Skin: Acne vulgaris genetics and molecular responses to bacterial challenges

Abstract

Skin is first line of defense against physical, chemical as well as biological environment. Human skin conditions are the 4 th non-fatal and 18 th fatal leading cause of global disease burden. Among skin diseases, bacterial infections are responsible for 66, 500 deaths annually. A number of the antibiotics have been produced to combat infectious agents; however, continuous emergence of resistant strains is a big challenge for human health and pharmaceutical industry. Skin is an ideal organ to study molecular responses to biological infections by virtue of diverse skin cells specialized in immune responses. Acne vulgaris is a top 8 th multifactorial skin disease of Propionibacterium acnes (P. acnes) infected pilosebaceous units with maximum prevalence in Asian population. Acne vulgaris is gaining importance as model disease to gain insight in molecular mechanisms of infectious and multifactorial skin diseases. Several factors involved in acne vulgaris pathogenesis has been investigated, however exact mechanisms and key regulators of acne initiation are still unidentified. The investigation of skin-microbe interaction using acne vulgaris as model disease would be helpful in understanding the host-microorganism relationship. Therefore, we used three approaches to investigate skin-microbe interactions in acne vulgaris model. First we investigated the role of heredity and dietary factors, as well as serum metabolites in acne pathogenesis. Second, skin responses to Gram positive bacteria-challenges were studied using microarray metaanalysis approach. Finally, differential gene expression in skin exposed to Gram positive bacterial strains was compared for further understanding of skin infections. Acne genetics was studied in 530 acne patients (329 Female and 201 male) recruited from outdoor patients department (OPD) of Islamabad, Rawalpindi and Lahore hospitals from Punjab province. For comparison 550 age, sex and ethnicity matched healthy controls (332 Female and 218 male) were also selected. The association of inflammatory cytokines single nucleotide polymorphisms (SNPs), including IL-6 (-174 G/C and -572 G/C), IL-1α (-889 C/T), TNF-α (-857 C/T, -863 C/A and -1031 T/C) and adipokine resistin (+299 G/A and -420 C/G), with acne vulgaris was investigated using PCR-RFLP method. Serum lipid profile was compared in 530 acne patients and 550 healthy controls using enzymatic endpoint spectrophotometric method to study importance of Skin: Acne vulgaris genetics and molecular responses to bacterial challenges dietary factors in acne pathogenesis. In addition serum levels of TNF-α, apolipoprotein-a (apo-a) and platelet activation marker platelet factor 4 (PF4) were measured in 89 acne patients using enzyme linked immunosorbent assay (ELISA) technique. The public repository “GEO Datasets” search for microarray data of skin responses to bacterial challenges in mammals presented two different study groups, first bovine primary mammary epithelial cells (PMECs) and udder responses to mastitis causing bacteria; second human systemic as well as skin and immune cells responses to S. aureus and S. aureus components. The more homogenous microarray data for bovine mastitis causing bacterial challenges was analyzed using RankProd, RMAExpress and DAVID software to optimize metaanalysis methodology. In addition, skin biopsies were taken from human skin exposed to P. acnes, Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis) and Toll-like receptors1/2 (TLR1/2) agonist (Pam3CSK4), and differentially expressed genes were identified using Affymetrix microarray chips. The current study revealed that the genetics background is very important in prediction of acne development risk in Pakistani population. The resistin (+299 G/A and -420 C/G), IL-6 (-572 G/C), IL-1α (-889 T/C) and TNF-α (-863 C/A) gene polymorphisms were strongly associated, whereas TNF-α gene polymorphism at - 1031 T/C was not associated with pathogenesis of acne vulgaris. Furthermore, IL-6 (- 174 G/C) and TNF-α (-857 C/T) gene polymorphisms showed protective role in acne risk development. In addition, haplotype analysis showed that resistin polymorphisms minor allele combination increased the risk of acne development whereas the presence of major allele combination of IL-6 polymorphisms reduced risk of acne development in Pakistani population. The lipid profile determination showed that levels of total cholesterol (TC) and triglycerides were significantly increased in patients than in controls, whereas high density lipoprotein cholesterol (HDL-C) was significantly decreased in patients than in respective controls. The apo-a levels did not show association with acne vulgaris. These results revealed importance of dietary lipids in acne pathogenesis. In addition, TNF-α and PF4 levels were significantly increased in acne patients with severe acne symptoms. The bovine microarray metaanalysis revealed that bovine mastitis causing bacteria suppress metabolic enzymes involved in milk production. Importantly, metaanalysis of microarray data from S. aureus- and S. aureus components- Skin: Acne vulgaris genetics and molecular responses to bacterial challenges challenged cells indicated that innate immune process genes were induced while adaptive immunity genes were suppressed in the S. aureus challenged cells. Conversely, S. aureus components induced adaptive immunity genes and suppressed innate immunity genes. In addition, this metaanalysis revealed different cytoprotective strategies adopted by S. aureus to evade host immune system mediated bactericidal activity. In this context, live S. aureus, inactivated S. aureus, and conditioned media from planktonic cultures of S. aureus induced cellular processes. However, S. aureus biofilms conditioned media induced anti-apoptotic genes as cytoprotective strategy. Importantly, the results for experiment of human skin challenged with different Gram positive bacterial strains revealed that P. acnes and S. aureus significantly induced cell cycle genes while suppressing keratinocytes differentiation. In addition, P. acnes and S. aureus significantly suppressed Golgi and endoplasmic reticulum (ER) specific bacterial components processing genes. S. epidermidis, a skin commensal, did not induce genes while it suppressed few membrane receptor genes. Interestingly, differentially expressed genes in Pam3CSK4-challenged cells were similar to those in P. acnes- and S. aureus-challenged cells, except that cell cycle genes were not induced and adaptive immunity genes were stimulated. This finding suggests that P. acnes and S. aureus induced skin cells proliferation genes through the receptors other than, or in addition to TLR1/2. The comparison of differential expression between P. acnes- and S. aureus- challenged cells showed that in contrast to the P. acnes, S. aureus significantly induced innate immunity system together with cell division genes. S. aureus induced innate immune processes and suppressed bacterial components processing genes more strongly than P. acnes. This finding may explain the pathogenic behavior of S. aureus. Finally, comparison of differential expression in P. acnes- vs. Pam3CSK4-challenged cells indicated that cell cycle and apoptosis genes were prominently induced by P. acnes while Pam3CSK4 induced innate immunity and wounding response genes similar to the changes in S. aureus- challenged cells. In conclusion, this study revealed importance of inflammatory cytokines and adipokines genetics, dietary factors and platelets in acne pathogenesis and proposed that resistin may be a key regulator of acne initiation. The role of resistin in acne vulgaris etiology needs to be further investigated to ascertain the current findings. Skin: Acne vulgaris genetics and molecular responses to bacterial challenges This study also explained the reason for reduced milk production in cows infected with mastitis-causing bacteria. The metaanalysis of S. aureus- and S. aureus components-challenged bacteria explained its pathogenic behavior and revealed bacterial strategies to induce disease while avoiding the host immune system. Furthermore, microarray analysis of skin treated with P. acnes and other Gram positive bacteria indicated different infectivity mechanisms of pathogenic, optimistic- pathogenic and commensal bacterial strains. This study described new insights in acne pathogenesis and skin-microbe interaction, and also proposed the questions for further research efforts to understand the interaction between a host and its microorganisms.