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

Abstract 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

Abnormal cerebellar development and ataxia in CARP VIII morphant zebrafish

Congenital ataxia and mental retardation are mainly caused by variations in the genes that affect brain development. Recent reports have shown that mutations in the CA8 gene are associated with mental retardation and ataxia in humans and ataxia in mice. The gene product, carbonic anhydrase-related protein VIII (CARP VIII), is predominantly present in cerebellar Purkinje cells, where it interacts with the inositol 1,4,5-trisphosphate receptor type 1, a calcium channel. In this study, we investigated the effects of the loss of function of CARP VIII during embryonic development in zebrafish using antisense morpholino oligonucleotides against the CA8 gene. Knockdown of CA8 in zebrafish larvae resulted in a curved body axis, pericardial edema and abnormal movement patterns. Histologic examination revealed gross morphologic defects in the cerebellar region and in the muscle. Electron microscopy studies showed increased neuronal cell death in developing larvae injected with CA8 antisense morpholinos. These data suggest a pivotal role for CARP VIII during embryonic development. Furthermore, suppression of CA8 expression leads to defects in motor and coordination functions, mimicking the ataxic human phenotype. This work reveals an evolutionarily conserved function of CARP VIII in brain development and introduces a novel zebrafish model in which to investigate the mechanisms of CARP VIII-related ataxia and mental retardation in humans

Mycobacterium marinum Causes a Latent Infection that Can Be Reactivated by Gamma Irradiation in Adult Zebrafish

The mechanisms leading to latency and reactivation of human tuberculosis are still unclear, mainly due to the lack of standardized animal models for latent mycobacterial infection. In this longitudinal study of the progression of a mycobacterial disease in adult zebrafish, we show that an experimental intraperitoneal infection with a low dose (~35 bacteria) of Mycobacterium marinum, results in the development of a latent disease in most individuals. The infection is characterized by limited mortality (25%), stable bacterial loads 4 weeks following infection and constant numbers of highly organized granulomas in few target organs. The majority of bacteria are dormant during a latent mycobacterial infection in zebrafish, and can be activated by resuscitation promoting factor ex vivo. In 5–10% of tuberculosis cases in humans, the disease is reactivated usually as a consequence of immune suppression. In our model, we are able to show that reactivation can be efficiently induced in infected zebrafish by γ-irradiation that transiently depletes granulo/monocyte and lymphocyte pools, as determined by flow cytometry. This immunosuppression causes reactivation of the dormant mycobacterial population and a rapid outgrowth of bacteria, leading to 88% mortality in four weeks. In this study, the adult zebrafish presents itself as a unique non-mammalian vertebrate model for studying the development of latency, regulation of mycobacterial dormancy, as well as reactivation of latent or subclinical tuberculosis. The possibilities for screening for host and pathogen factors affecting the disease progression, and identifying novel therapeutic agents and vaccine targets make this established model especially attractive.Public Library of Science open acces

Characterization of the innate immune response to Streptococcus pneumoniae infection in zebrafish.

Streptococcus pneumoniae (pneumococcus) is one of the most frequent causes of pneumonia, sepsis and meningitis in humans, and an important cause of mortality among children and the elderly. We have previously reported the suitability of the zebrafish (Danio rerio) larval model for the study of the host-pathogen interactions in pneumococcal infection. In the present study, we characterized the zebrafish innate immune response to pneumococcus in detail through a whole-genome level transcriptome analysis and revealed a well-conserved response to this human pathogen in challenged larvae. In addition, to gain understanding of the genetic factors associated with the increased risk for severe pneumococcal infection in humans, we carried out a medium-scale forward genetic screen in zebrafish. In the screen, we identified a mutant fish line which showed compromised resistance to pneumococcus in the septic larval infection model. The transcriptome analysis of the mutant zebrafish larvae revealed deficient expression of a gene homologous for human C-reactive protein (CRP). Furthermore, knockout of one of the six zebrafish crp genes by CRISPR-Cas9 mutagenesis predisposed zebrafish larvae to a more severe pneumococcal infection, and the phenotype was further augmented by concomitant knockdown of a gene for another Crp isoform. This suggests a conserved function of C-reactive protein in anti-pneumococcal immunity in zebrafish. Altogether, this study highlights the similarity of the host response to pneumococcus in zebrafish and humans, gives evidence of the conserved role of C-reactive protein in the defense against pneumococcus, and suggests novel host genes associated with pneumococcal infection

Zebrafish mortality, the development of bacterial load and the number of lesions have dose-dependent patterns.

<p>Adult zebrafish were i.p. infected with either a low (34±15 cfu) (n = 180) or a high dose (2029±709 cfu) (n = 104) of <i>M. marinum</i>. (A) Survival was followed for 32 weeks. * P<0.05 (B) The figure shows the average loads for 5 fish (except 32 wk high dose, n = 2). Low-dose statistics: * sig. diff. from 1 wk, ** sig. diff. from 1 and 2 wk. High-dose statistics: *** sig. diff. from 1, 2, 8, 11 and 20 wk. Low-dose vs. high-dose statistics: loads at time-points marked with † are sig. diff. (C) By default, 4 individuals per dose were analyzed by Ziehl-Neelsen staining (except 20 wk high dose, n = 3) per time-point The gonads, pancreas, liver, muscle, mesentery, spleen, gut and kidney were assessed and the number of organs with visible bacteria was determined. *P<0.05. (D) The total number of granulomas in a sample set for each individual was counted. * P<0.05.</p

<i>M. marinum</i> induces the formation of granulomas that mature into well-defined structures during an infection.

