Clinical, Cellular, and Molecular Aspects in the Pathophysiology Of

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Clinical, Cellular, and Molecular Aspects in the Pathophysiology of Rosacea Martin Steinhoff1,2,Jo¨rg Buddenkotte2, Jerome Aubert3, Mathias Sulk2, Pawel Novak2, Verena D. Schwab2, Christian Mess2, Ferda Cevikbas1, Michel Rivier3, Isabelle Carlavan3, Sophie De´ret3, Carine Rosignoli3, Dieter Metze2, Thomas A. Luger2 and Johannes J. Voegel3

Rosacea is a chronic inflammatory skin disease of may also be involved (ocular rosacea, subtype IV). Several unknown etiology. Although described centuries ago, additional variants have been described (Wilkin et al., 2002, the pathophysiology of this disease is still poorly 2004), although their subdivision into the entity rosacea is not understood. Epidemiological studies indicate a genetic yet clear. Rosacea prevalence is highest (between 2.7 and component, but a rosacea gene has not been identified 10%) in patients of northern European or Celtic heritage yet. Four subtypes and several variants of rosacea have (Webster, 2009; Abram et al., 2010b; McAleer et al., 2010). been described. It is still unclear whether these sub- Humans with fair skin (Fitzpatrick skin phenotypes I–II) are types represent a ‘‘developmental march’’ of different more likely to be affected, whereas Asians and African stages or are merely part of a syndrome that develops Americans are less affected, indicating a genetic component independently but overlaps clinically. Clinical and (Abram et al., 2010a). This is also supported by the finding that often African Americans are affected when one parent is histopathological characteristics of rosacea make it a of northern European origin (Steinhoff M, unpublished obser- fascinating ‘‘human disease model’’ for learning about vation). Rosacea is more common among female patients, the connection between the cutaneous vascular, nervous, and incidence peaks between the ages of 30 and 50 years. and immune systems. Innate immune mechanisms and Analyzing the molecular and protein profiles of the dysregulation of the neurovascular system are involved different rosacea subtypes has increased our understanding in rosacea initiation and perpetuation, although the of rosacea pathophysiology and will continue to do so. Gene complex network of primary induction and secondary array (Figure 1b and 2b) and subsequent real-time PCR reaction of neuroimmune communication is still unclear. analysis (not shown) indicate that the clinically defined Later, rosacea may result in fibrotic facial changes, subtypes of rosacea also differ in their gene profile. All suggesting a strong connection between chronic inflam- rosacea subtypes show a different gene profile compared matory processes and skin fibrosis development. This with healthy skin. Each subtype can be differentiated by a review highlights recent molecular (gene array) and selective gene profile (Figure 1b). Thus, the pathomechanisms cellular findings and aims to integrate the different of the different subtypes may vary with respect to the body defense mechanisms into a modern concept of molecular pathways and molecules involved. Our gene array rosacea pathophysiology. analysis indicates that certain genes overlap, which means that a developmental ‘‘march’’ among the different rosacea Journal of Investigative Dermatology Symposium Proceedings (2011) 15, 2–11; doi:10.1038/jidsymp.2011.7 subtypes can be assumed in most cases (Figure 1). Despite that, it is still unclear whether patients in whom rosacea becomes clinically apparent not earlier than in the PhR subtype undergo a subclinical form of ETR and PPR. CLASSIFICATION AND EPIDEMIOLOGY OF ROSACEA Rosacea is a common, almost exclusively facial inflammatory Rosacea’s clinical features and trigger factors indicate a skin disease characterized by erythema and telangiectasia complex dysregulation of inflammatory, vascular, and neuronal (erythematotelangiectatic rosacea, ETR, subtype I, RI), papu- systems at an early stage lopustular rosacea (PPR, subtype II, RII), and phymatous The impact of a dysregulatory vascular system in rosacea rosacea (PhR, subtype III, RIII; Figure 1a). Ocular structures patients has long been hypothesized (Flint and Wilkin, 1994;

1Departments of Dermatology and Surgery, University of California, San Francisco, San Francisco, California, USA; 2Department of Dermatology, University Hospital Mu¨nster, Mu¨nster, Germany and 3Department of In vitro Pharmacology, Galderma R&D, Sophia Antipolis, Valbonne, France Correspondence: Martin Steinhoff, Departments of Dermatology and Surgery, University of California, San Francisco, 513 Parnassus Avenue, Room S-1268, San Francisco, California 94143, USA. E-mail: [email protected] Abbreviations: ETR, erythematotelangiectatic rosacea; PhR, phymatous rosacea; PPR, papulopustular rosacea; TRPV1, transient receptor potential (TRP) vanilloid receptor 1 Received 20 June 2011; revised 19 August 2011; accepted 24 August 2011

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analysis and reverse transcriptase PCR we found that substantial upregulation of proinflammatory genes involved in vasoregulation (e.g., adrenergic receptors, tryptophan Clusters metabolites, proteases) and neurogenic inflammation (e.g., I II III IV transient receptor potential (TRP) vanilloid receptor 1 (TRPV1), pituitary adenylate cyclase-activating polypeptide), HS along with a marked inflammatory infiltrate (Th1 cells, macrophages, mast cells), can already be observed at a very early stage of rosacea, even before it is clinically visible in ETR terms of papules, nodules, or pustules (Figures 1–3). Thus, despite its origin, early rosacea has to be regarded as an PPR inflammatory disease characterized by neuroimmune dys- PhR function and neurovascular dysregulation.

