Ustekinumab Improves Psoriasis without Altering T Cell Cytokine Production, Differentiation, and T Cell Receptor Repertoire Diversity

<div><p>Ustekinumab is a fully human IgG1κ monoclonal antibody targeting interleukin (IL)-12/23 p40 subunit. The role of IL-12/23-mediated pathway in the mechanism of various inflammatory disorders especially psoriasis has been well recognized. Recently the long-term efficacy and safety of ustekinumab in patients with moderate-to-severe psoriasis has been evaluated in phase 2/3 clinical trials, and the results showed no significant risk for serious adverse effects, infections, or malignancies. Ustekinumab inhibits the function of the IL-12/23 p40 subunit, and therefore it is believed that inhibition of IL-12 p40 pathway decreases IFN-γ production. The major concern for the use of ustekinumab is the possibility of increased immunosuppression due to low IFN-γ production. However, the effects of ustekinumab on CD4⁺ T cell function have not been fully investigated so far. In this study, we explored changes in cytokine production by memory CD4⁺ T cells as well as in the differentiation of naïve T cells to helper T cell (Th) 1, Th2, or Th17 cells in psoriasis patients treated with ustekinumab. The effect of the treatment on T cell receptor repertoire diversity was also evaluated. The results showed that ustekinumab improves clinical manifestation in patients with psoriasis without affecting cytokine production in memory T cells, T cell maturation, or T cell receptor repertoire diversity. Although the number of patients is limited, the present study suggests that T cell immune response remains unaffected in psoriasis patients treated with ustekinumab.</p> </div

Differential Gene Expression in Thrombomodulin (TM; CD141)⁺ and TM⁻ Dendritic Cell Subsets

<div><p>Previously we have shown in a mouse model of bronchial asthma that thrombomodulin can convert immunogenic conventional dendritic cells into tolerogenic dendritic cells while inducing its own expression on their cell surface. Thrombomodulin⁺ dendritic cells are tolerogenic while thrombomodulin⁻ dendritic cells are pro-inflammatory and immunogenic. Here we hypothesized that thrombomodulin treatment of dendritic cells would modulate inflammatory gene expression. Murine bone marrow-derived dendritic cells were treated with soluble thrombomodulin and expression of surface markers was determined. Treatment with thrombomodulin reduces the expression of maturation markers and increases the expression of TM on the DC surface. Thrombomodulin treated and control dendritic cells were sorted into thrombomodulin⁺ and thrombomodulin⁻ dendritic cells before their mRNA was analyzed by microarray. mRNAs encoding pro-inflammatory genes and dendritic cells maturation markers were reduced while expression of cell cycle genes were increased in thrombomodulin-treated and thrombomodulin⁺ dendritic cells compared to control dendritic cells and thrombomodulin⁻ dendritic cells. Thrombomodulin-treated and thrombomodulin⁺ dendritic cells had higher expression of 15-lipoxygenase suggesting increased synthesis of lipoxins. Thrombomodulin⁺ dendritic cells produced more lipoxins than thrombomodulin⁻ dendritic cells, as measured by ELISA, confirming that this pathway was upregulated. There was more phosphorylation of several cell cycle kinases in thrombomodulin⁺ dendritic cells while phosphorylation of kinases involved with pro-inflammatory cytokine signaling was reduced. Cultures of thrombomodulin⁺ dendritic cells contained more cells actively dividing than those of thrombomodulin⁻ dendritic cells. Production of IL-10 is increased in thrombomodulin⁺ dendritic cells. Antagonism of IL-10 with a neutralizing antibody inhibited the effects of thrombomodulin treatment of dendritic cells suggesting a mechanistic role for IL-10. The surface of thrombomodulin⁺ dendritic cells supported activation of protein C and procarboxypeptidase B2 in a thrombomodulin-dependent manner. Thus thrombomodulin treatment increases the number of thrombomodulin⁺ dendritic cells, which have significantly altered gene expression compared to thrombomodulin⁻ dendritic cells in key immune function pathways.</p></div

Differentiation of naïve CD4⁺ T cells to cytokine-producing mature cells (Th1/Th2/Th17).

