Interleukin 10 mutant zebrafish have an enhanced interferon gamma response and improved survival against a Mycobacterium marinum infection

1 SCIEnTIFIC RepORts | (2018) 8:10360 | DOI:10.1038/s41598-018-28511-w www.nature.com/scientificreports Interleukin 10 mutant zebrafish have an enhanced interferon gamma response and improved survival against a Mycobacterium marinum infection Sanna-Kaisa E. Harjula1, Markus J. T. Ojanen1,2, Sinja Taavitsainen3, Matti Nykter3 & Mika Rämet1,4,5,6 Tuberculosis ranks as one of the world’s deadliest infectious diseases causing more than a million casualties annually. IL10 inhibits the function of Th1 type cells, and IL10 deficiency has been associated with an improved resistance against Mycobacterium tuberculosis infection in a mouse model. Here, we utilized M. marinum infection in the zebrafish (Danio rerio) as a model for studying Il10 in the host response against mycobacteria. Unchallenged, nonsense il10e46/e46 mutant zebrafish were fertile and phenotypically normal. Following a chronic mycobacterial infection, il10e46/e46 mutants showed enhanced survival compared to the controls. This was associated with an increased expression of the Th cell marker cd4-1 and a shift towards a Th1 type immune response, which was demonstrated by the upregulated expression of tbx21 and ifng1, as well as the down-regulation of gata3. In addition, at 8 weeks post infection il10e46/e46 mutant zebrafish had reduced expression levels of proinflammatory cytokines tnfb and il1b, presumably indicating slower progress of the infection. Altogether, our data show that Il10 can weaken the immune defense against M. marinum infection in zebrafish by restricting ifng1 response. Importantly, our findings support the relevance of M. marinum infection in zebrafish as a model for tuberculosis. Annually more than 10 million new tuberculosis cases are estimated to emerge, leading to over a million casualties1. The immune defense against the pathogen which causes tuberculosis, Mycobacterium tuberculosis, requires the elaborate collaboration of both the innate and adaptive immunity, as is demonstrated by the increased disease susceptibility in recipients of TNF-antagonist as well as in HIV-positive individuals1–3. Accordingly, deficient, but also excessive, macrophage mediated TNF production as well as the lack of T helper (Th) 1 type cell responses compromise the host’s ability to resist a mycobacterial infection and accelerate the disease pathogenesis4–7. IL10 is an anti-inflammatory cytokine that was originally identified as a protein secreted by Th2 cells able to inhibit cytokine production in Th1 cells8,9. Later, it was discovered that several other cell types, including both immune and nonimmune cells, produce IL1010. Genome-wide association studies in humans have linked IL10 polymorphisms to susceptibility and resistance towards tuberculosis, although the results vary depending on the polymorphism studied and the study subjects11,12. Furthermore, in vivo mouse studies have shown that IL10 impairs the immune defense against M. tuberculosis by impeding host immunity at an early13,14, but also during later stages of an infection15. In these studies, the lack of functional IL10 signaling resulted in enhanced protection 1Laboratory of Experimental Immunology, BioMediTech Institute and Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland. 2Laboratory of Immunoregulation, BioMediTech Institute and Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland. 3Laboratory of Computational Biology, BioMediTech Institute and Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland. 4Department of Pediatrics, Tampere University Hospital, Tampere, Finland. 5Department of Children and Adolescents, Oulu University Hospital, Oulu, Finland. 6PEDEGO Research Unit and Medical Research Center Oulu, University of Oulu, Oulu, Finland. Correspondence and requests for materials should be addressed to M.R. (email: [email protected]) Received: 22 August 2017 Accepted: 20 June 2018 Published: xx xx xxxx OPEN www.nature.com/scientificreports/ 2 SCIEnTIFIC RepORts | (2018) 8:10360 | DOI:10.1038/s41598-018-28511-w against mycobacteria and was attributed to an increased Th1 response13–16. As a consequence, for example, macrophages were more capable of presenting M. tuberculosis antigens and recruiting inflammatory cells13,14. Enhanced protection was demonstrated by a lower bacterial burden in the lungs and spleen13–15 and improved survival of the mice15. While IL10 deficiency or receptor