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• W3134228214 abstract "•Microplitis bicoloratus bracovirus (MbBV) induced apoptotic cell disassembly•Apoptotic body formation accompanied innexin hemichannel closure•MbBV suppressed PI3K/AKT signaling and activated caspase-3•Innexin cleavage by activated caspase-3 was involved in hemichannel closure Cell-cell communication is necessary for cellular immune response. Hemichannel closure disrupts communication between intracellular and extracellular environments during polydnavirus-induced immunosuppression in invertebrates. However, the effects of hemichannel closure on cellular immune response are unclear. Here, we examined apoptotic body formation triggered by hemichannel closure in hemocytes of Spodoptera litura infected with bracovirus from the parasitic wasp, Microplitis bicoloratus. We showed that Microplitis bicoloratus bracovirus (MbBV) induced apoptotic cell disassembly, accompanied by hemichannel closure. Hemocyte apoptotic body formation was caused by the dysregulation of the innexins (Inxs), Inx1, Inx2, Inx3, and Inx4, during the MbBV-mediated inhibition of pI3K/AKT signaling and activation of caspase-3, which cleaved gap junction Inx proteins. Our results showed that hemichannel opening or closure in response to various stimuli, which induces the modulation of Inx levels, could inhibit or activate apoptotic body formation, respectively. Therefore, the “hemichannel open and close” model may regulate the cellular immune response. Cell-cell communication is necessary for cellular immune response. Hemichannel closure disrupts communication between intracellular and extracellular environments during polydnavirus-induced immunosuppression in invertebrates. However, the effects of hemichannel closure on cellular immune response are unclear. Here, we examined apoptotic body formation triggered by hemichannel closure in hemocytes of Spodoptera litura infected with bracovirus from the parasitic wasp, Microplitis bicoloratus. We showed that Microplitis bicoloratus bracovirus (MbBV) induced apoptotic cell disassembly, accompanied by hemichannel closure. Hemocyte apoptotic body formation was caused by the dysregulation of the innexins (Inxs), Inx1, Inx2, Inx3, and Inx4, during the MbBV-mediated inhibition of pI3K/AKT signaling and activation of caspase-3, which cleaved gap junction Inx proteins. Our results showed that hemichannel opening or closure in response to various stimuli, which induces the modulation of Inx levels, could inhibit or activate apoptotic body formation, respectively. Therefore, the “hemichannel open and close” model may regulate the cellular immune response. Immunosuppression occurs during parasitization when endoparasitic wasps inject polydnaviruses into their caterpillar hosts (Bitra et al., 2012Bitra K. Suderman R.J. Strand M.R. Polydnavirus Ank proteins bind NF-kappaB homodimers and inhibit processing of Relish.PLoS Pathog. 2012; 8: e1002722Crossref PubMed Scopus (59) Google Scholar; Luo and Pang, 2006Luo K.-J. Pang Y. Spodoptera litura multicapsid nucleopolyhedrovirus inhibits Microplitis bicoloratus polydnavirus-induced host granulocytes apoptosis.J. Insect Physiol. 2006; 52: 795-806Crossref PubMed Scopus (7) Google Scholar; Thoetkiattikul et al., 2005Thoetkiattikul H. Beck M.H. Strand M.R. Inhibitor kappaB-like proteins from a polydnavirus inhibit NF-kappaB activation and suppress the insect immune response.Proc. Natl. Acad. Sci. U S A. 2005; 102: 11426-11431Crossref PubMed Scopus (126) Google Scholar). In some host-parasitoid systems, polydnaviruses induce apoptosis in the host hemocytes (Luo and Pang, 2006Luo K.-J. Pang Y. Spodoptera litura multicapsid nucleopolyhedrovirus inhibits Microplitis bicoloratus polydnavirus-induced host granulocytes apoptosis.J. Insect Physiol. 2006; 52: 795-806Crossref PubMed Scopus (7) Google Scholar; Strand and Pech, 1995Strand M.R. Pech L.L. Microplitis demolitor polydnavirus induces apoptosis of a specific haemocyte morphotype in Pseudoplusia includens.J. Gen. Virol. 1995; 76: 283-291Crossref PubMed Scopus (133) Google Scholar). Thus far, the fate of apoptotic hemocytes in this process remains unknown. It is well known that parasitoid polydnaviruses regulate the host's innate immune response. Humoral and cellular immunity are two arms of the insect innate immune systems, and their functions usually overlap (Stanley and Kim, 2014Stanley D. Kim Y. Eicosanoid signaling in insects: from discovery to plant protection.Crit. Rev. Plant Sci. 2014; 33: 20-63Crossref Scopus (81) Google Scholar). Humoral immunity mainly mediates the production of antimicrobial peptides, while cellular immunity destroys pathogens through phagocytosis, nodulation, and encapsulation (Ye et al., 2018Ye X.Q. Shi M. Huang J.H. Chen X.X. Parasitoid polydnaviruses and immune interaction with secondary hosts.Dev. Comp. Immunol. 