HDAC6 Mediates Poly (I:C)-Induced TBK1 and Akt Phosphorylation in Macrophages

Macrophages are derived from monocytes in the bone marrow and play an important role in anti-viral innate immune responses. Macrophages produce cytokines such as interferons and IL-10 upon viral infection to modulate anti-viral immune responses. Type I interferons (IFNs) promote anti-viral defense. IL-10 is a suppressor cytokine that down-regulates anti-viral immune responses. HDAC6 is a tubulin deacetylase that can modulate microtubule dynamics and microtubule-mediated cell signaling pathways. In the present study, we investigated the potential role of HDAC6 in macrophage anti-viral responses by examining poly (I:C)-induced IFN-β and IL-10 production in mouse bone marrow-derived macrophages (BMDMs). We also investigated the role of HDAC6 in poly (I:C)-induced anti-viral signaling such as TBK1, GSK-3β, and Akt activation in mouse BMDMs. Our data showed that HDAC6 deletion enhanced poly (I:C)-induced INF-β expression in macrophages by up-regulating TBK1 activity and eliminating the inhibitory regulation of GSK-3β. Furthermore, HDAC6 deletion inhibited poly (I:C)-induced suppressor cytokine IL-10 production in the BMDMs, which was associated with the inhibition of Akt activation. Our results suggest that HDAC6 modulates IFN-β and IL-10 production in macrophages through its regulation of TBK1, GSK-3β, and Akt signaling. HDAC6 could act as a suppressor of anti-viral innate immune responses in macrophages.

Type I interferons (IFNs) play an important role in anti-viral innate immune responses by upregulating anti-viral defense (13,14). Type I IFNs contain several subtypes such as IFN-α and IFN-β (13,14). Their expression is dependent on the stimuli and cell types (13,14). The expression of Type I IFNs is regulated at the transcriptional level by IFN regulatory factors (IRFs) (13,14). IL-10 is a suppressor cytokine during viral infection (15,16). IL-10 has been reported to down-regulate anti-viral immune responses and delay virus elimination (15,16). Macrophages are mighty anti-viral effector cells of innate immunity (7)(8)(9)(10). However, macrophages are also a major source of IL-10 production during viral infection (15,16). Viral infection could induce IL-10 production in macrophages (15,16). The increased suppressor cytokine IL-10 could then suppress anti-viral responses such as the type I IFN expression and allow the escape of virus from the immune defense (15,16). Therefore, the timing and balance of IL-10 and Type I IFN expression could be important to control viral infection (15,16).

Mouse Bone Marrow-Derived Macrophages
Wild type C57BL/6J mice and HDAC6 knockout C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, Maine). HDAC6 deletion in C57BL/6J mice is produced by CRISPR/Cas9-generated HDAC6 gene knock-out mutation. 9-to 15-week-old sex and age-matched HDAC6 knockout C57BL/6J mice and wild type C57BL/6J mice were used for the studies. All experiments and animal care procedures were approved by the Institutional Animal Care and Use Committee of the University of Kentucky. Mouse bone marrow-derived macrophages were prepared as described preciously (34). Briefly, bone marrow cells were flushed out from femurs and tibias of mice with cold DMEM medium containing 0.5 mM EDTA. The cell suspension was then passed through a cell strainer, centrifuged, and resuspended in DMEM medium containing 10% FBS and 20 ng/ml M-CSF. The cells were seeded into a cell culture plate and maintained in an incubator with 5% CO 2 at 37 • C for 5 d with fresh medium added every other day. After day 5, fully differentiated cells were stimulated with poly (I:C).

Immunoblotting and ELISA Assays
Immunoblotting assays were performed as described previously (35,36). Briefly, the protein samples were separated by SDS-PAGE electrophoresis and transferred to polyvinylidenedifluoride membranes. After probing with primary and secondary antibodies, the membranes were developed using a Clarity Western ECL Substrate (Cat#1705061; Bio-Rad, Hercules, California). IFN-β and IL-10 levels in the cell culture supernatant of BMDMs were examined by the mouse IFN-β ELISA kit (Cat# 42410; PBL Assay Science; Piscataway, New Jersey) and mouse IL-10 ELISA kit (Cat#431414; Biolegend; San Diego, California) according to the manufacturer's protocol.

