Intelectin 3 is dispensable for resistance against a mycobacterial infection in zebrafish (Danio rerio)

Abstract

Tuberculosis is a multifactorial bacterial disease, which can be modeled in the zebrafish (Danio rerio). Abdominal cavity infection with Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, leads to a granulomatous disease in adult zebrafish, which replicates the different phases of human tuberculosis, including primary infection, latency and spontaneous reactivation. Here, we have carried out a transcriptional analysis of zebrafish challenged with low-dose of M. marinum, and identified intelectin 3 (itln3) among the highly up-regulated genes. In order to clarify the in vivo significance of Itln3 in immunity, we created nonsense itln3 mutant zebrafish by CRISPR/Cas9 mutagenesis and analyzed the outcome of M. marinum infection in both zebrafish embryos and adult fish. The lack of functional itln3 did not affect survival or the mycobacterial burden in the zebrafish. Furthermore, embryonic survival was not affected when another mycobacterial challenge responsive intelectin, itln1, was silenced using morpholinos either in the WT or itln3 mutant fish. In addition, M. marinum infection in dexamethasone-treated adult zebrafish, which have lowered lymphocyte counts, resulted in similar bacterial burden in both WT fish and homozygous itln3 mutants. Collectively, although itln3 expression is induced upon M. marinum infection in zebrafish, it is dispensable for protective mycobacterial immune response.

Proprotein convertase subtilisin/kexin type 9 regulates the production of acute‐phase reactants from the liver

“…Our previous analysis of genes of the pcsk family in adult zebrafish tissues revealed high relative pcsk9 expression in the fish brain, 42 which is in accordance with an earlier report, where pcsk9 was inhibited with morpholinos and a critical role for PCSK9 in neural tissues and early fish development were described 35 . By creating targeted nonsense mutations, we have demonstrated that the CRISPR/Cas9 mutagenesis method can be efficiently used to knock out genes of interest in zebrafish 26,29 . Accordingly, we created 2 pcsk9 KO zebrafish lines; pcsk9 tpu‐13 and pcsk9 tpu‐2,+15 , with disrupted open reading frames (residual expression medians of the mutated mRNAs in the brain; 15.9% and 8.0%, respectively).…”

“…More than 70% of the human genes have zebrafish orthologues, 48 and consequently gene KO zebrafish accompanied with genome‐wide transcriptome analysis can be used to model both systemic and tissue‐specific functions of human genes 25,26 . Here, our genome‐wide transcriptomic analysis of pneumococcus infected pcsk9 tpu‐13/tpu‐13 mutants and WT fish revealed a substantial proportion (18/78 genes) of immunological genes among the down‐regulated transcripts in pcsk9 KOs.…”

“…Our previous work has demonstrated that CRISPR/Cas9‐mediated indel mutations not only affect the protein translation, causing inability to produce intact protein, but can also lead to changes in the transcription of the target gene 26 . In line with this, qPCR quantification of pcsk9 expression in adult steady‐state zebrafish revealed significantly lower pcsk9 mRNA levels in homozygous pcsk9 tpu‐13/tpu‐13 and pcsk9 tpu‐2,+15/tpu‐2,+15 mutants (residual expression medians of 15.9% and 8.0%; P = .008 in both comparisons) compared with their WT siblings (Figure 1D).…”

Serum amyloid A is a positive acute phase protein in Russian sturgeon challenged with Aeromonas hydrophila

“…In teleosts, the major family member expressed in the liver might be species-specific and has been only documented in a few fish species, such as D. rerio and Ictalurus punctatus. In D. rerio at least five ITLN isoforms are expressed in adult tissues, with itln-3 being the isoform most expressed and upregulated in the liver upon infection with A. salmonicida and Mycobacterium marinum 72,73 . In I. punctatus, two major ITLN genes were identified, and itln-2, but not itln-1, was found to be highly expressed and upregulated in the liver after E. ictaluri infection 49 .…”

Integrating fish models in tuberculosis vaccine development

“…The zebrafish- M. marinum model, in which both the active and latent phase of the mycobacterial infection can be studied, provides a feasible platform. Along with the above-mentioned studies on the adaptive immune response to M. marinum infection, several transcriptome analyses of wild-type ( Harjula et al, 2020 ; Ojanen et al, 2019 ; Hegedűs et al, 2009 ; van der Sar et al, 2009 ) and a hypersusceptible mutant ( Harjula et al, 2020 ) zebrafish have also provided invaluable insights into host responses and the factors that affect host susceptibility to mycobacterial infection. In addition to providing insights into the protective mechanisms in human TB, adult zebrafish can also be used for the direct assessment of novel TB vaccine candidates and delivery methods, as we discuss next.…”