2018; 83: 124-129Crossref PubMed Scopus (20) Google Scholar). For example, hemocytes kill parasitic eggs by forming a multilayer sheath and encapsulating them (Lavine and Strand, 2002Lavine M.D. Strand M.R. Insect hemocytes and their role in immunity.Insect Biochem. Mol. Biol. 2002; 32: 1295-1309Crossref PubMed Scopus (1068) Google Scholar; Stanley et al., 2009Stanley D. Miller J. Tunaz H. Eicosanoid actions in insect immunity.J. Innate Immun. 2009; 1: 282-290Crossref PubMed Scopus (77) Google Scholar); during this process, cell-to-cell communication occurs. The parasitoid polydnavirus is believed to inhibit cell-to-cell communication to protect the parasitic eggs from the immune response of the lepidopteran host. As early as 1995, it was discovered that the Microplitis demolitor bracovirus (MdBV) induced apoptosis and inactivated hemocytes (Strand and Pech, 1995Strand M.R. Pech L.L. Microplitis demolitor polydnavirus induces apoptosis of a specific haemocyte morphotype in Pseudoplusia includens.J. Gen. Virol. 1995; 76: 283-291Crossref PubMed Scopus (133) Google Scholar). Follow-up studies also found the same phenomenon in hemocytes infected by the bracoviruses from Cotesia congregata, Microplitis bicoloratus, and Snellenius manilae (Dong et al., 2017Dong S.-M. Cui J.-H. Zhang W. Zhang X.-W. Kou T.-C. Cai Q.-C. Xu S. You S. Yu D.-S. Ding L. et al.Inhibition of translation initiation factor eIF4A is required for apoptosis mediated by Microplitis bicoloratus bracovirus.Arch. Insect Biochem. Physiol. 2017; 96: e21423Crossref Scopus (14) Google Scholar; Le et al., 2003Le N.T. Asgari S. Amaya K. Tan F.F. Beckage N.E. Persistence and expression of Cotesia congregata polydnavirus in host larvae of the tobacco hornworm, Manduca sexta.J. Insect Physiol. 2003; 49: 533-543Crossref PubMed Scopus (42) Google Scholar; Tang et al., 2021Tang C.K. Tsai C.H. Wu C.P. Lin Y.H. Wei S.C. Lu Y.H. Li C.H. Wu Y.L. MicroRNAs from Snellenius manilae bracovirus regulate innate and cellular immune responses of its host Spodoptera litura.Commun. Biol. 2021; 4: 52Crossref PubMed Scopus (3) Google Scholar). Further studies have shown that polydnaviruses use gene products to induce host hemocyte apoptosis, such as the protein tyrosine phosphatase (PTP) of the bracovirus gene family (Ye et al., 2018Ye X.Q. Shi M. Huang J.H. Chen X.X. Parasitoid polydnaviruses and immune interaction with secondary hosts.Dev. Comp. Immunol. 2018; 83: 124-129Crossref PubMed Scopus (20) Google Scholar). PTP can dephosphorylate target proteins, thereby regulating intracellular signal transmission (Eum et al., 2010Eum J.H. Bottjen R.C. Pruijssers A.J. Clark K.D. Strand M.R. Characterization and kinetic analysis of protein tyrosine phosphatase-H2 from Microplitis demolitor bracovirus.Insect Biochem. Mol. Biol. 2010; 40: 690-698Crossref PubMed Scopus (15) Google Scholar; Pruijssers and Strand, 2007Pruijssers A.J. Strand M.R. PTP-H2 and PTP-H3 from Microplitis demolitor Bracovirus localize to focal adhesions and are antiphagocytic in insect immune cells.J. Virol. 2007; 81: 1209-1219Crossref PubMed Scopus (62) Google Scholar; Serbielle et al., 2012Serbielle C. Dupas S. Perdereau E. Hericourt F. Dupuy C. Huguet E. Drezen J.M. Evolutionary mechanisms driving the evolution of a large polydnavirus gene family coding for protein tyrosine phosphatases.BMC Evol. Biol. 2012; 12: 253Crossref PubMed Scopus (28) Google Scholar). The expression of PTP-H2 (MdBV) in the Sf21 cell line was found to induce apoptosis (Suderman et al., 2008Suderman R.J. Pruijssers A.J. Strand M.R. Protein tyrosine phosphatase-H2 from a polydnavirus induces apoptosis of insect cells.J. Gen. Virol. 2008; 89: 1411-1420Crossref PubMed Scopus (43) Google Scholar). Therefore, the regulation of PTP in host cell signaling pathways may be one of the ways by which polydnaviruses induce hemocyte apoptosis and inhibit host immune function. Early studies have described the viral ankyrin gene (vank), another member of the bracovirus gene family, as an inhibitor of NF-κB; and similar to IκB, it can inhibit the NF-κB signaling pathway of host cells (Bitra et al., 2012Bitra K. Suderman R.J. Strand M.R. Polydnavirus Ank proteins bind NF-kappaB homodimers and inhibit processing of Relish.PLoS Pathog. 2012; 8: e1002722Crossref PubMed Scopus (59) Google Scholar; Gueguen et al., 2013Gueguen G. Kalamarz M.E. Ramroop J. Uribe J. Govind S. Polydnaviral ankyrin proteins aid parasitic wasp survival by coordinate and selective inhibition of hematopoietic and immune NF-kappa B signaling in insect hosts.PLoS Pathog. 