blockade has been associated with an enhanced protection against mycobacteria in mice, it has also been reported that the lack of IL10 can eventually lead to harmful lung inflammation and to the progression of a mycobacterial disease in the mouse model16. The zebrafish is a small teleost, which is constantly gaining popularity as a model organism. The cellular components of the zebrafish immune system, such as mononuclear phagocytes17, dendritic cells18, T cells and B cells19–23 and eosinophils24 have been described and they resemble those of humans. To date, specific transcription factors expressed by different zebrafish Th as well as Treg cells have also been identified and characterized25,26. Previously, lymphocyte marker gene expression has been used to characterize immune response in adult zebrafish27 and for example foxp3a has been validated as a Treg marker28. As for the humoral components of the zebrafish immune system, the mammalian homologs of the complement system29, as well as the immunoglobulin isotypes IgD, IgM and the bony fish specific immunoglobulin Z/T30 have been found. Overall, the zebrafish is a suitable model for immunological research (reviewed in31). Mycobacterium marinum is a natural pathogen of the zebrafish and a close relative of Mycobacterium tuberculosis32. Comparably to M. tuberculosis, M. marinum infects macrophages33,34 and eventually causes a systemic disease in zebrafish, which shares pathological and histological features with human tuberculosis35–37. A M. marinum infection model in zebrafish larvae has been widely used to study the innate immune response in a mycobacterial infection, and it enables the real-time visualization and rapid screening of potential tuberculosis drugs38–40. Furthermore, the M. marinum infection model in adult zebrafish allows studying the adaptive response35–37. Zebrafish il10 has a mammalian-like gene organization and conserved IL10 signature motif 41,42. Furthermore, Grayfer and Belosevic43 have found IL10 receptor 1 in zebrafish and in goldfish (Carassius Auratus L.). Among their analyses, an alignment of these protein sequences with those of other vertebrates, the expression measurements in different tissues and immune cell populations at mRNA level and in vitro binding studies of recombinant goldfish IL10 receptor 1 and IL10 proteins, indicated conservation of the IL10 system throughout evolution. In order to study the role of Il10 in the immune defense against mycobacteria, we have here characterized an il10e46/e46 mutant zebrafish strain in relation to a M. marinum infection. Also, we aim to gain more information about M. marinum infection in zebrafish as a model for human tuberculosis. Results A nonsense il10e46 mutation creates an early stop codon in the zebrafish il10 gene. In order to study Il10 in the host response against mycobacteria the zebrafish line e46, carrying a nonsense il10 mutation, was obtained from the Wellcome Trust Sanger Institute44. In the il10e46 mutant zebrafish a specific adenosine (A) to thymidine (T) point mutation results in a stop codon (TAA) after the first 27 amino acids in the translated region of exon 1 (Fig.1a). As nonsense mediated decay degrades mRNA molecules producing non-functional proteins45, we first studied if the il10e46 mutation affects the levels of the il10 mRNA, and quantified the expression of il10 in different organs of the abdominal cavity by quantitative PCR (qPCR) (Fig.1b). However, in any of the studied tissues il10 mRNA expression did not differ between il10e46/e46 mutants and wild type (WT) zebrafish, suggesting that the effects of the mutation are only evident at the translational level. In fact, signal peptide prediction using SignalP 4.1 Server46 revealed that only five amino acids remain in the truncated protein, which consequently prevents the normal function of Il10 in the fish carrying the homozygous il10e46 mutation (Fig.1a). Unchallenged il10e46/e46 zebrafish are phenotypically normal and have similar blood cell populations and cytokine expression profiles compared to WT fish. IL10 knock-out (KO) mice have growth defects and suffer from chronic intestinal inflammation leading to 30% mortality before 3 months of age47,48. Like IL10 KO mouse strains, il10e46/e46 zebrafish are fertile and can be maintained by spawning homozygous mutant siblings47. However, in contrast to mice, il10e46/e46 mutant fish are phenotypically normal and do not have increased mortality compared to WT zebrafish. Our flow-cytometric analysis of the blood