The Non-Receptor Protein Tyrosine Phosphatase PTPN6 Mediates a Positive Regulatory Approach From the Interferon Regulatory Factor to the JAK/STAT Pathway in Litopenaeus vannamei

SH2-domain-containing protein tyrosine phosphatases (PTPs), belonging to the class I PTP superfamily, are responsible for the dephosphorylation on the phosphorylated tyrosine residues in some proteins that are involved in multiple biological processes in eukaryotes. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway transduce signaling responding to interferons and initiate cellular antiviral responses. The activity of the JAK/STAT pathway is generally orchestrated by the de-/phosphorylation of the tyrosine and serine residues of JAKs and STATs, in which the dephosphorylation processes are mainly controlled by PTPs. In the present study, an SH2-domian-contianing PTP, temporally named as LvPTPN6, was identified in Litopenaeus vannamei. LvPTPN6 shares high similarity with PTPN6s from other organisms and was phylogenetically categorized into the clade of arthropods that differs from those of fishes and mammals. LvPTPN6 was constitutively expressed in all detected tissues, located mainly in the cytoplasm, and differentially induced in hemocyte and gill after the challenge of stimulants, indicating its complicated regulatory roles in shrimp immune responses. Intriguingly, the expression of LvPTPN6 was regulated by interferon regulatory factor (IRF), which could directly bind to the LvPTPN6 promoter. Surprisingly, unlike other PTPN6s, LvPTPN6 could promote the dimerization of STAT and facilitate its nuclear localization, which further elevated the expression of STAT-targeting immune effector genes and enhanced the antiviral immunity of shrimp. Therefore, this study suggests a PTPN6-mediated regulatory approach from IRF to the JAK/STAT signaling pathway in shrimp, which provides new insights into the regulatory roles of PTPs in the JAK/STAT signaling pathway and contributes to the further understanding of the mechanisms of antiviral immunity in invertebrates.


INTRODUCTION
Protein tyrosine phosphatases (PTPs) are a sort of fundamental phosphatases responsible for the dephosphorylation of phosphorylated tyrosines in proteins that are involved in various biological processes including embryogenesis, organ development, tissue homeostasis, and the immune defenses in multicellular eukaryotes (1,2). To date, more than 100 PTPs have been identified in the human genome and most of them possess non-redundant functions verified by the unique phenotypes of many reported gene deletions in mice (3,4). According to the features of catalytic domains, PTPs can be classified into four subfamilies, among which classes I, II, and III are independently evolved PTPs that possess similar catalytic mechanisms and active targets, while class IV performs the catalytic function with a key aspartic acid and dependence on a cation (5)(6)(7)(8). Class I PTPs are cysteine-based and strictly tyrosine-specific PTPs that possess the largest number of subfamily members, including the classical PTPs, vaccinia virus H1-like (VH1-like) enzymes, and "dual-specific" protein phosphatases (DSPs), constituting the most diverse group in terms of substrate specificity (9,10). The classical PTPs can be further divided into receptor-like enzymes (RPTPs) and nonreceptor PTPs (NRPTPs), which are distinguished by the containing of the transmembrane domain, while both of them are essential for the dephosphorylation of the tyrosine residues in their specific targets (5,11).
The interferon (IFN) system plays a critical role in immunity, establishing a cellular antiviral state in cells of vertebrates and invertebrates (12,13). The IFN regulatory factor (IRF) family is a kind of transcriptional factors, responsible for the transcriptional regulation of IFNs that can bind to IFN receptors and activate the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway to promote the transcription of STAT-targeted genes (14,15). The JAK/STAT pathway has been proved to mediate the transcription of numerous genes that participate in many cellular processes, such as cell proliferation, differentiation, apoptosis, and immunity, via a series of intracellular signaling cascades (16,17). Briefly, after the binding of INFs, the transmembrane receptors are dimerized and trigger the auto-and transphosphorylation of JAKs to generate docking sites for the Src homology 2 (SH2) domains of latent STATs (18,19). STATs are recruited and reversible phosphorylated by JAKs on a crucial tyrosine residue in their C-terminal region and then homo-or heterodimerized through the SH2-domain-phospho-tyrosine interactions. The activated STAT dimers are translocated into the nucleus for establishing the cellular transcriptional landscape (20,21).
Pacific white shrimp Litopenaeus vannamei is the major cultured shrimp species around the world, which has considerable nutritional and economic values (25). With the rapid development of the shrimp farming industry and the popularization of intensive farming systems, shrimp aquaculture has been threatened by various pathogens that cause huge economic losses (26,27). Numerous studies had confirmed that L. vannamei possesses an IRF-Vago-JAK/STAT regulatory axis that is similar to the IRF-IFN-JAK/STAT axis of vertebrates, which establishes the important role of the JAK/ STAT signaling pathway in shrimp anti-viral immunity (12,28,29). However, the detailed regulatory mechanism of the shrimp JAK/STAT signaling pathway remains unclear. In this study, the non-receptor protein tyrosine phosphatases 6 gene, temporarily named as LvPTPN6, was identified in L. vannamei. Results intriguingly revealed that LvPTPN6 mediated an intracellular IRF-JAK/STAT positive regulatory axis. These findings may provide new insights into the regulatory mechanism of the IRF-JAK/STAT signaling pathway in shrimp and give a better understanding of PTP evolution.

