Lecture 4 PPT Notes: Innate Immunity and Pattern Recognition Signaling

Overview of the Innate Immune System

• Definition of Innate Immunity: A rapid response system to immunological challenges, whether they are exogenous (external) or endogenous (internal).

• Successive Layers of Defense: The system is organized into several layers of defense, which are often sufficient to clear a challenge before the adaptive system is required.

  • First Layer - Physical (Anatomical) Barriers: Includes the epithelial layers of the skin and various mucous membranes.

  • Second Layer - Chemical (Physiological) Barriers: These are soluble factors and environments with extreme pH levels encountered if physical barriers are breached.

  • Third Layer - Cellular Innate Immune Response (Phagocytic Barrier): Active within minutes of a challenge; primarily involves macrophages ($MF$) and polymorphonuclear neutrophils ($PMN$) engaging in phagocytosis.

Inflammation and Antimicrobial Proteins

• Inflammation: Triggered by signaling molecules; characterized by fluid accumulation at the site of challenge, the production of chemoattractants to recruit effector cells, and subsequent swelling.

• Antimicrobial Proteins: These may impede the growth of or actively kill organisms. Examples include:

  • Complement proteins.

  • Interferons.

  • Lysozyme.

  • Defensins.

• Adaptive Immune System Activation: If the innate system is subverted, it primes and activates the adaptive immune system, which provides a tailored, specific attack unique to the challenge.

Anatomic Barriers: The Skin

• Structure of the Skin: Consists of three primary layers:

  • Keratin: A thin outer layer composed of dead cells.

  • Epidermis: A thick inner layer of living cells.

  • Dermis: A layer of connective tissue containing fibroblasts.

• Protective Functions:

  • Sebaceous Glands: Produce an oily secretion called sebum, which contains fatty acids that lower the pH, inhibiting microbial growth.

  • Sweat Glands: Assist by washing away and diluting microbes; they also contain antimicrobial proteins.

Antimicrobial Proteins and Peptides of the Skin

• Secretory Protease Inhibitor: Inhibits infection by bacteria, fungi, and viruses.

• Psoriasin: Binds to the outer membrane of $E. coli$ and actively kills the bacteria.

• Calprotectin: Binds cell wall components of $S. aureus$ to kill the organism.

• Defensins (alpha and beta): Cyclic polypeptides that insert into bacterial cell walls to kill them.

• Dermcidin: An antimicrobial protein effective against bacteria and fungi; it functions as an ionophore, inserting into membranes to disrupt ion gradients.

Anatomic Barriers: Mucous Membranes

• Location: Protects the conjunctivae, alimentary tract, respiratory tract, gastrointestinal ($GI$) tract, and urogenital tract.

• Composition: An outer epithelial layer situated over connective tissue.

• Supportive Fluids: Tears, mucous, and saliva.

• Role of Normal Flora: Mutualistic symbionts providing non-specific immunity by:

  • Competing with pathogens for binding sites.

  • Competing for available nutrients.

Antimicrobial Substances in Mucous

• Goblet Cells: Produce and secrete the mucous layer containing several substances:

  • Lysozyme: An enzyme that cleaves the peptide link in the peptidoglycan component of bacterial cell walls, weakening them and killing the bacteria.

  • Lactoferrin: A chelator that binds and sequesters iron, impeding microbial growth.

  • Surfactant Proteins (SP-A and SP-D): Bind surface cell wall components to promote neutralization and clearance. $SP-A$ binds to polysaccharide capsule components, while $SP-D$ binds to lipopolysaccharides ($LPS$).

  • Cathelicidin: Inserts into membranes and enters the cytoplasm, where its toxic effects kill the cell.

• RegIII Proteins: Produced by intestinal epithelia; bind carbohydrates on microbial cell walls, creating pores to kill the microbe.

• Histatins: Lectins secreted by salivary glands; bind carbohydrates on fungal cells, penetrate the plasma membrane, and enter the cytosol to inhibit mitochondrial $ATP$ production.

Cell Signaling and Pattern Recognition

• Pathogen Associated Molecular Patterns (PAMP): Conserved macromolecules produced by most organisms in a species. Examples include:

  • Structural Components: Triacyl lipopeptides, peptidoglycan, lipoproteins, lipids, flagellin, mannans, glycoproteins, diacyl lipopeptides, and zymosan.

  • Nucleic Acids: Unmethylated $CpG$ islands, $rRNA$, and $ssRNA$.

• Damage Associated Molecular Patterns (DAMP): Host antigens released by damaged cells during infection.

• Pattern Recognition Receptors (PRR): Found on innate immune cells; they recognize PAMPs and DAMPs.

Toll-Like Receptors (TLR)

• Discovery: Identified in $D. melanogaster$.

• Structure:

  • Extracellular Domain: Features Leucine Rich Repeats ($LRR$) organized in a bent alpha helix to mediate PAMP binding.

