Opioid receptors expression and PGP 9.5 - positive epidermal nerve fiber density in psoriasis – relationship with itch. Opioid receptors expression and PGP 9.5 - positive epidermal nerve fiber density in psoriasis – relationship with itch. Piotr Kupczyk 1 , Adam Reich 2 , Edyta Wysokińska 3 , Mariusz Gajda 4 , Marcin Hołysz 5 , Thomas C. Hwang 6 , Anna Kobuszewska 1 , Beata Nowakowska 1,8. , Wanda Niepiekło-Maniszewska 1,7,9. , Małgorzata Kamińska 7,9. , Justyna Drukała 8 , Jacek C. Szepietowski 1 . Piotr Kupczyk 1 , Adam Reich 2 , Edyta Wysokińska 3 , Mariusz Gajda 4 , Marcin Hołysz 5 , Thomas C. Hwang 6 , Anna Kobuszewska 1 , Beata Nowakowska 1,8. , Wanda Niepiekło-Maniszewska 1,7,9. , Małgorzata Kamińska 7,9. , Justyna Drukała 8 , Jacek C. Szepietowski 1 . 1. Laboratory of Immunogenetic and Tissues Immunology, Department of Clinical Immunology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland. 2. Department of Dermatology, Venereology and Allergology, Wroclaw Medical University, Wroclaw, Poland. 3. Laboratory of Molecular Immunobiology of Cancer, Department of Experimental Oncology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland. 4. Department of Histology, Jagiellonian University Medical College, Cracow, Poland. 5. Department of Biochemistry and Molecular Biology, Medical University of Poznan, Poznan, Poland. 6. Department of Pharmacology, Medical University of Minnessota, MN, USA. 7. Laboratory of Tissues Immunology, Medical Center of Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland. 8. Laboratory of Cellular and Tissues Enginery, Department of Cell Biology, Division of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Cracow, Poland. 9. Laboratory of Clinical Immunogenetic and Pharmacogenetic, Department of Clinical Immunology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland. 1. Laboratory of Immunogenetic and Tissues Immunology, Department of Clinical Immunology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland. 2. Department of Dermatology, Venereology and Allergology, Wroclaw Medical University, Wroclaw, Poland. 3. Laboratory of Molecular Immunobiology of Cancer, Department of Experimental Oncology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland. 4. Department of Histology, Jagiellonian University Medical College, Cracow, Poland. 5. Department of Biochemistry and Molecular Biology, Medical University of Poznan, Poznan, Poland. 6. Department of Pharmacology, Medical University of Minnessota, MN, USA. 7. Laboratory of Tissues Immunology, Medical Center of Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland. 8. Laboratory of Cellular and Tissues Enginery, Department of Cell Biology, Division of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Cracow, Poland. 9. Laboratory of Clinical Immunogenetic and Pharmacogenetic, Department of Clinical Immunology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland. Introduction Opioid receptors (OR) are GCPR proteins involved in neurological dissorders, addiction, pain and itch. Itch (pruritus) is a sensation, which causes desire to scratch. Chronic and uncontrolled pathological itch is usually result of pharmacotherapy or it associated with many skin diseases. Psoriasis is a chronic inflammatory skin disease, with immunogenetic background, often associated with itch. Psoriasis patients experienced itch, which dramaticly reduces patients psychophysiological well-being. Molecular biology mechanisms of itch in psoriasis are poorly explained. Skin OR and unmyelinated C-nerve fiberes, afferent dorsal root ganglion neurons (nociceptors) are considered as linkers between skin and peripherial nervous system (part of neuroimmunoendocrine axis), participating in skin neurobiology, pain and itch control. While OPRM1/μ-MOR activates, OPRK1/κ-KOR is considered as inhibitor of itch signaling in skin and central nervous system (CNS). Imbalance of OR and downregulation of skin peripherial nervous system (SPNS) are