Light-based therapies, including photodynamic therapy (PDT), for acne are gaining popularity in dermatology. Based largely upon in vitro data, their beneficial outcome in acne is thought to be related to their bactericidal effects on Propionibacterium acnes. This randomised controlled study sought to determine the efficacy and tolerability of 610-950 nm IPL (administered as IPL-Placebo) and IPL-assisted methyl aminolaevulinate PDT (IPL-MAL) vs. adapalene 0.1% gel in the treatment of acne and to identify their mode of action, looking specifically at the effect on surface density of P. acnes. Thirty seven patients (31% of target due to slow recruitment) with mild to moderate facial acne were randomly allocated to IPL-MAL treatment, IPL-Placebo or adapalene. Both IPL groups received four treatments to the whole face, 2 weeks apart, while the third group was given adapalene nightly for 12 weeks. Assessments performed at baseline and weeks 8, 11, and 16 included inflamed, noninflamed and total lesion counts, Leeds grading, follicular porphyrin fluorescence, the Family Dermatology Life Quality Index and Dermatology Life Quality Index scores, and patient’s perspective of clinical improvement by the visual analogue scale (VAS). Cutaneous microflora was collected from all patients at similar intervals. Of the 37 patients randomised, only 30 completed the trial (10 in each group) and were included in the final analyses. Adapalene was found to be significantly superior to IPL-MAL and IPL-Placebo in reducing the noninflamed (adapalene 37.6% vs. IPL-MAL 3.4% vs. IPL-Placebo −9.7%) and total lesion counts (adapalene 35.7% vs. IPL-MAL 4.3% vs. IPL-Placebo −8.4%) at week 16. This was accompanied by a significant decrease (52.9%) in the DLQI score in this group (p = 0.031). The maximum improvement in inflamed lesion counts from baseline was seen at week 11 in the IPL-MAL (20.7%) and IPL-Placebo (13.4%) groups but occurred at week 16 in the adapalene group (26.5%). Statistical significance, however, was not reached in any group. There was no significant difference within or between the groups in the VAS, Leeds, FDLQI and porphyrin fluorescence results pre- and post- treatment. A significant increase in the density of propionibacteria (p = 0.021) and xxi coagulase-negative staphylococci (p = 0.039) was seen in the IPL-Placebo and IPL- MAL groups at week 16 and week 8, respectively; however, there was no significant difference between the groups. All the treatments were well tolerated. Adapalene remains an effective first line treatment in mild to moderate facial acne. However, the present study has remained indecisive (due to being underpowered) in drawing any firm conclusions regarding the efficacy of IPL and IPL-MAL on inflamed acne lesions. Further research is therefore warranted before their use can be advocated for acne treatment. An alternative mode of action for IPL and IPL-assisted MAL-PDT other than photodynamic destruction of P. acnes is suggested from the results of this study.

