Unc-13 homolog D mediates an antiviral effect of the chromosome 19 microRNA cluster miR-517a

Balanced maternal–fetal interactions at the placental interface are essential for a successful pregnancy (Sadovsky and Jansson, 2015). Although it has been commonly thought that the maternal immune response is compromised during gestation to avoid rejection of the allographic fetus, recent evidence challenges that dogma and provides alternative explanations to the balance between fetal tolerance and the ability to mount an inflammatory anti-microbial response (Racicot et al., 2014; Mor and Cardenas, 2010). MicroRNAs (miRNAs) have an established role in regulation of gene expression by degrading target mRNA through binding to complementary sequences in the target mRNA 3′-untranslated region or in the coding sequence, or by inhibiting translation (Bartel, 2004; Baek et al., 2008; Hausser et al., 2013). Mature miRNAs are assembled into the RNA-induced silencing complex (RISC) along with Argonaute 2 (Ago2) and cofactors as one of the common pathways in miRNA-mediated gene silencing (He and Hannon, 2004; Bartel, 2009).

Several groups, including our laboratory, have shown that miRNAs expressed from the chromosome 19 miRNA cluster (C19MC) are the most abundantly expressed miRNAs in human placental trophoblasts (Noguer-Dance et al., 2010; Bortolin-Cavaille et al., 2009; Bellemer et al., 2012; Donker et al., 2012; Mouillet et al., 2015; Sadovsky et al., 2015). Although the full spectrum of C19MC miRNA functions remains to be identified, several lines of evidence established the role of discrete C19MC members in villous trophoblast differentiation (Zhang et al., 2016; Kumar et al., 2013), migration and invasion of extravillous trophoblasts (Xie et al., 2014; Takahashi et al., 2017) or in natural killer (NK) cell function (Ishida et al., 2015). We recently showed that primary human trophoblast (PHT) cells are relatively resistant to infection by a range of viruses (Delorme-Axford et al., 2013). We also showed that C19MC miRNA or PHT conditioned medium (CM) containing C19MC miRNA can attenuate viral infection in non-trophoblastic cells (Delorme-Axford et al., 2013; Mouillet et al., 2015; Ouyang et al., 2014). Our data also implicated autophagy, a ‘self-eating’ autophagosome/lysosomal pathway (Lennemann and Coyne, 2015; Choi et al., 2018; Deretic et al., 2013), in the anti-viral effect of C19MC. This is supported by the following: (1) exposure of non-trophoblastic U2OS cells to PHT CM stimulates autophagy; (2) PHT CM causes vesicular stomatitis virus (VSV) particles to traffic to LC3B (MAP1LC3B)-positive autophagosomes; (3) expression of the entire C19MC cluster or its member miR-517a-3p (miR-517a) in U2OS cells stimulates autophagy; and (4) inhibition of autophagy blocks C19MC-mediated antiviral effects in U2OS cells (Delorme-Axford et al., 2013). The precise autophagic mediators that are targeted by C19MC miRNA remain unknown.

Toll-like receptors (TLRs) are a part of a family of pattern recognition receptors. Mouse TLR7 or human endosomal TLR8 are known to sense single-stranded RNA oligonucleotides, including viral RNA (Lan et al., 2007), and mediate viral degradation via autophagy (Lee et al., 2007). Recent data also implicated expression of human TLR8 in HEK293 cells in recognition of selected miRNAs and downstream signaling in cancer (Fabbri et al., 2012). Activation of TLR8 results in induction of NF-κB and/or interferon regulatory factors, leading to expression of cytokines or type I interferon, respectively (Kawasaki and Kawai, 2014). Furthermore, knockdown of TLR8 in U2OS cells increases replication of type B coxsackieviruses (Coyne et al., 2011).