Statistical Analysis
All experiments were repeated three times. Data are expressed as mean ± SEM. The Student's t-test was used for comparisons of two groups. ANOVA and post hoc multiple comparison tests were employed for multiple groups. Statistical significance was assigned to a P < 0.05.

RESULTS
To investigate the role of HDAC6 in poly (I:C)-induced type I IFN responses, we first examined the effects of HDAC6 deletion on poly (I:C)-induced IFN-β production in macrophages. BMDMs from the HDAC6 knockout and wild type mice were challenged with poly (I:C). Our data showed that HDAC6 deletion markedly enhanced poly (I:C)-induced IFN-β production in the BMDMs (Figure 1).
We then conducted experiments to assess the role of HDAC6 in poly (I:C)-induced suppressor cytokine IL-10 production in macrophages. BMDMs from the HDAC6 knockout and wild type mice were challenged with poly (I:C). Our data showed that HDAC6 deletion inhibited poly (I:C)-induced IL-10 production in the BMDMs (Figure 2).
Microtubule is involved in modulating a variety of cell signaling pathways (27,33). Tubulin acetylation status controls microtubule stability and dynamics (27,33,37). Tubulin is an endogenous substrate of HDAC6 (25,27). We conducted immunoblotting assays to examine the effects of HDAC6 deletion on tubulin acetylation status in macrophages. Our results showed FIGURE 1 | HDAC6 deletion enhances poly (I:C)-induced IFN-β production in BMDMs. BMDMs from the HDAC6 knockout and wild type mice (300,000 cells/well) were challenged without or with 100 µg/ml poly (I:C) for 12 h. Experiments were repeated three times. IFN-β production was assessed by ELISA. *P < 0.05. FIGURE 2 | HDAC6 deletion suppresses poly (I:C)-induced IL-10 production in BMDMs. BMDMs from the HDAC6 knockout and wild type mice (300,000 cells/well) were challenged without or with 100 µg/ml poly (I:C) for 12 h. Experiments were repeated three times. IL-10 production was assessed by ELISA. *P < 0.05. that HDAC6 deletion caused hyperacetylation of α-tubulin in the BMDMs (Figure 3).
TBK1 activation up-regulates type I IFN expression and anti-viral immunity (17,18). To assess the effects of HDAC6 deletion on anti-viral signaling in macrophages, we examined TBK1 modulation by HDAC6. HDAC6 deletion enhanced poly (I:C)-induced TBK1 activation in the BMDMs as showed by the increased TBK1 phosphorylation at Ser172 of its kinase activation loop (Figure 4).
Akt is another cell signaling molecule that interacts with microtubules (32,33). Akt has been reported to modulate IL-10 expression and GSK-3β activity (30,31). To assess the potential modulation of Akt by HDAC6 and tubulin acetylation, we conducted experiments to examine the effects of HDAC6 deletion on Akt activation by examining Akt phosphorylation at Ser473 that is known to mediate Akt activation. In our studies, HDAC6 deletion inhibited poly (I:C)-induced Akt phosphorylayion at Ser473 (Figure 6).