Mammalian skin is a highly-specialised organ, providing a robust barrier against environmental challenges. In order to maintain functional integrity and a healthy state, the skin’s constituent tissues undergo continuous renewal, controlled by finely-controlled homeostatic mechanisms. Skin disease is incredibly common, ranging from debilitating (but not life threatening) disorders such as acne or eczema to increasingly aggressive, pernicious disorders like psoriasis, through to neoplasms such as carcinomas and melanoma. Importantly, the skin may present manifestations of systemic disease such as in diabetes, obesity, cardiovascular diseases (CVD) and metabolic syndrome (Azfar and Gelfand, 2008, Van Hattem et al., 2008, Nawale et al., 2006). Such cutaneous alterations could be the first signs of the disease, and may precede diagnosis by many years. The integrity of the skin degrades with age, and numerous metabolic disorders lead to pathological conditions that exacerbate this process (Nikolakis et al., 2013). For example, the vast majority of individuals suffering from type 2 diabetes will experience skin complications during the natural history of their disease, especially with years of poorly- controlled plasma glucose (Romano et al., 1998, Levy and Zeichner, 2012). Problems range from the sub-clinical, such as neuropathy, chronic inflammation micro- and macrovascular damage and collagen disorganisation, which lead to potentially catastrophic events such as delayed wound healing, ulceration, infection and gangrene. Proactive control of the glycaemic state may prevent or delay many of these associated complications, but it may not reverse pathological changes once established. Obesity and related risk factors in humans are associated with increased fat deposition, with many studies documenting the abnormal structure and function of dermal adipose tissue. Impaired fibroblasts function may contribute to many dermatological changes, such as the loss of dermis or fibrosis, via mechanisms related to insulin signalling (Rivera-Gonzalez et al., 2014). However, the dysfunction of dermal adipocyte which is characterised by adipocyte hyperplasia and hypertrophy, hypoxia, elevated inflammation, and adipokines signalling are now emerging as a potential mediator of insulin-resistance (Klöting and Blüher, 2014). Thus it is possible that the chronic exposure of fibroblasts to adipokines underlies their impaired function. Although many reports describe the use of mouse skin to reveal insights into aspects of human disease pathology and aetiology, it remains a challenge to find a standard protocol that can create optimal sections, retaining the intrinsic structure of mouse skin effectively, and researchers tend to use human skin histology protocols. This is not necessarily acceptable when one considers the anatomical differences between human and mice (Treuting and Dintzis, 2011). Mouse skin is thinner and more fragile than human skin, and so loss of architecture during processing has implications for downstream applications. Obtaining good histology from diabetic mouse skin, and maintaining subcutaneous adipocytes architecture and the discrimination of components such as fine elastic fibres, present a considerable challenge. Therefore, great care must be taken when selecting the conditions, particularly fixative type, to ensure that meaningful conclusions may be drawn (Al-Habian et al., 2014). The skin sections from mouse models used in chapter 3 were prepared using best practice at the time for a retrospective study, but I was able to better histology at the cost of time and materials in sectioning and staining by using tissue macroarrays techniques. Thus, for prospective analysis of mouse skin histology I revised some histology methods. By evaluating the processing protocol and then four commonly used fixatives I enhanced IHC, and histomorphological analysis of normal mouse skin. Moreover, these histopathology techniques are applicable to different mouse tissues, and I recommend using these findings as a guideline not only for diabetic mouse skin histology but for many tissues derived from a variety of species. The dermatologic sequelae of type 2 diabetes mellitus are manifold. Murine models of insulin resistance are useful in elucidating molecular mechanisms of impaired wound healing, but little is known of other pathophysiological changes in these models. A variety of histological and in vitro techniques were used in this thesis to study skin organisation in environmental (diet-induced obesity) and genetic (C57BL/6 Lepob/Lepob and C57BLKS/J Leprdb/Leprdb) models of insulin resistance, type 2 diabetes mellitus, and in chronological age. An expanded subcutis was accompanied by progressive dermal erosion in which the papillary dermis was spared at the expense of the deeper reticular layer as insulin resistance increased. Elastosis was also observed in our models, but damage was not accompanied by an increase in immune infiltration, nor an increase in advanced glycation end products. Altered epidermal differentiation was associated only with the most extreme obese phenotype. Moreover, compromised fibroblast function was maintained in vitro, with C57BL/6 Lepob/Lepob cells displaying compromised growth and reduced collagen synthesis. While mouse skin does not necessarily model the range of cutaneous sequelae observed in human diabetic subjects, it is likely that underlying cellular mechanisms are shared. An improved understanding of the contribution of the layers of the skin, particularly the dermis during insulin resistance, and ageing and its pathological processes may provide new insights into the mitigation of damage. While impaired insulin signalling in skin is associated with disrupted skin homeostasis, and extra cellular matrix remodelling in ageing and type 2 diabetes mellitus (Nikolakis et al., 2013), a recent study has linked these changes to dermal fibroblast , and adipocyte dysfunction (Rivera-Gonzalez et al., 2014). Interestingly, our observation showed that both aged and Lepob/Lepob murine fibroblasts show lowered insulin receptor expression suggesting that this will be a fruitful area for future investigation for improving insulin sensitivity and glucose utilisation in the dermal layers could have important consequences for cutaneous health. This thesis reports a specific pattern of cutaneous damage that is initiated before the onset of frank diabetes and is exacerbated with increasing insulin resistance. This work provides a new insight into the consequences of metabolic disease on skin structure. Finally, further insights into how dermal damage in genetically modified and environmentally adapted diabetic mouse models and vice versa are likely to subserve in detecting early signs of cutaneous insulin resistance and may likely offer better approaches to prevent or at least cure the dermatological sequelae of type 2 diabetes mellitus.