<p>In fish infected with a low dose (34±15 cfu) of <i>M. marinum</i>, Ziehl-Neelsen staining at 2 wpi commonly reveals areas with free bacteria (C). Some slightly better formed and restricted areas containing bacteria, here referred to as early granulomas, are also seen (A), but as shown in (B) trichrome staining of the adjacent slide, encapsulation around the mycobacterial lesions is absent at the early stage of infection. At 20 weeks, fish that have survived have mature granulomas (D–F) many of which are multicentric surrounded by a fibrous capsule (D&E). (E) Trichrome staining shows the fibrous capsule in blue (F). The amount of bacteria inside granulomas has increased from the earliest time-points.</p

Bacterial dose and the presence of functional adaptive immunity define the outcome of mycobacterial infection.

<p>(A) The early cytokine response at 1 d post infection was measured from wt fish infected with a high (2029±709 cfu) or a low (34±15 cfu) dose or injected with sterile PBS buffer (n in each group 10–20). *P<0.05 (B) Wt fish were infected with a high or a low dose or sterile PBS buffer (for early time-points), and rag1 (−/−) fish were infected with a low dose Nos2b expression was measured with q-RT-PCR (n in each group was 9–20/time point). *P<0.05 (C) Fish were infected as in (B) and <i>IFNγ1–2</i> was measured with q-RT-PCR. *P<0.05. (D) Adult wt and rag1 (−/−) zebrafish were infected with a low dose (n = 30) and followed for survival. *P<0.05 (E) Adult wt and rag1 (−/−) fish were infected with a low dose. Average mycobacterial load was measured by qPCR at 2, 4, and 7 wpi (n = 10 per time point). *P<0.05.</p

A major part of the mycobacteria are in a dormant state in latent infection.

<p>(A) Parallel dilutions of fresh logarithmic or old plateau phase <i>M. marinum</i> cultures were plated +/− Rpf to show the resuscitating effect of <i>Micrococcus luteus</i> Rpf on dormant <i>M. marinum</i>. (B) Parallel homogenate sample dilutions from low-dose (34±15 cfu) infected fish (wt or rag1 (−/−)) were plated at different time points +/− Rpf to detect dormant mycobacteria. (C) <i>GltA1</i> expression was measured from low-dose infected rag1 (−/−) and wt fish and high-dose infected wt fish and normalized to the total <i>M. marinum</i> load in each fish measured by qPCR. *P<0.05.</p

Gamma irradiation induces reactivation resulting in increased mortality due to uncontrolled growth of mycobacteria.

<p>(A–C) Zebrafish (n = 17) with a latent <i>M. marinum</i> infection were irradiated twice with 25 Gy with one month between the irradiations. Twice irradiated, non-infected zebrafish (n = 23) as well as zebrafish with a latent infection (n = 14) were included as controls. (A) Survival was followed for 30 days after the second dose. *P<0.05. (B) During this period, moribund or recently dead fish were collected 15–22 days after the second radiation dose. Bacterial loads were compared with those of similarly infected, non-irradiated control fish that were collected at the end-point of the experiment. *P<0.05 (C&D) A representative Ziehl-Neelsen stained sample from a reactivated fish showing large numbers of free mycobacteria (purple areas) in the zebrafish body cavity (C). The sides of the body cavity are marked with arrowheads O = ovary, P = pancreas, L = liver, G = gut, K = kidney. (D) A picture taken with a higher magnification showing individual rods (few examples pointed out with arrows). (E) Four groups of 4 adult zebrafish (1 rag2-gfp, 1 lck-gfp and 2 wild-type groups) were γ-irradiated with 25 Gy. Similar control groups were left untreated. Kidneys were collected 8 d post irradiation, pooled and analyzed by FCM. FSC-SSC -plots were gated based on cell size and granularity as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002944#ppat.1002944-Traver2" target="_blank">[56]</a> (gates shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002944#ppat.1002944.s003" target="_blank">Figure S3</a>) to assess the effect of irradiation on leukocyte populations. *P<0.05. For further verification of the effect of radiation on lymphocytes, a GFP gate was used for the rag2 and lck groups expressing GFP in B and T cells, or T cells, respectively. (F) Adult non-infected wt zebrafish were irradiated with 25 Gy once (grey bars) (n = 3) or twice (n = 7) (black bars) with one month between the doses. Leukocyte recovery and re-depletion were assessed by FCM. Non-irradiated fish (n = 4) were used as controls. *P<0.05 (G) Fish with a latent infection (n = 7) were irradiated twice with 25 Gy with one month between the doses and plated +/− Rpf for 18 d after the second radiation dose. (H) Fish (n = 6) with a latent infection were plated +/− Rpf.</p