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. 2016 Feb;9(2):187-98.
doi: 10.1242/dmm.021881. Epub 2015 Dec 17.

Innate immune responses to gut microbiota differ between oceanic and freshwater threespine stickleback populations

Affiliations

Innate immune responses to gut microbiota differ between oceanic and freshwater threespine stickleback populations

Kathryn Milligan-Myhre et al. Dis Model Mech. 2016 Feb.

Abstract

Animal hosts must co-exist with beneficial microbes while simultaneously being able to mount rapid, non-specific, innate immune responses to pathogenic microbes. How this balance is achieved is not fully understood, and disruption of this relationship can lead to disease. Excessive inflammatory responses to resident microbes are characteristic of certain gastrointestinal pathologies such as inflammatory bowel disease (IBD). The immune dysregulation of IBD has complex genetic underpinnings that cannot be fully recapitulated with single-gene-knockout models. A deeper understanding of the genetic regulation of innate immune responses to resident microbes requires the ability to measure immune responses in the presence and absence of the microbiota using vertebrate models with complex genetic variation. Here, we describe a new gnotobiotic vertebrate model to explore the natural genetic variation that contributes to differences in innate immune responses to microbiota. Threespine stickleback, Gasterosteus aculeatus, has been used to study the developmental genetics of complex traits during the repeated evolution from ancestral oceanic to derived freshwater forms. We established methods to rear germ-free stickleback larvae and gnotobiotic animals monoassociated with single bacterial isolates. We characterized the innate immune response of these fish to resident gut microbes by quantifying the neutrophil cells in conventionally reared monoassociated or germ-free stickleback from both oceanic and freshwater populations grown in a common intermediate salinity environment. We found that oceanic and freshwater fish in the wild and in the laboratory share many intestinal microbial community members. However, oceanic fish mount a strong immune response to residential microbiota, whereas freshwater fish frequently do not. A strong innate immune response was uniformly observed across oceanic families, but this response varied among families of freshwater fish. The gnotobiotic stickleback model that we have developed therefore provides a platform for future studies mapping the natural genetic basis of the variation in immune response to microbes.

Keywords: Animal model; Fish; Germ-free; Gnotobiotic; Neutrophils; Stickleback.

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Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Bacterial isolates collected from lab-raised and wild-caught stickleback. Bacteria were isolated from the guts of lab-raised Alaskan adult stickleback and wild-caught Oregon freshwater and oceanic populations. (A) Colony types were based on physical characteristics like color, size and morphology. Representative plate shown. Red arrows point to five different colony types. (B) Relative abundance was estimated based on the number of colonies present in diluted samples. Colonies were identified based on sequencing of the 16S ribosomal RNA gene. Identification to the level of genus is provided. Colony types in the same genus that have different phenotypes or species identification are given letters. Colony types that had 16S sequences that could belong to two different genera are listed as such. Unknown indicates that the colony type could not be identified by 16S sequencing. Dark blue represents the most-abundant type in a population, light blue is the least abundant, and white squares indicate the absence of the colony type in that population. Oc, oceanic; F, freshwater.
Fig. 2.
Fig. 2.
Gut colonization and gnotobiotic manipulation. (A) Gut colonization was determined by plating dissected guts daily between 8 and 14 dpf. The average percent of gut colonized from each experiment are reported. Oc, oceanic stickleback (blue line); F, freshwater stickleback (green line). (B) Sterilization of germ-free (GF) stickleback eggs was confirmed with in situ hybridization with a pan-bacterial probe (red) and other methods described in the text; conventional (CV) fish shown for comparison. DAPI-stained DNA (blue) indicates epithelial gut cells. (C) Representative 14 dpf live oceanic fish colonized with Pseudomonas sp. isolate from a stickleback gut engineered to express red fluorescent protein (KMM0021:RFP). Top panel: differential interference contrast (DIC) image; middle panel: red channel; bottom panel: merged image of the DIC and red channel.
Fig. 3.
Fig. 3.
Stickleback gut development differs between populations but is not generally affected by the presence of microbes. (A) Scoring of the gut at 14 dpf. Representative images of scored guts are shown. Score 1=little to no intestinal folds; 2=small, undeveloped folds that are unevenly distributed; 3=unevenly distributed folds; 4=fully developed, evenly distributed folds. Scale bars: 25 μm. (B) Graph indicates the proportion of fish in each treatment with gut complexity levels 1 through 4 for conventional (CV), germ-free (GF) or monoassociated with Pseudomonas stickleback gut isolate KMM0021 (MA) for oceanic (Oc) and freshwater (F) populations. On average, Oc fish (blue groups) demonstrated lower complexity scores than F fish (green groups) (P=0.007). The only significant microbiota-related effect was a statistical interaction with host population in which complexity is higher in Oc-MA relative to Oc-GF fish but lower in F-MA relative to F-GF fish (P=0.02). Total numbers of fish scored per group are indicated below the group names. Lines above the figure indicate comparisons with differences in gut development, where the color of the line indicates the groups that were compared, and the width of the line indicates the direction and magnitude in the odds ratio for each comparison.
Fig. 4.
Fig. 4.
Oceanic populations mount a robust innate immune response to microbiota, whereas freshwater populations do not. (A) To examine the innate immune response to microbiota, myeloperoxidase-positive neutrophils (MPO+) were stained and counted. MPO+ cells are brown. Red arrows indicate black pigment at the periphery of the intestine; blue arrows indicate representative MPO+ cells within the intestinal epithelium. Oc, oceanic; CV, conventional; GF, germ-free. (B) The number of MPO+ cells per gut were counted per fish. Colored dots represent the number of MPO+ cells in individual fish, and boxplots represent distributions within conventional (CV), germ-free (GF) and Pseudomonas isolate KMM0021 monoassociation (MA) treatments. In Oceanic fish, the number of MPO+ cells differs across the three microbe treatments, as indicated by the non-overlapping letter groupings ‘A’, ‘B’ and ‘C’. In freshwater fish, all three microbe treatments are equivalent regarding MPO+ cell number (all three letter groupings overlap). This population-specific microbial effect on neutrophil abundance is reflected by a statistical interaction (P<0.0001). See text for details. (C) Neutrophil marker mpx transcript levels from an RNA-seq experiment involving CV and MA fish demonstrate the same population-by-microbiota interaction (P=0.005). Colored dots represent mpx expression levels in guts of individual fish (n=4 fish per population-treatment combination), and boxplots show within-group distributions.
Fig. 5.
Fig. 5.
Neutrophil differences in response to microbiota presence is stable between oceanic families, but variable between freshwater families. Ten oceanic and seven freshwater families were raised in the presence (CV) or absence (GF) of microbes. The number of MPO+ cells per fish was counted. Individual counts are represented by dots; bars on boxplots indicate median value, boxes represent first through third quartiles, and vertical lines represent outlier fences. For all oceanic families, a difference in MPO+ cells is observed in CV versus GF fish. In five of the seven freshwater families there is no difference in the number of MPO+ cells in CV versus GF fish, but in two families there is a difference. Red asterisks denote families in which the P-value is less than 0.05 for the number of MPO+ cells between CV and GF treatments. Families examined in the same experiment are denoted by the same number (i.e. Oceanic family 1 and Freshwater family 1). Diagram in the lower left corner depicts the crossing of a single male and a single female from the oceanic or freshwater population that resulted in a single family.

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