2013; 9: e1003580Crossref PubMed Scopus (55) Google Scholar). In our latest study, we showed that the Microplitis demolitor bracovirus (MbBV) vank protein interacts with dorsal interaction protein 3 and inhibits the transcription of the translation initiation factor eIF4E, thereby inducing the transcription of downstream target genes, such as inx2 and inx3 (Cai et al., 2021Cai Q.-C. Chen C.-X. Liu H.-Y. Zhang W. H Y.-F. Zhang Q. Zhou G.-F. Xu S. Liu T. Xiao W. et al.Interactions of Vank proteins from Microplitis bicoloratus bracovirus with host Dip3 suppress eIF4E expression.Dev. Comp. Immunol. 2021; 118: 103994Crossref PubMed Scopus (1) Google Scholar). This overexpression of Innexin (Inx) 2 and Inx3 promotes apoptosis in Sf9 and Spli221 cells by activating a low level of caspase-3 (Liu et al., 2013Liu T. Li M. Zhang Y. Pang Z. Xiao W. Yang Y. Luo K. A role for Innexin2 and Innexin3 proteins from Spodoptera litura in apoptosis.PLoS One. 2013; 8: e70456Crossref PubMed Scopus (25) Google Scholar). Inxs are the structural elements of hemichannels. Decrease in the transcription level of inx or increase in the number of Inx proteins affect hemichannel function, depending on the steady state levels of Inx on the cell surface (Pang et al., 2015Pang Z. Li M. Yu D. Zhang Y. Liu X. Ji X. Yang Y. Hu J. Luo K. Two innexins of Spodoptera litura influences hemichannel and gap junction functions in cellular immune responses.Arch. Insect Biochem. Physiol. 2015; 90: 43-57Crossref PubMed Scopus (10) Google Scholar). Findings from studies on vertebrates have shown that apoptotic cells form apoptotic bodies, which are rapidly cleared by neighboring phagocytic cells to prevent inflammation (Bellone et al., 1997Bellone M. Iezzi G. Rovere P. Galati G. Ronchetti A. Protti M.P. Davoust J. Rugarli C. Manfredi A.A. Processing of engulfed apoptotic bodies yields T cell epitopes.J. Immunol. 1997; 159: 5391-5399PubMed Google Scholar; Brock et al., 2017Brock C.K. Wallin S.T. Mandal A. Sumner E.A. Eisenhoffer G.T. Stem cell proliferation is induced by engulfment of apoptotic bodies from adjacent dying cells during epithelial tissue maintenance.Nat. Commun. 2017; 28: 1-11Google Scholar). However, the mechanism by which polydnavirus-induced apoptosis mediates cell disassembly is not clearly understood. Recently, we found that hemichannel closure involves an N-terminal, elongated Inx hemichannel (Chen et al., 2016Chen Y.-B. Xiao W. Li M. Zhang Y. Yang Y. Hu J.-S. Luo K.-J. N-terminally elongated SpliInx2 and SpliInx3 reduce baculovirus-triggered apoptosis via hemichannel closure.Arch. Insect Biochem. Physiol. 2016; 92: 24-37Crossref PubMed Scopus (5) Google Scholar; Guo et al., 2015Guo L.E. Zhang J.F. Liu X.Y. Zhang L.M. Zhang H.L. Chen J.H. Xie X.G. Zhou Y. Luo K.-J. Yoon J. Phosphate ion targeted colorimetric and fluorescent probe and its use to monitor endogeneous phosphate ion in a hemichannel-closed cell.Anal. Chem. 2015; 87: 1196-1201Crossref PubMed Scopus (35) Google Scholar). Cell-cell communication is inhibited upon hemichannel closure as suppressed immune cells cannot initiate encapsulation, nodulation, or phagocytosis. However, the mechanism underlying apoptosis induction due to blocked cell communication and the fate of apoptotic cells has not been determined. We investigated the formation of apoptotic bodies triggered by hemichannel closure following MbBV infection of hemocytes derived from the host, Spodoptera litura, or from cell lines derived from S. frugiperda pupal tissue. To investigate the disassembly of MbBV-induced apoptotic cells forming apoptotic bodies, we used hemocytes of the host, S. litura, its cell line, Spli221, and S. frugiperda-derived Sf9, which can undergo inducible apoptosis caused by MbBV infection (Figure 1). We unexpectedly observed that MbBV induced the disassembly of Sf9 cells in vitro during time-lapse microscopy. Using the same quantity of bracovirus for infection (three wasp equivalents) and increasing the incubation period, we observed that more cells formed apoptotic bodies, some of which could be labeled using Annexin V-FITC and some, like late apoptotic cells, using Annexin V-FITC and propidium iodide (PI) (Figures 1A‒1C). To determine whether this apoptotic induction occurred naturally in the wasp host, S. litura, we compared hemocytes from non-parasitized and parasitized hosts 6 days post-parasitization. We found a significantly higher number of apoptotic bodies in the hemocytes from parasitized hosts than in those from non-parasitized hosts (Figures 1D and 1E). We had previously reported hemichannel closure during reBac-virus infection (Chen et al., 2016Chen Y.-B. Xiao W. Li M. Zhang Y. Yang Y. Hu J.