Figure 1. Relationship between clinical characteristics and gene array Molecular and immunohistochemical (IHC) characterization profile of rosacea. Three clinical subtypes have been studied by gene reveals a role of innate and adaptive immunity in rosacea array analysis from facial skin biopsies and compared with healthy facial The PPR subtype of rosacea consists of an inflammatory infil- or uninvolved skin (healthy skin, HS). ETR, erythematotelangiectatic rosacea trate leading to papules, pustules, and, sometimes even, cysts. (subtype I, RI); PPR, papulopustular rosacea (subtype II, RII); PhR, phymatous Occasionally, neutrophils or B cells are found in rosacea rosacea (subtype III, RIII). (a) ETR is characterized by a prolonged flushing erythema and teleangiectasia. PPR is characterized by papules and, biopsies. Various microbial agents, including Demodex folli- more rarely, pustules, in addition to erythema and telangiectasia. PhR is culorum, Helicobacter pylori, Staphylococcus epidermidis, characterized by fibrotic changes and glandular hyperplasia, less clinically or Chlamydiae, have been implicated in the pathophysiology obvious inflammation, and erythema. Although flushing and discomfort of rosacea (reviewed by Powell, 2005; Lazaridou et al., 2011). are the predominant clinical features, and no papules are visible in ETR, þ The studies on H. pylori are contradictory (Mc Aleer et al., a marked inflammatory infiltrate of CD4 T lymphocytes, macrophages, 2009; Gallo et al., 2010; Tuzun et al., 2010; Lazaridou et al., and mast cells is visible already in ETR by immunohistochemistry (see also Figure 3). (b) Gene array analysis of biopsies from patients with 2011). As for D. folliculorum, recent studies support its role rosacea (subtypes I–III; n ¼ 6–8 patients per subtype) reveals differential as a key factor in at least certain subtypes of rosacea, regulation of various selective genes among the various subtypes predominantly subtype II, which is characterized by papules (RI ¼ ETR ¼ cluster II; RII ¼ PPR ¼ cluster III; RIII ¼ PhR ¼ cluster IV) as and pustules. Increased numbers of Demodex mites have compared with healthy or uninvolved skin (cluster I). Because certain been found in these patients as compared with healthy skin genes overlap, a developmental ‘‘march’’ from rosacea I to III can (Lazaridou et al., 2010). In addition, these mites may harbor be assumed, although it is not always clinically visible (arrows). bacteria that may exacerbate rosacea (Lacey et al., 2007; McAleer et al., 2010). Thus, it is reasonable to hypothesize that Demodex may activate immune mechanisms in pre- Huggenberger and Detmar, 2011). Substantial evidence disposed rosacea patients, in whom Demodex serves as a indicates that sun exposure is important in the pathophysiol- trigger factor leading to exacerbation of the papular and/or ogy of rosacea (reviewed by Powell, 2005; Marks, 2007; pustular phenotype. However, PPR is also found in patients McAleer et al., 2010; Webster, 2010). The impact of UV whose lesions have a normal number of Demodex; other yet radiation versus heat during sun exposure is still controver- unknown cofactors can be hypothesized. sial, although clinical experience suggests that both have an Our findings of upregulated genes involved in innate impact because flushing can develop from heat without sun immunity and enhanced immune cells (Figures 1–3) indicate exposure (e.g., hot steam from a coffee pot (Wilkin, 1981) the involvement of the adaptive and innate immune system and vice versa (sun exposure in a cold environment)). in all subtypes of rosacea, but to different extents. Although Moreover, temperature changes modulate vascular func- genes of the innate immune response are activated in all three tion (Wilkin, 1981, 1983; Crawford et al., 2004) and affect subtypes of rosacea, the adaptive immune response seems to bacterial protein production (Dahl et al., 2004). Both UV light be more distinct in PPR and PhR and less in ETR (Figures 1 and temperature changes can activate sensory nerves (Spiro and 2). This is supported by our IHC characterization of the et al., 1987; McArthur et al., 1998; Roosterman et al., 2006; inflammatory infiltrate within the various subtypes of rosacea, Aubdool and Brain, 2011). Thus, an activated nervous system suggesting that inflammatory cells of the innate and adaptive in the skin correlates well with the early phase of rosacea, immune system are involved (Figure 3). Our gene array and although it is still unclear whether neuronal activation morphometric data further reveal three important issues: First, precedes or follows the inflammatory infiltrate. The extent neutrophils and B cells are only rarely found in certain to which the autonomic and/or sensory nervous system patients, indicating that multiple trigger factors lead to the is involved in the neuronal dysregulation during rosacea same symptom (papule, pustule). IL-8 mRNA is significantly has received considerable attention, as modulation of upregulated in PPR, but the trigger that leads to upregula- a-adrenergic receptors or b-adrenergic blockers is helpful in tion of IL-8 and thus to neutrophil formation is currently some patients (Craige and Cohen, 2005; Shanler and Ondo, unknown. Second, gene array and quantitative real-time 2007; Gallo et al., 2010). It is noteworthy that by gene array RT-PCR data demonstrate upregulation of genes involved in

