pubmed.ncbi.nlm.nih.gov

Human Th17 cells produce a soluble mediator that increases podocyte motility via signaling pathways that mimic PAR-1 activation - PubMed

  • ️Tue Jan 01 2019

Human Th17 cells produce a soluble mediator that increases podocyte motility via signaling pathways that mimic PAR-1 activation

Carl J May et al. Am J Physiol Renal Physiol. 2019.

Abstract

The specific pathogenesis of idiopathic nephrotic syndrome (NS) is poorly understood, and the role of immune mediators remains contentious. However, there is good evidence for the role of a circulating factor, and we recently postulated circulating proteases as candidate factors. Immunosuppressive therapy with glucocorticoids (GCs) and T cell inhibitors are widely used in the clinical treatment of NS. Given that T helper (CD4+) cells expressing IL-17A (so-called Th17 cells) have recently been reported to be resistant to GC treatment, and GC resistance remains a major challenge in the management of NS, we hypothesized that Th17 cells produce a circulating factor that is capable of signaling to the podocyte and inducing deleterious phenotypic changes. To test this, we generated human Th17 cells from healthy volunteers and added the supernatants from these T cell cultures to conditionally immortalized human podocytes in vitro. This demonstrated that podocytes treated with Th17 cell culture supernatant, as well as with patient disease plasma, showed significant stimulation of JNK and p38 MAPK pathways and an increase in motility, which was blocked using a JNK inhibitor. We have previously shown that nephrotic plasma elicits a podocyte response via protease-activated receptor-1 (PAR-1). Stimulation of PAR-1 in podocytes elicited the same signaling response as Th17 cell culture supernatant treatment. Equally, protease inhibitors with Th17 cell culture treatment blocked the signaling response. This was not replicated by the reagents added to Th17 cell cultures or by IL-17A. Hence, we conclude that an undefined soluble mediator produced by Th17 cells mimics the deleterious effect of PAR-1 activation in vitro. Given the association between pathogenic subsets of Th17 cells and GC resistance, these observations have potential therapeutic relevance for patients with NS.

Keywords: T helper 17; circulating factor; nephrotic syndrome; podocyte; protease-activated receptor-1.

PubMed Disclaimer

Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

Fig. 1.
Fig. 1.

Podocyte signaling response to T helper (Th)17 cell culture supernatant. The addition of Th17 cell culture supernatant to podocytes elicited a significant response in both phosphorylated (p-)p38 MAPK (A) and pJNK (B) signaling pathways relative to Th0 cell culture supernatant treatment. Statistical significance was measured using one-way ANOVA and a post hoc Bonferroni’s multiple-comparison test.

Fig. 2.
Fig. 2.

JNK phosphorylation induced by T helper (Th)17 cell culture supernatant leads to phosphorylation of paxillin in podocytes. Both p38 MAPK and JNK have specific phosphorylation sites on paxillin: at S85 and S178, respectively. Only the JNK-specific site at S178 is phosphorylated (p). Results are representative of two blots.

Fig. 3.
Fig. 3.

T helper (Th)17 cell culture supernatant significantly increases podocyte motility relative to Th0 cell culture supernatant. A: podocytes treated with Th17 cell culture supernatant were significantly more motile than those treated with Th0 cell culture supernatant (n = 8, P = 0.0007 by an unpaired t-test). The area of the clear zone is measured in pixels. The area at 12 h was normalized to 0 h. Units are arbitrary. B: to establish the importance of the signaling events detected by Western blot, podocytes were treated with Th17 supernatant and inhibitors against p38 MAPK and JNK. Motility was measured in these podocytes using a wound healing assay. JNK inhibition significantly reduced the Th17-mediated increase in podocyte motility. Statistical significance was measured using one-way ANOVA and a post hoc Bonferroni’s multiple-comparison test. The area of the clear zone is measured in pixels. The area at 12 h was normalized to 0 h. Units are arbitrary. C: a typical set of images that were taken to measure podocyte motility. The area of the clear zone 12 h postscratch is expressed as a decimal of the area at 0 h. Scale bars = 500 μm.