We have identified PrimPol, a yet uncharacterized protein that can act in vitro as a low processivity polymerase able to bypasses thymine dimers in DNA. PrimPol localizes to both the nucleus and mitochondria. In order to investigate functional role of this enzyme we have generated genetrap mice and knock out mice where PrimPol expression is abolished. In cells lacking PrimPol, we can see increased genomic instability even in the absence of damage, indicating that it may suppress expression of common fragile sites. Upon UV damage and aphidicolin treatment, there was a significant increase in chromosomal aberrations. In genetrap embryos e14.5, PrimPol expression pattern was ectoderm and eye specific. This was confirmed in the skin of adult where PrimPol was most highly expressed in the skin compared to other tissues. PrimPol-/- UV damaged skin showed marked epidermal hyperplasia compared to WT. We have also observed in aged PrimPol-/- mice, and in young mice damaged by UV, upregulation of mitochondrial DNA copy number across different tissues, implying that PrimPol may be dealing with the repair of oxidative damage as well. In accordance with these findings, we hypothesize that PrimPol acts as a translesion polymerase that can bypass bulky lesions generated by UV and reactive oxygen to promote the integrity of nuclear and mitochondrial DNA and promote normal aging. / Hemos identificado PrimPol, una proteína no caracterizada que actua in vitro como una polimerasa de bajo procesamiento que es capaz de evitar los dímeros de timina del ADN y que se localiza en el núcleo y en las mitocondrias. Para poder investigar la función de este enzima, hemos generado ratones genetrap y knock out en los cuales se ha abolido la expression de PrimPol. En las células que no expresan PrimPol podemos observar un aumento de la inestabilidad genómica incluso en ausencia de daño, indicando que podría suprimir la expression de sitios frágiles comunes. Tras inducir daño mediante radiación ultravioleta y tratamiento con afidicolina se observó un aumento significativo de aberraciones cromosómicas. En embriones y ratones adultos mediante tinción de LacZ y pudimos ver que PrimPol está altamente expresado en piel y en los ojos en comparación con otros tejidos. La piel dañada con UV en ratones PrimPol-/- mostró una marcada hiperplasia epidérmica en comparación con los ratones salvajes. También hemos observado que en ratones PrimPol-/- de edad avanzada y en ratones jóvenes con lesiones por UV hay un aumento del número de copias de ADN mitocondrial en diferentes tejidos, lo que sugiere que PrimPol podría estar también implicada en la reparación del daño oxidativo. De acuerdo con estos descubrimientos nos planteamos la hipótesis de que PrimPol pueda actuar como una translesion polimerasa que puede saltarse grandes lesiones inducidas por UV y especies reactivas de oxígeno y así promover la integridad del ADN nuclear y mitocondrial además de un normal envejecimiento.