cell populations in kidney blood cell isolates revealed no differences in live cell, lymphocyte, myeloid cell or blood cell precursor cell counts in unchallenged il10e46/e46 zebrafish compared to the WT control fish (Fig.2a,b). Colitis in IL10 KO mice is attributed to the increased production of inflammatory mediators such as TNF and IL1b as well as to a hyper-activated Th1 response47,49,50. In order to study signs of inflammation and T cell homeostasis in unchallenged il10e46/e46 zebrafish, we extracted RNA from different adult zebrafish tissues and measured the expression levels of selected proinflammatory cytokines (il1b, tnfa and tnfb), Th cell cytokines (ifng1 and il4) as well as T cell markers cd4-1 and cd8a) and a B cell marker IgM (Supplementary Fig.S1). In contrast to the IL10 KO mice, qPCR analysis from the liver and spleen revealed no differences in the relative expression levels of the studied inflammatory markers tnfa, tnfb and il1b between il10e46/e46 and WT fish. The expression levels of the Th1 cytokine ifng1 and the Th2 cytokine il4 were also comparable between the fish groups. In addition, no differences in the mRNA expression of the T cell marker genes cd8a and cd4-1, between il10e46/e46 and WT fish, were observed. The expression of IgM in the liver of il10e46/e46 fish was decreased compared to controls (P = 0.029). Collectively, these data indicate that the il10e46/e46 mutant zebrafish have no apparent immune abnormalities under unchallenged conditions. In order to study in more detail the possible inflammation in the intestine of the Il10 deficient zebrafish, we collected the intestine from 1-year-old WT and il10e46/e46 fish and measured the expression levels of several inflammatory cytokines and immune cell markers (Supplementary Fig.S2a). No difference in the expression of the inflammatory cytokines (il1b, tnfa and tnfb), T lymphocyte markers (cd4-1 and cd8a), B lymphocyte marker IgM, Th2 and Treg markers, (gata3 and foxp3a, respectively), or in Th1 and Th2 hallmark cytokines (ifng1 and il4, www.nature.com/scientificreports/ 3 SCIEnTIFIC RepORts | (2018) 8:10360 | DOI:10.1038/s41598-018-28511-w or in il10) was observed between WT and il10e46/e46 fish. The expression of Th1 cell marker tbx21 was higher in WT fish compared to il10e46/e46 mutants (P = 0.023) but this is unlikely a sign towards colitis in il10e46/e46 mutants. Furthermore, the visual appearance of WT and il10e46/e46 intestine was similar with identifiable intestinal bulb, mid-intestine and posterior-intestine (Supplementary Fig.S2b). Survival of adult il10e46/e46 mutants is improved in a low-dose M. marinum infection. In mice, the absence of functional IL10 signaling leads to the improved control of a M. tuberculosis infection, and consequently, to better survival13,15. Other studies have, however, associated IL10 deficiency to increased inflammation leading to reduced survival16. To study the role of Il10 in the immune defense against a M. marinum infection in zebrafish, we first infected il10e46/e46 and WT embryos into the yolk at 0–6 hours post fertilization and followed the survival of the larvae for seven days (Fig.3a). After three days, mortality was observed in both of the groups resulting in ca. 70% mortality at the end of the follow-up with no significant difference between il10e46/e46 and WT larvae. Next, we injected a low-dose of M. marinum into the abdominal cavity of adult il10e46/e46 and WT zebrafish and followed the survival of the fish for 20 weeks (Fig.3b). During the first 8 weeks, we observed ca. 20% mortality in both of the fish groups, which was similar to our previously reported results from a low-dose mycobacterial infection35,51. However, from week eight onwards the mortality of the WT fish increased compared to the il10e46/e46 mutants with an endpoint survival of 44% and 63%, respectively (P = 0.037). In order to differentiate between resistance and tolerance against a M. marinum infection, we collected abdominal organ block samples from infected fish during the course of a mycobacterial infection. The samples were taken at 1 day post infection (dpi), 6 dpi, 4 weeks post infection (wpi) and 8/9 wpi, and bacterial amounts were analyzed. In our qPCR based bacterial quantification, detectable amounts of M. marinum in the infected zebrafish were primarily observed for the first time at the 6 dpi time point (Fig.3c). At 6 dpi, but also at 4 wpi, no differences in M. marinum counts between il10e46/e46 mutants and WT zebrafish were observed (at 6 dpi; bacterial