Human Microbiome Project Demonstration Study of Cutaneous Microbiome in Psoriasis

Psoriasis, a highly prevalent disease of humans of unknown cause, is a chronic inflammatory disorder primarily involving skin, with distinctive clinical characteristics. With the newly developed tools that facilitate microbiome research, it now is possible to assess whether the cutaneous microbiome plays a role in the pathogenesis of this disorder. Preliminary data from our studies suggest that the cutaneous microbiome in psoriasis is complex and possibly different from normal. To deal with this complexity, we propose to examine the cutaneous microbiome in relation to psoriasis with explorations at several taxonomic and informatic levels. Our overall objective is to examine how changes in the normal cutaneous microbiome contribute to the pathogenesis of psoriasis. Since causality is complex and often difficult to prove, and beyond the scope of this RFP, our overall hypothesis is that there are alterations in the cutaneous microbiome in areas of skin affected by psoriasis in comparison with the range observed in clinically unaffected areas, or in healthy persons. We also hypothesize that the characteristics of the microbiome may affect clinical responses to the immunomodulatory agents used to treat psoriasis. An alternative hypothesis is that effective treatment of psoriasis with systemic immunomodulatory agents will not substantially affect the disordered microbial ecosystem. Such observations would provide evidence for the roles of the microbiota in this disorder. Since an important consideration in microbiome research is the optimal level (e.g. phylum, genus, species, strain, gene) at which to examine a scientific question, and we are not yet certain what are the optimal levels for psoriasis, this also will be examined. Our studies of psoriasis should allow development of both approaches and tools that will have general utility for Microbiome research. To test our hypothesis, we propose the following specific aims: 1) To understand the cutaneous microbiome species composition overlaying psoriatic lesions; 2)To investigate differences in metagenome content for psoriatic lesions compared to normal skin; 3) To identify differences in the transcriptional profiles of the microbiome and the host between normal skin and psoriatic lesions using high-throughput sequencing; and 4) To estimate the effects of systemic immunomodulatory therapy for psoriasis on microbiome composition. In total, these studies should help us understand the role of the microbiome in psoriasis pathogenesis.

We sought to characterize and compare the cutaneous microbiota of psoriatic lesions (lesion), unaffected contralateral skin from psoriatic patients (normal), and similar skin loci in matched healthy controls (control) in order to discern patterns that govern skin colonization and their relationship to clinical diagnosis. Using high-throughput 16S rRNA sequencing, we assayed the cutaneous bacterial communities of 51 matched triplets and characterized these samples using community data analysis techniques.

• Study Design:
  • Case-Control
• Study Type:
  • Case-Control
• Total number of consented subjects: 93
• Subject Sample Telemetry Report (SSTR)

Between June 2008 and September 2011, we obtained consent (using the model consent forms for the HMP demonstration projects) and enrolled a total of 199 subjects (75 patients with psoriasis and 124 healthy controls) with ethical approval from New York University School of Medicine Institutional Review Board (IRB #08-709). Among the patients with psoriasis, 57 (76%) had not been exposed to antibiotics or received treatment relevant to psoriasis for at least one month before skin samples were obtained. Among the healthy controls, 112 (90.3%) had not been exposed to antibiotics or received treatments relevant to psoriasis for at least one month before skin samples were obtained. Psoriasis subjects receiving antibiotics less than one month before enrollment were excluded from further analysis, only six (11.8%) of the remaining subjects had taken any antibiotics in the preceding year. None of the matched control subjects had taken antibiotics in the 12 months prior to sampling. A total of 54 (72%) patients with psoriasis were studied by swabbing of the affected (Lesion) and unaffected (Normal) sites (see Specimen collection for details).

For these subjects, we sought control subjects of the same gender and ethnicity, and of similar age (+ 5 years), from whom a cutaneous specimen was obtained in a region proximate to the site of the psoriasis lesion. In total, we obtained matching specimens from 37 (29.8%) of the control subjects. One or more site from each of these controls were matched to the lesions in the 54 subjects with psoriasis. A control subject could be matched to more than one patient, since we also matched for cutaneous site. However, each control cutaneous site was uniquely mapped to only one triplet, thus there was no duplication of specimens in the analysis. The final analyses were performed on a set of 51 triplets, which had adequate depth of sequencing (>1000).

Inclusion Criteria: In order to be eligible for participation in this study, subjects must meet the following criteria:

• Eligible subjects will be people who have moderate to severe psoriasis and who are candidates for either UV phototherapy or systemic therapies for psoriasis.
• Male or female subjects 18 years of age, but not more than 75 years of age at the time of enrollment.
• Must be able to provide signed and dated informed consent.
• Healthy subjects willing and able to provide skin specimens.

