Innate Immunity

Lecture #4
 December 1882, Messina, Sicily,
Discovery of phagocytosis

Phagocytosis: 135 years after……………

Clearance of pathogens as a defence mechanism
PM as a sole supplier of membrane……..fusing with endo-, lysosomes
 Phagocytosis: engulfment of material by cells

Hydrolytic

MATURATION

-cell surface recognition: direct or via opsonin (large redundancy)

-de novo generated organelle: the phagosome, funct. in innate &adaptive immunity
-its composition depends on the receptors enagaged, mechanism of entry used,
nature of cargo, origin of the contributing membrane (NOT THE SAME!!!)
-difficult to study-phagocytes are not readily amenable to manipulations

Each phagosome is tailored for and by, its cargo

Phagocytosis of Mouse Peritoneal Macrophages
% of phagocytosing M-phages

100

80

60

40

20

0
0 15 30 60 120
Time (min)
 Nutrient acquisition by unicellular organisms

Nutrient acquisition is possible the evolutionary origin of the link

between phagocytosis and bacterial recognition
Pathogen recognition was advantageous before the evolution of multicellularity
Essential role of phagocytosis in innate immune sensing
 Insects have multiple levels of defence
against infection

Structural Proteolytic cascade Antimicrobial peptides

Antimicrobial peptides Plasmatocytes Lamellocytes

Innate Immune receptors in D. melanogaster

Similar to gp130 of IL-6R.

Activates JAK_STAT
 A model for antimicrobial gene regulation in insects
Fungi
Gram + Enterobacteria
serpin Gram- (LPS)
?
Lysine-PG NEC Fungi diaminopimelic
acid
seml PGRP-LC
(PGRP-SA) Persephone

dToll is not PRR!!!

Serine Spätzle imd
Protease dTAK1
BG4
MyD88
Toll (dFADD)
Degradation
Tube
Pelle Dredd IKKβ IKKγ
(Caspase
Protease)
Traf2
Cact Relish

Dl DIF Rel

ref2P
Dl DIF
aPKC Rel

Drosomycin Metchnikowin Diptericin

Their combination Defensin Drosocin
is important!!! CecropinA Attacin
-S2 cells: embryonic hemocytes derived
-proteomic analysis of latex beads-containing
phagosomes
-617 proteins identified
-70% of mammalian orthologs identified
-protein-protein interaction network generated
with 53,775 interactions and associations
-RNAi for 837 genes (617 identified) for their
role in phagocytosis of G- and G+ bacteria
-28% genes affected phagocytosis
-different genes regulate uptake and
internalization of G - and G+ bacteria

Phagosome interactome
Silencing exp.

F-actin staining
Exocyst is a functional component of the phagosome
 Innate Immune receptors in D. melanogaster

Proteomic data

4 classes of molecules involved in phagocytic recognition:

1st class: complement-like opsonins ; tioester containing proteins (TEP I-IV),

bind E.coli. S.aureus, C.albicans…….Receptors unknown
 2. Class: Scavenger receptors

Croquemort (french for pallbearer), human CD36 analog

-belongs to CD36 family, binds polyanionic ligands

-expressed only in hemocytes
-expressed immediately after the onset of apoptosis, embr. stage 11
-Important receptor for apoptotic cells in developing embryo
-in adult flies: binds S. aureus (diacylated lipopeptides)
Expanded in species lacking adaptive immunity (10 in d.m.)
SR-C class: insect specific, upregulated in larva after infection
3rd class: Receptors with multiple EGF-like repeats

-Emerging superfamily
-EMI:dimerization domain
-Eater: 32 EGFs. It is a major Receptor
for many pathogen in D.
N-terminus binds to G- G+
Toll and Imd-intact in Eater KO
Expressed in plasmatocytes
Not in embryonal Mfs (like Croqm)
Antibacterial immunity

Nimrod: for pathogens, hemocyte specific

cluster of 10 related genes
potential adhesion function
not in human

-Draper: removal of apoptotic cells in CNS

Human CD91, C.elegans CED-1 homolog
 Dscam receptors in flies

Somatic diversification of Ag receptors:

Jawed vertebrates: variable-diversity-joining recombination
Jawless vertebrates: gene conversion

Somatic diversification of antigen receptors is NOT limited to vertebrates!!!!

