Sphingosine kinase 1-specific inhibitor PF543 reduces goblet cell metaplasia of bronchial epithelium in an acute asthma model

Abstract

Sphingosine kinase 1 (SPHK1) has been shown to play a key role in the pathogenesis of asthma where SPHK1-generated sphingosine-1-phosphate (S1P) is known to mediate innate and adaptive immunity while promoting mast cell degranulation. Goblet cell metaplasia (GCM) contributes to airway obstruction in asthma and has been demonstrated in animal models. We investigated the role of PF543, a SPHK1-specific inhibitor, in preventing the pathogenesis of GCM using a murine (C57BL/6) model of allergen-induced acute asthma. Treatment with PF543 before triple allergen exposure (DRA: House dust mite, Ragweed pollen, and Aspergillus) reduced inflammation, eosinophilic response, and GCM followed by reduced airway hyperreactivity to intravenous methacholine. Furthermore, DRA exposure was associated with increased expression of SPHK1 in the airway epithelium which was reduced by PF543. DRA-induced reduction of acetylated α-tubulin in airway epithelium was associated with an increased expression of NOTCH2 and SPDEF which was prevented by PF543. In vitro studies using human primary airway epithelial cells showed that inhibition of SPHK1 using PF543 prevented an allergen-induced increase of both NOTCH2 and SPDEF. siRNA silencing of SPHK1 prevented the allergen-induced increase of both NOTCH2 and SPDEF. NOTCH2 silencing was associated with a reduction of SPDEF but not that of SPHK1 upon allergen exposure. Our studies demonstrate that inhibition of SPHK1 protected allergen-challenged airways by preventing GCM and airway hyperreactivity, associated with downregulation of the NOTCH2-SPDEF signaling pathway. This suggests a potential novel link between SPHK1, GCM, and airway remodeling in asthma.NEW & NOTEWORTHY The role of SPHK1-specific inhibitor, PF543, in preventing goblet cell metaplasia (GCM) and airway hyperreactivity (AHR) is established in an allergen-induced mouse model. This protection was associated with the downregulation of NOTCH2-SPDEF signaling pathway, suggesting a novel link between SPHK1, GCM, and AHR.

Keywords: PF543; airway epithelium; asthma; goblet cell metaplasia; sphingosine kinases.

Graphical abstract

Figure 1.

Allergen challenge increased the expression of SPHK1 in the airway. A: schematic representation of the triple antigen (DRA) allergic mouse asthma model protocol. The mice were sensitized initially by subcutaneous injection with the three allergens adsorbed to alum. This was followed by allergen challenge by intranasal insufflation. Treatment with PF543 was given on days 11, 12, 13, and 14, a day before each allergen challenge on days 12, 13, and 14. BAL and lung tissue were harvested on day 15. B: representative immunohistochemistry (IHC) of lung tissue sections showed increased expression of SPHK1 in mice challenged with allergen, which was reduced upon PF543 treatment. Secondary only controls are provided. n = 6 animals; 3 males, 3 females. Statistical test used is two-way ANOVA followed by Tukey’s multiple comparisons test. ***P < 0.001. BAL, bronchoalveolar lavage; DRA, dust mite, ragweed, Aspergillus sp.

Figure 2.

Inhibition of SPHK1 by PF543 treatment reduced allergen-induced inflammatory responses in mice. A: H&E images of the lung tissue sections showed an accentuated inflammatory response as can be seen with increased immune cell infiltration in the DRA-treated mice which was significantly reduced in the mice treated with PF543. B: the total BAL cell counts as well as individual cell count (differential staining) including eosinophils, lymphocytes, and macrophages were greater in the DRA-treated group as compared with the PF543-treated group. C: the cytokine profiles in the BAL fluid MCP-1, MIP-2, IL-4, and IL-13 were all significantly elevated in the Alum + DRA group. Inhibition of SPHK1 by PF543 in the DRA allergen-challenged mice reduced inflammatory cytokines in the BAL. n = 6 animals; 3 males, 3 females. Statistical test used is two-way ANOVA followed by Tukey’s multiple comparisons test. ****P < 0.0001 and **P < 0.01. BAL, bronchoalveolar lavage; DRA, dust mite, ragweed, Aspergillus sp; H&E, hematoxylin-eosin; MCP-1, monocyte chemoattractant protein-1; MIP-1, macrophage inflammatory protein 2; SPHK1, sphingosine kinase 1.

Figure 3.

