Innate immune system; part 1&2 4 de septiembre de 2018

INNATE IMMUNE SYSTEM

Most microbial invaders can be detected and destroyed within minutes or hours by the body’s
defense mechanisms of innate immunity, which do not rely on expansion of antigen-specific
lymphocytes. The innate immune system uses a limited number of secreted proteins and cell-
associated receptors to detect infection and to distinguish between pathogens and host tissues.
These are called innate receptors because they are inborn; they are encoded by genes directly
inherated from an individual’s parents.

The response to an initial infection occurs in three

phases. These are the innate phase, the early induced
innate response, and the adaptive immune response.
The first two phases rely on the recognition of pathogens
by germline-encoded receptors of the innate immune
system, whereas adaptive immunity uses variable
antigen-specific receptors that are produced as a result
of gene segment rearrangements. Adaptive immunity
occurs late, because the rare B cells and T cells specific
for the invading pathogen must first undergo clonal
expansion before they differentiate into effector cells
that migrate to the site of infection and clear the
infection. The effector mechanisms that remove the
infectious agent are similar or identical in each phase.

An infection starts when a pathogen breaches one of the

host’s anatomic barriers. The immediate defenses
include several classes of preformed soluble molecules
that are present in extracellular fluid, blood and
epithelial secretions and that can either kill the pathogen
or weaken its effect.

- Antimicrobial enzymes: such as lysozyme begin

to digest bacterial cell walls
- Antimicrobial peptides: such as the defensins
lyse baterial cell membranes directly
- Complement system: system of plasma proteing
that target pathogens both for lysis and for
phagocytosis by cells of the innate immune
system such as macrophages.

If these fail, innate immune cells become activated by pattern recognition receptors (PRRs) that
detect molecules called pathogen-associated molecular patterns (PAMPs) that are typical of
microbes. The activated innate cells can engage various effector mechanisms to eliminate the
infection.

Only if an infectious organism breaches these first two lines of defense will mechanisms be
engaged to introduce an adaptive immune response. This leads to the expansion of antigen-
specific lymphocytes that target the pathogen specifically and to the formation of memory cells
that provide long-lasting specific immunity.

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Anatomic barriers and initial chemical defenses

The anatomic barriers are fixed defenses against infection and consist of the epithelia that line
the internal and external surfaces of the body along with the phagocytes residing beneath all
epithelial surfaces. These phagocytes act directly by engulfing and digesting invading
microorganisms. Epithelia are also protected by many kinds of chemical defenses, including
antimicrobial enzymes and peptides.

Our body surfaces are defended by epithelia, which impose a physical barrier between the
internal milieu and the external world that contains pathogens. Epithelia comprise the skin and
the linings of the body’s tubular structures— the respiratory, urogenital, and gastrointestinal
tracts. Epithelia in these locations are specialized for their particular functions and possess
unique innate defense strategies against the microbes they typically encounter.

The epidermis has multiple layers of

keratinocytes in different stages of
differentiation arising from the basal
layer of stem cells. Differentiated
keratinocytes in the stratum spinosum
produce β-defensins and cathelicidins,
which are secreted into the intercellular
space to form a waterproof lipid layer
(the stratum corneum) containing
antimicrobial activity.

In the lung, the airways are lined by

ciliated epithelium. Beating of the cilia
moves a continuous stream of mucus
(green) secreted by goblet cells
outward, trapping and ejecting
potential pathogens. Type II
pneumocytes in the lung alveoli (not
shown) also produce and secrete
antimicrobial defensins.

In the intestine, Paneth cells—

specialized cells deep in the epithelial
crypts— produce several kinds of
antimicrobial proteins: α-defensins
(cryptdins) and the antimicrobial lectin
RegIII.

Most healthy epithelial surfaces are also associated with a large population of normally
nonpathogenic bacteria, known as commensal bacteria or the microbiota, that help keep
pathogens at bay. The microbiota can also make antimicrobial substances, such as the lactic acid
produced by vaginal lactobacilli, some strains of which also produce antimicrobial peptides
(bacteriocins). Commensal microorganisms also induce responses that help to strengthen the

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barrier functions of epithelia by stimulating the epithelial cells to produce antimicrobial

peptides. When commensal microorganisms are killed by antibiotic treatment, pathogens
frequently replace them and cause disease. Under some circumstances commensal microbes
themselves can cause disease if their growth is not kept in check or if the immune system is
compromised.