considered as possible molecular mechanism in psoriasis itch. The aim of the study: The aim of the project was to measure how genes and proteines of OR are expressed in skin of psoriasis patients with different itch sensation. Epidermal nerve fiber density (ENFD) was estimated for SPNS using neurobiological marker of C-nerve fibres, gene and proteine gene product 9.5 ( UCHL1/PGP 9.5). Clinical disease parametrs like psoriasis area and sevierity index (PASI) for psoriasis level and visual analoge scale (VAS) for itch intensity were estimated. Introduction Opioid receptors (OR) are GCPR proteins involved in neurological dissorders, addiction, pain and itch. Itch (pruritus) is a sensation, which causes desire to scratch. Chronic and uncontrolled pathological itch is usually result of pharmacotherapy or it associated with many skin diseases. Psoriasis is a chronic inflammatory skin disease, with immunogenetic background, often associated with itch. Psoriasis patients experienced itch, which dramaticly reduces patients psychophysiological well-being. Molecular biology mechanisms of itch in psoriasis are poorly explained. Skin OR and unmyelinated C-nerve fiberes, afferent dorsal root ganglion neurons (nociceptors) are considered as linkers between skin and peripherial nervous system (part of neuroimmunoendocrine axis), participating in skin neurobiology, pain and itch control. While OPRM1/μ-MOR activates, OPRK1/κ-KOR is considered as inhibitor of itch signaling in skin and central nervous system (CNS). Imbalance of OR and downregulation of skin peripherial nervous system (SPNS) are considered as possible molecular mechanism in psoriasis itch. The aim of the study: The aim of the project was to measure how genes and proteines of OR are expressed in skin of psoriasis patients with different itch sensation. Epidermal nerve fiber density (ENFD) was estimated for SPNS using neurobiological marker of C-nerve fibres, gene and proteine gene product 9.5 ( UCHL1/PGP 9.5). Clinical disease parametrs like psoriasis area and sevierity index (PASI) for psoriasis level and visual analoge scale (VAS) for itch intensity were estimated. Material: Skin punch biopsies from non-lesional and lesional skin of 20 psoriasis patients and 20 skin fragments from normal healthy skin was included in the study. Cell cultures of normal human keratinocytes was used to confirm OR expression on mRNA and protein level. Refference material have been intrudiced to study to confirm specificity of reagents. Comercially available total RNA from human brain (Agilent Technologies, USA) and total protein extracts from human temporal lobe (Abcam, UK) and hippocampus (Agilent Technologies, USA) were used for OR and PGP 9.5 expression in Western blot. Methods: Gene expression: Relative gene expression analysis was performed using multiplex real-time PCR with UPL probes (Light Cycler 480, Roche, Switzerland) before RNA isolation (Tissues Fibrosus Mini Kit, Qiagen, USA) and reverse transcription on cDNA (First Strand cDNA Synthesis Kit for RT-PCR (AMV), Roche, Switzerland) from total skin. ΔCt method was used for gene expression. Proteine expression: Direct immunofluorescence protocol have been performed on 12 μm skin frozen sections with rabbit polyclonal anti-MOR and -KOR (Santa Cruz-Biotechnology, USA) antibodies, while detection was done using donkey anti-rabbit Alexa 488 (MOR) and Alexa 594 (KOR) secondary conjugated Abs (Invitrogen, USA). Mouse monoclonal Abs mix of anti-human PGP 9.5 (13C4, 31A3, UltraClone and Abcam, UK) were used for ENFD and donkey anti-mouse conjugated secondary Ab with Alexa 546 was used for detection. All pictures were performed on fluorescence microscopre (Zeiss, Germany) and were analyzed on ImageJ Software (NIH, USA) for Fluorescence Intensity (FI) of OR protein expression. The project was approved by the Ethic Committee of Wroclaw Medical University. Material: Skin punch biopsies from non-lesional and lesional skin of 