Notes on Innate Immunity: PRRs, Inflammation, Neutrophils, Macrophages, ILCs, and NK Cells
Pattern Recognition Receptors (PRRs) and initial sensing
Microbes release PAMPs (pathogen-associated molecular patterns) such as sugars, proteins, and lipids on their surfaces.
Damaged host cells release DAMPs (damage-associated molecular patterns), e.g., ATP or misplaced organelle components.
PRRs detect PAMPs and DAMPs to recognize infection or tissue damage. A classic example is TLR4 (TLR 4) signaling in response to bacterial components.
Upon PRR recognition of PAMPs/DAMPs, two main signaling pathways are engaged:
MyD88-dependent pathway: activates nuclear factor kappa B (NF-κB) → transcription of pro-inflammatory cytokines.
MyD88-independent pathway (often referred to as TRIF-dependent): activates IRF (Interferon Regulatory Factor) axis → production of interferons (antiviral components).
Downstream, recognition leads to inflammasome activation, linking sensing to inflammatory effector functions.
Inflammasomes and cytokine maturation
Inflammasomes are multi-protein complexes whose structure can vary depending on the signal (PAMP or DAMP) detected.
Primary purpose: activate caspase-1 from its inactive zymogen procaspase-1.
Caspase-1 then cleaves inactive cytokine precursors to active cytokines:
IL-1β and IL-18 are pro-inflammatory cytokines that initiate and amplify the inflammatory response.
Caspase-1 activation can also induce a form of programmed cell death known as pyroptosis, a lytic, inflammatory cell death that helps contain infection.
The inflammasome can be activated via signals from PRRs (including the NF-κB-driven production of pro-IL-1β/IL-18) and also directly by DAMPs.
Key cytokinic players and their roles
IL-1β and IL-18: pro-inflammatory cytokines central to initiating and propagating inflammation.
Interferons (IFNs): antiviral components produced via the IRF pathway; help limit viral replication and modulate immune responses.
NF-κB: transcription factor activated via MyD88-dependent signaling; upregulates many inflammatory genes.
Pro-inflammatory context: Activation of inflammasomes and cytokines leads to recruitment and activation of additional immune cells.
Big picture: why inflammation matters
Presence of microbes in tissue triggers inflammatory signals to recruit leukocytes to the site of infection.
Inflammation involves both immune cell recruitment and vascular changes to allow cells and mediators to reach the tissue.
While necessary to control infection, excessive or prolonged inflammation can cause tissue damage; therapeutic blockade of PRRs or inflammasome components can reduce inflammation but increases infection risk.
In veterinary contexts (e.g., horses), pharmacologic blockade of these pathways has been used to control inflammation, with caveats about infection susceptibility when inflammation is suppressed.
The “dance” of inflammation: from recognition to recruitment
Recognized microbes or damaged cells release signals that initiate the inflammatory cascade.
Pattern Recognition Receptors (PRRs) are the primary sensors on immune cells.
Upon recognition, the NF-κB and IRF pathways are activated via the two main routes (MyD88-dependent and independent).
Activation leads to inflammasome formation and production of pro-inflammatory cytokines, particularly IL-1β and IL-18, which set the stage for inflammation.
Inflammation involves signaling to nearby blood vessels to become more permeable and to express adhesion molecules that facilitate leukocyte recruitment.
Recruitment of neutrophils: the first responders
The first immune cells to arrive at the site of infection are neutrophils (the “lawyers” of the immune system in this analogy).
Recruitment steps (extravasation) in order:
Tethering: neutrophils loosely attach to the endothelium.
Rolling: rolling along the vascular surface mediated by selectin–glycoprotein interactions.
Activation and stable adhesion: endothelial receptors (addressins) signal neutrophils; adhesion strengthened by integrins (e.g., LFA-1 which is composed of CD11 and CD18 subunits) binding to ICAM-1.
Arrest and transmigration (diapedesis): neutrophils squeeze through the endothelial barrier into tissue.
Chemotaxis: neutrophils migrate toward the infection using chemokine cues, chiefly IL-8 (CXCL8) and macrophage inflammatory protein-1α (MIP-1α).
Addresses on endothelium: endothelial cells express adhesion molecules that act as “addresses” to tell neutrophils where inflammation should occur.
CD18 (part of LFA-1) is critical for firm adhesion and stopping the neutrophil at the correct site; deficiency of CD18 can cause leukocyte adhesion deficiency in animals (e.g., cattle and dogs), reducing effective inflammatory responses.
The tethering–rolling–adhesion–transmigration sequence ensures that neutrophils exit the bloodstream specifically at sites of infection and inflammation.
