Chapter 3: Innate Immunity - The Induced Response

Three Lines of Defense

Induced Innate Immune Response

Once the pathogen has breached the physical barriers and
outrun the initial response, the induced innate response takes
place.

• Tissue Macrophages are the first to recognize and respond to the pathogen and secrete soluble molecules that lead to inflammation.

• Leukocytes, neutrophils at first, are called into the site of infection out of the blood.

• Soluble molecules activate cells, such as hepatocytes (liver cells), to make acute phase reactants, to aid in the clearance of the pathogen and these molecules also direct leukocytes to the site of inflammation.

Innate Immune Cell Receptors = Recognition of Pathogen or Damaged/Infected Host Cells

Two examples of Cells that use receptors to recognize self from non-self are Macrophages and NK cells

Both cell types need to be able to differentiate between host and pathogen.

There are limited types of innate receptors for recognition but because many pathogens have the same types of surface molecules this limited diversity works well.

• Ex: Gram negative bacteria have LPS in their outer membrane so it doesn’t matter what genus it is it will still be recognized by an LPS receptor on a macrophage.

Recognition Receptors

Phagocytosis by macrophages provides a first line of cellular defense against invading microorganisms

Pathogen invades  1st effector cells (resident macrophages)

• Resident in tissues

• Prevalent in connective tissues

  • Linings of GI, respiratory tract, lung alveoli, liver

• Innate, long-lived phagocytic cells

• Involved in both innate and adaptive immunity

Enhance phagocytosis → more efficient using macrophage
surface receptors

Enhance phagocytosis → more efficient using macrophage surface receptors

C3b is a ligand for Macrophage CR1

Pathogen C3b surface fragments + degraded by factor I → iC3b → now a ligand for macrophages CR3 and CR4 receptors

Combination of opsonization by complement and phagocytosis
by macrophages

• Pathogens recognized and destroyed at beginning of infection

Phagocytosis by macrophages is aided by receptors of innate immunity that bind directly to microbial surface components - Phagocytic Receptors

Surface components are characteristic of pathogens

• But absent from human cells

  • CR3 and CR4 recognize iC3b and other ligands such as:

    • LPS, lipopolysaccharide (Gram negative bacteria)

    • Lipophosphoglycan

    • Filamentous hemagglutinin

    • Cell-surface structures on yeast

  • Carbohydrate-binding proteins = lectins

    • Bind to particular carbohydrates (not found on human cells)

    • Mannose and glucan receptors are examples

  • Scavenger receptors = preference for molecules that are negatively charged.

    • Nucleic acids, phosphate-containing lipoteichoic acids (Gram positive bacteria) and LPS (Gram negative bacteria)

Macrophage Receptors (PRR’s) for Phagocytosis and Cytokine Signaling are Different

Receptors are found on:

Macrophages, monocytes, dendritic cells, granulocytes and NK cells.

~ 100 PRR’s are known

Recognition of Self vs. Non-self and altered self.

• Self – Normal healthy cell

• Non-self – Microbial cell

• Altered self – Cancer cell or apoptotic cell

Innate Leukocyte Receptors – Pattern Recognition Receptors (PRRs)

Receptors are not distributed equally on any given leukocyte. Two macrophages will not have the exact same receptors nor the same number of receptors.

This unequal distribution is thought to make sure that at least some of the leukocytes will be able to recognize the pathogen with their innate receptors.

These PRRs recognize Pathogen Associated Molecular Patterns (PAMPs), components common to many pathogens and/or altered self cells and molecules.

• Most PRRs recognize multiple different molecules

DAMPs – associated with cells that have been damaged

Binding of Microbe to Phagocytic Receptors Initiates Phagocytosis

Binding of the phagocytic receptors initiates receptor mediated endocytosis (phagocytosis).

