Koubourli DV , Yaparla A , Popovic M , Grayfer L .

	Figure 1. In silico analyses of CXCL8 phylogeny and protein sequence conservation.(A) The protein alignment was performed using ClustalW2 server. Fully conserved residues are indicated by an asterisk (*), partially conserved and semi-conserved substitutions are represented by “:” and “.”, respectively. Putative signal peptides are bolded, the ELR motif is boxed in gray and the conserved CXC motif is highlighted. (B) The phylogenetic tree was constructed using the neighbor joining method and bootstrapped 10,000 times (denoted as %s). The accession numbers for the respective protein sequences are: guinea pig CXCL8: NP_001166870.1; naked mole rat CXCL8: XP_004833980; rhesus monkey CXCL8: NP_001028137.1; chimpanzee CXCL8: NP_000575.1; human CXCL8: NP_000575.1; horse CXCL8: NP_001077420.2; hare CXCL8: ALG04568.1; pig CXCL8: NP_999032.1; cow CXCL8: NP_776350.1; zebrafish CXCL8_L2: XP_009305130.1; carp CXCL8_L2: XP_018936341.1; chicken CXCL8: NP_990349.1; painted turtle CXCL8: XP_005304195.1; soft shelled turtle CXCL8: ACP28489.1; alligator CXCL8: XP_006018817.1; kiwi CXCL8: XP_013807042.1; barn owl CXCL8: XP_009963343.1; chimney swift CXCL8: XP_010001929.1; Himalayan frog CXCL8: XP_018421489.1; Western clawed frog CXCL8a: XP_002942578.2; African clawed frog CXCL8a: OCU00045.1; Western clawed frog CXCL8b: XP_002942578.2; African clawed frog CXCL8b: NP_0010912223.1; zebrafish CXCL8_L1: XP_001342606.2; carp CXCL8_L1: XP_016375461; trout CXCL8: XP_020330727.1; salmon CXCL8: NP_001134182.1; haddock CXCL8: CAD97422.2; gilthead seabream CXCL8: AGS55343.1; fugu CXCL8: NP_001027759.1; snapper CXCL8: AGV99968.1; banded houndshark CXCL8: BAB79448.1; whale shark CXCL8: XP_020370926.1; elephant shark CXCL8: NP_001279539.1.
	Figure 2. Analyses of Cxcl8a and Cxcl8b gene expression in healthy and immune challenged animals. (A) Cxcl8a and Cxcl8b gene expression in tadpole and adult kidney (kid), liver (liv), muscle (mus), spleen (spl), skin (sk), bone marrow (bm), and intestine (int) tissues (N = 6). (B) Cxcl8a, Cxcl8b, and Gcsfr gene expression in tadpole and adult kidneys 3 days post FV3 infection (104 PFU/tadpole; 5 × 106 PFU/adult) (N = 5). (C) Cxcl8a and Cxcl8b gene expression in amputated tadpole tails (N = 5). (D) Cxcl8a and Cxcl8b gene expression in adult frog hind leg skin wounds (N = 5). All gene expression was compared relative to Gapdh control and all results are presented as means + SEM. Above-head letters denote statistical designations: experimental groups described by distinct letters are statistically different (P < 0.05) while those marked by the same letters are not.
	Figure 3. Analyses of CXCL8a and CXCL8b gene expression in frog macrophages and granulocytes. (A) Cytology of tadpole rG-CSF-elicited peritoneal granulocytes (G-CSF-Grn) and adult frog bone marrow-derived macrophages, differentiated with rM-CSF (M-CSF-MÏ•) or rIL-34-MÏ•s (L-34-MÏ•) and granulocytes (Grn), differentiated with rG-CSF (G-CSF-Grn). (B) The adult frog M-CSF-MÏ•, IL-34-MÏ•, and G-CSF-Grn were examined for their steady state gene expression of Cxcl8a and Cxcl8b. (C) The adult frog M-CSF-MÏ•, IL-34-MÏ•, and G-CSF-Grn were either mock infected (saline) or challenged with FV3 at a multiplicity of infection of 0.5 PFU/cell. After 24 h of challenge, cells were examined for Cxcl8a and Cxcl8b gene expression relative to Gapdh (N = 5). (D) M-CSF-MÏ•, IL-34-MÏ•, and G-CSF-Gran were mock challenged (saline) or challenged with heat-killed E. coli for 24 h before Cxcl8a and Cxcl8b gene expression analysis, relative to Gapdh (N = 5). All results are presented as means + SEM. Above-head letters denote statistical designations: experimental groups described by distinct letters are statistically different (P < 0.05) while those marked by the same letters are not.
