NK-4 exerts selective regulatory effects on the activation and function of allergy-related Th2 cells

<div><p>NK-4 is the main component of the antiallergic drug Lumin, which has been in popular usage since the early 1950s. In this study, we examined whether NK-4 exerts a regulatory effect on the activation and effector function of Th2 cells. NK-4 inhibited IL-4 production by anti-CD3ε mAb-stimulated BALB/c mouse spleen cells, whereas NK-4 had little effect on IFN-γ production. IL-4 and IL-5 secretion by anti-CD3ε mAb- or antigen-stimulated Th2 cells (D10.G4.1) was abrogated by NK-4 without affecting cell numbers, whereas IFN-γ secretion by activated Th1 cells was unchanged. Mechanistic analysis revealed that NK-4 inhibited mRNA expression of the Th2-associated transcription factors GATA-3 and NFATc1 in anti-CD3ε mAb-stimulated D10.G4.1 cells. Regarding the regulation of Th2 cell effector functions, NK-4 inhibited the secretion of eotaxin and thymus and activation-regulated chemokine (TARC) by normal human dermal fibroblasts in response to IL-4 and/or TNF-α. NK-4 achieved TARC attenuation comparable to what is observed with suplatast tosilate, an antiallergic drug that selectively inhibits Th2 cytokine production, at 14-fold lower concentrations of suplatast tosilate. Dexamethasone increased TARC production by 2.2- to 2.6-fold of control cultures. NK-4 successfully inhibited the STAT6 signaling pathway, suggesting a potential mechanism for down-regulating chemokines expression. In addition, NK-4 abrogated IL-4-driven modulation of cytokine production profile in human monocytic THP-1 cells from proinflammatory to anti-inflammatory response, as seen in the inverted ratio of TNF-α to IL-10 produced in response to LPS. These results suggest that NK-4 could prevent IL-4-driven polarization to alternatively activated macrophages, which are proposed to have pathogenic roles in allergic asthma. The importance of Th2 cytokines and chemokines in the development and progression of type 2 inflammatory disorders has been highlighted by recent advance in our understanding the immunological mechanism underlying allergic disease. Our results support the use of NK-4 as a reasonable therapeutic option to alleviate Th2-mediated allergic inflammation.</p></div

Chemical structure of NK-4.

<p>Chemical structure of NK-4.</p

NK-4 down-regulates secretion of TARC by NHDF stimulated with both IL-4 and TNF-α.

<p>NHDF were stimulated with 10 ng/ml IL-4 and 5 ng/ml TNF-α in the presence or absence (control) of varying concentrations of NK-4, dexamethasone or FK-506 (A) for 48 h at 37°C. In experiments comparing NK-4 with suplatast tosilate (B), NHDF were exposed to test articles for 15 min before stimulation with IL-4 and TNF-α. TARC levels in the cultures of NHDF without stimulation were below detectable limits. Results are the means ± S.D. of triplicate cultures. Results are representative of three independent experiments with similar results. **<i>p</i> < 0.01 compared with control cultures.</p

NK-4 suppresses the STAT6 signaling pathway in NHDF stimulated with IL-4 and TNF-α.

<p>NHDF were grown in confluent monolayer cultures in 12-well plates and were stimulated with 10 ng/ml IL-4 and 5 ng/ml TNF-α in the presence or absence of varying concentrations of NK-4 for 15 min. Phosphorylation of STAT6 was determined by immunoblotting whole-cell lysates using specific antibodies against the phosphorylated or total STAT6 protein. A representative blot is shown (A). The optical density ratio of phospho-STAT6 to total STAT6 is shown (B). Data from three independent experiments were combined and expressed as the means ± SD. **<i>p</i> < 0.01 compared with control cultures. Original uncropped and unadjusted Western blots were provided as supplementary files (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199666#pone.0199666.s001" target="_blank">S1 Fig</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199666#pone.0199666.s002" target="_blank">S2 Fig</a>).</p

NK-4 down-regulates secretion of eotaxin by NHDF in response to IL-4 and/or TNF-α.