If a microorganism crosses an epithelial barrier and begins to replicate in the tissues of the host,
in most cases, it is immediately recognized by resident phagocytic cells. The main classes of
phagocytic cells in the innate immune system are macrophages and monocytes, granulocytes,
and dendritic cells. Macrophages are the major phagocyte population resident in most normal
tissues at homeostasis. The second major family of phagocytes comprises the granulocytes,
which include neutrophils, eosinophils, and basophils. Of these, neutrophils have the greatest
phagocytic activity and are the cells most immediately involved in innate immunity against
infectious agents. The third class of phagocytes in the immune system is the immature dendritic
cells (DCs) that reside in lymphoid organs and in peripheral tissues.

The cells of the innate immune system serve as barriers against infections and as sentinels to
detect microbes and damaged cells in tissues and perform functions that are essential for
defense against microorganisms. Several cell types express the various PRRs, and after
recognizing PAMPs and DAMPs, the cells respond by producing inflammatory cytokines and
antiviral proteins.

Pattern recognition by cells of the innate immune system

The phagocytes that lie beneath the epithelial barriers and stand ready to engulf and digest
invading microorganisms also initiate the next phase of the innate immune response: inducing
an inflammatory response that recruits new phagocytic cells and circulating effector molecues
to the site of infection.

The innate immune system is able to recognize:

- Molecular structures that are produced by microbial pathogens. There are often share
by classes of microbes and are calles pathogen-associated molecular patterns (PAMPs).
Different types of microbes express different PAMPs
- Endogenous molecules that are produced by or released from damages and dying cells.
These substances are called damage-associated molecular patterns (DAMPs). DAMPs
may be produced as a result of cell damage caused by infections, but they may also
indicate sterile injury to cells caused by any myriad reasons, such as chemical toxins,
burns and trauma.
- Microbial products that are essential for survival of the microbes. An example of a
target of innate immunity that is indispensable for microbes is double-stranded viral
RNA. Similarly, LPS and lipoteichoic acid are structural components of bacteria cell calls
that are recognized by innate immune receptors.

The innate immune system uses several types of cellular receptors, present in different locations
in cells, and soluble molecules in blood and in mucosal secretions, to recognize PAMPs and
DAMPs. The cellular receptors for PAMPs and PAMPs are called pattern recognition receptors
(PRRs). They are expressed on the surface, in phagocytic vesicles, and in the cytosol of various
cell types. When these receptores bind to PAMPs and DAMPs, the activate signal transduction
pathways that promote the antimicrobial and proinflamatory functions of the cells in which they

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are expressed. In addition, there are many proteins present in the blood and extracellular fluids
that recognize PAMPs.

The receptors of the innate immune system are encoded by inherited genes, whereas the genes
encoding receptors of adaptive immunity are generated by somatic recombination of gene
segments in the precursors of mature lymphocytes. As a result, the diversity of innate immune
system receptors and the range of their specifities are small compared with those of B and T
cells of the adaptive immune system.

Cell-associated patterns recognition receptors

Most cell types express pattern recognition receptors and therefore are capable of paticipating
in innate immune responses. Phagocytes, especially macrophages, and DCs express the widest
variety and greatest number of these receptors. PRRs are linked to intracellular signal
transduction pathways that activate various cellular responses, including the production of
molecules that promote inflammation and molecules that destroy microbes.

TOLL-Like Receptors

Toll-like receptors (TLRs) are an evolutionarily conserved familiy of pattern recognition

receptors expressed on many cell types that recognize products of a wide variety of microbes,
as well as molecules expressed or released by stressed and dying cells. There are ten different
functional TLRs in humans, named TLR1 through TLR10.

TLRs are sensor for microbes present in extracellular spaces. Some mammalian TLRs are cell-
surface receptors, but others are located intracellularly in the membranes of endosomes,
where they detect pathogens or their
components that have been taken into
the cells by phagocytosis, receptor-
mediated endocytosis, or
macropinocytosis.

TLRs are single-pass transmembrane

proteins with an extracellular region
composed of 18-25 copies of a leucine-
rich repeat (LRR). Each LRR of a TLR
protein is composed of around 20-25
amino acids, and multiple LRRs create a
horseshoe-shaped protein scaffold that
is adaptable for ligan binding and
recognition on both the outer (convex)
and inner (concave) surfaces.

All mammalian TLR proteins have in

their citoplasmatic tail a TIR domain
(for Toll-IL-1 receptor), which interacts
with other TIR-type domains, usually in
other signaling molecules.

