NF-κB-coupled IL17 mediates inflammatory signaling and intestinal inflammation in Artemia sinica.

Nuclear factor-κB (NF-κB) plays a role as a rheostatic transcription factor in regulating intestinal inflammation, and its disruption or constitutive activation leads to inflammation and injury. However, the molecular mechanisms of NF-κB regulation remain largely unknown. In this study, the NF-κB-regulated host defenses against pathogen infections and facilitation of IL17 expression during stimulation with different bacteria were investigated. Intestinal inflammation was induced by dextran sulfate sodium, and NF-κB activity was inhibited in an intestinal injury model. Mannose receptor C type, ABF1/2, serpin B13, lysozyme, and ß-arrestin were significantly controlled by NF-κB in the inflamed intestinal tissue. High levels of NF-κB activation resulted in less pervasive intestinal damage and the maintenance of intestinal barrier integrity. Intestinal injury robustly increased the expression of IL17. NF-κB activation was enhanced by IL17 deficiency in the intestinal injury model. IL17 inhibition aggravated intestinal inflammation, leading to loss of epithelial architecture and the infiltration of inflammatory cells. These data suggest that NF-κB and IL17 play key mediator roles in the maintenance of gut epithelial integrity and immune homeostasis.

Expression pattern of mitogen-activated protein kinase kinase 4 and regulation to antibacterial factor ABF-1/2 in response to bacterial challenge from Artemia parthenogenetica.

Mitogen-activated protein kinase 4, MKK4, is a key upstream kinase in the JNK/p38 MAPK pathway that has been reported to participate in multiple immune responses. In this study, the gene that encodes ApMKK4 was isolated and identified from Artemia parthenogenetica. It was found to contain a 1134 bp open reading frame encoding 378 amino acids. The predicted protein contains D domain, DVD domain and kinase domain. Homology analysis revealed that ApMKK4 shares 38-69% identity with MKK4 homologs from other species. Results revealed that ApMKK4 was mainly expressed during early development of which highest at the gastrula stage. After challenged by Vibrio harveyi and Micrococcus lysodeikticus, ApMKK4 was remarkably upregulated at 10 and 103 cfu/mL bacterial concentrations, respectively. Through siRNAi, the transcript level of ApMKK4 was significantly decreased by 46-67%. Intriguingly, when the ApMKK4-knockdown nauplii faced with bacterial stimulation, the expression of ApMKK4 was completely restored in a short time. Moreover, this phenomenon also occurred in related antimicrobial peptide genes, ABF-1 and ABF-2. Our research reveals that ApMKK4 plays a pivotal role during early development and immune responses against bacterial infections.

Comparative analysis of two brine shrimps revealed differential expression pattern and functional characterization of CK2α under bacterial stimulation from different geographical distribution.

Understanding how the brine shrimp responds to different geographical populations can provide novel insights on response to bacterial stimulation. In the paper, Artemia sinica from lower altitudes and Artemia parthenogenetica from higher altitudes of the Tibetan Plateau, were used to illustrate different defense against bacteria mechanisms that these organisms used to adapt to different geographical environments. Protein kinase CK2 is a serine/threonine kinase with a multitude of protein substrates. It is a ubiquitous enzyme essential for the viability of eukaryotic cells, where its functions in a variety of cellular processes, including cell cycle progression, apoptosis, transcription, and viral infection. The gene encodes the same mRNA sequence in A. sinica and A. parthenogenetica, named AsCK2α and ApCK2α, respectively. The open reading frame was obtained, a 1047-bp sequence encoding a predicted protein of 349 amino acids. To systematically analyze the expression of AsCK2α and ApCK2α during embryonic development and bacterial challenge, real-time PCR, Western blotting and immunohistochemistry were performed. The results showed that AsCK2α was higher than ApCK2α at different developmental stages. Under bacterial challenge, the expression of ApCK2α was significantly higher than AsCK2α. Protein localization analysis showed that AsCK2α and ApCK2α were mainly distributed in the head and chest. Our research revealed that CK2α plays a vital role in the growth, development and bacterial stimulation of the brine shrimp.

Transcriptome analysis of Artemia sinica in response to Micrococcus lysodeikticus infection.

To enhance genomic resources and understand the molecular immune mechanisms underlying the response topathogens, we first performed a comparative gene transcription analysis from Micrococcus lysodeikticus-immunized Artemia sinica and from a control group through RNA-Seq technology, meanwhile the differentially expressed genes (DEGs) were investigated. In total, 80, 113, 984 clean reads were obtained and then assembled into 71,536 unigenes with an average length of 1115 bp and an N50 of 1783 bp. Unigenes were annotated by comparing against nr, Swiss-Prot\KEGG\ COG\ KOG\ GO and Pfam databases, and 27,689 unigenes (38.7%) were annotated in at least one database. After bacterial challenge, 183 and 298 genes were identified as remarkably up-regulated or down-regulated, respectively, amongst 481 were associated with 168 pathways, including classical immune-related pathways, such as 'Toll-like receptor signaling', 'the complement cascades', 'MAPK signaling pathway' and 'Apoptosis'. Besides, eight genes which were differently expressed immune-related were confirmed by using quantitative real-time PCR. This study characterized a gene expression pattern for normal and M. lysodeikticus -immunized A. sinica for the first time and sheds new light on the molecular mechanisms thus enabling future efforts on disease control programs in this valuable aquaculture species.

Cloning, expression pattern and functional characterization of interleukin-1 receptor-associated kinase 4, an important mediator of the Toll-like receptor signaling pathway, from Artemia sinica.

As a crucial component of Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) signaling pathways, IL-1R--associated kinase 4 (IRAK-4) plays a central role in innate immunity and embryonic development. Herein, we have characterized the full length cDNA of IRAK4 from Artemia sinica. Molecular characterization revealed that the sequence includes a 2550 bp open reading frame, encoding a predicted protein of 849 amino acids. The predicted protein contains a death domain in the N-terminus and two serine/threonine/tyrosine protein kinasedomains. Bioinformatics analysis showed that it belonged to a new member of the IRAK-4 family. The expression of AsIRAK-4 was researched in various stages during embryonic development by several molecular biology methods including real time PCR, Western blotting and immunohistochemistry. The results showed that AsIRAK-4 was constitutively expressed at all developmental stages from embryo to adult, and it was mainly expressed in the head and thorax at the early stages and on the surface of the alimentary canal at later stages. The highest expression level was at the 0 h, 15 h and 5 d stages of A. sinica. While challenged by Gram-positive and Gram-negative bacteria, AsIRAK-4 was remarkably upregulated with the rising concentration of bacteria. Our research revealed that AsIRAK-4 plays a vital role in growth, development and innate immunity of A. sinica.