Shrimps
L. vannamei (∼10 g) were obtained from an aquaculture farm in Zhuhai, acclimated in a recirculating water tank system filled with air-pumped seawater (2.0% salinity), fed with 3% body weight artificial diet for two times each day, and detected by PCR to ensure they are free of WSSV, Vibrio parahaemolyticus, and Staphylococcus aureus with 5% random sampling.

Quantitative Real-Time PCR
For the transcriptional-level analysis of specific genes in L. vannamei, cDNAs were synthesized from total RNAs using PrimeScript RT reagent kit with gDNA eraser (Takara, Japan) according to the manufacturer's instructions. Quantitative realtime PCRs (qRT-PCRs) were performed at a final volume of 10 ml containing 1 ml cDNA, 5 ml 2 × SYBR Premix Ex Taq II (Takara, Japan), and 0.5 ml each primer (10 mM) (

Immune Challenge
For immune challenge, V. parahaemolyticus and S. aureus were cultured to the logarithmic phase and diluted to 10 5 colonyforming units (CFUs) in 50 ml PBS after centrifugal collection, respectively. WSSV was prepared freshly from moribund shrimps infected with preserved WSSV stored at −80°C in our lab. Virus stock was quantified using absolute qRT-PCR and diluted to 10 6 copes in 50 ml PBS as described previously (30).
For expression pattern analysis of LvPTPN6, healthy shrimps were divided into six experimental groups in independent recirculating water tank systems. After acclimation for 1 week, shrimps of six groups were injected with 10 6 copies of WSSV, 10 5 CFU of V. parahaemolyticus 10 5 CFU of S. aureus, 5 mg of LPS, 5 mg of Poly(I:C), and PBS at the second abdominal segment, respectively. Hemocyte and gill were sampled from six randomly selected shrimps in each group at 0, 4, 12, 24, 48, 72, and 96 h post stimulant injection. After then, total RNAs were isolated and cDNAs were synthesized for qRT-PCR. For cumulative mortality analysis, shrimps (n = 40) were challenged with 10 6 copies of WSSV at 48 h after the injection of LvPTPN6 or green fluorescent protein (GFP, as negative control) dsRNA. The cumulative mortality was recorded every 6 h after WSSV challenge. The WSSV copy number in muscle was detected at 3 and 5 days after WSSV challenge in parallel experiments. In each group, six living shrimps were randomly selected and the total DNA of muscle was extracted using DNeasy Blood and Tissue Kit (QIAGEN, USA), then the ie1 (wsv069) DNA sequence (GenBank accession No. AY422228) was detected using relative qRT-PCR with shrimp EF-1a as internal control.

