Immune Sensing of Synthetic, Bacterial, and Protozoan RNA by Toll-like Receptor 8 Requires Coordinated Processing by RNase T2 and RNase 2.

Human toll-like receptor 8 (TLR8) activation induces a potent T helper-1 (Th1) cell response critical for defense against intracellular pathogens, including protozoa. The receptor harbors two distinct binding sites, uridine and di- and/or trinucleotides, but the RNases upstream of TLR8 remain poorly characterized. We identified two endolysosomal endoribonucleases, RNase T2 and RNase 2, that act synergistically to release uridine from oligoribonucleotides. RNase T2 cleaves preferentially before, and RNase 2 after, uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic oligoribonucleotides all required RNase 2 and T2 processing to activate TLR8. Uridine supplementation restored RNA recognition in RNASE2-/- or RNASET2-/- but not RNASE2-/-RNASET2-/- cells. Primary immune cells from RNase T2-hypomorphic patients lacked a response to bacterial RNA but responded robustly to small-molecule TLR8 ligands. Our data identify an essential function of RNase T2 and RNase 2 upstream of TLR8 and provide insight into TLR8 activation.

circCBL and its host gene CBL collaboratively enhance the antiviral immunity and antibacterial immunity by targeting MITA in fish.

IMPORTANCE: Increasing evidence indicates that circular RNAs exert crucial functions in regulating gene expression in mammals. However, the function of circRNAs in lower vertebrates still needs further exploration. Our research results demonstrated that circRNA, namely circCBL, is involved in modulating antiviral and antibacterial immune responses in lower vertebrates. In addition, our study also found that circCBL can serve as a competing endogenous RNA to facilitate MITA expression, thereby modulating MITA-mediated innate immunity. Further research has proved that the host gene CBL also promotes the expression of MITA, enhancing antiviral and antibacterial immune responses. Our study not only elucidated the underlying biological mechanism of the circRNA-miRNA-mRNA axis in the innate immune response of lower vertebrates but also unveiled the synergistic antibacterial and antiviral mechanisms between circRNA and its host gene in lower vertebrates.

CsIL-20, a tongue sole interleukin-20, negatively mediates leucocyte activity and antibacterial defense.

Interleukin-20 (IL-20), as an essential member of IL-10 family, plays vital roles in mammalian immunological response such as antimicrobial, inflammation, hematopoiesis, and immune diseases. In teleost, the study about immune antimicrobial function of IL-20 is largely scarce. In this article, we revealed the expression profiles and the immunological functions of the IL-20 (CsIL-20) in tongue sole Cynoglossus semilaevis. CsIL-20 is composed of 183 amino acid residues, with seven cysteine residues and a typical IL-10 domain which comprises six α-helices and two ß-sheets, and shares 34.4-71.2 % identities with other teleost IL-20. CsIL-20 was constitutively expressed in a variety of tissues and regulated by bacterial invasion, and the recombinant CsIL-20 (rCsIL-20) could bind to different bacteria. In vitro rCsIL-20 could interact with the membrane of peripheral blood leukocytes (PBLs), leading to the attenuation of reactive oxygen species (ROS) production and acid phosphatase activity in PBLs. In line with In vitro results, In vivo rCsIL-20 could obviously suppressed the host immune against bacterial infection. Furthermore, knockdown of CsIL-20 in vivo could markedly enhance the host antibacterial immunity. Collectively, these observations offer new insights into the negative effect of CsIL-20 on antibacterial immunity.

Selective autophagy receptor p62 promotes antibacterial and antiviral immunity in common carp (Cyprinus carpio).

Sequestosome 1 (SQSTM1/p62) is a selective autophagy adapter protein that participates in antiviral and bacterial immune responses and plays an important regulatory role in clearing the proteins to be degraded and maintaining intracellular protein homeostasis. In this study, two p62 genes were cloned from common carp (Cyprinus carpio), namely Ccp62-1 and Ccp62-2, and conducted bioinformatics analysis on them. The results showed that Ccp62s had the same structural domain (Phox and Bem1 domain, ZZ-type zinc finger domain, and ubiquitin-associated domain) as p62 from other species. Ccp62s were widely expressed in various tissues of fish, and highly expressed in immune organs such as gills, spleen, head kidney, etc. Subcellular localization study showed that they were mainly distributed in punctate aggregates in the cytoplasm. After stimulation with Aeromonas hydrophila and spring viraemia of carp virus (SVCV), the expression level of Ccp62s was generally up-regulated. Overexpression of Ccp62s in EPC cells could inhibit SVCV replication. Upon A. hydrophila challenge, the bacterial load in Ccp62s-overexpressing group was significantly reduced, the expression levels of pro-inflammatory cytokines and interferon factors were increased, and the survival rate of the fish was improved. These results indicated that Ccp62s were involved in the immune response of common carp to bacterial and viral infections.