20 psoriasis patients and 20 skin fragments from normal healthy skin was included in the study. Cell cultures of normal human keratinocytes was used to confirm OR expression on mRNA and protein level. Refference material have been intrudiced to study to confirm specificity of reagents. Comercially available total RNA from human brain (Agilent Technologies, USA) and total protein extracts from human temporal lobe (Abcam, UK) and hippocampus (Agilent Technologies, USA) were used for OR and PGP 9.5 expression in Western blot. Methods: Gene expression: Relative gene expression analysis was performed using multiplex real-time PCR with UPL probes (Light Cycler 480, Roche, Switzerland) before RNA isolation (Tissues Fibrosus Mini Kit, Qiagen, USA) and reverse transcription on cDNA (First Strand cDNA Synthesis Kit for RT-PCR (AMV), Roche, Switzerland) from total skin. ΔCt method was used for gene expression. Proteine expression: Direct immunofluorescence protocol have been performed on 12 μm skin frozen sections with rabbit polyclonal anti-MOR and -KOR (Santa Cruz-Biotechnology, USA) antibodies, while detection was done using donkey anti-rabbit Alexa 488 (MOR) and Alexa 594 (KOR) secondary conjugated Abs (Invitrogen, USA). Mouse monoclonal Abs mix of anti-human PGP 9.5 (13C4, 31A3, UltraClone and Abcam, UK) were used for ENFD and donkey anti-mouse conjugated secondary Ab with Alexa 546 was used for detection. All pictures were performed on fluorescence microscopre (Zeiss, Germany) and were analyzed on ImageJ Software (NIH, USA) for Fluorescence Intensity (FI) of OR protein expression. The project was approved by the Ethic Committee of Wroclaw Medical University. Results Results Research project: „Gene and proteine expression of opioid receptors OPRM1/MOR and OPRK1/KOR and neurobiological marker of nerve endings PGP 9.5 in psoriasis patients with and without pruritus. ”. National Science Center Grant (2011/01/N/NZ4/04946). Research project: „Gene and proteine expression of opioid receptors OPRM1/MOR and OPRK1/KOR and neurobiological marker of nerve endings PGP 9.5 in psoriasis patients with and without pruritus. ”. National Science Center Grant (2011/01/N/NZ4/04946). Normal Skin Normal Skin Lesional Skin Lesional Skin μ-MOR+PGP 9.5+DAPI → Skin μ-MOR+PGP 9.5+DAPI → Skin μ-MOR+DAPI μ-MOR+DAPI κ-KOR+DAPI κ-KOR+DAPI κ-KOR+PGP 9.5+DAPI → Skin κ-KOR+PGP 9.5+DAPI → Skin → OPRM1 and OPRK1 genes are signifficantly higer expressed in brain compared to the skin and keratinocytes. → OPRK1 gene expression have signifficantly lower expression in non- lesional and lesional skin with tendency to lower in itch group compared to healthy group. → OPRM1 gene expression is very low in the skin, but constantly expressed in all analyzed groups. → OPRM1 and OPRK1 genes are signifficantly higer expressed in brain compared to the skin and keratinocytes. → OPRK1 gene expression have signifficantly lower expression in non- lesional and lesional skin with tendency to lower in itch group compared to healthy group. → OPRM1 gene expression is very low in the skin, but constantly expressed in all analyzed groups. Non-lesional Skin Non-lesional Skin Keratinocyte in vitro Keratinocyte in vitro PGP 9.5 PGP 9.5 Itch Itch No Itch No Itch Itch Itch No Itch No Itch → OR protein expression are imbalanced in psoriasis patients. → While μ-MOR expression is constant in all analyzed groups,κ-KOR receptor is signifficantly downregulated in lesional skin of psoriasis patients. → Patients with high level of itch demonstrate significantly lower κ-KOR expression in lesional skin compared to non-lesional and control group skin, while those without itch do not demonstrate any signifficantly differences. → Epidermis of non-lesional and lesional skin have signifficantlly higher PGP 9.5 ENFD compared to normal skin. Non-lesional and lesional skin positively correlate with VAS, while lesional skin negativly correlate with PASI. → OR protein expression are imbalanced