receptor (NLR)

RIG-like receptor

(RLR)

Cytosolic DNA

sensor (CDS)

TLR

Endosomal

Cytosolic

Nucleic acids

of ingested

microbes

Plasma

membrane

Microbial

polysaccharide

C-type

lectin receptor

Endosomal

membrane

Extracellular

Microbial DNA

Fig. 2.2 Cellular locations of receptors of the innate immune system. Some receptors, such as certain

Toll-like receptors (TLRs) and lectins, are located on cell surfaces; other TLRs are in endosomes. Some receptors for viral nucleic acids, bacterial peptides, and products of damaged cells are in the cytoplasm. NOD and

RIG refer to the founding members of families of structurally homologous cytosolic receptors for bacterial

and viral products, respectively. (Their full names are complex and do not reflect their functions.) There are

five major families of cellular receptors in innate immunity: TLRs, CLRs (C-type lectin receptors), NLRs (NODlike receptors), RLRs (RIG-like receptors), and CDSs (cytosolic DNA sensors). Formylpeptide receptors (not

shown) are involved in migration of leukocytes in response to bacteria.

28 CHAPTER 2 Innate Immunity

changes associated with cell injury, and proteolytically

generate active forms of the inflammatory cytokines

IL-1ß and IL-18. IL-1ß and IL-18 are synthesized as inactive

precursors, which must be cleaved by the enzyme caspase-1

to become active cytokines that are released from the cell

and promote inflammation. Inflammasomes are composed

of oligomers of a sensor, caspase-1, and an adaptor that links

the two. There are many different types of inflammasomes,

most of which use 1 of 10 different NLR-family proteins

as sensors. These sensors directly recognize microbial

products in the cytosol or sense changes in the amount of

endogenous molecules or ions in the cytosol that indirectly

indicate the presence of infection or cell damage. Some

inflammasomes use sensors that are not in the NLR family,

such as AIM-family DNA sensors and a protein called pyrin.

After recognition of microbial or endogenous ligands, the

TLR-2 TLR-4 TLR-5

TLR-3

TLR-7

TLR-8

TLR-9

dsRNA

ssRNA

ssRNA

CpG DNA

TLR-1:TLR-2 TLR-2:TLR-6

LPS Bacterial

flagellin

Bacterial

peptidoglycan

Bacterial

lipopeptides

Bacterial

lipopeptides

Plasma

membrane

Endosome

Gram-positive bacteria

Gramnegative

bacteria

Fig. 2.3 Specificities of Toll-like receptors. Different TLRs recognize many different, structurally diverse

products of microbes. Plasma membrane TLRs are specific for cell wall components of bacteria, and endosomal TLRs recognize nucleic acids. All TLRs contain a ligand-binding domain composed of leucine-rich motifs

and a cytoplasmic signaling, Toll-like interleukin-1 (IL-1) receptor (TIR) domain. ds, Double-stranded; LPS,

lipopolysaccharide; ss, single-stranded.

CHAPTER 2 Innate Immunity 29

NLR sensors oligomerize with an adaptor protein and an

inactive (pro) form of the enzyme caspase-1 to form the

inflammasome, resulting in generation of the active form of

caspase-1 (Fig. 2.5). Active caspase-1 cleaves the precursor

form of the cytokine interleukin-1ß (IL-1ß), pro-IL-1ß, to

generate biologically active IL-1ß. As discussed later, IL-1

induces acute inflammation and causes fever.

One of the best characterized inflammasomes uses

NLRP3 (NOD-like receptor family, pyrin domain containing 3) as a sensor. The NLRP3 inflammasome is

expressed in innate immune cells including macrophages and neutrophils, as well as keratinocytes in the

skin and other cells. A wide variety of stimuli induce formation of the NLRP3 inflammasome, including crystalline substances such as uric acid (a by-product of DNA

breakdown, indicating nuclear damage) and cholesterol

crystals, extracellular adenosine triphosphate (ATP)

(an indicator of mitochondrial damage) binding to cell

surface purinoceptors, reduced intracellular potassium

ion (K+) concentration (which indicates plasma membrane damage), and reactive oxygen species. Thus, the

inflammasome reacts to injury affecting various cellular

components. How NLRP3 recognizes such diverse types

of cellular stress or damage is not clearly understood.

