Complement, Pattern Recognition Receptors, Phagocytosis, Other

infection compartments

pathogens are found in 2 basic compartments, each of which requires a different host defense mechanism

extracellular - complement, phagocytosis, antibodies, and on epithelial surfaces is antimicrobial peptides and antibodies

intracellular - NK and tox T cells (cytoplasmic), T cells and NK dependent macrophage activation in vesicles

barriers

effective physical barriers and chemical defenses

more effective against extracellular than intracellular

protective immunity

Ts and NKs better for intracellular threats

CDIF

takes over if commensal gut bacteria is wiped out by antibiotics

external epithelia

skin, great first line of protection, external watertight barrier

multiple layers of dead cells that can’t be infected covering the live cells

microbes seek alternative routes into host - wounds and abrasions, insect bites

mucosal surfaces (internal epithelia)

digestive, respiratory, and reproductive tissues are the main ones

mucosal layers more vulnerable than external, as they are a single layer of cells, no dead cell barrier, require more defenses

joined by tight junctions

mucous membrane, lines cavities inside the body

tight junctions exist in all epithelia

mucous - physical layer on top of epithelia as an extra barrier, made by goblet cells (not immune cell type), moved by air, fluid, cilia, etc - peristalsis, prevents bacterial colonization

mucous can have low pH and enzymes to destroy microbes - loaded with antimicrobials

normal commensal bacteria that inhabit us also pitch into the defensive effort by competing with invaders for resources

cystic fibrosis - mucous is thick and hard to move, forms plugs, stagnant mucous fills with bacteria and macrophages and neutrophils fighting it

self v nonself

immune system descriminates via molecular differences

phagocytes produce antimicrobial enzymes and peptides (focus on defensins)

gram of bacteria

gram positive have one membrane and a thick outer peptidoglycan wall

gram negative have a thinner peptidoglycan wall but two layer sandwiching it

lysozyme - secreted protein enzyme that digests bacterial gram pos cell wall polysaccharides (bacterial), on their own only effective against gram pos

• lysosomes help destroy bacteria after they have been engulfed

phospholipase A2 - destroys bacterial membrane phospholipids, effective against gram neg and pos

both lysozyme and phospholipase A2 secreted by phagocytes, paneth cells, in tears, and in saliva

defensins

amphipathic (polar one end, nonpolar on the other) peptides that disrupt bacterial and fungal cell membranes

amphipathic like phospholipids because they line up like them with polar ends facing out like the polar heads of the lipids - slot into the membrane

once 4 insert into the membrane, they create a pore complex by wrapping the membrane around themselves to create an opening - bleed microbes dry

produced by immune cells and epithelial cells (not endothelial blood vessel cells) - formed via cleavage (proteolysis), prodefensin isn’t dangerous, prevents autoimmunity issues

high affinity for bact and fungi cells

cannot get through cell walls

complement

system of soluble proteins present in the blood and bodily fluids

released to areas of infection along with immune cells through endothelial junctions, but some are often already present

30+ plasma proteins, mainly produced by the liver

interact with each other in a cascade-like way

triggered by recognition of microbial surface structures

recognizes pathogens via 3 diff pathways

destroys pathogens directly and promotes immune response

critical for protection against bacteria and viruses

stages of complement action

1. pattern recognition

2. protease cascade amplification/C3 convertase

3. inflammation

4. phagocytosis

5. membrane attack

complement nomenclature

proteins in cascade structure roughly numbered from C1-C9

once a protein is cleaved, the cleaved portions are given lowercase letter designations (a, b, c…)

enzymes that cleave complement are called C# convertases, the number corresponding to the parent protein

a complex of multiple proteins is often abbreviated as such:

C4b + C2a = C4b2a

C4b2a is the C3 convertase for both the lectin and classical pathways

complement pathways

all pathways proceed from microbial surface (pattern) recognition → activation cascade (all three paths share the same step) → effector function (all paths provoke all three effector measures)

• activation cascade is C3 to C3a and C3b, then C3b goes on to cleave c5 into C5a and C5b

• sensor molecule for lectin pathways is MBL

• sensor for classical pathway is C1

• lectin and classical both feed into C2 and C4 before reaching the C3 and 5 middle step