	Figure 4. The rCXCL8a and rCXCL8b elicit distinct chemotaxis responses in tadpole and adult granulocytes. (A,B) Medium or increasing concentrations of rCXCL8a or rCXCL8b were loaded into bottom chemotaxis chamber wells and (A) tadpole or (B) adult frog granulocytes (105 cells/well) were loaded into top wells, separated by 5 μm pore filters. After 3 h of incubation, the filters were stained with Giemsa, mounted bottom side up and the numbers of migrating cells per field of view enumerated. Cells from three individual animals were used for each chemokine concentration (N = 3) and the highest tadpole (10−4 ng/ml of rCXCL8a; 101 ng/ml of rCXCL8b) and adult frog (10−6 ng/ml of rCXCL8a; 10−4 ng/ml of rCXCL8b) granulocyte chemotactic activities were confirmed in two additional, independent experiments and the results combined and presented here (N = 9). Chemokinesis was measured by adding respective concentrations of rCXCL8 or rCXCL8b that elicited maximal cell migration, to both lower and upper chambers. (C) Tadpole and adult frog gene expression of Cxcr1 and Cxcr2 relative to Gapdh. All results are presented as means + SEM. Above-head letters denote statistical designations: experimental groups described by distinct letters are statistically different (P < 0.05) while those marked by the same letters are not.
	Figure 5. Roles of the frog CXCR1 and CXCR2 in the rCXCL8a- and rCXCL8b-elicited chemotaxis. (A) Tadpole and (B) adult frog granulocytes (105 cells/well) were subjected to chemotaxis assays in absence or presence of 1 nM or 100 nM of the CXCR1/CXCR2 inhibitor, reparixin and using the respectively most chemo-attractive concentrations of rCXCL8 or rCXCL8b. Cells from three individual animals were used for each chemokine concentration (N = 3). (C) Tadpole and (D) adult frog granulocytes (105 cells/well) were subjected to chemotaxis assays in absence or presence of 5 or 100 nM of the CXCR2 inhibitor, SB265610 and using the respectively most chemo-attractive doses of rCXCL8 or rCXCL8b. Cells from four individual animals were used for each chemokine concentration (N = 4). All results are presented as means + SEM. Above-head letters denote statistical designations: experimental groups described by distinct letters are statistically different (P < 0.05) while those marked by the same letters are not.
	Figure 6. Analysis of immune gene expression and cytology of rCXCL8a- and rCXCL8b-elicited tadpole cells. Tadpoles and adult frogs were injected intraperitoneally with rCXCL8a or rCXCL8b (1 μg/g of body weight) in 10 μl of saline or with an equal volume of the vector control (supernatants from empty vector-transfected Sf9II cell, processed in parallel to rCXCL8a and rCXCL8b production). After 4 h, animals were lavaged with saline and the cells were enumerated (A). The tadpole (B,C) and adult frog (not presented) rCXCL8a and rCXCL8b-elicited cell immune gene expression was examined relative to the Gapdh control (N = 6). All results are presented as means + SEM. Above-head letters denote statistical designations: experimental groups described by distinct letters are statistically different (P < 0.05) while those marked by the same letters are not. (D) Tadpole rCXCL8a and rCXCL8b-elicited cells were stained with a rabbit anti-frog rG-CSFR (or saline) Ab and secondary goat anti-rabbit Ab and examined by confocal microscopy.