Suppressed COP9 signalosome 5 promotes hemocyte proliferation through Cyclin E in the early G1 phase to defend against bacterial infection in crab.

Hemocytes are invertebrate immune cells that are similar to blood cells in vertebrates and play a crucial role in innate immunity. Previous work has found that mature circulating hemocytes lack the ability to proliferate. However, recent single-cell RNA sequencing and functional studies in invertebrate have challenged this view. Here, we report that bacteria induced hemocytes proliferation in the Chinese mitten crab, Eriocheir sinensis. Flow cytometry was used to collect non-proliferating and proliferating hemocytes populations, while the expression of EsCyclin E was highly expressed in proliferating hemocytes, but the expression of EsCsn5 was significantly suppressed in proliferating hemocytes. Subsequent studies have found EsCsn5 distributed in two fractions include holo-complex and monomeric form, whereas knockdown of EsCsn5 has little impact on the amount of the holo-complex. EsCsn5 was widely expressed in different crab tissues, while its expression was significantly reduced upon bacterial infection. Crab hemocytes showed significantly enhanced proliferation when EsCsn5 was genetically knocked down, suggesting a critical role for CSN5 in the negative regulation of crab hemocyte proliferation. Moreover, EsCSN5 but not the EsCSN8 was demonstrated to negatively regulate the early G1 phase of the cell cycle by controlling the degradation of EsCyclin E through ubiquitination steps, rather than affecting its transcription. Furthermore, in the EsCyclin E-suppressed crab there was a significantly reduced survival rate and an up-regulated hemolymph bacterial concentration. Taken together, this study provides evidence demonstrating that invertebrate hemocytes down-regulate the expression of EsCsn5 upon bacterial challenge, thus promoting proliferation in an EsCyclin E-dependent manner in order to protect the crab from infection.

TroTNFSF6, a tumor necrosis factor ligand superfamily member, promotes antibacterial immune response of golden pompano, Trachinotus ovatus.

Tumor necrosis factor ligand superfamily member 6 (TNFSF6), also known as FasL/CD95L, is essential for maintaining the body's immune homeostasis. However, the current reports on TNFSF6 in fish are relatively scarce. In the present study, we conducted functional analyses of a TNFSF6 (TroTNFSF6) from the teleost fish golden pompano (Trachinotus ovatus). TroTNFSF6 is composed of 228 amino acids and has a low similarity with other species (9.65%-58.79%). TroTNFSF6 was expressed in the 11 tissues tested and was significantly up-regulated after Edwardsiella tarda infection. In vivo, overexpression of TroTNFSF6 effectively stimulated the AKP and ACP activities, and reduced bacterial infection in fish tissues. Correspondingly, knockdown of TroTNFSF6 expression resulted in increasing bacterial dissemination and colonization in fish tissues. In vitro, recombinant TroTNFSF6 protein promoted the proliferation of T. ovatus head kidney lymphocytes (HKLs), and promoted the apoptosis of murine liver cancer cells (Hepa1-6). The results indicated that TroTNFSF6 plays an important role in the T. ovatus antibacterial immunity. These observations will facilitate the future in-depth study of teleost TNFSF6.

Two novel teleost calreticulins PoCrt-1/2, with bacterial binding and agglutination activity, are involved in antibacterial immunity.