Exclusion Criteria: Any subject who meets any of the following criteria will be excluded from participation in this study:

• Body Mass Index greater than or equal to 35 or less than or equal to 18.
• Vital signs outside of acceptable range at Screening Visit, i.e., blood pressure >140/90, oral temperature >100°F, pulse >100.
• Use of any of the following drugs within the last month:
• systemic antibiotics (intravenous, intramuscular, or oral);
• oral, intravenous, intramuscular, nasal or inhaled corticosteroids;
• cytokines; methotrexate or immunosuppressive cytotoxic agents;
• large doses of commercial probiotics consumed (greater than or equal to 108 cfu or organisms per day) - includes tablets, capsules, lozenges, chewing gum or powders in which probiotic is a primary component. Ordinary dietary components such as fermented beverages/milks, yogurts, foods do not apply.
• Use of topical antibiotics or topical steroids on the face, scalp, or neck or on arms, forearms, or hands within the previous 7 days.

Psoriasis: clinical features and course. The initial lesions of psoriasis often appear gradually. The natural history of psoriasis following initial symptoms includes unpredictable exacerbations and remissions, but no long-lasting freedom from disease (1, 2). After the initial presentation, the disease usually consolidates into a chronic condition that sometimes varies in intensity depending on season, worsening flares in colder, drier months, and the presence of concomitant infections. Specific drugs (lithium, beta-blockers, alcohol, and withdrawal of systemic corticosteroids) may cause exacerbations of disease (3). There are different types of psoriasis (2, 4). Chronic plaque psoriasis (psoriasis vulgaris [PV]), the most common form, is marked by relatively static, thick, erythematous plaques with overlying scale, that may bleed if removed (Auspitz sign). Although the plaques of psoriasis can appear anywhere on the skin, including trunk, extremities, genitals, and face, the most commonly affected areas are the elbows and knees, lower back, and scalp (5).

Other forms of psoriasis include pustular psoriasis, an acute generalized eruption of sterile pustules that may be accompanied by fever, lymphocytosis, hypocalcemia, and hypoalbuminemia. Pustular psoriasis may be limited to the hands and fingers or the palms and soles. This form also appears during pregnancy (referred to as "impetigo herpetiformis"). Guttate psoriasis is characterized by small psoriatic papules and plaques (0.5-1.5 cm) distributed diffusely over the trunk and proximal extremities. Streptococcal pharyngitis often precedes guttate psoriasis, which commonly occurs in children and adolescents. Erythrodermic psoriasis is characterized by large erythematous patches (involving > 2/3 of the body surface area) without much overlying scale. Both the plaque and pustular psoriasis variants may progress to this more severe form of the disease. Psoriasis may be associated with both nail disease (onychodystrophy) and arthritis. Psoriatic arthritis involves asymmetrical joints including the vertebral and sacroiliac joints. Psoriasis may be related to Reiter's syndrome, a post-infectious disease constellation characterized by urethritis, conjunctivitis, and arthritis. The frequency of inflammatory bowel disease is higher in patients with psoriasis, and many patients with psoriatic arthritis have asymptomatic bowel inflammation. Hypertension, obesity, and diabetes mellitus all are more common in psoriatic patients (6).

The lesions of psoriasis display several primary features: (i) keratinocyte hyperplasia, characterized by thickening of the epidermis (acanthosis) and downward expansion of the rete pegs, and associated with multiple overlying layers of scale (parakeratosis), and loss of the highest (granular) layer of the epidermis; (ii) inflammation, characterized by infiltrating neutrophils within the epidermis and in the scaly layer, and lymphocytes, monocytes, and dendritic cells (DC) in the dermis and epidermis; and (iii) ectatic blood vessels, scattered in the upper layers of the dermis (creating telangiectasias within the papillary tips).

Summary of the pathophysiology of psoriasis. Psoriasis is characterized by immune dysregulation within the skin, with the appearance of cutaneous autoreactive T cells (Th1) producing IFN-ϒ and TNF-β (2). Induction of an inflammatory milieu accelerates keratinocyte proliferation, leading to the development of epidermal hyperplasia (hyperkeratosis) and angiogenic tissue reactions, progressing to the increased skin vascularization and inflammatory cellular infiltrates that are the hallmarks of the psoriatic lesion. Normal skin contains both T lymphocytes and dendritic cells (7, 8) indicating a potential for triggering of recall immune responses to environmental agents. The lesions of psoriasis vulgaris contain large populations of leukocytes in addition to T cells and DCs, suggesting a function similar to organized lymphoid tissue that perpetuates immune infiltrates in psoriatic plagues. There is no good animal model for psoriasis; mouse and human skin are substantially different. Although some of the features of psoriasis (e.g. keritinocyte hyperplasia) can be obtained in transgenic or knock-out mice, the phenotypes include features of other dermatoses and neonatal lethality, which are not normally associated with human psoriasis (2). Thus, humans are the best experimental subjects in whom to study psoriasis. The availability of effective, systemic immunomodulatory agents in recent years has added resources for understanding pathogenesis (2, 3, 9, 10).