Immunofluorescence
For subcellular localization analysis, the LvPTPN6 coding sequence was cloned into the pAc5.1 vector to express the HAtagged LvPTPN6 protein. Drosophila Schneider 2 (S2) cells were plated on a siliconized coverslip with approximately 80% confluence, transfected with the pAc5.1-PTPN6-HA vector using FuGENE HD transfection reagent (Promega, Madison, WI, USA), and fixed with 4% paraformaldehyde for 5 min at 48 h post transfection. For nuclear translocation of L. vannamei STAT, shrimps were challenged with Poly(I:C) for 12 h as mentioned above, then hemolymph smear samples were made on siliconized slides and fixed with 4% paraformaldehyde for 10 min. For immunofluorescence assay, 4% paraformaldehyde fixed cells were infiltrated with 1% Triton X-100 for 20 min then successively incubated with rabbit Ab against HA (CST, Danvers, MA, USA) or shrimp STAT (GL Biochem, Shanghai, China) together with mouse Ab against b-actin (MBL, Tokyo, Japan) and Alexa Fluor 488-conjugated goat anti-rabbit Ab (CST, USA) together with Alexa Fluor 594-conjugated goat anti-mouse Ab (CST, USA). After staining with Hoechst 33342 (Invitrogen, Carlsbad, CA, USA) for the nuclei, sliders were observed using a TCS SP8 STED 3X Confocal Microscope (Leica, Munich, Germany). The immunofluorescence intensities of the cytoplasm-and nuclear-localized STAT were quantified and calculated using JACoP with an ImageJ plugin from four randomly selected microscopic vision fields in each group (32).

RNA Interference In Vivo
To investigate the transcription and function of LvPTPN6, the selected transcription factors as well as LvPTPN6 were knocked down through RNA interference (RNAi). The templates of shrimp genes or GFP for dsRNA synthesis were cloned from the L. vannamei cDNA or pAc5.1-GFP plasmid and incorporated with T7 RNA polymerase promoter at the 5′ end using specific primes listed in Table 1, respectively. The dsRNAs were synthesized in vitro using the T7 RiboMAX ™ Express RNAi system (Promega, USA), annealed from two independently transcribed single-strand RNAs, and then purified according to the manufacturer's instruction. Acclimated healthy shrimps were divided into several groups (n = 40) and intramuscularly injected with 10 mg specific dsRNAs. Hemocytes and gills were sampled from nine shrimps in each group for the following mRNA and protein analysis at 48 h post-dsRNA injection as described above.

Protein Preparation for Nuclear Localization Analysis of STAT
For nuclear localization analysis of STAT regulated by LvPTPN6 at the cellular level, S2 cells were co-transfected with pAc5.1-STAT and pAc5.1-PTPN6 or pAc5.1-STAT and pAc5.1-GFP (as control). Cells were collected at 48 h post-transfection followed by protein analysis as follows. For the analysis of nuclear localization of STAT, LvPTPN6 in L. vannamei were knocked down through RNAi as mentioned above. At 36 h post dsRNA injection, shrimps were challenged with Poly(I:C) for 12 h as mentioned above, then hemocytes were sampled from 30 randomly selected shrimps in each group followed by protein analysis. After preprocessing, the nuclear and cytoplasmic protein of S2 cells or hemocytes were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo, Waltham, MA, USA), respectively.