Neutrophil functions in the tissue: phagocytosis and extracellular defenses
After transmigration, neutrophils recognize microbes via opsonization and surface receptors (e.g., Fc receptors for IgG, complement receptors) that bind tagged pathogens.
Cytoplasmic extensions (pseudopods) attach to and engulf microbes, forming a phagosome (an intracellular vesicle).
Inside the phagosome, several antimicrobial mechanisms are deployed:
Respiratory burst: generation of reactive oxygen species (ROS) via NADPH oxidase, converting oxygen to superoxide and downstream ROS.
Approximate reactions:
Oxygen scavenging and reactive species destruction: hydrogen peroxide (H₂O₂) and other ROS damage microbial components.
Antimicrobial peptides (e.g., defensins) and digestive enzymes delivered by lysosomes fuse with the phagosome (phagolysosome) to digest microbes.
Lactoferrin in phagosomes chelates free iron (Fe³⁺), depriving microbes of iron needed for growth:
Phagosomal acidification: proton pumps acidify the phagosome, activating hydrolases and optimally functioning antimicrobial enzymes.
Lysosomal enzymes: proteases, nucleases, and other hydrolases digest microbial components.
The oxidative burst and enzymes together effectively kill most engulfed microbes within the phagosome.
PT: Hydrogen peroxide is also used externally in wound care and is generated internally via ROS production to kill microbes inside phagosomes.
It is critical that microbes are destroyed within phagosomes; excessive leakage of digestive contents can damage host tissue.
NETs (neutrophil extracellular traps): neutrophils can also deploy extracellular traps composed of DNA strands with histones and antimicrobial proteins (e.g., lactoferrin, defensins) to trap and kill microbes outside the cell.
Lifespan and limitations:
Neutrophils are rapidly mobilized and have a relatively short lifespan; they have limited capacity for repeated phagocytosis.
Monocytes/macrophages: longer-lived defenders and tissue remodelers
Circulating monocytes infiltrate tissues and differentiate into macrophages.
Macrophages provide a second wave of defense and are more potent than neutrophils, with longer life and the ability to perform repeated phagocytosis.
Macrophages can recruit other lymphocytes (T cells and B cells) and help initiate adaptive immune responses by presenting antigens and promoting antibody production.
Macrophage polarization:
M1 (classically activated, pro-inflammatory):
Generate nitric oxide (NO) via nitric oxide synthase (NOS) from arginine; NO contributes to vasodilation and antimicrobial activity.
Pro-inflammatory cytokine production helps sustain inflammation.
M2 (alternatively activated, anti-inflammatory/healing):
Express arginase, converting arginine to ornithine, reducing NO production and promoting tissue repair.
Nitric oxide production in macrophages:
NO effects: vasodilation, increased blood flow to the site, and antimicrobial activity; NO and reactive nitrogen species contribute to pathogen killing.
Macrophage roles beyond killing microbes:
Phagocytose dead neutrophils and debris to prevent collateral tissue damage.
Remodel connective tissue and aid in tissue repair after infection.
Shift from M1 to M2 phenotype over the course of inflammation to promote resolution and healing.
Innate lymphoid cells (ILCs) and natural killer (NK) cells: first responders beyond classic adaptive immunity
Innate lymphoid cells (ILCs): lymphocytes that reside in non-lymphoid tissues (e.g., subcutaneous tissues) and lack antigen-specific receptors. They respond to cytokine environments produced by infected or damaged tissues and influence other immune cells.
ILC1: secrete interferon-gamma (IFN-γ) and contribute to type 1 responses.
ILC2: secrete IL-5 and IL-13, promoting type 2 immunity and tissue repair processes.
ILC3: secrete IL-17 and IL-22, contributing to barrier defense and antimicrobial responses.
NK cells: specialized innate lymphocytes that kill infected or transformed cells, not by recognizing specific antigens, but by sensing stressed cells.
NK cells determine whether a cell is infected by detecting stress signals and/or reduced MHC class I expression on target cells.
Mechanism: degranulation releasing perforin and granzymes to induce apoptosis in the infected cell, thereby preventing replication and spread of pathogen.
NK cells can also secrete cytokines (e.g., IFN-γ) to further modulate the immune response.
MHC dependence and horse-specific notes:
NK recognition often involves interactions with MHC class I on target cells; healthy cells expressing normal MHC I inhibit NK activation via inhibitory receptors.
Infected or stressed cells often downregulate MHC I, removing inhibitory signals and allowing NK cell-mediated killing.
Horses differ in inhibitory receptors: they possess the LY-49 receptor (an NK-inhibitory receptor) and may lack certain other receptor types found in some other species; some farm animals have both, but horses rely on LY-49 for this inhibitory function.