The microbe is enveloped into a vesicle (phagosome)

The phagosome is joined to a lysosome = phagolysosome

The lysosome contains degradative enzymes and toxic substances to destroy the pathogen

Endocytosis - Phagocytosis

Macrophages have several types of surface receptor that bind to constituents of microbial surfaces and promote phagocytosis –

PRR (pattern recognition recpetors) on leukocytes bind to PAMPs (pathogen associated molecular patterns) on pathogens

Receptors that detect microbial products signal macrophage activation and cytokine secretion

Macrophage have receptors or sensors for pathogen components that signal macrophage to make and secrete cytokines

• Toll-like receptors (TLR)

  • 10 receptors

  • Specificities for different microbial products

  • TLR-4 senses ligand LPS

    • TLR-4 expressed on Macrophages

    • TLR-4 detects LPS and sends a signal to the nucleus of the macrophage to make and secrete inflammatory cytokines as well as cytokines that activate innate immune responses

    • LPS is a major Gram-negative bacteria component

    • LPS is an endotoxin responsible for septic shock

The 4 Lineages of TLR’s and Their Ligands

Some TLR’s need to be on the outside and some on the inside.

TLR-4 triggers a common pathway of intracellular signaling

1. LPS → LBP → CD14 → MD2 → TLR4

2. Adaptor protein → MyD88

3. Inhibitor of KappaB Kinase (IKK) → Phosphorylation of Inhibitor of Kappa B → degradation of IkB

4. Translocation of TF nuclear factor kB (NFkB) from cytoplasm to the nucleus

5. Directs the transcription of genes for inflammatory cytokines

   

NOD-Like Receptors

Cytoplasmic receptors for recognition of pathogens

NOD-1 – recognizes a degradation product of Gram (–) peptidoglycan

NOD-2 – recognizes a degradation product of most bacteria – muramyl dipeptide

Recognition leads to NFkB making
inflammatory cytokines

Not Covering and Not on Test – IL-1 Subfamilies or Inflammasome Details

Activation and release of IL-1B

Creation of inflammasome and cleavage of IL-1B

Release of IL-1B out of macrophage

Pyroptosis is a mechanism of death of the macrophage which allows a massive release of IL-1

• Large number of pores created for
  release

• Extreme circumstances

Most Macrophages probably don’t go through pyroptosis but rather release IL-1 more slowly.

• Less pores created for release

  

After Macrophage Recognition of PAMP

After recognition, macrophages form an Inflammasome to cleave the pro-form of IL-1 into an active form.

IL-1-pro form is kept in cytoplasm of Macrophages ready for cleavage.

• Quick response

IL-1 is an inflammatory cytokine made by macrophages after recognition of PAMPs.

IL-1 binding to its receptor IL-1R on macrophages initiates/activates the creation of the other inflammatory cytokines

Activation of resident macrophages induces inflammation at sites of infection

Development of inflammation in tissue leads to

• Local accumulation of fluid accompanied by

  • swelling, redness, heat and pain

Changes induced in blood capillaries

• → increase diameter (dilation) → reduction in the rate of blood flow → increased permeability
  to blood vessel wall → increased supply of blood = redness and heat

• → increased permeability of blood vessels → allows movement of fluid, plasma proteins and
  WBC (neutrophils primarily) from the blood capillaries into the tissue = swelling and pain

• Translocation of NFkB to macrophage nucleus initiates transcription of proinflammatory
  cytokines → IL-6, CXCL8, IL-12, CCL-2 and TNF-a

  • Look at fig. 3.9 with these cytokines and their functions.

At the site of infections, activated resident macrophages secrete inflammatory cytokines (IL-1 is the Master Regulator)

(in class* TNF-a: activates endothelial cells along with IL-1. CC or CXC = chemokine. Ending with L means ligands, so CXCL8 and CCL8.)

Chemokines => CXCL-8 and CCL-2

CCL-2 – recruits Monocytes from the blood to the infected tissue

• Binds to CCR-2

CXCL-8 attracts leukocytes (neutrophils) to site of tissue damage or infection

• bind to CXCR1 and CXCR2 on the neutrophil

Direct traffic of leukocytes during their development

• Small, (60-140 aa)

  • Two major families

    • Cysteine residues

    • CC or CXC

Cells are attracted from blood to infection site by following a concentration gradient of chemokine produced by cells at infection site

Chemokines interact with target cells by binding specific cell surface receptors → signal through associated GTP-binding proteins

