MicroRNA-30e-5p has an Integrated Role in the Regulation of the Innate Immune Response during Virus Infection and Systemic Lupus Erythematosus.

Precise regulation of innate immunity is crucial for development of appropriate host immunity against microbial infections and maintenance of immune homeostasis. MicroRNAs are small non-coding RNAs, post-transcriptional regulator of multiple genes, and act as a rheostat for protein expression. Here, we identified microRNA-30e-5p induced by hepatitis B virus and other viruses that act as a master regulator for innate immunity. Moreover, pegylated interferons treatment of patients with HBV for viral reduction also reduces miRNA. Additionally, we have also shown the immuno-pathological effects of miR-30e in patients with systemic lupus erythematosus (SLE) and mouse model. Mechanistically, miR-30e targets multiple negative regulators of innate immune signaling and enhances immune responses. Furthermore, sequestering of miR-30e in patients with SLE and mouse model significantly reduces type-I interferon and pro-inflammatory cytokines. Collectively, our study demonstrates the novel role of miR-30e in innate immunity and its prognostic and therapeutic potential in infectious and autoimmune diseases.

Introduction

Host innate immunity is an evolutionarily conserved defense system against microbial threats. These microbes express signature molecule known as pathogen-associated molecular patterns (PAMPs), which are sensed by host conserved sensors known as pattern-recognition receptors (PRRs) in various cellular compartments. The coordinated interactions among them activate a complex cascade of signaling pathways resulting in the development of innate immune responses, for the elimination of invading microbes, through production of pro-inflammatory cytokines, type I and III interferons (IFNs), chemokines, and recruitment of immune cells, and trigger cell death (Akira et al., 2006). These PRRs also sense host endogenous molecules known as damage/danger-associated molecular patterns (DAMPs) and trigger both heightened innate immune responses, including excesses interferon and cytokines production along with elevated TLRs activation, and cell death, resulting in the autoimmune disease, such lupus (Alvarez and Vasquez, 2017). These signatory features were reported to promote systemic lupus erythematous (SLE) (Yu et al., 2012; Devarapu and Anders, 2018). Elevated apoptosis and delay in clearance of apoptotic cellular bodies led to the accumulation of apoptotic debris, which is considered as a key process in the etiology of SLE, and it is also linked with the severity of SLE pathogenesis (Ronnblom and Leonard, 2019; Magna and Pisetsky, 2015; Mevorach, 2003; Munoz et al., 2008; Lovgren et al., 2004).

MicroRNAs (miRNAs) are a class of small non-coding RNAs (18–22 nucleotides) that fine-tune the protein expression through direct interaction with 3′ untranslated region (3′ UTR) of the gene transcript (He and Hannon, 2004). MicroRNA interacts with target transcript through base pairing and initiates degradation or blocking of translation machinery via multiprotein complex known as RNA-induced silencing complex (RISC) (Treiber et al., 2019). It has been reported that a single miRNA may have multiple mRNA targets and regulate cell signaling cascades and cellular responses during viral infections (Kim et al., 2015). Several viruses evade immune responses and establish infection by perturbing the host cellular miRNA expression or expressing viral(v)-miRNA upon infection (Zheng et al., 2013). In contrast, it has been reported that several host miRNAs restrict viral replication by targeting viral genome or those host genes that are essential for viral replication (Ingle et al., 2015). In this study, we have hypothesized that the dysregulated microRNAs during virus infections might have strong implication on innate immune responses to counter the viral infection. Therefore, we identified miR-30e, induced by different DNA and RNA viruses in primary human cells and various mammalian cell lines, which restrict virus replication. Introduction of miR-30e into the cells enhances the innate immune responses and therefore reduces viral load. Notably, higher levels of miR-30e were also detected in the serum of therapy-naive patients with HBV and vice versa effects after pegylated type I IFNs treatment. We identified several negative regulators of PRR-mediated signaling pathways, such as TRIM38, TANK, ATG5, ATG12, BECN1, SOCS1, and SOCS3, that were targeted by miR-30e post-transcriptionally through RNA-induced silencing machinery (Porritt and Hertzog, 2015; Kondo et al., 2012). Additionally, we demonstrated that miR-30e plays a pivotal role in the reduction of transcripts of negative regulators through Argonaute 2 (AGO2) protein pull-down assay. The miR-30e enhances innate immune responses by targeting key negative regulators; therefore, we chose the systemic lupus erythematosus (SLE) model to understand the role of miR-30e under physiological condition. Previously, it has been shown that miR-30e level enhances in SLE, in the Korean population, although this was not characterized (Moulton et al., 2017; Shen et al., 2012; Kim et al., 2016). We observed that patients with SLE and SLE mouse model have shown higher expression of miR-30e. We also demonstrated an ex vivo introduction of miR-30e antagomir into PBMCs of patients with SLE and an in vivo intra-orbital injection of locked nucleic acid (LNA)-based inhibitor for miR-30e in SLE-induced mice that reduces type-I interferons and pro-inflammatory cytokines and moderately enhances the negative regulators. Altogether, our study demonstrates the novel role of miR-30e in innate immune regulation and its probable prognostic and therapeutic potential in virus infection and an autoimmune disorder, SLE.

