3) Initiation and resolution of inflammation

PAMPS

pathogen associated molecular patterns

DAMPS

danger associated molecular patterns

What cells detect danger?

Sentinel cells (mast cells, dendritic cells, epithelial cells, macrophages)

What detects PAMPS/DAMPS?

PRR (pattern recognition receptors)

What are advantages and disadvantages of PAMPS?

A: Specific, conserved, rapid activation

D: Miss sterile injury, pathogen evasion, overactivation (sepsis)

What are advantages and disadvantages of DAMPS?

A:Detect sterile injury, amplify response, initiate repair

D:Non-specific, risk of auto-inflammation, collateral damage

Features of PAMPS

• shared by classes of organisms

• often essential for survival

• highly conserved

• absent from vertebrate host

• allow innate system to distinguish between self and non-self

What PAMP is found in gram-ve bacteria?

Lipopolysaccharide

What PAMP is found in gram+ve bacteria?

Lipoteichoic acid

What is germ-line encoded?

PRR + innate immune cells

Where are PRR’s present?

• cell membrane

• endosomes

What are the different functions of PRR?

-stimulate ingestion of microbes by phagocytosis

-act as chemotactic receptors and guide cells to sites of infection

-produce effector molecules to assist in innate and adaptive response

What are an example of PRR?

TLR (toll like receptors)

NLR (cytosolic NOD like receptors)

CLR (C-type lectin receptors)

RLR (RIG-I-like receptors)

When is CLR important?

Fungal infections

When is RLR important?

To detect viral RNA in cytoplasm

When is NLR important?

Cytoplasmic receptors to recognise PAMPS and DAMPS

What do NOD1 and NOD2 recognise and what is it’s clinical relevance?

NOD 1 & 2 recognise fragments of peptidoglycan from bacteria

NOD 2 detects muramyl dipeptide released from gut microbiota and important in gut homeostasis.

Mutations in NOD 2 can cause Crohn’s disease (type of IBS)

What is the importance of NLRP3?

It’s a type of NLR that has wide specificities recognising pieces of peptidoglycan,bacterial DNA, ATP,toxins and ds-RNA.

Forms part of a large cytosolic structure called the inflammasome

Linked to diseases like atherosclerosis,gout and type 2 diabetes.

Inflammasome induces inflammation by causing caspase 1 to activate IL-1 beta.

How do TLR function?

As dimers

Transmembrane proteins

Specific for a different set of pathogen products

10 functional TLR genes

Which TLR’s are found on the cell surface?

TLR 1,2,4,5,6,10

Recognise diverse microbial productBes

Which TLR’s are found in endosomes?

TLR 3,7,8,9

Recognise microbial nucleic acids

Besides ligand recognition what is another role of TLR?

Recruit adaptor molecules, mainly:

• MyD88

• TRIF

This triggers downstream signalling:

• Activation of NF-κB, AP-1, and IRFs (3 and 7).

Leads to release of:

• Inflammatory cytokines

• Type I interferons

• Chemokines

• Antimicrobial peptides

What do TLR’s recognise?

TLR’s recognise PAMPS

Some detect DAMPS

Eg: HMGB-1 (high mobility group box-1, a host nuclear protein) can induce signalling via TLR2 and TLR4.

What does TLR1:TLR2 recognise?

Lipoproteins

Glycosylphosphatidylinositol

What does TLR2:TLR6 recognise?

Lipoteichoic acid

Zymosan

What does TLR3 homodimer recognise?

Double stranded viral RNA

What does TLR4 homodimer recognise?

Lipopolysaccharide

What does TLR5 homodimer recognise?

Flagellin

What does TLR7 homodimer recognise?

Single stranded viral RNA

What does TLR8 homodimer recognise?

Single stranded viral RNA

What does TLR9 homodimer recognise?

Unmethylated CpG rich DNA

What does TLR10 recognise?

Unknown…

What is the role of DAMPS?

• Sterile inflammation can be triggered by self-molecules released during necrosis (not just by infection).

• DAMPs are typically intracellular molecules that are found in the wrong location (e.g. extracellular space).

What are examples of DAMP’s?

• HMGB-1 (High Mobility Group Box-1) — nuclear protein released from chromatin.

• ATP, DNA, RNA — normally intracellular molecules.

• Extracellular Matrix (ECM) components — exposed or released during tissue damage.

• Heat shock proteins — act as danger signals when extracellular.

• Oxidised LDL

  • Recognised by TLR4/TLR6.

  • Plays a role in atherosclerosis, a chronic inflammatory disease of the arterial wall.

  • Damaged mitochondria

    • Key source of DAMPs.

    • Due to their bacterial origin (endosymbionts), they can mimic PAMPs, triggering strong immune responses.

What happens in recruitment of the inflammatory exudate?

Multiple components work together to initiate and expand the acute inflammatory response.

• Vasodilation → increased blood flow to the affected area.

