Pattern Recognition Receptors (PRRs) – TLRs, NODs, RLRs; Viral Sensing and IFN Responses

Pattern Recognition Receptors (PRRs) and Toll-Like Receptors (TLRs)

• PRRs recognize pathogen-associated molecular patterns (PAMPs) and initiate innate immune signaling. Toll-like receptors (TLRs) are a major class of PRRs on the cell surface or in endosomes.
• Two broad receptor locations for TLRs:
  • Plasma membrane (external-facing): recognize microbial components on the surface (carbohydrates, lipids, proteins).
  • Endosomal: recognize nucleic acids from microbes, especially viruses.
• Key takeaway for study: focus on receptor, ligand, transcription factor, and gene expression outcomes when learning a TLR pathway.

TLRs: Overview and Organization

• TLRs are pattern recognition receptors (PRRs). For shorthand, note: TLRs are pattern-recognition receptors (PRRs).
• TLR4-LPS example highlights the canonical pathway: recognize Gram-negative bacteria via lipopolysaccharide (LPS) and trigger signaling leading to NF-κB–mediated cytokine production and adhesion molecule upregulation.
• Two common signaling outcomes for TLR pathways:
  • Cytokine production (inflammation, recruitment of new cells).
  • Increased adhesion of immune cells to endothelium.
• Color-coding theme used in teaching diagrams (helps study):
  • Receptors = blue
  • Ligands = pink
  • Signaling molecules = green
  • Transcription factors = purple

TLR4 and the LPS Pathway (Gram-Negative Bacteria)

• Gram-negative bacteria carry LPS on their outer membrane; LPS is the ligand for TLR4.
• Stepwise outline (as presented in the transcript):
  • Step $1$: The microbe is a Gram-negative bacterium containing LPS.
  • Step $2$: LPS is taken up by the cell and trafficked toward signaling partners.
  • Step $3$: LPS engages LPS at the cell surface (LPS is presented to the receptor/conduit at the plasma membrane side).
  • Step $4$: LPS binds the external (extracellular) domain of TLR4.
  • Step $5$: Internal signaling ensues, leading to activation of downstream kinases and transcription factors.
• Signaling components and outcome (as described):
  • A signaling cascade involving a protein named
  • a) a kinase-like molecule (often referred to in class as a kinase; the term in the talk sounds like a kinase with an ending “-k”, commonly IRAK/TBK family is involved in TLR signaling) and
  • b) a ubiquitin ligase (often TRAF6 in canonical TLR signaling) that links to downstream kinases such as IKK.
  • Activation of IKK (IκB kinase) and NF-κB (nuclear factor κB).
  • Outcome: gene expression changes that mediate cytokine production and increased adhesion molecules.
• Practical note: In this instructor’s framing, NF-κB is a central transcription factor for TLR signaling; others may use MyD88 as an adaptor in many TLRs (not deeply recited in the transcript).
• Emphasis for test prep: know the receptor (TLR4), the ligand (LPS), the transcription factor (NF-κB), and the major outcome (cytokines and adhesion). Signaling intermediates are less critical to memorize here.

TLR Localization and Ligand Specificity

• TLRs exist in two main locations with different ligand types:
  • Plasma membrane TLRs: recognize microbial surface components (carbohydrates, lipids, proteins).
  • Endosomal TLRs: recognize nucleic acids (DNA, RNA) often from viruses.
• Consequences of location:
  • Endosomal TLRs are particularly important for detecting viral nucleic acids.
• General rule from the lecture: knowing cellular location helps narrow possible ligands, but you don’t have to memorize exact membrane vs endosome placements for every TLR on the exam—focus on receptor, ligand, and outcome.

TLR Dimerization: Homodimers vs Heterodimers

• TLRs can form:
  • Homodimers: two identical TLR molecules.
  • Heterodimers: two different TLRs pairing together.
• Examples from the talk:
  • TLR1/2 heterodimer recognizes bacterial lipoproteins.
  • TLR2/6 heterodimer can form with TLR1/2 pairings to recognize different ligands.
  • TLR10 can interact with TLR1 or TLR2.
• The heterodimers expand ligand recognition diversity; the homodimers tend to be more specific and are more likely to be tested on.
• Viral recognition by endosomal TLRs is highlighted, with particular emphasis on endosomal TLRs forming the core sensing machinery for nucleic acids.