DISCUSSION
Macrophages play a central role in defending against viral infection (7)(8)(9)(10). We investigated the role of HDAC6 in poly (I:C)-induced anti-viral Type I interferon (IFN) and the suppressor cytokine IL-10 production in bone marrow-derived macrophages. Type I interferon (IFN) responses are fundamental in host innate immunity against viruses (13,14), whereas the production of IL-10 by macrophages during viral infection has been reported to downregulate type I interferon antiviral immune responses and hamper virus elimination (15,16). The interactions of the type I interferon-and IL-10mediated pathways could play an important role in modulating innate immune responses during viral infection (15,16). One previous study indicates that HDAC6 could modulate antiviral responses in different cell types including monocytes and fibroblasts (39). However, in our studies, poly (I:C)induced IFN-β production was increased by HDAC6 deletion in BMDMs, whereas poly (I:C)-induced IL-10 production was inhibited by HDAC6 deletion in BMDMs. Our data suggest that HDAC6 could function as a suppressor of anti-viral responses in macrophages by suppressing anti-viral cytokine IFN-β expression while elevating the suppressor cytokine IL-10 expression.
HDAC6 has been reported to modulate many signaling pathways that could modulate anti-viral immune responses  including NF-κB, ERK, and inflammasome activation (27,40,41). We examined some key signaling components in anti-viral responses. TBK1 is a key kinase in anti-viral innate immunity that activates the transcription factor IRF3 to induce type I IFN production (18,19). Autophosphorylation of TBK1 at Ser172 within its kinase activation loop is needed for its activation (18,19). TBK1-mediated anti-viral immune responses can be regulated by controlling Ser172 autophosphorylation (18,19). Poly (I:C) induces type 1 interferon (IFN) responses through its binding to TLR3 and activating TBK1 (18,19,28). In our studies, HDAC6 deletion led to a marked increase of poly (I:C)-induced TBK1 autophosphorylation at Ser172, which correlates with the increased IFN-β production in the macrophages. Our data indicate that HDAC6 could inhibit TBK1 activity during dsRNA viral infection to suppress type 1 interferon (IFN) responses.
GSK-3β is a major modulator of TBK1 activity (18,19). GSK-3β binds to TBK1 and enhances TBK1 activity by facilitating its auto-phosphorylation at Ser172 during viral infection, which then up-regulates type I IFN responses (18,19). Phosphorylation of GSK-3β at Ser9 has been known to inhibit GSK-3β function (30,42). HDAC6 deletion led to a reduction of the inhibitory GSK3β phosphorylation at Ser9 in macrophages during poly (I:C) challenge, which was associated with the increased TBK1 activation. Our results suggest that HDAC6 can regulate antiviral responses through GSK-3β. GSK3β has been reported to interact with microtubules (22)(23)(24). Our results showed that HDAC6 deletion led to a robust α-tubulin acetylation in macrophages. HDAC6, by modulating tubulin acetylation status, could suppress GSK-3β function and promote GSK-3β Ser9 phosphorylation through its control of microtubule dynamics.  Akt has been reported to modulate IL-10 expression and GSK-3β activity (30,31). Akt interacts with microtubules, and their interactions are regulated by tubulin acetylation (32,33). HDAC6 modulates a wide range of cellular responses and signaling through deacetylation of tubulin and microtubules (25)(26)(27). HDAC6 deletion inhibited poly (I:C)-induced Akt activation as demonstrated by the reduced Akt phosphorylation within the carboxy terminus at Ser473. Our data suggest that HDAC6 could modulate Akt activation in macrophages during viral infection. The inhibition of Akt activation by HDAC6 deletion could contribute to the decrease of poly (I:C)-induced IL-10 production in the macrophages. Furthermore, Akt-mediated phosphorylation of GSK-3β at Ser9 has been reported to inhibit GSK-3β activity (30). The inhibition of poly (I:C)-induced Akt activation by HDAC6 deletion could also contribute to the elimination of inhibitory phosphorylation of GSK-3β at Ser9.
In summary, our data indicate that HDAC6 deletion enhances poly (I:C)-induced INF-β expression by up-regulating TBK1 activity in macrophages, which is accomplished by eliminating the inhibitory regulation of GSK-3β. Furthermore, HDAC6 deletion inhibits poly (I:C)-induced suppressor cytokine IL-10 production in macrophages, which is associated with the decreased Akt activation. Our results suggest that HDAC6 could act as a suppressor of anti-viral innate immune responses in macrophages.

DATA AVAILABILITY STATEMENT
All datasets presented in this study are included in the article.

ETHICS STATEMENT
The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the University of Kentucky.

AUTHOR CONTRIBUTIONS
YW designed the study, performed the experiments, edited the manuscript, and analyzed the data. KW designed the study, analyzed the data, and edited the manuscript. JF designed the study, analyzed the data, and wrote the manuscript.