Receptor-Type Protein-Tyrosine Phosphatase ζ and Colony Stimulating Factor-1 Receptor in the Intestine: Cellular Expression and Cytokine- and Chemokine Responses by Interleukin-34 and Colony Stimulating Factor-1

<div><p>Differential intestinal expression of the macrophage growth factors colony stimulating factor-1 (CSF-1), interleukin (IL)-34, and their shared CSF-1 receptor (CSF-1R) in inflammatory bowel disease (IBD) has been shown. Diverse expression between CSF-1 and IL-34, suggest that IL-34 may signal via an alternate receptor. Receptor-type protein-tyrosine phosphatase ζ (PTPRZ1, RPTP-ζ), an additional IL-34 receptor, was recently identified. Here, we aimed to assess <i>PTPRZ1</i> expression in IBD and non-IBD intestinal biopsies. Further, we aimed to investigate cellular PTPRZ1 and CSF-1R expression, and cytokine- and chemokine responses by IL-34 and CSF-1. The expression of <i>PTPRZ1</i> was higher in non-IBD colon compared to ileum. <i>PTPRZ1</i> expression was not altered with inflammation in IBD, however, correlated to <i>IL34</i>, <i>CSF1</i>, and <i>CSF1R</i>. The expression patterns of PTPRZ1 and CSF-1R differed in peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and intestinal epithelial cell line. PBMCs and monocytes of the same donors responded differently to IL-34 and CSF-1 with altered expression of tumor-necrosis factor α (TNF-α), IL-1β, interferon γ (IFN-γ), IL-13, IL-8, and monocyte chemotactic protein-1 (MCP-1) levels. This study shows that <i>PTPRZ1</i> was expressed in bowel tissue. Furthermore, CSF-1R protein was detected in an intestinal epithelial cell line and donor dependently in primary PBMCs, monocytes, and macrophages, and first hints also suggest an expression in these cells for PTPRZ1, which may mediate IL-34 and CSF-1 actions.</p></div

PTPRZ1 is differentially expressed in normal human ileum and colon but not regulated with inflammation.

<p><b>(A)</b><i>PTPRZ1</i> relative mRNA expression in ileum and colon, presented as the mean per colon sites for each non-IBD subjects. <b>(B)</b> <i>PTPRZ1</i> relative mRNA in different sites of the colon from non-IBD subjects. <i>PTPRZ1</i> relative mRNA expression in <b>(C)</b> colon and <b>(D)</b> ileum from non-IBD, IBD, UC and CD patients. Comparisons between ileum and colon were calculated using Mann–Whitney <i>U</i> tests, and between different sites of colon by ANOVA with post-hoc LSD tests. N = 18 for ileum, n = 24 for colon. Results are means ± SEM *P<0.05; **P<0.01; ***P<0.001. <b>(E)</b> <i>PTPRZ1</i> relative mRNA expression compared between non-inflamed and inflamed in IBD patients. <i>PTPRZ1</i> relative mRNA expression in colon presented as the mean per colon sites for each patient in IBD patients subdivided into CD and UC. Correlations of <i>PTPRZ1</i> with <b>(F)</b> <i>IL34</i>, <b>(G)</b> <i>CSF1</i>, <b>(H)</b> <i>CSF1R</i>, <b>(I)</b> <i>TNFA</i> and <b>(J)</b> <i>CD68</i> in IBD patients. Comparisons were evaluated using Mann–Whitney U tests. Correlations were assessed by Spearman’s correlation coefficients. N = 18 for non-inflamed IBD, n = 23 for inflamed IBD, n = 6 for non-inflamed CD, n = 6 for inflamed CD, n = 11 for non-inflamed UC, n = 16 for inflamed UC. Results are mean ± SEM *P<0.05; **P<0.01; ***P<0.001.</p

Diverse regulation of IL-10, IL-1β, TNF-α and MCP-1 in macrophages differentiated in the presence of IL-34 and CSF-1.