-S. Luo K.-J. N-terminally elongated SpliInx2 and SpliInx3 reduce baculovirus-triggered apoptosis via hemichannel closure.Arch. Insect Biochem. Physiol. 2016; 92: 24-37Crossref PubMed Scopus (5) Google Scholar; Guo et al., 2015Guo L.E. Zhang J.F. Liu X.Y. Zhang L.M. Zhang H.L. Chen J.H. Xie X.G. Zhou Y. Luo K.-J. Yoon J. Phosphate ion targeted colorimetric and fluorescent probe and its use to monitor endogeneous phosphate ion in a hemichannel-closed cell.Anal. Chem. 2015; 87: 1196-1201Crossref PubMed Scopus (35) Google Scholar). Hence, to determine whether hemichannel closure occurred during MbBV infection of Sf9 cells, we used TO-PRO-3, which can pass through open hemichannels (Figure 1F), and found that MbBV significantly decreased TO-PRO-3 uptake in a viral-dose-dependent manner (Figures 1H and 1I). Carbenoxolone (CBX) is a pannexin and connexin hemichannel/gap junction inhibitor, which inhibits Sf9 hemichannel opening (Luo and Turnbull, 2011Luo K. Turnbull M.W. Characterization of nonjunctional hemichannels in caterpillar cells.J. Insect Sci. 2011; 11: 6Crossref PubMed Scopus (28) Google Scholar). We determined that both MbBV and CBX inhibited Sf9 hemichannels to similar extents (Figures 1J and 1K). Based on the information regarding MbBV-induced apoptosis (Luo and Pang, 2006Luo K.-J. Pang Y. Spodoptera litura multicapsid nucleopolyhedrovirus inhibits Microplitis bicoloratus polydnavirus-induced host granulocytes apoptosis.J. Insect Physiol. 2006; 52: 795-806Crossref PubMed Scopus (7) Google Scholar), we tested our hypothesis that hemichannel closure in MbBV-infected cells would persist throughout the apoptotic process triggered by the virus. We performed a set of assays to detect hemichannels in the different stages of apoptosis using PI and Annexin V-labeled FITC in MbBV-infected cells. PI can pass through hemichannels without endocytosis at 4°C for 5 min (Luo and Turnbull, 2011Luo K. Turnbull M.W. Characterization of nonjunctional hemichannels in caterpillar cells.J. Insect Sci. 2011; 11: 6Crossref PubMed Scopus (28) Google Scholar) (Figure 1G). Similar to hemichannel closure by CBX, we observed hemichannel closure during MbBV infection at 12, 24, and 48 hr post-infection (p.i.) (Figures 1L and 1M). Flow cytometric analysis revealed an increase in early apoptotic cells at 12 and 24 hr but not at 48 hr p.i. and a significant increase in late apoptotic cells from 12 to 48 hr p.i. but not 24 hr (Figures 1N and 1O). Next, we investigated the relationship between hemichannel closure and apoptotic body formation by comparing the effects of CBX, zeocin, an inducer of apoptosis and DNA double-strand breaks (Delacôte et al., 2007Delacôte F. Deriano L. Lambert S. Bertrand P. Saintigny Y. Lopez B.S. Chronic exposure to sublethal doses of radiation mimetic Zeocin™ selects for clones deficient in homologous recombination.Mutat. Res. 2007; 615: 125-133Crossref PubMed Scopus (8) Google Scholar), and MbBV. CBX induced membrane blebbing (Figure 1P, Video S1) but did not induce cellular disassembly. Zeocin killed cells but did not induce cellular disassembly (Figure 1P, Video S2). Interestingly, only MbBV induced cell apoptosis and promoted apoptotic cell disassembly (Figure 1P; Video S3). These findings indicate that MbBV induced apoptosis in cells along with the formation of apoptotic bodies, followed by hemichannel closure and the disassembly of cells (Figure 1Q). These results suggest that apoptosis and hemichannel closure are required for apoptotic cell disassembly. https://www.cell.com/cms/asset/6bc7134a-60e8-4086-b0ac-ffd7df182120/mmc2.mp4Loading ... Download .mp4 (0.76 MB) Help with .mp4 files Video S1. CBX induced membrane blebbing, related to Figure 1 https://www.cell.com/cms/asset/5643d5ed-ade0-4c8d-8c40-964a5b0d880d/mmc3.mp4Loading ... Download .mp4 (0.05 MB) Help with .mp4 files Video S2. Zeocin induced cell death but not cellular disassembly, related to Figure 1 https://www.cell.com/cms/asset/a6bbb7d0-a3fa-4147-b23a-1f160b69955f/mmc4.mp4Loading ... Download .mp4 (0.18 MB) Help with .mp4 files Video S3. MbBV induced cell apoptosis and promoted the disassembly of apoptotic cells, related to Figure 1 Inxs form hemichannels in invertebrates (Güiza et al., 2018Güiza J. Barría I. Sáez J.C. Vega J.L. Innexins: expression, regulation, and functions.Front. Physiol. 2018; 9: 1414Crossref PubMed Scopus (22) Google Scholar; Luo and Turnbull, 2011Luo K. Turnbull M.W. Characterization of nonjunctional hemichannels in caterpillar cells.J. Insect Sci. 2011; 11: 6Crossref PubMed Scopus (28) Google Scholar). To examine the regulation of Inx proteins by MbBV for hemichannel closure, we performed genome analysis (Figure 2A) of M. bicoloratus-parasitized hemocytes of S. litura and MbBV-infected Spli221 cell line hemocytes by searching for host integration motifs (HIMs) (Beck et al., 2011Beck M.H. Zhang S. Bitra K. Burke G.R. Strand M.R. The encapsidated genome of Microplitis demolitor bracovirus integrates into the host Pseudoplusia includens.J. Virol. 