Results

Virus Infection Induces miRNA-30e to Inhibit Viral Infection through Enhancing Innate Antiviral Responses

To investigate the miRNAs involved in the regulation of innate immune response during viral infections, we performed unbiased data analyses on previously published reports and miRNA microarray GEO datasets as shown in the schematic workflow (Figure S1A). In particular, the miRNA reports in H5N1 (Vela et al., 2014) or Epstein-Barr virus (Gao et al., 2015) were analyzed for upregulated miRNAs. These upregulated miRNAs were compared with our previous miRNA profiling dataset from NDV infection in HEK293T cells (NCBI's Genbank_GSE65694). Upon comparison with NDV infection we selected miR-30e-5p, miR-27a-3p, and mir-181a/2-3p as the common miRNAs across miRNA profiles related to viral diseases (Figure S1B). Our analysis identified miR-30e as a unique miRNA that was predicted to target various PRR-mediated signaling regulators during negative regulation of innate immune responses (Figure S1C) and was upregulated in viral infections; moreover, its mature form was highly conserved among the wide range of species (Figure S1D). Additionally, datasets for H1N1 infection in mice (NCBI's Genbank_GSE69944), H5N1 infection in human lung carcinoma cells (A549 cells, NCBI's Genbank_GSE96857), and HBV-infected liver tissues of patients with hepatitis (NCBI's Genbank_GSE21279) were also analyzed by GEO2R package for upregulated miRNAs, and among all upregulated miRNAs, miR-30e upregulation is represented here (Figure S1E). The expression of miR-30e was upregulated during viral infections or stimulation with PAMPs in vitro in various cell lines (Figures S2A–S2F). At the transcriptional level, miR-30e promoter activity was moderately enhanced by NDV but was unaffected by rh-IFNβ or rh-TNFα stimulation, which activated the ISRE, IFNβ, and NF-κB promoters, respectively, suggesting that miR-30e expression might be induced by the viral infections but not by the cytokines produced during infection (Figures S2G–S2K).

To understand the clinical relevance, induction of miR-30e was tested in the cohort of 51 non-treated patients with HBV (demographic details mentioned in Table S1). Notably, the expression of miR-30e was evaluated from serum samples of therapy-naive patients with chronic hepatitis B (CHB) in comparison with healthy controls, and significant elevated levels of miR-30e were detected in patients with HBV (Figure 1A). Similar results were obtained with HepG2 cell line treated with serum from patients with HBV (HBV PS) for different time points; as shown (Figure 1B), the induction of miR-30e enhanced at 2 and 3 dpi (days post infection) with the highest expression at 3 dpi. Additionally, miR-30e mimic (miR-30e) inhibited HBV infection in HepG2 cells treated with serum from patients with HBV or in HepG2215 cells, stably expressing HBV replicon cells as compared with control miRNA (miR-NC1)-treated cells, which was tested by HBV-specific RNA and DNA quantification (Figures 1C and 1D) (Figure S3A). Interestingly, ectopic expression of miR-30e significantly reduced the HBV replication in HepG2-NTCP cells as tested by HBV RNA and HBV pgRNA quantification (Figure S3B). Notably, we found significant elevated levels of HBV DNA and HBV covalently closed circular DNA in HBV patient serum infected HepG2 cells and stably expressing HBV replicon HepG2215 cells compared with uninfected HepG2 cells (Figures S3C and S3D) to show the infection status in the cells. On the other hand, expression of IFNλ1 and IFIT1 was enhanced in HepG2 and HepG2215 cells, respectively, in the presence of miR-30e (Figures 1C and 1D), and IFNλ1 in HepG2215 cells (Figure S4A). Similarly, HBV infection in HepG2-NTCP cells (liver hepatoma cell line permissive for HBV infection through NTCP receptor) overexpressing miR-30e (ectopic) elevated IFNλ1 transcript (Figure S4B). To study whether miR-30e was involved in controlling RNA virus infection, we infected human PBMCs (hPBMCs) with NDV to quantify the expression of miR-30e. We found that NDV infection elevated the expression of miR-30e in a time-dependent manner (Figure 1E). Additionally, PBMCs infected with NDV in the presence of miR-30e showed a significant reduction in NDV replication with a concomitant elevation of IL6 expression compared with control (miR-NC1), whereas miR-30e inhibitor (AmiR-30e) reversed this phenomenon (Figure 1F). Similar inhibition of viral replication was observed in multiple cell lines infected with NDV in the presence of miR-30e or miR-NC1 (Figures S3E–S3G). Comparable results for antiviral responses were obtained after NDV infection in different cell types at the transcript and protein levels (Figures S4C–S4I) in the presence of miR-30e, and it also activated the ISRE, IFNβ, and NF-κB promoters as tested by luciferase assay (Figures S4J–S4L). Furthermore, miR-30e presence reduces NDV replication in terms NDV protein as tested by NDV-specific antibody estimated by western blot, microscopy, and FACS analysis, respectively (Figure 1G, 1H, and S3H). To further validate the function of miR-30e in controlling viral infections, we quantified the expression of miR-30e upon Sendai virus (SeV) infection in A549 cells. SeV induced the expression of miR-30e (Figure 1I), and ectopic expression of miR-30e (miR-30e mimic) inhibited the SeV replication as shown by qRT-PCR and FACS analysis (Figures 1J and 1K) through enhancing antiviral genes, such as the expression of IFIT1 (Figure 1J).

Figure 1

Viral Infection Induces miRNA-30e that Inhibits Virus Replication by Promoting Innate Immunity

(A) Quantification (as determined by qRT-PCR analysis) of the fold changes in the abundances of miR-30e as indicated, in the serum collected from patients with hepatitis B (n = 51) compared with healthy controls (n = 24).

(B–F, I, and J) Quantification of the fold changes in the relative abundances of miR-30e, viral transcripts, and respective innate immune transcripts (IFNλ1, IL6, and IFIT1) at the indicated times after treatment or infection with (B) HBV patient’s serum (HBV PS) in HepG2 cells, (C) HepG2 cells were transfected with miR-30e (50 nM) or miR-NC1 (50 nM) prior to infection (D) HepG2215 cells, stably expressing HBV replicon HepG2 cells transfected with miR-30e or miR-NC1, (E) NDV (MOI 5) in human(H) PBMCs, (F) hPBMCs transfected with miR-30e or AmiR-30e (50 nM) or miR-NC1prior to infection, and (I) SeV (MOI 5) in A549 cells (J) A549 cells transfected with miR-30e or AmiR-30e or miR-NC1 prior to infection.