Inflammasome activation is tightly controlled by posttranslational modifications such as ubiquitination and

phosphorylation, which block inflammasome assembly or activation, and some micro-RNAs, which inhibit

NLRP3 messenger RNA.

Inflammasome activation also causes an inflammatory form of programmed cell death of macrophages and

DCs called pyroptosis, characterized by swelling of cells,

loss of plasma membrane integrity, and release of inflammatory cytokines. Activated caspase-1 cleaves a protein

called gasdermin D. The N-terminal fragment of gasdermin D oligomerizes and forms a channel in the plasma

membrane that initially allows the egress of mature IL-1ß,

and eventually permits the influx of ions, followed by cell

swelling and pyroptosis.

The inflammasome is important not only for host

defense but also because of its role in several diseases.

Gain-of-function mutations in NLRP3, and less frequently,

loss-of-function mutations in regulators of inflammasome

activation, are the cause of autoinflammatory syndromes, characterized by uncontrolled and spontaneous

inflammation. IL-1 antagonists are effective treatments for

these diseases. The common joint disease gout is caused

by deposition of urate crystals and subsequent inflammation mediated by inflammasome recognition of the

crystals and IL-1ß production. The inflammasome may

also contribute to atherosclerosis, in which inflammation

caused by cholesterol crystals may play a role.

Cytosolic RNA and DNA Sensors

The innate immune system includes several cytosolic

proteins that recognize microbial RNA or DNA and

respond by generating signals that lead to the production of inflammatory and antiviral cytokines.

• The RIG-like receptors (RLRs) are cytosolic proteins

that sense viral RNA and induce the production of the

antiviral type I IFNs. RLRs recognize features of viral

Leucine-rich

repeats

Toll-IL-1 receptor (TIR)

signaling domain

IRFs (interferon

regulatory factors)

TLR engagement

by bacterial or

viral molecules

Increased expression of:

Cytokines, adhesion

molecules, costimulators

Production of

type 1 interferon

(IFN a, ß)

-Acute inflammation

-Stimulation of

 adaptive immunity

Antiviral state

NF-?B

Recruitment of

adaptor proteins

Activation of

transcription

factors

Fig. 2.4 Signaling functions of toll-like receptors. TLRs activate similar signaling mechanisms, which involve adaptor proteins

and lead to the activation of transcription factors. These transcription factors stimulate the production of proteins that mediate

inflammation and antiviral defense. NF-?B, Nuclear factor ?B.

30 CHAPTER 2 Innate Immunity

RNAs not typical of mammalian RNA, such as dsRNA

that is longer than dsRNA that may be formed transiently in normal cells, or RNA with a 5' triphosphate

moiety not present in mammalian host cell cytosolic

RNA. (Host RNAs are modified and have a 5’ 7methyl-guanosine “cap.”) RLRs are expressed in many cell

types that are susceptible to infection by RNA viruses.

After binding viral RNAs, RLRs interact with a mitochondrial membrane protein called mitochondrial

antiviral-signaling (MAVS), which is required to initiate signaling events that activate transcription factors

that induce the production of type I IFNs.

+

+

Acute inflammation

Pathogenic bacteria

Extracellular ATP

Innate signals

(e.g., TLRs)

Bacterial products

Crystals

K+ efflux

Reactive oxygen species

Pro-IL1ß

NLRP3

inflammasome

Caspase-1 (active)

IL-1ß

Nucleus Secreted

IL-1ß

Plasma

membrane

Pro-IL1ß gene

transcription

K+

K+

NLRP3

(sensor)

Adaptor

Caspase-1

(inactive)

Fig. 2.5 The inflammasome. Shown is the activation of the NLRP3 inflammasome, which processes pro–

interleukin-1ß (pro–IL-1ß) to active IL-1. The synthesis of pro–IL-1ß is induced by various PAMPs or DAMPs

through pattern recognition receptor signaling. Subsequent production of biologically active IL-1ß is mediated

by the inflammasome. The inflammasome also stimulates production of active IL-18, which is closely related

to IL-1 (not shown). Other forms of the inflammasome exist which contain sensors other than NLRP3, including NLRP1, NLRC4, or AIM2. ATP, Adenosine triphosphate; NLRP3, NOD-like receptor family, pyrin domain

containing 3; TLRs, Toll-like receptors.