copy number median 65,000 vs 74,000, at 4 wpi; median 126,000 vs 90,000, Fig.3c). However, at 8/9wpi there was a smaller bacterial amount in the il10e46/e46 fish compared to the WT fish (median 86,000vs 645,000, P = 0.039). Figure 1. il10e46 point mutation in the exon 1 of the il10 results in a premature stop codon but does not alter il10 expression. (a) A schematic presentation of the il10e46 mutation; a adenosine (A) to thymidine (T) point mutation results in a stop codon (TAA) after the first 27 amino acids in exon 1. (b) Relative il10 expression was measured with qPCR in unchallenged il10e46/e46 mutant and WT zebrafish, spleen and liver from the same individuals (n = 10 in both groups) and is presented as a scatter dot blot and median. Note the divided y axis. Gene expressions were normalized to the expression of eef1a1l1. A two-tailed Mann-Whitney test was used for the statistical comparison of differences between il10e46/e46 zebrafish and WT controls. www.nature.com/scientificreports/ 4 SCIEnTIFIC RepORts | (2018) 8:10360 | DOI:10.1038/s41598-018-28511-w In addition, bacterial counts in il10e46/e46 fish remained stable between 4 and 8/9 weeks, whereas there was a clear increase in bacteria in WT fish (median 65,000 vs 645,000, P < 0.001) (Fig.3c). A Ziehl-Neelsen staining of formalin fixed and paraffin embedded zebrafish confirmed the presence of mycobacteria in both infected il10e46/e46 and WT fish at 4 and 9 wpi (Fig.3d). The histopathological analysis did not reveal any differences in granuloma morphology or in their tissue distribution (Supplementary Fig.S3). In conclusion il10e46/e46 mutant zebrafish showed improved survival but no apparent changes in histopathology. In addition, there was a decrease in bacterial burden of il10e46/e46 mutants at 8/9 wpi fish indicating enhanced resistance rather than a higher tolerance against low dose M. marinum infection. il10e46/e46 mutant zebrafish have an enhanced ifng1 response in a low-dose mycobacterial infection. To study the mechanisms underlying enhanced survival in il10e46/e46 mutant zebrafish against a low-dose M. marinum infection we quantified the expression of selected cytokines and immune cell markers and conducted a flow cytometric analysis in the zebrafish kidney blood cells at different time points post infection (Figs4–8). The expression levels of the studied genes in the kidney and abdominal organ block samples of unchallenged WT and il10e46/e46 fish were similar. However, the relative IgM expression in the il10e46/e46 mutant kidney was increased 2.5-fold compared to WT zebrafish (P = 0.004) but otherwise the mutants did not show any differences compared to WT. First, we quantified the expression of il10, tnfa, tnfb, and il1b in the abdominal organ blocks of zebrafish at early time points during an infection (Fig.4). Until 6 dpi, M. marinum infection did not alter the expression of tnfb or il1b either in il10e46/e46 mutants or in WT fish compared to corresponding PBS injected controls. However, similarly to previous reports about the upregulation of Il10 upon immunogenic stimulation52,53, il10 was significantly upregulated in the il10e46/e46 mutants at 6 dpi in comparison to PBS controls (P = 0.009). WT fish, in turn, had a slight reduction in tnfa expression at 1 dpi compared to PBS injected fish (P = 0.045). As in unchallenged zebrafish, we did not see any differences in the expression levels of the cytokine genes between the il10e46/e46 mutants and WT fish at 1 dpi or 6 dpi. At these early time points, expression levels of the T cell markers cd4-1 (CD4 + cells) or cd8a (CD8 + cells) or the B cell marker IgM did not differ between il10e46/e46 and WT fish either (Supplementary Fig.S4). These data indicate that a low-dose mycobacterial infection does not cause acute systemic inflammation in zebrafish and that the il10e46/e46 zebrafish have a transcriptional innate cytokine response similar to the WT controls in the low-dose infection. In order to study the role of Il10 later in a M. marinum infection, we conducted a flow cytometric analysis in the zebrafish kidney blood cells at 4 and 8 wpi and analyzed the relative amounts of lymphocyte, precursor cell and myeloid cell populations in il10e46/e46 mutants and WT controls (Fig.5). There were no differences in the relative lymphocyte or precursor cell counts between the groups. However, the relative proportion of myeloid cells was significantly lower in WT fish compared to the il10e46/e46 mutants (median 21.6% vs. 26.2%, P = 0.014). Of