(G, H, and K) Quantification of viral infection as indicated in (G) HEK293 cells (transfected with miR-30e or AmiR-30e for 24 h then infected with GFP-tagged NDV (NDV-GFP) (MOI 5) for 36 h and subjected to immunoblot analysis using antibodies specific for GFP (anti-GFP antibody) and γ-tubulin (used as a loading control), (H) HeLa cells transfected with miR-30e or infected with NDV-GFP and subsequently subjected to confocal microscopic analysis for NDV particles tagged with anti-GFP antibody (green), and nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI; blue) and (K) A549 cells were transfected with miR-30e or AmiR-30e for 24 h then infected with RFP tagged SeV (MOI 5) for 24 h and analyzed by flow cytometry. Ctrl represents control untreated sample. Data are mean ± SEM of triplicate samples from single experiment and are representative of two (B–K) independent experiments. ∗∗∗p < 0.001, ∗∗p < 0.01 and ∗p < 0.05 by one-way ANOVA Tukey test, Mann-Whitney test and unpaired t test. See also Figures S1–S6 and Table S1.

Next, the role of miR-30e on DNA virus replication was determined; to this end, HFF cells were infected with DNA virus, HCMV, alone or along with miR-30e or miR-NC1. The viral replication was significantly reduced in the presence of miR-30e compared with the miR-NC1-treated HFF cells as quantified by HCMV transcript encoding viral glycoprotein gene (Gly B) by real-time PCR and analyzed by microscopy (Figure S5A), and additionally, the transcript levels of IL6 was enhanced (Figure S5B).

To investigate how miR-30e influences antiviral responses upon treatment with pure viral ligand such as poly I:C and ssRNA, different cells including human PBMCs were transfected and stimulated, respectively, along with miR-30e.The expression of various ISGs and cytokines such as IFNβ, CXCL10, and IL6 (Figures S6A–S6F) were elevated upon poly IC or ssRNA treatment along with miR-30e transfection, and similar results were found for the promoter activity of ISRE and IFNβ in the presence of miR-30e (Figures S6G and S6H). Collectively, our results demonstrate that miR-30e is upregulated during virus infection and miR-30e inhibits viral replication by promoting the expression of innate antiviral genes.

miR-30e Globally Enhances Innate Immune Responses during Virus Infection

To investigate the effect of miR-30e on innate immune responses upon virus infection, A549 cells were either mock transfected or transfected with miR-NC1 and miR-30e for 24 h, followed by infection with NDV for 24 h, and finally subjected to whole-transcriptome sequencing using an Illumina next-generation sequencer (NGS) and analyzed for differentially expressed genes as shown in the schematic (Figures 2A and S7A). Notably, the transfection efficiency of miR-30e in both replicates was confirmed by qRT-PCR using miR-30e-5p TaqMan assay and reduction in viral infection was confirmed by quantifying the NDV RNA in both the replicates to establish further analysis as per our previous findings (Figure 2B). Principal component analysis of the RNA-seq data for the samples resulted in the formation of three distinct groups (miR-30e, miR-NC1, and uninfected) according to their treatment (Figure 2C). Additional analysis of transcriptomic data showed that 1,179 genes were significantly upregulated and 1,206 genes were significantly downregulated upon miR-30e transfection in comparison with miR-NC1, represented by volcano plot (Figure 2D) and represented by MA plot (Bland-Altman plot where “M” represents log ratio and “A” represents mean average as plotting on MA scales) (Figure S7B). Moreover, KEGG pathway analysis of significantly upregulated genes, upon miR-30e treatment and NDV infection, indicated enrichment of genes belonging to key cellular machineries, namely, cell cycle, NOD-like receptor signaling pathway, MAPK signaling pathway, TLR signaling pathway, RLR signaling pathway, PI3K-AKT signaling pathway, cytokine-cytokine receptor interaction pathway, NF-κB signaling pathways, and TNF signaling pathway (Figure 2E). The relative expression levels of the highest expressing genes involved in these pathways is represented by heatmap (Figure S7C). Intriguingly, we noticed that a significant number of interferons-stimulated genes (ISGs) like IFITM2, ISG20, IFIT5, MX1, IFIT2, IRF8, IFNB1, IRF1, IRF2, IFNL1, IFNL4, IRF7, CXCL10 (IP10), ISG15, IL6, IRF3, OAS2, IFIT1, IFIT3, and OASL were also predominantly upregulated (Figure 2F), which combines with the initial findings of the study. Furthermore, our NGS results were verified by quantifying the expression level of type 1 interferon IFNβ, interferon stimulated genes IFIT1 and OAS2, and pro-inflammatory cytokines IL6 and CXCL10 (Figure 2G). These outcomes strongly suggest that miR-30e reduces the viral replication by enhancing the innate immune responses upon activation of various signaling cascades. Additionally, miR-30e impact on innate immune responses during viral infection prompted us to investigate transcriptome and gain mechanistic insight for the target of miR-30e.

Figure 2

Transcriptomic Analysis Shows miR-30e Enhances Innate Immune Responses during NDV Infection

(A) Schematic outline of experimental setup, transfection with control (miR-NC1) or miR-30e and NDV infection (MOI 5) in A549 cells at indicated time, samples were subjected to whole-transcriptome sequencing and differential gene analysis.

(B) Quantification of the fold changes in the abundances of miR-30e is measured by qRT-PCR and normalized with U6 control, and NDV viral transcripts in both the replicate samples used for transcriptome sequencing and analysis.

(C) Plot showing first two components from principal component analysis of all the six samples, distance between samples indicate how different they are from each other in terms of gene expression.

(D) Volcano plot represents differential expression of genes between two groups of samples (miR-30e and miR-NC1 overexpression) during NDV infection in A549 cells. For each gene: p value is plotted against fold change (miR-30e vs miR-NC1). Genes significantly changed (>1.5-fold) are colored in red (upregulated) and blue (downregulated).

(E) KEGG pathway analysis of upregulated genes, outer circle indicates top upregulated pathways and the inner circle represents corresponding combined score (a derivative of p value and Z score).