Ig domain/fold

Ig fold: 100AA:Ig, MHC, β2microglobulin,

Thy-1: IgSF

2 categories: V-domain: Ag binding

C-domain: Effector f.

Structure: sandwich of 2 β-sheets

Hypervariable regions: in the loops at one

end of the domain

C-C link: (Β-F strands)

Evolutionary success: versatility!!!

stable fold/ resistant to proteolysis/ forms
homo:heterodimers/interacts via diff.
Residues/single exon coding for most IgSF
domains
 Genes with IgSF are among the most common domain types in human

1. There has been disproportionate increase in IgSF number in the evolution from
invertebrates to vertebrates: IgSF proteins constitute 2% of human genes

2. This increase parallels the evolution of the adaptive immune system

(Ig receptors, regulatory proteins) but also other functions.
Shape, size, association and interaction of the IgSF containing molecules
6 classes of IgSF proteins
 24 exons
Mutually exclusive alternative splicing occurs for exons 4, 6, 9 and 17
 Dscam disrupts Axon
Tracts in the embryo
(CNS of stage 16 embryo)

Mild to severe disorganization of a ladder-

Homophilic
like pattern within ventral interactions
nerve cord are critical !!!
First 8 Ig domains are required
1

2
3
4
Dscam expression in fat body tissue
Global assessment of alternative splicing in different cell types
 Comparison of Dscam Exon 4-6
combinations in brain tissue, fat
body and hemocytes

….most exon 4-6 combinations are

Individual cDNAs (1-55)

unique suggesting that a large

diversity is expressed not only in
the nervous system but also in
immune-competent cells
 24 exons
Mutually exclusive alternative splicing occurs for exons 4, 6, 9 and 17
Dscam protein is expressed in immunocompetent cells
1. Crude cell lysates

2. Immunoprecipitation
S2
Dscam is required for efficient phagocytosis and binds to E.coli
 Conservation of Dscam diversity in insects

250 million years separating the fly, the mosquito and the bee.

The mouse and Human Dscam do not undergo extensive alternative splicing
Q:
-Immune system vs. Development (axon guidance)

-ligands: endogenous & exogenous

-repertoire of Dscam receptors:14-50 splice-form per cell:

combinatorial association of Dscam receptors for pathogen
recognition?

-risk of autoimmunity as they are prone to interact with

each other (homotypic interaction)

-selection through the regulation of splicing?

-Is Dscam expression induced during immune responses?

Negative stain electron microscopy:
a.) Dscam D1-D8 1.34.30-different shapes

HOWEVER
b.) D1-D4 retains horse-shoe shape ( >90%)

Horse-shoe
shape
 Crystal structure of D1-D4 domains

Homophilic interaction: by symmetric antiparallel interaction of identical peptides !!!

 24 exons
Mutually exclusive alternative splicing occurs for exons 4, 6, 9 and 17
 Crystal structure of D1-D4 domains

Homophilic interaction: by symmetric antiparallel interaction of identical peptides !!!

Q:
-Immune system vs. Development (axon guidance)

-ligands: endogenous & exogenous

-repertoire of Dscam receptors:14-50 splice-form per cell:

combinatorial association of Dscam receptors for pathogen
recognition?

-risk of autoimmunity as they are prone to interact with

each other (homotypic interaction)

-selection through the regulation of splicing?

-Is Dscam expression induced during immune responses?

Similarities between
Dscam and Ig
Q:
-Immune system vs. Development (axon guidance)

-ligands: endogenous & exogenous

-repertoire of Dscam receptors:14-50 splice-form per cell:

combinatorial association of Dscam receptors for pathogen
recognition?

-risk of autoimmunity as they are prone to interact with

each other (homotypic interaction)

-selection through the regulation of splicing?

-Is Dscam expression induced during immune responses?

 INVERTEBRATE’S “ADAPTIVE IMMUNE RESPONSES”:

Somatic diversification of antigen receptors is NOT limited to vertebrates!!!!

Summary
Phagosome is a multifunctional organelle
Toll pathway is required for the primed response

PBS

S.p.

PBS

S.p.
Primed flies exhibit an enhanced phagocytic response
Mammals: phagocytosis is essential for Ag presentation