To address the role of C19MC miRNA in stimulating autophagy and attenuating viral infection, we identified unc-13 homolog D (UNC13D), regulatory-associated protein of mTOR Complex 1 (RPTOR) and integrin β4 (ITGB4) as autophagy-related, direct mRNA targets of the C19MC miR-517a. While silencing of UNC13D, RPTOR and ITGB4 enhanced autophagy, inactivation of UNC13D, but not RPTOR and ITGB4, reduced VSV viral RNA (vRNA) levels. Importantly, overexpression of UNC13D reduced the antiviral activity of miR-517a, suggesting that UNC13D mediates miR-517a's effect on viral replication. Although miR-517a activated TLR8-mediated signaling, the effect was not restricted to C19MC miRNA.

We previously showed that PHT cells highly express C19MC miRNA and exhibit an elevated level of baseline autophagy (Delorme-Axford et al., 2013). We also showed that the highly expressed C19MC member miR-517a exerts its antiviral activity via enhanced autophagy (Delorme-Axford et al., 2013). To identify direct targets of C19MC miRNA that might stimulate autophagy and mediate the antiviral activity of C19MC miRNA, we conducted crosslinking, ligation and sequencing of hybrids (CLASH) using PHT cells, and screened for autophagy inhibitors that were bound by miR-517a. We identified three relevant proteins as putative targets for miR-517a: UNC13D, a regulator of endosomal vesicle trafficking (Feldmann et al., 2003); RPTOR, an autophagy inhibitory protein that is a part of the mTORC1 complex; and ITGB4 (Xie and Sun, 2019; Ge et al., 2011). To validate the silencing effect of miR-517a on these targets, we used U2OS cells that do not endogenously express miR-517a, as we previously showed (Delorme-Axford et al., 2013). Indeed, overexpression of miR-517a mimics in U2OS cells resulted in a significant reduction in UNC13D, ITGB4 and RPTOR mRNA expression compared to the control miRNA mimics (Fig. 1A,B).

Overexpression of C19MC miRNA (Delorme-Axford et al., 2013) or miR-517a enhanced expression of the autophagic protein LC3B (Fig. 1C). Because miR-517a silences UNC13D, we investigated the impact of UNC13D silencing on LC3B expression. As shown in Fig. 1D and Fig. S1A, CRISPR-Cas9-mediated knockout (KO) or silencing of UNC13D enhanced LC3B expression. Similarly, we found that miR-517a silenced the expression of RPTOR and ITGB4 proteins (Fig. S1B). Knockdown of ITGB4 or RPTOR increased LC3B expression (Fig. S1C). Given that RPTOR is known as a negative regulator of autophagy (Ali and Sabatini, 2005; Kim et al., 2002; Chiang and Abraham, 2005), we confirmed that RPTOR knockdown decreased the levels of phosphorylated mechanistic target of rapamycin (mTOR) and phosphorylated p70 S6 kinase (also known as RPS6KB; Fig. S1D), consistent with induction of autophagy.

To assess the effect of reduced UNC13D on viral replication, we knocked out or silenced UNC13D expression and examined the levels of VSV vRNA, frequently used to study the interaction of virus with host cells, by reverse transcription qPCR (RT-qPCR). As shown in Fig. 1E and Fig. S1E, reduced UNC13D expression lowered VSV vRNA levels at a range of different multiplicities of infection (MOIs) in U2OS cells. In contrast, silencing of RPTOR, ITGB4, or both ITGB4 and RPTOR, had an insignificant effect on VSV vRNA levels (Fig. 1F). Taken together, these results suggest that among the three miR-517a targets we identified, UNC13D supports viral replication.

To assess the possibility that UNC13D might mediate, at least in part, the antiviral effect of miR-517a, we examined whether overexpression of UNC13D affects viral replication. As shown in Fig. 2A, when compared to control GFP-expressing U2OS cells, overexpression of UNC13D significantly increased VSV vRNA levels. Moreover, consistent with our data on UNC13D silencing (Fig. 1D), overexpression of UNC13D led to a reduction in LC3B levels (Fig. 2B). Importantly, in cells expressing the antiviral miR-517a, overexpression of UNC13D increased VSV vRNA levels (Fig. 2C), suggesting that the antiviral effect of miR-517a is mediated, at least in part, by silencing the pro-viral UNC13D.