TNF-a released by macrophages induces protection at the local level

Causes and consequences of the release of TNF- and IL-1 within a local area

• Endothelium (venules) →

  1. TNF-a, IL-1 and IL-6 raise temperature - Pyrogens

  2. Increased blood flow (Vasodialation)

  3. Increased permeability

  4. Endothelial adhesiveness for wbc and platelets (adhesion molecules induced)

     • ICAM-1 and ICAM-2 on endothelial cells and LFA-1 on Neutrophils

Causes blood in venules to clot

Prevents spread of infection to the blood (sepsis or septicemia)

IL-12 and IL-6

IL-6 – acts on local muscle and fat cells to increase temperature and Signals to liver hepatocytes to make acute phase reactants

• Mannose binding lectin (MBL) and C-reactive protein (CRP)

IL-12 activates NK cells

• Lymphocyte of the innate immune system

• NK cells secrete cytokines to maintain the macrophages activity

• Protect against viral infections

Distinct but complementary properties of Macrophages and Neutrophils

Macrophages

• Long-lived

• Reside in tissues

• Work as infection begins

• Raise alarm

Neutrophils

• Short-lived dedicated killers

• Circulate in blood

• Polymorphonuclear Leukocytes
  (PMNs)

• Wait for macrophage to sound
  the alarm

• To enter tissue

Neutrophils are dedicated phagocytes that are summoned to sites of infection

Granulocytes

• Granules containing antimicrobials

  • Primary (azurophilic), secondary (specific) granules and tertiary (gelatinase) granules Make sure to know what is underlined

• Polymorphonuclear leukocytes

  • Variable and irregular shapes of their nuclei

  • Microphages

  • Smaller sized than macrophages

  • Most abundant wbc (50 billion in circulation)

  • Life span < 2 days

  • 60% of hematopoietic activity of bone marrow

  • Large reserve kept in bone marrow (~5 days worth)

  • 1st leukocyte called to infected area in mass
    27

Neutrophils are excluded from healthy tissue

Neutrophils are excluded from healthy tissue

• Release of inflammatory mediators at infection sites is what attracts neutrophils

• Become dominant phagocytic cells

  • 3×10^9 enter mouth and throat each day

    • Arrival of neutrophils is the 1st of a series of rxns

      • Inflammatory response

Inflammatory Response

Involves recruitment of cells and molecules of innate
immunity into sites of infection

• Neutrophils die after 1 round of phagocytosis by apoptosis and form pus at the site of infection

• Extracellular bacteria that cause superficial wounds lead to pus formation

  • Ex: S. aureus → superficial infections and abscesses that neutrophils tackle in large numbers

    • Ex: Pus-forming = pyogenic bacteria

Leukocyte adhesion molecules

The four structural classes of adhesion molecule present on
white blood cells and the cells with which they interact are:

1. Selectins – are carbohydrate-binding lectins – L-Selectin

2. Vascular addressins - contain carbohydrate groups to which
   selectins bind – CD34, GlyCAM-1, MAdCAM-1

3. Integrins – typically bind to Ig superfamily proteins – LFA-1

4. Immunoglobulin superfamily – ICAM-1

   (1&2, 3&4 go together)

   

The homing of neutrophils to infected is induced by inflammatory mediators

Neutrophil receptors bind molecules that have chemoattractant capability

• Inflammatory mediators - Chemoattractants

  • C5a and C3a - cleaved during complement activation

  • CXCL8 - secreted by activated macrophages

  • Peptides containing N-formylmethionine (not human)

• Ligand binding to neutrophil surface receptor → changes in neutrophil adhesion molecules

  • Assist neutrophils in migrating out of blood capillaries

  • Extravasation

The homing of neutrophils to infected tissues is induced by inflammatory mediators

Inflammatory mediators (Cytokines) secreted by macrophages change the ligand expression for the neutrophil receptors on the surface of endothelial cells near the site of infection.

This allows the neutrophils to exit the blood near the site of infection = Extravasation

• 4 steps

  • Note that the WBC’s at some point will do something analogous to this process, we are just using neutrophil homing as our model.

Nascent - newly formed

Extravasation - Rolling adhesion → Tight binding → Diapedesis → Migration to infection site.