• Increased vascular permeability →

  • Allows protein-rich fluid to enter tissues.

  • Leads to fibrin web formation.

  • Recruitment of complement and C-reactive protein (CRP).

• Leukocyte recruitment from circulation into tissues:

  • Neutrophils arrive first (early responders).

  • Monocytes follow → mature into tissue macrophages.

  • Lymphocytes arrive later in the response.

  • Eosinophils and plasma cells can be abundant in certain types of infection (e.g. parasitic or chronic).

What happens in activation of sentinel mast cells and macrophages?

Mast cells and macrophages express numerous PRRs and are activated by a wide range of PAMPs and DAMPs.

Mast Cells

• Contain granules with preformed inflammatory mediators.

• Also synthesise prostaglandins and leukotrienes, but this is slower.

  • These are collectively known as Slow Reacting Substance of Anaphylaxis (SRS-A).

• Can also be activated by neurogenic inflammation, responding to Substance P released from nerve fibres

Macrophages

• Phagocytose microbes at the site of inflammation.

• Produce a range of cytokines, including:

  • Endogenous pyrogens → induce fever.

    • Examples: IL-1, IL-6, TNF-α.

• Cytokines exert:

  • Local effects on vascular endothelium.

  • Systemic effects — e.g. acute phase protein response (especially IL-6).

• Also synthesise prostaglandins and leukotrienes.

What happens in the recruitment of leulocytes to sites of infection?

1) Rolling (Weak Tethering)

2) Tight Adhesion

3) Diapedesis (Extravasation)

4) Migration (Chemotaxis)

Rolling (Weak Tethering)

• P-selectin rapidly induced on endothelial cells by:

  • Thrombin (from the blood).

  • Histamine (from resident mast cells).

• P-selectin is released from Weibel–Palade bodies (intracellular stores).

• E-selectin appears 1–2 hours later, induced by IL-1 and TNF-α from tissue macrophages.

• Selectins (P- and E-selectin) bind to Sialyl-Lewis X glycoprotein ligands on neutrophils.

• This binding is low affinity and easily broken by shear stress, resulting in rolling of cells along the endothelium.

Tight Adhesion

• Mediated by integrins on leukocytes:

  • LFA-1 (integrin) on neutrophils binds to ICAMs (Intercellular Adhesion Molecules) on endothelium.

• Initially, the interactions are weak.

• Chemokines (e.g. CXCL8) bind to neutrophil chemokine receptors → conformational change in LFA-1 → high affinity binding.

• This leads to firm adhesion of the neutrophil to the endothelium.

Diapedesis (Extravasation)

• Leukocytes squeeze through gaps between endothelial cells to enter tissues.

• This involves:

  • LFA-1 / ICAM interactions.

  • CD31 on leukocytes binding to CD31 on endothelial cells.

• Neutrophils secrete enzymes (e.g. elastase) to degrade the basement membrane, aiding migration.

Migration (Chemotaxis)

• Neutrophils follow a chemokine gradient to the site of infection:

  • CXCL8, secreted by activated macrophages, binds to the extracellular matrix to create a stable gradient.

• This directs neutrophils accurately to the infection site.

What is LAD?

• Leulocyte adhesion deficiency is a rare immunodeficiency caused by defective neutrophil recruitment.

• LAD1:

  • Caused by a defect in CD18, the β-chain of LFA-1.

  • Results in recurrent, life-threatening bacterial infections in infants.

• BLAD (Bovine Leukocyte Adhesion Deficiency) is an analogous condition in cattle.

Why are neutrophils recruited first then monocytes?

• Monocytes are recruited later because:

  • Their receptor ligand VCAM-1 (on endothelial cells) is upregulated more slowly (~24 hours).

  • VCAM-1 binds to VLA-4 (very late antigen-4) on monocytes.

• Lymphocytes and other leukocytes use similar multistep mechanisms (rolling → adhesion → diapedesis → migration) to exit the circulation and enter tissues throughout the body.

What is sepsis?

• Sepsis occurs when pathogens enter the bloodstream.

• Macrophages in the liver and spleen respond by secreting TNF-α into the circulation.

What chemical signal is involved with sepsis?

• If infection becomes widespread (e.g. severe burns → loss of skin barrier), pathogens can spread into the circulation.

• Endotoxins such as LPS (lipopolysaccharide) can provoke widespread TNF-α release.

• TNF-α plays a critical role in local containment of infection.

• Blood clotting and local TNF-α expression help:

  • Prevent pathogens from entering the bloodstream.

  • Limit their spread to other tissues.

What are systemic effects of TNF-alpha?

• Widespread vasodilation and fluid movement into tissues →

  • Drop in blood pressure.

  • Can progress to septic shock and heart failure.

• TNF-α also triggers coagulation in small vessels throughout the body →

  • Disseminated intravascular coagulation (DIC).

• Failure of major organs — including:

  • Kidney

  • Liver

  • Heart

  • Lungs
    → caused by impaired blood perfusion.