Psoriasis is a common, debilitating systemic inflammatory disorder that is characterised by sharply demarcated, thick, erythematous scaly skin plaques. Such plaques commonly appear on skin that is subjected to repeated tensile trauma, such as elbows, knees and flexures. The mechanism by which these inflammatory lesions are spatially restricted is not known and yet knowledge of this could be of critical importance for our understanding of this disease. Chemokines are the principal regulators of leukocyte migration and play a critical role in the initiation and maintenance of inflammation. The atypical chemokine receptor ACKR2 (formerly D6) binds inflammatory CC-chemokines, but does not signal upon ligand binding; instead ACKR2 internalises and helps degrade such chemokines, after which it continues to cycle back to the cell surface. ACKR2 acts, through this mechanism, as a high-capacity scavenger of chemokines, and plays an important role in regulating inflammation. It is known that ACKR2 expression is high in unaffected skin in patients with psoriasis (remote from inflammatory plaques) and concurrently deficient in the plaques themselves. Additionally, human studies have shown that simple skin trauma in psoriasis patients causes a reduction in cutaneous ACKR2 expression at the site of trauma. However, the functional significance and the molecular mechanism by which it occurs are not understood. This thesis explored the role of ACKR2 in the spatial restriction of psoriasiform inflammation and the molecular mechanisms for its differential regulation. Through the use of disease relevant mouse models, primary human cell cultures and novel cell migration assays, the results presented here show that localised psoriasiform inflammation upregulates ACKR2 in remote tissues through the systemic release of cytokines. This remotely upregulated ACKR2 expression protects tissues from the further spread of inflammation. This protective effect is mediated by stromally expressed ACKR2 that acts to control inflammatory T-cell positioning within the skin. Tensile trauma of keratinocytes however, acted to reduce ACKR2 expression in the context of inflammation, which in turn provides a novel mechanism for the well-characterised phenomenon that occurs in psoriasis (and a range of skin condition) termed ‘koebnerisation’. Koebnerisation refers to the phenomenon by which relatively simple skin trauma induces the development of disease-specific skin lesions. Furthermore, this thesis defines novel disease-relevant regulators of ACKR2 expression. In silico analyses identified psoriasis-associated microRNAs that bound to the 3’-UTR of ACKR2, and reduced its expression at transcriptional and protein level. Importantly, trauma of keratinocytes induced ACKR2 downregulation concurrent with a substantial and significant increase in the expression of the identified ACKR2 targeting microRNAs. Together, this thesis defines a novel mechanism by which ACKR2-mediated regulation of chemokine function, cutaneous trauma, microRNAs and systemic cytokines, co-ordinately modulate the predisposition of remote tissue sites to the development of new lesions. Importantly, the results presented here have profound implications for how spatial restriction is imposed on inflammation. The data also highlight therapeutic ACKR2 induction as a plausible novel strategy for the limitation and treatment of psoriasiform- and potentially other forms of inflammation.

Erosive lichen planus affecting the vulvovaginal region (ELPV) is a rare chronic inflammatory condition causing painful raw areas that can lead to scarring, at the vaginal entrance. Symptoms considerably impact upon daily function and quality of life. There is risk of cancerous change in affected skin of 1-3%. A Cochrane Systematic Review, published in 2012, found no randomised controlled trials (RCT) on which to base treatment for ELPV. Retrospective case series suggest that super-potent topical corticosteroids are frequently used as first-line therapy, although one third of patients fail to respond adequately and require escalation of therapy. There is clinical uncertainty regarding which second-line therapies should be used. The following steps were taken to inform the design of an RCT to determine optimal second-line therapy for EVLP resistant to topical steroids: • A multi-centre retrospective review and audit of case notes to assess current clinical management in the UK. • A qualitative investigation with UK clinicians to establish their views and principles of management of ELPV. • An international multi-disciplinary electronic-delphi consensus exercise to agree a set of diagnostic criteria for ELPV. • A systematic review to assess existing outcome measure tools that have been used in randomised controlled trials of vulval skin disorders. • Assessment of patients views through a survey of a national patient group and subsequent focus groups with patients. The resulting multi-centre, four-armed, open-label, pragmatic randomised controlled trial will compare hydroxychloroquine, methotrexate and mycophenolate mofetil against a standard care group of clobetasol propionate 0.05% plus a short course of oral prednisolone. This will be the first RCT to test systemic agents for patients with ELPV and will add to the existing evidence base. The methodologies employed to develop the RCT protocol, and the trial design itself, may act as a template for clinical research into the therapeutic management of other rare inflammatory conditions.

