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Dr. Amelia Woolums, Mississippi State University ISVMA Annual Conven;on, November 2016

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How Does The Immune System Do It? Inflamma;on and Innate Immunity

Amelia R. Woolums, DVM MVSc PhD DACVIM DACVM Department of Pathobiology and Popula;on Medicine, Mississippi State University

[email protected]


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Immune System: Overview

•  Innate immune response –  Immediately and always ac;ve – Doesn’t improve with repeated exposure

•  Acquired immune response – Takes several days-‐weeks to be fully ac;ve –  Improves with repeated exposure: “memory” – Target of vaccina;on

Immune System: Overview

•  Innate and acquired responses are ac;ve in 2 major sites

– mucosal surfaces

– blood and ;ssue fluids

•  Host is thus protected from aYack on any front

Innate immune system: components

•  Physical or chemical barriers – Skin, mucociliary elevator, gastric pH, urine flow

•  Soluble factors –  in serum, secre;ons, excre;ons, ;ssue fluids

•  Cellular factors – granulocytes, macrophages, natural killer cells, gamma delta T cells, epithelial cells


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•  Mechanisms important in the ini;al response to infec;on are also involved in the response to non-‐infec;ous ;ssue injury

–  trauma – burns or frostbite – bites

•  All induce inflamma;on

•  Inflamma;on ac;vates the immune response

Infec&on Tissue injury

PAMPs DAMPs (“alarmins”)

Sen&nel cells displaying pathogen recogni;on receptors

Influx of innate immune cells, vascular change associated with inflamma&on

cytokines, vasoac&ve molecules

Infec&on or injured &ssue is REMOVED OR

acquired immune response is ac&vated in con&nued effort to remove infec&on

PAMPs and PRRs

•  PAMPs: pathogen associated molecular patterns – Highly conserved molecules found in many

different microorganisms

– Host response evolved to recognize these

•  relatively few molecules can initiate immunity to the limitless microbial world


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Important PAMPs •  Peptidoglycan and lipoteichoic acid

–  Gram positive bacteria (Staph., Strep., and others) •  Lipopolysaccharide

–  Gram negative bacteria (E. coli, Salmonella, and others) •  Glycolipids

–  acid-fast bacteria (mycobacteria: Johne’s disease, TB) •  Mannan-rich carbohydrates

–  fungi (Aspergillus and others) •  Unmethylated CpG nucleotide motifs

–  bacteria and viruses •  dsRNA

–  viruses

Some important DAMPs

•  DAMPs are components of host tissues •  Extracellular DAMPs

–  Extracellular matrix components

•  hyaluronic acid

•  fibronectin

•  collagen-derived peptides

•  elastin

•  Release of small and/or soluble fragments during tissue injury allows PRR activation

•  Intracellular DAMPs

–  High mobility group box protein-1 (HMGB1)

•  Associated with DNA in normal cells: ensures proper folding

•  Triggers inflammation when released from damaged cells

–  If DNA associated: anti-DNA response can occur

•  Can be secreted by macrophages

–  Unmethylated CpG DNA from mitochondria

–  Adenosine

•  In cAMP, ATP, nucleic acids

–  Uric acid

•  Breakdown product of purines (e.g. adenine, guanine)


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•  Sentinel cells in tissues are the first cells to “see” PAMPs or DAMPs

– dendritic cells – macrophages – mast cells – epithelial cells –  fibroblasts

These cells can also be called into regions of inflammation

Dendri;c Cells

•  Present in most ;ssues •  Have 3 major func;ons:

– Survey ;ssues for PAMPs and DAMPs

– Present an;gen to T cells and B cells •  The only APC that can ac;vate naïve TC

–  Influence the type of immune response that occurs in response to PAMPs or DAMPs

•  TH1, TH2, TH17, or T regulatory (Treg)

– More in the next hour

Macrophages

•  Iden;fy and kill pathogens via many PRRs and other surface receptors – phagocytosis – secrete an;microbial products

•  Produce proinflammatory cytokines –  IL-1, TNF-α, and IL-6

– ac;vate inflamma;on •  kick off the immune response


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•  Given s;muli that lead to “classical ac;va;on”, macrophages become M1 macrophages – Phagocytosis – Microbial killing – Proinflammatory cytokine produc;on

•  Given s;muli that lead to “alterna;ve ac;va;on”, macrophages become M2 macrophages – Suppression of inflamma;on – Promo;on of blood vessel forma;on – Promo;on of ;ssue remodeling and repair

“Accumulated evidence indicates that macrophages are func;onally plas;c cells with the poten;al to alter their ac;vi;es progressively and reversibly in response to changes in the ;ssue environment.” Sang et al., 2011