In light of the recent finding that transcription factor EB (TFEB), an indispensable autophagy-promoting factor, is activated in mice deficient in Unc13D (Zhang et al., 2019), we hypothesized that overexpression of UNC13D might inactivate TFEB, thus attenuating autophagy and promoting viral replication. Because TFEB is inactivated by mTORC1 via phosphorylation of Ser122 or Ser211 (Vega-Rubin-de-Celis et al., 2017; Martina et al., 2012), we examined the phosphorylation of endogenous TFEB after UNC13D overexpression. As expected, we found that cells overexpressing UNC13D showed higher levels of phospho-Ser122 of TFEB (Fig. 2D). Moreover, treatment of cells with the mTOR inhibitor Torin 2, which dephosphorylates TFEB and thus promotes its activity (Vega-Rubin-de-Celis et al., 2017; Roczniak-Ferguson et al., 2012; Napolitano et al., 2020), caused a reduction in VSV vRNA levels compared to levels in untreated control cells (Fig. 2E), suggesting that TFEB activation by Torin 2 reduced the virus-promoting activity of UNC13D. Taken together, our data support a role for UNC13D in mediating the antiviral activity of miR-517a in non-trophoblastic target cells.

We recently found that trophoblastic small extracellular vesicles (sEV, exosomes) enter target cells through endosomal pathways and deliver their miRNA cargo, including the C19MC miR-517a, to RISC complex proteins (Li et al., 2020). In addition, trophoblastic C19MC miRNA-containing sEVs attenuate VSV replication (Ouyang et al., 2016, 2014). In light of these observations, and the ability of selected mature miRNAs to activate the human endosomal toll-like receptor TLR8 through NF-κB p65 signaling (Diebold et al., 2004; Fabbri et al., 2012; Heil et al., 2004; Coyne et al., 2011), we hypothesized that miR-517a might initiate downstream signaling via stimulation of TLR8. To test this hypothesis, we used a 293XL cell line overexpressing human TLR8 (293XL/8; Claudepierre et al., 2014; Pépin et al., 2017; Sarvestani et al., 2015), or parental 293XL (293XL/0) lines as controls. Using RT-qPCR we confirmed the proper TLR expression pattern in these lines (Fig. S2). Because stimulation of TLR8 leads to downstream activation of NF-κB p65 (also known as RELA) and subsequent increase in cytokine or interferon expression (Kawai and Akira, 2007; Lim and Staudt, 2013; Hiscott, 2007), we used the p65-Luc reporter (luciferase under control of the RELA promoter) to assess the effect of miR-517a and two other autophagy-promoting miRNAs, miR-512 and miR-516b (Donker et al., 2012; Delorme-Axford et al., 2013). Using a concentration of mature miR-517a similar to the range used by others (Lehmann et al., 2012; Fabbri et al., 2012) and the TLR8 ligand TL8-506, we initially conducted a timecourse experiment to optimize the p65-Luc activity (Fig. S3A). As shown in Fig. 3A, the C19MC miRNAs miR-517a, -512 and -516b activated p65-Luc in 293XL/8 cells that express TLR8, but not in 293XL/0 cells that are devoid of TLR8, or in cells overexpressing TLR7 (data not shown). However, this effect was not restricted to C19MC miRNAs, because miR-630, which did not induce autophagy in our previous studies (Delorme-Axford et al., 2013), or miR-29a, which activates NF-κB (Fabbri et al., 2012), also upregulated p65-Luc in 293XL/8 cells (Fig. 3A). In contrast, the control fragment RNA41 had no effect in 293XL/8 cells. In addition, only the TLR3-recognizing poly I:C, but not miR-517a, activated ISRE-Luc [luciferase under control of a minimal promoter inserted with an interferon-stimulated response element (ISRE)] and IFNβ-Luc (luciferase under control of the IFN-β promoter) in 293XL/8 cells (Fig. S3B), suggesting that the effect of mature miRNA was unique to TLR8-mediated NF-κB signaling.