(F) Heatmap represents the relative abundance of top upregulated interferon stimulated genes across different samples. Green color indicates downregulation and red color indicates upregulation of genes.

(G) Validation and quantification (measured by qRT-PCR) of the fold changes in the abundances of type 1 interferon and pro-inflammatory cytokines in the samples of A549 cells transfected with miR-NC1 or miR-30e and infected with NDV as indicated (NDV + miR-NC1) and (NDV + miR-30e), analyzed by RNA sequencing.

See also Figure S7.

miR-30e Targets Negative Regulators of PRR-Sensing and Interferon Signaling Pathways

To investigate the underlying molecular mechanism for the reduction of viral burden and enhanced antiviral innate immune responses by miR-30e, we conducted unbiased rigorous screening using various bioinformatic tools for the identification of innate immune genes. To filter the genes transcript targeted by miRNA, certain criterion for screening were applied. First of all, common genes involved in innate immune regulation upon viral infections and targeted by miR-30e were identified and subjected to KEGG pathway analysis. The analysis revealed that a majority of genes (TRIM38, TANK, ATG5, ATG12, BECN1, SOCS1, SOCS3, TRIM13 and EPG5) were involved in negative/down regulation of PRRs-mediated signaling pathways (Figure 3A). Additionally, our NGS analysis demonstrated that expression of identified negative regulators during NDV infection were reduced upon miR-30e treatment as compared with the miR-NC1 group (Figure 3B). This further concludes that the negative regulators are the potential targets of mir-30e. The binding efficiency for miR-30e and identified targets genes are significantly high to alter any physiological functions by the miRNA that was tested by different in silico tools such as miRanda, DIANA, Target Scan, miRDB, and BiBiServ2_RNAhybrid as reported (Table S2). Although few targets of miR-30e are not negative regulators, the binding energy for those target transcript and miRNA assembly is low (Figure S8) and, therefore, it may not significantly alter the cellular function. To test the specificity of miRNA with the 3′ UTR of identified negative regulator genes, the 3′ UTR of the gene was cloned downstream of luciferase gene under the CMV promoter to perform the luciferase assay. It was found that miR-30e significantly reduced the luciferase activity of investigated genes compared with control miR-NC1 (Figure 3C). In contrast, introduction of mutation (3′ UTR_MUT) in cloned 3′ UTR/3′ UTR_WT by site-directed mutagenesis did not change the luciferase activity in the presence of miR-30e and it was comparable with 3′ UTR_WT (wild-type). miR-NC1 was used as a control for the experiment (Figure S9A); moreover, it could be noted here that the target sequence in each negative regulator genes was the same. Additionally, we knocked down these negative regulators in HEK-293T cells and infected them with NDV to further estimate the level of IFNβ, which clearly elucidated their inhibitory effect on the mRNA levels of IFNβ within a cell; this effect was found to be significant with respect to the majority of the targets (Figure 3D). And we observed that the production of IFNβ is comparable after knockdown of target genes and introduction of miR-30e into the cells during viral infection suggesting the pivotal role of miR-30e in the suppression of negative regulators transcripts. Furthermore, we scanned the 3′ UTRs of identified negative regulators for RNA-binding site for AGO2 protein, in CLIP database, which is a key component of the miRNA-mediated silencing complex (RISC) and found that the miR-30e strongly complexes with the target genes as shown in (Table S3). To validate miR-30e and negative regulator transcripts (TRIM38, TANK, ATG5, ATG12, BECN1, SOCS1, and SOCS3) interaction, AGO2 pull-down assay was performed as shown in schematic diagram (Figure 3E), and we found that introduction of miR-30e significantly enriches the transcript of negative regulators compared with the NDV alone infection or NDV infection along with control miR-NC1 treated cells suggesting that miRNA-30e directly interacts with the transcript through the formation of RISC.

Figure 3

miR-30e Targets 3′ UTR of Negative Regulators of Innate Immune Signaling Pathways

(A) Screening pipeline used for identification of miR-30e target genes, based on the indicated schematic workflow, final hits of 09 genes corresponds to negative regulators targeted by miRNA-30e.

(B) RNA-seq analysis of transcriptome data for the identification of negative regulators (targeted by miR-30e) upon miR-30e transfection and NDV infection (MOI 5) compared with miR-NC1 in two replicate samples.

(C) HEK293 cells were transfected with 50 ng of pRL-TK and 300 ng of 3′ UTR_WT (of indicated genes) together with 25 nM miR-30e or miR-NC1 mimics; 24 h after transfection, the cell was lysed and subjected to luciferase assay.

(D) HEK293T cells were transiently transfected with 1.5 μg of sh-clones of indicated genes or scrambled control for 48 h then infected with NDV (MOI 5) for 24 h and subjected to the quantification of the indicated transcripts or genes and IFNβ.

(E) Schematic for RNA immunoprecipitation assay. HEK293 cells were transfected with plasmid encoding FLAG-AGO2 in presence of miR-30e (50 nM) and miR-NC1 (50 nM) and then infected with NDV (MOI 5). Twenty-four hours after transfection cells were subjected to RNA immunoprecipitation with anti-FLAG antibody and quantified for TRIM38, TANK, ATG5, ATG12, BECN1, SOCS1, and SOCS3 transcripts.

(F) A549 cells were transfected with miR-30e or miR-NC1 mimic and then infected with NDV (MOI 5) for 36 h before being subjected to immunoblot analysis with antibodies specific for indicated protein or γ-tubulin (used as a loading control). Percentage represents the area under the curve/peak and/or intensity of the bands calculated by densitometry analysis using ImageJ software. Difference in the percentage of density between the three groups is statistically significant (performed using unpaired t test).

Data are mean ± SEM of triplicate samples from single experiment and are representative of three (C) and two (D–F) independent experiments. ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05 by one-way ANOVA Tukey test and unpaired t test. See also Figures S8–S10 and Tables S2 and S3.

