfactant opsonins, TOX(ROX)

• Microbiological

• normal protective flora competition, antimicrobial colicin

• Inductive

• receptors that recognize pathogens and signal other

innate and adaptive immune response

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• Neutrophils

• Eosinophil's

• Basophils/Mast Cells

• Monocytes

• Macrophages

• Natural Killer Cells

• Platelets

CELLS OF INNATE IMMUNITY

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LEUKOCYTE TERMINOLOGY

TWO SYSTEMS

• Nuclear Morphology

• Mononuclear Cells

• Monocytes/Macrophages

• Lymphocytes

• Polymorph nuclear Cells

• Polymorphonuclear Leukocytes, PMNLs, PMNs

• Granule Morphology

• Granulocytes

• Neutrophils (neutral), Eosinophil's (orange), Basophils (blue)

• Agranulocytes

• Lymphocytes, Macrophages,

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COMPARATIVE MORPHOLOGY OF

GRANULOCYTES

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Leukocyte Players of Innate Immune Responses

Innate Immune Receptors

Innate immune receptors are not clonally distributed

Binding of receptors results in rapid response

Innate immune receptors mediate three functions:

- phagocytic receptors to stimulate pathogen uptake

- chemotactic receptors that guide phagocytes to site of

infection

- stimulate production of effector molecules and cytokines

that induce innate responses and also influence

downstream adaptive immune responses

Most microorganisms express repeating patterns of molecular structures termed Pathogen Associated Molecular Patterns (PAMPs)

Innate immune system has evolved mechanisms capable of recognizing these repeating patterns termed Pattern Recognition Receptors (PRRs)

Examples of Pattern Recognition Receptors: - Mannose-Binding Lectin (MBL) - Macrophage Mannose Receptor - Scavenger Receptors - Toll-like Receptors (TLRs) - Nod-like Receptors (NLRs) - RNA helicases (RIG-I, MDA-5)

Pathogen Recognition

PHAGOCYTOSIS • Phagocytosis

• Definition: uptake of large particles (>0.5 mm)

• Actin-dependent, clathrin-independent

• High rate & efficiency of internalization • Professional phagocytic cells

• Macrophages

• Neutrophils • These cells have phagocytic receptors

• External receptors

• FcR, CR3, Mannose receptor

• Internal receptors

• TLRs

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• Blood - Called monocytes (1-6%

WBC)

• Tissues - Called macrophages

• mature form of monocytes

• normally found in tissues such

as gastrointestinal tract, lung,

liver and spleen

• Functions:

• Phagocytose and kills after

bactericidal mechanisms are

activated (T cells)

• Produce cytokines/chemokines

(initiates inflammation)

• Is an antigen presenting cell

(co-stim. Molecules)

MACROPHAGES

(MQ)

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• Present in blood (55-60%

of WBC)

• Not normally present in

tissues

• Short lifespan - 12 hours

• Functions:

• First at the site of

infection/injury

• Ingest and kill microbes

after bactericidal

mechanisms are activated

(binding to pathogen)

NEUTROPHILS

(PMN)

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• Granulocytic Leukocyte

• Most Abundant White Blood Cell

• 2-6 x 103 cells/μL

• 40 –75 % of

leukocytes

• Very Short Lifetime

• t1/2 = 6 hours

• 55 % of Bone Marrow Weight

Devoted to Neutrophil Production

HUMAN NEUTROPHIL

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Human Neutrophil Size • In blood:

• Volume = 300 μm3 sphere (300 fl vs. 90 fl for RBC)

• Diameter = 8.3 μm

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EOSINOPHIL

EM MORPHOLOGY

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• Receptors

• recognition of pathogens

• chemical signals

• Transduction pathways

• G proteins, Kinases

• Effector activation

• gene induction

• motility, secretion

• adherence, phagocytosis

SIGNAL TRANSDUCTION

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G PROTEIN CYCLE

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Toll-Like Receptors (TLRs)

Cellular Localization: - Lysosomal localization (i.e. subcellular) of TLR-3 and TLR7-9

- TLR-3 and 7-9 recognize viral/bacterial nucleic acids

- lysosomal expression isolates pathogen nucleic acid recognition away

from potential cross-reaction with host mammalian nucleic acid motifs

TNFa

IFNab

G PROTEIN-COUPLED RECEPTORS

• Largest receptor family appx ~1000 types

• Bind proteins, peptides, absorb light

• Highly homologous in structure

• Gab protein exchange factors

• G protein splitters ==> Ga-GTP & Gb

• Primary transducers are Ga-GTP & Gb

• Activate membrane phospholipases and cyclases

GTP

a b

GTP

a b GDP

GDP

a b a

GDP b

GTP GDP

P

L

C

INTERNALIZED

GDP

a

P110

PIP2 PIP3

Receptor - G protein

Coupling

R R

R R

R

L

L

L

L

L L

L

L

L

L

L

L

L

a

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GPCR OF INNATE IMMUNITY

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• Peptide receptors

• fMLF receptor-- chemotaxis toward bacteria

• Complement receptors C5a, C3a -- chemotaxis toward sites of

complement activation

• Lipid receptors

• Leukotriene (LTB4), Eiosanoid (LPXA4), PAF, PG

• Chemokine receptors

• CXC (IL-8), CC (MCP), CXXXC(Fractalkine)

