Keratinocyte

The cells that form the keratinizing squamous epithelium in layers ( stratum basale, stratum spinosum, stratum granulosum). Keratinocytes are the predominant (>90 percent) cell type in the epidermis (epidermis). Keratinocytes are formed in the basal cell layer, mature over the course of two weeks, undergo a sudden transformation from a living cell to dead corneocytes, which remain in the horny layer for a further two weeks before sloughing off on the surface.

For a long time, the epidermis was regarded as a simple structural membrane whose main task is to protect internal homeostasis. Keratinocytes achieve this by continuously undergoing processes of proliferation, differentiation and keratinization in a highly orchestrated process. In addition to the production of various structural proteins, keratinocytes express and secrete a large number of biologically important molecules through which they actively participate in many inflammatory and immunological reactions. These are cytokines and their receptors (e.g. pro-inflammatory cytokines of the interleukin-1 family - such as interleukin-1alpha, interleukin-1beta, interleukin-18 as well as the interleukins -6, -7, -8, -10, -12, -15, and -20 and TNF-alpha/Grone A 2002), surface molecules and growth factors (e.g. GM-CSF, GM-CSF, TNF-1beta, TNF-1beta, TNF-1beta, TNF-1beta and TNF-1beta).e.g. GM-CSF), hormones, neuropeptides, arachidonic acid molecules and various enzymes (collagenase, proteases, plasminogen activator). These mediators are available to the keratinocytes in a finely orchestrated interaction with each other as well as with cells of the dermis and the immune system.

Concept of the activated keratinocyte

During undisturbed homeostatic proliferation, the keratinocyte is active in the sense that it performs its specific metabolic functions and also accumulates reserves of biologically relevant substances (e.g. interleukin-1), but does not release them. It therefore behaves neutrally in immunological terms. This concept shows that the keratinocyte has a dual functionality - proliferation and immune activation. Although these are not coupled with each other in principle, they can influence each other in inflammatory processes (e.g. acanthosis in lichenoid reactions, etc.).

However, if the keratinocyte is exposed to internal or external noxae, it changes its functionality in principle. A physiologically proliferative keratinocyte now becomes an immunologically active keratinocyte that is initially capable of antigen-unspecific inflammation. The activated keratinocyte expresses and secretes a rich set of mediators with which it sends signals to itself and to the cellular non-keratinogenic environment.

As a first line of defense, keratinocytes can recognize specific small molecular motifs on bacteria and other microorganisms called pathogen-associated molecular patterns(PAMPs). PAMPs, such as bacterial lipopolysaccharides (LPS), endotoxins or virus-derived nucleic acids, are explicitly recognized by pattern recognition receptors(PRRs). Keratinocytes express various PRRs, including Toll-like receptors (TLRs), C-type lectin receptors, nucleotide-binding oligomerization domain-like receptors and retinoic acid-inducible gene-1 (RIG-I)-like receptors. Ligand recognition by these specific PRRs and their binding leads to the activation of various signaling pathways and the production of inflammatory cytokines, chemokines and antimicrobial molecules. After their release from the keratinocytes, these factors fulfill an important function in the activation of the immune cells in the skin and the recruitment of circulating immune cells, e.g. in the case of an epithelial defect.

The activated keratinocyte in epithelial defects

The integrity of the epidermis is essential for the homeostasis of the organism. Epithelial defects of any kind also lead to activation of the keratinocyte. On the one hand, an epithelial defect leads to a growth impulse of the keratinocytes. They proliferate, differentiate and migrate in order to restore the epidermal barrier. On the other hand, keratinocytes now express various mediators (cytokines, chemokines, antimicrobial peptides/AMPs) and thus mediate extensive interactions between keratinocytes and hematopoietic immune cells, which play an essential role in wound healing.

