1.

1: Intro

The innate immune system includes anatomical barriers against infection both physical
and chemical as well as cellular responses.
o Anatomical Barriers:
The main physical barriers are the epithelial layers of the skin and the
epithelial layers of the mucosal and glandular tissue surfaces. Epithelial
barriers prevent infection by blocking pathogens from entering the body.
The main chemical barriers at these surfaces are specialized soluble
substances that possess antimicrobial activity AND acid pH.
o Cellular Responses:
The cellular innate immune response occurs after an infectious agent
overcomes the initially epithelial barriers.
This response is rapid.
It is triggered by cell surface or intracellular receptors that recognize
conserved molecular components of pathogens.
What does it constitute? Macrophages and neutrophils (white blood cells) are
activated to engulf and destroy microbes via phagocytosis. Other receptors
induce production of proteins and other substances that have various effects
such are direct antimicrobial activity or recruitment of fluid, cells and
molecules.
The influx of fluid, cells, and molecules causes swelling inflammation.
REFER TO FIGURE 5-1

1.2: Anatomical Barriers to Infection

REFER TO FIGURE 5-2
Lets go into more detail about these barriers to infection. The epithelial barriers include the skin
and the tissue surfaces connected to the bodys opening: the mucous epithelial layers that line the
respiratory, gastrointestinal, and urogenital tracts and the ducts of the secretory glands such as
the salivary, lacrimal, and mammary glands

A. Epithelial Barriers Prevent Pathogen Entry into the Bodys Interior

Epithelial Barrier #1: Skin

Skin has two layers: thin outer layer (epidermis) and thick inner layer (dermis)
Epidermis:
o Contains tiers of epithelial cells
o Consists of mostly dead cells filled with a waterproofing protein called keratin.
Dermis:
o Composed of connective tissues and contains blood vessels, hair follicles, sebaceous
glands, sweat glands, and scattered myeloid leukocytes such as dendritic cells,
macrophages, and mast cells.

Epithelial Barrier #2: Tissue Surfaces

Instead of skin, the respiratory, gastrointestinal, and urogenital tracts, as well as the ducts of
the salivary, lacrimal, and mammary glands are lined by strong barrier layers of epithelial
cells stitched together by tight junctions that prevent pathogens from squeezing between
them to enter the body.

Other Barriers

Secretions of secretory tissue (mucus, urine, saliva, tears, and milk) wash away potential
invaders and also contain antibacterial and antiviral substances.
Mucus from mucosal epithelial layers entraps foreign microorganisms.
Cilia in the respiratory tract are hairlike protrusions of the cell membrane which cover
epithelial cells. The movement of cilia propers mucus-entrapped microorganisms from the
respiratory tract.
The flow of urine sweeps bacteria from the urinary tract.
Saliva has antimicrobial compounds that attack microbes the moment we ingest food.
The vagina has vaginal secretions with an acidic pH.

Some organisms evolved to evade these barriers. For instance, influenza virus has a surface
molecule that enables it to attach firmly to cells in mucous membranes of the respiratory
tract, preventing the virus from being swept out by the ciliated epithelial cells.

B. Antimicrobial Proteins and Peptides Kill Would-be Invaders

*Side note: A peptide is anything with less than 100-amino acids linked together. A protein is
anything with more than 100-amino acids linked together. They are basically the same thing.

Epithelial cells secrete a broad spectrum of proteins and peptides that provide protection
against pathogens.

Antimicrobial Proteins

Among the antimicrobial proteins produced by the skin and other epithelia in humans (Table
5-2), several are enzymes and binding proteins that kill or inhibit the growth of bacterial and
fungal cells.
o Lysozyme is an enzyme found in saliva, tears, and fluids of the respiratory tract that
cleaves the peptidoglycan components of bacterial cells walls.
o Lactoferrin and calprotectin are two proteins that bind and sequester metal ions
needed by bacteria and fungi, limiting their growth.
o Psoriasin is an antimicrobial protein that kills E.coli on the skin. However it cannot
kill S.auerus. This highlights the fact that antimicrobial proteins show some
specificity toward particular pathogens.