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Epidermal activation

Alcohol metabolism

Organic acid metabolism 5 7 - Carboxylic acid metabolic process Others: the non-enriched 9

44 % Response to stimulus: 9 - Response to biotic stimulus - Antimicrobial humoral response 10

Immune system process 16

Lipid metabolism (36 genes) - Steroid metabolism - Fatty acid metabolism - Lipid biosynthesis b Chemokines, hits ETR PPR PhR WFDC12 WAP four-disulfide core domain 12 3.09 3.33 4.45 CRIP1 Cysteine-rich protein 1 (intestinal) –4.47 –4.1 –4.26 IL7R Interleukin 7 receptor 3.33 9.66 6.31 KLRB1 Killer cell lectin-like receptor subfamily B, member 1 3.61 7.68 4.04 LTF Lactotransferrin 8.84 53.36 25.43 OAS1 2’,5’-oligoadenylate synthetase 1, 40/46 kDa 3.08 4.73 5.57 IL26 Interleukin 26 4.14 22.5 6.94 CCL5 Chemokine (C-C motif) ligand 5 3.56 7.22 4.77 CCL18 Chemokine (C-C motif) ligand 18 (pulmonary and activation-regulated) 5.7 17.86 9.92 CCL19 Chemokine (C-C motif) ligand 19 4.63 10.39 8.25 CCL20 Chemokine (C-C motif) ligand 20 3.96 5.6 5.54

Figure 2. Analysis of inflammatory and immune changes in rosacea on the basis of gene array analysis. (a) Biological interpretation of genes upregulated in cluster II (erythematotelangiectatic rosacea, ETR). Of the 221 known genes that clustered in various dominant biological processes, about 20% can be implicated in inflammation and immune defense. A surprising and thus far unacknowledged increase of genes involved in alcohol and lipid metabolism was observed. About 5% of the genes are involved in epidermal activation or development, indicating the involvement of keratinocytes at an early stage of rosacea. (b) Gene array analysis of cytokines and chemokines involved in the pathophysiology of rosacea indicates a role of interleukin (IL)-7, IL26, and various chemokines already in early subtypes of rosacea (see Gerber et al., 2011, for details). RI, ETR; RII, papulopustular rosacea (PPR); RIII, phymatous rosacea (PhR). WAP, whey acidic protein.

innate and adaptive immunity, including cathelicidin, which phages (Marks and Harcourt-Webster, 1969), mast cells has been implicated in rosacea pathophysiology by interact- (Bamford, 2001; Aroni et al., 2008), and neutrophils (Ramelet ing with kallikrein-5 (Yamasaki et al., 2007; Morizane et al., and Perroulaz, 1988; Akamatsu et al., 1990; Millikan, 2003) 2010). Third, the inflammatory infiltrate in ETR is predomi- has been described. Plasma cells have been described in PhR nantly perivascular, not periglandular. Accordingly, our gene (Aloi et al., 2000). array studies and histological data do not support an Our IHC and morphometric analyses of rosacea subtypes important role for known microbial agents in the early phases I–III suggest that the dominating activated cells in rosacea are of rosacea. However, further investigations about the impact CD4 þ Th1 cells, macrophages, and mast cells (Figure 3; of the innate immune system on neurovascular and neuroim- Steinhoff et al., 2010; Schwab V et al., 2011). It is noteworthy mune function are warranted to fully understand the that the early stage of rosacea already presents as a Th1-cell- neuroimmune and immune defense connection in the mediated inflammatory skin disease accompanied by macro- pathophysiology of rosacea. phages and mast cells, without enhancement of Langerhans cells, eosinophils, or natural killer cells (Figure 3). Addi- Molecular and morphological characterization reveals differ- tional trigger factors not yet known seem to activate neutro- ences in the inflammatory infiltrate within different rosacea phils and B cells in rosacea patients under certain conditions, subtypes resulting in pustules (neutrophils) and probably development Although various studies indicate that a lymphomonocytic into a hyperglandular–phymatous stage (increased density of infiltrate dominates in rosacea, few examined the whole B cells). spectrum of involved inflammatory cells in all subtypes of In contrast with other inflammatory skin diseases such as rosacea by immunohistochemistry, including morphometry. psoriasis, lupus erythematosus, or atopic dermatitis, the Involvement of T cells (Rufli and Buchner, 1984), macro- crucial cytokines and chemokines that orchestrate the

