1. Critical Role for a Subset of Intestinal Macrophages in Shaping Gut Microbiota in Adult Zebrafish 
Alison M. Earley, Christina L. Graves, Celia E. Shiau 
Cell Reports 
Volume 25, Issue 2, Pages (October 2018)
DOI: /j.celrep
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2. Cell Reports 2018 25, 424-436DOI: (10.1016/j.celrep.2018.09.025)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1
Adult irf8−/− Zebrafish Have a Severe Loss of Tissue-Resident Intestinal and Brain Macrophages but Normal Numbers of Peripheral Macrophages
(A) Image analysis was performed on whole-mount gut, brain, and fin dissected from transgenic adult zebrafish expressing the macrophage-specific reporter mpeg1:GFP. Left column shows images of dissected organs and the general region from which several areas were quantified and analyzed (dotted box). Each image represents a quantified 0.045 mm2 field of view. High-magnification insets show an inverted image of a GFP+ macrophage. No clear microglia were detected in irf8 mutant brains. Scale bars in GFP image panels are 50 μm and in insets are 10 μm.
(B) Scatterplot shows macrophage number per field of view in each tissue region. Numbers below plot represent the number of areas analyzed from 4 or more animals.
Statistical significance was determined by a two-tailed t test. ∗∗p < 0.01, ∗∗∗∗p < 0.0001; n.s., not significant. See also Figure S1.
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4. Figure 2
Significant Reduction of Intestinal Macrophages but No Change in Neutrophil Number in irf8−/− Mutants
(A) Representative maximum intensity projections of confocal z stacks of the adult S7 intestinal region. Double transgenic irf8 mutants and siblings carrying the macrophage reporter mpeg1:GFP (green) and neutrophil reporter lyz:mCherry (red) were analyzed.
(B) Schematic of the S1–S7 segmentation of the adult zebrafish intestine. The green square inside of S7 represents the approximate region where images in (A) were taken.
(C) Quantification of the number of macrophages (mpeg1+) from a representation of all intestinal segments S1–S7. ∗p < 0.05.
(D) Quantification of the number of neutrophils (lyz+) from a representation of all intestinal segments S1–S7.
(E) qPCR analysis of neutrophil marker mpx expression shows no significant difference between irf8 mutants and siblings.
3 or more independent animals were used per mutant and sibling groups for all quantifications. Error bars show SEM. Student’s t test was used to determine statistical significance. SIB, wild-type and heterozygous siblings.
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5. Figure 3 Significant Gut Microbiota Alteration in irf8−/− Mutants
(A) Three-dimensional principal coordinates analysis (PCoA) plot showing significantly different gut microbial communities in the irf8 mutants (n = 8 animals) compared with their siblings (n = 10 animals) using the jackknifed unweighted pair-group method of analysis (UPGMA) clustering based on weighted UniFrac distances of 16S rRNA gene sequences. Each sphere represents a gut microbial community from an independent animal. Statistical significance comparing all 3 genotype groups was determined by a p of using the PERMANOVA test with 1,000 permutations.
(B) Hierarchical jackknifed UPGMA clustering of the different genotypes based on relative bacterial class abundance as analyzed by 16S rRNA gene sequencing. irf8st95/st95 mutants (asterisk) have an aberrant expansion of Deltaproteobacteria (rose pink) at the expense of Alphaproteobacteria (purple), Fusobacteriia (light blue), and Gammaproteobacteria (dark green). Each bar represents an individual fish gut. Scale bar shows substitutions per site. Confidence level is shown at the nodes using a sampling depth of 7,000 sequences.
(C) qPCR validation of the microbial shift in irf8 mutants. Statistical significance was determined by a two-tailed t test. ∗∗∗p <
(D) Diagram summarizing the dysbiosis in irf8 mutants.
See also Figures S2 and S3 and Table S1.
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6. Figure 4
RNA-Seq Reveals Gut Transcriptomic Changes in irf8−/− Mutants but No Apparent Intestinal Structural Defects
(A) Volcano plot showing fold change and the statistical significance of all genes analyzed by RNA-seq; each dot represents a different gene. Significantly altered genes with a false discovery rate (FDR) adjusted p ≤ 0.05 are highlighted in red.