Fig. 4.
Fig. 4.

IL-23 can significantly increase podocyte motility, although not at the dose present initially in the T helper (Th)17 cell culture supernatant. A: IL-17 had no significant effect on podocyte motility at any of the doses tested (n = 3). B: IL-23 was the only supplementary cytokine that was capable of significantly increasing podocyte motility (six replicates, P = 0.0041 by an unpaired t-test). However, podocytes were treated with a 10 ng/ml dose of IL-23, which is the concentration at which IL-23 is added to the Th17 cell culture medium. By the time the supernatant was applied to the podocytes, the medium was in the presence of Th17 cells for 48 h and through one freeze-thaw cycle. C: therefore, an ELISA was performed to determine the concentration of IL-23 that remained in the Th17 cell culture supernatant. There was 0.7 mg/ml IL-23 in the Th17 cell culture supernatant. D: at this dose, IL-23 had no significant effect on podocyte motility.

Fig. 5.
Fig. 5.

The protease-activated receptor-1 (PAR-1) agonist signaling response mimics the T helper (Th)17 cell culture supernatant response. Conditionally immortalized podocytes were treated with 15 μM PAR-1 agonist for the indicated time course. Control lysate was generated from untreated podocytes. The data shown are representative blots (A) with combined densitometry normalized to the β-actin load control (B−E). B: phosphorylated (p-)JNK signaling. C: p-p38 MAPK signaling. D: p-paxillin S178. E: p-paxillin S85. Signaling responses for each were dynamically like those elicited by Th17 cell culture supernatant treatment. Data represent four independent experiments. Statistical significance was measured using one-way ANOVA and a post hoc Bonferroni’s multiple-comparison test.

Fig. 6.
Fig. 6.

Protease-activated receptor-1 (PAR-1) inhibition blocks the signature T helper (Th)17 cell culture supernatant response. Th17 cell culture supernatant treatment of podocytes significantly increased phosphorylation of JNK [phospho-JNK (p-JNK); A], VASP S157 [phospho-VASP (p-VASP) 157; B], p38 MAPK [phospho-p38 MAPK (p-p38 MAPK); C], and paxillin S178 [phospho-paxillin (p-paxillin) S178; D]. Conversely, inhibition of PAR-1 by FR171113 significantly reduced each Th17 response (densitometry based on four blots, by one-way ANOVA and a post hoc Bonferroni multiple-comparison test). Representative blots of proteins were studied (E). VPX, vorapaxar.

Similar articles

Cited by

References

    1. Abrahamson DR. Role of the podocyte (and glomerular endothelium) in building the GBM. Semin Nephrol 32: 342–349, 2012. doi:10.1016/j.semnephrol.2012.06.005. - DOI - PMC - PubMed
    1. Birkenkamp KU, Tuyt LM, Lummen C, Wierenga AT, Kruijer W, Vellenga E. The p38 MAP kinase inhibitor SB203580 enhances nuclear factor-kappa B transcriptional activity by a non-specific effect upon the ERK pathway. Br J Pharmacol 131: 99–107, 2000. doi:10.1038/sj.bjp.0703534. - DOI - PMC - PubMed
    1. Correia AC, Moonen JR, Brinker MG, Krenning G. FGF2 inhibits endothelial-mesenchymal transition through microRNA-20a-mediated repression of canonical TGF-β signaling. J Cell Sci 129: 569–579, 2016. doi:10.1242/jcs.176248. - DOI - PubMed
    1. Deakin NO, Turner CE. Paxillin comes of age. J Cell Sci 121: 2435–2444, 2008. doi:10.1242/jcs.018044. - DOI - PMC - PubMed
    1. Deegens JK, Dijkman HB, Borm GF, Steenbergen EJ, van den Berg JG, Weening JJ, Wetzels JF. Podocyte foot process effacement as a diagnostic tool in focal segmental glomerulosclerosis. Kidney Int 74: 1568–1576, 2008. doi:10.1038/ki.2008.413. - DOI - PubMed

Publication types

MeSH terms

Substances

Supplementary concepts