Human skin cells recognize the presence of the skin microbiome through pathogen recognition receptors. Epidermal keratinocytes are known to activate toll-like receptors (TLRs) 2 and 4 in response to the commensal Cutibacterium acnes (C. acnes, formerly known as Propionibacterium acnes) bacterium and subsequently to induce innate immune and inflammatory events. These events may lead to the appearance of macroscopic inflammatory acne lesions in puberty: comedos, papules and, pustules. Healthy skin does not exhibit inflammation or skin lesions, even in the continuous presence of the same microbes. As the molecular mechanism for this duality is still unclear, we aimed to identify factors and mechanisms that control the innate immune response to C. acnes in keratinocytes using a human immortalized keratinocyte cell line, HPV-KER, normal human keratinocytes (NHEK) and an organotypic skin model (OSM). TNIP1, a negative regulator of the NF-κB signaling pathway, was found to be expressed in HPV-KER cells, and its expression was rapidly induced in response to C. acnes treatment, which was confirmed in NHEK cells and OSMs. Expression changes were not dependent on the C. acnes strain. However, we found that the extent of expression was dependent on C. acnes dose. Bacterial-induced changes in TNIP1 expression were regulated by signaling pathways involving NF-κB, p38, MAPKK and JNK. Experimental modification of TNIP1 levels affected constitutive and C. acnes-induced NF-κB promoter activities and subsequent inflammatory cytokine and chemokine mRNA and protein levels. These results suggest an important role for this negative regulator in the control of bacterially induced TLR signaling pathways in keratinocytes. We showed that all-trans retinoic acid (ATRA) induced elevated TNIP1 expression in HPV-KER cells and also in OSMs, where TNIP1 levels increased throughout the epidermis. ATRA also reduced constitutive and bacterium-induced levels of TNFα, CCL5 and TLR2, while simultaneously increasing CXCL8 and TLR4 expression. Based on these findings, we propose that ATRA may exhibit dual effects in acne therapy by both affecting the expression of the negative regulator TNIP1 and attenuating TLR2-induced inflammation. Overall, TNIP1, as a possible regulator of C. acnes-induced innate immune and inflammatory events in keratinocytes, may play important roles in the maintenance of epidermal homeostasis.

our skin provides a physical barrier to separate the internal part of our body from the environment. Maintenance of complex barrier functions is achieved through anatomical structures in the skin, the stratified squamous epithelium specialized junctional organelles, called tight junctions (TJs). Several members of our microbial communities are known to affect the differentiation state and function of the colonized organ. Whether and how interactions between skin cells and cutaneous microbes, including Cutibacterium acnes (C. acnes), modify the structure and/or function of our skin is currently only partly understood. Thus, in our studies, we investigated whether C. acnes may affect the epidermal barrier using in vitro model systems. Real-time cellular analysis showed that depending on the keratinocyte differentiation state, the applied C. acnes strains and their dose, the measured impedance values change, together with the expression of selected TJ proteins. These may reflect barrier alterations, which can be partially restored upon antibiotic-antimycotic treatment. Our findings suggest that C. acnes can actively modify the barrier properties of cultured keratinocytes, possibly through alteration of tight cell-to-cell contacts. Similar events may play important roles in our skin, in the maintenance of cutaneous homeostasis. One of the most important properties of our skin is the complex barrier it provides to separate the internal part of our body from the environment, limiting contact with harmful chemicals, microbes, allergens and radiation 1-3. The major building blocks of the skin barrier are the keratinocytes, which are capable of recognizing the everchanging environmental conditions and mounting appropriate responses to maintain the integrity of the human body 4,5. Maintenance of complex barrier functions is achieved through anatomical structures in the skin. The stratified squamous epithelium is the uppermost skin layer that contains live keratinocytes and contains specialized junctional organelles, called tight junctions (TJs), which are localized between the cells of the second and third layer of the stratum granulosum 6. TJs provide intimate links between adjacent cells and play major roles in establishing the epidermal barrier, as well as act as important determinants of transepidermal transport 7-9. The complex, multi-protein structure of TJs includes more than 40 proteins 10,11. Claudin (CLDN) protein family members are some of the most important TJ components, as they are critical for the regulation of barrier functions, including permselectivity, which determines the size, ionic charge and electric resistance of molecules that may be transported through the barrier 12,13. Keratinocytes are also in constant contact with various members of the cutaneous microbiota. One of the most well-known members of this community is the Cutibacterium acnes (C. acnes) bacterium, which, beginning with puberty, is a dominant species and preferentially inhabits sebum-rich skin regions 14,15. Current research is eluci...

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 control of cutaneous microbiota-induced immune activation in keratinocytes is key to the maintenance of epidermal homeostasis. Tumour necrosis factor alpha-induced protein 3, a regulator of nuclear factor kappa B signalling pathways, has important roles in these processes. It forms a negative regulatory feedback loop, controls the activity of toll-like receptor-induced signalling pathways, but is also regulated by different toll-like receptor ligands and by Cutibacterium acnes, a member of our skin microbiota. The levels of tumour necrosis factor alpha-induced protein 3 greatly influence levels of bacterium-induced inflammatory mediators in keratinocytes. This study found elevated expression of tumour necrosis factor alpha-induced protein 3 mRNA in acne lesions. These findings suggest a general role of tumour necrosis factor alpha-induced protein 3, not only in the regulation of toll-like receptor-induced immune activation, but also in cutaneous immune homeostasis.Human epidermal keratinocytes sense the presence of human skin microbiota through pathogen recognition receptors, such as toll-like receptors, and induce innate immune and inflammatory events. In healthy epidermis there is an absence of inflammation despite the continuous presence of cutaneous microbes, which is evidence of an effective immune regulatory mechanism. The aim of this study was to investigate tumour necrosis factor alpha-induced protein 3 (TNFAIP3), a negative regulator of toll-like receptor and nuclear factor kappa B signalling pathways, and its role in these regulatory events. A broad spectrum of tolllike receptor ligands induced TNFAIP3 expression, as did live Cutibacterium acnes, which is involved in the pathogenesis of acne. Changes in bacterium-induced, dose-dependent TNFAIP3 expression were Jun kinaseand nuclear factor kappa B-dependent, and resulted in altered cytokine and chemokine levels in in vitro cultured human keratinocytes. In acne lesions, TNFAIP3 mRNA expression was elevated compared with nonlesional skin samples from the same individuals. These results suggest that TNFAIP3 may have a general role in fine regulation of microbiota-induced cutaneous immune homeostasis.

Dendritic cells (DCs) are important APCs and responsible for the induction of immune responses as well as preserving peripheral tolerance. We showed that targeting of antigens via CLRs to different specialized DC subpopulations induced either CD4+ or CD8+ T cell responses in vivo. Besides C-type lectin receptors, Fc receptors are highly endocytic and expressed on many APCs. Here, we show that primary DCs are able to take up Fc receptor antigen targeting antibodies leading to the induction of both, potent CD4+ and CD8+ T cell responses in vivo. Despite the broad expression profile of Fc receptors on APCs and non-APCs, we found DCs to be able to induce primary immune responses. Although some of the Fc receptors were expressed on several DC subsets, the activation of CD4+ or CD8+ T cell responses was again only dependent on the intrinsic properties of the DC subsets. Notably, the induction of these T cell responses was independent on the signalling properties of the targeted receptors. We therefore, we suggest antigen targeting to Fc receptors as useful tool for the induction of de novo as well as the modulation of immune responses for future therapeutic applications.