Western Blot
For analysis of STAT localization, protein extractions were directly treated with SDS loading buffer to obtain protein samples, which were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes using the Trans-Blot Transfer system (Bio-Rad, Hercules, CA, USA) for the determination of the STAT level. For analysis of the STAT dimer level, S2 cells were lysed using Pierce IP Lysis buffer (Thermo, USA) at 48 h post-transfection to obtain the native proteins, which were separated with 10% native polyacrylamide gel electrophoresis and transferred to PVDF membranes in native transfer buffer. Western blot was performed using rabbit antibodies against shrimp STAT (GL Biochem, China), HistoneH3 (CST, USA), and b-actin (MBL, Japan), as previously described (33). HistoneH3 or b-actin was detected as an internal control of cytoplasmic or nuclear protein to rule out the possible contamination of nuclear or cytoplasmic protein, respectively. The protein levels were determined by analyzing the gray values of specific protein bands using Quantity One 4.6.2 software (Bio-Rad, USA) by the Gauss model and normalized to those of internal control.

Co-Immunoprecipitation
The interactions between LvPTPN6 and L. vannamei JAK or STAT were analyzed using co-immunoprecipitation (Co-IP). The ORFs of STAT and L. vannamei JAK (JAK) were cloned into the pAc5.1/V5-His A plasmid (Invitrogen, USA) to generate V5-tagged STAT and JAK expression vectors, respectively. An HA-tagged GFP expression vector was used as control. HAtagged LvPTPN6 or GFP and V5-tagged STAT or JAK were cotransfected into S2 cells. After 48 h, cells were harvested and lysed in IP Lysis Buffer (Thermo, USA) with a protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Co-IPs were performed using anti-HA affinity agarose (Sigma, USA), and Western blot was performed with a rabbit anti-V5 primary antibody (Merck, Burlington, MA, USA) and a horseradish peroxidase (HRP)conjugated goat anti-rabbit secondary antibody (Promega, USA).

Electrophoretic Mobility Shift Assay
To verify the regulatory role of LvIRF on LvPTPN6 transcription, the interaction between the LvIRF protein and LvPTPN6 promoter was detected through electrophoretic mobility shift assay (EMSA) in vitro.
LvIRF ORF was amplified using primers of IRF-32a-EcoRIF/ NotIR, then cloned into the pET32a (Invitrogen, USA) prokaryotic expression vector; the original pET32a plasmid which expresses the thioredoxin (TRX) protein was set as negative control. The recombinant plasmids were transformed into Escherichia coli Rosetta (DE3) cells and induced with 1 mM isopropyl-beta-D-thiogalactoside (IPTG) in the logarithmic phase after enlarged cultivation. The His-tagged LvIRF or TRX protein was purified using Ni-NTA Agarose (QIAGEN, USA) and quantified using BCA Protein Assay Kit (Beyotime, Shanghai, China) according to the manufacturer's instruction.
The 5′ biotin-labeled probe that contains the predicted IRFbinding motif sequence (GGAAAGGGGAAGGATCAA) was synthesized by Invitrogen (Shanghai, China) ( Table 1). EMSA was performed using a LightShift Chemiluminescent EMSA Kit (Thermo, USA) as described previously (35). In general, the purified proteins (10 mg) were incubated with 20-fmol probes for the binding reactions between probes and proteins, separated by 5% native PAGE, transferred to positively charged nylon membranes, and cross-linked by UV light (256 nm). Then the biotin-labeled DNAs on the membrane were detected by chemiluminescence and captured by Amersham Imager 600 (GE, USA).

Bioinformatics and Statistical Analysis
The IRF-binding motif in the LvPTPN6 promoter was predicted using JASPAR 2016 (http://jaspar2016.genereg.net/cgi-bin/jaspar_ db.pl). The multiple-sequence alignment of PTPN6 homologs was performed using ClustalX 2.1, and the phylogenetic tree was constructed using MEGA 5.0. All data were presented mean ± SD. The significance of difference between groups of numerical data was calculated using Student's t-test. The cumulative mortalities were analyzed using GraphPad Prism 5.01 to generate the Kaplan-Meier plot (log-rank c 2 test).