	Supplemental Figure 1. Western blot of the produced, purified, and eluted rCXCL8a and rCXCL8b fractions. The eluted recombinant (r)CXCL8a and rCXCL8b were resolved by SDS PAGE, transferred onto nitrocellulose membranes and western blots were performed using an HRP-conjugated mouse anti-V5 (Sigma) to determine which elution fractions contained rCXCL8a (15 kDa) and rCXCL8b (16 kDa).
	Supplemental Figure 2. Analyses of the anti-G-CSFR polyclonal antibody and the rCXCL8a- and rCXCL8b-elicited granulocyte expression of the Cxcr1 and Cxcr2 genes. (A) Rabbits were immunized with rG-CSFR and the resulting serum was applied to a HiTrap Proetin A HP column (GE Health) to isolate the IgG fraction, and to a rG-CSFR-bound Sulfo-Link Protein column to purify the IgG fraction that cross-reacted with the rG-CSFR. To confirm the specificity of this reagent, this anti-rG-CSFR IgG farction was used to perform a western blot of the rG-CSFR before (lane 1) or after (lane 2) pre-absorbing the Ab against the rG-CSFR. (B) Tadpoles and adult frogs were injected intraperitoneally with rCXCL8a or rCXCL8b (1 μg/g of body weight) in 10 μl of saline or with an equal volume of the vector control. After 4 h, animals were lavaged with saline and the cells were enumerated and examined for their expression of Cxcr1 and Cxcr2. Above-head letters denote statistical designations: experimental groups described by distinct letters are statistically different (P < 0.05) while those marked by the same letters are not.
	cxcl8 expression in macrophages and granulocytes

Auten, Nonpeptide CXCR2 antagonist prevents neutrophil accumulation in hyperoxia-exposed newborn rats. 2001, Pubmed

Auten, Nonpeptide CXCR2 antagonist prevents neutrophil accumulation in hyperoxia-exposed newborn rats. 2001, Pubmed
Baggiolini, Neutrophil-activating peptide-1/interleukin 8, a novel cytokine that activates neutrophils. 1989, Pubmed
Balkwill, Cancer and the chemokine network. 2004, Pubmed
Bayley, Susceptibility of the European common frog Rana temporaria to a panel of ranavirus isolates from fish and amphibian hosts. 2013, Pubmed
Bazzoni, Understanding the molecular mechanisms of the multifaceted IL-10-mediated anti-inflammatory response: lessons from neutrophils. 2010, Pubmed
Bertini, Noncompetitive allosteric inhibitors of the inflammatory chemokine receptors CXCR1 and CXCR2: prevention of reperfusion injury. 2004, Pubmed
Bird, Teleost Chemokines and Their Receptors. 2015, Pubmed
Brinkmann, Neutrophil extracellular traps kill bacteria. 2004, Pubmed
Brugman, T lymphocyte-dependent and -independent regulation of Cxcl8 expression in zebrafish intestines. 2014, Pubmed
Cai, Functional characterization of the ELR motif in piscine ELR+CXC-like chemokine. 2009, Pubmed
Clark-Lewis, Structure-activity relationships of interleukin-8 determined using chemically synthesized analogs. Critical role of NH2-terminal residues and evidence for uncoupling of neutrophil chemotaxis, exocytosis, and receptor binding activities. 1991, Pubmed
Clark-Lewis, Platelet factor 4 binds to interleukin 8 receptors and activates neutrophils when its N terminus is modified with Glu-Leu-Arg. 1993, Pubmed
Corripio-Miyar, Cloning and expression analysis of two pro-inflammatory cytokines, IL-1 beta and IL-8, in haddock (Melanogrammus aeglefinus). 2007, Pubmed
de Oliveira, Cxcl8 (IL-8) mediates neutrophil recruitment and behavior in the zebrafish inflammatory response. 2013, Pubmed
de Oliveira, Cxcl8-l1 and Cxcl8-l2 are required in the zebrafish defense against Salmonella Typhimurium. 2015, Pubmed
DeVries, Defining the origins and evolution of the chemokine/chemokine receptor system. 2006, Pubmed
