ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Cxcr3.3 has features of both conventional Cxcr3 receptors and ACKRs. Phylogenetic analyses including CXCR3 (green) and ACKR sequences (blue) of relevant species revealed that Cxcr3.3 is closely related to its paralogs Cxcr3.1 and Cxcr3.2 (A) (ZF, zebrafish; COE, coelacanth; HU, human; MO, mouse; ES, elephant shark; LAM, lamprey) despite having structural features of ACKRs (B), such as an altered E/DRY‐motif (orange) and microswitches (green). The predicted primary ligand‐binding site of both Cxcr3.2 and Cxcr3.3 is highly conserved and structural predictions suggest that they share several ligands (Supplementary Table 2). (C and D) The whole predicted structure of the Cxcr3.2 and Cxcr3.3 receptors (a), the ligand binding site of both proteins (b) and the binding of one of the shared predicted ligands (0NN) by each receptor (c)

cxcr3.3 mutant larvae do not show morphological aberrations or major differences in macrophage development. A 46 bp deletion was induced in the cxcr3.3 gene using CRISPR‐Cas9 technology (A). The deletion is located in the first exon (orange), at the very end of the first TM domain (TM1).The mutation shifts the reading frame and results in a premature stop codon (B). Nonsense‐mediated decay assessment suggests that the cxcr3.3 mutant gene codes a truncated Cxcr3.3 protein (C). No evident morphological aberrations were observed in cxcr3.3–/– larvae within the first 5 dpf and the mutant allele segregated following Mendelian proportions (D). Macrophage development was faster in cxcr3.3–/– embryos at 2 dpf but reverted to WT and cxcr3.2–/– pace after day 3 (E). Fewer macrophages were found in the head area of cxcr3.2–/– larvae only at day 4 (F), whereas there were more macrophages in the tail region in cxcr3.3–/– (G). The cell numbers corresponding to each day are the average of 35 larvae of each of the 3 groups (genotypes). Data were analyzed using a two‐way ANOVA and are shown as mean ± sem (ns P > 0.05, ^*P ≤ 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001, ^****P ≤ 0.0001)

Depletion and overexpression of cxcr3.3 result in opposite M. marinum infection outcomes. Cxcr3.3‐deficient larvae had a higher bacterial burden than their WT siblings at 4 days following blood island (BI) infection with 300 CFU of M. marinum (Mm) (A and B). We transiently overexpressed cxcr3.3 in AB/TL embryos by injection of a CMV: cxcr3.3 construct at 0 hpf and observed that bacterial burden was lower in larvae overexpressing the gene than in noninjected controls at 4 dpi (C and D). To rescue the cxcr3.3–/– phenotype, we restored the expression of the gene by transiently overexpressing it (CMV: cxcr3.3) in one‐half of the cxcr3.3 mutants (cxcr3.3–/– rescued). The bacterial burden was lower in the rescued group than in noninjected cxcr3.3 mutants (cxcr3.3–/–) and similar to the bacterial burden in WT controls (E and F). Results from qPCR show that cxcr3.2 expression remained unaltered in the cxcr3.3 mutants and that it was induced upon infection (G), whereas cxcr3.3 expression was lower in cxcr3.2–/– and was moderately induced upon Mm infection (H). The ligand cxcl11aa was induced upon infection independently of any of the cxcr3 genes. In all cases, systemic infection was done at 28 hpf in the BI with 300 CFU of Mm. The bacterial burden data were analyzed using a two‐tailed t‐test (A–C) and a one‐way ANOVA (E). Results are shown as mean ± sem (ns P > 0.05, ^*P ≤ 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001, ^****P ≤ 0.0001) and combine data of 3 independent replicates of 20–30 larvae each. The qPCR data were analyzed with the 2^–∆∆Ct method and a one‐way ANOVA. Results are plotted as mean ± sem (ns P > 0.05, ^*P ≤ 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001)

Macrophages lacking Cxcr3.3 efficiently clear IC bacteria. Cxcr3.3‐deficient larvae and their WT siblings were infected in the BI at 28 hpf with 200 CFU of the ΔERP M. marinum‐wasabi strain that is unable to survive and replicate inside acidic compartments and can be easily cleared by macrophages. The total number of bacterial clusters in every fish was quantified (A). We divided the bacterial clusters into 3 groups based on the number of bacteria they contained (1–5, 1–6, and >10) to assess bacterial clearance at 44 hpi (B). No difference between WT and cxcr3.3–/– cluster size distributions (frequency in %) was found (C). A Mann‐Whitney test was conducted to analyze the overall bacterial burden of the pooled data of 3 independent replicates of 9 fish each. Data are shown as mean ± sem (A). A Kolmogorov‐Smirnov test was used to analyze the distribution of bacterial cluster sizes (C) (ns P > 0.05)