Cloning and Bioinformatics Analysis of LvPTPN6
The full length of LvPTPN6 mRNA is 2,746 bp with a 5′ untranslated region (UTR) of 140 bp, an ORF of 2,010 bp encoding a protein of 669 amino acids, and a 3′ UTR of 596 bp (GenBank accession no.: OL652660). The LvPTPN6 protein has a calculated molecular weight of 75.62 kDa and a theoretical isoelectric point of 8.60 (Supplemental figure 1A). LvPTPN6 was predicted to contain two Src homology 2 (SH2) domains and a protein tyrosine phosphatase catalytic (PTPc) domain covering residues of 4-87, 110-193, and 248-519 (Supplemental figure  1B), which share the identical domain character as human PTPN6. Multiple-sequence alignment showed that LvPTPN6 shared high homology to the PTPN6s from Crustacea, Insect, and Arachnoidea, especially in the SH2 and PTPc domains, which shared higher identities of 98.2%, 96.15 and 92.2% with those of PTPN6s from Penaeus japonicus, Penaeus monodon, and Homarus americanus, and lower identities of 68.1%, 68.1%, 68.5%, and 69.9% from Stegodyphus dumicola, Trichonephila clavata, Nephila pilipes, and Nymphon striatum, respectively (Figure 1). In the constructed phylogenetic tree, the analyzed PTPN6s could be categorized into three clades of arthropods, fishes, and mammals (Figure 2), and LvPTPN6 was most closely clustered with the Armadillidium vulgare PTPN6 in the arthropod clade.

Tissue Distribution and Subcellular Localization of LvPTPN6
The expression level of LvPTPN6 in 12 tissues of L. vannamei was detected by qRT-PCR ( Figure 3A). Results showed that LvPTPN6 could be detected in all selected tissues, and the lowest expression level was revealed in pyloric cecum. The expression level of LvPTPN6 was gradually increased in the epithelium, heart, eyestalk, hepatopancreas, scape, gill, hemocyte, and stomach, which was 1.22-, 1.39-, 1.53-, 1.62-, 1.71-, 1.85-, 1.87-, and 2.01-fold compared with it in pyloric cecum, respectively, and was roughly increased in the intestine, muscle, and the highest of nerve, which was 3.74-, 4.69-, and 9.80-fold compared with it in pyloric cecum. The subcellular localization of LvPTPN6 was detected using immunofluorescence assay in S2 cells which expressed the HA-tagged LvPTPN6 protein ( Figure 3B). Results showed that LvPTPN6 was mainly located in the cytoplasm.

Expression Profiles of LvPTPN6 After Immune Stimulation
The expression profiles of LvPTPN6 were investigated through qRT-PCR in hemocyte and gill from immune stimulated L. vannamei (Figure 4). In hemocyte, the expression levels of LvPTPN6 were generally upregulated after the stimulation of all stimulants. It was gradually increased from 1.18-fold at 4 h to 16.30-fold at 96 h after the stimulation of WSSV. However, it was roughly up-regulated of 3.2-, 2.05-, 1.71-, and 1.71-fold at 4 h, then gradually calmed down at the following time points, and up-regulated again of 1.43-, 1.90-, 2.29-, and 1.82-fold at 96 h after the stimulation of V. parahaemolyticus, S. aureus, LPS, and poly(I:C), respectively. Notably, the expression pattern of LvPTPN6 in gill was totally different from that in hemocyte, which was generally suppressed after the stimulation of WSSV, V. parahaemolyticus, S. aureus, and poly(I:C), while it did not significantly change, except being up-regulated of 1.57-fold at 4 h, after the stimulation of LPS.