Dimitriou, Putting out the fire: coordinated suppression of the innate and adaptive immune systems by SOCS1 and SOCS3 proteins. 2008, Pubmed
Fukazawa, Suppression of the immune response potentiates tadpole tail regeneration during the refractory period. 2009, Pubmed , Xenbase
Gallant, Developmental expression profiles and thyroidal regulation of cytokines during metamorphosis in the amphibian Xenopus laevis. 2018, Pubmed , Xenbase
Grayfer, The amphibian (Xenopus laevis) type I interferon response to frog virus 3: new insight into ranavirus pathogenicity. 2014, Pubmed , Xenbase
Harun, The biological effects of rainbow trout (Oncorhynchus mykiss) recombinant interleukin-8. 2008, Pubmed
Hébert, Scanning mutagenesis of interleukin-8 identifies a cluster of residues required for receptor binding. 1991, Pubmed
Hodgkinson, Biology of Bony Fish Macrophages. 2015, Pubmed
Hoverman, Anuran susceptibilities to ranaviruses: role of species identity, exposure route, and a novel virus isolate. 2010, Pubmed
Juffermans, Expression of the chemokine receptors CXCR1 and CXCR2 on granulocytes in human endotoxemia and tuberculosis: involvement of the p38 mitogen-activated protein kinase pathway. 2000, Pubmed
Koubourli, Immune roles of amphibian (Xenopus laevis) tadpole granulocytes during Frog Virus 3 ranavirus infections. 2017, Pubmed , Xenbase
Kulbe, The chemokine network in cancer--much more than directing cell movement. 2004, Pubmed
Landsberg, Co-infection by alveolate parasites and frog virus 3-like ranavirus during an amphibian larval mortality event in Florida, USA. 2013, Pubmed
Li, IL-8 directly enhanced endothelial cell survival, proliferation, and matrix metalloproteinases production and regulated angiogenesis. 2003, Pubmed
Mescher, Inflammation and immunity in organ regeneration. 2017, Pubmed , Xenbase
Mills, Macrophage arginine metabolism to ornithine/urea or nitric oxide/citrulline: a life or death issue. 2001, Pubmed
Morales, Innate immune responses and permissiveness to ranavirus infection of peritoneal leukocytes in the frog Xenopus laevis. 2010, Pubmed , Xenbase
Munn, IDO in the Tumor Microenvironment: Inflammation, Counter-Regulation, and Tolerance. 2016, Pubmed
Peatman, Evolution of CC chemokines in teleost fish: a case study in gene duplication and implications for immune diversity. 2007, Pubmed
Picciocchi, C-terminal engineering of CXCL12 and CCL5 chemokines: functional characterization by electrophysiological recordings. 2014, Pubmed , Xenbase
Raman, Role of chemokines in tumor growth. 2007, Pubmed
Rebl, The proximal promoter of a novel interleukin-8-encoding gene in rainbow trout (Oncorhynchus mykiss) is strongly induced by CEBPA, but not NF-κB p65. 2014, Pubmed
Reeve, Natural stressors and ranavirus susceptibility in larval wood frogs (Rana sylvatica). 2013, Pubmed
Sarris, Inflammatory chemokines direct and restrict leukocyte migration within live tissues as glycan-bound gradients. 2012, Pubmed
Seppola, Characterisation and expression analysis of the interleukin genes, IL-1beta, IL-8 and IL-10, in Atlantic cod (Gadus morhua L.). 2008, Pubmed
Shoeibi, Important signals regulating coronary artery angiogenesis. 2018, Pubmed
Teshima, Neofunctionalization of duplicated genes under the pressure of gene conversion. 2008, Pubmed
van der Aa, CXCL8 chemokines in teleost fish: two lineages with distinct expression profiles during early phases of inflammation. 2010, Pubmed
Waterston, On the sequencing of the human genome. 2002, Pubmed
Yaparla, Differentiation-dependent antiviral capacities of amphibian (Xenopus laevis) macrophages. 2018, Pubmed , Xenbase
Yaparla, The unique myelopoiesis strategy of the amphibian Xenopus laevis. 2016, Pubmed , Xenbase