Membrane-Permeable Calpain Inhibitors Promote Rat Oral Mucosal Epithelial Cell Proliferation by Inhibiting IL-1α Signaling

<div><p>To standardise regenerative medicine using cultured cells, the use of serum-free, chemically defined media will be necessary. We have reported that IL-1α inhibits the growth of epithelial cells in culture and that recombinant IL-1 receptor antagonist (IL-1RA) significantly promotes epithelial cell growth in no feeder layer condition. In this study, we examined inhibitors of calpain, a cysteine proteinase that plays crucial roles in various cellular functions, including IL-1α maturation and secretion. The culturing of epithelial cells in serum-free media supplemented with a membrane-permeable calpain inhibitor significantly promoted growth while suppressing IL-1α maturation and secretion. By contrast, non-membrane-permeable calpain inhibitor treatment did not have these effects. Interestingly, immunoblotting analysis revealed that immature, untruncated, IL-1α expression was also downregulated by cell-permeable calpain inhibitor treatment, and the difference in IL-1α gene expression increased from day 2 to day 6. Although IL-1RA has been reported to promote epithelial cell growth, we detected no synergistic promotion of epithelial cell growth using a calpain inhibitor and IL-1RA. These findings indicate that calpain inhibitors promote epithelial cell proliferation by inhibiting IL-1α maturation at an early phase of epithelial cell culture and by suppressing the positive feedback-mediated amplification of IL-1α signalling.</p></div

Carbohydrate-binding modules influence substrate specificity of an endoglucanase from <i>Clostridium thermocellum</i>

<p>Most cellulases contain carbohydrate-binding modules (CBMs) that largely contribute to their activity for insoluble substrates. <i>Clostridium thermocellum</i> Cel5E is an endoglucanase having xylanolytic activity. The Cel5E originally has a family 11 CBM preferentially binding to β-1,4- and β-1,3-1,4-mixed linkage glucans. In this study, we replaced the CBM with a different type of CBM, either a family 3 microcrystalline cellulose-directed CBM from <i>Clostridium josui</i> scaffoldin, or a family 6 xylan-directed CBM from <i>Clostridium stercorarium</i> xylanase 11A. Chimeric endoglucanases showed enhanced activity that was affected by CBM binding specificity. These chimeric enzymes could efficiently degrade milled lignocellulosic materials, such as corn hulls, because of heterologous components in the plant cell wall, indicating that diverse CBMs play roles in degradation of lignocellulosic materials.</p> <p>Substrate specificities of an endoglucanase, Cel5E from <i>Clostridium thermocellum</i> were affected by fusion of different type CBMs.</p

Scheme of calpain and IL-1α signalling in regulating epithelial proliferation by cell permeable calpain inhibitors.

<p>Scheme of calpain and IL-1α signalling in regulating epithelial proliferation by cell permeable calpain inhibitors.</p

Time-course of gene expression in cultured oral mucosal epithelial cells.

<p>The gene expression levels of IL-1α, TNF-α, IL-1RA, calpain I, calpain II, IL-1 receptor 1 (IL-1r1), IL-1 receptor 2 (IL1r2), p63, and Bmi1 were quantified via qRT-PCR. Primary cultures of rat oral mucosal epithelial cells were harvested on day 2, 4, 6, 8, or 10 (<i>n</i> = 4). β-2-microglobulin gene expression was used as an internal control. The error bars indicate the SD.</p

Expression levels of cellular IL-1α and calpains.

<p>The expression levels of the 33-kDa pro-IL-1α, the 17-kDa mature IL-1α, calpain I, and calpain II were detected in cultured rat oral mucosal epithelial cells via immunoblotting. GAPDH expression was used as an internal control. The cultured epithelial cells were harvested on day 8.</p

IL-1α levels in the supernatant of epithelial cell cultures.

<p>IL-1α was measured in supernatants of rat oral mucosal epithelial cell cultures via ELISA. The amount of supernatant assessed on day 8 or 9 was adjusted based on the number of cells for each condition. Calpeptin, <i>n</i> = 3; calpain inhibitor III, <i>n</i> = 3; calpastatin, <i>n</i> = 2; and B27-WT, <i>n</i> = 2. White blocks, controls; blue blocks, inhibitors. The error bars indicate the SD. * <i>P</i> < 0.05 using Student's <i>t</i>-test.</p

The proliferation and attachment of oral mucosal epithelial cells in the presence of calpain inhibitors.

<p>(A) Phase contrast microscopic images of cultured rat oral mucosal epithelial cells. The bars indicate 200 μm. (B) The cells were collected and counted at 8–11 days after seeding (calpeptin, <i>n</i> = 4; calpain inhibitor III, <i>n</i> = 3; calpastatin, <i>n</i> = 3; B27-WT, <i>n</i> = 3; and cystatin C, <i>n</i> = 2). White blocks, controls; blue blocks, inhibitors. * <i>P</i> < 0.05 using Student's <i>t</i>-test. (C) The number of initially attached epithelial cells was determined 48 h after seeding (<i>n</i> = 4). The error bars indicate the SD. * <i>P</i> < 0.05 using Student's <i>t</i>-test.</p

Treatment of cell cultures with a calpain inhibitor (A) and/or IL-1RA (B) and/or IL-1α.

<p>The reagents were applied upon seeding the cells, and the cells were collected and counted on (A) day 7 or 11 (<i>n</i> = 3) (B) day10 (n = 3). The error bars indicate the SD. (A) No significant differences were detected between any of the reagent-treated cultures. (B) * <i>P</i> < 0.05 using Student's <i>t</i>-test. N.S. indicates non-significant difference.</p

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

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