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CD31 ETR Podopl ETR NF ETR CD1a ETR

CD4 ETR CD8 ETR CD56 ETR C68 ETR

Vim ETR NF PPR Ela PPR Vim PPR

CD79 PPR/PhR PGP PhR CD20 PhR CD79 PhR

Figure 3. Histopathological characteristics of rosacea subtypes (erythematotelangiectatic rosacea (ETR), papulopustular rosacea (PPR), phymatous rosacea (PhR)). All three subtypes were stained for inflammatory and immune markers to determine the inflammatory infiltrate and skin structures involved in inflammation and fibrosis (T cells: CD4, CD8; B cells: CD20, CD79a; Langerhans cells: CD1a; macrophages: CD68; natural killer cells: CD56; neutrophils: CD15, elastase; mesenchymal cells: vimentin (VIM); nerves: PGP9.5, NF200; blood vessels: CD31; lymphatic vessels: podoplanin (Podpl)) in the various subtypes. An increased mast cell infiltrate can also be observed in all subtypes of rosacea (Schwab et al., 2011). (b)Bar¼ 200 mm; (a, g, i, j, l–o), bar ¼ 100 mm; (c, d, f, h, k, p), bar ¼ 50 mm.

initiation and perpetuation of rosacea are not fully known. leakage of blood vessels and rosacea is characterized by Our molecular characterization of the different subtypes of edema derived from blood and lymphatic vessels. Edema rosacea as compared with healthy human skin by gene array intensity varies in rosacea, and the maximal clinical stage is analysis and real-time PCR indicates that a variety of represented by morbus morbihan, in which lymphedema is cytokines, chemokines, metalloproteinases, proteases, and the predominant clinical appearance. reactive oxygen species molecules, as well as lipid mediators, Blood and lymphatic vessels have an extraordinary role in contribute to dysregulation of the inflammatory responses in skin development, body homeostasis, and wound repair rosacea (Figure 2a; Gerber et al. 2011). Still lacking, (reviewed by Roosterman et al., 2006; Huggenberger and however, is a systematic profile of the inflammatory media- Detmar, 2011; Meyer-Hoffert and Schro¨der, 2011). Both are tors involved in rosacea pathophysiology, both at the gene apparently involved in chronic inflammatory diseases such as and protein level. psoriasis. Recent data indicate that blood and lymphatic vessels are also involved in rosacea (Gomaa et al., 2007; Molecular and morphological characterization indicates pre- Smith et al., 2007; Aroni et al., 2008; Bender et al., 2008). dominantly vasodilatation, but not angiogenesis in early rosacea The crucial growth factors, vasoregulatory molecules, and The importance of the vascular cutaneous system in rosacea receptors contributing to the development of vasodilatation, is supported by the clinical and histopathological character- edema formation, or lymphedema are poorly understood in istics of flushing, erythema, and telangiectasia. Furthermore, the context of rosacea (Nakamura and Rockson, 2008; edema results from plasma extravasation; i.e., the vascular Ogunbiyi et al., 2011). Although immunoreactivity for