To provide further evidence for TLR8-mediated stimulation of NF-κB signaling, we examined NF-κB p65 nuclear translocation in response to mature miRNA (Fabbri et al., 2012; Claudepierre et al., 2014; Pépin et al., 2017; Sarvestani et al., 2015). Whereas in control cells p65 was found only in the cytoplasm, exposure to TL8-506 led to robust nuclear translocation of p65 by 2 h (Fig. 3B). Importantly, miR-517a enhanced the nuclear translocation of p65 at 6 h, albeit to a lesser extent. A similar effect was seen in response to mature miR-630, but not RNA41. Finally, we assessed the effect of miR-517a on several cytokines, as previously shown (Gorden et al., 2005; Ghosh et al., 2006), and found that the expression of IL-8 (also known as CXCL8) and CXCL10, but not TNFα (TNF) mRNAs, was weakly increased by miR-517a in 293XL/8 cells (Fig. 3C). Using a more physiologically-relevant analysis by ELISA, we confirmed the effect of miR-517a on IL-8 release to the medium (but did not observe an effect on CXCL10 release, Fig. 3D). This effect was specific to cells expressing TLR8, because there was no effect in 293XL/0 cells (Fig. S3C,D). Taken together, our experiments showed that miR-517a activated NF-κB through TLR8 in target cells. However, this activation was not restricted to C19MC miRNA, and other mature RNAs also induced TLR8 downstream pathways.

UNC13D was initially shown to regulate exocytosis via direct binding to endosomal Rab27 in diverse cell types, including hematopoietic cells (Shirakawa et al., 2004; Neeft et al., 2005; Ménager et al., 2007; Boswell et al., 2012; Zhang et al., 2013; Elstak et al., 2011), and mutations in UNC13D result in impaired granule exocytosis and cause familial hemophagocytic lymphohistiocytosis subtype 3 (FHL3) (Feldmann et al., 2003; Yamamoto et al., 2004; Meeths et al., 2011; Zur Stadt et al., 2006). UNC13D was recently found to attenuate macroautophagy through interaction with endolysosomal trafficking (Zhang et al., 2019). Consistent with this finding, we have demonstrated that human UNC13D KO cells exhibit increased lipidated LC3B, indicative of increased autophagy. Importantly, our data suggest that UNC13D mediates, at least in part, the pro-autophagy, antiviral effect of miR-517a in non-trophoblastic target cells: (1) miR-517a silences UNC13D; (2) VSV vRNA levels are lower in UNC13D KO cells; (3) VSV vRNA levels are increased upon overexpression of UNC13D in cells expressing miR-517a; and (4) UNC13D enhances the phosphorylation and, hence, inactivation of the autophagy-promoting factor TFEB (Settembre et al., 2011; Napolitano et al., 2020), and the TFEB activator Torin 2 attenuates VSV levels. Whereas our data support a role for miR-517a in regulation of RPTOR and ITGB4, siRNA-mediated knockdown of these proteins did not reduce VSV vRNA levels. Although both RPTOR and TFEB regulate autophagy, RPTOR indirectly affects autophagy by recruiting the mTORC1 substrates p70 S6 kinase and 4E-BP1 (also known as EIF4EBP1). In contrast, activated TFEB directly enhances the expression of autophagy genes (Settembre et al., 2011; Napolitano et al., 2020). Notably, the inability of overexpressed UNC13D to fully restore VSV levels that were suppressed by miR-517a suggests that other miR-517a targets contribute to its antiviral effect.

