The effects of CsA and Tac on cytokine production from memory CD4⁺ T cells.

<p>Memory CD4⁺ T cells were stimulated with PMA and ionomycin in the absence or presence of CsA or Tac. Fluorescence profiles showed distinct cytokine productions from mature cells, and the production of all cytokines investigated here was strikingly suppressed with the addition of CsA or Tac, even at the lower concentration. Representative figures of ten independent experiments are shown (A). The percentages of IFN-γ⁺CD4⁺ T cells (B), IL-4⁺CD4⁺ T cells (C) or IL-17⁺CD4⁺ T cells (D) within the CD4⁺ T cell population were significantly suppressed with the addition of CsA or Tac. Data are expressed as the mean ± SEM.</p

The effects of CsA and Tac on the differentiation of naïve CD4⁺ T cells into cytokine-producing mature cells (Th1/Th2/Th17).

<p>Flow cytometric analysis showed abundant cytokine production from CD45RA⁻CD45RO⁺CD4⁺ T cells. Representative figures of ten independent experiments are shown (A). Addition of CsA or Tac lowered the percentage of CD45RA⁻CD45RO⁺IFN-γ⁺ cells significantly compared with the control group (B). The percentages of CD45RA⁻CD45RO⁺IL-4⁺ cells and CD45RA⁻CD45RO⁺IL-17⁺ cells within the CD4⁺ T cell population were also decreased compared with the control group (C,D). Data are expressed as the mean ± SEM.</p

Changes in cytokines mRNA expression levels in the ears of AD mice by vaccination of rhPIV2/Ag85B.

<p>Cytokines: IL-4 (panel A), IFN-γ (panel B), IL-10 (panel C), TGF-β (panel D), TNF-α (panel E), MIP2-α (panel F), IL-2 (panel G), IL-17 (panel H), mRNA expression in the ear lesions measured with Quantitative RT-PCR. Expressions of IL-4, TNF-α and MIP2-α mRNA were significantly decreased in the ear skin treated with intra-nasally rhPIV2/Ag85B treated group compared to those of control groups. Meanwhile, the expression levels of mRNA of IFN-γ, IL-10, TGF-β and IL-2 were significantly elevated in rhPIV2/Ag85B intra-nasally treated group compared to those of control groups. *P<0.05, **P<0.01, ***P<0.001.</p

Expression of EGFP from rhPIV2/EGFP.

<p><b>A.</b> HaCat cells were infected with rhPIV2/EGFP at an MOI of 0.5. Three days after, EGFP was clearly visualized using a fluorescence microscopy (x100). <b>B.</b> The rhPIV2/EGFP (5×10⁶ TCID₅₀) were administered to a wild type BALB/c mice intranasally EGFP was visualized clearly in the airway epithelial cells 4 days after administration (x200, upper right box, x400).</p

Anti-inflammatory effects of vaccination with rhPIV2/Ag85B.

<p><b>A.</b> Clinical manifestation of the ear skin at 6 hours after OX challenge on day 21. The control groups (PBS, PIV2 ear, and PIV2 nasal on the panel) showed severe edema with erythema, however the intranasal and/or subcutaneous administration of the rhPIV2/Ag85B (Ag nasal and Ag ear on the panel, respectively) clearly reduced skin reactions in OX-sensitized mice. <b>B.</b> Ear thickness measured before and 6 hours after each OX application on day 21. The ear swelling was suppressed significantly in rhPIV2/Ag85B treated groups in two ways compared to those in the placebo treated groups. (*P<0.05, **P<0.01.) <b>C.</b> Histopathological changes of the ear skin obtained on day 21 in paraffin embedded sections stained with hematoxylin and eosin. The placebo treated groups (PBS, PIV2 ear and PIV2 nasal on the panel) revealed marked inflammatory reactions with acanthosis and ulceration in epidermis, and marked edema with cellular infiltration including mononuclear cells and neutrophils in the dermis. The skin infiltration of inflammatory cells and epidermal thickness were decreased in rhPIV2/Ag85B treated group (Ag85B ear and Ag85B nasal on the panel). Original magnification × 100. <b>D.</b> Plasma IgE levels on day 21. Plasma IgE level was decreased in rhPIV2/Ag85B treated groups (Ag ear and Ag nasal). *P<0.05, **P<0.01.</p

Immunostaining for Tregs in the inflamed ear skin lesions.

<p><b>A.</b> CD4⁺ T cells are displayed with green fluorescence (1), and Foxp3⁺ T cells are with red (2). Merged yellow color means Foxp3⁺CD4⁺ T cells (3) (x100). <b>B.</b> The number of skin infiltrating CD4⁺ T cells is less in Ag nasal group and Ag ear group compared to that of PBS-treated group. Although it does not reach statistical significance, CD4⁺ T cell number is less in Ag nasal group compared to that of Ag ear group. <b>C.</b> The number of Foxp3⁺CD4⁺ T cells in the inflamed ear skin is significantly increased in both of the intra-nasal and ear-subcutaneous rhPIV2/Ag85B application groups.</p

IL-1α and IL-1β trigger fat tissue remodeling.

