miR-146a regulates TLR1/2 and 4 induced inammation and links it with proliferation in human SZ95 sebocytes

Activation of Toll-like receptors (TLR) 1/2 and 4 are central in inducing inammation in sebocytes and in the pathogenesis of acne by regulating the expression of protein coding mRNAs, however the microRNA (miRNA) prole in response to TLR activation and thus the possible role of miRNAs in regulating sebocyte functions has not been elucidated. In this work therefore, we aimed to identify the miRNA with the most abundant induction and to reveal its role in TLR1/2- and 4-activated SZ95 sebocytes. We found that miR-146a, detected with increased expression also in sebaceous glands of acne samples, showed the highest induction levels in the activated sebocytes. When exploring its role, we found that the increased levels of miR-146a led to the down-regulation of IL-8 secretion, decreased the chemoattractant potential and stimulated the proliferation of sebocytes, whereas the latter may be affected by GNG7 down-regulation. According to our results miR-146a may be a potential player in acne pathogenesis by regulating inammation and by providing a link between inammation and sebocyte proliferation.


Introduction
Toll-like receptors (TLRs) belong to the family of pattern-recognition receptors and are key players in the innate immune system to selectively sense the presence of various microorganisms [1][2][3] . Importantly, elevated expression of TLR2 and TLR4 were reported in acne-involved skin 4,5 , suggesting that these pathways may also be involved in acne pathogenesis. While TLR2 recognizes peptidoglycans, lipoproteins, lipoarabinomannans and short-chain fatty acids from Gram-positive bacteria, TLR4 is activated by the Gram-negative bacterial component lipopolysaccharide (LPS) 6 . Interestingly lipids, such as saturated fatty acids, are also able to activate both TLR2 and TLR4 [7][8][9] . Indeed, products of Propionibacterium acnes (P. acnes), a Gram-positive, anaerobic bacterium, which behaves both as commensal and pathogen in acne skin, and several sebaceous lipids, among them palmitic acid, whose altered ratios were detected in acne patients, are possible TLR activators in acne lesions [10][11][12][13][14][15][16][17] .
Sebaceous glands are known for their primary role to secrete and metabolize lipids leading to the production of sebum to moisturize the hair and the skin, which feature is regulated by a wide repertoire of stimuli such as hormones, lipids and pathogens both in physiological as well as in disease settings 18 . However, with a great number of proteins and lipids that exert in ammatory properties, sebocytes are also actively involved in shaping the in ammatory environment [19][20][21] , in which their activation through TLRs 14,22,23 might play a central role. Supporting this pustulate, our previous genome wide gene expression study has shown that sebocytes are able to rapidly gain and prioritize an immune-competent status in response to TLR1/2 and/or TLR4 activation at the level of mRNA expression 22 .
MicroRNAs (miRNA) are small non-coding RNAs also selectively transcribed from the genome under various conditions. In contrast to mRNAs they do not encode proteins but control gene expression by binding and destabilizing their target mRNA 24 . Therefore, they can regulate transcriptional and post-transcriptional gene expression. In human sebocytes, miRNA presence was con rmed and was proven essential for lipogenesis 25 , but without providing any disease-speci c conclusions on their possible role.
In the present study, we aimed to extend our knowledge on the gene expression regulation and pro le of TLR-activated sebocytes and investigated the role of miRNAs in it. We found that in TLR1/2-and 4activated sebocytes, miRNAs had altered expression levels, with miR-146a showing the most prominent upregulation. Con rming that sebaceous glands of acne samples also exhibited high expression levels of miR-146a, we aimed to de ne a possible pathophysiological role for miR-146a in sebocytes. Our results suggest that miR-146a may not only regulate TLR-induced in ammation in sebocytes but could be a missing link in connecting it with hyperproliferation, which may have both pathological and therapeutic implications in sebaceous gland-associated diseases, such as acne.