The study of skin microbiota has always been focused from a clinical point of view. However, an ecological approach to the skin microbiota is impeded by different methodological limitations, including the high host/microbial DNA ratio or the low microbial content. In contrast with the burgeoning field of gut metagenomics, skin metagenomics has been hindered by the absence of an efficient methodology to work with skin microbial DNA. This thesis aims to settle the basis for further human skin microbiome studies from a systems approach. I have set four different important approaches for a ecological and systematic view of skin: 1) I have tested and compared a method to construct NGS libraries from trace amounts of DNA, allowing to work with very rare samples, and performing multiple functional experiments on the same sample; 2) I have defined a method to isolate the microbial DNA from a skin sample, to perform actual metagenomic studies. We have proved the utility of the method by constructing 2 metagenomic libraries from mouse skin biopsies; 3) I have tested the relationship between microbial diversity and unrelated phenotypes in mouse skin samples; 4) I have analyzed the host phenotypical spectra, characterized what it is metabolic health, and assessed the relationship between health and microbiota. / L’estudi de la microbiota de la pell ha estat sempre enfocat cap a un punt de vista clínic. No obstant, una aproximació ecològica a la microbiota cutània és impossibilitada per multiples limitacions metodològiques, que inclouen la baixa ràtio de DNA hospedador/DNA comensal o la baixa quantitat absoluta de DNA microbià. En contrast al pròsper camp de la metagenòmica intestinal, la metagenòmica cutània ha estat obstaculitzada metodològicament, i per això, aquesta tesi intenta sentar les bases per al futur de l’estudi del microbioma cutàni i la aplicació d’una aproximació de sistemes. He aplicat quatre aproximacions importants per a la observació sistemàtica i ecològica de la pell: 1) He testat i comparat un mètode per construir llibreries de NGS a partir de traces de DNA, permetent treballar amb mostres de difícil obtenció i aplicar-hi multiples experiments funcionals a la mateixa mostra, sense haver d’usar tot el material en la seqüenciació; 2) He definit un mètode per a aïllar el DNA microbià d’una mostra de pell completa per tal de dur a terme una anàlisi metagenòmica de la mostra. Hem demostrat la seva utilitat construint dues llibreries independents a partir de pell de ratolí; 3) He analitzat la relació entre la diversitat microbiana i un fenotip aïllat de l’hospedador; 4) He analitzat l’espectre fenotípic de l’hoste des d’un punt de vista sistèmic, he caracteritzat l’estat de salut metabòlica i la seua relació amb la microbiota.

The mechanism of action of IPL in acne treatment is not clearly understood, but an immunomodulatory role has been suggested. Furthermore, inflammatory cytokines and matrix degrading enzymes play a key role in acne pathogenesis. Therefore, curbing the production of these mediators may assist acne resolution. In photorejuvenation studies, IPL has been shown to induce the expression of a key immunomodulatory cytokine, TGF-β. Interestingly, TGF-β has been demonstrated to mediate immunosuppression, inhibition of keratinocyte proliferation and MMP-1 repression through a Smad3-mediated signalling pathway. Therefore, we sought to investigate the in vivo effects of IPL used for acne treatment. Biopsies obtained from 20 patients with inflammatory acne vulgaris at baseline and post-IPL treatment (48 hrs after the first treatment and 1 week after the final treatment) were immunohistochemically analysed to investigate the expression of TGF-β1, TGF-β2, TGF-β3, Smad3, MMP-1 and IL-8. Digital images were semi-qualitatively assessed using image analysis software. In addition, quantitative PCR analysis of TGF-β1, Smad3 and IL-8 was performed on biopsies from seven cases. Immunohistochemical analysis demonstrated that IPL elicited a statistically significant increase in epidermal TGF-β1 expression. However, no statistically significant difference was observed in the expression of TGF-β2/β3. Increased nuclear immunolocalisation of Smad3 was demonstrated in the post-IPL biopsies, which was statistically significant. Although not statistically significant, both IL-8 and MMP-1 expression showed a downward trend in the majority of cases. No statistically significant change was detected in the gene expression of TGF-β1, Smad3 and IL-8, which may be attributed to the small sample in which PCR was carried out. The data from this study suggests that Smad3-mediated TGF-β1 signalling may play a role in IPL-induced resolution of acne vulgaris. The therapeutic effect of TGF-β1 in inflammatory acne vulgaris could be attributed to its immunosuppressive effect and its ability to inhibit matrix degradation and keratinocyte proliferation.