Calreticulin (Crt), a conserved lectin-like pleiotropic protein, plays crucial roles in mammalian immune response. In fish, the immunological function of Crt is limited investigated. Herein, we studied the antibacterial immunity of two type of Crt homologues (i.e. PoCrt-1 and PoCrt-2) in Japanese flounder (Paralichthys olivaceus). PoCrt-1 and PoCrt-2 are composed of 419 and 427 amino acid residues respectively, with 69.09% overall sequence identities with each other. Both PoCrt-1 and PoCrt-2 contain a signal peptide and three functional domains i.e. N-, P- and C-domains. Both PoCrt-1 and PoCrt-2 were constitutively expressed at various tissues with highest expression level in liver, and obviously regulated by Edwardsiella tarda and Vibrio harveyi. Furthermore, recombinant PoCrt-1 and PoCrt-2 (rPoCrt-1 and rPoCrt-2) could bind to different Gram-negative bacteria with highest binding index with E. tarda. At same time, in vitro rPoCrt-1 and rPoCrt-2 could agglutinate E. tarda, V. harveyi, and Vibrio anguillarum, and inhibit the bacterial growth. Similarly, in vivo rPoCrt-1 and rPoCrt-2 could significantly suppress the dissemination of E. tarda. Overall, these observations add new insights into the antibacterial immunity of Crt in P. olivaceus.

Effects of the tumor necrosis factor on hemocyte proliferation and bacterial infection in Chinese mitten crab (Eriocheir sinensis).

Tumor necrosis factor (TNF) is an important cytokine that can regulate a variety of cellular responses by binding tumor necrosis factor receptor (TNFR). We studied whether the TNF of Eriocheir sinensis can regulate hemocyte proliferation. The results showed that the EsTNF and EsTNFR were constitutively expressed in all tested tissues, including the heart, hepatopancreas, muscles, gills, stomachs, intestines, and hemocytes. We found that low levels of EsTNF and EsTNFR transcripts were present in hemocytes. The gene expression levels were significantly increased in the hemocytes after being stimulated by Staphylococcus aureus or Vibrio parahaemolyticus. We also found some genes related to cell proliferation were expressed at a higher level in pulsing rTNF-stimulated hemocytes compared with the control group. We also knocked down the EsTNFR gene with RNAi technology. The results showed that the expression level of these genes related to cell proliferation was significantly down-regulated compared with the control group when the TNF does not bind TNFR. We used Edu technology to repeat the above experiments and the results were similar. Compared with the control group, the hemocytes stimulated by rTNF showed more significant proliferation, and the proliferation rate was significantly down-regulated after knocking down the EsTNFR gene. Therefore, we indicate that TNF binding TNFR can affect the proliferation of E. sinensis hemocytes, which might be manifested by affecting the expression of some proliferation-related genes.

Thymosin beta-4 participate in antibacterial immunity and wound healing in black tiger shrimp, Penaeus monodon.

Thymosin beta-4 (Tß4) is a ubiquitous protein with multiple and diverse intracellular and extracellular functions in vertebrates, which play fundamental roles in innate immune against pathogens and wound healing. In this study, the full-length cDNA of Tß4 was cloned from Penaeus monodon (designated as PmTß4), using the technology of rapid amplification of cDNA ends (RACE). The cDNA of PmTß4 was 1361 bp with an open reading frame (ORF) of 501 bp, which encoding a polypeptide of 166 amino acid. The Quantitative Real-time PCR (qRT-PCR) analysis results showed that PmTß4 was ubiquitously expressed in all the tested shrimp tissues, with the highest expression level was detected in the hemolymph, while the lowest expression level in the muscle. The expression level of PmTß4 was significantly up-regulated in hepatopancreas after challenged by Vibrio parahaemolyticus, Vibrio harveyi and Staphylococcus aureus. In vitro antimicrobial test showed that the recombinant protein of PmTß4 (rPmTß4) had broad-spectrum of antimicrobial activity, which could inhibit both the growth of gram-negative bacteria and gram-positive bacteria, including Vibrio vulnificus, V. parahaemolyticus, Streptococcus agalactiae, S. aureus and Aeromonas hydrophila. Moreover, rPmTß4 had a certain binding ability to different bacteria, and this binding ability exhibits a strong dose-dependent effect. In vivo, PmTß4 could facilitate external bacterial clearance in shrimp, and have beneficial to shrimp survival post V. parahaemolyticus infection. Furthermore, wound-healing assay was carried out to study the role of PmTß4 in the process of wound healing. The results showed that the PmTß4 expression was significantly up-regulated by injury treatment, and exerted positive effects to promote wound healing. In addition, PmTß4 can significantly increase the expression level of superoxide dismutase (SOD) and Catalase (CAT) after injury treatment in shrimp, which would involve in scavenging reactive oxygen species (ROS) caused by the wound. In conclusion, these results indicated that PmTß4 may play important roles in antibacterial immunity and wound healing in Penaeus monodon.

