The survival of multicellular organisms requires mechanisms for defense against microbial infections and the elimination of damaged and necrotic cells. The mechanisms that evolved first in invertebrates and persist in all higher vertebrates are always present and functional within the organism, ready to recognize and eliminate microbes and dead cells. Therefore, this type of host defense is known as innate immunity, also called natural immunity or native immunity. The cells and molecules that are responsible for innate immunity make up the innate immune system .
Innate immunity is the first line of host defense against infections. It blocks microbial invasion through epithelial barriers, destroys many microbes that do enter the body, and is capable of controlling and even eradicating infections. The innate immune response is able to combat microbes immediately upon infection; in contrast, to defend against a microbe not previously encountered, the adaptive immune response needs to be stimulated by antigen to undergo cell proliferation and differentiation steps and therefore is delayed. Innate immunity provides essential protection against infections during this delay. The innate immune response also instructs the adaptive immune system to respond to different microbes in ways that are effective for combating these microbes. In addition, innate immunity is a key participant in the clearance of dead tissues and the initiation of repair after tissue damage.
Before we consider adaptive immunity, the main topic of this book, we discuss the early defense reactions of innate immunity in this chapter. The discussion focuses on the following three questions:
How does the innate immune system recognize microbes and damaged cells?
How do the different components of innate immunity function to combat different types of microbes?
How do innate immune reactions stimulate adaptive immune responses?
General Features and Specificity of Innate Immune Responses
The innate immune system performs its defensive functions with a small set of reactions, which are more limited than the varied and specialized responses of adaptive immunity. The specificity of innate immunity is also different in several respects from the specificity of lymphocytes, the antigen-recognizing cells of adaptive immunity ( Fig. 2.1 ).
The two principal types of reactions of the innate immune system are inflammation and antiviral defense. Inflammation consists of the accumulation and activation of leukocytes and plasma proteins at sites of infection or tissue injury. These cells and proteins act together to kill mainly extracellular microbes and to eliminate damaged tissues. Innate immune defense against intracellular viruses, even in the absence of inflammation, is mediated by natural killer (NK) cells, which kill virus-infected cells, and by cytokines called type I interferons (IFNs), which block viral replication within host cells.
The innate immune system responds in essentially the same way to repeat encounters with a microbe, whereas the adaptive immune system mounts stronger, more rapid and thus more effective responses on successive encounters with a microbe. In other words, for the most part, the innate immune system does not remember prior encounters with microbes and resets to baseline after each such encounter, whereas memory is a cardinal feature of the adaptive immune response. There is emerging evidence that some cells of innate immunity (such as macrophages and natural killer cells) are altered by encounters with microbes such that they respond better upon repeat encounters. But it is not clear if this process results in improved protection against recurrent infections or is specific for different microbes.
The innate immune system recognizes structures that are shared by various classes of microbes and are not present on normal host cells . The cells and molecules of innate immunity recognize and respond to a limited number of microbial structures, much less than the almost unlimited number of microbial and nonmicrobial antigens that can be recognized by the adaptive immune system. Each component of innate immunity may recognize many bacteria, viruses, or fungi. For example, phagocytes express receptors for bacterial endotoxin, also called lipopolysaccharide (LPS), and other receptors for peptidoglycans, each of which is present in the outer membranes or cell walls of many bacterial species but is not produced by mammalian cells. Other receptors of phagocytes recognize terminal mannose residues, which are typical of bacterial and fungal but not mammalian glycoconjugates. Receptors in mammalian cells recognize and respond to double-stranded ribonucleic acid (dsRNA), which is produced during replication of many viruses but is not produced in mammalian cells, and to unmethylated CG-rich (CpG) oligonucleotides, which are common in microbial DNA but are not abundant in mammalian DNA. The microbial molecules that stimulate innate immunity are often called pathogen-associated molecular patterns (PAMPs) to indicate that they are present in infectious agents (pathogens) and shared by microbes of the same type (i.e., they are molecular patterns). The receptors of innate immunity that recognize these shared structures are called pattern recognition receptors.
Innate immune receptors are specific for structures of microbes that are often essential for the survival and infectivity of these microbes . This characteristic of innate immunity makes it a highly effective defense mechanism because a microbe cannot evade innate immunity simply by mutating or not expressing the targets of innate immune recognition. Microbes that do not express functional forms of these structures lose their ability to infect and colonize the host. In contrast, microbes frequently evade adaptive immunity by mutating the antigens that are recognized by lymphocytes, because these antigens are usually not required for the life of the microbes.
The innate immune system also recognizes molecules that are released from damaged or necrotic host cells. Such molecules are called damage-associated molecular patterns ( DAMPs ). Examples include high mobility group box protein 1 (HMGB1), a histone protein that is released from cells with damaged nuclei, and extracellular ATP, which is released from damaged mitochondria. The subsequent responses to DAMPs serve to eliminate the damaged cells and to initiate the process of tissue repair. Thus, innate responses occur even following sterile injury, such as infarction, the death of tissue due to loss of its blood supply.
The receptors of the innate immune system are encoded by inherited genes that are identical in all cells. The pattern recognition receptors of the innate immune system are nonclonally distributed; that is, identical receptors are expressed on all the cells of a particular type, such as macrophages. Therefore, many cells of innate immunity may recognize and respond to the same microbe. This is in contrast to the antigen receptors of the adaptive immune system, which are encoded by genes formed by rearrangement of gene segments during lymphocyte development, resulting in many clones of B and T lymphocytes, each expressing a unique receptor. It is estimated that there are about 100 types of innate immune receptors that are capable of recognizing about 1000 PAMPs and DAMPs. In striking contrast, there are only two kinds of specific receptors in the adaptive immune system (immunoglobulin [Ig] and T cell receptors [TCRs]), but because of their diversity they are able to recognize millions of different antigens.