Thyroid dysfunction is frequently associated with skin and hair diseases; however, the underlying pathogenic mechanisms are poorly understood. Pathological activation of the thyroid stimulating hormone receptor (TSHR) is the key feature of both hyper- and hypo-thyrodism. Expression of the (TSHR) has been reported in several extra-thyroidal locations including adipose tissue, bone and skin fibroblasts. TSHR expression may explain the association between the thyroid and skin disease. The TSHR can also be activated by a newly discovered glycoprotein hormone, known as thyrostimulin. This hormone is composed of a dimer of unique α 2 and β 5 subunits. Although thyrostimulin has not been detected in the circulation. However, both subunits have been shown to be expressed in different tissues including the skin. The aim of this study is to examine the expression of the TSHR and thyrostimulin in the skin. In addition, to investigate the expression of a variant form of the TSHR in human and mouse skin and, other mouse tissues. RT-PCR using primers specific for the full length receptor and the truncated variant revealed that although the variant was widely expressed in mouse tissues including skin, it was not found in human skin. The full length receptor and thyrostimulin were found to be co-expressed in eye, testis and skin. Immunohistochemistry of frozen skin and thyroid sections using commercially available antibodies against the extracellular (A9) and transmembrane domains (A7) of TSHR demonstrates that TSHR is not expressed in the epidermis but expressed in dermal fibroblasts and in myoepithelium around sweat glands. A new β5 antibody was characterised by western blotting and immunohistochemistry for future investigation of β5 expression in the skin. These data suggest a functional role for TSHR signalling possibly via thyrostimulin in the skin and that the variant form,although potentially present in some tissues, is unlikely to be important in human skin.

Molluscum contagiosum virus (MCV) is a significant human pathogen causing benign tumours in the human skin. Molluscum contagiosum (MC) infection is most common in children, young adults and immunodeficient individuals. MCV replicates well in the human skin in vivo, but conditions that support MCV replication in vitro are unknown. The lack of in vitro cell culture system has significantly limited progress in MCV research. The aims of this study were, (i) To develop a reporter assay for the sensitive detection and quantitation of MCV infections in vitro, (ii) To express suitable MCV virion surface antigens to develop a sensitive MCV ELISA assay, and (iii) To raise and characterize monoclonal antibodies (mAbs) against these antigens for the detection of MCV in infected human skin. All goals were achieved. A reporter assay based on simultaneous transfection of luciferase/EGFP reporter plasmids and infection with live MCV worked well and was used to test the infectivity of MCV in several human and animal cells. A novel C-terminal MC084 ELISA was established and used to determine MCV seroprevalence in two European populations. mAbs against MC084 and MC133 were raised and partially characterized. Interesting findings were that MCV not only infects human keratinocytes, but also a wide range of animal and human cells in vitro, which, however, do not support replication. Our MCV ELISA gives seroprevalences in UK and German general populations comparable with previous Australian and Japanese studies (<40 years of age). Surprisingly, in older age groups (vaccinated against smallpox), a significantly higher MCV seroprevalence was observed. This was not due to crossreactivity with VACV. We propose this increase may be due to childhood MCV antibodies being boosted by subsequent vaccination or vice versa. Finally, the mAbs raised are unique reagents and will be used in future work to test a hypothesis that MCV replicates in human epidermal stem cells.

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