• effector functions are inflammation promotion, indirect killing, and direct killing

microbial surface recognition

sensor proteins bind unique structures to microbial surfaces or recognize specific antibodies

many of the structures picked up are polysaccharides (carbs) specific to bacteria

• we don’t display bacterial carbs, so complement usually doesn’t attack our own cells

• complement system also highly regulated by inhibitor proteins expressed on the surface of healthy self cells

ficolins are the sensor proteins - MBL from lectin pathway is one

lectin pathway

MBL (manose, referring to part of bact polysaccharide, BL) binds microbial surface

once bound, MBL cleaves C4 to C4a and b

C4b binds to the surface, binds and cleaves C2 to C2a and b

C4b forms C4b2a complex - active C3 convertase

C4b2a cleaves C3 to a and b, which bind to the microbe surface or the convertase

classical pathway

initiated by the activation of C1 complex

very similar to lectin pathway

sensor protein called C1q

can bind pathogen surface structures OR antibodies bound to the surface (this is the pathway for antibody activated complement and for repeat infections)

antibody binding connects to the adaptive immune response

• usually binds antibodies released early on in recurring infection

• antibody more strongly activates the classical pathway

alternative pathway

amplification loop for C3b formation that is accelerated by specific co-factors in the presence of pathogens

can be triggered by classical or lectin pathways, but can also spontaneously activate

ends in C3 activation

tag and bag

complement tags pathogens (opsonization - the coating of a pathogen by complement and/or antibodies to mark for destruction)

tagged pathogens are ingested by phagocytes

ingestion of tagged pathogens is mediated by receptors for the bound complement proteins

bacterium becomes coated with C3b → phagocytes recognizes and binds (via surface receptors) to C3b on pathogen but does not immediately ingest (C3b helps grab) → C5a binds to phagocyte receptor CR1 and induce ingestion

• CR means complement receptor

inflammation effect

small fragments of some complement proteins initiate a local inflammatory response

soluble complement fragments signal to immune and endothelial cells to promote inflammation - increase endothelial permeability and attract immune cell

mainly C5a and C3a

direct attack

terminal complement proteins polymerize to form pores in membranes that kill certain pathogens

C5b binds C6 and C7

C5b67 binds to pathogen membrane via C7

C8 binds to complex and inserts into membrane

C9 proteins bind to complex and polymerize into tube

pore is formed, called membrane attach complex

evading complement

some pathogens can inactivate complement

mostly extracellular bacteria that can do this, which makes sense since they are more vulnerable to complement than intracellular pathogens

complement autoimmunity

improper activation of complement can overrun cell complement inactivation measures and target the self

innate v adaptive receptor characteristics

innate - specificity inherited in genome, not expressed by all cells, triggers immediate response, recognize broad pathogen classes, interact with a range of structures of a given type

adaptive - random splicing, no immediate response, recognizes specific pathogens and interacts with a specific structure

both - able to discriminate between closely related molecular structures

Toll

fruit fly pathogen receptor gene

immunodeficiency - compromised by fungal infection

human homolog = toll-like receptors (TLRs)

TLRs

all in extracellular environments (either on outside of cell or in endosomes, pockets formed by endocytosis that contain external environment components

some single, some dimers

hook-like extracellular domain binds ligand

intracellular domain handles signal transduction

RTKs

what sense what

SURFACE - microbial surface components (bacteria mostly)

• TLR 2-6 dimer - bact membrane

• TLR 2-1 dimer - bact membrane

• TLR 5 - bacteria flagella

• TLR 4 + MD-2 - Gram neg bact membranes

ENDOSOME - microbial nucleic acids (viruses and internal bacteria)

• TLR 3 - virus dsRNA (3ds)

• TLR 7 - virus ssRNA

• TLR 8 - virus ssRNA

• TLR 9 - bacteria CpG DNA (and herpesviruses)

knowing what is foreign

weird RNA/DNA locations (in the endosome, host DNA wouldn’t be there, know its foreign)

• sometimes our dying cells release RNA and that flags the immune system through toll even though is us

double-stranded RNA - we don’t have this

unmethylated CpG DNA - indicates bacteria, ours is methylated

main thing - location and structure

TLR considerations for mRNA vaccine

mRNA has to survive long enough to be expressed before its flagged and jumped on/destroyed by the cytoplasm

can chemically modify inbound mRNA to make it far harder to trip TLRs

bacteria sensing TLRs activate NFkB

TLR major response to infection is to trigger gene expression via induction of TFs