CpG ODN 1668 as TLR9 agonist mediates humpback grouper (Cromileptes altivelis) antibacterial immune responses.

Cromileptes altivelis (humpback grouper) is the main farmed species in the southern coastal area of China owing to its important economic value. Toll-like receptor 9 (TLR9) belongs to the toll-like receptor (TLR) family and functions as a pattern recognition receptor, recognising unmethylated oligodeoxynucleotides containing the CpG motif (CpG ODNs) in bacterial and viral genomes, thereby activating host immune response. In this study, the C. altivelis TLR9 (CaTLR9) ligand CpG ODN 1668 was screened and found to significantly enhance the antibacterial immunity of humpback grouper in vivo and head kidney lymphocytes (HKLs) in vitro. In addition, CpG ODN 1668 also promoted the cell proliferation and immune gene expression of HKLs and strengthened the phagocytosis activity of head kidney macrophages. However, when the CaTLR9 expression was knocked down in the humpback group, the expression levels of TLR9, myeloid differentiation factor 88 (Myd88), tumour necrosis factor-α (TNF-α), interferon γ (IFN-γ), interleukin-1ß (IL-1ß), IL-6, and IL-8 were significantly reduced, and the antibacterial immune effects induced by CpG ODN 1668 were mostly abolished. Therefore, CpG ODN 1668 induced antibacterial immune responses in a CaTLR9-dependent pathway. These results enhance the knowledge of the antibacterial immunity of fish TLR signalling pathways and have important implications for exploring natural antibacterial molecules in fish.

CpG ODN 2102 promotes antibacterial immune responses and enhances vaccine-induced protection in golden pompano (Trachinotusovatus).

CpG oligodeoxynucleotides (ODNs) are oligodeoxynucleotides containing CpG motifs and can be recognized by toll-like receptor 9 (TLR9), activating the host's immune responses. In this study, ten different CpG ODNs were designed and synthesized to study the antibacterial immune responses of CpG ODNs in golden pompano (Trachinotus ovatus). Results showed that CpG ODN 2102 significantly improved the immunity of golden pompano against bacteria. Besides, CpG ODN 2102 promoted the proliferation of head kidney lymphocytes and activated the head kidney macrophages. When TLR9-specific small interfering RNA (siRNA) was used to interfere with TLR9 expression, the immune responses were decreased. Moreover, the expression levels of myeloid differentiation primary response 88 (Myd88), p65, tumor necrosis factor receptor-associated factor 6 (TRAF6), and tumor necrosis factor-alpha (TNF-α) in the TLR9-knockdown golden pompano kidney (GPK) cells were significantly reduced. The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) promoter activity of the TLR9-knockdown GPK cells was also significantly reduced. In vivo, the antibacterial immune effects induced by CpG ODN 2102 in golden pompano were mostly abolished when TLR9 expression was knocked down. These results suggested that TLR9 was involved in the immune responses induced by CpG ODN 2102. CpG ODN 2102 also enhanced the protective effect of the Vibrio harveyi vaccine pCTssJ, where the survival rate of golden pompano was significantly improved by 20%. In addition, CpG ODN 2102 enhanced the messenger RNA (mRNA) expression levels of TLR9, Myxovirus resistance (Mx), interferon γ (IFN-γ), TNF-α, interleukin (IL)-1ß, IL-8, major histocompatibility complex class (MHC) Iα, MHC IIα, Immunoglobulin D (IgD), and IgM. Therefore, TLR9 was involved in the antibacterial immune responses induced by CpG ODN 2102 and CpG ODN 2102 possessed adjuvant immune effects. These results enlarged our knowledge of the antibacterial immunity of fish TLRs signaling pathway and had important implications for exploring natural antibacterial molecules in fish and developing new vaccine adjuvants.

Caspar negatively regulates anti-bacterial immunity by controlling the nuclear translocation of Relish in Chinese mitten crab (Eriocheir sinensis).

Caspar, a homolog of the Fas-associated factor 1 (FAF1) family, contains an N-terminal ubiquitin interaction domain, a ubiquitin-like self-association domain, and a C-terminal ubiquitin regulatory domain. Caspar has been reported to be involved in the antibacterial immunity of Drosophila, which is unclear whether it is involved in the antibacterial immune process of crustaceans. In this article, we identified a Caspar gene in Eriocheir sinensis and named it EsCaspar. EsCaspar positively respond to bacterial stimulation and downregulate the expression of certain associated antimicrobial peptides by inhibiting the nuclear translocation of EsRelish. Thus, EsCaspar might be a suppressor of the immune deficiency (IMD) pathway that prevents over-activation of the immune system. Indeed, excess EsCaspar protein in crabs reduced resistance to bacterial infection. In conclusion, EsCaspar is a suppressor of the IMD pathway in crabs that plays a negative regulatory role in antimicrobial immunity.