Toll-like receptors and epithelial defects

Toll-like receptor activation is a crucial element in the initiation and amplification of inflammation following epithelial defects. Keratinocytes express several TLRs, including TLR-1, -2, -4, -5 and -6 as well as the endosomal TLR-3 and -TLR-9. TLR-2 forms a heterodimer with either TLR1 or TLR6 to recognize bacterial peptides. TLR2{{article-url::27291}} can also recognize bacterial peptidoglycan (PGN) and lipoteichoic acid. TLR4, together with CD14, recognizes LPS from Gram-negative bacteria. TLR5 is known to specifically sense and recognize flagellin, an important structural protein of bacterial flagella. TLR9 is a nucleotide-sensing TLR that recognizes bacterial unmethylated 5′-cytosine phosphate guanosine-3′ (CpG)-containing DNA and host-derived denatured DNA from apoptotic cells. TLR3 recognizes viral double-stranded RNA (dsRNA) as well as dsRNA derived from damaged cells, leading to the expression of cytokines by keratinocytes, including IFN-beta, interleukins -8, -18, -36gamma (IL-8, IL-18, IL-36γ), tumor necrosis factor alpha (TNF-α) (Köllisch G et al. 2005; Lebre MC et al. 2003; Liu S et al. 2019) and chemokines, such as the C-C motif chemokine ligands 20 and 27 (CCL20 and CCL27). Exposure to dsRNA in keratinocytes also leads to an upregulation of the expression of receptors that recognize dsRNA, such as TLR3, but also of the receptor TLR7, which recognizes single-stranded RNA (ssRNA) and is hardly expressed in keratinocytes under homeostatic conditions. Furthermore, it was shown that dsRNA can form a complex with a human antimicrobial peptide (AMP), cathelicidin LL-37, to amplify TLR-mediated inflammatory signals.

In addition to TLRs, RIG1-like receptors (retinoic acid-inducible gene-1/ RIG-1) are also important cytosolic nucleic acid sensors in keratinocytes (Kimura K et al. 2012). When bound to dsRNA or dsDNA, they trigger an antiviral defense by activating the production of type I IFNs. Type I IFNs are critical regulators of host antiviral defense as they can promote the maturation of myeloid dendritic cells, the proliferation of T cells and the priming of CD8+ T cells. Furthermore, they stimulate the differentiation of B cells into antibody-secreting plasma cells. RIG-1 and the MDA5 protein respond efficiently to dsRNA and trigger the activation of IRF3 and the subsequent expression of type I IFN in keratinocytes. Other cytosolic nucleic acid sensors are cyclic guanosine monophosphate adenosine monophosphate (cGAMP) synthase or cGAS (cyclic GMP-AMP synthase) and AIM2, which is absent in melanomas and recognizes dsDNA.

The STING signaling pathway (Stimulator of Interferon Genes) also plays an important role in the inflammatory response of keratinocytes (Piipponen M et al. 2020). While the receptor proteins cGAS, RIG-1 and MDA5 all contribute to the type I IFN response in keratinocytes, the receptor protein AIM2(AIM2 stands for "Absent In Melanoma 2") recognizes cytosolic DNA and triggers interleukin-1beta release in keratinocytes (Pipponen M et al. 2020).

Other receptors expressed by keratinocytes to receive signals from immune cells.

By secreting cytokines and chemokines, keratinocytes can recruit, activate and regulate immune cells. On the other hand, keratinocytes also receive signals from immune cells and react with altered cell functions. For example, keratinocytes express receptors for TNF-alpha and IFN-gamma. Stimulation with these cytokines induces keratinocyte expression of a broad spectrum of pro-inflammatory genes, such as CXCL5, CXCL8 (= interleukin-8) and ICAM-1 (intercellular adhesion molecule 1). In addition, IFN-gamma upregulates the expression of major histocompatibility complex(MHC) class II in keratinocytes, which are important for antigen presentation to T cells. Interleukin-1alpha and interleukin-1beta are important proinflammatory cytokines that are also expressed by keratinocytes. For example, the expression of interleukin-1 increases rapidly with skin injuries, and the binding of interleukin-1alpha to its receptor causes keratinocytes to express CCL20. CCL20 attracts immune cells. Interleukin-1beta induces the skin's antimicrobial defense and, together with interleukin-6 and interleukin-23, is crucial for interleukin-17-producing T cells. In skin injuries, the expression of interleukin-6 and its receptor is increased in keratinocytes. In addition, keratinocytes express a soluble interleukin-6 receptor (sIL-6R) that responds to interleukin-6 and promotes skin barrier repair.

During undisturbed homeostatic proliferation, the (non-activated) keratinocyte has been shown to express a number of chemokine receptors (C-C chemokine receptor (CCR)1, CCR3, CCR4, CCR6, CCR10 and C-X-C motif chemokine receptor (CXCR)1, CXCR2, CXCR3 and CXCR4. For CCR1, CCR10, CXCR1, CXCR2 and CXCR3). Upon binding to their corresponding monospecific ligands, the migration and proliferation of keratinocytes is stimulated. These chemokines are released, for example, by both immune cells and keratinocytes after epithelial injury (Pipponen M et al. 2020). Furthermore, keratinocytes produce proinflammatory cytokines (e.g. TNF-alpha and interleukin-1) and chemokines (e.g. CXCL1, -5, -8) after an injury. Factors secreted by keratinocytes such as interleukins and chemokines are also necessary for homeostasis and the recruitment of so-called TRM cells during an infection. On the other hand, CD8+ TRM cells secrete IFN-gamma to modulate the chemokine synthesis of keratinocytes - for example the induction of CXCL9 and CXCL10 (Rauschenberger T et al. 2019).