<p><b>A</b>) KCASP1Tg mice were treated once-a-week with an intra-peritoneal injection of 10 µg anti-IL-1α and/or IL-1β neutralizing antibodies between 4 to 24 weeks of age. PBS-treated KCASP1Tg littermates were used as controls. The body weight loss was ameliorated by either anti-IL-1α, anti-IL-1β or anti-IL-1α/β administration, (n = 7, each group). <b>B</b>) Emaciation was reproduced by administering 1 µg of recombinant IL-1α or IL-1β protein 3 times per week from 6 to 16 weeks of age to wild type mice compared to PBS-injected mouse controls from 6 to 16 weeks (n = 6, each group). <b>C</b>) H&E staining of abdominal adipose tissue revealed that the adipocytes were large and plump in shape in normal control and 6-months old KIL-18Tg(−) mice; they were small and round, however, in 6-month-old KCASP1Tg and 18-months old KIL-18Tg(+) mice. The number of infiltrating mononuclear cells was similar among these groups (n = 7, each group). <b>D</b>) Cytokine levels in the skin culture supernatant were measured by flow cytometry. IL-1α and IL-1β were detected in conditioned medium from the skin culture of normal control mice and KIL-18Tg(−) mice, but was significantly higher in medium from skin culture of KCASP1Tg mice (n = 7, each group). <b>E</b>) Mouse adipose cells cultured in regular medium contained abundant lipid drops on day 14. The addition of supernatant from normal skin culture revealed a decrease in the number of plump adipocytes containing lipids as stained with oil red O, which was reversed by supplementing with supernatant from KCASP1Tg mice skin culture. The pretreatment of KCASP1Tg mice skin culture medium with anti-IL-1α or anti-IL-1β neutralizing antibodies partially ameliorated the inhibitory effects on adipose cells, which were almost abrogated by simultaneous treatment with both antibodies (n = 7, each group).</p

KCASP1Tg and KIL-18Tg(+) mice showed emaciation and altered lipid metabolism in addition to dermatitis.

<p><b>A</b>) KCASP1Tg mice had erosive dermatitis at week 8, which spread across the entire face and trunk when mice were 5-months old. KIL-18Tg mice showed no dermatitis at 6 months of age, KIL-18Tg(−). Weight loss began at 10 weeks in KCASP1Tg mice, but not in age matched KIL-18Tg mice (n = 10, each group). The left Y-axis shows body weight and the right Y-axis shows the percentage of dermatitis. KIL-18Tg mice developed dermatitis at 1-year old, followed by weight loss, KIL-18Tg(+) (n = 7, each group) (*p<0.05, **p<0.001, ***p<0.0001). <b>B</b>) CT scan of KCASP1Tg mice at 6 months of age revealed a dramatic decrease in visceral fat as shown in yellow compared to normal control or KIL-18Tg(−) mice. Eighteen-year old KIL-18Tg(+) showed decreased visceral fat. Subcutaneous fat (in orange color) was also decreased in KCASP1Tg and KIL-18Tg(+). <b>C</b>) A comparison of the somatic fat ratio across the three groups determined by CT scan at 2, 4 and 6 months of age, and a decrease was observed in KCASP1Tg mice compared to the other two groups (n = 6, each group). <b>D</b>) Six-month-old KCASP1Tg mice showed decreased plasma HDL cholesterol and leptin levels, as well as increased triglyceride levels. LDL cholesterol and adiponectin levels remained normal. Eighteen-months old KIL-18Tg(+) mice showed decreased leptin levels. No significant change was identified in the triglyceride, HDL and LDL cholesterol, and adiponectin levels in KIL-18Tg(+) mice. <b>E</b>) Plasma IL-1α and β levels were elevated in 6-months old KCASP1Tg mice. IL-1 levels were under the detection limit in KIL-18Tg(−) mice, but were elevated in 18-months old KIL-18Tg(+) mice. Plasma IL-18 levels were increased in both KCASP1Tg and KIL-18Tg mice (n = 10).</p

KCASP1Tg and KIL-18Tg(+) mice developed amyloidosis in the liver, kidney and spleen.

<p><b>A</b>) Histological analyses showed loss of normal architecture: hepatocytes were replaced by dense deposits in the liver, the glomeruli and renal tubules were damaged in the kidney, and lymph-follicles were absent in the spleen. Dense amyloid deposition was detected in KCASP1Tg and KIL-18Tg(+) mice by Congo-red staining. <b>B</b>) KCASP1Tg mice showed mild liver and kidney dysfunction while renal function had significantly deteriorated in KIL-18Tg(+) mice (n = at least 7).</p

High serum amyloid A protein and organomegaly in KCASP1Tg and KIL-18Tg(+) mice.

<p><b>A</b>) Serum amyloid A protein (SAA) levels were significantly higher in KCASP1Tg mice than in normal control and 6-months old KIL-18Tg(−) mice. Eighteen-months old KIL-18Tg(+) mice also showed elevated SAA concentration (n = at least 7). <b>B</b>) <b>C</b>) The liver, kidney, and spleen of both KCASP1Tg and KIL-18Tg(+) mice were significantly enlarged compared to control and KIL-18Tg(−) mice.</p