SUPPLEMENTARY ONLINE MATERIAL: BONNEAUD ET AL.
Innate immunity and the evolution of resistance to an emerging infectious disease in a wild
bird
Online Gene Function ................................................................................................................... 1
Supplementary table S1................................................................................................................ 5
References ...................................................................................................................................... 6
Online Gene Function
Among the known genes that were significantly differentially expressed (tables S1), we detected
6 that have been identified with primary immune function and hence may have been directly
involved in the immune response to MG. Note that functions were mostly determined from
studies on humans and mice, so although they are likely to be conserved, we cannot rule out that
proteins have evolved to serve different roles in house finches. T-cell immunoglobulin and
mucin domain containing (TIM) 4 is a trans-membrane receptor that is expressed at the surface
of antigen-presenting cells (e.g., macrophages and dendritic cells) and that ligates TIM1 on the
cell-surface of naive CD4+ T-cells to activate their differentiation into Th2 cells (Liu et al.
2007); it also mediates the clearance of apoptotic (phosphatidylserine-expressing), antigenspecific T-cells after infection to avoid autoimmunity (Albacker et al. 2010; Kobayashi et al.
2007). MHC class II-associated invariant chain Ii (CD74) is a chaperone molecule that plays
a role during the assembly of MHC class II molecules within and transport out of the
endoplasmic reticulum (Bertolino & RabourdinCombe 1996; Stumptner-Cuvelette & Benaroch
2002).
Programmed death ligand 1 (CD274; B7-H1; PDL-1) can be expressed on
macrophages, T- and B-cells and enhances T-cell proliferation and secretion of interleukin 10,
interferon ɣ and granulocyte macrophage colony stimulating factor, and preferentially affects Thelper (CD4+) cell functions (Tamura et al. 2001); it has also been shown to limit effector T-cell
responses and protect tissues from immune-mediated tissue damage (Francisco et al. 2010; Keir
et al. 2008; Sharpe et al. 2007). Lectin galactoside-binding soluble 2 protein (galectin 2) is
part of a family of proteins differentially expressed in various immune cells and up-regulated
during infections (Rubinstein et al. 2004); galectins are involved in the regulation of cellular
immune responses and immune cell homeostasis (Liu 2005), and galectin 2 is thought to control
inflammation and regulate activated CD8+ T cells by inducing their apoptosis (Loser et al.
2007).
Neutrophil cytosolic factor 4 encodes a cytosolic regulatory component of the
superoxide-producing phagocyte NADPH-oxidase and was found to be essential for a key host
innate immune defence mechanism: phagocytosis-induced oxidant production in neutrophils
(Matute et al. 2009). hCG40889 (complement factor H) is secreted in the plasma to regulate
complement-mediated immunity, which plays a key role in microbial killing; it serves to protect
host cells and tissues by preventing excessive activation of the complement cascade (Boon et al.
2009; de Cordoba & de Jorge 2008).
We also identified 3 genes with auxiliary immune function: Thioredoxin, Rho GTPase and
Lymphocyte cytosolic protein. Although these genes were implicated in several biological
processes, we highlight here their implication in processes associated with immune functioning.
The production of reactive oxygen species (ROS) by phagocytic cells during oxidative bursts is
an important antibacterial mechanism that has been found to take place during infections with
MG (Fang 2004; Jenkins et al. 2008). ROS are free radicals (e.g., superoxide O2-, hydroxyl
radicals OH, hydrogen peroxide H2O2) that are produced at high levels to kill internalized
pathogens (Swindle & Metcalfe 2007). ROS act non-specifically and as they accumulate, they
damage both host tissues and pathogens indiscriminately, e.g. by inducing DNA or cell damage
through lipid peroxidation (Droge 2002; Valko et al. 2007). ROS scavenging mechanisms, such
as the enzyme superoxide dismutase, which catalyzes the dismutation of superoxide (O2-) into
oxygen (O2) and hydrogen peroxide (H2O2), have evolved to minimize such costs.