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vascular endothelial growth factor, CD31 (blood vessel and may be a target for anti-inflammatory therapies, even marker), and D2-40 (lymphatic vessel marker) has been topically. Activation of lymphatic vessel function via described as enhanced in rosacea (Gomaa et al., 2007), topically applied vascular endothelial growth factor-C may the impact of angiogenetic or vasoregulatory mechanisms be a promising pathway to block inflammation in rosacea appears to be irrelevant in the pathophysiology of ETR and other diseases (Huggenberger et al., 2010). and PPR. Our combined IHC and morphometric analyses (Figure 3), together with gene array and RT-PCR studies NEUROVASCULAR CHANGES IN ROSACEA: THE TRPV1 (Figures 1 and 2), indicate that blood vessels and lymphatic HYPOTHESIS vessels are dilated in ETR and PPR and that angiogenesis The clinical features of rosacea indicate that several body and lymphangiogenesis are part of the rosacea patho- defense mechanisms are involved in its complex pathophysiology only in the PhR subtype (Schwab et al., 2011). physiology, reflecting the inflammatory, vascular, neuronal, Flushing can be activated by the autonomic or the sensory and fibrotic components of the different disease subtypes nervous system, although both systems are no longer (Figure 4). The role of the skin nervous system in the control completely divided but communicate with each other of inflammation, immunity, and vascular regulation is now (Gibbons et al., 2010; Mousa et al., 2011). The kinetics of well established (Roosterman et al. 2006). A direct involve- flushing, however, resemble the pattern of sensory C-fiber ment of neurovascular dysregulation in rosacea skin is also activation more. Moreover, non-neuronal mediators such as supported by the characteristics of certain cell surface lipid metabolites and tryptophan derivates are ultimately receptors that are intriguingly activated by trigger factors of involved in vasoregulation (Wang et al., 2010). Thus, rosacea—namely, spicy food, heat, noxious cold, exercising, vasodilatation may be induced by neuronal stimulation, but and ethanol—and are simultaneously involved in inflamma- could also result from inflammatory mediators released tion, vasoregulation, and neuronal function, notably the during the early phase of rosacea, in which inflammatory TRPV1 (Figure 5; see Aubdool and Brain, 2011 for details). It cells are already abundantly present (Figure 3; Roosterman is noteworthy that rosacea patients present with an enhanced et al., 2006; Graepel et al., 2011). susceptibility to trigger factors such as ‘‘hot’’ spicy food, In rosacea, which is characterized by marked infiltration noxious heat and cold, UV radiation, physical exercise, of T cells, macrophages, and occasionally neutrophils or B stress, alcohol (ethanol), and medications (sympathomi- cells, an important role of blood vascular endothelial cells metics, niacin). Many of these trigger factors activate ion lies in their capacity to express selectins and cell adhesion channels such as TRPV1 (Figure 5; see also Aubdool and molecules, which are important for the recruitment of Brain, 2011; Schwab et al., 2011). TRPV1 is expressed by leukocytes to the site of inflammation (Hua and Cabot, sensory nerves (Figure 5) and is ultimately involved in 2010; Muller, 2011). The chemokines involved in the vasoregulation (Caterina et al., 1997; Earley, 2010) and recruitment of these cells in rosacea are still unknown. nociception. Thus, neurovascular dysregulation through Recently, inhibition of blood vessel activation was found TRPV1 might contribute to the early symptoms of rosacea to exert potent anti-inflammatory properties (reviewed by characterized by transient flushing (‘‘pre-rosacea’’). This is Huggenberger et al., 2010; Huggenberger and Detmar, 2011) also supported by our finding that the density of TRPV1 þ

Stimuli: Color code: - Hot environments Healthy skin - Hot beverages and meals Rosacea skin TRPV1 - Spicy food, ethanol Intervention Over expression?

Capsaicin Heat Channel sensitization Reduced activation threshold Na+ Ca2+

TRPV1 Therapeutic intervention: hyperactivation TRPV1 inactivation Depolarization Block

Neuropeptide Normal response F inflammation Pathological response TRPV1

Figure 4. Potential role of neurogenic inflammation in the early phase of rosacea. Genetic predisposition, along with exogenous or endogenous trigger factors, stimulates peripheral nerve endings of the skin. Central transmission of neuronal activation leads to facial discomfort (stinging, burning pain), perceived by the central nervous system. The sensory axon reflex of primary afferents in the dermis and epidermis releases vasoactive neuropeptides such as pituitary adenylate cyclase-activating polypeptide or vasoactive intestinal peptide (VIP) into the microenvironment. Binding of neuromediators to high-affinity neuropeptide receptors on arterioles or venules leads to vasodilatation (flushing, erythema) or plasma extravasation (edema). Activation of T cells, macrophages, and mast cells by neuropeptides results in activation or aggravation of inflammatory responses. It is unknown to what extent neuromediators may also exert anti-inflammatory capacities in human skin diseases. Bi-directional communication between the innate immune and nervous systems may aggravate early rosacea leading to chronic disease. Ca2 þ , calcium; E, epidermis; F, nerve fibre; Na2 þ , sodium; TRPV1, transient receptor potential (TRP) vanilloid receptor 1.

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Exogenous triggers: Genetic Early rosacea, ETR + Heat, toxins, UV ?, predisposition microbes ?

Innate immunity IL-1, IL-8, Chemokines, VEGF, FGF? LL37, KLK5 TLR2, PAR-2?