2011; 85: 11685-11696Crossref PubMed Scopus (38) Google Scholar; Chevignon et al., 2018Chevignon G. Periquet G. Gyapay G. Vega-Czarny N. Musset K. Drezen J.-M. Huguet E. Cotesia congregata Bracovirus circles encoding PTP and Ankyrin genes integrate into the DNA of parasitized Manduca sexta hemocytes.J. Virol. 2018; 92 (e00438-00418)Crossref PubMed Scopus (11) Google Scholar) to identify the sites of viral integration into host DNA. Four inx genes were found in three chromosomes, 2, 9, and 29, among the 31 pairs of chromosomes in S. litura (Cheng et al., 2017Cheng T. Wu J. Wu Y. Chilukuri R.V. Huang L. Yamamoto K. Feng L. Li W. Chen Z. Guo H. et al.Genomic adaptation to polyphagy and insecticides in a major East Asian noctuid pest.Nat. Ecol. Evol. 2017; 1: 1747-1756Crossref PubMed Scopus (139) Google Scholar). The HIMs, HIM-C16, and HIM-F157 (Figure S1A) were found near inx1 and inx2 in chromosome 2, indicating that MbBV DNA was not inserted into inx1 and inx2 (Figure 2B). Similarly, HIM-C16, HIM-C14, and HIM-F157 were found near inx3 in chromosome 9 (Figure 2B), and HIM-C16 and HIM-F157 were found near inx4 in chromosome 29 (Figure 2B). Therefore, MbBV DNA was not inserted into any of the four inx genes, which led us to question whether this location affected inx expression (Table S1). In addition, we performed proteome analysis (Figure 2C) of the hemocytes of S. litura. The proteomics data analysis revealed an absence of Inx1 and Inx4 proteins, normal levels of Inx2, and decreased levels of Inx3 (Table S2). To confirm these data, we measured the mRNA levels in hemocytes from S. litura larvae after parasitization by the wasp and in Spli221 cells infected by MbBV. inx1 mRNA could be detected in both the larvae and the hemocytes, albeit inconsistently, and the expression of inx2 and inx3 mRNAs was similar, whereas that of inx4 mRNA was consistently downregulated by MbBV (Figure 2D). The hemocyte expression of inx1, inx2, and inx3 was similar in MbBV-infected Spli221 cells; however, no inx4 mRNA was detected (Figure 2D). Combining the results of the mRNA and protein analyses, we concluded that MbBV downregulated inx1 and inx4 expression and inhibited Inx1 and Inx4 synthesis in hemocytes during parasitization and infection by MbBV, whereas inx2/3 mRNA and protein continued to be expressed. As these findings led to further questions regarding the roles of Inx1 and Inx4 in apoptotic body formation, we designed siRNAs to knock down the expression of all 4 inx genes (Figure S1B). Unexpectedly, none of the siRNAs triggered apoptotic body formation when used alone (Figure 2E, Video S4); however, the treatment of MbBV-infected cells with the combination of siRNAs against inx1 and inx4 significantly increased apoptotic body formation (Figures 2F and 2G). To confirm these results, we employed the CRISPR/Cas9 system using inx2 gRNA (Figure 2H) and found that although no apoptotic bodies were formed when we used gInx2-Target1 and gInx2-Target2, both alone and together (Figures 2I and 2J), apoptotic body formation increased in the presence of both the gRNAs and MbBV infection (Figures 2K and 2L). These results suggest that cooperation between MbBV infection and loss of inx is necessary for apoptotic body formation. https://www.cell.com/cms/asset/519868fb-f286-4c21-8552-c3652672a1ed/mmc5.mp4Loading ... Download .mp4 (0.1 MB) Help with .mp4 files Video S4. Inx siRNA did not trigger the formation of apoptotic bodies, related to Figure 2 To identify the factors responsible for Inx-mediated apoptotic body formation, we generated dsRNAs against all four inx genes (Figure S2) by feeding S. litura larvae and also determined the levels of apoptosis (Figure 3A) and apoptotic body formation (Figure 3B). We found that all four inx dsRNAs increased the number of apoptotic cells (Figure 3A), and flow cytometry analysis (Figure S3) revealed a significant increase in apoptotic body formation (Figures 3B and 3C), which suggests that Inx depletion can trigger apoptosis even in uninfected larvae. Cleaved caspase-3, which has been found to cleave connexin45.6 (Cx45.6) (Yin et al., 2001Yin X. Gu S. Jiang J.X. The development-associated cleavage of lens connexin 45.6 by caspase-3-like protease is regulated by casein kinase II-mediated phosphorylation.J. Biol. Chem. 2001; 276: 34567-34572Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), was detected in the