INNATE IMMUNITY

NOBEL PRIZE WINNING TOPIC - 2011

Dr.T.V.Rao. MD

DR.T.V.RAO MD

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 bodys immune system that can recognize invading microorganisms as being foreign and try to destroy them.
DR.T.V.RAO MD

THE NOBEL PRIZE IN PHYSIOLOGY OR MEDICINE 2011

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".
DR.T.V.RAO MD

THE IMMUNE SYSTEM

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.
DR.T.V.RAO MD

OVERVIEW OF THE IMMUNE SYSTEM

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
DR.T.V.RAO MD

DR.T.V.RAO MD

INNATE IMMUNITY :DEFINITION

no need for prolonged induction

no clonal expansion of Ag specificity

act quickly immediate direct response 04 hrs rapid induced 4-96 hrs

failure ==> adaptive immune response dependence on germ line encoded receptors high discrimination of host and pathogen
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What happens when the physical and chemical barriers are breached?

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Innate ImmunityFirst Line of Defense

Characteristics:
- rapid - does not generate immunologic memory - dependent upon germ line encoded receptors recognizing structures common to many pathogens

Innate Immunity

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Leukocyte Players of Innate Immune Responses

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INNATE (NON-SPECIFIC) IMMUNITY

The elements of the innate (nonspecific) 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.

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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
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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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HOW INNATE IMMUNITY ELIMINATES PATHOGENS

Identifies and eliminates pathogens

Non-adaptive recognition systems Activates molecules that target the microbe and aid in its identification.
These factors may be expressed at the surface or within cells, released from immune cells or are secreted and present within circulatory system

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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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Macrophage Microbial Killing

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

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

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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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CELLS OF INNATE IMMUNITY

Neutrophils Eosinophil's Basophils/Mast Cells

Monocytes
Macrophages Natural Killer Cells Platelets
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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

Pathogen Recognition

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)

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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MACROPHAGES

(MQ)
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 DR.T.V.RAO MD (co-stim. Molecules)
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NEUTROPHILS (PMN)
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 DR.T.V.RAO MD (binding to pathogen)

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HUMAN NEUTROPHIL
Granulocytic Leukocyte

Most Abundant White Blood Cell

2-6 x 103 cells/L 40 75 % of leukocytes

Very Short Lifetime t1/2 = 6 hours

55 % of Bone Marrow Weight Devoted to Neutrophil Prod uction

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Human Neutrophil Size

In blood:
Volume = 300 m3 sphere (300 fl vs. 90 fl for RBC) Diameter = 8.3 m

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EOSINOPHIL EM MORPHOLOGY

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SIGNAL TRANSDUCTION
Receptors

recognition of pathogens
chemical signals Transduction pathways G proteins, Kinases Effector activation gene induction motility, secretion

adherence, phagocytosis

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G PROTEIN CYCLE

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Toll-Like Receptors (TLRs)

TNFa IFNab

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

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

L L L

L L

L L

Receptor - G protein Coupling

L
L

R
a b GDP a

R
b

L L L

GTP

GDP

a b GDP

a GDP P L C

R
b GDP

PIP2

PIP3 P110

GTP
GTP

INTERNALIZED
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GPCR OF INNATE IMMUNITY

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

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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

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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)

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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 41 complex.

DR.T.V.RAO MD

ENZYME REACTIONS OF RESPIRATORY BURST

Respiratory Burst

NADPH
+ 2 O2 2O

NADP+

Superoxide
dismutase

H 2O 2

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
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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

 Programme created by Dr.T.V.Rao MD for Medical and Paramedical students in the Developing World

Email
doctortvrao@gmail.com

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