We also showed that mature miRNA members of C19MC, which in total account for nearly 40% of trophoblastic miRNAs in term human pregnancy (Donker et al., 2012), activated NF-κB through TLR8 in target cells. However, this activation was not restricted to C19MC miRNA, and other mature RNAs also induced TLR8 downstream pathways. A previous study reported that a miR-517a mimic induced the expression of NF-κB targets by promoting the nuclear localization of p65 (Olarerin-George et al., 2013). We highlight that we used HEK293 cells that express TLR3 at baseline and overexpress TLR8. These cells were also engineered to overexpress the anti-apoptotic Bcl-XL protein (encoded by BCL2L1), designed to enhance cell viability (Chao and Korsmeyer, 1998; Janumyan et al., 2003). By interacting with Beclin-1, Bcl-2 family members, including Bcl-XL, can reduce the level of autophagy (Kim et al., 2014; Gordy and He, 2012), which might have attenuated some of our autophagy-related measurements using these cells. Our data emphasize the significance of understanding the mechanisms underlying the apoptotic and antiviral action of C19MC miRNA in target cells.

The Institutional Review Board at the University of Pittsburgh approved all placental procurement protocols used in these studies. Tissue for preparation of PHT cells was collected under an exempt protocol, by which patients give consent for use of de-identified, discarded tissue for education and research purposes upon admission to the hospital. PHT cells were dispersed from term placentas using a modification of previously published protocols (Kliman et al., 1986; Nelson et al., 1999), and maintained for up to 72 h in Dulbecco Modified Eagle medium (DMEM; Corning; New York, NY) containing 10% bovine growth serum (BGS; HyClone, Logan, UT) and antibiotics at 37°C in a 5% carbon dioxide–air atmosphere. Human osteosarcoma cells (U2OS; ATCC, Manassas, VA) were maintained in DMEM containing 10% BGS and antibiotics. HEK293XL (herein referred to as 293XL/0, which express TLR3), HEK293XL/TLR7 (293XL/7) and HEK293XL/TLR8 (293XL/8) were purchased from Invivogen (San Diego, CA) and were maintained in DMEM containing 10% BGS and 50 U/ml penicillin, 50 μg/ml streptomycin with the addition of 100 μg/ml Normocin (Invivogen) and 10 μg/ml of selection antibiotic Blasticidin (Invivogen). Infection of U2OS cells with vesicular stomatitis virus (VSV, Indiana, 7 h, MOI=0.02, 0.1 or 0.3), a non-segmented negative-strand RNA virus, was performed as previously described (Delorme-Axford et al., 2013; Bayer et al., 2018). The trophoblast BeWo cell line (ATCC) was maintained in F12K Kaighn's modified medium (Gibco, Gaithersburg, MD) supplemented with 10% BGS and antibiotics. Human uterine microvascular endothelial cells (HUtMECs; PromoCell, Burlington, Ontario, Canada) were cultured in EBM medium (Lonza, Walkersville, MD). Primary placenta fibroblasts (PPF) were isolated during standard placental cell isolation, as we routinely perform in our lab, with further purification of PPF cells using CD9 antibody (BS3022, Bioworld), as commonly performed and following established protocols (Manoussaka et al., 2005; Yui et al., 1994), and maintained in DMEM with 10% BGS and antibiotics.

For mRNA analysis, total RNA was extracted using TRI reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocol. RNA samples were further purified using on-column RNase-free DNAse (Qiagen, Germantown, MD). Total RNA was reverse transcribed using a HiCapacity cDNA synthesis kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol and using a StepOnePlus real-time PCR system (Applied Biosystems). Quantitative PCR was performed using SYBR select in a ViiA 7 system (Applied Biosystems), and results were calculated using the ΔΔCt method (Livak and Schmittgen, 2001) and normalized to the expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). In some cases, values were presented as log₂ (40−Ct) values (corrected for expression of GAPDH), where maximal cycle was set as 40 and the sample Ct value was subtracted from this value (Mouillet et al., 2010). All primer sequences were checked using BLAST (Basic Local Alignment Search Tool) for specificity and are presented in Table S1. Dissociation curves were run on all reactions to ensure amplification of a single product with the appropriate melting temperature. Control samples of H₂O were included in each qPCR experiment.