hemocytes of larvae after dsRNA administration (Figure 3D). Indeed, cleaved caspase-3 levels were significantly higher (Figure 3E) in hemocytes after parasitization, whereas p85 levels had decreased considerably (Figure 3F). The reduction in p85 levels by treatment with dsRNA directed against the p85 gene increased the number of apoptotic hemocytes (Figures 3G and 3H). These results led to the investigation of the interaction between the pI3K/AKT signaling pathway, specifically via Ser473 and Thr308 of AKT, and MbBV. We found a viral-dosage-dependent decrease in the levels of AKT-Ser473 phosphorylation (Figures 3I and 3J), AKT-Thr308 phosphorylation (Figure 3K), and p85 (Figure 3L), accompanied by an increased level of cleaved caspase-3 (Figure 3M). To confirm whether p85 phosphorylated AKT-Ser473 and Thr308, we overexpressed p85 in the Spli221 cell line and found an increase in the levels of AKT-pSer473 but not of AKT-pThr308 (Figures 3N and 3O), suggesting the involvement of an MbBV-mediated decrease in p85-catalyzed phosphorylation of Ser473 in the observed apoptotic effects. Next, we investigated the factors from MbBV that could dephosphorylate Thr308. As PTP is known to dephosphorylate Thr308 in AKT and the levels of MbBV PTP109 are high during parasitization, we overexpressed PTP109 in Spli221 cells and found that AKT-Thr308 was dephosphorylated (Figures 3P and 3Q) to a greater extent than Ser473 (Figure 3R). Taken together, these results indicate that activated (cleaved) caspase-3, generated by MbBV, regulated Inx protein levels, which decreased p85-mediated phosphorylation of AKT-Ser473 and, along with viral PTP109-mediated dephosphorylation of AKT-Thr308, led to the inhibition of the pI3K/AKT signaling pathway. As Cx45.6 is a substrate of caspase-3 (Yin et al., 2001Yin X. Gu S. Jiang J.X. The development-associated cleavage of lens connexin 45.6 by caspase-3-like protease is regulated by casein kinase II-mediated phosphorylation.J. Biol. Chem. 2001; 276: 34567-34572Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), we examined whether Inx proteins are also substrates of caspase-3. Interestingly, Inx2 and Inx3 have cleavage sites for caspase-3 (Figures 3S and 3T), and immunoprecipitation results revealed that Inx2 and Inx3 were cleaved by activated caspase-3 (Figures S4A and S4B). Immunoprecipitation results revealed that both activated caspase-3 and MbBV infection led to the cleavage of Inx2 and Inx3 in Spli221 cells (Figures S4C and S4D) to ∼34 kDa fragments. The Spli221 cells showed basal levels of cleaved Inx2 and Inx3 (Figures S4E and S4F) and the cleavage of Inx2 and Inx3 followed a vial-dosage-dependent pattern (Figures 3U–3X). These results indicate that Inx proteins are substrates of activated caspase-3, which is generated by MbBV-mediated cleavage, and that the cleavage of Inx proteins decreased the p85-mediated effects and increased the PTP109-mediated effects on AKT-Ser473 and AKT-Thr308 phosphorylation, respectively. Based on the above results, we sought to confirm our conclusion that MbBV closed hemichannels by activating caspase-3 to trigger apoptotic cell disassembly. We used reBac-TEV-Inx2 and reBac-TEV-Inx3, which had shown hemichannel closure in infected cells (Chen et al., 2016Chen Y.-B. Xiao W. Li M. Zhang Y. Yang Y. Hu J.-S. Luo K.-J. N-terminally elongated SpliInx2 and SpliInx3 reduce baculovirus-triggered apoptosis via hemichannel closure.Arch. Insect Biochem. Physiol. 2016; 92: 24-37Crossref PubMed Scopus (5) Google Scholar; Guo et al., 2015Guo L.E. Zhang J.F. Liu X.Y. Zhang L.M. Zhang H.L. Chen J.H. Xie X.G. Zhou Y. Luo K.-J. Yoon J. Phosphate ion targeted colorimetric and fluorescent probe and its use to monitor endogeneous phosphate ion in a hemichannel-closed cell.Anal. Chem. 2015; 87: 1196-1201Crossref PubMed Scopus (35) Google Scholar), and used a tobacco etch virus (TEV) protease to cleave the TEV sites of the two reBac-TEV-Inxs (Figure 4A). Specifically, cells infected by reBac-TEV-Inxs showed an increase in AKT-pSer473 levels, highly stable AKT-pThr308 levels, and reduced cleavage of caspase-3; resultantly, Inx cleavage was limited (Figures 4B‒4G). Next, in the process of Inx recovery, cell co-infection with reBac-TEV-Inxs and reBac-TEVp resulted in the cleavage of the TEV sites and the loss of a 6×His fragment from the N-terminal ends of the Inx proteins (Figure 4H). All four recovered Inx proteins were detected when the cells were co-infected with both reBac-TEV-Inxs and reBac-TEVp (Figures 4I‒4L). Notably, the recovered Inx proteins led to the opening of the Inx hemichannels (Figures 4M and 4N). Simultaneously, the number of apoptotic cells was confirmed to decrease significantly (Figures 