Antimicrobial Peptides

These are also antimicrobial components secreted by skin and other epithelial layers.
Antimicrobial peptides generally are cysteine-rich, cationic, and amphipathic (containing
both hydrophilic and hydrophobic regions).
Because of their positive charge and amphipathic nature, they interact with acidic
phospholipids in lipid bilayers, disrupting the membranes of bacteria, fungi, parasites, and
viruses.
They then can enter the microbes where they have other toxic effects, such as inhibiting the
synthesis of DNA, RNA, or proteins, and activating antimicrobial enzymes, resulting in
death.

Types of antimicrobial peptides: alpha-defensins, beta-defensins, and cathelicidin.

Antimicrobial peptides also attack the lipoprotein envelope of enveloped viruses.
Defensins and cathelicidin LL-37 (the only cathelicidin expressed in humans) are secreted
constitutively by epithelial cells in many tissues, as well as stored in neutrophil granules
where they contribute to killing phagocytosed microbes.
As we will see later, production of antimicrobial peptides can be induced in many epithelial
and other cell types by the binding of microbial components to cellular receptors.

Surfacants

The epithelium of the respiratory tract secretes a variety of lubricating lipids and proteins
called surfacants.
There are two: SP-A and SP-D. They are members of a class of microbe-binding proteins
called collectins.
SP-A and SP-D bind differentially to sets of carbohydrate, lipid, and protein components
of microbial surfaces and help to prevent infection by blocking and modifying surface
components and promoting pathogen clearance.

Now that weve covered the physical and chemical anatomical barriers, lets discuss the cellular
response that results when anatomic barriers fail.

1.3: Phagocytosis
A. Overview

Once pathogens penetrate through the epithelial barrier layers into the tissue spaces of the
body, an array of cellular membrane receptors and soluble proteins that recognize
microbial components play the essential roles of detecting the pathogen and triggering
effective defenses against it.
The next line of defense after the anatomic barriers are the phagocytic cells. Phagocytosis is
the cellular uptake (eating) of particulate materials such as bacteria. Phagocytes are nonspecific.
o In the tissues, the phagocytic cells are macrophages, neutrophils, and dendritic cells.
o In the blood, the phagocytic cells are the monocytes.

How Do Macrophages Work?:

o Most tissue contain macrophages that are on standby for the innate response.
o Through various cell surface receptor, the macrophages recognize microbes such as
bacteria, extend their plasma membrane to engulf them, and internalize them in
phagosomes (endosomes resulting from phagocytosis)
o Lysozomes then fuse with the phagosomes, delivering agents (proteases,
glycopeptidases, primarily acidic protein enyzmes) that kill and degrade the microbes.
These agents are produced in the golgi of the macrophage. The lysosome becomes
acidic when it fuses with the phagosome because of a proton flow into the lysosome
which lowers the pH. This change in acidity makes the digestive agents acidic as
well.

Neutrophils are a second major phagocyte.

Dendritic cells are the third phagocyte. Uptake a degradation of microbes by dendritic cells
play key roles in the initiation of adaptive immune response.
In addition to triggering phagocytosis, various receptors on phagocytes recognize microbes
and activate the production of a variety of molecules that contribute in other way to
eliminating infection, as will be described later.

B. Microbes are Recognize by Receptors on Phagocytic Cells

How does a phagocytic cell recognize microbes, triggering their phagocytosis?

Phagocytes have a variety of surface receptors. We can organize them into two categories (1)
Pattern recognition receptors, OR (2) Opsonin receptors.
REFER TO TABLE 5-3
REFER TO FIGURE 5-6

Pattern Recognition Receptors

o Microbes have specific conserved molecular components on their surfaces.
o These conserved molecular components (or motifs) are known as pathogenassociated molecular patterns (PAMPs)
o Phagocytes have receptors that recognize the PAMPs. These receptors are called
pattern recognition receptors (PRRs).

o PRRs can operate in two ways:

(1) They can bind the microbes (PAMP > PRR) and trigger phagocytosis of
the bound microbe. The PAMPs that cause this response are usually cell-wall
components of the microbes (complex carbs such as mannans and betaglucans, lipopolysaccharides, peptidoglycans, surface proteins)
(2) They can bind the microbes, and instead of triggering phagocytoses, they
trigger a different type of response.

Opsonin Receptors
o Instead of directly activating phagocytosis (the receptor on the phagocyte (the PRR)
directly binds to the PAMPs on the microbe and results in phagocytosis), we can
activate phagocytosis indirectly.
o There are soluble proteins that can bind to microbial surface and can be easily
recognized by phagocytes. These soluble phagocytosis-enhancing proteins (called
opsonins) will bind to carbohydrate structures, lipopolysaccharides, and viral proteins
on the microbe.
o Once bound to microbe surfaces, opsonins are recognized by membrane opsonin
receptors on phagocytes, thereby activating phagocytosis.