CHAPTER 2 Innate Immunity 31

• Cytosolic DNA sensors (CDSs) include several

structurally related proteins that recognize microbial double-stranded (ds) DNA in the cytosol and

activate signaling pathways that initiate antimicrobial responses, including type 1 IFN production and

autophagy. DNA may be released into the cytosol

from various intracellular microbes. Since mammalian DNA is not normally in the cytosol, the innate

cytosolic DNA sensors will see only microbial DNA.

Most innate cytosolic DNA sensors engage the

stimulator of IFN genes (STING) pathway to induce

type 1 IFN production (Fig. 2.6). In this pathway, cytosolic dsDNA binds to the enzyme cyclic GMP-AMP

synthase (cGAS), which activates the production of

a cyclic dinucleotide signaling molecule called cyclic

GMP-AMP (cGAMP), which binds to an endoplasmic

reticulum membrane adaptor protein called stimulator of interferon gene (STING). In addition, bacteria

P

DNA

Viruses,

bacteria

cGAS

TBK1 STING

ER

IRF3

Type I IFN gene

transcription

Type I

Interferons

P

Induction of

antiviral state

Bacteria

Cyclic

dinucleotides

IRF3

Fig. 2.6 Cytosolic DNA sensors and the STING pathway. Cytoplasmic microbial dsDNA activates the

enzyme cGAS, which catalyzes the synthesis of cyclic GMP-AMP (cGAMP) from ATP and GTP. cGAMP binds

to STING in the endoplasmic reticulum membrane, and then STING recruits and activates the kinase TBK1,

which phosphorylates IRF3. Phospo-IRF3 moves to the nucleus, where it induces type I IFN gene expression.

The bacterial second messenger molecules cyclic di-GMP (c-di-GMP) and cyclic di-AMP (c-di-AMP) are directly

sensed by STING. STING also stimulates autophagy and lysosomal degradation of pathogens associated with

cytoplasmic organelles. cGAS, Cyclic GMP-AMP synthase; IFN, interferon; IRF3, interferon regulatory factor 3.

32 CHAPTER 2 Innate Immunity

themselves produce other cyclic dinucleotides that

also bind to STING. Upon binding these cyclic dinucleotides, STING initiates signaling events that lead

to transcriptional activation and expression of type I

IFN genes. STING also stimulates autophagy, a mechanism by which cells degrade their own organelles

in lysosomes. Autophagy is used in innate immunity

to deliver cytosolic microbes to the lysosome, where

they are killed by proteolytic enzymes. Other cytosolic

DNA sensors besides cGAS can also activate STING.

Other Cellular Receptors of Innate Immunity

Many other receptor types are involved in innate

immune responses to microbes (see Fig. 2.2).

Some lectins (carbohydrate-recognizing proteins) in the

plasma membrane are receptors specific for fungal glucans

(these receptors are called dectins) or for terminal mannose residues (called mannose receptors); they are involved

in the phagocytosis of fungi and bacteria and in inflammatory responses to these pathogens. A cell surface receptor

expressed mainly on phagocytes, called formyl peptide

receptor 1, recognizes polypeptides with an N-terminal

formylmethionine, which is a specific feature of bacterial

proteins. Signaling by this receptor promotes the migration

as well as the antimicrobial activities of the phagocytes.

Although our emphasis thus far has been on cellular receptors, the innate immune system also contains

several circulating molecules that recognize and provide

defense against microbes, as discussed later.

COMPONENTS OF INNATE IMMUNITY

The components of the innate immune system include epithelial cells; sentinel cells in tissues (resident macrophages,

dendritic cells, mast cells, and others); circulating and

recruited phagocytes (monocytes and neutrophils); innate

lymphoid cells; NK cells; and a number of plasma proteins.

We next discuss the properties of these cells and soluble

proteins and their roles in innate immune responses.

Epithelial Barriers

The major interfaces between the body and the external environment—the skin, gastrointestinal tract,

respiratory tract, and genitourinary tract—are protected by layers of epithelial cells that provide physical and chemical barriers against infection (Fig.

