Tuftsin signals through its receptor neuropilin-1 via the transforming growth factor beta pathway

Abstract

Tuftsin (Thr-Lys-Pro-Arg) is a natural immunomodulating peptide found to stimulate phagocytosis in macrophages/microglia. Tuftsin binds to the receptor neuropilin-1 (Nrp1) on the surface of cells. Nrp1 is a single-pass transmembrane protein, but its intracellular C-terminal domain is too small to signal independently. Instead, it associates with a variety of coreceptors. Despite its long history, the pathway through which tuftsin signals has not been described. To investigate this question, we employed various inhibitors to Nrp1's coreceptors to determine which route is responsible for tuftsin signaling. We use the inhibitor EG00229, which prevents tuftsin binding to Nrp1 on the surface of microglia and reverses the anti-inflammatory M2 shift induced by tuftsin. Furthermore, we demonstrate that blockade of transforming growth factor beta (TGFβ) signaling via TβR1 disrupts the M2 shift similar to EG00229. We report that tuftsin promotes Smad3 phosphorylation and reduces Akt phosphorylation. Taken together, our data show that tuftsin signals through Nrp1 and the canonical TGFβ signaling pathway. Despite the 40-year history of the tetrapeptide tuftsin (TKPR), a macrophage and microglial activator, its mechanism of action has not been defined. Here, we report that the tuftsin-mediated anti-inflammatory M2 shift in microglia is caused specifically by tuftsin binding to the receptor neuropilin-1 (Nrp1) and signaling through TGFβ receptor-1, a coreceptor of Nrp1. We further show that tuftsin signals via the canonical TGFβ pathway and promotes TGFβ release from target cells.

Keywords: anti-inflammatory; cytokines; microglia; neuropilin 1; tuftsin.

Figure 1. EG00229 reduces tuftsin binding to the surface of microglia in a dose-dependent manner

Microglial cells were pre-blocked with the indicated concentrations of EG00229, and incubated with biotinylated tuftsin at room temperature as described in Methods. Cells were then fixed and stained for Nrp1 (FITC) and with streptavidin-conjugated Cy3 to visualize biotinylated tuftsin on the cell surface (A). Fluorescence intensity of tuftsin staining was quantified in (B). n= 3; ***, p<0.001.

Figure 2. The tuftsin-mediated M2 shift in microglia is disrupted by EG00229

Quantitative RT-PCR was performed to analyze changes in gene expression of the M1 marker TNFα (A) and the M2 marker IL10 (B). Primary microglia were treated 100 μg/ml tuftsin or 100μg/ml tuftsin and NCM. Some groups were additionally treated with increasing concentrations of EG00229 as shown. (C) The ratio of the fold change of IL10 (M2) to the fold change of TNFα (M1). n= 3, *, p<0.05.

Figure 3. Blockade of TβR1 prevents the tuftsin-mediated M2 shift in microglia

Quantitative RT-PCR was performed to analyze changes in gene expression of the M1 marker TNFα (A) and the M2 marker IL10 (B). Primary microglia were treated 100 μg/ml tuftsin or 100μg/ml tuftsin and NCM. Some groups were additionally treated with 5nM c-Met inhibitor or 10μM TβR1 inhibitor. (C) The ratio of the fold change of IL10 (M2) to the fold change of TNFα (M1). n= 3–5, *, p<0.05; **, p<0.01; ns, not significant.

Figure 4. Tuftsin signals through the canonical TGFβ signaling pathway, which is prevented by TβR1 inhibition

Western blots were performed on primary microglia extracts that were exposed to NCM, 100 μg/ml tuftsin, 5 nM c-Met inhibitor or 10 μM TβR1 inhibitor for 10 hours. 1 hour prior to harvesting, cells were restimulated with their designated treatment to better observe phosphorylation changes. Blots were probed for Smad3 and phospho-Smad3 (A), and fold change relative to NCM treatment alone was quantified in (B), and adjusted to reflect changes in total Smad3 levels. Blots were probed for Akt and phospho-Akt in (C), and fold change relative to NCM alone is quantified in (C), and adjusted to reflect changes in total Akt levels. n= 3, *, p<0.05; **, p<0.01; ns, not significant.

Figure 5. Blockade of TβR1 prevents tuftsin-mediated TGFβ upregulation

An ELISA assay probing for TGFβ levels (A) or a nitrite assay using DAN reagent (B) was performed on media isolated from primary microglial cells treated 100 μg/ml tuftsin or 100μg/ml tuftsin and NCM for 10 hours. Some groups were additionally treated with 5 nM c-Met inhibitor, 10 μM TβR1 inhibitor, or 30 μM EG00229, also for 10 hours. n= 3, **, p<0.01; ***, p<0.001, ns, not significant.