Learning Objectives • Understand basic divisions of the immune system

THE INVERTEBRATE IMMUNE SYSTEM

– Innate vs. Adaptive – Humoral vs. Cellular

• Provide examples of each division • Provide examples of how pathogens subvert or evade the immune system • Understand how these divisions work independently and together • Exploitation of immunity with immunostimulants

The Immune System: Function • All organisms have a natural tolerance to infection, which varies with life stage, physiology, stress, genetics immune response • Protects animals from infectious disease and their toxic products. • Powerful mechanisms to locate, neutralize, and eliminate foreign cells or macromolecules

Invertebrate Immune System

– – – – –

Innate Immunity (non-specific) Evolutionarily old system First line of defense Rapid response Distinguishes broad classes of pathogen No long-term memory

Potential for (Quasi-)Adaptive Immunity (Acquired/ Specific) – Apparent specific response to previously encountered foreign antigen – Hypervariable and/or alternate histocompatibility complex (fusion gene Fu) – Ig Superfamily – Not well understood

Immune System in Action Fig 1. Litman et al. 2005 Nat Rev Immunol. Nov 2005; 5(11): 866–879. doi: 10.1038/nri1712

Pathogen Invades/ Breaches Barriers

RECONSTRUCTING IMMUNE PHYLOGENY: NEW PERSPECTIVES

Signals Quasiadaptive immune response???

Innate immune system responds (phagocytosis)

Eliminates Contains Or does not contain

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Immune Organs • External

• Internal Lymphoid Organs Pathogen Invades/ Breaches Barriers

– Exoskeleton* – Epithelium*

– Lymphoid organ (crustaceans) – Connective tissue or other • Gills tissue suspected to be • Gut Mucus cell: Mucus contains humoral factors source of hemocytes (=hematopoietic tissue) – Hemocytes Mantle Epithelium – Hemolymph

What happens when a pathogen breaches the outside barriers? Innate immune system responds

Lymphoid Organ

Innate Humoral Factors

Cellular and Humoral Effector Molecules Innate

Cellular

• Substances that non-specifically inhibit or destroy pathogens • Found in hemolymph, mucus, hemocytes and other tissues

Adaptive

Humoral

Cellular ?

Humoral?

• Cellular: mediated by cells • Humoral: mediated by a secreted substance usually a protein or glycoprotein

Viruses Cell

Interferon is a type of cytokine

Type I Interferons

Antiviral State Upregulate genes/proteins to produce an anti-viral state

– Some examples: • • • •

Messengers: cytokines and chemokines Enzyme Inhibitors: Anti-proteases Lysins: Lysozyme, antimicrobial peptides Opsonins: Lectins

No evidence of interferon in invertebrates but can induce an antiviral state with dsRNA

Rapid cell suicide to prevent viral replication

Returns to Normal

Robalino et al. 2004

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Robalino et al. 2005

Robalino et al. 2005

• 1a. Sequence-specific protection from WSSV ds-RNA; magnitude of antiviral state varied with WSSV gene sequence used (IM)

• 1c. dsRNA protection likely sequence dependent as antisense RNA to same gene failed to protect

• 1b. Sequence-independent innate IR failed to protect (poly(CG)) from high dose WSSV but WSSV dsRNA protected (per os)

Robalino et al. 2005

Innate Humoral Factors: Lysins • 1d. dsRNA protection is both sequence and dose dependent

• Lytic enzymes/cytotoxic molecules – Lysozyme in some fish and molluscan species – Antimicrobial peptides

• Hemocyanin

• 1e. Viral-specific motifs afford transient innate stimulation and a delay in onset of mortality but not full protection as did dsRNA from the challenge virus

– In crustaceans/gastropods/chitons – And some bivalves (not oysters, scallops, mussels) www.csulb.edu/~gcampus/science/ bio/gastropod/figure5.1.jpg science.msfc.nasa.gov/.../ dewar_pix/pcg_gallery.htm

Lysozyme

Peptidoglycan

Antimicrobial peptides

• Defense mechanism against bacterial infections – Found in mantle, mucus and serum of Crassostrea spp. and in the digestive gland and mantle of Haliotis spp. – Released during degranulation in phagocytosis – Season and health affect levels • Hydrolysis of bacterial cell walls – Cleaves the glycosidic bond between carbon number 1 of N-acetylmuramic acid and carbon number 4 of N-acetylD-glucosamine – = carbohydrates that polymerize to form the cell wall polysaccharide