• surface TLR binds antigen and then dimerizes

• intracellular domain activates adaptor proteins which activate ubiquitin

• phosphorylation cascade ends up marking NFkB inhibitor, IkB, for destruction

• IkB is degraded, NFkB is free to enter the nucleus and act as a TF (transcription factor), upregulates cytokine transcription (and other antimicrobial genes)

  • NFkB leads to the transcription of hundreds of pro-inflammatory genes (TNFa, IL-6, IL-1B - the big three)

  • also ROS (reactive oxygen species) genes and antimicrobial peptides

NFkB is Nuclear Factor

virus sensing to IRF transcription factors

• same receptor dimerization upon binding antigen

• IRF is Interferon Regulatory Factor - TF of key antiviral factors

• TLR3 activation by dsRNA leads to phosphorylation of IRF3, which enters the nucleus as a TF to upregulate type I interferon genes

• TLR7 is activated by ssRNA and paths to IRF7, which also upregs type I interferon genes

• Type I interferons are IFNa and B, specific cytokines that alert neighboring cells to the presence of a viral infection

why have multiple stimulatory pathways?

the intracellular pathways and extracellular pathways need different tools to sort our their respective jobs (antibacterial v antiviral)

NOD-like receptors

NLRs

intracellular cytoplasmic sensors for bacterial infection and cellular damage

ligands from intracellular cytoplasmic bacteria/can be transported in by endosomes

NFkB pathway to activate inflammatory genes

• NFkB can also induce the expression of other TFs to fine-tune cell response

NLRPs

Larger name - larger complex (inflammasome)

NLRPs are proteins that react to infection/cellular damage through an inflammasome (multi-protein signaling complex) to induce cell death and inflammation

bacteria can cause a cells K+ levels to drop, which can trigger the dissociation of the chaperones that keep NLRP inactive

• ROS, particulates, and ofc bacterial components can all activate NLRPs

inflammasome formation causes cleavage of caspase 1s, which go on to cleave cytokines IL-18 and 1B from their proproteins to ship them out to the fight

• caspase 1 clear effector

• production of IL-1(B) is key to pro-inflammatory response

• inflam response is main job, cell death is secondary

RIG-I-like receptors

RLRs

detect cytoplasmic microbial RNAs or DNA (mainly viral)

• catch viral components (uncapped ss viral RNA, distinguished from our capped RNA) in the middle of them replicating and assembling newely-built parts

• binding causes RLR change in conformation and aggregation

induce type I interferon production and pro-inflammatory cytokines

• activation of BOTH IRF-3 (interferon) and NFkB (pro-inflam cytokines)

• mostly IRF activation

TLRs and DC stimulation

TLR activation causes changes in their DCs (this is the cell they are on)

• TLR signaling informs the cell of microbial presence - there is a danger

DCs stimulated to migrate to lymph nodes and initiate adaptive immune response

• enhanced migration and upref co-stimulatory molecules CD80/86 - enhance presentation of antigen to T cells

engulfing

macrophages and neutrophils mainly engulf to destroy - DCs engulf to deconstruct and present antigens

• recall macrophages are tissue residents and are waiting to spring to action

recall that complement helps bind pathogen and promote phagocytosis

neutrophil phagocytotic response

first wave of cells that cross the endothelial vessel wall to enter inflamed tissue

potent respiratory burst (rapid increase in the production of reactive oxygen species, ROS, during phagocytosis)

• generate oxygen and nitrogen radicals toxic to life - induce oxidation and damage - superoxide, hydrogen peroxide, etc. NO, etc.