Macrophages lacking Cxcr3.3 show an enhanced recruitment to sites of infection, toward Cxcl11aa, and to sites of mechanical damage. Significantly fewer cells were recruited to the hindbrain ventricle in cxcr3.2–/– at 3 hpi with Mm and more macrophages were recruited to the same site in cxcr3.3–/– compared to WT controls (A and B). The same trend was observed when 1 nl of Cxcl11aa protein (10 ng/ml) was injected in the same experimental setup (C and D). To assess macrophage recruitment to sites of injury, we used the tail‐amputation model and observed enhanced recruitment of macrophages in cxcr3.3–/– larvae and attenuated recruitment of macrophages in cxcr3.2–/– relative to WT at 4 h postamputation (E and F). The PBS‐injected control group combines WT, cxcr3.2, and cxcr3.3 mutants and shows no cell recruitment at 3 hpi. In all cases, statistical analyses were done with pooled data of 3 independent replicates (20–30 larvae per group each). A Kruskal‐Wallis test was used to assess significance (^*P ≤ 0.05, ^***P ≤ 0.001, ^****P ≤ 0.0001) and data are shown as mean ± sem

Neutrophil recruitment to sites of infection and injury is not altered in cxcr3.3 mutants. A total of 100 CFU of Mm‐mCherry were injected in the hb ventricle of 2 day‐old WT, cxcr3.2, and cxcr3.3 mutant larvae to assess neutrophil (mpx: eGFP) recruitment to the infection site at 3 hpi. The number of cells that infiltrated the cavity was lower in cxcr3.2 mutants but remained unchanged in WT and cxcr3.3 mutants (A and B). The tail fin of WT larvae and cxcr3.2 and cxcr3.3 mutants was amputated and neutrophil recruitment was assessed at 4 h postamputation. There were fewer recruited neutrophils in the cxcr3.2 mutants, whereas there was no difference between cxcr3.3 mutants and WT. The PBS‐injected control group PBS combines WT, cxcr3.2, and cxcr3.3 mutants and shows no cell recruitment at 3 hpi. In all cases, statistical analyses were done with pooled data of 3 independent replicates (20–30 larvae per group each). A Kruskal‐Wallis test was used to assess significance (ns P > 0.05, ^***P ≤ 0.001, ^****P ≤ 0.0001) and data are shown as mean ± sem

Cxcr3.3‐depleted macrophages move faster than WT cells under basal conditions and upon mechanical damage and have a lower CI. Panel A shows representative images of tracks of macrophages of 3‐day‐old larvae from the 3 genotypes under unchallenged conditions (random patrolling). Macrophages were tracked for 3 h and images were taken every 2 min. Graphs in B show the total displacement of all cells tracked shortly after amputation in each group throughout 3 h (B‐2) and the average speed of each cell (B‐2). In this case, macrophages were tracked for 1.5 h and images were acquired every 1 min. There was no significant difference between the groups in terms of total cell displacement (B‐1.), however cxcr3.3–/– macrophages did move faster than the remaining groups as indicated by the dot‐plots in (B‐2.). Panel C shows representative images of macrophage tracks of the 2 groups directly after a tail amputation. The tracks of cxcr3.2–/– macrophages were shorter than those of the remaining groups (D‐1.) and cxcr3.3–/– macrophages moved faster than the other 2 groups when mechanical damage was inflicted (D‐2.). Data of unchallenged larvae were collected from 2 independent replicates (5 larvae per group each) and for the tail‐amputation model data from 3 independent replicates (4 larvae per group each) were pooled together for analysis One‐way ANOVA was performed to test for significance (ns P > 0.05, ^*P ≤ 0.05, ^**P ≤ 0.01) and data are shown as mean ± sem. The CI distributions of both cxcr3.2–/– and cxc3.3–/– differ from the WT control but are skewed in opposite directions as low CI values are more frequent in cxcr3.3 mutants than in WT and high CI values are more frequent in cxcr3.2 mutants as shown by the curves (E). Panel F shows representative images of the most frequent CI interval in each group and the bar displays the percentage of each CI category within each genotype. A Kolmogorov‐Smirnov test was used to evaluate the CI value distributions using the WT data as reference distribution (^**P ≤ 0.01, ^****P ≤ 0.0001)