  • Cytosolic Domain: Homologous to the $IL-1$ receptor, termed the Toll/$IL-1R$ ($TIR$) domain; it transduces the signal.

• Functional Mechanics:

  • TLRs must dimerize for activity, usually forming homodimers (some heterodimers).

  • Localization: May reside on the plasma membrane (recognizing surface PAMPs like flagellin or $LPS$) or endosomal membranes (recognizing degraded PAMPs like bacterial/viral nucleic acids).

  • TLR4: An exception that is expressed on both plasma and endosomal membranes.

  • Humans and mice possess alleles $TLR1$ through $TLR13$.

TLR Signaling Pathways

Plasma Membrane Signaling

1. PAMP-mediated dimerization promotes binding of $MyD88$ to $TIR$ domains.

2. $MyD88$ is bound by an $IRAK4/IRAK1$ heterodimer.

3. $IRAK1$ autophosphorylates and recruits $TRAF6$.

4. Phosphorylated $TRAF6$ creates a docking point for the trimeric complex $TAK1/TAB1/TAB2$.

5. $IRAK1$ phosphorylates $TAK1$, and $TAB2$ is also phosphorylated.

6. The activated $TAK1$ complex binds the $IKK$ complex ($NEMO$, $IKKa$, $IKKb$).

7. $TAK1$ phosphorylates $IKKb$, which in turn phosphorylates $IkB$.

8. $IkB$ is ubiquitinated and degraded, releasing $NF-kB$.

9. $NF-kB$ translocates to the nucleus to activate transcription.

10. MAP Kinase Pathway: $TAK1$ phosphorylates $MEK$, activating a cascade where $cElk$ activates $cFos$, and $Jun$ kinase ($JNK$) phosphorylates $cJun$. The $cJun/cFos$ heterodimer ($AP1$) activates transcription.

Endosome Membrane Signaling

• Specific to $TLR3$ (recognizing viral $dsRNA$):

1. $TIR$ on $TLR3$ recruits $TRIF$.

2. $TRIF$ recruits $TRAF3$, which recruits a kinase complex containing $NEMO$, $TANK$, $IKKe$, and $TBK1$.

3. $TBK1$ phosphorylates transcription factors $IRF3$ and $IRF7$, inducing the expression of $IFNa$ and $IFNb$.

C-Type Lectin Receptors (CLR) and NOD-Like Receptors (NLR)

• CLR (e.g., Dectin-1): Recognize carbohydrate antigens (mannose, fucose, glucan). Dimerization activates a tyrosine kinase that phosphorylates an Immunoreceptor Tyrosine-based Activation Motif ($ITAM$). This recruits $PLCd$, forming $CARD$ complexes, which activate $AP1$, $NF-kB$, and release $Ca^{++}$ via $IP3$ to activate $NFAT$.

• NLR (Cytosolic PRRs):

  • Structure: Leucine rich region ($LRR$) for binding, Nucleotide Binding Domain ($NBD$), and variable amino-terminal domains: $BIR$ ($NLRB$), $CARD$ ($NLRC$), or $PYD$ ($NLRP$).

  • NOD1: Recognizes diaminopimelic acid ($DAP$) from Gram-negative bacteria.

  • NOD2: Recognizes muramyl dipeptide from Gram-positive bacteria.

  • RIP2: Recruited by $CARD$ domains to activate $MAP$ kinase and $NF-kB$ signaling.

The Inflammasome

• Formation: $NLRP3$ binds PAMPs via $LRR$, promoting oligomerization. The $PYD$ domain binds the adaptor protein $ASC$, which uses its $CARD$ domain to recruit $Caspase-1$.

• Activation:

  1. $PAMP$ binding to plasma membrane PRRs induces expression of $pro-IL-1b$ and $pro-IL18$.

  2. In the cytosol, $NEK7$ promotes $NLRP3$ oligomerization.

  3. $Caspase-1$ cleaves $pro-IL-1b$ and $pro-IL-18$ into active forms.

  4. $Caspase-1$ cleaves Gasdermin D; the amino-terminal fragment causes cell death (pyroptosis), releasing the cytokines to promote inflammation.

Detection of Cytosolic Nucleic Acids

• Viral dsRNA: Recognized by RIG-I-Like Receptors ($RLR$) like $RIG-I$ (binds $5'$ terminal phosphate) and $MDA5$ (binds internally). They associate with mitochondrial protein $MAVS$, activating $NF-kB$ and interferon expression.

• Cytosolic dsDNA: Recognized by Cyclic $GMP-AMP$ Synthase ($cGAS$). $cGAS$ produces cyclic $GMP-AMP$ ($cGAMP$) with a $2'$ to $5'$ linkage. $cGAMP$ binds STING (STimulator of INterferon Genes) on the $ER$/Golgi, recruiting $TBK1$ to activate $NF-kB$ and interferon production.