<p>NHDF were stimulated with varying concentrations of IL-4 (A) or TNF-α (B) in the presence or absence (control) of 10 μM NK-4 for 48 h at 37°C. The effect of NK-4 on the secretion of eotaxin from NHDF stimulated with 10 ng/ml IL-4 and 5 ng/ml TNF-α was assessed (C and D). Concentrations of eotaxin in culture supernatants were measured by ELISA (A to C). Cell numbers of NHDF were determined by cell counting kit-8 (D). Results are the means ± S.D. of triplicate cultures. Results are representative of three independent experiments with similar results. *<i>p</i> < 0.05, **<i>p</i> < 0.01 compared with control cultures.</p

NK-4 suppresses the mRNA expression of the Th2-associated transcription factors GATA-3 and NFATc1.

<p>Total RNA was extracted from D10.G4.1 cells (2 x 10⁶ cells) stimulated with immobilized anti-CD3ε mAb (8 μg/ml) in the presence or absence (control) of NK-4 for 6 h at 37°C. First-strand cDNA was synthesized using reverse transcriptase, as described in the Materials and Methods. Expression levels of GATA-3 (A), NFATc1 (B) and c-Maf (C) were analyzed by real-time PCR. 18S rRNA expression levels were used for normalization. Results are given as changes in gene expression relative to the mean values of the control. Results are the means ± S.D. of three independent experiments. *<i>p</i> < 0.05, compared with control cultures.</p

NK-4 selectively down-regulates Th2 cytokine production by antigen-stimulated established Th2 cells.

<p>Th1 clone #4 (2.5 x 10⁴ cells/well) were stimulated with OVA (200 μg/ml) and MMC-treated BALB/c mouse spleen cells (1.2 x 10⁶ cells/well) in the presence or absence (control) of varying concentrations of NK-4 for 48 h at 37°C in 96-well plates (A). D10.G4.1 cells (2.5 x 10⁴ cells/well) were stimulated with MMC-treated C57BL/6 mouse spleen cells (1.2 x 10⁶ cells/well) (B and C). Concentrations of IFN-γ (A), IL-4 (B) and IL-5 (C) in culture supernatants were measured by ELISA. Results are the means ± S.D. of triplicate cultures. Results are representative of three independent experiments with similar results. **<i>p</i> < 0.01 compared with control cultures.</p

NK-4 selectively down-regulates Th2 cytokine production by anti-CD3ε mAb-stimulated established Th2 cells.

<p>Th1 clone #4 (A and B) and Th2 clone D10.G4.1 cells (C to E) (2.5 x 10⁴ cells/well) were stimulated with immobilized anti-CD3ε mAb (8 μg/ml) in the presence or absence (control) of varying concentrations of NK-4 for 48 h at 37°C in 96-well plates. Concentrations of IFN-γ (A), IL-4 (C) and IL-5 (D) in culture supernatants were measured by ELISA. Cell numbers of #4 (B) and D10.G4.1 (E) were determined by cell counting kit-8. Results are the means ± S.D. of triplicate cultures. Results are representative of three independent experiments with similar results. *<i>p</i> < 0.05, **<i>p</i> < 0.01 compared with control cultures.</p

Primers used for quantitative real-time PCR analysis.

<p>Primers used for quantitative real-time PCR analysis.</p

NK-4 selectively down-regulates IL-4 production by primary splenic T cells in response to anti-CD3ε mAb.

<p>BALB/c moue spleen cells (1.2 x 10⁶ cells/well) were stimulated with 5 μg/ml anti-CD3ε mAb in the presence or absence (control) of varying concentrations of NK-4 for 48 h at 37°C in 96-well plates. Concentrations of IFN-γ (A) and IL-4 (B) in culture supernatants were measured by ELISA. The ratios of IFN-γ to IL-4 are shown (C). Results are the means ± S.D. of triplicate cultures. Results are representative of two independent experiments with similar results. **<i>p</i> < 0.01 compared with control cultures.</p