Various insults can trigger innate immune activation through pattern recognition receptors(PRRs) in different cell types. These can leave lasting epigenetic changes, and affected cells undergo cellular reprogramming, resulting in altered responsiveness to subsequent encounters. This is an adaptive response of the innate immune system called innate immune memory(IIM). We were interested in whether Cutibacterium acnes (C. acnes) may initiate similar events in keratinocytes. We used C. acnes for primary training, and after five days of rest, Pam3Csk4 (TLR1/2 agonist) for secondary induction in normal human epidermal keratinocyte (NHEK) and HaCaT cells. The mRNA expression of several immune-related genes (e.g., TNFa, IL-8, SOCS1, TNFAIP3, TNIP1) increased in Pam3Csk4-induced trained HaCaT cells, compared to Pam3Csk4-induced, untrained ones, suggestive of innate training-like IIM processes. Expression differences were also observed in NHEK cells, but their direction was not the same in cells originating from different body parts. We found statistically significantly higher TNFa levels in cells from the breast region (NHEK-B), but lower ones in abdominal (NHEK-A) samples, indicative of innate training vs., tolerance events, respectively. The global 5-methylcytosine (5-mC) content of the genomic DNA isolated from these samples was higher in control (untrained, uninduced) NHEK-B cells compared to NHEK-A ones even after the 5 days of resting. Previous C. acnes treatment led to a marked and sustained decrease in NHEK-A cultures and no changes in NHEK-B ones. Members of our microbiota may modify keratinocyte immune events and through that profoundly affects the cutaneous immune responses in vitro and possibly in vivo. Epigenetic differences of NHEK cells representing different skin regions may arise due to the variations in the inhabiting microbiota composition.

The disease-residual transcriptomic profile (DRTP) within psoriatic healed/resolved skin and epidermal tissue-resident memory T (TRM) cells have been proposed to be crucial for the recurrence of old lesions. However, it is unclear whether epidermal keratinocytes are involved in disease recurrence. There is increasing evidence regarding the importance of epigenetic mechanisms in the pathogenesis of psoriasis. Nonetheless, the epigenetic changes that contribute to the recurrence of psoriasis remain unknown. The aim of this study was to elucidate the role of keratinocytes in psoriasis relapse. The epigenetic marks 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) were visualized using immunofluorescence staining, and RNA sequencing was performed on paired never-lesional and resolved epidermal and dermal compartments of skin from psoriasis patients. We observed diminished 5-mC and 5-hmC amounts and decreased mRNA expression of the ten-eleven translocation (TET) 3 enzyme in the resolved epidermis. SAMHD1, C10orf99, and AKR1B10: the highly dysregulated genes in resolved epidermis are known to be associated with pathogenesis of psoriasis, and the DRTP was enriched in WNT, TNF, and mTOR signaling pathways. Our results suggest that epigenetic changes detected in epidermal keratinocytes of resolved skin may be responsible for the DRTP in the same regions. Thus, the DRTP of keratinocytes may contribute to site-specific local relapse.

The exact pathogenesis of acne-related post-inflammatory hyperpigmentation (PIH) is unknown. It is thought that epidermal inflammation induces the release of arachidonic acid and its oxidation products, leading to melanocyte activation and pigment production. Cutibacterium acnes (C. acnes) contributes to keratinocyte inflammation in acne pathogenesis. In severe cases inflamed sebaceous follicles may be ruptured, thereby bacteria and their metabolites can reach the melanocytes in the basal epidermal layer. We aimed to investigate whether, in addition to inducing epidermal immune activation, C. acnes may also directly affects melanocytes, thus contributing to PIH development. In our study we established C. acnes-treated normal human melanocyte and melanocyte-keratinocyte co-cultures and analyzed the properties of melanocyte immune activation and melanogenesis. We observed that direct C. acnes treatment increased the mRNA expression level of tumor necrosis factor alpha and the secreted protein levels of interleukin 6 and 8, together with the mRNA levels of tyrosinase and dopachrome tautomerase, playing roles in melanin biosynthesis. Tyrosinase enzyme activities and the intracellular melanin levels were also elevated, which we confirmed using real-time RT-PCR, L-DOPA staining, direct melanin measurement, and silver-nitrate staining. Based on our results, we propose that C. acnes or bacterial components may directly interact with melanocytes during the formation of severe acne lesions due to the disruption of intact follicles. They may induce melanocyte immune activation and increased melanogenesis, contributing to PIH symptoms.