Cxcr3.3‐depleted macrophages move faster than WT cells under basal conditions and upon mechanical damage and have a lower CI. Panel A shows representative images of tracks of macrophages of 3‐day‐old larvae from the 3 genotypes under unchallenged conditions (random patrolling). Macrophages were tracked for 3 h and images were taken every 2 min. Graphs in B show the total displacement of all cells tracked shortly after amputation in each group throughout 3 h (B‐2) and the average speed of each cell (B‐2). In this case, macrophages were tracked for 1.5 h and images were acquired every 1 min. There was no significant difference between the groups in terms of total cell displacement (B‐1.), however cxcr3.3–/– macrophages did move faster than the remaining groups as indicated by the dot‐plots in (B‐2.). Panel C shows representative images of macrophage tracks of the 2 groups directly after a tail amputation. The tracks of cxcr3.2–/– macrophages were shorter than those of the remaining groups (D‐1.) and cxcr3.3–/– macrophages moved faster than the other 2 groups when mechanical damage was inflicted (D‐2.). Data of unchallenged larvae were collected from 2 independent replicates (5 larvae per group each) and for the tail‐amputation model data from 3 independent replicates (4 larvae per group each) were pooled together for analysis One‐way ANOVA was performed to test for significance (ns P > 0.05, *P ≤ 0.05, **P ≤ 0.01) and data are shown as mean ± sem. The CI distributions of both cxcr3.2–/– and cxc3.3–/– differ from the WT control but are skewed in opposite directions as low CI values are more frequent in cxcr3.3 mutants than in WT and high CI values are more frequent in cxcr3.2 mutants as shown by the curves (E). Panel F shows representative images of the most frequent CI interval in each group and the bar displays the percentage of each CI category within each genotype. A Kolmogorov‐Smirnov test was used to evaluate the CI value distributions using the WT data as reference distribution (**P ≤ 0.01, ****P ≤ 0.0001)

Enhanced motility of cxcr3.3 mutant macrophages facilitates bacterial dissemination. Four days after local infection with 200 CFU of Mm in the hb, cxcr3.3 mutants developed more distal granulomas (22%) than WT (12.7%) and cxcr3.2 mutants (5%), whereas the latter developed fewer than the other 2 groups (A). Embryos from the 3 genotypes were infected at 28 hpf and imaged under the stereo fluorescence microscope (whole body and zoom to the tail) at 4 dpi. Panel B illustrates the imaging process of a representative cxcr3.3 mutant larvae. Cxcr3.3‐depleted larvae developed more distal granulomas per fish (C) and these granulomas were also larger in cxcr3.3 mutants than the other 2 groups, whereas cxcr3.2 mutants showed an opposite trend (D). Graphs show pooled data from 4 independent replicates, each of 12–15 infected larvae per group. The number and size of distal granulomas were determined using the “analyze particle” function in Fiji. A χ² test was conducted to assess differences in the proportion of larvae that developed distal granulomas within the 3 groups and a one‐way ANOVA to compare the number and size of distal granulomas (ns P > 0.05, ^*P ≤ 0.05, ^***P ≤ 0.001 and ^****P ≤ 0.001). Data are shown as mean ± sem

Chemical inhibition of both Cxcr3 receptors affects only macrophages expressing Cxcr3.2 and renders a similar bacterial burden and macrophage recruitment efficiency as cxcr3.2 mutants. After bath exposure of 3‐day‐old larvae to either the CXCR3‐specific inhibitor NBI 74330 (50 µM) or vehicle (DMSO 0.01%), before and after tail‐amputation showed that cell recruitment to the site of injury was reduced in macrophages expressing Cxcr3.2, namely, WT and cxcr3.3–/– (A and C), whereas no further decline in cell recruitment was observed in cxcr3.2 mutants (B and D). Chemical inhibition of both Cxcr3 receptors with NBI 74330 (25 µM) before and after systemic infection with Mm resulted in a lower bacterial burden at 4 dpi than in the vehicle‐treated control and resembles the cxcr3.2 mutant phenotype (E and F). The data of 3 independent replicates were pooled and are presented as mean ± sem. A Kruskal‐Wallis test was conducted to assess significance (ns P > 0.05, ^****P ≤ 0.0001) in the macrophage recruitment assay. Only the P‐values among each condition (vehicle/NBI 74330) within each group are shown (D). Bacterial burden data were analyzed using a two‐tailed t‐test and data are shown as mean ± sem (^****P ≤ 0.0001)