Regulation of LvPTPN6 Expression by IRF
A 1,609-bp promoter sequence (Supplemental Figure 2A) of LvPTPN6 was cloned into the pLG3-Basic vector and the transcriptional activity regulated by L. vannamei immune-related transcriptional factors, including Dorsal, Relish, STAT, and IRF, were analyzed through dual-luciferase assay. Results demonstrated that IRF could significantly up-regulate the transcription of the LvPTPN6 promoter of 2.03-fold compared with the GFP control ( Figure 5A). To search the IRF-binding motif, the LvPTPN6 promoter was cleaved to -13,60, -737, -517, and -297 bp upstream of the transcriptional initiation site ( Figure 5B) and cloned into the pGL3-Basic promoter. Results of dual-luciferase assay unveiled that the IRF-mediated transcriptional activation was vanished after the deletion of the -1,609 to -1,360 regions of the LvPTPN6 promoter ( Figure 5C). In this region, a potential IRF-binding motif (GGAAAGGGGAAGGATCAA) was predicted through bioinformatics analysis (Supplemental Figure 2B), and its combination with IRF was verified in vitro. EMSA results showed that the IRF protein bound the biotin-labeled probe of the LvPTPN6 promoter to form a retarded shift band, which was gradually eliminated when 200 × and 400 × unlabeled probes were added to competitively bind IRF. In contrast, there was no shift band in the incubation between TRX protein and the biotinlabeled probe ( Figure 5D). This suggested that LvPTPN6 could be directly regulated by IRF. To further verify the regulatory mechanism of LvPTPN6, IRF was silenced using the RNAi strategy in vivo ( Figure 5E). Consistent with our expectation, silencing of IRF significantly inhibited the transcription of LvPTPN6 in both hemocyte and gill ( Figure 5F), confirming that LvPTPN6 was transcriptionally regulated by IRF in L. vannamei.

Interaction Between LvPTPN6 and JAK/STAT
To explore the mechanisms of LvPTPN6 promoting nuclear translocation of STAT, the interaction between LvPTPN6 and JAK/STAT was analyzed using Co-IP ( Figure 6). Results showed that both V5-tagged JAK and STAT could co-precipitate with HA-tagged LvPTPN6 but not GFP protein, confirming the interaction between LvPTPN6 and JAK, and LvPTPN6 and STAT, respectively.

Nuclear Localization of STAT Regulated by LvPTPN6
To reveal the regulatory role of LvPTPN6 on the JAK/STAT signaling pathway, nuclear localization of STAT was investigated after the down-or up-regulation of LvPTPN6 in in vivo or S2 cells, respectively. Western blot analysis showed that, after knockdown of LvPTPN6 and stimulation of Poly(I:C) in vivo ( Figure 7A), cytoplasm-localized STAT was increased by 59.9%, and nuclear-localized STAT was decreased by 38.4% compared with the negative control in hemocyte, respectively ( Figure 7B). This result was confirmed by immunofluorescence assay, which demonstrated that silencing of LvPTPN6 suppressed the nuclear translocation of STAT by 29.3% compared with the GFP control in hemocytes (Figures 7C, D). The facilitated role of LvPTPN6 on STAT nuclear localization was further verified through   Figure 7E). Besides, overexpression of LvPTPN6 significantly promoted the dimerization of STAT, the essential procedure of STAT activation ( Figure 7F). These data indicated that LvPTPN6 could facilitate the activation of the JAK/STAT signaling pathway.

Involvement of LvPTPN6 in Antiviral Immunity
Before investigating the regulatory effects on the expression of immune effector genes mediated by LvPTPN6, the transcriptional activation of STAT on the promoters of L. vannamei immune effector genes was investigated by dualluciferase assays. Results revealed that STAT could significantly activate the transcription of ALF2, ALF5, CTL4, and Lys-IT2 ( Figure 8A). Correspondingly, the expressions of ALF2, ALF5, CTL4, and Lys-IT2 were generally suppressed in hemocyte and gill after knockdown of LvPTPN6 in vivo ( Figure 8B). The antiviral role of LvPTPN6 was further investigated in shrimp. After knockdown of LvPTPN6, the cumulative mortality of shrimps after 3 days of WSSV infection was significantly increased compared with the control (Figure 8C), and the difference between the two treatments reached a peak of 33.5% at 4 days post WSSV infection. Consistently, the virus load of WSSV in muscles was significantly increased in the LvPTPN6silenced group at 3 and 5 days post WSSV infection ( Figure 8D). These data indicate that LvPTPN6 could facilitate antiviral immunity through the JAK/STAT singling pathway in shrimp.