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Signaling by mammalian TLRs is activated when binding of a ligan induces formation of a dimer
or induces conformational changes in a preformed TLR dimer.

Direct recognition of pathogen-associated molecular patterns by TLR-1 and TLR-2 induces dimerization of
the TLRs and signaling. TLR-1 and TLR-2 are located on cell surfaces (left panel), where they can directly
recognize bacterial triacyl lipoproteins (middle panel). The convex surfaces of their extracellular domains
have binding sites for the lipid side chains of triacyl lipopeptides. In the crystal structure (right panel), the
ligand is a synthetic lipid that can activate TLR1:TLR2 dimers; it has three fatty-acid chains bound to a
polypeptide backbone. Two fatty-acid chains bind to a pocket on the convex surface of the TLR-2
ectodomain, and the third chain associates with a hydrophobic channel in the convex binding surface of
TLR-1, inducing dimerization of the two TLR subunits and bringing their cytoplasmic Toll–IL-1 receptor
(TIR) domains together to initiate signaling.

TLR signaling can activate the transcription factor NFκB, which induces the expression of pro-
inflammatory cytokines. First panel: TLRs signal via their cytoplasmic TIR domains, which are brought into
proximity to each other by ligand-induced dimerization of their ectodomains. Some TLRs use the adaptor
protein MyD88, and others use the MyD88/MAL pair to initiate signaling. The MyD88 death domain
recruits the serine–threonine kinases IRAK1 and IRAK4, in association with the ubiquitin E3 ligase TRAF-6.
IRAK undergoes autoactivation and phosphorylates TRAF-6, activating its E3 ligase activity. Second panel:
TRAF-6 cooperates with an E2 ligase (UBC13) and a cofactor (Uve1A) to generate polyubiquitin scaffolds
(yellow triangles) by attachment of ubiquitin through its lysine 63 (K63). This scaffold recruits a complex
of proteins composed of the kinase TAK1 (transforming growth factor-β-activated kinase 1) and two
adaptor proteins, TAB1 (TAK1-binding protein 1) and TAB2. TAB1 and TAB2 function to bind to
polyubiquitin, bringing TAK1 into proximity with IRAK to become phosphorylated (red dot). Third panel:
activated TAK1 activates IKK, the IκB kinase complex. First, the IKKγ subunit (NEMO) binds to the
polyubiquitin scaffold and brings the IKK complex into proximity to TAK1. TAK1 then phosphorylates and
activates IKKβ. IKKβ then phosphorylates IκB, the cytoplasmic inhibitor of NFκB. Fourth panel:

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phosphorylated IκB is targeted by a process of ubiquitination (not shown) that leads to its degradation.
This releases NFκB, which is composed of two subunits, p50 and p65, into the nucleus, driving the
transcription of many genes including those encoding inflammatory cytokines. TAK1 also stimulates
activation of the mitogen-activated protein kinases (MAPKs) JNK and p38, which phosphorylate and
activate AP-1 transcription factors (not shown).

NOD-Like Receptors
NOD-like receptors (NLRs) are a family of more than 20 different cytosolic proteins, some of which
recognize PAMPs and DAMPs and recruit other proteins to form signaling complexes that promote
inflammation. Typical NLRs include C-terminal leucine-rich repeat domain that senses the presence of
ligan, a central NOD (nucleotide oligomerization domain), domain required for NLR proteins to bind to
one another and form oligomers; and an N-terminal effector domain, which recruits other proteins to
form signaling complexes.

There are three NLRs subfamilies that serve as innate immune receptors, each using a different effector
domain to initiate signaling:

- NLRB: uses the BIR (baculovirus inhibition of apoptosis protein repeat)

- NLRC: uses CARDs (caspase recruitment and activation domains)
- NLRP: uses Pyrin domains (so called because they are found in proteins involved in causing fever)

Intracellular NOD proteins sense the presence of bacteria by recognizing bacterial peptidoglycans and
activate NFκB to induce the expression of pro-inflammatory genes. First panel: NOD proteins reside in
an inactive state in the cytoplasm, where they serve as sensors for various bacterial components. Second
panel: degradation of bacterial cell-wall peptidoglycans produces muramyl dipeptide, which is recognized
by NOD2. NOD1 recognizes γ-glutamyl diaminopimelic acid (iE-DAP), a breakdown product of Gram-
negative bacterial cell walls. The binding of these ligands to NOD1 or NOD2 induces aggregation, allowing
CARD-dependent recruitment of the serine– threonine kinase RIP2, which associates with E3 ligases,
including XIAP (X-linked inhibitor of apoptosis protein), cIAP1 (cellular inhibitor of apoptosis 1), and cIAP2.
This recruited E3 ligase activity produces a polyubiquitin scaffold, as in TLR signaling, and the association
of TAK1 and the IKK complex with this scaffold leads to the activation of NFκB, as shown in Fig. 3.15. In
this pathway, RIP2 acts as a scaffold to recruit XIAP, and RIP2 kinase activity is not required for signaling.