Next, the ectopic expression of miR-30e reduced the expression level of these targets (TRIM38, TANK, ATG5, ATG12, BECN1, SOCS1, and SOCS3) in A549 cells compared with the control after NDV infection (Figure S9B). Consistent with these results induction of targets was also reduced in HBV-patient serum-treated HepG2 cells, HepG2215 cells stably expressing HBV replicon cells, and HepG2-NTCP cells infected with HBV, in the presence of miR-30e compared with the control miRNA transfection (Figures S10A–S10C), suggesting that miR-30e targets these genes during HBV and NDV infection in HepG2, HepG2215, HepG2-NTCP, and A549 cells, respectively. Similar results were obtained due to miR-30e transfection in NDV-infected and poly I:C-treated HeLa cells (Figures S10D and S10E). We not only confirmed the expression of negative regulators by analyzing transcripts but also tested for protein expression using specific antibodies by western blot analysis. The introduction of miR-30e significantly reduced the expression of TRIM38, TANK, ATG12, BECN1, SOCS3, and SOCS1 as shown (Figure 3F). Therefore, our results strongly suggest that these key negative regulators of innate immunity are targeted by miR-30e, which are induced during virus infection and resulted in enhanced antiviral responses.

dsDNA (DAMP) Induces miR-30e and Enhances Innate Immune Responses

Our observation for induction of miR-30e and subsequent heightened innate immune responses upon viral infection or pure PAMPs stimulation prompted us to investigate the ability of host DAMPs for induction of miR-30e because sustained DAMPs production in the host can lead to enhanced sterile inflammation and subsequently it might establish autoimmune disease (Alvarez and Vasquez, 2017). To this end, ex vivo experiment was performed using hPBMCs from three healthy volunteers. The genomic DNA was extracted from a portion of hPBMCs and sonicated to make small fragments (approximately 110–150 bps) for efficient transfection into the cells (Figure 4A). The cultured hPBMCs were stimulated with the extracted small DNA fragments and tested for the induction of miR-30e and innate immune cytokines. The dsDNA (DAMP) stimulation significantly induces the miR-30e (Figure 4B) and expression of IFNα, IFNβ, IFIT1, and IL6 genes in the cells of all three healthy volunteers (Figures 4C–4E). Next, to understand physiological significance of DAMP-induced innate immune cytokines, the dsDNA (DAMP)-stimulated cells were infected by NDV and NDV replication was measured. The dsDNA stimulation significantly reduced the NDV replication (Figure 4F) suggesting that inflammatory cytokines and type I interferons induced by dsDNA inhibited the viral replication, although inhibition of viral replication could be the collective result of both dsDNA and virus-mediated induction of proinflammatory cytokines, type I interferons, and type I inducible genes. Finally, we examined the levels of apoptosis induced by autologous dsDNA in hPBMCs by Annexin-PI assay using FACS analysis as shown in Figure S11A; dsDNA stimulation enhanced apoptosis compared with the mock stimulation, and it is comparable with the positive control treated cells, by Camptothecin. Additionally, the levels of TLRs 3/7/9 were estimated upon dsDNA treatment as previously it has been reported that DAMPs enhance the levels of these TLRs. Consistent with previous observation, we obtained similar results (Figures S11B–S11D). Taken together, these results showed that DAMPs, particularly dsDNA, enhance miR-30e and innate immune responses as well as promote apoptosis and induce TLRs; these important characteristic features for the development of autoimmune disorder in co-occurrence with DAMPs (dsDNA) and miR-30e might play a pivotal role in autoimmune diseases.

Figure 4

Human PBMCs Stimulated with dsDNA (DAMP) Induces miRNA-30e and Enhances Innate Immune Responses

PBMCs from three healthy individuals were transfected with their own genomic DNA (ds DNA).

(A) Isolated genomic DNA sonicated into small fragments of dsDNA of approximately 100–150 bps each (as shown) and transfected using Lipofectamine 2000.

(B–F) Quantification (by qRT-PCR analysis) of the fold changes in the relative abundances of (B) miR-30e, (C–E) respective innate immune transcripts (IFNα, IFNβ, IFIT1, and IL6) in all three individuals, and (F) NDV viral transcripts in presence of dsDNA.

Data are mean ± SEM of triplicate samples from single experiment and are representative of three independent experiments in three different individuals. ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05 by one-way ANOVA Tukey test and unpaired t test; N.D., not detected. See also Figure S11.

Ex Vivo and In Vivo Dissemination of miR-30e

To investigate the role of miR-30e under physiological condition, an autoimmune disease, SLE, was selected because patients with SLE show enhanced inflammatory cytokines, type I interferons, and type I interferon-inducible cytokines production (Moulton et al., 2017). Patients with SLE also had elevated levels of several autoantibodies particularly antinuclear and anti-dsDNA antibodies. Therefore, PBMCs were isolated from clinically verified patients with SLE (P:n = 13) as shown (Table S4) and healthy controls (HC:n = 13) and cultured for 72 h, as shown in the schematic workflow (Figure 5A). The expression levels of IFNβ, IFIT1, and IL6 were compared and quantified by qRT-PCR. As expected, we found that the expression levels of IFNβ, IFIT1, and IL6 were significantly enhanced in patients with SLE compared with healthy controls (Figure 5B). The enhanced innate immune responses prompted us to investigate the expression levels of miR-30e. Interestingly, the expression of miR-30e is significantly enhanced (several fold) in patients (n = 13) compared with healthy controls (n = 13) (Figure 5C). Further to confirm our observations in patients with SLE, the SLE mouse model was used. The New Zealand white/black (NZW/B) mice were extensively used for SLE studies as shown in the schematic workflow (Figure 5D). The splenocytes from both parents NZB and/or NZW mice (n = 7) mice and lupus prone F1 (F1: NZW/B n = 7) generation mice were tested for the expression levels of Cxcl10, Tnfα, and Il6 by qRT-PCR (Figure 5E). The F1 mice showed a significantly high level of inflammatory responses compared with non-SLE parent mice. Consistent with human SLE results, the expression of miR-30e is enhanced manyfold both in F1 mouse splenocytes and serum compared with the parents (Figures 5F and 5G). Additionally, GEO dataset: NCBI's Genbank_GSE79240 (Wang et al., 2016) was utilized to observe the differential expression of microRNAs during SLE, especially miR-30e expression level, which further revealed that expression of miR-30e modestly enhanced in dendritic cells of patients with SLE (n = 5) compared with healthy controls (n = 5) (Figure S12A). To understand the relevance of miR-30e in another autoimmune disease, we reanalyzed the previously submitted GEO dataset (NCBI's Genbank_GSE55099) for patients with type 1 diabetes mellitus. The reanalysis unveils that the expression of miR-30e significantly enhanced in PBMCs of patients (n = 12) compared with healthy controls (n = 10) (Figure S12B). Collectively, these results suggest that enhanced innate immune responses are strongly linked with miR-30e expression under physiological condition and it might also play a pivotal role in immuno-pathogenesis of SLE in both human and mouse model.