DISCUSSION
Tyrosyl phosphorylation plays a critical role in multiple signaling pathways regulating innate and acquired immunity, which is mainly regulated by the dynamic equilibrium between protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs) (3). Non-receptor PTPs are a classical type of PTPs, which consist of  several subtypes distinguished with different domain features (11). SH2-domain-containing PTPs (SHPs) are characterized by composition of an N-terminal SH-2 domain, a C-terminal SH-2 domain, a classical PTP domain, and a C-terminal tail that contains two tyrosyl phosphorylation sites (36). In mammals, there are two kinds of SHPs, SHP1 (PTPN6) and SHP2 (PTPN11), which possess similar overall structures and regulatory mechanisms but differ in detailed structure, expression pattern, and, most importantly, physiological functions (3). In this study, an SH2 domaincontaining PTP (LvPTPN6) sharing a higher homology with PTPN6 from other animals was identified in L. vannamei, which contains the classical domain features of SHPs. Different from the distribution characters of human PTPN6 which is most abundantly expressed in hematopoietic cells and low in some epithelial, endothelial, and central nervous system cells (37), LvPTPN6 exits in all detected tissues. Besides, human PTPN6 is most abundantly expressed in the nucleus of epithelial cells and the cytoplasm of hematopoietic cells (38), while the subcellular localization analysis in S2 cells showed that LvPTPN6 was mainly located in the cytoplasm. These features corroborate the assertion that LvPTPN6 may have specific functions that differ from those of mammalian PTPN6s.
In addition to the posttranscriptional approaches, such as truncation, phospholipid binding, or tyrosine phosphorylation of the C-terminal tail, the activity of mammalian PTPN6 can also be regulated from transcriptional levels (39,40). In the current study, LvPTPN6 was transcriptionally regulated after the challenge of stimulants, which share different expression patterns in hemocyte and gill, implying its complicated regulatory role in the shrimp immune responses.
In the canonical IFN systems, IRF activates the JAK/STAT signaling pathway to establish the cellular antiviral state through regulating the secretion of IFNs recognized by INF receptors and initiating the phosphorylation cascade of JAK and STAT (12,28,41). The IRF/Vago/JAK-STAT axis in shrimp is known to be similar to the interferon system of mammals and also plays an essential role in antiviral response (26,42). Except the extracellular signal axis mediated by IFNs, very few studies revealed the intracellular activation of the JAK/STAT pathway regulated by IRF. For instance, in children with autosomal recessive homozygous IRF9 deficiency, IFN-stimulated response element (ISRE)-regulated transcription was attenuated for the disruption of the STAT1/STAT2/IRF9 heterotrimer; this finding presented an instance of IRF that could directly facilitate the transcriptional activation function of STAT (43). In this study, we found that IRF could activate the expression of LvPTPN6, further facilitating the nuclear localization of STAT and promoting the antiviral immunity of shrimp. Our results revealed that in addition to the canonical IRF/Vago/STAT axis, LvPTPN6 mediated an alternative regulatory approach from IRF to STAT. This novel regulatory strategy between IRF and STAT may provide a basis for further research on the IFN systems.