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Other cell-associated pattern recognition receptors

Several other types of plasma membrane and cytoplasmatic receptors transmit activating signals
similar to TLR that promote inflammatory responses and enhance killing of microbes, or mainly
participate in the uptake of microbes into phagocytes.

C-type lectin receptors

Cellular receptors that recognize carbohydrates on the surface of microbes facilitate the
phagocytosis of the microbes and the secretion of cytokines that promote inflammation and
subsequent adaptive immune responses. They are called C-type because they bind
carbohydrates in Ca2+-dependent manner. There are different types:

o Mannose receptor: is involved in phagocytosis of microbes. This receptor

recognizes certain terminal sugars on microbial surface carbohydrates, such as
D-mannose, L-fuctose, which are often present on the surface of
microorganisms; or galactose and sialic acid, which are present on the
eukaryiotic cell surface. Thus, the terminal sugars on microbes can be
considered PAMPs.
o Dectins: are expressed on DCs and macrophages, and play important roles in
antifungal immunity, as well as in responses to certain bacteria. Dectin-1 blinds
β-glucan, which is a major cell wall component of many fungal species.
o Langerin and DC-SIGN

Scanvenger receptors

recognize various anionic polymers and acetylated low-density lipoproteins. These receptors are
structurally heterogeneous, consisting of at least six different molecular families. Class A
scavenger receptors are membrane proteins composed of trimers of collagen domains. They
include SR-A I, SR-A II, and MARCO (macrophage receptor with a collagenous structure), which
all bind various bacterial cell-wall components and help to internalize bacteria, although the
basis of their specificity is poorly understood. Class B scavenger receptors bind high-density
lipoproteins, and they internalize lipids. One of these receptors is CD36, which binds many
ligands, including long-chain fatty acids.

Formyl-Peptide receptors

It is a G-protein-coupled receptor that senses the presence of bacteria by recognizing a unique

feature of bacterial polypeptides. Protein synthesis in bacteria is typically initiated with an N-
formylmethionine (fMet) residue, an amino acid present in prokaryotes but not in eukaryotes.
The fMLF receptor is named after a tripeptide, formylmethionyl-leucyl phenylalanine, for which
it has a high affinity, although it also binds other peptide motifs. Bacterial polypeptides binding
to this receptor activate intracellular signaling pathways that direct the cell to move toward the
most concentrated source of the ligand. Signaling through the fMLF receptor also induces the
production of microbicidal reactive oxygen species (ROS) in the phagolysosome.

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Epithelial cells and phagocytes produce several kinds of

antimicrobial proteins
Our surface epithelia are more than mere physical barriers to infection; they also produce a wide
variety of chemical substances that are microbicidal or that inhibit microbial growth. For
example, the acid pH of the stomach and the digestive enzymes, bile salts, fatty acids, and
lysolipids present in the upper gastrointestinal tract create a substantial chemical barrier to
infection. One important group of antimicrobial proteins comprises enzymes that attack
chemical features specific to bacterial cell walls. Such antibacterial enzymes include lysozyme
and secretory phospholipase A2, which are secreted in tears and saliva and by phagocytes.
Lysozyme is a glycosidase that breaks a specific chemical bond in the peptidoglycan component
of the bacterial cell wall. Peptidoglycan is an alternating polymer of N-acetylglucosamine
(GlcNAc) and N-acetylmuramic acid (MurNAc), strengthened by cross-linking peptide bridges.
Lysozyme selectively cleaves the β-(1,4) linkage between these two sugars and is more effective
in acting against Gram-positive bacteria, in which the peptidoglycan cell wall is exposed, than
against Gram-negative bacteria, which have an outer layer of LPS covering the peptidoglycan
layer.

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The second group of antimicrobial agents secreted by epithelial cells and phagocytes is the
antimicrobial peptides. Epithelial cells secrete these peptides into the fluids bathing the
mucosal surface, whereas phagocytes secrete them in tissues. Three important classes of
antimicrobial peptides in mammals are defensins, cathelicidins, and histatins.

