Two ACTH analogs exert differential effects on monocytes/macrophages function regulation in ayu (Plecoglossus altivelis).

Adrenocorticotropic hormone (ACTH), a bioactive peptide of the family of melanocortins, is generated from pro-opiomelanocortin (POMC). So far, the research on the specific functions of ACTH in the immune system of teleosts is limited. We determined two complementary DNA (cDNA) sequences of POMC in ayu (Plecoglossus altivelis), termed PaPOMC-A and PaPOMC-B. PaPOMCs transcripts occurred in all examined tissues, and their expression in immune tissues changed following experimental infection with Vibrio anguillarum. PaACTH-B, but not PaACTH-A, suppressed the phagocytosis of monocytes/macrophages (MO/MФ). Two isoforms of PaACTH increased the bactericidal capacity of MO/MФ. PaACTH-A increased anti-inflammatory cytokine expression, while PaACTH-B decreased pro-inflammatory cytokine expression in MO/MФ. Compared with PaACTH-B treatment, the PaACTH-A treatment improved survival rate and reduced the bacterial load in V. anguillarum-infected ayu through interleukin (IL)-10. Our results indicate that the two PaACTH isoforms exert different effects in the host defense against bacterial infection.

Two paralogs of CXCR4 in the Japanese sea bass (Lateolabrax japonica) are involved in the immune response of B lymphocytes.

CXC chemokine receptor 4 (CXCR4), a member of the G-protein-coupled receptor family, plays an important role in host immune responses. Within the teleost lineage, there are two paralogs of CXCR4; however, the role of CXCR4 in teleost B cells is poorly understood. In this study, we determined the cDNA sequences of the two CXCR4 paralogs from the Japanese sea bass (Lateolabrax japonica; LjCXCR4a and LjCXCR4b). Sequence and phylogenetic tree analyses revealed that LjCXCR4a and LjCXCR4b are most closely related to CXCR4a and CXCR4b, respectively, in the large yellow croaker (Larimichthys crocea). CXCR4 transcripts were mainly expressed in the gills, and their expression in different tissues was altered upon infection with Vibrio harveyi. LjCXCR4a and LjCXCR4b protein levels were upregulated in infected B cells. Knockdown of LjCXCR4a and LjCXCR4b in B cells by RNA interference, the phagocytic activity of B cells was not affected. Furthermore, knockdown of LjCXCR4a, not of LjCXCR4b, was observed to inhibit LjIgM expression in lipopolysaccharide-stimulated B cells. In addition, knockdown of LjCXCR4a, not of LjCXCR4b, was found to reduce reactive oxygen species levels in B cells. Our results indicate that LjCXCR4a and LjCXCR4b modulate the immune response of Japanese sea bass B cells against bacterial infection, albeit via different pathways.

Matrix metalloproteinase-25 from Japanese sea bass (Lateolabrax japonicus) is involved in pro-inflammatory responses.

Matrix metalloproteinases (MMPs) are highly expressed in leukocytes and macrophages, which play a role in the innate immune response. Here, the cDNA sequence of MMP25 from Japanese sea bass (Lateolabrax japonicus) (LjMMP25) was identified. Phylogenetic analysis revealed that LjMMP25 was most closely related to large yellow croaker MMP25. Multiple sequence alignment of LjMMP25 with MMP25 sequences from other teleosts revealed that regions of known functional importance were highly conserved. Expression analysis revealed that LjMMP25 was highly expressed in the head kidney and widely expressed in other tissues including gill, spleen, and liver. LjMMP25 was found to regulate inflammatory cytokine production and promote phagocytosis and bacterial killing in monocytes/macrophages (MO/MФ). Furthermore, LjMMP25 regulated the inflammatory response by modulating NF-κB signaling. These findings reveal new information about the role of LjMMP25 in regulating pro-inflammatory responses in this species.

Meteorin links the bone marrow hypoxic state to hematopoietic stem/progenitor cell mobilization.

Hematopoietic stem/progenitor cells (HSPCs) are supported and regulated by niche cells in the bone marrow with an important characterization of physiological hypoxia. However, how hypoxia regulates HSPCs is still unclear. Here, we find that meteorin (Metrn) from hypoxic macrophages restrains HSPC mobilization. Hypoxia-induced factor 1α and Yin Yang 1 induce the high expression of Metrn in macrophages, and macrophage-specific Metrn knockout increases HSPC mobilization through modulating HSPC proliferation and migration. Mechanistically, Metrn interacts with its receptor 5-hydroxytryptamine receptor 2b (Htr2b) to regulate the reactive oxygen species levels in HSPCs through targeting phospholipase C signaling. The reactive oxygen species levels are reduced in HSPCs of macrophage-specific Metrn knockout mice with activated phospholipase C signaling. Targeting the Metrn/Htr2b axis could therefore be a potential strategy to improve HSPC mobilization for stem cell-based therapy.

Hypoxia-inducible factor-1α involved in macrophage regulation in ayu (Plecoglossus altivelis) under hypoxia.

Hypoxia-inducible factor-1α (HIF-1α) plays a critical role in immune and inflammatory responses and is important in controlling a variety of processes in monocytes and macrophages. However, the role of HIF-1α in the teleost immune system remains less known. In this study, we cloned the cDNA sequence of HIF-1α from the ayu (Plecoglossus altivelis, PaHIF-1α). Sequence and phylogenetic tree analysis showed that PaHIF-1α clustered within the fish HIF-1α tree and was closely related to that of Northern pike (Esox lucius). PaHIF-1α was expressed in all tested tissues and expression increased in liver, head kidney, and body kidney upon Vibrio anguillarum infection. PaHIF-1α was found to regulate the expression of cytokines in ayu monocytes/macrophages (MO/MФ). PaHIF-1α mediated hypoxia-induced enhancement of MO/MФ phagocytic and bactericidal activities to enhance host defenses. Compared with the control, intermittent hypoxia further increased the expression of PaHIF-1α mRNA, improved the survival rate, and reduced the bacterial load of V. anguillarum-infected ayu. Therefore, PaHIF-1α may play a predominant role in the modulation of ayu MO/MФ function.

Two transcription factors PU.1a and PU.1b have different functions in the immune system of teleost ayu.

Transcription factor PU.1 is a regulator of macrophage function, however, the specific function of PU.1 in teleost monocytes/macrophages (MO/MФ) remains unknown. We determined the cDNA sequence of two PU.1 genes from ayu (Plecoglossus altivelis; PaPU.1a and PaPU.1b). Sequence comparisons showed that PaPU.1 were most closely related to the PU.1 of rainbow smelt (Osmerus mordax). The PU.1 transcripts were mainly expressed in the spleen, and their expression was altered in various tissues upon infection with Vibrio anguillarum. PaPU.1a and PaPU.1b proteins were upregulated in MO/MФ, after infection. RNA interference was employed to knockdown PaPU.1a and PaPU.1b to investigate their function in MO/MФ. The expression of inflammatory cytokines was regulated by PaPU.1a, but not PaPU.1b, in ayu MO/MФ upon V. anguillarum infection. Both PaPU.1a and PaPU.1b knockdown lowered the phagocytic activity of MO/MФ. Furthermore, PaPU.1b knockdown attenuated MO/MФ bacterial killing capability. Our results indicate that two PaPU.1 genes differentially modulate the immune response in ayu MO/MФ against bacterial infection.