• antimicrobial peptides and enzymes - cathelicidin in macrophages and a and B defensins and others in neutrophils

• pH drop - 3.5-4

• To summarize, inside the phagosome they are raising pH, releasing antimicrobial peptides, and releasing ROS

• primary granule with enzymes, secondary with ROS, and lysozome with lysozyme - digests cell walls of some gram-pos bacteria

• granules and lysosomes deliver contents to phagosome - cause compartment to acidify (adds lysozyme)

• phagocytosis needed if complement isn’t enough/is inactivated

GPCR on phagocytes link microbe recognition with increased efficiency of intracellular killing

short-lived, ingest and kill, no antigen presentation

can release Neutrophil Extracellular Traps (NETs) - sticky nuclear DNA with antimicrobila coating

interleukins

type of cytokine

cytokines

activated macrophages (just encountered a pathogen, TLRs) activated begin to express inflammation genes - generate cytokines (DCs also do this) to recruit effector cells to infection site

• some of these genes are antimicrobial and others are cytokines

• IL-1B, TNF-a, and IL-6 are also expressed early, known as the big three

• cytokines organized into families of structurally-related proteins

cytokines are extracellular ligands, bind to the surface receptors on targets, can promote gene expression and changes in cell cytoskeleton (migration),

a means for intercellular communication

cytokines aren’t only made by immune cells but interleukins, a division of cytokines, are mostly made just by immune cells for intercell communication

JAK-STAT pathway

common pathway for cytokine signal transduction

most cytokine receptors in general are RTKs

• binding causes RTKs with JAK cytoplasmic domains to dimerize (bind to parts of the same ligand)

• JAKs autophosphorylate and phosphorylate STAT dimer - 6 different types (variety of outcomes)

• activated STAT dimer enters nucleus to act as a TF

easy to make drugs to regulate their activity

cytokine nomenclature

cyto = cell / kine = movement

IL refers to interleukin

cytokines mainly classified as:

• pro-inflammatory - IL-1, IL-6, TNF (tumor necrosis factor)

• anti-inflammatory - IL-10, TGFbeta

• cell differentiation factors

• chemokines - cytokines that attract cells (bread crumbs)

• interferons - cytokines that are crucial for antiviral response

acute phase response

cytokines made by macrophages and DCs induce a systemic reaction known as the acute phase response

• given a strong enough infection, there are enough cytokines produced to enter the vasculature

• this is the point at which you begin to feel sick

• IL-1 especially responsible for the fever response, which enhances the immune metabolism and hinders bacteria reproduction

• pyrogens - molecules that promote fever

• mobilize more immune cells, starting with neutrophils

mainly driven by reception of big 3 proinflammatory cytokines- IL-1B, IL-6, TNF-a

affects liver, bone marrow and endothelium, hypothalamus, fat, muscle, and DCs

• release of fat - have to have more energy available to fuel the immune system

triggered by high level of infection - “all hands on deck” state

• IL-6 acts on liver hepatocytes to produce acute phase proteins (e.g. MBL from lectin pathway) - enhance opsonization (tagging pathogens for phagocyte elimination), antimicrobial defenses, ramps up complement activity

  • liver also produces clotting factors, other complement components

  • e.g. some acute phase proteins act as opsins, binding to bacteria and activating complement, resulting in a phagocytosis assist

  • CRP, another PRR (pattern recog receptor), directs opsonization and/or activation of complement cascade when bound

microbial recognition and tissue damage initiate the inflammatory response

1. microphages activated by PRR binding, release cytokines and chemokines, cause the dilation of local small blood vessels

   1. increased vascular diameter and permiability

   2. endothelial cells loosen tight junctions due to cytokine signaling

   3. 4 cardinal signs of inflammation - pain, redness, heat, swelling

2. leukocytes traveling through the blood are slowed (attracted by chemokines), move to the periphery of the blood vessel as a result of the increased expression of adhesion molecules by the endothelium

3. leukocytes extravasate (leaking into) at site of infection

4. clotting blocks the dissemination of microbes into the blood (because a system infection is really really bad)

The role of TNFa

important cytokine that triggers local containment of infection but induces shock when released systemically

local TNFa release by macrophages is very rapid

activates endothelium and leukocytes - local edema (fluid leaks into tissues, swelling)

infection is then contained and cleared

if released systemically by macrophages activated in the liver, causes systemic edema, drop in blood pessure due to so many leaky vessels, clotting, which can lead to vessel collapse, systemic clotting and coagulation can lead to wasting and multiple organ failure - sceptic shock

• a quarter of afflicted people die from this

• not enough to block TNFa, by the time the patient has gotten into hospital the cytokine has already been released

migration of monocytes from blood to inflamed tissues

1. monocyte binds adhesion molecules on vascular endothelium near site of infection and receives chemokine signal