Innate cytokine signaling by ILCs: three major ILC types secrete characteristic cytokines (as listed above), enabling rapid, antigen-independent regulation of inflammation and tissue homeostasis.
Putting it all together: the sequence of innate immune responses to infection
Step 1: Recognition
PRRs detect PAMPs and DAMPs (e.g., TLR4 detects LPS; other PRRs detect various microbial components and damaged self-signals).
Activation of MyD88-dependent and MyD88-independent pathways leads to NF-κB–driven cytokine transcription and IRF-driven interferon production, respectively.
Inflammasomes are activated, caspase-1 is activated, and IL-1β/IL-18 are produced (and can induce pyroptosis in infected cells).
This recognition represents the initial “alarm signal” that infection is present and needs to be contained.
Step 2: Inflammation and vascular changes
Inflammation is initiated; cytokines promote vasodilation and increased vascular permeability, allowing fluid and leukocytes to exit the bloodstream and enter tissue.
Endothelial cells express “addresses” (adhesion molecules) that guide neutrophil extravasation to the site of infection.
NO production by macrophages and other cells contributes to vasodilation and further enhances tissue perfusion.
Step 3: Recruitment of neutrophils and macrophages
Neutrophils arrive first and perform rapid phagocytosis and extracellular defense (NETs).
Monocytes follow and differentiate into macrophages (M1 then M2 shift as inflammation resolves).
Step 4: Effector antimicrobial actions
Neutrophils: phagocytose microbes into phagosomes; kill via ROS (NADPH oxidase) and antimicrobial enzymes; acidify phagosomes to optimize enzyme activity; release lactoferrin to sequester iron; can deploy NETs for extracellular killing.
Macrophages: continue phagocytosis, generate NO (via iNOS) to kill microbes and promote vasodilation; M2 macrophages promote tissue repair and anti-inflammatory functions; recruit lymphocytes and coordinate adaptive responses.
Step 5: Bridging to adaptive immunity
Macrophages present antigens and recruit T and B cells; this supports antibody production and a targeted adaptive response.
NK cells monitor for infected cells; they kill stressed cells and secrete cytokines to shape subsequent immune responses.
Step 6: Resolution and repair
M2 macrophages promote tissue repair, remodeling, and suppression of excessive inflammation.
Inflammation declines as pathogens are cleared and tissue integrity is restored; excessive persistence of inflammatory signals can cause collateral tissue damage.
Practical implications and examples
Therapeutic modulation: Drugs that block PRRs or inflammasome components can reduce inflammation (e.g., in horses), but at the cost of increased susceptibility to infections.
Balance of inflammation: Early neutrophil responses are essential for containment, but prolonged neutrophil activity and cytokine production can cause tissue damage if not resolved.
Relevance to clinical disease: Dysregulation of these pathways can contribute to inflammatory diseases, sepsis, and chronic inflammatory conditions; understanding the sequence helps tailor interventions (e.g., timing of anti-inflammatory therapy).
Species differences: Receptor repertoires vary among species (e.g., the LY-49 NK receptor in horses vs other mammals); these differences influence how innate immunity operates in different animals.
Key takeaways (big picture)
PRRs detect PAMPs and DAMPs and initiate two main signaling axes: MyD88-dependent NF-κB activation for cytokines and MyD88-independent IRF activation for interferons.
Inflammasomes activate caspase-1, which matures IL-1β and IL-18, driving inflammation and potentially pyroptotic cell death.
Inflammation proceeds through neutrophil recruitment (tethering, rolling, adhesion via CD18/LFA-1, transmigration, chemotaxis) and phagocytosis, with phagosomal killing via ROS, proteases, lactoferrin, and acidification.
NETs provide extracellular antimicrobial defense when neutrophils cannot contain infection intracellularly.
Monocytes/macrophages follow neutrophils, differentiating into M1 (pro-inflammatory, NO production) and M2 (anti-inflammatory, tissue repair) phenotypes, and bridging innate and adaptive immunity by recruiting T and B cells.
Innate lymphoid cells (ILCs) and NK cells furnish rapid, non-antigen-specific responses; NK cells kill infected cells via granule release and can be regulated by MHC I interactions and stress signals.
No single component works alone; the coordinated interplay among PRR signaling, inflammasomes, cytokines, phagocytes, macrophages, and NK/ILCs constitutes the core of the innate immune response and its transition to adaptive immunity.
Note: The content above reflects the lecture’s emphasis on pathways, cell types, and their interactions, including the practical implications of anti-inflammatory therapies and species-specific differences in innate immunity.