Figure 5

Enhanced Innate Immune Responses and miRNA-30e Expression Suppress Negative Regulators in the Patients with SLE and SLE Mouse Model

(A and D) Schematic representation of the workflow for quantification of relative expression by qRT-PCR analysis of indicated transcripts IFNβ, IFIT1,IL6, TRIM38, TANK, SOCS1, and SOCS3 (in patients with SLE) and Cxcl10, Tnfα, Il6, Socs1, Socs3, Atg5, and Atg12 (in SLE mice) and miR-30e at indicated time points in (B and C) PBMCs from SLE diagnosed patients (P) (n = 13) and healthy controls (HC) (n = 13). (E) Splenocytes from parent (New Zealand White and Black-NZW and NZB) (PM) (n = 7) and lupus induced mice (NZW/B -F1 progeny) (F1) (n = 7) and (F and G) Splenocytes from PM (n = 9) and F1 (n = 12) and serum from PM (n = 16) and F1 (n = 21). Data are mean ± SEM of triplicate samples from single experiment and are representative of single (B, C and H) two independent experiments (E–G and I). ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05 by unpaired t test Mann-Whitney test. See also Figures S12 and S13 and Table S4.

The enhanced expression of inflammatory cytokines, type I IFNs, and type I IFN-inducible genes along with elevated miR-30e in patients with SLE and SLE mouse model prompted us to examine the levels of our previously identified negative regulators as shown (Figures 3A and 3B). Additionally, it has been reported that several negative regulators of innate immunity play a crucial role in the development of autoimmune diseases. As expected, the expression of negative regulators of PRR-mediated signaling pathways, namely, TRIM38, TANK, SOCS1, and SOCS3 was significantly reduced in the PBMCs of patients with SLE compared with healthy controls (Figure 5H). To support our observation, reanalysis of previously submitted GEO dataset, NCBI's Genbank_GSE11909 (Chaussabel et al., 2008) for patients with SLE (n = 103) and healthy controls (n = 12) in PBMCs reveal that, in SLE, the identified negative regulators of innate immune signaling pathway that may be targeted by miR-30e were significantly reduced in patients (n = 103) compared with healthy controls (n = 12) (Figure S13A). To confirm our observations, splenocytes from mouse model were analyzed for identified negative regulators. Consistent with human result for the expression of negative regulators, the expression of Atg5 and Atg12 was significantly reduced, whereas expression of Socs1 and Socs3 was moderately reduced in SLE mouse (F1-NZW/B mice: n = 7) compared with parent mice (PM-NZW-NZB: n = 7) (Figure 5I). Collectively, these results suggest that, in SLE pathogenesis, in both human and mouse, enhanced expression of miR-30e might play a crucial role by suppressing the expression of negative regulators of innate immune signaling pathway, which in turn enhances innate immune cytokines and contributes to the development or severity of the disease. Therefore, manipulation of miR-30e expression might prove to be a key factor for controlling SLE pathogenesis.

Prognostic and Therapeutic Potential of miR-30e

To explore the prognostic and therapeutic potential of miR-30e, which modulates innate immune responses during infection and autoimmune diseases, particularly HBV infection and SLE, we tested the prognostic potential of miR-30e. We validated the miR-30e expression by comparing the serum levels in pre- and post 6 months therapy (pegylated type I interferon) samples of patients with HBV (demographic details mentioned in Table S5). Strikingly, we found significant reduction of miR-30e expression after interferon therapy (Figure 6A), suggesting that HBV infection triggers miR-30e over-expression in the host, which combats the virus infection, that is, otherwise ablates upon Peg-IFN treatment, a well-established therapy that reduces the hepatitis B virus titer in patients. Additionally, GEO dataset NCBI's Genbank_GSE104126 (Yang et al., 2018) supports the above finding in the context of miR-30e expression level, as its reanalysis revealed, that there was significant reduction in the level of miR-30e in pegylated type I interferons treated and HBsAg-loss (reduction in viral titer) patients compared with pegylated type I interferons treated and non-HBsAg loss (no change in viral titer) patients (Figure S14).

Figure 6

Prognostic and Therapeutic Potential of miR-30e

For a Figure360 author presentation of this figure, see https://doi.org/10.1016/j.isci.2020.101322.

(A) Schematic representation of the workflow for quantification of the fold changes by qRT-PCR analysis of miR-30e as indicated, in the serum collected from hepatitis B (HBV) naive patients (n = 7) compared with HBV treated (with pegylated IFNs) patients (n = 7).

(B) Schematic representation of workflow for quantification of the fold changes in the relative abundances of miR-30e and IFNβ as indicated, in the PBMCs from patients with SLE treated with/without AmiR-30e (miR-30e inhibitor).

(C) Schematic representation of the ex vivo experiment workflow for quantification of the fold changes in the relative abundances of Il6 and Tnfα as indicated, in the splenocytes from SLE mice model (as described previously) treated with AmiR-30e (miR-30e inhibitor) and AmiR-NC1.