2.7). Microbes come into contact with vertebrate hosts

mainly at these interfaces by external physical contact,

ingestion, inhalation, and sexual activity. All these portals of entry are lined by continuous epithelia consisting

of tightly adherent cells that form a mechanical barrier

against microbes. Keratin on the surface of the skin and

mucus secreted by mucosal epithelial cells prevent most

microbes from interacting with and infecting or getting

through the epithelia. Epithelial cells also produce antimicrobial peptides including defensins and cathelicidins, which kill bacteria and some viruses by disrupting

their outer membranes. Thus, antimicrobial peptides

provide a chemical barrier against infection. In addition, epithelia contain lymphocytes called intraepithelial T lymphocytes that belong to the T cell lineage but

express antigen receptors of limited diversity. Some of

these T cells express receptors composed of two chains,

? and d, that are similar but not identical to the aß T

cell receptors expressed on the majority of T lymphocytes (see Chapters 4 and 5). Intraepithelial lymphocytes

often recognize microbial lipids and other structures.

Intraepithelial T lymphocytes presumably react against

infectious agents that attempt to breach the epithelia,

but the specificity and functions of these cells are poorly

understood.

Peptide

antibiotics

Intraepithelial

lymphocyte

Physical barrier

to infection

Killing of microbes

by locally produced

antibiotics

Killing of microbes

and infected cells

by intraepithelial

lymphocytes

Fig. 2.7 Functions of epithelia in innate immunity. Epithelia present at the portals of entry of microbes provide physical

barriers formed by keratin (in the skin) or secreted mucus (in

the gastrointestinal, bronchopulmonary, and genitourinary systems) and by tight junctions between epithelial cells. Epithelia

also produce antimicrobial substances (e.g., defensins) and harbor lymphocytes that kill microbes and infected cells.

CHAPTER 2 Innate Immunity 33

Phagocytes: Neutrophils and Monocytes/

Macrophages

The two types of circulating phagocytes, neutrophils

and monocytes, are blood cells that are recruited to

sites of infection, where they recognize and ingest

microbes for intracellular killing (Fig. 2.8).

• Neutrophils, also called polymorphonuclear leukocytes (PMNs), are the most abundant leukocytes in

the blood, numbering 4,000 to 10,000 per µL (Fig.

2.9A). In response to certain bacterial and fungal

infections, the production of neutrophils from the

bone marrow increases rapidly, and their numbers

in the blood may rise up to 10 times the normal.

The production of neutrophils is stimulated by cytokines, known as colony-stimulating factors (CSFs),

which are secreted by many cell types in response

to infections and act on hematopoietic cells to stimulate proliferation and maturation of neutrophil

precursors. Neutrophils are the first and most

numerous cell type to respond to most infections,

particularly bacterial and fungal infections, and

thus are the dominant cells of acute inflammation,

as discussed later. Neutrophils ingest microbes in

the circulation, and they rapidly enter extravascular

tissues at sites of infection, where they also phagocytose (ingest) and destroy microbes. Neutrophils

express receptors for products of complement activation and for antibodies that coat microbes. These

receptors enhance phagocytosis of antibody- and

complement-coated microbes and also transduce

activating signals that enhance the ability of the

neutrophils to kill ingested microbes. The process

of phagocytosis and intracellular destruction of

microbes is described later. Neutrophils are also

recruited to sites of tissue damage in the absence of

infection, where they initiate the clearance of cell

Feature Neutrophils Macrophages

Origin HSCs in bone marrow HSCs in bone marrow (in inflammatory reactions)

Many tissue-resident macrophages: stem cells in

yolk sac of fetal liver (early in development)

Phagocytosis Rapid ingestion of microbes Prolonged ability to ingest microbes, apoptotic

cells, tissue debris, foreign material

Responses to

activating stimuli

Rapid, short lived,

enzymatic activity

More prolonged, slower, often dependent

on new gene transcription

Life span in tissues 1–2 days Inflammatory macrophages: days or weeks

Tissue-resident macrophages: years

Reactive oxygen

species

Rapidly induced by assembly

of phagocyte oxidase

(respiratory burst)

Less prominent