note, the total live cell numbers were also lower in the WT controls compared to il10e46/e46 mutant fish at 8 wpi (P < 0.001). Furthermore, in the WT control group the median of the relative myeloid cell count was 14.5% lower compared to the median of unchallenged fish at 8 wpi (P = 0.001). Figure 2. Unchallenged il10e46/e46 zebrafish have kidney blood cell populations similar to those of WT control fish. (a,b) The relative proportions of live cells, lymphocytes, myeloid cells and precursor cells were determined with flow cytometry in il10e46/e46 mutant zebrafish and in WT fish (n = 10 in both groups) based on granularity (SSC) and cell size (FSC). Representative flow cytometry plots are shown in panel (a). Gated populations are outlined, and the cell counts inside the gates are given as the percentages of the total viable cell population. The median of the relative proportions of different blood cell populations is presented as a scatter plot in panel (b). A two-tailed Mann-Whitney test was used for the statistical comparison of differences between il10e46/e46 zebrafish and WT controls. www.nature.com/scientificreports/ 5 SCIEnTIFIC RepORts | (2018) 8:10360 | DOI:10.1038/s41598-018-28511-w Figure 3. Adult il10e46/e46 zebrafish have enhanced survival compared to WT controls in a low-dose M. marinum infection. (a) il10e46/e46 (n = 158) and WT (n = 181) zebrafish larvae were microinjected before 6 hours post fertilization with M. marinum (3–29 CFU) and their survival was monitored for 7 days. The experiment was done six times and the data presented here is collected from one representative experiment. (b) The survival of adult il10e46/e46 (n = 172) and WT (n = 149) zebrafish was monitored for 16–20 weeks after a low-dose (2–156 CFU) mycobacterial infection. The data were collected from four experiments. (c) The M. marinum burden in abdominal organ blocks (including kidney in the first and the second experiment, without kidney in the third experiment) of adult il10e46/e46 mutant zebrafish (n = 22–27) and WT controls (n = 22–30 fish) was quantified with qPCR at 4 and 8/9 weeks post a low-dose infection (1–18 CFU). The bacterial load is presented as a scatter dot plot and as the median of total bacterial copies (log10). The data were collected from three experiments. (d) M. marinum granulomas were detected with Ziehl-Neelsen staining (n = 4 in both groups at both time points) at 4 and 9 weeks post a low-dose infection (2–9 CFU). Representative individuals from each group are shown. Granulomas are indicated with arrows. For panels (a) and (b) a log-rank (Mantel-Cox) and for panel (c) A twotailed Mann-Whitney test was used for the statistical comparison of differences. www.nature.com/scientificreports/ 6 SCIEnTIFIC RepORts | (2018) 8:10360 | DOI:10.1038/s41598-018-28511-w To obtain more quantitative data on T and B lymphocytes in il10e46/e46 mutant and WT fish at 4 and 8 wpi, we next extracted RNA from the unsorted kidney cell samples used for the flow cytometry and determined relative expression levels of il10, the T cell markers cd4-1 and cd8a as well as the B cell marker IgM (Fig.6). No differences in the relative il10 expression levels between the il10e46/e46 mutant and the WT groups were observed. Interestingly, a qPCR analysis showed approximately 34.3% higher relative expression of cd4-1 in il10e46/e46 mutants compared to WT control fish at 4wpi (P = 0.052, NS) and a significantly higher expression at 8 wpi (P = 0.017). No differences in the kidney blood cell cd8a nor the B lymphocyte marker IgM expression were detected between il10e46/e46 and WT zebrafish at 4 and 8 wpi. Notably, however, cd8a expression was significantly lower in il10e46/e46 abdominal organ blocks compared to WT controls at 4 wpi (P = 0.032) (Fig.6). Abdominal organ blocks did not have statistically significant differences in their cd4-1 or IgM expression levels between il10e46/e46 mutants and WT fish, although a trend towards the upregulation of cd4-1 was seen at 4 wpi (P = 0.053, NS, Fig.6). Altogether, the upregulated cd4-1 expression in the il10e46/e46 mutant kidneys is a possible consequence of an enhanced Th cell response in the infected il10e46/e46 zebrafish. In addition to the effects of Il10 in regulating the production of proinflammatory cytokines in mice, human IL10 is known to suppress the activation of Th cells by inhibiting the production of IFNG and IL454. Hence, we quantified the relative expression of the Th1, Th2 and Treg cell transcription factors, tbx21, gata3 and