(D) Schematic representation of the in vivo experiment workflow for quantification of the fold changes of miR-30e, Il6, Cxcl10, Socs1, and Socs3 as indicated, in the splenocytes and serum from SLE mice model (as described previously); four mice distributed in each group were subjected to LNA-miR-30e-antagomir (LNA Amir-30e) and LNA-negative control antagomir (LNA NC) treatment (explained in materials and methods). Data are mean ± SEM of triplicate samples from single experiment (A, B, and D) and are representative of two independent experiments (C). All the p values/∗∗∗p < 0.001 defined by paired t test.

(E) Viral PAMPs (green) and DAMPs (red) sensed by pattern recognition receptors (PRRs) to activate cascade of innate immune signaling pathways, which induce pro-inflammatory cytokines (yellow), type I and type III interferons (sky blue), and miRNA-30e (purple). miRNA-30e regulates both PAMPs and DAMPs induced immune responses by targeting the 3′ UTR of negative regulators (dark blue) of innate immune signaling pathways and reducing the expression of these negative regulators (gray). During viral infection, miR-30e is induced, which reduces the cellular abundance of negative regulators to enhance innate immune responses and facilitate viral clearance. The endogenous host DNA induces miR-30e and subsequently enhances innate immune responses for the development of autoimmune disease, SLE. SLE signatory inhibition is shown by LNA-AntigomiR-30e and endogenous miR-30e complex.

See also Figure S14 and Table S5.

Next, we investigated the therapeutic potential of miR-30e modulator and have shown the importance of AmiR-30e (miR-30e inhibitor) that sequesters the activity of endogenous miR-30e. Our results showed that patients with SLE and mouse model produce high inflammatory responses in terms of innate cytokines and it is linked to the elevated miR-30e expression (Figures 5A–5G) and contributes to the reduction of negative regulators (Figures 5H and 5I). Therefore, we introduce AmiR-30e into PBMCs of patients with SLE. Interestingly, we found that ectopic expression of AmiR-30e sequesters the expression of miR-30e in PBMCs of patients with SLE and reduces the IFNβ expression as quantified by qRT-PCR (Figure 6B). Next, we examined the role of AmiR-30e in SLE mouse model, NZW/B. In an ex vivo experiment, transfection of splenocytes, derived from the F1 mice (NZW/B), with AmiR-30e significantly reduces the expression levels of Il6 and Tnfα compared with the control AmiR-NC1 (Figure 6C). Furthermore, to show the stable and specific sequestering effects of microRNAs for therapeutic relevance, we found that previously it was reported that locked nucleic acid (LNA)-based chemistry to design a potent inhibitor against the microRNA has showed promising effects in in vivo studies (Garchow et al., 2011). Therefore we finally performed in vivo experiments to test the effects of LNA-anti-mir-30e-5p (LNA Amir-30e) on innate immune responses in SLE mice. The two groups of mice, each consisting of four mice, were injected with LNA Amir-30e or LNA negative control (LNA NC) through retro-orbital route thrice with 1-day interval to sequester the endogenous expression of mir-30e, to measure the abundance of mir-30e and related genes. The expression of mir-30e in serum and splenocytes of SLE induced F1 (NZW/B) mice was significantly reduced compared with the LNA NC treated mice. Additionally, innate immune cytokines, namely, Il6 and Cxcl10, significantly reduced in LNA Amir-30e-treated mice compared with the LNA NC. In contrast, the expression of negative regulators, Socs1 and Socs3, significantly enhanced in LNA Amir-30e-treated mice compared with the LNA NC (Figure 6D). Taken together these findings conclude that LNA Amir-30e was stable under in vivo conditions and significantly inhibits the activity of mir-30e in SLE mouse model, which further reduces the inhibition/targeting of negative regulators, contributing toward controlling of SLE phenomena. Overall, our study presents the involvement of miR-30e and its inhibition in two of the challenging diseases, virus infections and autoimmune disorder, through a common axis of mechanism, summarized in Figure 6E.

Discussion

Innate immune responses to viral infection induce the production of pro-inflammatory cytokines and type I interferons (IFNs) through a cascade of complex signaling pathways that play critical roles in development of appropriate anti-viral immunity. In contrast, dysregulation of these signaling pathways results in inefficient clearance of microbial infection, immunopathology, or autoimmune diseases (Akira et al., 2006; He and Hannon, 2004). Therefore, the expression and activation of signaling molecules in signaling pathways are tightly regulated at transcriptional, post-transcriptional, translational, and post-translational levels. The non-coding small (micro) RNAs play a pivotal role in fine-tuning of protein coding genes through affecting the stability and modulating the translation of gene transcripts. Here, our study identifies a novel role of miR-30e in the regulation of innate immune signaling pathway during virus infections (chronic infection, such as HBV) and immuno-pathogenesis of autoimmune disorder, SLE. We also demonstrated the therapeutic and prognostic potential of miR-30e inhibition by AmiR-30e (antagomir-30e) and miR-30e in SLE and HBV infection, respectively.

The miR-30e was identified through unbiased in silico screening using GEO datasets obtained from cell lines, primary cells, mice, or human patients challenged by different viruses. Although other miRNAs such as miR-27a-3p and miR-181a/2-3p were also induced, however, their complementation potency toward mRNA-miRNA targets was manyfold lower than that of miR-30e. The miR-30e has been reported to be associated with cancer (Ning et al., 2017; Feng et al., 2017; Liu et al., 2017)-, cardiac dysfunction (Su et al., 2018), kidney malfunction (Wu et al., 2015), fatty acid deregulation, as a biomarker for SLE (Kim et al., 2016), a dysregulated micro RNA during Zika virus infection (Kozak et al., 2017), and suppressor for Dengue virus (Zhu et al., 2014). However, its role in innate immune signaling pathway and innate immune responses during virus infections and pathogenesis of autoimmune diseases such as SLE remains unclear.