Pro-inflammatory effects: vasodilatation plasma extravasation edema T cell Macrophage Neuropetide release Sensory nerve immunomodulation + autonomic nerve ? Mast cell Neural sensitization immunomodulation vasoregulation

Genetic predisposition + endogenous triggers: Spicy food pH changes Pain lipids burning exercise etc. stinging

Figure 5. Neurovascular transient receptor potential (TRP) vanilloid receptor 1 (TRPV1) hypothesis in rosacea. TRPV1 localized in abundance on epidermal and dermal perivascular sensory nerve endings (lower right picture) can be stimulated by various trigger factors of rosacea such as spicy food, ‘‘hot’’ temperature changes, ethanol or inflammatory acidosis, or prostanoids. In healthy humans (black), stimulation of TRPV1 on nerve endings leads to a transient dilatation, followed by rapid recovery. In rosacea patients (red), overexpression or overstimulation of TRPV1 by a possibly genetic predisposition leads to neuropeptide release and thereby neurogenic inflammation (see Figure 4 for details), characterized by flushing, erythema, edema, and inflammation. It is unknown whether TRPV1 dysregulation is based on increased ion channel sensitization or a reduced activation threshold. Thus, therapeutic intervention of TRPV1 overstimulation may be a new therapeutic strategy in rosacea (green). ETR, erythematotelangiectatic rosacea; FGF, fibroblast growth factor; KLK5, kallikrein-related peptidase-5; PAR-2, proteinase-activated receptor-2; TLR-2, Toll-like receptor 2; VEGF, vascular endothelial growth factor. nerve fibers is increased in ETR as compared with healthy epidermal and dermal TRPV1 þ nerve fibers (Figure 5), as skin (Steinhoff M and Voegel JJ, unpublished observation). well as enhanced expression levels of TRPV1 mRNA in skin Transient flushing later often results in persistent erythema biopsies of rosacea patients (Steinhoff M and Voegel JJ, with dysesthesia (burning, prickling, pain, rarely itch). unpublished observation). Thus, TRPV1 may be a therapeutic Dysesthesia is controlled by sensory A-delta and/or target for rosacea treatment. Other TRPV ion channels are C-fibers (Steinhoff et al., 2003; Ma, 2010). On this basis, likely to have a role in rosacea, but further exploration is one may hypothesize that rosacea patients are subjected to a needed (see Aubdool and Brain, 2011, for details). dysregulation of TRPV1, which is activated by spicy food, heat, and pH changes (Figure 5). In healthy humans, TRPV1 THE RELATIONSHIP OF CHRONIC INFLAMMATION activation by these trigger factors leads to a short experience AND FIBROSIS IN ROSACEA of flushing and burning pain. In rosacea patients, however, The PhR rosacea subtype is more common in male patients TRPV1 is eventually hyperactive, resulting in sustained and develops mostly from PPR. However, in a certain flushing, a feeling of warmth, and ‘‘neurogenic’’ inflam- subgroup of rosacea patients, PhR is present without obvious mation with edema and inflammatory cell infiltration clinical signs of ETR or PPR, probably derived from a (Figure 5), which can be extensive (Scharschmidt et al., subclinical form of rosacea (Figure 1). The genes and 2011). In accordance with this hypothesis, our own data molecules that may regulate the interaction between the confirm that rosacea patients demonstrate a high density of inflammatory system and the extracellular matrix during the