4O and 4P), suggesting that the opening of the Inx hemichannels reduced cell disassembly. Taken together, our results show that MbBV dephosphorylated AKT and activated caspase-3, which cleaved the Inx proteins, closed hemichannels, and promoted apoptotic cell disassembly; additionally, opening of the closed hemichannels reduced the formation of apoptotic bodies (Figure 4Q). The results of this study enable the advancement of several concepts. First, we identified that MbBV induced hemichannel closure to trigger apoptosis and promote apoptotic cell disassembly. Second, the cleavage of Inxs by activated caspase-3 was responsible for hemichannel closure mediated by MbBV via the suppression of pI3K/AKT signaling. Third, the opening of hemichannels formed by Inxs suppressed apoptosis via the modulation of Inx levels and inhibition of cell-cell communication, which attenuated immunosuppression in invertebrates and vertebrates. Contrary to the view that the opening of hemichannels mediates apoptosis (Chandrasekhar and Bera, 2012Chandrasekhar A. Bera A.K. Hemichannels: permeants and their effect on development, physiology and death.Cell Biochem. Funct. 2012; 30: 89-100Crossref PubMed Scopus (51) Google Scholar; Hur et al., 2003Hur K.C. Shim J.E. Johnson R.G. A potential role for cx43-hemichannels in staurosporin-induced apoptosis.Cell Commun. Adhes. 2003; 10: 271-277Crossref PubMed Scopus (27) Google Scholar), our study showed that MbBV promotes the unconventional apoptosis pathway of infected cells. We propose that, in the invertebrate host, the hemichannel switching mechanism is related to cell disintegration, which is consistent with the finding that pannexin1 channel activity is negatively correlated with the number of apoptotic bodies (Poon et al., 2014Poon I.K. Chiu Y.H. Armstrong A.J. Kinchen J.M. Juncadella I.J. Bayliss D.A. Ravichandran K.S. Unexpected link between an antibiotic, pannexin channels and apoptosis.Nature. 2014; 507: 329-334Crossref PubMed Scopus (144) Google Scholar). It is now well known that connexin (Yin et al., 2001Yin X. Gu S. Jiang J.X. The development-associated cleavage of lens connexin 45.6 by caspase-3-like protease is regulated by casein kinase II-mediated phosphorylation.J. Biol. Chem. 2001; 276: 34567-34572Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) and pannexin (Ruan et al., 2020Ruan Z. Orozco I.J. Du J. Lu W. Structures of human pannexin 1 reveal ion pathways and mechanism of gating.Nature. 2020; 584: 646-651Crossref PubMed Scopus (41) Google Scholar) are regulated by caspase, and our results also show that Inxs are regulated by caspase-3, completing the mechanism of interaction between the connexin and caspase families. Meanwhile, we also found that MbBV relies on pI3K/AKT signaling to release caspase to induce and accelerate cell apoptosis; this result is different from viruses that also use pI3K/AKT and need to replicate. For example, the enterovirus EV71 activates AKT to inhibit cell apoptosis in the early stages of infection and inhibits AKT phosphorylation to promote cell apoptosis until the late stage of infection (Zhang et al., 2015Zhang F. Liu Y. Chen X. Dong L. Zhou B. Cheng Q. Han S. Liu Z. Peng B. He X. et al.RASSF4 promotes EV71 replication to accelerate the inhibition of the phosphorylation of AKT.Biochem. Biophys. Res. Commun. 2015; 458: 810-815Crossref PubMed Scopus (8) Google Scholar). This suggests that MbBV may have a different infection mechanism from ordinary viruses and needs to be explored further. The intercellular transmission of small molecules plays a key role in the regulation of cell tissue homeostasis (Chen et al., 2021Chen C.-X. Luo K.-J. Yang J.-P. Huang Y.-C. Cardenas E.R. Nicholson B.J. Jiang J.X. Connexins and cAMP cross-talk in cancer progression and metastasis.Cancers (Basel). 2021; 13: 58Crossref Scopus (2) Google Scholar). For the immune system, cellular communication mediated by small molecules is particularly important due to the lack of gap junctions. Panx1 has been proven to be widely present in mammalian macrophages (Marina-Garcia et al., 2008Marina-Garcia N. Franchi L. Kim Y.G. Miller D. McDonald C. Boons G.J. Nunez G. Pannexin-1-mediated intracellular delivery of muramyl dipeptide induces caspase-1 activation via cryopyrin/NLRP3 independently of Nod2.J. Immunol. 2008; 180: 4050-4057Crossref PubMed Scopus (132) Google Scholar), neutrophils (Chen et al., 2010Chen Y. Yao Y. Sumi Y. Li A. To U.K. Elkhal A. Inoue Y. Woehrle T. Zhang Q. Hauser C. et al.Purinergic signaling: a fundamental mechanism in neutrophil activation.Sci. Signal. 