# Lecture 5: Innate Immunity II: Pattern Recognition Receptors and PAMPs
## Immunology
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## Learning Objectives
By the end of this lecture, students will be able to:
1. Define pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs)
2. Describe the major families of pattern recognition receptors (PRRs) and their ligands
3. Explain the signaling pathways downstream of Toll-like receptors
4. Describe the function of cytoplasmic PRRs (NLRs, RLRs, cGAS-STING)
5. Discuss the concept of trained immunity
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## Lecture Content
### I. Pathogen-Associated Molecular Patterns (PAMPs)
PAMPs are conserved molecular structures shared by groups of microorganisms but absent from host cells. They have several important properties: they are essential for microbial survival and therefore cannot be easily mutated to escape recognition, they are shared by entire classes of pathogens rather than being unique to individual species, and they are distinct from host molecules, enabling self/non-self discrimination. Examples of PAMPs span all major categories of pathogens. **Bacterial** PAMPs include lipopolysaccharide (LPS, found on Gram-negative bacteria), lipoteichoic acid (Gram-positive bacteria), peptidoglycan, flagellin, and unmethylated CpG DNA. **Viral** PAMPs include double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), viral DNA, and viral glycoproteins. **Fungal** PAMPs include β-glucan, mannan, and zymosan, while **parasitic** PAMPs include glycosylphosphatidylinositol (GPI) anchors and profilin-like proteins.
### II. Damage-Associated Molecular Patterns (DAMPs)
DAMPs are endogenous molecules released from damaged, stressed, or dying host cells. They signal "danger" to the immune system even in the absence of infection, thereby triggering sterile inflammation. Important examples include HMGB1 (high mobility group box 1), a nuclear protein released from necrotic cells; ATP, which is released from damaged cells and activates the P2X7 receptor and the NLRP3 inflammasome; uric acid crystals (monosodium urate), which trigger inflammation in gout; heat shock proteins (HSPs); mitochondrial DNA, which contains CpG motifs similar to bacterial DNA; S100 proteins; and IL-1α, which is released from necrotic cells.
### III. Pattern Recognition Receptors (PRRs) -- Overview
PRRs are germline-encoded receptors that detect PAMPs and DAMPs. They are found on innate immune cells such as macrophages, dendritic cells, and neutrophils, as well as on some non-immune cells including epithelial cells and fibroblasts. The major families of PRRs include membrane-bound receptors -- **Toll-like receptors (TLRs)**, **C-type lectin receptors (CLRs)**, **scavenger receptors**, and **N-formyl peptide receptors** -- as well as cytoplasmic sensors -- **NOD-like receptors (NLRs)**, **RIG-I-like receptors (RLRs)**, and the **cGAS-STING pathway** for cytoplasmic DNA sensing.
A schematic of a macrophage cell showing the locations and ligands of all major PRR families. On the cell surface: TLR1/2, TLR2/6, TLR4, TLR5 with their respective ligands (lipopeptides, LPS, flagellin). On the endosomal membrane: TLR3, TLR7/8, TLR9 with their ligands (dsRNA, ssRNA, CpG DNA). In the cytoplasm: NOD1 and NOD2 detecting peptidoglycan fragments; RIG-I and MDA5 detecting viral RNA; cGAS detecting cytoplasmic DNA and producing cGAMP to activate STING on the ER. CLRs (Dectin-1, DC-SIGN, Mannose receptor) on the cell surface detecting fungal and microbial carbohydrates. Downstream signaling pathways are indicated with arrows leading to NF-kB activation, IRF3/7 activation, and inflammasome assembly.
### IV. Toll-Like Receptors (TLRs)
Toll-like receptors were first identified in Drosophila, where the Toll gene is involved in dorsoventral patterning and antifungal defense. Humans have 10 TLRs (TLR1 through TLR10), while mice have 12 (TLR1-TLR9 and TLR11-TLR13). Structurally, TLRs are type I transmembrane proteins with a leucine-rich repeat (LRR) extracellular domain and an intracellular TIR domain.
The **cell surface TLRs** detect extracellular microbial components. The TLR1/TLR2 heterodimer recognizes triacyl lipopeptides from bacteria, while TLR2/TLR6 recognizes diacyl lipopeptides, lipoteichoic acid, and zymosan. TLR4 detects lipopolysaccharide (LPS) and requires the co-receptors MD-2 and CD14. TLR5 recognizes flagellin from bacterial flagella, and TLR10 has an unknown ligand but may have a regulatory function. The **endosomal TLRs** detect nucleic acids following phagocytosis: TLR3 recognizes double-stranded RNA (a viral replication intermediate), TLR7 and TLR8 recognize single-stranded RNA from viral genomes, and TLR9 detects unmethylated CpG DNA from bacteria and viruses.
TLR signaling proceeds through two major pathways. The **MyD88-dependent pathway**, used by all TLRs except TLR3, involves MyD88 adaptor recruitment leading to IRAK4, IRAK1/2, TRAF6, TAK1, and the IKK complex, ultimately activating NF-κB, which induces pro-inflammatory cytokines such as TNF-α, IL-1, IL-6, and IL-12. The **TRIF-dependent pathway**, used by TLR3 and TLR4, signals through the TRIF adaptor to TRAF3, TBK1, and IRF3 phosphorylation, inducing type I interferons (IFN-α and IFN-β) that are critical for antiviral defense. TLR4 is unique in that it signals through both pathways.
