Dev Comp Immunol 2026 Jun 29;181:105668. doi: 10.1016/j.dci.2026.105668.

Functional analysis of IRF1/2 homolog in immune regulation of the sea urchin Strongylocentrotus intermedius.

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As one of the critically important transcription factors, interferon regulatory factors (IRFs) modulate the host transcriptional program triggered by pathogen-associated molecular patterns in variety of marine invertebrates. Their activation is indispensable for initiating and modulating innate immune defenses across animal taxa. However, the functional characteristics and regulatory mechanisms of IRFs in echinoderms remain poorly understood. In this study, a newly identified IRF family member, Si-IRF1/2, was cloned and characterized in the sea urchin Strongylocentrotus intermedius. Si-IRF1/2 comprises a full-length ORF of 1668 bp, corresponding to a 555-amino-acid protein. Computational analysis identified a conserved IRF domain at the N-terminal region. This conserved signature was identified through integrated domain prediction and corroborated by multiple sequence alignment with orthologs from diverse species. Phylogenetic analysis showed that Si-IRF1/2 clusters closely with members of the vertebrate IRF1/2 proteins and molluscan IRF1/2 homologs. The tissue-specific expression profile of Si-IRF1/2 was assessed by qRT-PCR. Although transcripts were ubiquitously present, expression was markedly enriched in coelomocytes, indicating a specialized role in this immunologically relevant tissue. Moreover, expression of Si-IRF1/2 was upregulated when stimulated with lipopolysaccharide (LPS) and poly (I:C). Subcellular localization assays showed that Si-IRF1/2 is predominantly localized in the nucleus. The results of the RNAi experiment indicate that after Si-IRF1/2 was knocked down, the mRNA expressions of Si-strongylocins and Si-IL17s in coelomocytes changed significantly at 12 h after LPS stimulation. The transcriptional regulatory capacity of Si-IRF1/2 was further confirmed by dual-luciferase reporter assays, which demonstrated its ability to enhance the activity of promoters from multiple immune-related genes, including interleukin-6 (IL-6), Interferon-α/β (IFNα/β), Signal transducer and activator of transcription 3 (STAT3), activator protein-1 (AP-1), interferon-stimulated response element (ISRE), nuclear factor-κB (NF-κB), and tumor necrosis factor α (TNFα). Under LPS stimulation, overexpression of Si-IRF1/2 promoted activation of mitogen-activated protein kinase pathways, specifically c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2). Taken together, these findings provide new insights into the immune-regulatory role of Si-IRF1/2 to establish a conceptual framework for breeding sea urchin lines with improved resistance to disease, while simultaneously providing new knowledge of innate immune mechanisms mediated by IRFs in invertebrates.

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