2010; 3: ra45Crossref PubMed Scopus (148) Google Scholar), T cells (Orellana et al., 2013Orellana J.A. Velasquez S. Williams D.W. Saez J.C. Berman J.W. Eugenin E.A. Pannexin1 hemichannels are critical for HIV infection of human primary CD4+ T lymphocytes.J. Leukoc. Biol. 2013; 94: 399-407Crossref PubMed Scopus (51) Google Scholar), B cells, and dendritic cells (Saez et al., 2014Saez P.J. Shoji K.F. Aguirre A. Saez J.C. Regulation of hemichannels and gap junction channels by cytokines in antigen-presenting cells.Mediators Inflamm. 2014; 2014: 742734Crossref PubMed Scopus (45) Google Scholar). Some studies have reported that the main function of the Panx channel is to release ATP (Pelegrin and Surprenant, 2006Pelegrin P. Surprenant A. Pannexin-1 mediates large pore formation and interleukin-1beta release by the ATP-gated P2X7 receptor.EMBO J. 2006; 25: 5071-5082Crossref PubMed Scopus (1082) Google Scholar, Pelegrin and Surprenant, 2007Pelegrin P. Surprenant A. Pannexin-1 couples to maitotoxin- and nigericin-induced interleukin-1beta release through a dye uptake-independent pathway.J. Biol. Chem. 2007; 282: 2386-2394Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Extracellular ATP is closely involved in the immune response and is usually a pro-inflammatory factor (Faas et al., 2017Faas M.M. Sáez T. de Vos P. Extracellular ATP and adenosine: the Yin and Yang in immune responses?.Mol. Aspects. Med. 2017; 55: 9-19Crossref PubMed Scopus (83) Google Scholar); however, it may also have anti-inflammatory properties under certain conditions, and its role in the immune response depends on the relative balance between its inflammatory properties (Faas et al., 2017Faas M.M. Sáez T. de Vos P. Extracellular ATP and adenosine: the Yin and Yang in immune responses?.Mol. Aspects. Med. 2017; 55: 9-19Crossref PubMed Scopus (83) Google Scholar). Therefore, hemichannel closure blocks the transmission of immune signals and inhibits the immune response. In conclusion, this study revealed a mechanism whereby MbBV-mediated hemichannel closure was activated in MbBV infection-induced immunosuppression during the parasitization of S. litura via the inhibition of pI3K/AKT signaling; additionally, apoptosis was also promoted by the activation of caspase-3, a manifestation of the “hemichannel open and close” theory of regulated cellular immune response. In terms of the limitations, our data are primarily based on the Microplitis bicoloratus bracovirus (MbBV)-Microplitis bicoloratus-Spodoptera litura model. Although we confirmed MbBV cellular immunity via hemichannel closure in innate immunity, the regulation of humoral immunity by MbBV may affect hemichannels. Given that the novel “hemichannel opening and closure” model proposes the global regulation between cellular immunity and humoral immunity, further investigation will be necessary to fully understand the molecular mechanisms underlying the link between cellular immunity-hemichannels-humoral immunity in innate immunity during MbBV infection. Further information and requests for resources and reagent should be directed to and will be fulfilled by the lead contact, Kai-Jun Luo ( [email protected] ). This study did not generate new unique reagents. This study did not generate data sets. All methods can be found in the accompanying transparent methods supplemental file. We thank Dr. Andrew J. Saurin (Aix-Marseille University, IBDML) for generously providing the protocol for the determination of a high-titer Bac-to-Bac virus based on cell cycle arrest. This study was funded by the Science and Technology Planning Project in Key Areas of Yunnan Province, China [grant numbers 202001BB050002 ], the National Natural Science Foundation of China, China [grant numbers 31772225 , 31471823 , 31260448 , 31060251 ], and the NSFC- NRF, China [grant number 31411140238 ] to K.L. This study received funding from the Yunnan Department of Science and Technology, China [grant number 2013FA003 to K.L. and 2018IA100 to W.X.]. K.L. was also supported by the Donglu Scholar Program of Yunnan University, China . K.L., C.C., H.H., Q.C., T.K., W.Z., W.X., and Q.Z. designed experiments. C.C., W.Z., and X.Z. constructed the L4440 vector and prepared dsRNA feeding. H.H., S.Y., and T.L. constructed CRISPR/Cas9 plasmids. H.H., C.C., and X.Z. constructed bacmids and generated viruses. Q.C. performed genome analysis and proteome analysis. C.C. and Y.C. performed flow cytometry. C.C., H.H., T.K., and S.Y. performed time-lapse experiments. C.C. organized the data. K.L., C.C., Q.C., and X.W. wrote the manuscript. K.L. and W.X. conceived the project. The authors declare no competing interests. Download .pdf (.63 MB) Help with pdf files Document S1. Transparent methods, Figures S1–S4, and Tables S1 and S2" @default.
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