Opsonins
o A variety of soluble proteins function as opsonins; many play other roles as well in
innate immunity. Produced elsewhere and found floating around.
o Lets look at an example: SP-A and SP-D.
We said earlier that SP-A and SP-D are anatomic barriers, specifically,
surfacants that lubricate.
They are ALSO found in the blood in addition to the mucosal secretions
throughout the body.
In the blood, they can function as opsonins.
After binding to microbes, they are recognized by the CD91 opsonin receptor
on alveolar and other phagocyte (in this case macrophages) populations.
Binding activates phagocytosis.
This function of SP-A and SP-D contributes to the clearance of the fungal
respiratory pathogen Pneumocystis carinii, a major cause of pneumonia.
o Lets look at another example: Mannose-binding lectin (MBL)
This is a third collectin with opsonizing activity.
It is found in the blood and respiratory fluids.
o Lets look at another example: L-fociolin
A member of the ficolin family that is related to MBL and other collectins.
It is found in the blood where it binds to acetylated sugars on microbes,
including some streptococcal bacteria.
o Lets look at another example: C1q

The complement component C1q also functions as an opsonin, binding

bacterial cell wall components such as lipopolysaccharides and some viral
proteins.

o MBL (and other collectins), ficolins, and C1q share structural features, including
similar polymeric structures with collagen-like shafts, but have recognition regions
with different binding specificities
Because of their structural similarities, they are all received by the same
receptor on a phagocyte (the CD91 opsonin receptor):
INSERT FIGURE 5-7
o Another opsonin, C-reactive protein (CRP), regnozies phosphorylcholine and
carbohydrates on bacteria, fungi, and parasites, and is then bound by Fc receptors
(FcRs) for IgG found on most phagocytes.
FcRs are also important for the opsonizing activity of IgA antibodies and
some IgG antibody subclasses.
After binding specifically to antigens on microbe surfaces, the Fc regions of
these antibodies can be recognized by specific FcRs, triggering phagocytosis.

Brief Mentioning of the Complement System

o Among the most effective opsonins are several components of the complement
system.
o In vertebrates, complement consists of more than 30 binding proteins and enzymes
that function in a cascade of sequential activation steps.
o It can be triggered by several innate soluble pattern-recognition proteins (including
the first complement component, C1q, and the structurally related lectins MBL and
ficolins, C-reactive protein, and properdin), as well as by microbe-bound antibodies
generated by the adaptive immune response.
o Phagocytosis is one of the many important antimicrobial effects resulting from
complement activation.
o MBL is important in that it is both an opsonizer and an inducer of complement
activation.

C. Phagocytosed Microbes are Killed by Multiple Mechanism

So now that we know how phagocytes detect microbes, how exactly are the microbes killed?

The binding of microbes to phagocytes via pattern recognition receptors or opsonins/opsonin

receptors activates signaling pathways.
o The signaling pathways trigger actin polymerization, resulting in the membrane of the
phagocyte extending around the microbe particles and engulfing/internalizing it,
ultimately forming phagosomes.
o The phagosomes then fuse with lysosomes. In the case of neutrophils, the
phagosomes fuse with the lysosomes as well as primary and secondary granules.
o The resulting phagolysosomes contain an arsenal of antimicrobial agents that kill and
degrade the internalized microbes.
o These agents include antimicrobial proteins and peptides, low pH, acid-activated
hydrolytic enzymes (including lysozyme and proteases) , and specialized molecules
that mediate oxidative attack.

Lets discuss oxidative attack in detail:

o Oxidative attack on the phagocytosed microbes employs highly toxic reactive

oxygen species (ROS) and reactive nitrogen species (RNS), which damage
intracellular components.
o The reactive oxygen species are generated by the phagocytes NADPH oxidase
enzyme complex (aka phagosome oxidase). This complex is activated when the
microbes bind to the phagocyte receptors.
NADPH oxidase converts oxygen (which is supplied via a metabolic process
known as respiratory burst) converts oxygen to superoxide ion (O2-). You
also get other enzymes producing H2O2 and HClO.
o The reactive nitrogen species are generated in a different way. Generation of RNS
requires the transcriptional activation of the gene for an enzyme known as inducible
nitric oxide synthase (iNOS or NOS2).

o Collectively the ROS and RNS are highly toxic to phagocytosed microbes due to the
alteration of microbial molecules through oxidation, hydroxylation, chlorination,
nitration, and S-nitrosylation, along with formation of sulfonic acids and destruction
of iron-sulfur clusters in proteins.
o For example, oxidation by ROS of cysteine sulfhydryls that are present in the active
site of many enzymes in pathogens results in the inactivation of those enzymes.