<p><b>(A-C)</b><i>IL1B</i>, <i>TNFA</i> and <i>IL10</i> mRNA expression in IL-34 and/or CSF-1 differentiated macrophages and polarized to an M1-like phenotype, an M2-like phenotype or non-polarized were analysed by q-PCR and normalized to <i>GAPDH</i>. Data represent mean + SEM, *P<0.05; **P<0.01; ***P<0.001, ANOVA, n = 5–6 donors. <b>(D-F)</b> Secreted IL-10, MCP-1 and IL-1β in the supernatant from IL-34 and/or CSF-1 differentiated macrophages and polarized to an M1-like phenotype, an M2-like phenotype or non-polarized were analysed by ELISA. Data represent mean + SEM, *P<0.05; **P< 0.01; ***P<0.001, ANOVA, n = 9 donors.</p

CSF-1R dependent regulation of pro-inflammatory cytokines and chemokines in PBMCs and monocytes <i>IL1B</i>, <i>TNFA</i>, <i>IFNG</i>, <i>IL8</i> and <i>MCP1</i> relative mRNA expression from PBMCs.

<p><b>(A, B)</b> and monocytes <b>(C, D)</b> stimulated with IL-34, PBMCs <b>(E, F)</b> and monocytes <b>(G, H)</b> stimulated with CSF-1, after blocking CSF-1R for 6 h. IgG1 was used as a control. Gene expression was analysed by q-PCR and normalized to <i>GAPDH</i>. Data represent mean + SEM, *P<0.05; **P<0.01; ***P<0.001, Student’s T-test, n = 5–6 donors. (<b>I-J</b>) Secreted MCP-1 in supernatants from PBMCs <b>(I)</b> and monocytes <b>(J)</b> stimulated with IL-34 or CSF-1 after blocking CSF-1R for 24 h, IgG1 was used as a control, were analysed by ELISA. Data represent mean + SEM, *P<0.05; **P<0.01; ***P<0.001, Student’s T-test, n = 6 donors.</p

Expression of CSF-1R and PTPRZ1 in PBMCs, monocytes, macrophages and colonic epithelial cells.

<p>Immunoblotting of lysates from PBMCs, monocytes, CSF-1 monocyte derived and non-polarized macrophages, Caco-2 and A549 cells analysed for <b>(A)</b> CSF-1R and <b>(B)</b> PTPRZ1. β-actin was used as a loading control (10 and 15 μg protein per lane, respectively).</p

Regulation of pro—inflammatory cytokines and chemokines through IL-34 and CSF-1 in PBMCs and monocytes <i>IL1B</i>, <i>TNFA</i>, <i>IFNG</i>, <i>IL8</i> and <i>MCP1</i> relative mRNA expression in PBMCs.

<p><b>(A, B)</b> and monocytes <b>(C, D)</b> stimulated with IL-34, CSF-1 for 1 h or left untreated were analysed by q-PCR and normalized to <i>GAPDH</i>. <i>IL1B</i>, <i>TNFA</i>, <i>IFNG</i>, <i>IL8</i> and <i>MCP1</i> relative mRNA expression from PBMCs <b>(E, F)</b> and monocytes <b>(G, H)</b> stimulated with IL-34, CSF-1 for 6 h or left untreated were analysed by q-PCR and normalized to <i>GAPDH</i>. Data represent mean + SEM, *P<0.05; **P<0.01; ***P<0.001, Student’s T-test, n = 5–6 donors. <b>(I)</b> Secreted MCP-1 in supernatants from Caco-2 cells, PBMCs and monocytes stimulated with IL-34 or CSF-1 for 24 h, as analysed by ELISA. Data represent mean + SEM, *P< 0.05; **P< 0.01; ***P< 0.001, Student’s T-test, n = 5–6 donors. <b>(J)</b> Secreted MCP-1 in supernatants from PBMCs stimulated with IL-34 or CSF-1 (10 or 50 ng/ml for 24h) were analysed by ELISA. Data represent mean + SEM, *P<0.05; **P<0.01; ***P<0.001, Student’s T-test, n = 6 donors.</p

The antimicrobial peptide psoriasin (S100A7) mediates cytokine-dependent caspase regulation and IL-1β release by epidermal keratinocytes.