(B) Interferon-responsive genes and immune-related genes were prominent groups significantly altered. Unknown represents uncharacterized genes without an informative gene name. Other represents genes of diverse functions and pathways that do not belong in a common category. Pancreas and/or intestine represents genes that are known to be expressed or have functions in pancreas, intestine, or both.
(C) Heatmaps listing the downregulated and upregulated genes. Two independent biological samples per genotype are represented. Color range from −3 to +3 is based on normalized fragments per kilobase of transcript per million mapped reads (FPKM) values using relative deviation per gene over all samples centered on 0.
(D) Differentially expressed gene list generated from DESeq2 using a broader cutoff of a p < 0.05 was used to determine significantly changed pathways in the PANTHER database. Number of genes identified in each pathway is shown in parentheses.
(E and F) Adult intestinal morphology was assessed by (E) H&E and (F) AB-PAS staining on 5-μm sections of the proximal gut regions (S1 and S2).
See also Figures S4 and S5 and Table S2.
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7. Figure 5
irf8−/− Guts Have a Significant Deficiency in Expression of Complement C1q Genes
(A) Relative RNA expression levels by qPCR of C1q genes, alkaline phosphatase 3 (alp3), and irf1b in whole gut of irf8 mutants (red) and control siblings (blue). N = 3–12 independent gut samples per bar graph. Statistical significance determined by two-tailed t test and FDR-adjusted p value. ∗∗∗p < ∗p < 0.05.
(B) Relative levels of target genes of interest by qPCR in FACS-sorted macrophages (mpeg1: GFP+) from wild-type whole gut compared with remaining cells that were GFP negative. Each bar represents an average expression from two independent experiments.
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8. Figure 6
Mosaic Rescue of Macrophages in irf8−/− Mutants Is Sufficient to Restore Commensal Microbiota and Complement c1q Expressions
(A) Schematic illustrating the genetic constructs used to generate rescue of macrophages in irf8 mutants.
(B) In contrast to the negative controls (uninjected irf8 mutants), which have no brain macrophages, several brain macrophages (arrows) are recovered in the pu.1:Gal4-UAS-GFP+/UAS-irf8-injected embryos.
(C) pu.1:Gal4-UAS-GFP+/UAS-irf8-injected embryos were raised to adulthood and were found to exhibit recovery of intestinal macrophages (arrows) in the irf8 mutant adult guts. Images show gut segment S7 visualized from the lumen side.
(D) Relative abundance of gut microbes in the adult intestine was assayed by qPCR in irf8 mutants and their siblings with the rescue construct and at baseline without the rescue construct.
(E) Comparison of relative bacterial levels between irf8 mutants and macrophage-rescued irf8 mutants.
(F) Fold difference in target c1q genes. Irf8 mutants with the rescue construct were compared to baseline irf8 mutants (control data are represented from Figure 5A). Each symbol represents an individual animal.
Scale bars show 50 μm. Statistical significance was determined by a Student’s t test. 3 or more animals per group were analyzed for all experiments. Error bars show SEM. GH, cmlc2:GFP expression (GFP+ heart). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < mut, mutant; sib, heterozygous or WT sibling; WT, wild-type. See also Figures S6 and S7 and Table S3.
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9. Figure 7
Proposed Function of irf8 in Intestinal Macrophages for Shaping the Gut Microbiota
Two subsets of intestinal macrophages can be classified based on their differential requirement for irf8 to establish in the intestine: most intestinal macrophages (purple) are “irf8 dependent,” as they are eliminated in the absence of irf8, and “irf8-independent” macrophages (lavender) remain in the irf8 knockout zebrafish, although they may be functionally regulated by irf8. For example, irf8 may directly or indirectly activate the transcription of c1q genes in both macrophage subsets. The intestinal macrophages may be the primary source of C1q production important for preventing outgrowth of rare or opportunistic bacteria, thereby influencing the assembly and maintenance of the gut commensal microbiota.
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