Neutrophils are potent pathogen killers but are themselves programmed to die

Phagocytosis by neutrophils

• Fc receptors – bind Abs

• Complement receptors – bind complement component = opsonins

• Phagocytosis of complement opsonized pathogens

• On availability of specific Abs

  • Opsonized with antibody and complement

Neutrophil process of phagocytosis is similar to
macrophages, but

• > range of particulate engulfed

• > microbicidals

• 4 types of granules

• devoted to storage & delivery of antimicrobial weaponry

4 Granular Types

Focus on 1-3, on the top

Neutrophil Granules for Killing

Bacterial agents produced or released by phagocytic cells on the ingestion of microorganisms (macrophage & neutrophil)

Neutrophil Granules

Primary/Azurophilic granules

• Lysozyme, defensins, myeloperoxidase, neutral proteases, elastase, proteinase 3...

Secondary/Specific granules

• Lactorferrin – metal sequestering protease

• Lysozyme

• NADPH oxidase proteins

Tertiary/Gelatinase granules

• Gelatinase – metal sequestering protease – restricts bacteria from gaining access to iron

• Secretory Vesicles

  • Surface molecule to attach to endothelial cells and home to site of infection

Respiratory Burst

Pathogen engulfed by neutrophil → degradative enzymes/toxins → fusion of phagosomes with neutrophil granules

• Granules = modified lysosomes of hydrolytic degradative enzymes, NADPH-dependent oxidases
  & a-defensins

NADPH oxidase produces Superoxide radicals which are converted to Hydrogen peroxide (h2o2) by superoxide dismutase.

• Reaction causes an increased consumption of hydrogen ions → raised pH → activation of primary and secondary granule contents.

Respiratory burst = Transient increase in oxygen consumption = purpose is to raise the pH of the phagosome so the granule contents can become active to kill pathogen

Lysozymes release contents → lower pH → continue the destruction of engulfed pathogens

Respiratory Burst → Kill Pathogens

Toxic Oxygen species produced during the respiratory burst can diffuse out and damage host cells

To limit damage by respiratory burst

• Phagocytes synthesize enzymes to inactivate toxic oxygen species

• Ex: catalase

  • Catalase degrades H2O2 → H2O + O2

What happens to these neutrophils?

• Neutrophils can’t replenish granule contents, so they die.

  Neutrophils → apoptosis → phagocytosed by macrophage

Neutrophil NET’s

Netosis – a way neutrophils die that leads to capture and destruction of the pathogen

NET’s – neutrophil extracellular traps - The neutrophil’s nucleus swells and leads to the cell bursting (netosis) leaving behind all the antimicrobial components of the granules as well as DNA and histones that serve to trap the pathogen.

Chronic Granulomatous Disease

NADPH oxidase is non-functional

• No oxidative burst

• pH isn’t raised in the phagolyosome → no activation of granular contents → no killing of pathogen

• Neutrophils phagocytosed by macrophages but can’t be destroyed

• Infected macrophages are surrounded by other leukocytes (walling them off) to stop the spread of the pathogen → granuloma formation

Question on this slide, disease-like state.

Macrophages produce cytokines: TNF-a, IL-1 and IL-6 = Pyrogens

A spectrum of biological activity to coordinate the body’s response to infection

• “Heat cytokines” alter energy mobilization to generate increased temperature and they act on:

  • hypothalamic temperature-control sites

  • muscle

  • fat cells

Result in body temperature increase → fever

What’s the role of fever?

Helps immune system fight infection

• Most bacterial and viral pathogens grow better at T’s lower than body
  temperature

• Elevated T’s → bacteria, viral replication decreases

• Ag processing increases

• Human cells become more resistant to TNF-a deleterious effects

Systemic effect of IL-6 changes soluble plasma proteins secreted by the hepatocytes of the liver → acute phase response → acute phase proteins → MBL and CRP

Inflammatory cytokines raise body temperature and activate hepatocytes to make the acute-phase response

Acute Phase Proteins

Produced in response to inflammatory cytokines

Produced by hepatocytes in liver

C-reactive protein and MBL are present at low levels in plasma

Levels increase during acute-phase response

• C-reactive and MBL both bind structures that are common to pathogens but absent on human cells

• Initiate complement activation

• Fixation by a pathway almost identical to classical pathway used by Abs

  • Abs of the adaptive immune response = similar role to that of C-reactive protein and MBL in innate immunity

  • Expand the range of pathogen-recognition molecules

  • Complement components used in the classical and lectin pathway are structurally and functionally related to components of the alternative pathway

C-reactive protein (CRP)

Pentamer of identical subunits (pentraxin family)

• Binds to phosphorylcholine component of LPS of bacterial and fungal cell walls

  • But not to phosphorylcholine in human cells

  • Acts as an opsonin independent of its complement activity

  • Can bind C1q to initiate classical pathway of complement fixation in absence of Abs

  • Enhance complement fixation to pathogen surfaces

C-reactive protein binds with C1q → stalks

Abs binds with C1q → globular heads

but then...