foxp3a, respectively, as well as the canonical Th1 and Th2 cell cytokine genes ifng1 and il4 in the kidney blood cells (Fig.7). Indicative of an enhanced Th1 type immune response, our qPCR analysis showed that il10e46/e46 mutant fish had higher relative expression levels of tbx21 at 8wpi (P = 0.042), whereas the expression of gata3 was lower compared to WT controls at the same time point (P = 0.033). Additionally, expression of the Th1 type cytokine gene ifng1 was upregulated in il10e46/e46 fish compared to WT zebrafish 8 wpi (P = 0.025). The expression of the canonical Th2 cytokine gene il4 was instead similar in both il10e46/e46 and WT fish the unsorted kidney cells at 4 and 8 wpi. Nor were any differences seen in the expression levels of foxp3a between the groups at either of the time points, suggesting a similar transcriptional Treg cell response in both mutants and WT fish during an infection. Mutation in il10 can also cause differential tissue and cell type specific regulation of Th type gene expression since upregulation of il4 was observed in the abdominal organ blocks of the il10e46/e46 mutants compared to WT zebrafish at 9 wpi (P = 0.023, Fig.7). Figure 4. Nonfunctional il10 does not increase the expression of proinflammatory cytokines at the early stages of a low-dose (2–9 CFU) mycobacterial infection in zebrafish. (a–d) The relative expression levels of il10 and proinflammatory cytokine genes (il1b, tnfa and tnfb) were measured in the abdominal organ blocks (including kidney) of il10e46/e46 mutant fish (n = 6–12) and WT controls (n = 5–12) at 1 dpi and 6 dpi and are presented as a scatter dot plot and median. Note the different scales of the y axes. Gene expressions were normalized to the expression of eef1a1l1. The data were collected from a single experiment. A two-tailed Mann-Whitney test was used for the statistical comparison of differences. www.nature.com/scientificreports/ 7 SCIEnTIFIC RepORts | (2018) 8:10360 | DOI:10.1038/s41598-018-28511-w A mycobacterial infection elicits the host’s immune cells to produce humoral effectors such as complement components, reactive oxygen and nitrogen intermediates as well as proinflammatory cytokines to fight the infection55. In general, the magnitude of this response can be used to assess the severity of the prevalent bacterial disease. To compare the expression of mediators of inflammation in il10e46/e46 mutants and WT fish in a chronic M. marinum infection, we quantified the expression of tnfa, tnfb and il1b in the kidney blood cells at 4 and 8 wpi (Fig.8). qPCR results showed that il10e46/e46 mutants had significantly lower expression levels of tnfb (P = 0.048) and il1b (P = 0.029) compared to WT fish at 8 wpi. No differences were observed in the expression of tnfa between il10e46/e46 mutants and WT fish. Similar results were seen in the abdominal organ blocks as the relative expression of il1b was downregulated in il10e46/e46 mutant fish compared to WT controls (P = 0.043, Fig.8). Together, the elevated tbx21 and ifng1 expressions as well as the reduced expression of gata3 in the il10e46/e46 mutants indicate that a nonsense mutation in il10 leads to a Th1 cell type immune response in zebrafish. In addition, this Th1 cell response associates with lower tnfb and il1b expression presumably indicating the slower progress of a mycobacterial infection as the expression of these cytokines have been shown to associate with the bacterial burden during the reactivation of M. marinum infection56. The il10e46/e46 mutants are produced by ENU mutagenesis and thus likely contain also other mutations in their background in addition to the e46 mutation. To address this, we outcrossed il10e46/e46 mutants to wild type AB Figure 5. il10e46/e46 mutation associates with a higher proportion of myeloid cells at 8 weeks post a low-dose (1–18 CFU) M. marinum infection. (a,b) The relative proportions of live cells, lymphocytes, precursor cells and myeloid cells were determined with flow cytometry from the kidneys of il10e46/e46 mutants (n = 10–15) and WT control fish (n = 10–12) at 4 and 8 wpi based on granularity (SSC) and cell size (FSC). The data were collected from a single experiment and are presented as a scatter dot plot and median. A two-tailed Mann-Whitney test was used for the statistical comparison of differences between il10e46/e46 zebrafish and WT controls. www.nature.com/scientificreports/ 8 SCIEnTIFIC RepORts | (2018) 8:10360 | DOI:10.1038/s41598-018-28511-w zebrafish and incrossed their heterozygous progeny. Thereafter, we