in psoriasis patients. → While μ-MOR expression is constant in all analyzed groups,κ-KOR receptor is signifficantly downregulated in lesional skin of psoriasis patients. → Patients with high level of itch demonstrate significantly lower κ-KOR expression in lesional skin compared to non-lesional and control group skin, while those without itch do not demonstrate any signifficantly differences. → Epidermis of non-lesional and lesional skin have signifficantlly higher PGP 9.5 ENFD compared to normal skin. Non-lesional and lesional skin positively correlate with VAS, while lesional skin negativly correlate with PASI. Normal Skin Normal Skin Glands dermis Glands dermis Summary and perspectives Summary and perspectives Normal Skin Normal Skin PGP 9.5 PGP 9.5 Non-lesional Skin Non-lesional Skin Lesional Skin Lesional Skin PGP 9.5 PGP 9.5 → μ-MOR receptor and PGP 9.5 are commonly expressed in skin nerve endings, especially in non-lesional skin. → We did not observed common expression of κ-KOR and PGP 9.5 within the skin nervous system. → μ-MOR receptor and PGP 9.5 are commonly expressed in skin nerve endings, especially in non-lesional skin. → We did not observed common expression of κ-KOR and PGP 9.5 within the skin nervous system. 1 → Hippocampus 2 → Temporal Lobe 3 → Normal Skin 1 → Hippocampus 2 → Temporal Lobe 3 → Normal Skin 1 1 1 2 2 3 3 1 KOR normal KOR non-lesional (+) KOR non-lesional (-) KOR lesional (+) KOR lesional (-) 0 10 20 30 40 50 60 70 80 90 100 KOR normal KOR non-lesional (+) KOR non-lesional (-) KOR lesional (+) KOR lesional (-) 0 10 20 30 40 50 60 70 80 90 100 MOR normal MOR non-lesional (+) MOR non-lesional (-) MOR lesional (+) MOR lesional (-) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% MOR normal MOR non-lesional (+) MOR non-lesional (-) MOR lesional (+) MOR lesional (-) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Normal Skin Non-lesional Skin (+) Non-lesional Skin (-) Lesional Skin (+) Lesional Skin (-) 0 2 4 6 8 10 12 14 16 18 Normal Skin Non-lesional Skin (+) Non-lesional Skin (-) Lesional Skin (+) Lesional Skin (-) 0 2 4 6 8 10 12 14 16 18 Normal skin Non-lesional skin (+) Non-lesional Skin (-) Lesional Skin (+) Lesional Skin (-) 0 2 4 6 8 10 12 14 16 18 Normal skin Non-lesional skin (+) Non-lesional Skin (-) Lesional Skin (+) Lesional Skin (-) 0 2 4 6 8 10 12 14 16 18 ΔCt ΔCt ΔCt ΔCt MOR: Normal vs. Non-Lesional (+) p=0,69 Normal vs, Non-Lesional (-) p=0,12 Normal vs. Lesional (+) p=0,85 Normal vs. Lesional (-) p=0,26 MOR: Normal vs. Non-Lesional (+) p=0,69 Normal vs, Non-Lesional (-) p=0,12 Normal vs. Lesional (+) p=0,85 Normal vs. Lesional (-) p=0,26 KOR: Normal vs. Non-lesional (+) p <0,05 Normal vs. Non-lesional (-) p <0,01 Normal vs. Lesional (+) p <0,001 Normal vs. Lesional (-) p= 0,74 KOR: Normal vs. Non-lesional (+) p <0,05 Normal vs. Non-lesional (-) p <0,01 Normal vs. Lesional (+) p <0,001 Normal vs. Lesional (-) p= 0,74 OPRM1: Normal vs. Non-Lesional (+) p= 0,96 Normal vs, Non-Lesional (-) p= 0,45 Normal vs. Lesional (+) p= 0,2 Normal vs. Lesional (-) p= 0,98 OPRM1: Normal vs. Non-Lesional (+) p= 0,96 Normal vs, Non-Lesional (-) p= 0,45 Normal vs. Lesional (+) p= 0,2 Normal vs. Lesional (-) p= 0,98 OPRK1: Normal vs. Non-lesional (+) p <0,01 Normal vs. Non-lesional (-) p <0,01 Normal vs. Lesional (+) p <0,001 Normal vs. Lesional (-) p = 0,04 OPRK1: Normal vs. Non-lesional (+) p <0,01 Normal vs. Non-lesional (-) p <0,01 Normal vs. Lesional (+) p <0,001 Normal vs. Lesional (-) p = 0,04 1 → Skin 2 → Keratinocyte 3 → Brain 1 → Skin 2 → Keratinocyte 3 → Brain 1 → Skin 2 → Keratinocyte 3 → Brain 1 → Skin 2 → Keratinocyte 3 → Brain OPRM1/ACTB OPRM1/ACTB 1 2 2 3 3 1 OPRK1/ACTB OPRK1/ACTB Opioid receptors expression in skin with (+) and without itch (-). Opioid receptors expression in skin with (+) and without itch (-). Opioid receptors gene expression in skin with (+) and without itch (-). Opioid receptors gene expression in skin with (+) and without itch (-).