Such
antioxidant properties have been demonstrated for thioredoxin, which is an oxido-reductase
system induced by oxidative stress (Nordberg & Arner 2001) that can influence downstream
immune functions through the regulation of transcription factors and cytokines (Bubici et al.
2006; Sen & Packer 1996). Rho GTPase proteins belongs to a family of small signalling G
proteins that are involved in several signal transduction pathways and cellular functions; for
example, they have been shown to play an important role in the regulation and coordination of
the innate immune response (reviewed in (Bokoch & Knaus 2003; Scheele et al. 2007)). Indeed,
Rho GTPase proteins are involved in Toll-like receptor signalling, a key line of defense against
microbial pathogens (Aderem & Ulevitch 2000), and they form a subunit of the NADPH oxidase
complex where they regulate the formation of ROS during oxidative bursts (Kao et al. 2008).
Another important role of Rho GTPase proteins is their implication in actin and microtubules
regulation and cytoskeletal rearrangements mediating leukocytes chemotaxis and motility,
phagocytosis as well as lymphocyte cytotoxicity (Cicchetti et al. 2002; Khurana & Leibson
2003; Scott et al. 2005). Lymphocyte cytosolic proteins (L-Plastin) are actin-binding proteins
that have been found to be expressed exclusively in the hemopoietic cell lineages. They have
been shown to stabilize actin filaments during T-lymphocyte migration (Morley et al. 2010;
Samstag et al. 2003), while the interaction between actin filaments and myosin, and the
phosphorylation of myosin regulatory light chain, generate the contractile force necessary for
cell migration (Mizutani et al. 2006). They have also been shown to play a role in eosinophil
priming for chemotaxis and degranulation (Pazdrak et al. 2011), in B-cell motility and
development (Todd et al. 2011), and T-cell activation (Wabnitz et al. 2007; Wang et al. 2010).
Supplementary table S1
Fold change (FC) and p values for the 25 clones found to be significantly differentially expressed
in at least one of the four comparisons in the microarray (significance level: P<0.05), and for
which we identified a vertebrate homologue.
Genes
Arizona
Infected day 3
vs. control
FC
p
Alabama
Infected day 3
vs. control
FC
p
Arizona
Infected day 14
vs. control
FC
p
T-cell immunoglobulin and mucin
domain
-6.17
0.011
Programmed death ligand 1
-3.57
0.028
Lectin galactoside-binding soluble 2
protein
-4.13
0.028
MHC class II-associated invariant
chain Ii
-5.89
0.011
Neutrophil cytosolic factor 4, 40kDa
Complement factor H
Thioredoxin
-3.00
0.040
Cytochrome oxidase subunit I
-6.63
0.037
Cytochrome c oxidase subunit VIIa
2
Phospholipase D family, member 4
-2.49
0.033
2.66
0.022
-2.52
0.046
-2.97
0.028
-1.95
0.028
RhoA GTPase
MLTK-beta
-2.23
0.046
Tyrosine 3-monooxygenase
activation protein
Pleckstrin homology domain
-1.81
0.043
3.29
0.018
2.84
0.013
Protein 4.1-G
-4.30
0.044
-3.39
0.043
Cytoplasmic beta-actin gene
-2.66
0.041
-3.36
0.013
Lymphocyte cytosolic protein 1
-2.86
0.026
-1.95
0.046
Destrin
-2.34
0.033
Heat shock protein 90a
2.06
0.039
-2.22
0.021
Translation initiation factor EIF4G2
-4.25
0.042
Ribosomal protein S15
-2.21
0.039
Alabama
Infected day 14
vs. control
FC
p
3.90
0.044
2.89
0.017
1.94
0.049
11.71
0.021
2.08
0.033
2.11
0.043
-2.71
0.017
2.12
0.045
-2.47
0.033
Eukaryotic translation initiation
factor 4E
2.26
0.041
1.93
0.040
-2.76
0.023
SEC61 gamma
-6.28
0.012
-5.14
0.007
3.71
0.016
Hemoglobin alpha
-7.28
0.021
-3.29
0.04
-4.23
0.045
Epidermal differentiation-specific
protein
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