We applied a recently developed CLASH protocol (Helwak et al., 2013; Gay et al., 2018). Briefly, after culture for 72 h, PHT cells were washed with ice cold phosphate-buffered saline (PBS) and placed in UV crosslinker (Spectroline UV Crosslinker Select, Westbury, NY) at a wavelength of 250 nm at 400 mJ/cm². Cell pellets were lysed using a lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40 and 10 mM MgCl₂) plus protease inhibitor cocktail mini EDTA-free (Roche, Mannheim, Germany) and PhoSTOP (Roche). DNA was removed using RQ1 RNase-free DNase (1 U/µl, 1:50 dilution; Promega, Madison, WI) followed by partial RNase T1 digestion (Thermo Fisher; 1000 U/µl at final dilution of 1:20,000). Lysates were immunoprecipitated using rat anti-Ago2 antibody (Sigma, Saint Louis, MO, USA; MABE253)-conjugated magnetic beads. RNA was isolated on-column using a Qiagen RNeasy kit by following the kit instructions. RNA libraries were constructed using the Clontech SMARTER smRNA kit (TaKaRa Bio USA Inc., Mountain View, CA) according to the manufacturer's instructions. Sequencing data were analyzed using the bioinformatics pipelines developed by Moore et al. (Moore et al., 2015). The CLASH-seq data were deposited to the NIH Sequence Read Archive with BioProject accession #PRJNA659526.

Human UNC13D (NM_199242) sgRNA plasmid (pUNC13D sgRNA hereafter), which harbors the sgRNA sequence 5′-ATGAAGGTCTCGTCCCAGAC-3′ targeting exon 6 of the UNC13D genomic region, was purchased from Dharmacon (#GSGH11838-247008925; Dharmacon). For KO we followed Feng Zhang's protocol (Ran et al., 2013), with minor modifications. Briefly, U2OS cells in a 24-well plate were co-transfected using 0.5 µg of pUNC13D sgRNA along with the Cas9-encoding plasmid VP12 (Addgene #72247) using Lipofectamine 3000 (Thermo Fisher) according to the manufacturer's instructions. Single cells were sorted into ten 96-well plates to produce monoclonal KOs. We screened for KO clones by genomic DNA PCR using the primers shown in Table S1. Genomic DNA was extracted from the wells using QuickExtract DNA solution (QE09050; Lucigen, Madison, WI) and used for Q5 (M0494S; New England Biolabs) PCR reaction. Derived amplicons were heated at 95°C for 10 min and then cooled down to room temperature using the following parameters: 95–85°C (ramp rate of −2°C/s); 85–25°C (ramp rate of −0.3°C/s). DNA heteroduplex strands were then processed with T7 endonuclease I (M0302S; New England Biolabs) at 37°C for 1 h. Wild-type clones generated one band of 674-bp amplicon, and KO clones produced cleaved bands with varied sizes according to distinctive insertion/deletion (indel) positions. Identified KO clones were further expanded and validated by western blotting, as described below.

A lentiviral plasmid expressing UNC13D was purchased from Horizon (pLV-UNC13D, #OHS5898-202624196). Lentivirus was produced by transfection of HEK293T cells with pLV-UNC13D and two lentiviral packaging plasmids of pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260) according to the standard procedure (see Addgene website; https://www.addgene.org/protocols/lentivirus-production/). After 60 h, lentivirus-containing supernatants were centrifuged to remove cell debris, and used for transduction of U2OS cells in the presence of 8 µg/ml polybrene (Sigma; #TR-1003-G). At 48 h post transduction, U2OS cells were selected with 25 µg/ml blasticidin (Thermo Fisher; #A1113903) to clear any non-transduced cells. U2OS cells overexpressing UNC13D were exposed to 500 nM Torin 2 (Cell Signaling Technology; #14385S) at 37°C for 1 h prior to VSV infection.