Results miR-146a shows the most prominent induction in TLR1/2 and 4-stimulated SZ95 sebocytes Sebocytes are able to sense and respond to different TLR stimuli 22 , making them an active player in a pathogen-associated in ammatory environment. To investigate the change in their miRNA pro le in such a response, we applied two different TLR activators, PAM3CSK4 (TLR1/2 activator) and LPS (TLR4 activator), to treat human SZ95 sebocytes. Performing genome wide expression studies in samples treated for 24 h, 23 microRNA responded to PAM3CSK treatment, while 54 miRNA were signi cantly upregulated after LPS treatment (Supplementary Table 1). Both TLR activators induced common signi cant elevation of 14 miRNA expression levels (Fig. 1a), of which miR-146a showed the most abundant values (Fig. 1b). This signi cant increase in the levels of miR-146a could also be detected by in situ hybridization in TLR1/2-and 4-activated SZ95 sebocytes (Fig. 1c). miR-146a is elevated in sebaceous glands of human acne tissue samples To provide a biological relevance for our nding, we performed in situ hybridization against miR-146a in 5 acne vulgaris and 5 normal skin samples from the back of young male adults. While in normal skin, miR-146a was detectable with low-intensity homogeneous staining, a more intense, mainly granular staining was observed in sebaceous glands of acne tissue samples (Figure 2.). miR-146a decreases IL-8 secretion and negatively regulates the chemoattractant potential of SZ95 sebocytes To assess the possible role of miR-146a, we focused on the functional analysis of SZ95 sebocytes transfected with the hsa-miR-146a inhibitor, hsa-miR-146a mimic, or negative controls for 72 h.
As IL-8 is an important chemokine in immune cell migration, we examined the migratory capacity of peripheral blood monocytes towards supernatants of transfected SZ95 sebocytes. We observed a lower migration to mimic-transfected and an increased migration towards miR-146a inhibitor treated SZ95 sebocyte supernatants, showing that miR-146a may have a negative regulatory role on monocyte chemoattraction under in ammatory conditions (Fig. 3b).
miR-146a regulates cell proliferation but not lipid production of SZ95 sebocytes To examine the role of miR-146a in sebocyte proliferation and lipid production of cells treated with hsa-miR-146a inhibitor, hsa-miR-146a mimic, or negative controls for 72 h, we analyzed the cell numbers in different cell cycle phases using DNA content histograms and applied Oil Red O staining to detect lipids.
When incubated with miR-146a mimic sequence, the proportion of cells in G2 and M phases increased with a signi cant decrease in S phase (P<0.05) and a slight decrease in G0 and G1 phases in comparison with control ( Fig. 3c), showing that cell proliferation is stimulated by higher miR-146a levels. Transfecting SZ95 sebocytes with miR-146a inhibitor, the cell proportion in the S phase increased and the G0/G1 population decreased, suggesting that the mitotic machinery itself was activated but might be blocked in S phase. According to the DNA histogram, a peak of apoptotic cells was also observed following miRNA inhibitor treatment.
Lipid measurements found a slight, but statistically not signi cant decrease in the lipid content of miRNA inhibitor-treated cells and no change in cells treated with the mimic sequence (Fig. 3d).
Whole transcriptome analysis with clustering revealed changes in genes related to in ammation and cell proliferation in miR-146a inhibitor-treated SZ95 sebocytes To detect how levels of miR-146a could impact changes at the level of gene expression in sebocytes, we performed whole transcriptome analysis of inhibitor-treated cells at 72 h and found that 330 genes were upregulated, while 311 genes were downregulated. Functional gene clustering con rmed that the altered levels of miR-146a in sebocytes may lead to changes in immune response, in apoptosis and cell proliferation-related pathways. Importantly, genes could not be clustered into lipid production, which was in line with our lipid detection studies (Fig. 4).