Our results showed that miR-30e induced manyfold in the serum of (n = 51) therapy-naive patients with chronic hepatitis B (CHB) compared with healthy control (n = 24). This finding was also supported by other DNA and RNA virus, such as HCMV, NDV, and SeV, infection or stimulation with TLRs or RLRs viral PAMPs, such as ssRNA and poly I:C of various cell lines. Ectopic expression of miR-30e in primary cells or cell lines upon subsequent DNA or RNA virus infection significantly reduces the viral load through global enhancement of innate immune responses in terms of pro-inflammatory cytokines, type I interferons, and type I interferon-inducible genes as shown by NGS data analysis. The enhanced miR-30e expression in therapy-naive HBV (CHB) patients might elevate innate immune responses to combat HBV infection; however, it might be insufficient to control HBV infection. Therefore, patients with HBV receiving pegylated interferon were sampled after 6 months of therapy and were found to show significant reduction of viral load and miR-30e expression highlighting the link between HBV, miR-30e, and innate immune responses. To establish the role of miR-30e under physiological condition, we selected SLE as a disease model. Patients with SLE show enhanced expression of miR-30e, pro-inflammatory cytokines, type-I interferons, or type-I interferon-inducible genes. Further to confirm our observations, we exploited SLE mouse model (NZW/NZB F1) (Morel, 2010) and obtained similar results for the expression of miR-30e that were consistent with the results of patients with SLE, suggesting the correlation of miR-30e with innate immune responses in SLE under physiological condition.

The transcriptomic analysis of our NGS data and GEO datasets using various bioinformatics tools shows that miR-30e directly targets several signal transducers and the negative regulators such as TRIM38 (Versteeg et al., 2014; Zhao et al., 2012; Hu et al., 2014), TANK (Kawagoe et al., 2009), ATG5, ATG12 (Li et al., 2018; Jounai et al., 2007), BECN1 (Cui et al., 2016), SOCS1, and SOCS3 (Pothlichet et al., 2008) validated in the study and few others TRIM35 (Wang et al., 2015), TRIM13 (Narayan et al., 2014), and EPG5 (Lu et al., 2016) discussed in Table S2. The microRNA targeting reduces the expression of negative regulators and enhances the innate immune responses that play a pivotal role in TLR, RLR, NLR, and type I interferon signaling pathways. Although miR-30e also binds with few other gene transcripts that are not the negative regulators in the innate immune signaling pathways, the combined mean free energy for these transcripts are lower than the threshold binding energy necessary for significant change in the expression of transcripts, for subsequently affecting the outcome of signaling pathways. Notably, the expression of negative regulators such as TRIM38, TANK, SOCS1, and SOCS3 in patients with SLE are significantly reduced. Moreover, SLE mice also showed similar results for the expression of Socs1, Socs3, Atg5, and Atg12. Collectively, human and mouse results illustrate that miR-30e targets negative regulators to elevate innate immune responses and dysregulation of miR-30e expression might be one of the factors for the establishment of SLE or probably other autoimmune diseases.

Previously, it has been shown that patients with SLE have reduced ability to degrade DNA and cellular chromatin (Gheita et al., 2018). DAMPs, specially the double-stranded (ds)DNA, are considered to be associated with the lupus as mentioned in the introductory section of the study and could be utilized to study the SLE-related cellular phenomena by adopting simple experimental techniques. The dsDNA could mimic the self-DNA of lupus-associated outcomes and results in modulating the inflammatory/immune responses, cell death, which were conventionally considered as the signatory and/or onset features associated with SLE pathogenesis. Therefore, PBMCs from healthy donors were stimulated with partially degraded self-DNA, and these cells showed enhanced miR-30e expression and innate immune responses suggesting the pathogenic role of miR-30e, which suggests the association of self-DNA with the pathogenesis of SLE. In contrast, sequestering the endogenous miR-30e in PBMCs of patients with SLE by introducing antagomir/inhibitor or miR-30e significantly reduced the levels of miR-30e and innate immune responses in terms of IFNβ production. Additionally, the introduction of locked nucleic acid-based inhibitor of miR-30e (a stable form of antagomir) through retro-orbital route into SLE mice model significantly reduced mir-30e and innate immune genes expression in splenocytes, whereas expression of some of the negative regulators was moderately enhanced, demonstrating the ability of mir-30e antagomir/inhibitor to reduce the innate immune responses under physiological condition. Overall, our study identified miR-30e as a post-transcriptional regulator of negative regulation of PRR-mediated innate immune signaling pathways and its prognostic potential in virus infections, such as HBV infection. Additionally, our study demonstrated the therapeutic implications of miR-30e antagomir/inhibitor in immunologically complex autoimmune disease, SLE, or possibly other autoimmune diseases.

Limitations of the Study

The limitation of the study is the exclusion of in vivo experiments for the mechanism of induction of miR-30e-5p, using various knockout mice such as TLR/MyD88/TRIF/IPS-1 or STING. Knockout mice inclusion will provide the mechanistic insight for miR-30e-5p induction in viral infections and/or SLE. Another limitation is the exclusion of data demonstrating the reduction of the negative regulator at protein levels in several primary cells because of the limitation of survival of primary cell due to the introduction of miR-30e-5p and subsequent virus infections. Though in this work, we have demonstrated the reduction of negative regulators in vivo at the transcript levels. Additionally, serum samples collected from the patients could also be normalized, for the quantification of microRNAs, by using more than one internal control along with conventional RNU6 (U6) internal control to avoid any challenge in disseminating the patient-related data.

Resources Availability

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Himanshu Kumar (hkumar@iiserb.ac.in).

Material Availability

The study did not generate any unique reagent/s.

Data and Code Availability

The NGS (RNA sequencing [RNA-seq]) data for expression profiling reported in this paper have been deposited in the GenBank database (accession no. NCBI's Genbank_GSE130005).

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.