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fibrotic process are poorly understood. Candidates include that aggravate inflammatory cascades such as cytokines, reactive oxygen species (Bakar et al., 2007), metalloprotei- chemokines (Gerber et al. 2011), antimicrobial peptides nases (Maatta et al., 2006), serine proteases (Yamasaki et al., (Yamasaki and Gallo, 2011), radical oxygen species (Meyer- 2011), and growth factors and cytokines/chemokines (re- Hoffert and Schro¨der, 2011), neuromediators (Aubdool viewed by Gallo et al., 2010; Meyer-Hoffert and Schro¨der, and Brain, 2011; Schwab et al., 2011), or proteases (Meyer- 2011; Gerber et al., 2011). On the basis of our knowledge Hoffert and Schro¨der, 2011). Moreover, Toll-like receptor-2 from other skin diseases or animal models, it appears that expression is enhanced in rosacea but not in atopic dermatitis certain receptors for cytokines, chemokines, growth factors, or psoriasis (Yamasaki et al., 2011), indicating a role of or proteases are involved in the switch from an inflammatory Toll-like receptors in rosacea. Among antimicrobial peptides, to a fibrotic stage (see also Gerber et al., 2011; Meyer-Hoffert cathelicidin (LL37) appears to have an important role in and Schro¨der, 2011). The cellular mechanisms underlying innate immunity by regulating inflammation, reactive oxygen this switch from inflammation to fibrosis are in general poorly species production, angiogenesis, and protease function understood, and for rosacea, completely unknown. Good (Yamasaki et al., 2006, 2007). In support of this finding, our candidates for such a ‘‘link’’ between the chronic inflamma- gene array data confirm that mRNA expression levels of LL37 tory and fibrotic stages appear to be fibroblast growth were found to be significantly increased in all subtypes of factors (Shaw et al., 2010; Craik et al., 2011; Meyer-Hoffert rosacea (Schwab et al., 2011). How the various trigger factors and Schro¨der, 2011), cytokines (Gerber et al. 2011), and compromise the innate immune system and skin nervous proteases (Steinhoff et al., 2000, 2005). Future molecular and system needs to be explored. Understanding the role of the cellular characterization of fibroblast growth factors, extra- innate immune system and its key factors in rosacea may lead cellular matrix proteins, and proteases in the context with to new strategies for treatment of rosacea at an early stage inflammatory changes in rosacea development will shed new (Meylan et al., 2006; Yamasaki and Gallo, 2009). Recent light on disease pathophysiology and potential new treatment findings strongly indicate an important role of antimicrobial options against rosacea. peptides and proteases as ‘‘alarmins’’ (mediators activated in Our immunohistological and gene array studies indicate the skin at an early stage of tissue injury) and are thereby that fibroblasts are already activated in the earliest stages of part of the innate immune system. Upon activation by rosacea (Schwab et al., 2011). It remains unclear which cells trigger factors, they can respond to ‘‘danger’’ molecules and mediators upregulate the function and number of that may impair epidermal homeostasis (reviewed by fibroblasts and myofibroblasts in ETR and PPR before the Meyer-Hoffert and Schro¨der, 2011; Yamasaki and Gallo, phymatous phenotype becomes clinically evident. It is 2011). As peripheral nerves are also part of the noteworthy that the observed increased number of fibroblasts defense system in the epidermal skin barrier, the relationship correlates well with enhanced numbers of mast cells (Schwab among nerves, antimicrobial peptides, and proteases et al., 2011), which are known to modulate fibroblast func- needs to be further deciphered (Figure 5 and 6; Schwab tion and fibrosis (Hermes et al., 2001; Chujo et al., 2009). et al., 2011). Thus, mast cells (Aroni et al., 2008; Schwab et al., 2011) along with T cells (Rufli and Buchner, 1984; Figure 3) and CONCLUSIONS AND FUTURE DIRECTIONS macrophages (Marks, 1973, Figure 3) may be ultimately The pathophysiology of rosacea is still poorly understood. involved in the development of PhR. Moreover, further Recent cellular, IHC, molecular, and pharmacological studies indicate that keratinocytes are no longer passive approaches helped to further decipher new molecules and bystanders in the pathobiology of rosacea but actively pathways in the pathobiology of rosacea. Our current contribute to inflammation and fibrosis by releasing cyto- hypothesis (Figure 6) is that a genetic predisposition, together kines, chemokines, growth factors, metalloproteinases, and with trigger factors, leads to the clinical occurrence of proteases, and regulate expression of intergrins, occludins, transient flushing, which may be because of overstimu- and claudins (Eslami et al., 2009; Liu et al., 2009; lation of the sensory and/or autonomic nervous system Huggenberger and Detmar, 2011; Meyer-Hoffert and in the skin and induction of innate immune responses. Schro¨der, 2011). However, the exact role of keratinocytes, The concrete relationship between the skin nervous system innate immunity, and epidermal barrier dysregulation in the and the innate immune system is still unclear. Neurovas- context of rosacea still needs to be explored. cular mechanisms are very likely involved in vascular dysregulation characterized by flushing, persistent erythema, Innate immunity and neural receptors as sensors for ‘‘outside and telangiectasia. Sustained chronic inflammation charac- danger’’: potential role in rosacea terized by a mainly Th1 macrophage and mast cell-driven Recent evidence indicates that the innate immune system infiltrate further leads to the release of incompletely and epidermal barrier are impaired in rosacea, leading to understood mediators involved in vasoregulation, immunity, increased susceptibility of the skin (‘‘hypersensitive skin’’) and fibrosis. toward toxins, temperature changes, microbe invasion, Many details of the new cellular and molecular observa- tissue injury, UV radiation, and endogenous mediators tions are still unexplained and raise new critical questions: (e.g., pH changes, medication, and so on). This activation (1) What are the roles of the innate and adaptive immune of cells involved in skin innate immune regulation and barrier systems, especially Th1 cells, mast cells, and macrophages, function may subsequently stimulate the release of mediators in the various subtypes? (2) What is the impact of mast cell

8 Journal of Investigative Dermatology Symposium Proceedings (2011), Volume 15 M Steinhoff et al. Pathophysiology of Rosacea

Blood vessels: vasodilatation (erythema, teleangiectasia)