miR-146a levels in sebocytes lead to changes in the expression of GNG7
According to the transcriptome analysis, G protein gamma 7 (GNG7), found also to contribute to a great number of the functional clusters, had one of the most robust upregulation following miR-146a inhibition (Fig. 5a). The miR-146a mediated expression changes of GNG7 could be further con rmed by qPCR and in situ hybridization in SZ95 sebocyte cultures transfected with mimic, inhibitor and control sequences ( Fig. 5b-c), suggesting a central role for GNG7 in miR-146a-induced signaling.
To assess if GNG7 mRNA could be also detected in vivo, in situ hybridization was performed on human acne and normal skin samples. The presence of GNG7 mRNA could be visualized in sebaceous glands of normal skin, while in acne samples, in which elevated miR-146a levels were shown, it could not be detected (Fig. 5d).

Discussion
The application of a system-based approach of whole genome sequencing of SZ95 sebocytes, treated with speci c and selective TLR1/2 and TLR4 activators, provided evidence that miRNAs are selectively induced in sebocytes upon TLR activation. Moreover, identifying miR-146a as the miRNA with the most abundant induction with an increased expression also in the sebaceous glands of acne samples, our work is the rst to identify a miRNA, which is increased in sebaceous glands in a disease setting. Characterizing sebocytes with altered levels of miR-146a, we showed that miR-146a is not only a marker for activation, but could have a regulatory role on basic cellular functions as well.
In line with our previous results, showing that TLR1/2 and TLR4 pathways induced a similar change in the mRNA pro le of sebocytes, miRNAs also changed similarly in response to the used activating agents.
This nding further supports our previously raised hypothesis that these pathways and the related changes are not stimulus-/pathogen-speci c in sebocytes as these receptors can be activated with a wide range of stimuli both of pathogenic and of non-pathogenic origin 22 . In other words, sebocytes use these receptors to sense changes in their environment, such as an altered microbiome or the presence of lipids, which activation needs to be further modulated to gain its disease speci c role. Based on our results miR-146a could represent a negative regulator, just as it is observed in various cell types of lymphoid, myeloid and of non-immune origin, where miR-146a decreases the production of in ammatory cytokines 27 .
Regarding dermatological diseases, increased levels of miR-146a was already con rmed in keratinocytes of atopic dermatitis and psoriasis samples, with a suggested role to regulate in ammation. In psoriasis, its genetic alterations even showed an association with disease severity 28-30 . Importantly, higher levels of miR-146a was also detected in keratinocytes of acne samples, where it may down-regulate P. acnesinduced production of IL-6, -8, and TNF-α by inhibiting the TLR2/IRAK1/TRAF6/NF-κB and MAPK pathways 31 . Our ndings that miR-146a was also highly expressed in sebaceous glands of acne samples, con rms that miR-146a may be involved in acne also at the level of sebocytes and adds further important details on the immune-competence of this cell type. Therefore, the most interesting nding of the induction of the TLR-miR-146a axis in sebocytes may result in a decreased production of IL-8, a cytokine characteristic in acne-related in ammation, and a decreased chemoattractant potential of sebocytes, a feature that was recently reported by our group 19 . Speculating on the in vivo relevance of this nding, it is reasonable to put forward that the increased levels of miR-146a in sebocytes could serve as a negative regulator of in ammation in acne lesions with an impact on the production of in ammatory cytokines and the number of in ltrating immune cells.
Further important result of our studies was the role of miR-146a to regulate the proliferation of sebocytes.
By showing that the proliferation and apoptosis of sebocytes were dependent on the levels of miR-146a, the increased levels led to an increased proliferation while decreased ones to apoptosis, may bring us closer to understand the morphological and functional changes of sebaceous glands observed under in ammation. Although there was no evidence for an altered lipid metabolism in our results, a recent publication 32 reported that the pathogenetic basis of acne is the alteration in sebocyte differentiation.
The less differentiated and with that the more proliferating the sebocytes are the more they are responding to regulatory stimuli. It is intriguing to put forward that the promoted cell proliferation due to the increased levels of miR-146a may sensitize sebocytes to regulatory stimuli both of pathogenic and of therapeutic relevance that need further characterization.