<p>A, E, Keratinocytes stimulated with IFNγ, IL-17A, transfected with dsDNA and indicated siRNA, and the psoriasin-dependent IL-1β release was analyzed by ELISA. Data represent mean + SEM, **, <i>p</i> < 0.01, Student’s <i>t</i> test, n = 7–8. B, D, Regulation of IL-1β, caspase-1, caspase-5 in cytokine-stimulated keratinocytes, transfected with psoriasin-targeting siRNA was analyzed by RTqPCR and normalized to β-actin. Data represent mean + SEM, *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001 determined by Student’s <i>t</i> test, n = 9. C, Induction of psoriasin in cytokine-stimulated keratinocytes analyzed by RTqPCR and normalized to β-actin. Data represent mean + SEM, *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001 determined by ANOVA, n = 9.</p

IFNγ and cytosolic dsDNA regulate inflammatory caspase-5 and IL-1β release by epidermal keratinocytes.

<p>A-B, Regulation of IL-1β and caspase-5 in Th1/Th17 cytokine-stimulated keratinocytes analyzed by RTqPCR and normalized to β-actin. Data represent mean + SEM, *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001, Student’s <i>t</i> test, n = 9. C, Keratinocytes stimulated with IFNγ, transfected with dsDNA [Poly(dA:dT)] ± DNase and the DNA-dependent IL-1β release was analyzed by ELISA. D, Representative immunoblotting of corresponding supernatants analyzed for DNA-dependent activation of caspase-5 (exposure times; caspase-5, 30s; cleaved caspase-5, 30min) compared to loading control (Ponceau staining), three independent experiments. E, Keratinocytes stimulated with IFNγ, transfected with dsDNA and indicated siRNA and the caspase-5 dependent IL-1β release was analyzed by ELISA. C, E, Data represent mean + SEM, **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001, Student’s <i>t</i> test, n = 3–6.</p

IL-17A amplifies caspase-5 induction and controls NLRP1-mediated IL-1β release by epidermal keratinocytes.

<p>A-D, Regulation of IL-1β, caspase-1, caspase-5, NLRP1in IFNγ- and IL-17A -stimulated keratinocytes analyzed by RTqPCR and normalized to β-actin. Data represent mean + SEM of three independent experiments performed in triplicates *, p < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001 determined by ANOVA. E, dsDNA-transfected keratinocytes stimulated with IFNγ, IL-17A, and the cytokine-dependent IL-1β release was analyzed by ELISA. Data represent mean + SEM *, p < 0.05; *** p< 0.001 determined by ANOVA, n = 6. F, IFNγ and IL17A-treated keratinocytes, transfected with dsDNA and with siRNA targeting NLRP1 (N1) or non-coding siRNA (Co), and the supernatant was analyzed for NLRP1-dependent activation of caspase-5 (exposure time 20s; active p10, p20 subunits; exposure time, 120s) and caspase-1 (exposure time 35s). Corresponding protein levels were quantified by densitometry, n = 3 and compared to Ponceau staining (loading control). G, Keratinocytes stimulated with IFNγ, IL-17A transfected with dsDNA and indicated siRNA, and the NLRP1 inflammasome-dependent IL-1β release was analyzed by ELISA. Data represent mean + SEM, ***, p < 0.001 determined by ANOVA, n = 6.</p

Inflammatory caspase-5 is increased and active in psoriatic skin.

<p>A-C, Increased expression of IL-1β, caspase-1, caspase-5 in healthy (H) and psoriatic (P) skin analyzed by RTqPCR and normalized to PBGD. Data represent mean + SEM **, <i>p</i> < 0.01, Student’s <i>t</i> test (n = 6–8). D, Immunofluorescent staining of caspase-5 in healthy skin which was enhanced in psoriasis (n = 3), scale bar = 50μm. Representative control section stained with secondary antibody (ab) only and DAPI. E, Representative immunoblot analysis of healthy and psoriatic skin stained for caspase-5, indicated are pro-caspase-5 and active caspase-5 in psoriasis compared to healthy skin versus β-actin (n = 3).</p