Kallikreins (PAR2 ?) Neuropeptides, Neuronal sensation 5 Genetic 4 LL37, (Pain, burning, stinging) predisposition Epinephrin, NO Neuropeptides, pH (TRPV1 ?), pH (TRPV1 ?) Neutrophins, bradykinin ? + Neuro vascular dysregulation 1 Angiogenesis (flush) 6 (only PhR) Environmental trigger factors Growth factors, LL37, proteases Podoplanin Dysregulation of Inflammatory mediators, 2 lymphatic system Receptor dysregulation, (lymphedema) Leukocyte recruitment 7 ROS, growth factors, 3 cytokines, proteases Immune defense: - Defensins 8 - AMPs Inflammation: Fibrosis - Chemokines - Vasodilatation (rhinophyma) - Cytokines - Plasma extravasataion - Interferons (erythema, edema, - ROS papules, pustules) Glandular hyperplasia

Figure 6. Potential pathophysiology of rosacea integrating clinical, immunological, neurovascular, and molecular characteristics. (1) Genetic predisposition plus trigger factors activate hypersensitive sensory nerve endings in the skin. Alternatively (2), trigger factors may stimulate innate immune mechanisms in the skin, thereby ‘‘alarming’’ the neuronal defense system in the skin, which is hypersensitive (3) Neural activation results in vasodilatation, edema (4), and discomfort (pain, burning) (5). (6) Chronic neurogenic inflammation may lead to persistent erythema, and, at later stages, to angiogenesis, which is probably limited to the phymatous rosacea (PhR) subtype. (7) It is unknown whether dilation of lymphatic vessels is dependent or independent on neuronal stimulation. (8) Chronic inflammation characterized by Th1 cells, macrophages, and mast cells because of sustained innate immune and neurogenic stimulation results in induction of profibrotic growth factors, probably via mast cells, and thereby activation of myofibroblasts, rearrangement of the extracellular matrix, and finally fibrosis. AMP, antimicrobial peptide; NO, nitric oxide; PAR-2, proteinase-activated receptor-2; ROS, reactive oxygen species; TRPV1, transient receptor potential (TRP) vanilloid receptor 1.

infiltration and activation in all subtypes of rosacea? (3) What REFERENCES are the roles of the sensory and autonomic nervous system Abram K, Silm H, Maaroos HI et al. (2010a) Risk factors associated with and which receptors and mediators are involved in inflam- rosacea. J Eur Acad Dermatol Venereol 24:565–71 mation, vasoregulation, and the hypersensitive skin of Abram K, Silm H, Oona M (2010b) Prevalence of rosacea in an Estonian rosacea patients? (4) How are the immune and neurovascular working population using a standard classification. Acta Derm Venereol systems linked to each other in terms of disease initiation and 90:269–73 aggravation? (5) Which are the crucial factors initiating the Akamatsu H, Oguchi M, Nishijima S et al. (1990) The inhibition of free radical generation by human neutrophils through the synergistic transient flushing and vasodysregulation? (6) What are the effects of metronidazole with palmitoleic acid: a possible mechanism crucial factors modulating vasodilatation of blood and of action of metronidazole in rosacea and acne. Arch Dermatol Res lymphatic vessels—are these the same factors and can they 282:449–54 be targeted therapeutically? (7) What is the impact of Aloi F, Tomasini C, Soro E et al. (2000) The clinicopathologic spectrum of keratinocyte and fibroblast activation in rosacea, and could rhinophyma. J Am Acad Dermatol 42:468–72 they be targets for therapy to control inflammation and thus Aroni K, Tsagroni E, Kavantzas N et al. (2008) A study of the pathogenesis of prevent fibrosis? The many questions reflect our poor rosacea: how angiogenesis and mast cells may participate in a complex multifactorial process. Arch Dermatol Res 300:125–31 understanding of rosacea and emphasize the need for Aubdool AA, Brain SD (2011) Neurovascular aspects of skin neurogenic advanced scientific approaches, adequate in vivo and ex inflammation. J Investig Dermatol Symp Proc 15:33–9 vivo models, and preclinical proof-of-principle studies in Bakar O, Demircay Z, Yuksel M et al. (2007) The effect of azithromycin on rosacea research. reactive oxygen species in rosacea. Clin Exp Dermatol 32:197–200 Bamford JT (2001) Rosacea: current thoughts on origin. Semin Cutan Med CONFLICT OF INTEREST Surg 20:199–206 The authors state no conflict of interest. Bender A, Zapolanski T, Watkins S et al. (2008) Tetracycline suppresses ATP This review was published as part of a supplement sponsored by Galderma. gamma S-induced CXCL8 and CXCL1 production by the human dermal microvascular endothelial cell-1 (HMEC-1) cell line and primary human ACKNOWLEDGMENTS dermal microvascular endothelial cells. Exp Dermatol 17:752–60 This work was supported by the National Rosacea Society, West Havens Caterina MJ, Schumacher MA, Tominaga M et al. (1997) The capsaicin Foundation (UCSF), DFG 1014/2-2, and the National Institutes of Health (to receptor: a heat-activated ion channel in the pain pathway. Nature MS), and DFG Ce165/1-1 (to FC). 389:816–24

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