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The Pennsylvania State University The Graduate School Physiology Graduate Program IMMUNOMODULATORY EFFECTS OF ISOTRETINOIN IN VIVO A Dissertation in Physiology by Melanie Claire Dispenza 2011 Melanie Claire Dispenza Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2011 The dissertation of Melanie Claire Dispenza was reviewed and approved* by the following: Diane M. Thiboutot Professor of Dermatology Dissertation Advisor Chair of Committee Robert H. Bonneau Professor of Microbiology and Immunology and Pediatrics Charles H. Lang Distinguished Professor of Cellular and Molecular Physiology Program Director for Physiology Gary A. Clawson Professor of Pathology Stephen R. Rannels Associate Professor of Cellular and Molecular Physiology *Signatures are on file in the Graduate School ii ABSTRACT Acne is a common human ailment that can cause significant physical and psychological morbidity. Though it is thought to be partially caused by Propionibacterium acnes colonization, studies suggest that patients‟ specific immune responses to P.acnes may play a larger role in acne than the pathogenicity of P.acnes itself. Isotretinoin, a pro-drug for retinoic acid, is the only agent that induces a permanent remission of acne, but the mechanism underlying its long-term efficacy is unknown. We hypothesized that modulation of the immune response to P.acnes is key to isotretinoin‟s ability to induce long-term or permanent remissions of acne. In this Thesis, we show that acne patients‟ monocytes express higher levels of TLR-2 and secrete more IL-1β, IL-6, IL-8, IL-10, and IL-12 in response to P. acnes than monocytes from normal volunteers. Isotretinoin therapy significantly decreases TLR-2 expression and inflammatory cytokine secretion by acne patients‟ monocytes compared to baseline. These changes continue through 20 weeks of isotretinoin therapy and persist for at least six months after the cessation of therapy, indicating that modification of patients‟ immune responses to P. acnes may represent a long-term mechanism by which isotretinoin can cure acne. Because the exogenous administration of retinoids causes severe side-effects including birth defects, studies characterizing the action of isotretinoin in humans in vivo are lacking. This is the first study to show that isotretinoin has immunomodulatory effects in acne patients in vivo. Findings from this study extend to other diseases for which isotretinoin is used as a therapy as well as inflammatory disorders characterized by TLR-2 over-expression. iii TABLE OF CONTENTS List of Figures .................................................................................................................................... vii List of Tables ...................................................................................................................................... xiv List of Abbreviations .......................................................................................................................... xv Acknowledgements ............................................................................................................................ xvii Chapter 1: Literature Review ........................................................................................................ 1 1.1 Introduction ......................................................................................................................... 1 1.2 Skin ...................................................................................................................................... 2 1.2.1 Structure of the skin .................................................................................................. 2 1.2.1.1 Epidermis ....................................................................................................... 2 1.2.1.2 Dermis and hypodermis ................................................................................. 3 1.2.1.3 Sebaceous glands ........................................................................................... 4 1.2.2 Immune function of the skin ..................................................................................... 5 1.2.2.1 Innate immunity in the skin ........................................................................... 6 1.2.2.2 Antigen presenting and phagocytic cells........................................................ 10 1.2.2.3 T cells ............................................................................................................. 12 1.2.2.4 Regulatory T cells .......................................................................................... 14 1.3 Acne ..................................................................................................................................... 16 1.3.1 Epidemiology ............................................................................................................ 16 1.3.2 Pathophysiology........................................................................................................ 17 1.3.2.1 Follicular hyperkeratinization ........................................................................ 17 1.3.2.2 Excess sebum production ............................................................................... 18 1.3.2.3 P.acnes and inflammation .............................................................................. 19 1.3.3 Current treatments for acne ....................................................................................... 22 1.3.3.1 Topical treatments .......................................................................................... 22 1.3.3.2 Systemic treatments ....................................................................................... 23 1.3.3.3 Isotretinoin ..................................................................................................... 24 1.3.4 Model systems for acne research .............................................................................. 25 1.3.4.1 Cell lines ........................................................................................................ 25 1.3.4.2 Animal models ............................................................................................... 26 1.3.4.3 Human subjects .............................................................................................. 27 1.4 Retinoids .............................................................................................................................. 27 1.4.1 Retinoid biology, function, and metabolism ............................................................. 27 1.4.2 Retinoid effects on the immune system .................................................................... 33 1.4.2 Mechansims of retinoids in acne therapy.................................................................. 37 1.5 Significance of research project ........................................................................................... 40 Chapter 2: Systemic Isotretinoin Treatment Modulates Acne Patients’ Immune Response to P. acnes .................................................................................................................................. 42 2.1 Chapter abstract ................................................................................................................... 42 2.2 Introduction ......................................................................................................................... 42 2.3 Results ................................................................................................................................. 43 2.3.1 Subject demographics ............................................................................................... 43 iv 2.3.2 All enrolled patients responded clinically to isotretinoin therapy ............................ 45 2.3.3 Serum concentrations of retinoids do not differ between acne patients and normal volunteers .................................................................................................................... 46 2.3.4 Acne patients have higher serum concentrations of IL-10, but not inflammatory cytokines, compared to healthy volunteers ................................................................. 46 2.3.5 Isotretinoin therapy down-regulates surface expression of TLR-2 on acne patients‟ monocytes in vivo ....................................................................................................... 49 2.3.6 TLR-2 expression on peripheral blood monocytes does not correlate with age ....... 51 2.3.7 Acne patients‟ monocytes express lower levels of TLR-4 than normal volunteers‟................................................................................................................... 53 2.3.8 Isotretinoin therapy decreases inflammatory cytokine production by acne patients‟ monocytes in response to P. acnes.............................................................................. 54 2.3.9 Isotretinoin therapy does not affect TLR-2 expression in the epidermis of acne patients as determined by immunohistochemistry ...................................................... 56 2.3.10 Isotretinoin therapy does not affect proportions of Treg in peripheral blood ........... 59 2.3.11 Lymphocyte proliferation in response to P. acnes is blunted in acne patients compared to normal volunteers ................................................................................... 61 2.3.12 Isotretinoin therapy does not affect