- DEFENSINS. They are short cationic peptides of

around 30–40 amino acids that usually have three
disulfide bonds stabilizing a common amphipathic
structure—a positively charged region separated
from a hydrophobic region. Defensins act within
minutes to disrupt the cell membranes of bacteria
and fungi, as well as the membrane envelopes of
some viruses. The mechanism is thought to involve
insertion of the hydrophobic region into the
membrane bilayer and the formation of a pore that
makes the membrane leaky. Three subfamilies of
defensins—α-, β-, and θ-defensins—are distinguished
on the basis of amino acid sequence, and each family
has members with distinct activities, some being
active against Gram-positive bacteria and some
against Gram-negative bacteria, while others are specific for fungal pathogens. All the
antimicrobial peptides, including the defensins, are generated by proteolytic processing
from inactive propeptides. In humans, developing neutrophils produce α-defensins by
the processing of an initial propeptide of about 90 amino acids by cellular proteases to
remove an anionic propiece, generating a mature cationic defensin that is stored in so-
called primary granules. The primary granules of neutrophils are specialized
membraneenclosed vesicles, rather similar to lysosomes, that contain a number of other
antimicrobial agents as well as defensins. These primary granules within neutrophils are
induced to fuse with phagocytic vesicles (phagosomes) after the cell has engulfed a
pathogen, helping to kill the microbe.
- CATHELICIDINS. The lack the disulfide bonds that stabilize the defensins. Cathelicidins
are made constitutively by neutrophils and macrophages, and are made in response to
infection by keratinocytes in the skin and epithelial cells in the lungs and intestine. They
are made as inactive propeptides composed of two linked domains and are processed
before secretion. In neutrophils, the inactive cathelicidin propeptides are stored in
another type of specialized cytoplasmic granule called secondary granules.
- HISTATINS. They are constitutively produced in the oral cavity by the parotid, sublingual,
and submandibular glands.

Another type of bactericidal proteins made by epithelia is carbohydrate binding proteins, or

lectins. C-type lectins require calcium for the binding activity of their carbohydrate-recognition
domain (CRD), which provides a variable interface for binding carbohydrate structures. C-type
lectins of the RegIII family include several bactericidal proteins expressed by intestinal
epithelium in humans and mice, comprising a family of ‘lecticidins.’ RegIII family proteins
preferentially kill Gram-positive bacteria, in which the peptidoglycan is exposed on the outer
surface. In fact, the LPS of Gram-negative bacteria inhibits the pore-forming ability of RegIIIα,
further enforcing the selectivity of RegIII proteins for Gram-positive bacteria.

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The inflammatory response

The principal way by which the innate immune system deals with infections and tissue injury is
to stimulate acute inflammation, which is the accumulation of leukocytes, plasma proteins, and
fluid derived from the blood at an extravascular tissue site of infection or injury.

Inflammation has three essential roles in combating infection. The first is to deliver additional
effector molecules and cells from the blood into sites of infection, and so increase the
destruction of invading microorganisms. The second is to induce local blood clotting, which
provides a physical barrier to the spread of the infection in the bloodstream. The third is to
promote the repair of injured tissue.

Inflammatory responses are characterized by pain, redness, heat, and swelling at the site of an
infection, reflecting four types of change in the local blood vessels. The first is an increase in
vascular diameter, leading to increased local blood flow—hence the heat and redness—and a
reduction in the velocity of blood flow, especially along the inner walls of small blood vessels.
The second change is the activation of endothelial cells lining the blood vessel to express cell-
adhesion molecules that promote the binding of circulating leukocytes. The combination of
slowed blood flow and adhesion molecules allows leukocytes to attach to the endothelium and
migrate into the tissues, a process known as extravasation. All these changes are initiated by
the pro-inflammatory cytokines and chemokines produced by activated macrophages and
parenchymal cells.

The third major change in local blood vessels is an increase in vascular permeability. Thus,
instead of being tightly joined together, the endothelial cells lining the blood vessel walls
become separated, leading to an exit of fluid and proteins from the blood and their local
accumulation in the tissue. This accounts for the swelling, or edema, and pain—as well as the
accumulation in tissues of plasma proteins such as complement and MBL that aid in host
defense. The changes that occur in endothelium as a result of inflammation are known generally
as endothelial activation. The fourth change, clotting in microvessels in the site of infection,
prevents the spread of the pathogen via the blood.

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