Endosomal TLRs (Nucleic Acid Recognition)

• Endosomal TLRs primarily recognize nucleic acids, which is characteristic of viral pathogens.
• Endosomal TLRs that recognize viruses in the lecture: TLR3, TLR7, TLR8, and TLR9.
  • TLR3 recognizes dsRNA (double-stranded RNA).
  • TLR7 and TLR8 recognize ssRNA (single-stranded RNA).
  • TLR9 recognizes DNA (unmethylated CpG DNA).
• The lecture notes emphasize the distinction that endosomal TLRs largely detect viral genetic material, whereas surface TLRs recognize components of the microbial surface.
• Note on methylation distinction (as stated): unmethylated DNA (e.g., viral or bacterial DNA) can be distinguished by TLR9; methylation patterns help differentiate self from non-self DNA.

Non-Like (NOD-Like) Receptors: Cytosolic Sensing of Bacteria

• NOD-like receptors (NLRs) are cytoplasmic (intracellular) receptors, not membrane-spanning.
• Two major NLRs discussed: NOD1 and NOD2.
• Ligands (from bacterial cell wall breakdown products) as described in the transcript:
  • NOD1: gamma-glutamyl diaminophenolic acid (the transcript’s phrasing; note in standard biology this corresponds to a peptidoglycan fragment, often iE-DAP).
  • NOD2: neuraminol dipeptide (the transcript’s phrasing; standard biology describes muramyl dipeptide, MDP).
• Peptidoglycan (PG) is the bacterial cell wall component: PG is abbreviated as PG in the notes.
• Specificity: because these ligands are PG breakdown products, the NOD receptors target bacteria (bacterial PG fragments) rather than viruses, fungi, or parasites.
• Signaling pathway excerpt (as presented):
  • Internal PG products are transported to the cytoplasm and activate the NLR signaling axis.
  • A cascade involving RIPK2 (RIPK2) and TAK (TAK1; the transcript says “TAC” which likely refers to TAK1) leads to the IKK complex activation and ultimately NF-κB activation.
• Outcome: gene expression changes that promote macrophage activation, including increased killing and increased phagocytosis.
• The transcription notes emphasize that the strongest readouts are receptor (NOD1/NOD2) and ligand (PG breakdown products), and the downstream transcription factor NF-κB; the intermediate signaling molecules are less critical to memorize for the exam.
• Macrophage activation outcomes include:
  • Increased killing (of bacteria).
  • Increased phagocytosis through upregulated surface receptors.

Interferon (IFN) Responses to Viral Infection

• When virus infection occurs, a cytokine called interferon is produced, with two main forms:
  • Interferon-α (IFN-α)
  • Interferon-β (IFN-β)
• The transcript emphasizes IFN-β as the predominant cytokine produced in this pathway.
• Induction and response sequencing:
  • In virus-infected cells: IFN-β (and some IFN-α) is produced and released. The IFN-β acts in neighboring cells to establish an antiviral state (paracrine signaling) while IFN-α can act in an autocrine fashion to amplify IFN production in the same cell.
  • The antiviral state is achieved by upregulating hundreds of interferon-stimulated genes (ISGs) that block viral replication and enhance antiviral defenses.
• Analogy used in the talk: IFN-β acts like a signal to neighboring villages, warning them to get their weapons ready before a raid; the neighboring cells prepare their defenses.
• Key ISGs mentioned (examples):
  • OAS1 (2'-5'-oligoadenylate synthetase 1): degrades viral RNA.
  • PKR (protein kinase R): binds viral RNA and shuts down translation by phosphorylating eIF2α (global translation inhibition in infected and neighboring cells).
• The ISG program expands beyond OAS1 and PKR; hundreds of ISGs exist, contributing to broad antiviral defenses.
• The signaling axis for IFN response involves IFN-α/β binding their receptor (IFNAR) on cells, triggering JAK-STAT signaling and ISG transcription.
• Important concept: the antiviral state protects both infected and neighboring cells by limiting viral entry and replication, though in infected cells, this signaling can contribute to controlled cell death to prevent viral spread.