miRNA mimics and siRNA SmartPool constructs were purchased from Dharmacon (Lafayette, CO), mature (single-stranded) miRNAs and RNA41 [a derivative of the U5 region of HIV-1 RNA, which does not stimulate TLR7 or TLR8 (Heil et al., 2004)] were custom synthesized by IDT (Coralville, IA). miR-517a, miR-512-3p, miR-516b-5p, miR-29a-5p, miR-630 and RNA41 included a phosphorothioate bond modification of all bases and were purified by HPLC. For experiments with miRNA, cells were transfected with Lipofectamine RNAiMAX (Thermo Fisher) in Opti-MEM (Thermo Fisher; #31985062). For regulation of autophagy-related proteins, U2OS cells were plated on a 12-well plate and transfected with miR-517a mimic or the relevant siRNA (40 nM each) and delivered with Lipofectamine RNAiMAX. The medium was changed after 24 h to fresh DMEM and the cells were cultured for a total of 48 h for RNA analysis or 72 h for proteins analysis. For VSV vRNA levels, we tested amplicons derived from the Glycoprotein (G) region in the VSV genome, as previously described (Delorme-Axford et al., 2013). We also performed qPCR to detect RNA of VSV Nucleoprotein (N). VSV RNA expression changes of either viral G or N RNA were comparable.

Luciferase reporter plasmids for Renilla (Promega), p65-Luc, ISRE-Luc and IFNβ-Luc were previously described (Delorme-Axford et al., 2013). Reporter plasmids including p65-, ISRE- or IFNβ-firefly luciferase plasmid (200 ng each) were mixed with 1 ng of control Renilla luciferase plasmid, and transfected into 293XL/0, 293XL/7 or 293XL/8 cells using Lipofectamine 3000 for 24 h in a 12-well plate. For assessment of TLR activation, cells were transfected with mature miRNA at concentration of 150 nM for 24 h or were exposed to the TLR ligands CL264 (TLR7 ligand, 10 µg/ml), TL8-506 (TLR8 ligand, 0.4 µg/ml) or free poly I:C (TLR3 ligand, 10 µg/ml) (all from Invivogen) in serum-free medium for 8 h. The cells were lysed with passive lysis buffer (Promega), and Firefly and Renilla luciferase activities were measured consecutively with the Dual Luciferase Reporter system (Promega) using a Veritas Microplate Luminometer (Turner BioSystems, Promega). Firefly luciferase activity was normalized to Renilla luciferase to control for cell number and transfection efficiency, and data expressed as relative luciferase units (RLU).

Cells were lysed on ice for 30 min in lysis buffer (20 mM HEPES, pH 7.5, 150 mM NaCl and 1% Triton X-100) supplemented with protease inhibitor cocktail mini EDTA-free and PhoSTOP (Roche) and centrifuged at 16,000 g at 4°C for 10 min to remove cell debris. Whole-cell lysates (160 µg) were mixed with 5 µl TFEB rabbit monoclonal antibody (Cell Signaling Technology; #37785S) at 4°C overnight. Protein G magnetic beads (30 µl; Thermo Fisher #88847) were then added and incubated at room temperature for 2 h. After washing beads with lysis buffer, TFEB was eluted by adding sample buffer containing 100 mM DTT and resolved on a 10% SDS–PAGE gel. PVDF membranes were blotted with rabbit anti-TFEB antibody (Cell Signaling Technology; #37785S; 1:500) or rabbit anti-phospho-Ser122-TFEB (Cell signalling Technology; #86843S; 1:200). A mouse anti-rabbit IgG (conformation specific, L27A9) HRP-conjugated antibody (Cell Signaling Technology; #5127S; 1:2000) was used as a secondary antibody to recognize the native rabbit IgG rather than the denatured and reduced heavy or light chains of rabbit IgG.

