Psoriasis is an immune-mediated skin disease affecting approximately 2% of the population;1 its pathogenesis is characterized by a complex interplay of cells and cytokines.2 Recently, attention has focused on two main subpopulations of dendritic cells (DC), myeloid DC (mDC) and plasmacytoid DC (pDC), which represent a critical link between the innate and the adaptive immune systems.3
In the inflamed psoriatic dermis, there is an increase in mDC,4 probably derived from circulating DC precursors which migrate into the skin in response to chemotactic signals and synthesize high levels of pro-inflammatory cytokines (e.g. interleukin [IL]-12 and IL-23).5 pDC have been identified in the lesional and in the non-lesional psoriatic skin. Activated pDC produce interferon (IFN)-α in the early phase of the development of a psoriasis lesion with a strong amplification of a pathogenetic role of T lymphocytes.6 The antimicrobial peptide LL37 is expressed in high concentrations in psoriatic skin and through its binding with self-DNA is a potent activator of pDC.7
In a previous work, we have reported a reduction of both mDC and pDC in psoriatic lesions upon treatment with biologic therapies,8 but to date no studies have compared blood DC before and upon biologic treatment. Our purpose was to monitor the blood DC compartment in psoriatic patients before and during biologic therapy with infliximab (an anti-tumor necrosis factor-α molecule). Our analysis could better focus the activity of this drug on the number and phenotype of DC.
Eight patients (four male and four female; aged 44–80 years, mean 46.25 years) affected by severe psoriasis (Psoriasis Area and Severity Index [PASI] score >10) treated at the PsoCare center of the Dermatological Unit of Florence University, were evaluated before and after 6 months of infliximab therapy (Shering-Plough, Levallois-Perret, France), given at a dose of 3 mg/kg i.v. then every 8 weeks. In addition, four healthy controls (matched with patients for sex and age) were included in the study.
The study was approved by the local ethics committee, and all patients gave informed consent.
Peripheral blood mononuclear cells were isolated from 3 mL of peripheral blood by density gradient centrifugation with Lymphoprep (Axis-Shield, Oslo, Norway) and washed in phosphate-buffered saline (PBS; EuroClone, Whetherby, UK). Multiple cell staining was performed with anti-BDCA-1-FITC conjugated (AD5-8E7, IgG2a) and BDCA-2-PE (AC144, IgG1) from Miltenyi Biotec (Bergisch, Gladbach, Germany), anti-HLA-DR-Pe/Cy7 (L243, IgG2a) from BioLegend (San Diego, CA, USA) and CD86-Pe/Cy5 (2331 FUN-1, IG1) from BD-Pharmingen (San Diego, CA, USA) for 30 min at 4°C. All monoclonal antibodies dilutions and wash steps were done in PBS (EuroClone) containing 2% FCS (EuroClone) and 2 mm/L ethylenediamine tetraacetic acid (Sigma, Milan, Italy). Isotype-matched antibodies were used as negative controls. Cells were acquired using a FACSCanto flow cytometer (Becton-Dickinson, San Jose, CA, USA). The mDC and pDC were identified by the concomitant HLA-DR expression and mutually exclusive membrane expression of BDCA-1 or BDCA-2, respectively. The expression of CD86 required for T-cell activation was also analyzed.
Statistical analysis was performed using Microsoft Excel software. The mean ± standard deviation of the percentage of DC and their subsets were calculated. Student’s t-test was used to compare the DC populations identified. Correlations were considered significant at P < 0.05.
We detected both mDC and pDC in peripheral blood of untreated psoriatic patients and controls. The proportions of DC subsets are given in Table 1. Interestingly, psoriatic peripheral blood was characterized by a decreased number of both mDC and pDC subsets: mDC constituted 3.2 ± 0.3% of total peripheral blood mononuclear cells in untreated psoriatic patients versus 6.8 ± 4.5% in the healthy control group (P < 0.05) and pDC constituted 1.3 ± 0.4% in untreated psoriatic patients versus 2.3 ± 41.1% in the healthy control group. We concluded that untreated psoriatic patients are characterized by a quantitative deficit in their peripheral circulating DC. MDC and pDC subsets were also evaluated after infliximab therapy. Responders (patients who had 75% improvement or more in PASI after 6 months of therapy) showed a substantial increase in their number of circulating DC (Table 1). Specifically, mDC frequency increased from 3.2% ± 0.3% to 7.8% ± 1.8% (P < 0.05), whereas there was a blood pDC level increase, but was not statistically significant (from 1.3% ± 0.4% to 3.8% ± 2.7%, P = 0.16). Next, we set out to determine if numerical alterations of blood DC were associated also with phenotypic or functional abnormalities. Because the expression of co-stimulatory molecules on the surface of DC has been correlated to their immunostimulatory capacity, we evaluated the expression of CD86 in these patients, before and during infliximab therapy and we compared it to the controls. The expression of this antigen is shown in Table 1. Patients before and after treatment did not show a significant variation in the expression of CD86.
|Average % (mean ± SD)||Controls (n = 4)||Untreated patients (n = 8)||Treated patients (n = 8)||P-value|
|mDC||6.8 ± 4.5||3.2 ± 0.3||7.8 ± 1.8||0.043*||0.021**|
|CD86+||84.1 ± 14||78.2 ± 0.2||96.6 ± 1.2|
|pDC||2.3 ± 1.1||1.3 ± 0.4||3.8 ± 2.7||0.181*||0.161**|
|CD86+||70.3 ± 16.3||77.2 ± 0.3||88.9 ± 13.9|
Our preliminary results, although in a limited number of patients, suggest that the increased recruitment of DC into cutaneous plaques might be responsible for decreased peripheral DC and their relative lower percentage in untreated patients. If this is the case, it seemed likely that effective therapy might block this migration and increase the blood DC level. According to our results there is an alteration in circulating DC which could correlate with the persistence of the disease and the response to biologic therapy.