Our unbiased strategy of whole transcriptome analysis performed on sebocytes treated with a speci c miR-146a inhibitor, revealed that miR-146a may also in uence the gene expression pro le of sebocytes.
Although the exact mechanisms remain to be elucidated, in inhibitor-treated sebocytes, pathways with pivotal roles in sebocyte functions, such as Wnt, EGF or insulin signalling, might be altered at the level of transcription 33 . These results showed that the complex changes induced by miR-146a may go beyond altering the in ammatory properties of sebocytes. Based on the fold change values, the functional clustering of the differentially expressed genes and the in situ hybridization studies, GNG7 came into the focus showing an opposite regulation with miR-146a both in sebocytes and in sebaceous glands of acne samples, in which the expression of miR-146a increased while GNG7 had a lower expression. GNG7, predominantly expressed in nervous tissue 34 , inhibits cell proliferation, promotes cell differentiation and induces cell death by inhibiting mTOR signalling 35,36 , with so far little data on its involvement in skin. Considering that the activation of mTOR pathway is also central in the regulation of sebocyte proliferation and maturation, and its induction by various agents is central in the development of acne [37][38][39][40] , further studies are needed to identify in more details the contribution of GNG7 to sebocyte biology and how its decreased levels may be involved in disease pathogenesis (Fig 6).
In summary, our ndings deliver novel data, that in the active role of sebocytes to shape the in ammatory environment, miRNAs are also utilized. Moreover, our results also point on the therapeutic relevance which the modulation of miR-146a levels may deliver to acne therapy by targeting in ammation and cell proliferation at the same time.

Cells, transfection and treatment
Immortalized human SZ95 sebocytes were maintained as adherent culture at 37°C in a humidi ed chamber containing 5% (v/v) CO 2 in Sebomed basal medium (Sigma-Aldrich, St. Louis, MO, USA) as previously described 41 . To obtain global transcriptome data high throughput mRNA sequencing analysis was performed on Illumina sequencing platform as described previously 22,42,43 . Total RNA sample quality was checked on Agilent BioAnalyzer using Eukaryotic Total RNA Nano Kit according to the manufacturer's protocol. Samples with RNA integrity number value >7 were accepted for library preparation process. RNA-Seq libraries were prepared from total RNA using Ultra II RNA Sample Prep kit (New England BioLabs) according to the manufacturer's protocol. Brie y, poly-A RNAs were captured by oligo-dT conjugated magnetic beads then the mRNAs were eluted and fragmented at 94˚C for 15 minutes. First strand cDNA was generated by random priming reverse transcription and after second strand synthesis step doublestranded cDNA was generated. After repairing ends and adapter ligation steps, adapter-ligated fragments were ampli ed in enrichment polymerase chain reaction and nally, sequencing libraries were generated. The sequencing run was executed on Illumina NextSeq500 instrument using single-end 75 cycle sequencing.

RNA-Seq data analysis
Raw sequencing data was aligned to human reference genome version GRCh37 using HISAT2 algorithm and BAM les were generated. Downstream analysis was performed using StrandNGS software (version 2.8, build 230243; Strand Life Sciences, Bangalore, India). BAM les were imported into the software, DESeq1 algorithm was used for normalization. Sequencing data have been deposited into the GEO database (PRJNA673828). mRNA levels with 1.3/-1.3 fold-change or higher/less were stated as signi cantly changed. Pathway analysis was performed with the PANTHER Classi cation System Chemotaxis cell migration assay Monocytes were isolated from whole blood of healthy donors by density centrifugation (Ficoll, Paque Plus, GE Healthcare, Chicago, IL, USA), CD14 microbeads (Miltenyi Biotech, Bisley, UK), resuspended in RPMI 1640 medium (Invitrogen) supplemented with 1 v/v% L-glutamine (Sigma-Aldrich) and 0,5 v/v% Antibiotic-Antimycotic (penicillin, amphotericin-B, streptomycin, BioSera, Nuaille, France). 1x10 5 isolated monocytes were added to the top of a 5 µm pore cell migration chamber plate (Chemicon QCM 96-well chemotaxis cell migration assay, Temecula, CA, USA). Feeder trays were loaded with supernatants of SZ95 cells transfected with miR-146a-5p mimic, inhibitor or control sequences in triplicates. After 24 h at 37°C with 5% CO 2 migrated cells were harvested, lysed and stained with CyQuant GR (Chemicon).