• Usually + charged • Destabilize membranes of targets and create pores  lyse cells • Pre-pro-peptide: limited lysis to activate Prepro

peptide

– E.g from protein with other use (recycle) • Hemocyanin (net negative charge): antifungal activity in Penaeus vannamei • Cleavage results in 50x increase in antifugal activity! http://www.kingsu.ab.ca/~han k/Course%20files/Biochemistr y/lysozyme.htm ; wikipedia

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Pattern recognition proteins (PRPs) • LPS and beta glucan binding protein (LGBP) • Important in innate IR of many invertebrates • Up-regulated upon exposure to bacteria, fungi and viruses • Activates Prophenyloxidase cascade • Melanin production • Also suggested role in stimulating antimicrobial peptide production and release

Innate Cellular Mediators • Function in nutrient digestion, metabolite transport, wound & shell repair • Main role is in internal defense:

Adaptive

Innate

Cellular

Humoral

Cellular

Innate Cellular Mediators: Phagocytes

Bacterium

Phagocytosis 1. Pseudopodia capture bacteria (ROI stimulation) 2. Ingestion to form phagosome 3. Fusion to lysosome containing enzymes and toxins 4. Digestion via enzymes such as lysozyme, acid hydrolases, lipase, b-glucuronidase, aminopeptidase

5. Release of digestion products

Humoral

• Cellular: mediated by cells • Humoral: mediated by a secreted substance usually a protein or glycoprotein

Hemocyte

– chemotaxis, lectin-mediated pathogen recognition, phagocytosis, encapsulation and elimination of invaders via enzymatic destruction and/or by the production of antimicrobial peptides – How do they signal other cells?

Figure 1-3. Kuby Immunology 4.0 CD ©2000 W.H. Freeman and Sumunas Multimedia Development Services, Sumanas Inc.

Cellular and Humoral Effector Molecules

Bacterium is engulfed and enters phagosome

Cytokines Chemokines

lysosome

Lysosome fuses with phagosome and lysosomal contents degrades the bacterium

MAP Kinase (MAPK) signal transduction pathway

Releases degraded particles and glycogen into hemolymph or commits suicide by exiting across an epithelium (diapedesis)

A Good Place To Hide • Some bacteria or protists use phagocytosis to gain entry into the cell • Bacteria/protists can escape the phagosome or phagolysosome • Once in the hemocyte, the pathogen is hidden from the host immune system

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Cellular and Humoral Effector Molecules Adaptive

Innate

Cellular

Humoral

Cellular

Humoral

• Cellular: mediated by cells • Humoral: mediated by a secreted substance usually a protein or glycoprotein

Possible Cellular Mediators of the Quasi-adaptive Immune System • Hemocytes • ? Do they retain memory of a past exposure

Quasi-adaptive system of crustaceans

Venegas et al. 2000… Penaeus rod-shaped DNA virus (PRDV) = WSSV

Cellular and Humoral Effector Molecules Adaptive

Innate

Cellular

Humoral

Cellular

Humoral

• Cellular: mediated by cells • Humoral: mediated by a secreted substance usually a protein or glycoprotein

• Confirmed that challenged shrimp were ‘immune’ by rechallenge with virus up to 40d post initial challenge • To examine for presence of neutralizing serum factor, took serum from survivors of a natural exposure 17d and 40d post challenge and coinjected with virus into naive shrimp • Protection at 17d only – ’quasi-acquired immune response’

• Neutralizing factor 17d • Protection vs re-challenge at least 40d

NS = serum of natural survivors 40d NES= from NS+rechallenge 17d

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Adaptive Humoral Mediators: What are they? • No antibodies

Summary of Responses Humoral

• But have/secrete: – PRPs – AMPs – IgSF-domain-containing fibrinogenrelated proteins (FREPs)

Innate Opsonins Lysins Cytokines/chemokines

• Yet to identify/understand a full spectrum of mediators and their role in the observed memory Cellular

Adaptive ? Polymorphis m in immune gene loci IgSF domains (FREPs) AMPs (penaeidin) ?

Phagocytes

Immunostimulants

Summary • Invertebrates have an efficient immune response

• Beta Glucans (long chain polysaccharide found in many plant, bacterial and fungal cell walls) • Structurally resemble bacteria to the immune system – Mimic the microbial cell capsules and cell walls • Stimulate hemocytes to synthesize and release antimicrobial peptides • Probiotic bacteria in feed • Once activated  increased metabolic needs

– Considered more primitive than that of vertebrates

• Many analogs to higher animals • But lack antibodies – Quasi-adaptive IR seen in shrimp

• Relies on less specific innate immune response – Humoral and cellular facets • Also seen in higher animals

: www.cat.cc.md.us/.../lecguide/ unit1/bacpath/capc3b.html

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