Cells were lysed on ice in RIPA buffer (TaKaRa Bio USA) supplemented with protease inhibitor cocktail mini EDTA-free and PhoSTOP (Roche), and sonicated for 3 s. Lysate concentrations were determined with a Pierce BCA protein assay kit (Thermo Fisher) using a Versa Max microplate reader (Molecular Devices LLC, San Jose, CA). Protein samples (10 µg) were separated using SDS–PAGE and transferred onto 0.2 µm PVDF membrane (Bio-Rad, Hercules, CA) using standard procedures. Membranes were immunoblotted with a mouse monoclonal anti-actin antibody (Sigma; #MAB1501; 1:40,000) and rabbit anti-UNC13D (Abcam, Cambridge, MA; ab109113; 1:1000), rabbit anti-ITGB4 (Cell Signaling Technology; #14803; 1:1000), rabbit ant-LC3B (Cell Signaling Technology; #2775; 1:2000; preferentially immuno-reactive to the lipidated LC3B-II form rather than the unmodified LC3B form), rabbit anti-RPTOR (Cell Signaling Technology; #2280; 1:1000), rabbit anti-phospho mTOR (Cell Signaling Technology; #2974; 1:1000) or rabbit anti-phospho p70 S6 kinase (Cell Signaling Technology; #9234; 1:1000). A horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA; #115035146; 1:40,000) or goat anti-rabbit IgG (Cell Signaling Technology; #7074; 1:10,000) was used as a secondary antibody. The blots were processed for chemiluminescence using SuperSignal West Pico or Dura (Thermo Fisher) and developed using an SRX-101A film processor (Konica Minolta, Tokyo, Japan), and imaged with a UVP BioImaging system (UVP, Upland, CA).

Cells were plated on coverslips (Thermo Fisher) coated with poly-D-Lysine (Thermo Fisher). After transfection with miRNA or incubation with specific TLR ligands, the cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS, blocked with a blocking buffer (5% Normal Donkey Serum and 0.3% Triton X-100 in PBS) for 1 h at room temperature. Subsequently, samples were stained for LC3B (using the same antibodies as for immunoblotting; final concentration 281 ng/ml) or p65 (rabbit anti-p65; Cell Signaling Technology; #8242; final concentration 530 ng/ml) diluted in buffer containing 1% BSA and 0.3% Triton X-100 in PBS at 4°C, overnight. The samples were washed and incubated with donkey anti-rabbit Alexa Fluor 488 secondary antibodies (Invitrogen, Carlsbad, CA; final concentration 2 µg/ml) at room temperature for 1 h. After washing, the coverslips were mounted using ProLong Diamond Antifade Mount with DAPI (Thermo Fisher). Pictures were taken at 60× magnification using a Nikon A1 Confocal Laser Microscope (Nikon Instruments Inc. Melville, NY).

293XL/8 and 293XL/0 cells were plated in a 12-well plate in duplicate at 5×10⁵ cells per well in complete DMEM medium. The next day, when the cells were near 80% confluence, the medium was changed to serum-free medium, and mature miRNAs (150 nM) or specific ligands were added. After 24 h, the conditioned medium from each of the two wells was pooled and concentrated to a final volume of 400 µl with a CentriVap Concentrator (Labconco, Kansas City, MO). Cytokine levels were determined using the Human CXCL10/IP-10 and IL-8/CXCL8 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) using standard protocols.

All statistical analyses were performed with Prism software (GraphPad Software, San Diego, CA). Statistical significance for multiple comparisons was calculated by one-way ANOVA and Tukey post-hoc test, or using two-tailed unpaired t-test, where appropriate. Significance was determined as P <0.05. Values are presented as mean±s.d., derived from at least three independent experiments, as indicated in each figure legend.

We thank Elena Sadovsky, Hui Li and Tiffany Coon for technical assistance; Lori Rideout for assistance during manuscript preparation; and Bruce Campbell for editing.

The peer review history is available online at https://jcs.biologists.org/lookup/doi/10.1242/jcs.246769.reviewer-comments.pdf