Bactericidal permeability-increasing protein/LPS-binding protein (BPI/LBP) enhances resistance of golden pompano Trachinotus ovatus against bacterial infection.

Antimicrobial peptides are crucial components of innate immunity against microbial invasions. As a kind of antimicrobial peptides, bactericidal permeability-increasing protein (BPI)/lipopolysaccharide-binding protein (LBP) play vital roles in defending the host against gram-negative bacteria. In the current study, a novel BPI/LBP from Trachinotus ovatus (TroBPI/LBP) was characterized. The full length of TroBPI/LBP cDNA sequence is 1434 bp, which contained 477 amino acids. Multiple amino acid alignments of TroBPI/LBP shows 34.07%-84.49% identity with other fish BPI/LBP. Similar to other BPI/LBP, TroBPI/LBP also possesses an N-terminal signal peptide, a BPI/LBP/CETP N-terminal domain, and a BPI/LBP/CETP C-terminal domain. In vitro, the recombinant protein of TroBPI/LBP showed effective bacterial depression activity and binding activity to gram-negative bacteria. In vivo, TroBPI/LBP was constitutively expressed in tested tissues, and the highest expression level was in liver. Following Vibrio alginolyticus stimulation, the mRNA expression of TroBPI/LBP was significantly upregulated in immune-related tissues, and peaked at 12 h post-infection, which confirmed that TroBPI/LBP was highly sensitive to V. alginolyticus stimuli. Furthermore, functional analyses showed that the overexpression of TroBPI/LBP could enhance the ability of fish to against V. alginolyticus infection, and the knockdown of TroBPI/LBP significantly diminished bacterial clearance capacity post-infection. Therefore, these results suggest that TroBPI/LBP may play an important role in host defense against bacterial infection.

A new SVWC protein functions as a pattern recognition protein in antibacterial responses in Chinese mitten crab (Eriocheirsinensis).

Because invertebrates lack acquired immunity, they rely primarily on the innate immune system to defend themselves against viral and bacterial infections. SVWC, also called Vago, is a class of small-molecule proteins characterized by a single von Willebrand factor C-domain and appears to be restricted to arthropods. It has been reported that SVWC is involved in antiviral immunity in invertebrates, but whether it is involved in antimicrobial immunity and the mechanism of its involvement in antimicrobial immunity remains unclear. In this study, we identified a novel SVWC gene in Eriocheir sinensis and named it EsSVWC. EsSVWC was found to respond positively to bacterial stimulation and to regulate the expression of related antimicrobial peptides (AMPs). The EsSVWC protein recognized and bound to a variety of pathogen-associated molecular patterns (PAMPs) but did not exhibit direct bactericidal effects. Thus, the EsSVWC protein in crabs helps resist bacterial infection and improve survival rates. In summary, EsSVWC may regulate the innate immune system of crabs in response to microbial invasion in an indirect manner.

The role of ficolin as a pattern recognition receptor in antibacterial immunity in Eriocheir sinensis.

Ficolin, a member of the fibrinogen-related proteins family (FREPs), functions as a pattern recognition receptor (PRR) in vertebrates and in invertebrates as a novel lectin. In this study, we discovered the Ficolin homolog of Chinese mitten crab (Eriocheir sinensis), which we named EsFicolin. The obtained sequence showed that it has a highly conserved C-terminal fibrinogen-related domain (FReD) and a coiled-coil structure for trimer formation. EsFicolin was up-regulated in hemocytes after being stimulated by bacteria. Recombinant EsFicolin protein binds to gram-negative and gram-positive bacteria and agglutinates bacteria through pathogen-associated molecular patterns. In-depth study found that recombinant EsFicolin could effectively remove bacteria and showed direct antibacterial activity. EsFicolin could also promote the phagocytosis of hemocytes to enhance bacterial clearance. These findings suggest that EsFicolin plays an important role in the crab antibacterial immune response.

Cathepsin C (CTSC) contributes to the antibacterial immunity in golden pompano (Trachinotus ovatus).