Th1 T cell-inducing cytokines from dendritic cells (DC) drive the Th1 response observed in psoriasis, and thus are prime targets for disease control with anti-cytokine reagents (11). IL-12 and IL-23 are related molecules sharing a common p40 chain. Whereas IL-12 induces IFN-ϒ and TNF-β, IL-23 induces IL-17 (12), TNF-β, and IL-6; both play important roles in the psoriatic inflammatory response. IL-12/IL-23 production by cutaneous DC, and thus to Th1-driven inflammation, may be due to Toll-like receptor (TLR) stimulation in the psoriatic lesions (via NF-kB stimulation). The role of TLRs in recognizing microbes implies a possible pathway for microbial involvement, at least in the final steps of psoriatic inflammation. The formation of a psoriatic plaque requires the continued presence of activated T cells specific for an unknown inciting antigen, and persistent T cell expression of plaque-inducing cytokines or cell-surface factors that form a "cytokine network." Thus, by the release of specific cytokines that act as growth factors or by cell-to-cell interactions, activated intra-dermal T cells trigger keratinocyte hyperproliferation. Subsequently, cytokines produced by stimulated nonlymphoid cells in the skin, such as keratinocyte-derived IL-8, chemotactic for neutrophils, and vascular endothelial growth factor (VEGF), inducing the proliferation of the dermal vasculature, contribute to the inflammatory process. The triggers that initiate the inflammatory pathways are unknown, however, immunomodulatory agents are effective therapies (3, 9, 13).

Small subunit ribosomal RNA diversity. Investigation of ssrRNA gene sequences has revolutionized the study of microbial ecology, leading to fundamental new insights into the diversity present in the prokaryotic world. Further, it has underscored the major difference between the numbers of microbes cultured in the laboratory versus those present in the environment; thus, it has become a well-accepted method for interrogating diversity in environmental samples (14, 15). Diversity measurements using ssrRNA analyses have become an important complement to whole community or metagenomic sequencing. New approaches are constantly being developed to address diversity questions with respect to microbial communities, by focusing on the regions of the ribosomal operons that have the highest information content (see for example 16-19).

Molecular analysis of the human microbiome has great potential (20-23). It has focused on fecal samples from the human GI tract (24) at the J. Craig Venter Institute (JCVI), and other centers (25-28), and in relation to disease (29, 30). Such studies have highlighted the extent of diversity associated with the human body. Other studies of the human Microbiome have focused on the oral cavity (31), upper gastrointestinal tract (32, 33), vagina (34-36), and on the skin (29, 37-41). Previous studies using ssrRNA approaches to study the diversity of the skin microbiome indicate that a community of microorganisms is consistently associated with the skin and is dominated by a median level of ~30-50 operational taxonomic units (OTUs) from the bacterial domain in individual hosts at any time in a single site (37). In total, based on previous studies, >400 OTUs are predicted for superficial skin. The metabolic potential of these microbes has not been well-studied despite their fundamental importance to health and disease states of skin. Molecular studies of the fungal biota of the skin are in their infancy (99-41), but also are based on SSU sequences, especially the ITS1 and ITS2 loci (42-44).

Psoriasis is a chronic inflammatory disease of unknown cause affecting 3% of the U.S. population. Its cause is unknown but there is evidence for extensive immune dysregulation. The role of the cutaneous microbiota in psoriasis has not been extensively examined which is surprising based on the evidence for inflammatory changes in tissues. Important questions that can be addressed by the proposed work include: Addressing such questions should enhance our knowledge about the diagnosis, prevention, natural history, and treatment of psoriasis.

Are there organisms whose presence or absence is a marker for psoriasis?
Could a host's status with respect to an organism reflect propensity to develop psoriasis?
Could specific organisms or their metabolic pathways correlate with psoriasis risk, and possibly explain the altered immunity?
Since immunomodulatory therapy is not always successful, could the nature of the microbiota be a predictor for treatment success or failure?
Will treatment of psoriasis with systemic therapy clear any abnormal microbiota, or do the abnormalities persist, suggesting a primary microbiological role in the disease?

• Primary Phenotype: Psoriasis

• BioProject
• PubMed
• Sequence Read Archive
• BioSample
• MeSH
• PMC

• Principal Investigator
  • Martin J. Blaser, MD. New York University Langone Medical Center, New York, NY, USA.
• Co-Investigators
  • Alexander V. Alekseyenko, PhD. New York University Center for Health Informatics and Bioinformatics, New York, NY, USA.
  • Bruce Strober, MD, PhD. New York University Langone Medical Center, New York, NY, USA.
  • Guillermo Perez-Perez, DSc. New York University Langone Medical Center, New York, NY, USA.
  • Aieska De Souza, MD. New York University Langone Medical Center, New York, NY, USA.
  • Stuart Brown, PhD. New York University Langone Medical Center, New York, NY, USA.
  • Barbara A. Methé, PhD. J Craig Venter Institute, Rockville, MD, USA.
• Research Assistants
  • Elizabeth Camacho. New York University Langone Medical Center, New York, NY, USA.
  • Trisha Ali-Shaw. New York University Langone Medical Center, New York, NY, USA.
  • Ari Blumberg. New York University Langone Medical Center, New York, NY, USA.
• Researcher Associate
  • Zhan Gao, MD. New York University Langone Medical Center, New York, NY, USA.
• Funding Sources
  • UH2AR057506-01S1 (Human Microbiome Project of the NIH Common Fund). National Institutes of Health, Bethesda, MD, USA.
  • UL1 TR000038. National Center for the Advancement of Translational Science (NCATS), National Institutes of Health, Bethesda, MD, USA.