The innate immune system does not react against healthy cells. Several features of the innate immune system account for its inability to react against an individual’s own cells and molecules. First, the receptors of innate immunity have evolved to be specific for microbial structures (and products of damaged cells) but not for substances in healthy cells. Second, some pattern recognition receptors can recognize substances such as nucleic acids that are present in normal cells, but these receptors are located in cellular compartments (such as endosomes; see below) from where components of healthy cells are excluded. Third, normal mammalian cells express regulatory molecules that prevent innate immune reactions. The adaptive immune system also discriminates between self and nonself; in the adaptive immune system, lymphocytes capable of recognizing self antigens are produced, but they die or are inactivated on encounter with self antigens.
The innate immune response can be considered as a series of reactions that provide defense at every stage of microbial infections:
At the portals of entry for microbes: Most microbial infections are acquired through the epithelial barrier of the skin and gastrointestinal, respiratory and genitourinary systems. The earliest defense mechanisms active at these sites are epithelia, providing physical barriers and antimicrobial molecules, and lymphoid cells.
In the tissues: Microbes that breach epithelia, as well as dead cells in tissues, are detected by resident macrophages, dendritic cells, and mast cells. Some of these cells react by secreting cytokines, which initiate the process of inflammation, and phagocytes residing in the tissues or recruited from the blood destroy the microbes and eliminate the damaged cells.
In the blood: Plasma proteins, including proteins of the complement system, react against microbes that enter the circulation and promote their destruction.
We will return to a more detailed discussion of these components of innate immunity and their reactions later in the chapter. We start with a consideration of how microbes, damaged cells, and other foreign substances are detected and how innate immune responses are triggered.
Cellular Receptors for Microbes and Damaged Cells
The pattern recognition receptors used by the innate immune system to detect microbes and damaged cells are expressed on phagocytes, dendritic cells, and many other cell types and are located in different cellular compartments where microbes or their products may be found. These receptors are present on the cell surface, where they detect extracellular microbes; in vesicles (endosomes) into which microbial products are ingested; and in the cytosol, where they function as sensors of cytoplasmic microbes and products of cell damage ( Fig. 2.2 ). These receptors for PAMPs and DAMPs belong to several protein families.
Toll-like receptors ( TLRs ) are homologous to a Drosophila protein called Toll, which was discovered for its role in the development of the fly and later shown to be essential for protecting flies against fungal infections. In vertebrates, there are 10 different TLRs specific for different components of microbes ( Fig. 2.3 ). TLR-2 recognizes several glycolipids and peptidoglycans that are made by gram-positive bacteria and some parasites; TLR-3 is specific for double-stranded RNA, and TLR-7 and TLR-8 are specific for single-stranded RNA; TLR-4 is specific for bacterial LPS (endotoxin), made by gram-negative bacteria; TLR-5 is specific for a bacterial flagellar protein called flagellin; and TLR-9 recognizes unmethylated CpG DNA, which is abundant in microbial genomes. TLRs specific for microbial proteins, lipids, and polysaccharides (many of which are present in bacterial cell walls) are located on cell surfaces, where they recognize these products of extracellular microbes. TLRs that recognize nucleic acids are in endosomes, into which microbes are ingested and where they are digested and their nucleic acids are released.
Signals generated by TLRs activate transcription factors that stimulate expression of cytokines and other proteins involved in the inflammatory response and in the antimicrobial functions of activated phagocytes and other cells ( Fig. 2.4 ). Among the most important transcription factors activated by TLR signals are members of the nuclear factor κB (NF-κB) family, which promote expression of various cytokines and endothelial adhesion molecules that play important roles in inflammation, and interferon regulatory factors (IRFs), which stimulate production of the antiviral cytokines, type I interferons.
Rare autosomal recessive diseases characterized by recurrent infections are caused by mutations affecting TLRs or their signaling molecules, highlighting the importance of these pathways in host defense against microbes. For example, individuals with mutations affecting TLR-3 are susceptible to herpes simplex virus infections, particularly encephalitis, and mutations in MyD88, the adaptor protein downstream of several TLRs, make individuals susceptible to bacterial pneumonias.
The NOD-like receptors (NLRs) are a large family of innate receptors that sense DAMPs and PAMPs in the cytosol of cells and initiate signaling events that promote inflammation. All NLRs contain a nucleotide oligomerization domain (NOD, named because of the activity it was originally associated with) but different NLRs have different N-terminal domains. Two important NLRs, NOD1 and NOD2, have N-terminal caspase related domains (CARDs), and are expressed in several cell types including mucosal barrier epithelial cells and phagocytes. NOD1 and NOD2 both recognize peptides derived from bacterial cell wall peptidoglycans, and in response, they generate signals that activate the NF-κB transcription factor, which promotes expression of genes encoding inflammatory proteins. NOD2 is highly expressed in intestinal Paneth cells in the small bowel, where it stimulates expression of antimicrobial substances called defensins in response to pathogens. Some polymorphisms of the NOD2 gene are associated with inflammatory bowel disease, perhaps because these variants have reduced function and allow luminal microbes to penetrate the intestinal wall and trigger inflammation.
Inflammasomes are multiprotein complexes that assemble in the cytosol of cells in response to microbes or changes associated with cell injury, and proteolytically generate active forms of the inflammatory cytokines IL-1β and IL-18. IL-1β 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 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-1β (IL-1β), pro-IL-1β, to generate biologically active IL-1β. 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-1β, 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-1β 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 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.
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 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.
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, γ and δ, that are similar but not identical to the αβ 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.
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 debris. Neutrophils live for only several hours in tissues, so they are the early responders, but they do not provide prolonged defense.