Psoriasis is a common relapsing autoimmune skin disease. Effective treatment resolves cutaneous symptoms, but they often relapse in previously involved regions. Apart from T-cell-driven disease memory, a pathogenic genetic and epigenetic memory of the cutaneous structural cells has been suggested, however the nature of such a plaque-site memory is still under investigation. We aimed to perform a pairwise comparison of psoriatic never-lesional skin (PSO-NES) and resolved skin (PSO-RES) to identify genomic and epigenomic differences, which may contribute to the formation of localized memory in clinically healed skin samples. To address these questions, we performed immunofluorescence staining to visualize the general pattern of 5-Methylcytosine (5-mC) and 5-Hydroxymethylcytosine (5-hmC) epigenetic marks in the skin biopsy sections. We also applied high-throughput RNA-seq to profile the transcriptional status of the epidermal and dermal compartments. With confocal microscopic analysis, we found that 5-mC and 5-hmC intensities were overall lower in PSO-RES compared with PSO-NES epidermis samples. Our transcriptomic data showed diminished suppressor of cytokine signaling 1 (SOCS1) expression in PSO-RES vs. PSO-NES epidermis. Our observations suggest the possibility of epigenetic reprogramming in PSO-RES skin due to the reduction of 5-hmC as functionally active epigenetic modifications. These may be a consequence of greater sensitivity of these keratinocytes to inflammatory stimuli due to the reduced levels of a downregulating factor, SOCS1.

Keratinocytes are important gatekeepers of our body, forming a complex barrier. They distinguish between friendly microbes and harmful invaders and initiate effective immune responses if necessary. Tight regulation of these innate immune events are crucial, as they may quickly become deleterious, if not properly controlled. We aimed to analyze the regulation of innate immune reactions of keratinocytes initiated upon the recognition of Cutibacterium acnes (C. acnes) bacterium. We identified TNIP1 as a factor exhibiting negative regulatory effects on C. acnes-induced TLR signaling pathways. Transcriptional regulation of this gene is complex, JNK and MAPKK-dependent signaling pathways are important for basal TNIP1 expression, whereas C. acnes-induced expression changes diminish in response to JNK, MAPKK, NF-κB and p38 inhibition. The promoter region of TNIP1 also contains functional retinoic acid response elements (RARE) and all-trans retinoic acid (ATRA) treatment significantly increased TNIP1 protein expression levels. As a consequence, C. acnes-induced mRNA expression of TLR-2 and the pro-inflammatory TNFα and CCL5 decreased, TLR-4 and CXCL8 levels increased, whereas TLR3 and IL-6 mRNAs were not affected by the treatment. C. acnes is a key member of the postadolescent cutaneous microbiome of healthy skin, but also inducing innate immune and inflammatory events in acne pathogenesis in teenagers. Retinoids, apart from their know effects (decreasing sebaceous gland size and sebum secretion, leading to the control of C. acnes load) may attenuate C. acnes-induced inflammation by directly affecting TNIP1 expression and also by attenuating TLR2 expression and signaling.

Propionibacterium acnes (P. acnes) is a resident microbe of the healthy human skin microbiome, but also know to simulate immune and inflammatory events via the activation of Toll-like receptors (TLRs). These molecular pathways are well characterized, but little is known about the endogen negative regulatory mechanisms in the keratinocytes that counteract the bacterium-induced signaling events and thus may protect the host from the prolonged, uncontrolled and often destructive inflammation. In our studies we aimed to characterize the keratinocyte expression of endogen negative regulators of TLR signaling pathways previously identified in other cell types, and to analyze their possible role in the attenuation of the P. acnes–induced molecular events. For that, we studied the basal mRNA and protein expression of selected genes (SIGIRR, TOLLIP, TNFAIP3, TNIP1) in a human, in vitro cultured immortalized keratinocyte cell line (HPV-KER) by real time RT-PCR and western blot analysis. Our results suggest that all the investigated negative regulators are expressed in HPV-KER cells and the TNFAIP3 and TNIP1 mRNA expressions significantly and dose dependently increase in response to the bacterium. At the protein level, we found increased TNFAIP3 and decreased SIGIRR expressions following the bacterial treatment, and these events also appeared to be dose dependent. Next, we compared the effect of two P. acnes strains (889, 6609) belonging to different phylogenetic groups within the species (IA and IB, respectively), but no major differences have been observed in the induced expression changes. Our study suggests that in our in vitro model system P. acnes causes a dose-dependent activation of downstream TLR signaling processes. However, parallel to that specialized, endogen negative regulators are also expressed in these cells, which may control the bacterial-induced molecular events, and thus can be important for the maintenance of epidermal homeostasis.