Mast Cells •  Originate from myeloid precursors in bone

marrow

•  Migrate to ;ssues

– Mucosal mast cells: under mucosal surfaces

–  Connec;ve ;ssue mast cells: in skin, peritoneal cavity

•  Granules contain enzymes and vasoac;ve mediators

•  Degranulate when surface IgE molecules cross-‐linked an;gen binding

–  Key mediator of allergy and anaphylaxis


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•  It is now recognized that mast cells can respond to PAMPs and DAMPs via PRRs – Help ini;ate early inflammatory response to many s;muli

– Granules are released “piecemeal” •  Provides a more ;trated response than when an;gen binds surface IgE

Pathogen recogni;on receptors (PRR)

•  Sen;nel cells ac;vate the inflammatory/immune response when their PRR bind PAMPs or DAMPs

•  PRR are found – On cell surface –  Inside endosomes inside the cell –  In cell cytoplasm

Sites where cellular pathogen recogni&on receptors (PRR) can bind to PAMPs or

DAMPs

Sentinel cell

TLR-‐6 TLR-‐2

TLR-‐5 TLR-‐4

TLR-‐2 TLR-‐1 On the cell surface

TLR-‐3

TLR-‐9

TLR-‐7

Within endosomes inside of cell

Within the cytoplasm NLR

RIG-‐I MDA5

CLR


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•  Binding of PRR by a PAMP or DAMP ini;ates a signal transduc;on sequence in the cell

•  This will cause the cell to produce cytokines that will in turn ac;vate the inflammatory/immune response

•  The mixture of cytokines produced will determine the kind of immune response ac;vated

LPS

MYD88

TLR-‐4

MKK IRAK

TRIF

MAPK NF-‐κB IRF3

TNF-‐α

IL-‐1β

IL-‐6 IFN-‐β

Consequences of PAMP binding to PRR Example: LPS binding to TLR-‐4

Lipopoly

saccharid

e

(LPS)

TLR-‐4

Macrophage

A. Woolums

•  Consequence of TLR binding by PAMP –  Initiation of signal transduction sequence – Expression of genes for

•  cytokines • other antimicrobial molecules

•  Different TLRs trigger different combinations of gene expression – Response appropriate for defense against the

initiating microbe


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Cytokines

•  All cells influence other cells by release of cytokines •  Cytokines...

– Ac;vate the immune response

– Direct specific types of responses •  An;-‐viral, an;-‐bacterial, an;-‐parasi;c

– Contribute to inflamma;on and some;mes death •  “Sep;c shock”

So…why does this matter??


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M. haemolytica CD18/CD11a (LFA 1) leukotoxin

proteases

elastases

reactive O2

tissue necrosis

TLR-4 + CD 14

LPS

TNF-α, IL-1β endothelial cell

activation,

leaky blood vessels

edema,

hemorrhage,

thrombosis

Fibrinous, necrotizing bronchopneumonia

neutrophils

macrophage

Other Players in the Innate Immune Response

Innate immunity, soluble factors

•  Defensins and cathelicidins – kill or inac;vate bacteria, fungi, enveloped viruses

•  Lysozyme – degrades pep;doglycan

•  Lec;ns – bind microbial carbohydrates (e.g., to opsonize)

•  Iron-‐binding proteins •  Complement


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•  Soluble factors are not only an;microbial

•  Some also ac;vate immune cells – act as “natural adjuvants”

– a cathelicidin included in an experimental vaccine improved immune responses in caYle

Kovacs-‐Nolan et al., 2009

Innate immunity, cells

•  Granulocytes: immediate responders • Neutrophils: any infec;on or injury •  Eosinophils: parasites and hypersensi;vity • Basophils: func;ons not well characterized


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Neutrophils

•  “First responders” – Among the first immune cells to come to site of infec;on

•  Mediate an;-‐pathogen defense in many ways –  Engulf (phagocytose) and destroy pathogens with

•  reac;ve oxygen species (ROS) via the respiratory burst •  proteoly;c enzymes

•  an;microbial pep;des

Natural killer cells

•  Major mediator of innate anti-viral defense

•  Large, granular, nonphagocytic lymphocytes

•  Respond to balance of inhibitory and activating signals on target cells

–  MHC I on healthy cell: inhibitory signal transmitted

–  Lack of MHC I: activating signal


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Tizard, 2009

natural killer cell

virus-‐infected cell, before killing

virus-‐infected cell, aeer killing

natural killer cell

•  NK cells kill target cells by

–  Releasing perforin •  forms pores in target cell membrane, then

–  Releasing granzymes from cytotoxic granules •  enter target cell through perforin pores

•  granzymes induce caspase ac;va;on

•  caspases induce target cell apoptosis (programmed cell death)

Gamma Delta (γδ) T cells

•  “In between” innate and acquired immunity

•  Can be active immediately –  Produce cytokines, kill other cells, survey mucosa

•  Also seem to exhibit some memory –  Improved response following vaccination in some cases

•  Gamma delta T cells make up major proportion of circulating cells in young ruminants and swine –  Significance?


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Acknowledgement

•  Mr. William (Kip) Carter, University of Georgia – classier anima;ons

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