Interleukin-1β induces blood-brain barrier disruption by downregulating Sonic hedgehog in astrocytes

Abstract

The blood-brain barrier (BBB) is composed of capillary endothelial cells, pericytes, and perivascular astrocytes, which regulate central nervous system homeostasis. Sonic hedgehog (SHH) released from astrocytes plays an important role in the maintenance of BBB integrity. BBB disruption and microglial activation are common pathological features of various neurologic diseases such as multiple sclerosis, Parkinson's disease, amyotrophic lateral sclerosis, and Alzheimer's disease. Interleukin-1β (IL-1β), a major pro-inflammatory cytokine released from activated microglia, increases BBB permeability. Here we show that IL-1β abolishes the protective effect of astrocytes on BBB integrity by suppressing astrocytic SHH production. Astrocyte conditioned media, SHH, or SHH signal agonist strengthened BBB integrity by upregulating tight junction proteins, whereas SHH signal inhibitor abrogated these effects. Moreover, IL-1β increased astrocytic production of pro-inflammatory chemokines such as CCL2, CCL20, and CXCL2, which induce immune cell migration and exacerbate BBB disruption and neuroinflammation. Our findings suggest that astrocytic SHH is a potential therapeutic target that could be used to restore disrupted BBB in patients with neurologic diseases.

Figure 1. IL-1β abolishes the protective effect of astrocytes on BBB integrity.

MEBC4 cells were treated for 24 h with 2 ng/ml IL-1β, ACM, or IL-1β–treated ACM for 24 h. FITC-BSA was loaded onto the luminal side of the insert for 1 h, and then the FITC-BSA levels on the abluminal side were analyzed. All quantitative data are expressed as means ± SEM (n = 5), normalized to the corresponding values from untreated cells. *, p<0.001.

Figure 2. IL-1β downregulates SHH production in astrocytes.

(A) Shh mRNA levels in astrocytes, determined by qPCR. Astrocytes were treated with IL-1β for 6 h. Values are means ± SEM (n = 5). *, p<0.05; †, p<0.01. (B) Protein levels of SHH in ACM, determined using ELISA. Astrocytes were treated with IL-1β for 24 h. Values are means ± SEM (n = 5). *, p<0.001.

Figure 3. SHH signaling is critical for maintenance of BBB integrity.

(A) MBEC4 cells were treated with SHH or the Smo agonist purmorphamine for 24 h. (B) MBEC4 cells were treated with ACM or the Smo antagonist cyclopamine for 24 h. FITC-BSA was loaded onto the luminal side of the insert for 1 h, and then the FITC-BSA levels on the abluminal side were analyzed. All quantitative data are expressed as means ± SEM (n = 5), normalized to the corresponding values from untreated cells. *, p<0.001.

Figure 4. Astrocytic SHH signaling regulates expression of tight junction proteins in BBB.

Western blotting of claudin-5 (A), occludin (B) and ZO-1 (C) in MBEC4 cells. Cells were treated for 24 h with ACM, SHH (100 ng/ml), the Smo agonist purmorphamine (1 µM), or the Smo antagonist cyclopamine (30 µM). All quantitative data are expressed as means ± SEM (n = 5), normalized to the corresponding values from untreated cells. *, p<0.05.

Figure 5. IL-1β upregulates production of pro-inflammatory chemokines in astrocytes.

(A) Cxcl2, Ccl2, and Ccl20 mRNA levels in astrocytes, determined by qPCR. Astrocytes were treated with IL-1β for 6 h. Values are means ± SEM (n = 5). *, p<0.05; †, p<0.01. (B) Protein levels of CXCL2, CCL2, and CCL20 in astrocytes, determined by ELISA. Astrocytes were treated with IL-1β for 24 h. Values are means ± SEM (n = 5). *, p<0.001.

Figure 6. Model of the roles of the SHH and IL-1β pathways in the BBB.

(A) Model of SHH signaling pathway in brain capillary endothelial cells. Secreted SHH binds and inactivates its receptor Patched-1, which allowed Smoothened to activate the transcription factor Gli-1. Gli-1 upregulates tight junction proteins and enhances BBB integrity. (B) Model of BBB breakdown by IL-1β. Under healthy conditions (left), astrocytes secrete SHH to upregulate tight junction proteins in endothelial cells and maintain BBB integrity. Under pathologic conditions (right), pathogenic stimuli such as amyloid β, DAMPs, or cytokines induce microglia to release IL-1β. IL-1β suppresses astrocytic SHH production, leading to downregulation of tight junction proteins in endothelial cells and disintegrity of the BBB. IL-1β also activates astrocytes to release pro-inflammatory chemokines such as CXCL2, CCL2, and CCL20. These chemokines induce migration of immune cells, thereby worsening BBB disruption and neuroinflammation. Neut, neutrophils; Mo, monocytes; MΦ, macrophage; DC, dendritic cells; T, T cells.