The same sequence of complement reactions occurs whether C-reactive protein or Ab interacts with pathogen and C1q binds

Mannose-binding lectin (MBL)

Binds mannose-containing carbohydrates of bacteria and yeast

• Calcium-dependent lectin

• MBL molecule is similar in structure to C1q

  • Acts as an opsonin independent of its complement activity

  • Activates the Lectin complement cascade

  • Enhance complement fixation to pathogen surfaces

  • 15 to 18 potential binding sites to attach to the pathogen surface

    • Even weak individual interactions with a carbohydrate structure can be developed into a high-avidity using multipoint attachments

    • Mannose containing carbohydrates on human cells do not bind MBL because their geometry does not permit multipoint attachment to MBL

MBL, CRP Opsonization

Acute-phase response increases the supply of the recognition molecules of innate immunity

Shows opsonization of pathogen by MBL and CRP

MBL activates a proteolytic enzyme complex: MBL- associated serine protease = MASP

MASP-2 (serine protease) cleaves C4 and C2

Initiates complement activation

• Lectin pathway of complement activation

• MBL serves as an opsonin to facilitate bacteria uptake by monocytes in the blood

• Monocytes do not express the macrophage mannose receptor but have receptors that can bind to MBL molecules coating a bacterial surface

• Leads to the Classical C3 convertase forming

  

C4 and C3 are similar in function,
C4 and C3 are similar in function, structure and the thioester bond and C2 and factor B are similar.

C3 convertases:

• Alternative – C3bBb

• Classical – C4b2a

  

Classical Complement Pathway with C1 binding to CRP

Classical Pathway Initiated by Ab’s

Figure 9.28

1 IgM or 2 IgG’s bound by C1q → C1r activation and cleavage of C1s

C1s cleaves C4 and C4 similar to MASP2 in lectin pathway and just like in the Classical started by CRP

Classical C3 convertase = C4b2a

MASP and C1 – Similar Structure and Function

Close structural relationships between components of the alternative, lectin-mediated & classical pathways of complement activation

Innate Lymphocytic Cells

ILCs provide a quick response to pathogen prior to activation of the adaptive T cells.

CD4 T helper cells and CD8 cells secrete the same cytokines/cytotoxins as the ILCs

Intracellular Infection: Type I interferons inhibit viral replication and activate host defenses

Human cell infected with virus → response is secretion of type I interferons (interferon)

• Family of interferons different from → IFN-y which is secreted from NK cells, CD8 T cells and CD4 TH1 cells (type II interferon)

• Type I interferon

  • Interfere with viral replication in infected cells

  • Alert immune system cells that infection is present

  • Make virus-infected cells more vulnerable to killer lymphocyte attack

• All nucleated cells are susceptible to viral infections

• All nucleated cells have the capacity to make type I interferon as well as its cell-surface receptor

Type I interferons inhibit viral replication and activate host defenses

The type I interferon receptor is always present on cell surfaces

• Ready to bind interferon made in response to infection

Type I interferon is barely detectable in healthy people’s blood

• But at infection, type I interferon becomes abundant

Many isotypes of type I interferons – main two are Alpha and Beta
• Interferon-B (IFN-B) = single form in humans
• Interferon-a (IFN-a) = multiple forms
• Interferon-S, -k -y, -t and -w - FYI – will not test on these

Intracellular (viral) Infection

RIG-1 and MDA-5 – cytoplasmic sensors for viral RNA’s  type 1 Interferon production

RIG-1 – retinoic-acid-inducible gene 1

MDA-5 – melanoma differentiation-associated protein 5

Use fig. 3.15 below as the beginning point for fig. 3.16 (next two slides)