Exchange of keratinocytes with immune cells via extracellular vesicles

Keratinocytes can extend their communication with other cells by transporting bioactive cargo, such as proteins, lipids, DNA and RNA, in specific membrane-enclosed vesicles called extracellular vesicles (EVs). EV secretion by keratinocytes is triggered by various stimuli such as hypoxia, irradiation and starvation.

Interaction of keratinocytes with T cells via antigen presentation

In addition to producing cytokines and EVs, keratinocytes can also act as atypical antigen-presenting cells (APC) and interact with T lymphocytes in an antigen-specific manner. The expression of MHC-II has been detected in keratinocytes in a variety of skin diseases, e.g. lupus erythematosus, vitiligo, lichen planus, cutaneous T-cell lymphoma, various infectious dermatoses, allergic contact dermatitis, granulomatous dermatoses, lichen sclerosis, erythema nodosum and psoriasis. It is known that keratinocytes can synthesize HLA-DR under the stimulation of IFN-gamma (Wikner NE et al 1986). In the presence of alloantigen or nominal antigen, interaction between MHC-II+ keratinocytes and T cells leads to T cell anergy or tolerance, whereas bacterial superantigens presented by keratinocytes activate T cells that produce mainly IL-4, IL-5 and IL-10, but not IFN-gamma (Goodman RE et al. 1994).

AMPs and keratinocytes

In addition to cytokines and chemokines, activated keratinocytes also produce large amounts of AMPs, such as β-defensins, cathelicidins, S100 proteins (e.g. psoriasin and calprotectin) and RNase 7, which are positively charged amphiphilic molecules that can directly kill various microbial pathogens (Chessa C et al.2020). For example, Escherichia coli induces the secretion of psoriasin (S100A7) by keratinocytes, while S. aureus stimulates keratinocytes to produce beta-defensins, and these AMPs protect the skin from infection. Keratinocytes constitutively produce beta-defensin 1 (hBD1), while hBD2, -3 and -4 are induced in response to injury to promote keratinocyte migration and proliferation, but also to stimulate the production of proinflammatory cytokines and chemokines (e.g. IL-6, IL-10, CCL-5) (Rivas-Santiago B et al. 2012). Perforin-2 (or macrophage expressed gene 1, MPEG1) is an antibacterial effector protein that is constitutively expressed in the epidermis. It is upregulated after injury.

Epigenetic regulation of keratinocyte immune functions during wound healing (trained immunity of epithelial stem cells)

"Trained immunity" (TI) or "innate immune memory" is a new concept that describes the ability of an organism to develop an enhanced responsiveness to secondary stimuli independent of adaptive immunity. TI was first discovered in cells of the innate immune system, e.g. monocytes, macrophages and natural killer cells; however, these cells have a shorter lifespan in the bloodstream than the duration of TI. TI has also been found in cells with long life spans, such as stem cells and fibroblasts. Interestingly, it has been shown that epithelial stem cells (EpSCs) can harbor a long-lasting memory of previous inflammatory stimuli, such as topical imiquimod treatment, enabling the skin to rapidly respond to subsequent attacks and accelerate wound healing (Hamada A et al. 2018). After the initial stimulus, EpSCs retain chromosomal accessibility of several critical genes for the inflammatory response, such as AIM2, enabling rapid transcription of AIM2 and its downstream effector genes upon a secondary stimulus, i.e. skin injury (Naik S et al. 2017). This memory is mediated by the Aim2 gene encoding an activator of the inflammasome. The absence of the AIM2 protein or its downstream effectors, caspase-1 and interleukin-1beta, abolishes the memory of EpSCs for inflammation (Naik, S et al. 2017).

MicroRNAs and keratinocytes

MiR-146 has been identified as an important regulator of innate immunity of keratinocytes. miR-34a and miR-34c were significantly increased in the wound edge keratinocytes of patients with venous ulcers. These two miRNAs promote the production of inflammatory chemokines and cytokines in keratinocytes by targeting the leucine rich repeat-containing G protein-coupled receptor 4 (LGR4) (Wu J et al. 2020).

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