Cathepsins, as a class of protein hydrolases, are widely found in the lysosomes of many tissues and play an essential role in various physiological activities. Cathepsin C (CTSC), a lysosomal cysteine protease, is an essential component of the lysosomal hydrolase family. In this study, we identified a CTSC from Trachinotus ovatus (TroCTSC) and analyzed its function. TroCTSC contained an ORF of 1368 bp and encoded 455 amino acids, which included three conserved catalytically active sites (Cys251, His397, and Asn419). It shares high homology (69.47%-90.77%) with the other known CTSC sequences of teleosts, which was most closely related to Seriola dumerili. TroCTSC was most abundant in the muscle, liver, and head kidney. After Vibrio harveyi infection, the expression levels of TroCTSC in liver, spleen, and head kidney were significantly up-regulated. TroCTSC was found in the cytoplasm with some of which were co-located with the lysosome. After V. harveyi stimulation, TroCTSC was translocated to nucleus in golden pompano snout (GPS) cells. In vitro, results revealed that the optimal hydrolase activity of the recombinant protein, rTroCTSC, was at 40 °C and pH 5.5. The activity of rTroCTSC was promoted by Zn2+ and Ca2+ but inhibited by Fe2+ and Cu2+. However, three mutant proteins, rTroCTSC-C251A, rTroCTSC-H397A, rTroCTSC-N419A, were dramatically reduced the proteolytic activity. Furthermore, in vivo results showed that overexpression of TroCTSC could significantly enhance body's ability to resist V. harveyi and promote the expression of proinflammatory cytokines, including interleukin 1-beta (IL-1ß), IL-6, IL-8, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). In contrast, the interference of TroCTSC expression induced a significant increase in the number of bacteria after V. harveyi infection. Our results suggested that TroCTSC was an essential effector of the innate immune system and played a pivotal role in antibacterial immunity.

Membrane orthologs of TLR5 of tongue sole Cynoglossus semilaevis: Expression patterns, signaling pathway and antibacterial property.

Mammalian toll-like receptor 5 (TLR5) is crucial for recognizing bacterial flagellin and initiating the inflammatory signaling cascades via myeloid differentiation factor 88 (MyD88) signaling pathway, which plays vital roles in innate immune against pathogenic bacteria. Herein, we reported the signaling pathway and antibacterial property of tongue sole (Cynoglossus semilaevis) membrane forms of TLR5 (i.e. CsTLR5M1and CsTLR5M2). CsTLR5M1/M2 contain 936 and 885 amino acid residues respectively. CsTLR5M1 shares 86.7% overall sequence identities with CsTLR5M2. CsTLR5M1/M2 possess the same extracellular domain (ECD) and transmembrane domain (TMD), but the different toll-interleukin-1 receptor (TIR) domain. CsTLR5M1/M2 expression occurred constitutively in multiple tissues and regulated by bacterial stimulation. Recombinant CsTLR5M1/M2 (rCsTLR5M) could bind to flagellin and Gram-negative/positive bacteria, which could suppress bacterial growth. Stimulation of the CsTLR5M pathway by flagellin resulted in increased expression of MyD88-dependent signaling molecules and inflammatory cytokines. Blocking rCsTLR5M by antibody markedly reduced the phagocytosis and ROS production of peripheral blood leukocytes (PBLs), which in turn in vivo promoted the dissemination of bacteria. Overall, these observations add new insights into the signaling pathway and immune function of teleost TLR5M.

Antheraea pernyi Suppressor of Cytokine Signaling 2 Negatively Modulates the JAK/STAT Pathway to Attenuate Microbial Infection.

The Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway has been shown to govern various physiological processes, including immune responses, hematopoiesis, cell growth, and differentiation. Recent studies show that suppressors of cytokine signaling (SOCS) proteins attenuate JAK-STAT signaling in mammals; however, their functions are less clear in lepidopteran insects. Here, we report a full-length sequence of SOCS-2 from the Chinese oak silkworm Antheraea pernyi (designated as ApSOCS-2) and study its biological role in immune responses via the JAK-STAT pathway. ApSOCS-2 expression was high in the fat bodies and hemocytes of A. pernyi fifth instar larvae. After pathogen infection with nucleopolyhedrovirus, Beauveria bassiana, Escherichia coli, and Microccus luteus, ApSOCS-2 mRNA was strongly increased compared to the control group. To elucidate the possible involvement in innate immunity, we measured antimicrobial peptide genes expression profiles in the fat body of A. pernyi. In contrast, recombinant ApSOCS-2 protein administration significantly reduced the AMPs transcription, while the depletion of ApSOCS-2 by RNAi increased their expression. Furthermore, we observed higher antibacterial activity and lower bacterial replication in dsApSOCS-2-treated larvae. The ApSOCS-2 transcription level was reduced in STAT depleted A. pernyi larvae challenged by M. luteus. The ApSOCS-2 RNAi data sets were also subjected to transcriptomic analysis, which suggests that ApSOCS-2 is a key regulator of immune function. Taken together, our data suggest that ApSOCS-2 is required for the negative regulation of AMPs transcripts via the JAK-STAT pathway in the insect.

[Rosmarinic acid bolsters antibacterial immunity activity of macrophages by up-regulating PINK1/Parkin-mediated mitophagy].

This study aims to explore the molecular mechanism through which rosmarinic acid up-regulates mitophagy and enhances antibacterial immunity activity of macrophages. To be specific, RAW264.7 macrophages were treated with rosmarinic acid and then infected with Staphylococcus aureus. The total mRNA and proteins of the cells were then extracted. The mRNA and protein levels of phosphatase and tensin homolog(PTEN)-induced putative kinase 1(PINK1) were detected by q-PCR and Western blot, respectively. Cell mitochondria isolation kit was employed to isolate mitochondria in macrophages. Recruitment of E3 ubiquitin ligase Parkin to mitochondria and the phosphorylation of Parkin were detected by Western blot. Co-immunoprecipitation and laser confocal microscopy were employed to observe the co-localization of PINK1 and Parkin. Mitochondrial division inhibitor 1(Mdivi-1), small interfering RNA(siRNA)-directed gene knockdown, and plate-colony counting were used to detect the levels of inflammatory cytokines and the intracellular antibacterial ability, in an attempt to confirm that rosmarinic acid promotes antibacterial immunity activity of macrophages through strengthening PINK1/Parkin-mediated mitophagy. The results showed that rosmarinic acid up-regulated the mRNA and protein expression of PINK1, promoted the recruitment of Parkin from cytoplasm to mitochondria and the phosphorylation, and enhanced the interaction between PINK1 and Parkin and their co-localization in macrophages. Blocking mitophagy or knocking PINK1 significantly abrogated the promotion of macrophage antibacterial immune response by rosmarinic acid. In summary, rosmarinic acid enhances antibacterial immunity activity of macrophages through up-regulating PINK1/Parkin-mediated mitophagy.

Differential immune responses of immunoglobulin Z subclass members in antibacterial immunity in a zebrafish model.

Immunoglobulin Z (IgZ) or its equivalent immunoglobulin T (IgT) is a newly identified immunoglobulin (Ig) class from teleost fish. This Ig class is characterized by its involvement in mucosa-associated lymphoid tissues (MALTs) for mucosal defence against pathogen infection. Recently, several subclass members of IgZ/IgT, such as IgZ, IgZ2, Igτ1, Igτ2 and Igτ3, have been further identified from zebrafish, common carp and rainbow trout. However, the functional diversity and correlation among these subclasses remain uncertain. Here, we explored the differential immune reactions of the IgZ and IgZ2 subclasses in antibacterial immunity in a zebrafish model. IgZ was extensively distributed in the peripheral serum and skin/gill MALTs and showed a rapid induction upon bacterial infection. IgZ2 was specialized in skin/gill MALTs and showed a strong induction following IgZ production. Correspondingly, the IgZ+ B cells had a wider distribution in the systemic primary/secondary lymphoid tissues and MALTs than the IgZ2+ B cells, which were predominant in MALTs. IgZ and IgZ2 exhibited a complementary effect in antibacterial immunity by possessing differential abilities. That is, IgZ is preferentially involved in bactericidal reaction that is in part C1q-dependent, and IgZ2 participates in neutralization action through bacteria-coating activity. The production of IgZ largely depended on the αß T/CD4+ T cells, whereas that of IgZ2 did not, suggesting the different dependencies of IgZ and IgZ2 on systemic immunity. Our findings demonstrate that the functional behaviour and mechanism of the IgZ/IgT family are more diverse than previously recognized and thus improve the current knowledge about this ancient Ig class.