infected ungenotyped offspring of the heterozygous il10e46/+ mutants with a low-dose of M. marinum and collected zebrafish kidneys for gene expression analysis (cd4-1, tbx21, gata3 and ifng1) 8 wpi. Also in this setting, ifng1 expression was enhanced in il10e46/e46 compared to controls (Fig.9). We did not see significant differences in the expression levels of cd4-1, tbx21 or gata3. However, the elevated ifng1 expression further supports the notion that a nonsense mutation in il10 leads to a Th1 cell type immune response in zebrafish. Figure 6. il10e46/e46 mutant zebrafish have an elevated Th cell marker, cd4-1, expression level at 8 weeks post a M. marinum infection. (a–d) The relative expressions of il10 and lymphocyte markers (cd4-1, cd8a and IgM) in kidneys of unchallenged il10e46/e46 mutants (n = 9–10) and WT control fish (n = 9–10) and in the unsorted kidney cell populations of il10e46/e46 mutants (n = 10–14) and WT control fish (n = 10–12) at 4 and 8 weeks post a low-dose (1–18 CFU) infection (on the left) as well as in the abdominal organ blocks (including kidney) of unchallenged il10e46/e46 mutants (n = 12) and WT control fish (n = 11–12) and at 4 and 9 weeks post a low-dose (2–9 CFU) infection (n = 9–12 and n = 11–12, respectively) (on the right) were measured with qPCR. Data are presented as a scatter dot plot and median. Note the different scales of the y axes and the divided y axis in panels a and c. Gene expressions were normalized to the expression of eef1a1l1. Each dataset was collected from a single experiment. A two-tailed Mann-Whitney was used for the statistical comparison of differences. www.nature.com/scientificreports/ 9 SCIEnTIFIC RepORts | (2018) 8:10360 | DOI:10.1038/s41598-018-28511-w Figure 7. il10e46/e46 mutant zebrafish have an enhanced Th1 cell mediated immune response in a mycobacterial infection. (a–e) The relative expressions of Cd4 + lymphocyte transcription factors (tbx21, gata3, foxp3a) and Th cell cytokines (ifng1 and il4) in kidneys of unchallenged il10e46/e46 mutants (n = 10) and WT control fish (n = 10) and in the unsorted kidney cell populations of il10e46/e46 mutants (n = 10–14) and WT control fish (n = 10–12) at 4 and 8 weeks post a low-dose (1–18 CFU) infection (on the left) as well as in the abdominal organ blocks (including kidney) of unchallenged il10e46/e46 mutants (n = 12) and WT control fish (n = 11–12) and at 4 and 9 weeks post a low-dose (2–9 CFU) infection (n = 12 and n = 12, respectively) (on the right) were measured with qPCR. Data are presented as a scatter dot plot and median. Gene expressions were normalized to the expression of eef1a1l1. Each dataset was collected from a single experiment. 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T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011). Acknowledgements This study was financially supported by the Academy of Finland (M.R., 277495), the Sigrid Juselius Foundation (M.R.), the Jane and Aatos Erkko Foundation (M.R.), the Competitive State Research Financing of the Expert Responsibility Area of Tampere University Hospital (M.R.), Competitive State Research Financing of the Expert Responsibility area of Oulu University Hospital (M.R.) and the Tampere Tuberculosis Foundation (M.R., S.-K.H.), the Emil Aaltonen Foundation (S.-K.H), Foundation of the Finnish Anti-Tuberculosis Association (S.-K.H.), the City of Tampere Science Foundation (S.-K.H), the Väinö and Laina Kivi Foundation (S.-K.H.), the Finnish Cultural Foundation, the Central Foundation (S.-K.H.), the Finnish Concordia Fund (S.-K.H.), Orion Research Foundation sr (S.-K.H) and University of Tampere Doctoral Programme in Biomedicine and Biotechnology (M.O.). We thank the Tampere Zebrafish Core Facility, partly funded by Biocenter Finland, for maintaining and providing the zebrafish, Hannaleena Piippo, Jenna Ilomäki, Leena Mäkinen, Tuula Myllymäki and Sami Leino for technical assistance, Marko Pesu, Mataleena Parikka and Hannu Turpeinen for scientific advice and support, Heini Huhtala for the statistical advice and Helen Cooper for proof-reading this manuscript. Author Contributions S.-K.H., M.O., M.N. and M.R. designed the experiments. S.-K.H., M.O., S.T. and M.R. wrote the paper. S.-K.H. and M.O. performed the experiments. S.-K.H., M.O., S. T and M.N. analyzed the data. All authors reviewed and approved the manuscript. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-28511-w. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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