MicroRNAs are regulatory molecules that can be packaged into exosomes to modulate cellular response of recipients. While the role of exosomes during viral infection is beginning to be appreciated, the involvement of exosomal miRNAs in immunoregulation in invertebrates has not been addressed. Here, we observed that exosomes released from WSSV-injected mud crabs could suppress viral replication by inducing apoptosis of hemocytes. Besides, miR-137 and miR-7847 were found to be less packaged in mud crab exosomes during viral infection, with both miR-137 and miR-7847 shown to negatively regulate apoptosis by targeting the apoptosis-inducing factor (AIF). Our data also revealed that AIF translocated to the nucleus to induce DNA fragmentation, and could competitively bind to HSP70 to disintegrate the HSP70-Bax (Bcl-2-associated X protein) complex, thereby activating the mitochondria apoptosis pathway by freeing Bax. The present finding therefore provides a novel mechanism that underlies the crosstalk between exosomal miRNAs and apoptosis pathway in innate immune response in invertebrates.
21MicroRNAs are regulatory molecules that can be packaged into exosomes to 22 modulate recipient's cellular response, while their role during viral infection is 23 beginning to be appreciated. However, the involvement of exosomal miRNAs during 24 immunoregulation in invertebrates has not been addressed. Here, we found that 25 exosomes released from WSSV-injected mud crabs could suppress viral invasion by 26 inducing apoptosis of hemocytes. Besides, miR-137 and miR-7847 were found to be 27 less packaged in mud crab exosomes during viral infection, with both miR-137 and 28 miR-7847 shown to be negative apoptosis regulators by targeting the 29 apoptosis-inducing factor (AIF). Moreover, our data revealed that AIF did not only 30 translocate to the nucleus to induce DNA fragmentation, but could also competitively 31 bind to HSP70 to disintegrate the HSP70-Bax (Bcl-2-associated X protein) complex, 32 which eventually activated the mitochondria apoptosis pathway via free Bax.
33Therefore, our findings provides a novel mechanism underlying the crosstalk between 34 exosomal miRNAs and apoptosis pathway in innate immunity in invertebrates. 35 36 109 exosome-WSSV (Fig. 1E). These results suggest that the secreted exosomes could be 110 internalized in hemocytes, which might play an important role in the immune 111 response of hemocytes in mud crab against virus infection.112 Exosome-mediated viral suppression is relevant to apoptosis activation 113 To investigate the involvement of apoptosis during exosome-mediated virus 114 suppression in mud crab, exosome-PBS or exosome-WSSV and WSSV were 115 co-injected into mud crabs. The number of apoptotic hemocytes (at 48 hpi) were 116 evaluated by Annexin V/PI staining. The results showed higher apoptosis in the 117 exosome-WSSV and WSSV co-injected group compared with the other groups 118(control and exosome-PBS groups) ( Fig. 2A). Caspase 3/7 activity was found to 119 significantly increase in the exosome-WSSV and WSSV co-injected group compared 120 to the control group (Fig. 2B). To better understand the role of exosomes in mediating 121 mitochondrial membrane potential, mud crabs were co-injected with either 122 exosome-PBS or exosome-WSSV and WSSV, and hemocytes analyzed using 123 confocal microscopy. The confocal microscopic observation revealed that the 124 mitochondrial membrane potential showed weak red fluorescence (based on JC-1 125 aggregates) and strong green fluorescence (JC-1 monomers) in both 126exosome-injected groups, compared with the controls (Fig. 2C). Moreover, the 127 proapoptotic protein, BAX, was upregulated, while prosurvival Bcl-2 was 128 downregulated in hemocytes of exosome co-injected mud crabs compared with 129 controls (Fig. 2D). To reveal the interactions between apoptosis and virus infection in 130 mud crab, the apoptosis inducer cycloheximide and apoptosis inhibitor Z-VAD-FMK 131 were used to evaluate their effects on WSSV proliferation in hemocytes of mud crab.
132The results showed significantly lower WSSV copy number in the cycloheximide and 133 ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.