RIG-I–Like Receptors (RLRs): Cytosolic dsRNA Sensors for RNA Viruses

• Cytosolic RNA sensors discussed: RIG-I and MDA5 (RLRs).
• Ligands: double-stranded RNA (dsRNA).
  • RIG-I recognizes shorter dsRNA.
  • MDA5 recognizes longer dsRNA.
• RLRs reside in the cytoplasm because many RNA viruses replicate there, so cytosolic sensing is strategic for early detection.
• Structural features:
  • Both RIG-I and MDA5 contain CARD domains (signaling domains) and helicase domains that bind dsRNA; the C-terminal domain helps keep the receptor in an inactive state until dsRNA binding occurs.
  • The CAR (CARD) domains mediate downstream signaling; without proper auto-inhibition by the C-terminal domain, constitutive signaling could occur, causing chronic inflammation and tissue damage.
• Signaling adaptor: MAVS (mitochondrial antiviral signaling protein; sometimes referred to as IPS-1 or VISA in literature) on mitochondria coordinates downstream signaling with TBK1 and IKKε, leading to activation of transcription factors IRF3 and IRF7.
• Transcription factors and outcomes:
  • IRF3 and IRF7 are activated; IRF7 is particularly important in immune cells, while IRF3 is more broadly expressed.
  • Activated IRFs drive production of interferons, especially IFN-β and IFN-α, contributing to the antiviral state.
• When dsRNA is detected and signaling proceeds, transcription factors induce IFN production, feeding into the IFN signaling cascade described above (autocrine and paracrine signaling to establish the antiviral state).
• Note on chronic signaling risk: constitutive or unregulated RLR signaling can contribute to chronic inflammation and disease; proper regulatory control is essential.

Interferon Signaling Pathways (JAK-STAT and ISGs)

• Interferon signaling connects innate sensing to an antiviral gene program via JAK-STAT signaling:
  • IFN-β and IFN-α bind IFN receptors on cells, activating the JAK-STAT pathway.
  • Downstream transcription factors drive expression of ISGs (interferon-stimulated genes).
• Example ISGs and antiviral effects:
  • OAS1 degrades viral RNA.
  • PKR inhibits translation by phosphorylating eIF2α.
• The ISG network fosters an antiviral state in both infected and neighboring cells, restricting viral replication and spread.

Practical and Conceptual Takeaways

• Focus areas for studying these pathways:
  • Receptor identity (TLR, NOD, RLR), the ligand they recognize, and the transcription factor(s) they activate.
  • The primary biological outcomes: cytokine production, adhesion molecule upregulation, phagocytosis, and antiviral ISG expression.
• For TLR pathways, remember:
  • Location matters: plasma membrane TLRs vs endosomal TLRs.
  • Dimerization: homodimers vs heterodimers broaden ligand recognition.
  • The end result is often NF-κB–driven inflammatory gene expression; for viral recognition, IRF3/IRF7–driven interferon production is central.
• For NOD-like receptors, remember:
  • Intracellular sensing of PG fragments leads to NF-κB activation and macrophage activation (increased killing and phagocytosis).
• For RLRs and interferon signaling:
  • dsRNA sensing in the cytosol triggers IRF3/IRF7 activation and IFN production, which then signals via JAK-STAT to upregulate ISGs and establish an antiviral state.
• Exam strategy tips (as per the lecturer):
  • Memorize receptor, ligand, and transcription factor for each pathway.
  • Understand the type of organism each receptor is most likely to recognize (bacteria vs viruses) and the cellular location that dictates ligand types.
  • Be able to describe the antiviral state and give examples of ISGs (e.g., OAS1, PKR).

Quick Glossary (Aligned with Transcript Content)

• PRR: Pattern Recognition Receptor
• PAMP: Pathogen-Associated Molecular Pattern
• PAMPs recognized by TLRs include: LPS (lipopolysaccharide), lipoproteins, flagellin, nucleic acids, etc.
• TLR: Toll-like receptor
• NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; central transcription factor for inflammatory cytokines and adhesion molecule genes.
• IRF3/IRF7: Interferon regulatory factors; drive IFN production in response to viral sensing.
• IFN-β, IFN-α: Interferons; induce ISGs and antiviral state.
• ISG: Interferon-stimulated gene
• JAK-STAT: Janus kinase–signal transducer and activator of transcription signaling cascade
• OAS1: 2'-5'-oligoadenylate synthetase 1; degrades viral RNA
• PKR: Protein kinase R; inhibits translation by phosphorylating eIF2α
• RIG-I: Retinoic acid-inducible gene I; cytosolic dsRNA sensor
• MDA5: Melanoma differentiation-associated protein 5; cytosolic dsRNA sensor
• MAVS: Mitochondrial antiviral-signaling protein; adaptor for RLR signaling
• TAK1: Transforming growth factor beta-activated kinase 1 (often downstream of TLR signaling)
• TRAF6: TNF receptor–associated factor 6; ubiquitin ligase involved in TLR signaling
• LPS: Lipopolysaccharide; a component of Gram-negative bacteria recognized by TLR4
• PG: Peptidoglycan; bacterial cell wall component; breakdown products recognized by NOD1/NOD2
• iE-DAP: γ-D-glutamyl-meso-diaminopimelic acid; a PG fragment recognized by NOD1 (as described in the transcript)
• MDP: Muramyl dipeptide; a PG fragment recognized by NOD2 (as described in the transcript)