PF543 therapy during allergen exposure protects mouse lung function. Allergen exposure triggered an increase of both baseline respiratory system resistance and AHR following intravenous methacholine (MCh) challenge. FlexiVent ventilator measurements showed increased respiratory system resistance (R_rs; A) and Newtonian resistance (R_n; B) in the allergen-treated mice compared with control mice exposed to alum. Mice challenged with allergen but treated with PF543 had reduced constrictor responsiveness to methacholine challenge. Allergen-exposed mice had sustained lung tissue injury causing increased tissue impedance (C) and elastance (D), which was rescued by PF543 therapy. n = 6 animals; 3 males, 3 females. Statistical test used is two-way ANOVA followed by Tukey’s multiple comparisons test. ****P < 0.0001. AHR, airway hyperreactivity.

Figure 4.

SPHK1 inhibits allergen-induced goblet cell metaplasia in vivo. Representative images of PAS‐stained lung sections from the indicated treatment groups. Quantification showed an increased percentage of PAS‐positive cells (purple) that was reduced by PF543. n = 6 animals; 3 males, 3 females. Statistical test used is two-way ANOVA followed by Tukey’s multiple comparisons test. ****P < 0.0001. PAS, Periodic acid-Schiff; SPHK1, sphingosine kinase 1.

Figure 5.

SPHK1 inhibition by PF543 attenuated the goblet cell metaplasia (GCM) signaling pathway. Representative images of lung sections from the indicated treatment groups (6 mice/group) stained for acetylated α-tubulin (A) MUCIN 5AC (B), NOTCH2 (C), and SPDEF (D). Quantified data from DRA allergen-induced mouse lungs revealed an increased expression of NOTCH2, SPDEF, and MUCIN 5AC. The immunofluorescence staining by acetylated α-tubulin showed a decrease in percentage of ciliated cells in the allergen-treated lung tissue which was restored upon PF543 treatment. Secondary only controls are provided. n = 6 animals; 3 males, 3 females. Statistical test used is two-way ANOVA followed by Tukey’s multiple comparisons test. ****P < 0.0001, ***P < 0.001 and **P < 0.01. DRA, dust mite, ragweed, Aspergillus sp.; SPHK1, sphingosine kinase 1.

Figure 6.

SPHK1 inhibitor, PF543, attenuates HDM-induced increase in expression of SPHK1, NOTCH2, and SPDEF in primary human small airway epithelial cells (SAECs). A: immunoblots showing an increase in SPHK1, NOTCH2, and SPDEF upon allergen treatment which got reduced with PF543 treatment. B–D: quantitative data showing the same. n = 3 replicates. Statistical test used is two-way ANOVA followed by Tukey’s multiple comparisons test. ***P < 0.001, **P < 0.01, and *P < 0.05. HDM, house dust mite; SPHK1, sphingosine kinase 1.

Figure 7.

Allergen-induced responses to HDM is activated through SPHK1- NOTCH 2 -SPDEF pathway: HDM-induced increase in expression of NOTCH2 (A and B) and SPDEF (A and C) in human primary small airway epithelial cells (SAECs) was reduced significantly by siRNA silencing SPHK1 expression. Silencing NOTCH2 did not affect SPHK1 (D and E) but reduced SPDEF expression (D and F). Western blots show the effect of SPHK1 (G and H) and NOTCH2 (I and J) siRNA silencing in SAECs. n = 3 replicates. Statistical test used is t test. ***P < 0.001, **P < 0.01, *P < 0.05, and ns is not significant. HDM, house dust mite; SPHK1, sphingosine kinase 1.

Figure 8.

Proposed model of DRA allergen-induced asthma. DRA allergen causes an increase in SPHK1, NOTCH2, and SPDEF expression leading to increased goblet cell metaplasia (GCM) and inflammation which was inhibited by PF543 treatment. Mechanistically, this increase in NOTCH2 and SPDEF expression was inhibited by SPHK1 inhibitor, PF543, establishing a potential link between SPHK1 and GCM through NOTCH2/SPDEF signaling pathway. The regulation of GCM by SPHK1 inhibitors was previously reported to be cytokine (IL-13) driven. NOTCH 2 and SPDEF that we investigated could be upstream or downstream of IL-13 or an independent pathway which needs further investigation. DRA, dust mite, ragweed, Aspergillus sp.; SPHK1, sphingosine kinase 1. [Image created with a licensed version of BioRender.com.]