Fluorescence was measured with Epoch microplate reader (BioTek) at the wavelength of 520 nm.

Statistical evaluation
Values are presented as mean ± SEM. All experiments have been performed in triplicate. P values were calculated using Mann-Whitney test, *P < 0.05; **P < 0.01; ***P < 0.001 considered as statistically signi cant.
Data availability RNA sequencing data have been deposited into the GEO database under accession number PRJNA673828. Authors can con rm that all relevant data are included in the article and/or its supplementary information les.
Declarations situ hybridization, NBT/BCIP blue chromogen reaction with nuclear Fast Red background staining. Original magni cation x200. Experiments were carried out in triplicates.  treated with inhibitor, more cells were found in S phase and less in G2/M phase. Note the sub-G0/G1 peak, representing apoptotic cells (blue arrow). (d) Oil Red O staining of SZ95 sebocytes. Treatment with mimic or with inhibitor sequence caused no signi cant change in the lipid content of SZ95 sebocytes when normalized to control. Graphs show the mean optical density normalized to control. RFU: relative uorescence unit. All experiments were carried out in triplicates.
Pathway analysis of signi cantly regulated genes resulted in clusters such as in ammation and cell proliferation. Detection of GNG7 mRNA in miR-146a inhibited sebocytes and in sebaceous glands of histological specimens. (a) Genes with the highest fold change values in their expression levels in miR-146a inhibitor treated SZ95 sebocytes as revealed by our RNAseq measurements. Note that GNG7 was contributing also to a large number of the functional clusters. (b) Changes of relative GNG7 mRNA levels measured by qRT-PCR in SZ95 sebocytes normalized to PPIA. GNG7 mRNA levels decreased in SZ95 sebocytes transfected with mimic and increased in the inhibitor treated cells. FC: fold change value, compared to relative GNG7 expression values of sebocytes transfected with miR-146a mimic or inhibitor sequences. (c) in situ hybridization for the detection of GNG7 mRNA in SZ95 sebocytes. Note, that when compared to miR-146a mimic (a), a more intense blue hybridization signal was seen in the cytoplasm of the inhibitor treated (b) SZ95 sebocytes. Representative photomicrograph, n=3. (d) in situ hybridization for the detection of GNG7 in sebaceous glands of normal (A) and acne vulgaris (B) FFPE human tissue samples. Note that while in acne samples, GNG7 mRNA could not be detected, in healthy skin samples obtained from the back, weak hybridization signals were observed. Arrows show GNG7 mRNA-positive sebocytes. Representative photomicrograph, n=5. Chromogenic in situ hybridization, NBT/BCIP blue chromogen with nuclear Fast Red background staining. Original magni cation x200. Figure 6 miR-146a regulates in ammation and cell proliferation in TLR-activated sebocytes. Our data showed that in response to TLR1/2 and -4 activation the expression of miR-146a, a microRNA leading to the degradation of its target mRNAs, is abundantly induced in sebocytes. The increased levels of miR-146a may have a negative regulatory role on cytokine secretion and chemotaxis and induce cell proliferation while decrease apoptosis. GNG7 is down-regulated in parallel with the elevation of miR-146a levels in human SZ95 sebocytes and sebaceous glands of human acne samples which altogether may have pathological as well as therapeutical relevance.

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