D. Phagocytosis Contributes to Cell Turnover and the Clearance of Dead Cells

As the body;s main scavenger cells, macrophages also utilize their phagocytic receptors to take
up and clear cellular debris, cells that have died from damage or toxic stimuli (necrotic cell
death) or from apoptosis (programmed cell death), and aging red blood cells.

Collectively the components of dead/dying cells and damaged tissues that are recognized by
PRRs leading to their clearance are referred to as damage-associated molecular patterns
(DAMPs).
Phagocytosis is the major mode of clearance of cells that have undergone apoptosis as part of
developmental remodeling of tissues, normal cell turnover, or killing of pathogen-infected or
tumor cells by innate or adaptive immune responses.
Apoptic cells attract phagocytes by releasing the lipid mediator lysophosphatidic acid, which
functions as a chemoattractant.
o These dying cells facilitate their own phagocytosis by expressing on their surfaces an
array of molecules not expressed on healthy cells, including phospholipids (such as

phosphatidyl serine and lysophosphatidyl choline), proteins (annexin I), and altered
carbohydrates.
An important additional activity of macrophages in the spleen and those in the liver (Kupffer
cells) is to recognize, phagocytose, and degrade aging and damaged red blood cells.
o As the RBCs age, specific molecules accumulate on their membranes.
o For instance, phosphatidyl serine flips from the inner to the outer leaflet on the
bilayer and is recognized by phosphatidyl serine receptors on the phagocytes.

Something important to note is that there is a mechanism to prevent phagocytosis of normal

self-cells.
o Whether or not a cell is phagocytosed is controlled by sets of eat me signals the
altered membrane components (DAMPs) described above and dont eat me
signals expressed by normal cells.
o Young, healthy erythrocytes avoid being phagocytosed by not expressing eat me
signals, such as phosphatidyl serine, and also by expressing a dont eat me signal,
the protein CD47.
o CD47 is recognized by the SIRP-alpha receptor on macrophages, which transmits
signals that inhibit phagocytosis.

1.4: Induced Cellular Innate Responses

In addition to triggering their own uptake and killing by phagocytic cells, microbes induce a
broad spectrum of cellular innate immune responses by a wide variety of cell types

There are several families of pattern recognition receptors (PRRs) that bind to PAMPs as
well as to some endogenous (self) DAMPs and trigger signal-transduction pathways that turn
on the expression of genes with important functions in innate immunity.
The proteins that are encoded by these genes are:
o Antimicrobial peptides and interferons
o Chemokines and cytokines that recruit and activate other cells, enzymes such as
iNOS that generate antimicrobial molecules
o Proinflammatory mediators (i.e., components that promote inflammation.

A. Cellular Pattern Recognition Receptors Activated Responses to Microbes and Cell Damage
PRRs are expressed either on the surface of the plasma membrane OR inside the cell
itself (in the endosomes/lysosomes or in the cytosol).

By having so many different PRRs in two locaations it ensure that the cell can recognize
PAMPs on both extracellular and intracellular pathogens.
Also, its not just PAMPs that are recognized. You can have DAMPs that are released by
damaged cells or tissue which can be recognized.

There are multiple cells that express these intracellular and extracellular PRRs:
o The myeloid white blood cells:
Monocytes
Macrophages
Neutrophils
Eosinophils
Mast Cells
Basophils
Dendritic Cells
o And Subsets of the tree types of lymphocytes:
B cells
T cells
NK cells
o As well as other cell types, especially those exposed to infectious agents:
Skin cells
Mucosal cells
Glandular epithelial cells
Vascular endothelial cells that line blood vessels
Fibroblasts and stromal support cells in various tissues.

We will now look at one of four main families of mammalian PRRs. We wont look at the
signaling pathways that these PRRs activate and ultimate cause a protective response.