• nomenclature from amino terminal cysteines

• IL-8, MCP induce extravasation of neuts, M

• Fractalkine - monocyte /endothelial adhesion

KILLING MECHANISMS • Phagosome - membrane bounded vesicle that becomes acidified

• Lysozome - granules that contain products that damage or kill pathogens

• Enzymes

• Lysozyme - dissolves cell walls of some bacteria

• Acid hydrolases - digests bacteria

• Proteins

• Lactoferrin - binds Fe++ needed for bacterial growth

• Vitamin B12-binding protein

• Peptides

• Defensins and cationic proteins - direct antimicrobials

DR.T.V.RAO MD 38

Activated macrophages secrete proteins that drive innate response

Cytokines

- induce response by binding to specific receptors

- can function in autocrine or paracrine manner

- cytokines (and their receptors) are clustered according to structural similarities

- critical cytokines secreted by macrophages following activation include TNFa, IL-1, IL-6, IL-12 to stimulate inflammation and phagocytosis/killing

Chemokines

- diverse family of chemotactic cytokines, induce directed chemotaxis of cells

- all related in amino acid structure

- certain chemokines induce cell activation in addition to cell recruitment

- promiscuous in receptor usage, each can bind more than one receptor

- likewise, receptors are promiscuous

KILLING MECHANISMS - CONT. • Respiratory Burst

• Activated following phagocytosis

• Stimulated by PRR

• Requires increased oxygen consumption

• Produces substances that are directly toxic to the bacteria

• Oxygen-derived products

• O2-, H2O2 & Myeloperoxidase

• Nitrogen-derived products

• NO (nitrogen oxide)

• Produced by inducible NO synthase (iNOS) enzyme

• Enzyme is induced by cytokines (LT, TNFb)

DR.T.V.RAO MD 40

NADPH OXIDASE Mitochondrial-independent

respiratory burst

P47phox & p67phox

normally resides in the

cytoplasma.

P47phox becomes

hyperhposphorylated

following phagocytosis and

binds to p67phox.

These components move

to the membrane and bind

the NADPH complex

resulting in an active

complex.

DR.T.V.RAO MD 41

ENZYME REACTIONS OF

RESPIRATORY BURST • Respiratory Burst

NADPH NADP+ Superoxide

+ dismutase

2 O2 2 O-

H2O2

• Myeloperoxidase

• Enzyme which is stored in primary granules of PMN & MQ and uses the products of the

respiratory burst.

• H2O2 + C1- Chloramines

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Clinical symptoms of inflammation: pain, redness, heat, swelling

1. Increased vascular diameter, increased blood flow (heat, redness)

2. Activation of vascular endothelium to express adhesion molecules, increases leukocyte binding

3. PMNs are first cell type recruited to site, followed later by monocytes

4. Increased vascular permeability results in local swelling and pain

Microvascular coagulation helps prevent pathogen spread into bloodstream (physical barrier)

CHEMOKINE'S

• Infection induces the

release of various

chemokine's

• Theses substances bind

specific and sometimes

shared receptors to

recruit various types of

immune cells to the site

of infection

DENDRITIC CELLS

• DCs link innate and adaptive immunity

• DCs are immature as they circulate waiting to encounter pathogens

• At this point, they are highly phagocytic, but not good stimulators of adaptive T cell responses

• Once they are activated by pathogens and activation of their PRRs, they secrete cytokines to initiate inflammation and then they migrate to lymph nodes and mature

• As mature DCs they are excellent APCs for T cell stimulation DR.T.V.RAO MD 45

THE CURRENT KNOWLEDGE HELPS FOR

NEWER VACCINE TRENDS

• The detailed understanding of the immune

system provided by the new Nobel laureates has

given other researchers the ability to improve

vaccines and to attempt to stimulate immune

reactions to cancer. Their insights also inform

efforts to damp down the immune system when

it becomes too zealous, which can lead to

excessive inflammation and autoimmunity.

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FOR FURTHER INFORMATION . . . • Immunology Project Resources –

• Understanding Autoimmune Disease

• http://www.niaid.nih.gov/publications/autoimmune/work.htm

• Antibody descriptions [IgG, IgM, IgA]

• http://sprojects.mmi.mcgill.ca/immunology/Ig_text.htm

• Immunology Hyperlinked History & Molecular Movies

• http://www.bio.davidson.edu/courses/Immunology/Bio307.html

• Nature Magazine & Immunology

• http://www.nature.com/nature/view/030102.html

• NCBI Genome

• http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=1589796

• NCBI Genome Base

• http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=1589796

• Immune System Animation Links through Anatomy & Physiology Groups

• http://science.nhmccd.edu/biol/ap2int.htm

• Pier,G. , Lyczak,J., Wetzler,L; Immunology, Infection, and Immunity; American Society for Microbiology Press,2004,p.12.

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• Programme created by Dr.T.V.Rao MD

for Medical and Paramedical students in

the Developing World

• Email

• doctortvrao@gmail.com