   1. adhesion molecules upped in expression, sort of grab on to the passing cell and pull it down towards the endothelial wall, where its chemokine receptor then binds the cytokine

   2. adhesion molecules also work for lymphocytes

   3. job is to slow cells to give them a chance to receive other signals - control interactions between leukocytes and endothelial cells during inflammatory response

   4. stored in cells to be put out for fast expression

2. The monocyte migrates into the surrounding tissue

3. The monocyte differentiates into an inflammatory monocyte at the cite of infection

chemokine nomenclacture

name example - CXCL8

• CXC - class

• L - its a ligand

• 8 - the receptor it binds

chemokine receptors are GPCRs

cytokines mostly produced by innate immune cells

remember that chemokines induce chemotaxis - directed movement of cells towards a source (of the chemical gradient)

more on adhesion and protein-protein interactions

healthy cells lack/don’t have many of these receptors

endothelium - adhesion molecules - selectins - P-selectin

• weak adhesion, causes monocytes to sort of roll over the inner face of the vessel

• upregulated within minutes of inflammatory stimulus

on leukocytes - integrins

• strong adhesion (selectins are weak adhesion)

• bind specific adhesion molecules, stops immune cells

• bind ICAMs

on endothelium - ICAMs (intercellular adhesion molecules)

• strong adhesion, bind integrins (LFA-1)

• bind specific integrins, stop leukocytes

integrin-ICAM interactions and chemokines direct tighter adhesion and extravasation into infected region

expression of integrins on leukocytes and ICAMs on the endothelium acts as a sort of zip code to direct leukocytes to inflamed tissues

viral sensing cytokines

mainly IFN-a and B (type I interferons)

also TNF-a and IL-12

production leads to activation of NKs for mediated killing of infected cells, then another spike in activity for T-cell mediated killing of the infected cells

virus sensing

activation of virus-sensing TLRs leads to the activation of IRFs of some type (dsRNA IRF3, ssRNA IRF7)

IRF TFs help alter gene expression to activate the IFN response

IFN = interferon, interferes with virus ability to colonize cells

IFN receptors expressed on a lot of different cells, induces viral refractory (antiviral) state

• induces alarm, tells cells virus is present and to become resistant

• starts JAK-STAT pathway

• activates innate resistance and activates NK and cytotox Ts

• halts translation

IRF3 mutations associated with HSV

IRF7 and 9 mutations associated with life-threatening flu infections

IFN contributions to host defense

1. virus infected host cells produce INF-a and B (type I IFNs)

   1. can be produced and sensed by most cells (including non-immune)

   2. induce local antiviral resistance - antiviral state

2. IFNa and B received by their receptor, causes induction of interferon stimulated genes

   1. induction of antiviral cellular state - innate resistance to viral replication

      1. protein translation shutdown

      2. cells become more sensitive to apoptosis

   2. does so in immune and nonimmune cells

   3. increases MHC-I expression and antigen presentation in all cells

3. activation of adaptive and NK cells

type I IFN and NKs

type I IFNs (IFN a and B) activate NK cells (and also by macrophage-derived cytokines)

NKs armed with granules of cytotoxic chemicals and ligands that induce apoptosis

• trying to control the situation and prevent loss of too many host cells while waiting for innate immune

NKs considered to be innate lymphoid cells - LACK variable antigen receptors

NK targeting

NKs express activating and inhibitory receptors to distinguish between healthy and infected cells

use these receptors to inspect other cells:

• sense cell stress signals (caused by virus pushing cell machinery to the limit to replicate) - activate NK

• sense expression of MHC class I (CD8) - inhibits NK

NK won’t kill a cell if it is expressing low levels of stress ligands (upregulated in stressed cells) and normal levels of MHC-1 (virus-infected cells remove MHC-1 to avoid detection by T-cells, but consequently get found out by NKs)

• all nucleated cells in the body express MHC-1 so T-cells can find them if they become problematic

NKs and antibodies

NKs can also use antibodies from the adaptive immune sys to recognize targets

• uses a different NK receptor but same cytotox machinery

• another example of an adaptive immune response triggering an innate immune response (also antibodies triggering classical complement)