B. Toll-Like Receptors Recognize Many Types of Pathogen Molecules

Toll-like receptors (TLRs) were the first family of PRRs to be discovered.
o First, it was discovered that mutations of the toll gene (a gene that coded for the Toll
membrane protein) in Drosphila made the flies highly susceptible to lethal infection
with Aspergillus fumigatus, a fungus to which wild-type flies were usually immune.
o This observation let to other studies showing that Toll and related proteins are
involved in the activation of innate immune responses in invertebrates.
o Then, scientists discovered a human gene for a protein similar to Toll that activated
the expression of innate immunity genes in human cells. The proteins came to be
known as Toll-like receptors.
o A scientists, Beutler, obtained the important proof that TLRs contribute to normal
immune function in mammals.

Mice homozygous for a mutant form of a gene called lps were resistant to the
harmful responses induced by lipopolysaccharide (aka endotoxin), a major
component of the cell walls of Gram-negative bacteria.
In humans, a buildup of endotoxin from severe bacterial infection can induce
too strong of an innate immune response, causing septic shock, a lifethreatening condition in which vital organs such as the brain, heart, kidney,
and liver may fail.
Beutler found that the defective mouse lps gene encodaed a mutant form of
one TLR, TLR4, which differed from the normal formb y a single amino acid
so that it was no longer activated by LPS.
Thus, he showed that TLR4 is the cellular innate pattern recognition receptor
that recognizes LPS.

C. Expression of Innate Immunity Proteins is Induced by PRR Signaling

NOT SURE IF WE NEED TO KNOW THIS
(1)
(2)
(3)
(4)
(5)

Antimicrobial peptides
Type I Interferons
Cytokines
Chemokines
Enzymes: iNOS and COX2

1.5: Inflammatory Responses

A. Overview

When an innate immune response occurs it can induce a complex cascade of events known as
the inflammatory response.
Inflammation can be acute
o This means there are short-term effects contributing to combating infection, followed
by healing.
Inflammation can also be chronic
o This means that it is long term and not resolved.
o It can contribute to conditions such as arthritis, inflammatory bowel disease,
cardiovascular disease, type 2 diabetes.
What does the inflammatory response consists of?
o For one, there is an increase in vascular diameter (vasodilation), which results in a
rise of blood volume in the area of injury/infection.
o Higher blood volume heats the tissue and causes it to redden.
o Vascular permeability also increase, leading to leakage of fluid from the blood
vessels, resulting in accumulation of fluid (edema) that swells the tissue.

o Within a few hours, leukocytes also enter the tissue from the local blood vessels.
These leukocytes area activated to phagocytose bacteria and debris and to amplify the
response by producing additional mediators.
How does the inflammatory response end?
o Resolution of acute inflammatory response includes the clearance of invading
pathogens, dead cells, and damaged tissue; the activation of the systemic acute phase
response and additional physiological responses, including the initiation of wound
healing; and the induction of adaptive immune responses.
o If the infection or tissue damage is not resolved, it can lead to a chronic inflammatory
state that can cause more local tissue damage and potentially have systemic
consequences for the affected individual.

B. Inflammation Results from Innate Responses Triggered by Infection, Tissue Damage or

Harmful Substances

When there is local infection, tissue damage, or exposure to some harmful substances,
sentinel cells residing in the epithelial layer (basically, macrophages, mast cells, and dendritic
cells) are activated by PAMPs, DAMPs, crystals, and so on to starts phagocytosing the
offending invaders.
o The cells are also activated to release innate immunity mediators that trigger a series
of processes that collectively constitute the inflammatory response.
The recruitment of various leukocyte populations to the site of infection or damage is a
critical early component of inflammatory responses.
o PRR signaling activates resident macrophages, dendritic cells, and mast cells to
release the initial components of cellular innate immune responses.
o These components act on the vascular endothelial cells of local blood vessels,
increasing the vascular permeability and the expression of cell adhesion molecules
(CAMs) and chemokines. This epithelium is said to be inflamed or activated.
o Cells flowing through local capillaries are induced by chemoattractants and adhesion
molecule interaction to adhere to vascular endothelial cells in the inflamed region and
pass through walls of capillaries and into the tissue spaces, a process called
extravasation.
o Neutrophils are the first to be recruited to a site of infection where they enhance local
innate responses, followed by monocytes that differentiate into macrophages that
participate in pathogen clearance and help initiate wound healing.