### V. C-Type Lectin Receptors (CLRs)
C-type lectin receptors are calcium-dependent carbohydrate-binding receptors. **Dectin-1** recognizes β-glucan on fungal cell walls and activates NF-κB via Syk kinase, making it important for antifungal immunity. **Dectin-2** recognizes high-mannose structures on fungi. **DC-SIGN**, found on dendritic cells, binds mannose and fucose on pathogens to facilitate antigen capture and presentation, though it is exploited by HIV for DC-mediated trans-infection of T cells. The **mannose receptor (CD206)** recognizes terminal mannose, fucose, and GlcNAc on pathogens and mediates phagocytosis. **Mincle** recognizes mycobacterial trehalose dimycolate (cord factor) as well as damaged self-molecules (SAP130).
### VI. NOD-Like Receptors (NLRs)
NLRs are cytoplasmic sensors for intracellular PAMPs and DAMPs. **NOD1** detects γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) from Gram-negative bacterial peptidoglycan, while **NOD2** detects muramyl dipeptide (MDP) from all bacterial peptidoglycan. Mutations in NOD2 are associated with Crohn's disease. Both NOD1 and NOD2 activate NF-κB via RIP2 kinase, leading to pro-inflammatory cytokine production.
The **NLRP3 inflammasome** is the most extensively studied inflammasome. It is activated by remarkably diverse stimuli, including ATP, uric acid crystals, bacterial toxins, alum adjuvant, and potassium efflux. The inflammasome assembles from three components: the NLRP3 sensor, the ASC adaptor, and pro-caspase-1. Once assembled, activated caspase-1 cleaves pro-IL-1β into active IL-1β (a potent pro-inflammatory cytokine), pro-IL-18 into active IL-18 (which promotes IFN-γ production), and gasdermin D, whose N-terminal fragment forms pores in the cell membrane leading to pyroptosis, a form of inflammatory cell death. Inflammasome activation follows a two-signal model: Signal 1 (priming) involves TLR activation of NF-κB, which drives transcription of NLRP3 and pro-IL-1β, while Signal 2 (activation) triggers the actual assembly of the inflammasome complex and caspase-1 activation. Other inflammasomes include NLRC4 (activated by flagellin and type III secretion systems), AIM2 (activated by cytoplasmic dsDNA), and pyrin.
Diagram of NLRP3 inflammasome assembly and activation. Panel A: Signal 1 (Priming) -- TLR4 on the cell surface detects LPS, activating NF-kB, which translocates to the nucleus to upregulate transcription of NLRP3, pro-IL-1beta, and pro-IL-18. Panel B: Signal 2 (Activation) -- various danger signals (ATP, K+ efflux, ROS, lysosomal damage, crystal phagocytosis) activate NLRP3, which oligomerizes and recruits ASC adaptor (forming ASC specks) and pro-caspase-1. Caspase-1 becomes active and cleaves pro-IL-1beta into mature IL-1beta, pro-IL-18 into mature IL-18, and gasdermin D into N-terminal fragments that form pores in the plasma membrane, leading to pyroptosis. Released IL-1beta and IL-18 are shown acting on neighboring cells.
### VII. RIG-I-Like Receptors (RLRs)
RLRs are cytoplasmic sensors for viral RNA. **RIG-I (retinoic acid-inducible gene I)** detects short dsRNA with a 5'-triphosphate cap, a feature absent from host mRNA. **MDA5 (melanoma differentiation-associated gene 5)** detects long dsRNA, such as picornavirus replication intermediates. Upon activation, both RIG-I and MDA5 signal through MAVS (mitochondrial antiviral signaling protein, located on the outer mitochondrial membrane), which activates TBK1/IKKε, leading to IRF3/IRF7 activation and the production of type I interferons (IFN-α and IFN-β). This pathway also activates NF-κB, driving pro-inflammatory cytokine production.
### VIII. cGAS-STING Pathway
The cGAS-STING pathway detects cytoplasmic double-stranded DNA, whether foreign or self in origin. **cGAS (cyclic GMP-AMP synthase)** binds cytoplasmic dsDNA and produces cyclic GMP-AMP (cGAMP). cGAMP then activates **STING (stimulator of interferon genes)**, an ER-resident adaptor, which signals through TBK1 to IRF3, ultimately inducing type I interferons. This pathway is important for defense against DNA viruses, intracellular bacteria, and retroviruses. However, aberrant activation by self-DNA can cause autoinflammatory diseases such as Aicardi-Goutieres syndrome and STING-associated vasculopathy.
### IX. Trained Immunity
Traditionally, innate immunity was thought to lack memory, but the concept of **trained immunity** has challenged this view. Trained immunity refers to the long-term functional reprogramming of innate immune cells following an initial stimulation. The underlying mechanisms involve epigenetic modifications (histone methylation and acetylation) in monocytes and macrophages, along with metabolic rewiring toward glycolysis. For example, BCG vaccination enhances non-specific protection against other infections, and β-glucan exposure trains monocytes for enhanced cytokine responses. This reprogramming is mediated at the level of bone marrow progenitors (HSCs). Importantly, trained immunity is distinct from adaptive memory: it is not antigen-specific and does not involve clonal expansion.
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