Biological Impacts of Interferon (IFN) Production

• Interferon Signaling: $IFNa$ and $IFNb$ bind the $IFNAR1/IFNAR2$ receptor, activating the $Janus$ kinases $JAK1$ and $TYK2$. These phosphorylate $STAT1$ and $STAT2$, which dimerize and translocate to the nucleus.

• Antiviral Effects:

  • 2′-5′ poly A synthetase: Degrades viral RNA.

  • PKR: A kinase that phosphorylates $eIF2$ to shut down protein synthesis.

  • Mx Proteins: Form hexamers to prevent viral transcription and assembly.

  • IFIT Proteins: Complex with viral $dsRNA$ or $eIF3$ to inhibit translation.

Cytokine Production and TNF Signaling

• Key Cytokines:

  • IL-1: Pyogenic (fever), leukocytosis, acute phase proteins.

  • IL-6: Pro-inflammatory, hematopoiesis, acute phase proteins.

  • TNFa: Activates $MF$, induces apoptosis, acute phase proteins.

  • GM-CSF: Induces hematopoiesis of myeloid progenitor cells.

  • IL-12/IL-18: Propagate $TH1$ responses.

  • IL-10: Regulatory cytokine; suppresses pro-inflammatory responses.

• TNF Receptor Signaling:

  • Activation: $TNF$ trimerizes $TNFAR$. Death Domains ($DD$) recruit $TRADD$, $TRAF2$, and $RIP1$. This activates $TAK1$ and $IKK$, stimulating $NF-kB$ and expression of $cFLIP$ to inhibit apoptosis.

  • Apoptosis: In cells lacking protective adaptors (like some tumor cells), $TRADD$ associates with $FADD$, which recruits $pro-caspase 8$. This converts to caspase 8, initiating an apoptotic cascade.

Phagocytosis and Killing Mechanisms

• Stages:

  1. Chemotaxis: Movement toward microbial products or complement components ($C5a$, $C3a$).

  2. Adherence: Attachment, often enhanced by opsonization.

  3. Ingestion: Pseudopods fuse to form a phagosome.

  4. Digestion: Fusion with a lysosome creates a phagolysosome.

• Killing Mechanisms:

  • Oxygen-Dependent: Oxidase converts $O_2$ into $2O^-$; Myeloperoxidase generates $Cl^-$; Nitric Oxide Synthase ($iNOS$) creates $NO$.

  • Oxygen-Independent: Lysozyme, defensins (pore formation).

• Phagocytic Receptors:

  • $CD91$/calreticulin: Binds surfactants, $MBL$, and $C1q$.

  • Complement Receptors ($CR1$, $CR3$, etc.): Bind $C3b$ and $C1q$.

  • $FcaR$ and $FcgR$: Bind $IgA$ and $IgG$.

Innate Lymphoid Cells (ILC) and NK Cells

• Innate Lymphoid Cells: Progenitor-derived cells lacking antigen-specific receptors.

  • ILC1 (NK and ILC1): Produce $IFNg$ and $TNF$.

  • ILC2: Produced $IL-4$, $IL-5$, $IL-9$, $IL-13$; involved in parasite responses.

  • ILC3: Produce $IL-17$, $IL-22$; thwart extracellular bacteria.

• Natural Killer (NK) Cells:

  • Express Activation Receptors (AR) that bind Activation Receptor Ligands (ARL) on aberrant cells.

  • Express Inhibitory Receptors (IR) that recognize $MHC$ molecules on healthy cells.

  • Use granzyme, perforin, and $FasL$ for killing.

Regulation and Clinical Relevance

• Septic Shock: Overproduction of $TNF-a$, $IL-1b$, and $IL-6$ triggered by $LPS$ binding to $TLR4$. Results in systemic clotting, drop in blood pressure, and respiratory failure.

• Negative Regulation: To prevent over-inflammation, cells produce:

  • Truncated MyD88: Competes for docking points.

  • Phosphatases: Remove signaling points.

  • Transcription Inhibitors ($I-kB$): Block $NF-kB$.

  • Soluble Receptors: Released by proteolysis to bind and neutralize cytokines.

Adjuvants

• Substances that enhance the immunogenic potential of an immunogen.

• Freund’s Incomplete Adjuvant: Antigen in oil/emulsifier; retards dispersion.

• Freund’s Complete Adjuvant: Contains heat-killed mycobacteria; muramyl dipeptide activates macrophages.

• Alum: Activates the $NLRP3$ inflammasome and facilitates slow release through granuloma formation.

Questions & Discussion

Q: How does the innate immune system interact with the adaptive system?A: Dendritic cells act as the bridge. They recognize PAMPs via PRRs, migrate to lymph nodes, and present antigens in $MHC$ molecules. The specific PRR activated on the dendritic cell dictates the differentiation of T cells (e.g., $TLR4$ leads to $TH1$ via $IL-12$, while $TLR2/1$ heterodimers lead to $TH2$ via $IL-10$).