• Massive consumption of clotting proteins leads to:

  • Depletion of clotting factors.

  • Bleeding tendency (inability to clot properly).

What is the resolution of inflammation?

• The acute inflammatory response must be tightly controlled to minimise damage to host tissue.

• Many inflammatory mediators have short half-lives and degrade rapidly after release → leads to natural decline of the response.

• Neutrophils are short-lived — undergo apoptosis after a few hours in the tissues.

What are the stop signals of inflammation?

• As inflammation progresses, it triggers active resolution mechanisms:

  • Lipid mediator class switch:

    • Instead of macrophages producing pro-inflammatory leukotrienes from arachidonic acid,

    • The lipoxygenase system switches to producing anti-inflammatory lipoxins.

  • Switch to anti-inflammatory cytokines:

    • IL-10 and TGF-β are produced.

  • This switch is triggered by:

    • Macrophages engulfing apoptotic neutrophils.

How do we repair and heal damaged tissue?

• After tissue damage, the body attempts to replace dead tissue with healthy cells.

• The repair tissue formed is called granulation tissue.

• Main features:

  • Recruitment of endothelial cells → formation of new blood vessels.

  • Recruitment of fibroblasts → deposition of extracellular matrix (ECM).

  • Remodelling of ECM → formation of strengthened scar tissue.

What are the main mediators in the repair and healing of damaged tissue?

-Macrophages

-Fibroblast

-Angiogenesis

What is the role of macrophages in the repair and heal of tissues?

• Macrophages are pivotal in controlling the repair process:

  • Phagocytose debris (RBCs, apoptotic neutrophils, dead microbes, etc.).

  • Produce reactive oxygen intermediates and nitric oxide to kill microbes.

  • Recruit fibroblasts via FGF (fibroblast growth factor) → ECM deposition.

  • Recruit endothelial cells via VEGF (vascular endothelial growth factor) → angiogenesis.

  • Secrete metalloproteinases → remodelling of ECM.

What is the role of fibroblast in the repair and healing of tissues?

• Recruited to the injury site by FGF from macrophages.

• Induced to:

  • Increase collagen synthesis.

  • Produce other ECM proteins.

• Result: formation of collagen scar tissue.

What is the role of angiogenesis in repair and healing of tissues?

• Angiogenesis is rare in adults except in pathological states (and the female reproductive tract).

• Stimulated by VEGF and other cytokines produced by macrophages.

• Process:

  • Pre-existing vessels send out capillary sprouts into the damaged area.

  • Endothelial cells:

    • Break off from the basement membrane of existing vessels.

    • Migrate to the site of injury.

    • Proliferate and differentiate to form a lumen.

  • Acquire pericytes and smooth muscle → form a mature blood vessel.

What is chronic inflammation?

• Occurs when the injury or causative agent persists and is not removed.

• Inflammation and repair processes continue simultaneously, often leading to tissue damage and scarring.

What may cause the persistent injury for chronic inflammation?

• Endogenous injurious agents

  • e.g. stomach acid in peptic ulcers.

• Non-degradable agents

  • e.g. silica, dust particles.

• Pathogens that evade host defences

  • e.g. Mycobacterium tuberculosis.

• Autoimmune reactions

  • e.g. rheumatoid arthritis.

What is the role of macrophages in chronic inflammation?

• Macrophages are major players in chronic inflammation.

• Chronically activated macrophages:

  • Accumulate at the site.

  • Release cytokines that stimulate:

    • Fibroblast proliferation.

    • Collagen production.

  • Leads to scar tissue formation (fibrosis).

What is a granuloma?

• Granulomas may form during chronic inflammation.

• Can be caused by infectious or non-infectious agents.

• Classic example: tuberculosis.

What is tuberculuous granuloma?

• Mycobacteria resist macrophage killing → not eliminated.

• Instead, they are “walled off” from surrounding tissue.

• Structure:

  • Central core of macrophages.

  • Surrounded by T cells.

  • Macrophages can:

    • Fuse → multinucleated giant cells.

    • Become epithelioid cells (large, activated).

  • Central necrosis is common.

• Granuloma formation:

  • Often seen as bacterial hiding, but actually a programmed host response.

  • Allows containment rather than sterilisation of infection.

When may granulomas occur besides infection?

• Granulomas can also occur without infection:

  • e.g. Blau syndrome — caused by gain-of-function mutations in NOD2 (a PRR).

  • Drives chronic inflammation in the absence of pathogens.

• Biological purpose of granuloma formation:

  • Decision by immune system to:

    • Not fully sterilise the infection site.

    • Shift to a long-term, local immune reaction.

  • This can benefit the host by containing damage and limiting systemic inflammation.

How is the adaptive immune response induced?

• Dendritic cells (DCs):

  • Internalise antigens from pathogens at the infection site.

  • Carry both PAMP information and specific antigens to draining lymph nodes.