The JAK/STAT pathway is one of the important immune signaling pathways and exists wildly from vertebrates to invertebrates, which is regulated by different posttranscriptional mechanisms including acetylation, demethylation, and serine or tyrosine de-/phosphorylation (21). One of the major mechanisms t h a t a t t e n u a t e J A K / S T A T p a t h w a y a c t i v a t i o n i s dephosphorylation of the tyrosine residues by PTPs (22). In mammals, most of the PTP members, such as PTP receptortype D (PTPRD), PTP receptor-type T (PTPRT), PTP receptortype K (PTPRK), PTPN2, PTPN6, PTPN9 and PTPN11, had been proved to suppress the signal transduction of the JAK/STAT pathway by dephosphorylating on JAKs or STATs (44)(45)(46)(47)(48)(49)(50).  Accumulating evidence has established the important role of PTPN6 in the regulation of the JAK/STAT signaling pathway in different organisms (51). The absence of SPH1 (PTPN6) activates JAK/STAT signaling pathways via the enhanced phosphorylation of JAK1, JAK2, JAK3, STAT3, STAT5, or STAT 6 (52), while its induction by chemical compounds could reduce the phosphorylation of STAT3, STAT5, or STAT 6 to block its signal transduction (22). Interestingly, our present finding demonstrated that LvPTPN6 could promote the unclear localization of STAT, activate the expression of STAT-regulated genes, and ultimately initiate the antiviral status of shrimp. Although most of the PTPs exhibit negative regulatory roles through their dephosphorylation activity in the JAK/STAT signaling pathway, some exceptions can promote it in specific The relative viral load in muscle was analyzed by detecting the DNA of the WSSV ie1 gene in six randomly selected shrimps using qRT-PCR with three repetitions, and the shrimp EF-1a gene was used as the internal control. The level in dsRNA-GFP group at 3 days post WSSV infection was set as baseline (1.0). *p < 0.05 by two-tailed unpaired Student's t-test. circumstances. For example, the EGF-and IFN-g-induced STAT activation was suppressed by expressing a catalytically inactive form of SHP1 (PTPN6) in HeLa cells, while overexpression of the native SHP1 had no effect on EGF-induced STAT activation but showed a positive effect on IFN-g-induced STAT activation. This suggests that SHP-1 can function as a positive regulator in the activation of STAT (23). Inhibition of SHP2 expression initially enhanced and later inhibited STAT5 phosphorylation and reduced the expression of the antiapoptotic genes of MCL1 and BCLXL (53). In mouse mammary gland cells, SHP2 plays a positive role in the prolactin-induced JAK2 activation pathway. JAK2 tends to associate with suppressor of cytokine signaling 1 (SOCS1), which targets JAK2 through a ubiquitin-dependent degradation pathway and serves as a negative regulator for the JAK2/STAT5 pathway. The interaction between JAK2 and SOCS1 is mediated by phosphorylation of Tyr1007 in JAK2. In vitro studies demonstrated that SHP2 was able to dephosphorylate this Tyr site and prevent the formation of the JAK2-SOCS1 complex and subsequent degradation of JAK2. Upon being released from the inhibitory effects of SOCS1, JAK2 is recruited to the prolactin receptor (PrlR) and phosphorylates STAT5 (24). However, the precise mechanism of how these molecules achieve positive functions in different systems remains to be clarified. In this study, LvPTPN6 can both combine with JAK and STAT and elevate the dimerization of STAT, providing us with clues that LvPTPN6 may belong to catalytically inactive PTPs, which could enhance the stability of the JAK and STAT dimers by blocking their tyrosyl phosphorylation sites from other PTPs. To the best of our knowledge, it is the first report of a PTP positively regulating JAK/STAT pathway in invertebrates. These findings indicated that the structures and functions of PTPs vary from different organisms that required further excavation to reveal their nonnegligible regulatory roles in invertebrates and vertebrates.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

AUTHOR CONTRIBUTIONS
HZ and JH supervised the overall project and designed the experiments. HZ wrote the manuscript. ML and XX performed the experiments and analyzed the data with the help from XL, LY, WS, and SW. ZZ offered the assistance to revise the manuscript. All authors contributed to the article and approved the submitted version.  Figure 7C.