<p>Representative flow cytometry data are shown. (A) The percentage of CD45RA⁻CD45RO⁺IFN-γ⁺ cells in the CD4⁺ T cell population (B) The percentage of CD45RA⁻CD45RO⁺IL-4⁺ cells in the CD4⁺ T cell population (C) The percentage of CD45RA⁻CD45RO⁺IL-17⁺ cells in the CD4⁺ T cell population. T cell maturation was not influenced by ustekinumab treatment.</p

Background of five patients and five healthy controls.

<p>The PASI score of the patients was high before ustekinumab therapy, and improved dramatically after the treatment. However, the PASI score of case 5 was increased at one month after the third therapy. WBC counts and the ratio of lymphocytes in all patients and controls were preserved during all the course of the study.</p

Gene expression changes in DCs following sTM treatment or sorting into TM⁺ or TM⁻ DCs.

<p>(A) The mean (black) and 95th percentile (red) of PWF values for each probe rank from 500 permutations of the sample-labels as described in Materials and Methods show that the ‘sorted’ term PWF values (blue) were extraordinarily large compared to the simulations. For the ‘treatment’ term, the PWF values of fewer than 1000 probes were well above the simulated values, and for the ‘sorted:treatment’ interaction term, few probes showed a significantly higher PWF than by permuted sample-label computations. (B) PWF statistics permitted identification of genes exhibiting the strongest association with sorting into TM⁺ and TM⁻ DC, for example <i>Alox15</i>. Similarly, genes exhibiting strong association with TM treatment included <i>Copa</i>. <i>Serpinb8</i> exhibited a large PWF value for the sorted:treatment interaction, as seen by the contrasting effect of treatment on gene expression in TM⁻ and TM⁺ DCs. <i>Fdft1</i> gave large values of PWF for both treatment and sorted:treatment interaction, and shows both down-regulation with TM treatment and notably higher expression in TM⁺ DCs, specifically in untreated cells. (C) Correlation between gene expression changes determined by microarray to those determined by qPCR. A panel of genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072392#pone-0072392-t001" target="_blank">Table 1</a>) whose expression was changed when analyzed by microarray, their fold change was also determined by qPCR. The qPCR data was normalized to GAPDH resulting in this equation for the best fit line: log y = (0.99 *log x) +1.16 with R² = 0.79.</p

T cell receptor repertoire diversity.

<p>TCR BV subfamilies were preserved during treatment with ustekinumab as compared with normal volunteer.</p

Naturally occurring regulatory T cells.

<p>The percentage of nTreg (FoxP3⁺CD127^lowCD25^highCD4⁺ T cells/CD4⁺ T cells) was similar among the seven volunteers. Flow cytometry data from four patients and three healthy controls are shown.</p

List of miRNAs modulated in TM⁺ DCs compared to TM⁻ DCs.

<p>List of miRs whose expression is altered, identified from microarray analysis by comparison of levels in TM⁺ and TM⁻ DCs which change >2 fold with p<0.0001.</p

Cytokine production by memory CD4⁺ T cells and differentiation of naïve CD4⁺ T cells to mature cytokine-producing T cells in psoriasis patients during treatment with ustekinumab and in healthy controls.

<p>(A) Memory CD4⁺ T cells were stimulated with PMA and ionomycin. Distinct cytokine production by mature cells was observed, and the production of all cytokines was not suppressed in patients with psoriasis treated with ustekinumab. (B) Naïve CD4⁺ T cells were differentiated to mature T cells (Th1/Th2/Th17). Ustekinumab treatment did not change the percentage of cytokine-producing mature T cells compared to the control group.</p

Characterization of TM⁺ DCs.

<p>Mouse bone marrow cells were differentiated in GM-CSF for 6 days in the presence of 200 nM sTM from day 4 to 6. DCs were then analyzed for the expression of cell surface markers by flow cytometry cells gated on CD11c⁺ and TM⁺ for the expression of other DC markers. Isotype control is shown in gray.</p