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. 2018 Aug 13:9:1871.
doi: 10.3389/fimmu.2018.01871. eCollection 2018.

Host Defense Peptide LL-37-Mediated Chemoattractant Properties, but Not Anti-Inflammatory Cytokine IL-1RA Production, Is Selectively Controlled by Cdc42 Rho GTPase via G Protein-Coupled Receptors and JNK Mitogen-Activated Protein Kinase

Mahadevappa Hemshekhar  1 , Ka-Yee Grace Choi  1   2 , Neeloffer Mookherjee  1   2
Affiliations

Host Defense Peptide LL-37-Mediated Chemoattractant Properties, but Not Anti-Inflammatory Cytokine IL-1RA Production, Is Selectively Controlled by Cdc42 Rho GTPase via G Protein-Coupled Receptors and JNK Mitogen-Activated Protein Kinase

Mahadevappa Hemshekhar et al. Front Immunol. .

Abstract

The human host defense peptide LL-37 promotes immune activation such as induction of chemokine production and recruitment of leukocytes. Conversely, LL-37 also mediates anti-inflammatory responses such as production of anti-inflammatory cytokines, e.g., IL-1RA, and the control of pro-inflammatory cytokines, e.g., TNF. The mechanisms regulating these disparate immunomodulatory functions of LL-37 are not completely understood. Rho GTPases are GTP-binding proteins that promote fundamental immune functions such as chemokine production and recruitment of leukocytes. However, recent studies have shown that distinct Rho proteins can both negatively and positively regulate inflammation. Therefore, we interrogated the role of Rho GTPases in LL-37-mediated immunomodulation. We demonstrate that LL-37-induced production of chemokines, e.g., GRO-α and IL-8 is largely dependent on Cdc42/Rac1 Rho GTPase, but independent of the Ras pathway. In contrast, LL-37-induced production of the anti-inflammatory cytokine IL-1RA is not dependent on either Cdc42/Rac1 RhoGTPase or Ras GTPase. Functional studies confirmed that LL-37-induced recruitment of leukocytes (monocytes and neutrophils) is also dependent on Cdc42/Rac1 RhoGTPase activity. We demonstrate that Cdc42/Rac1-dependent bioactivity of LL-37 involves G-protein-coupled receptors (GPCR) and JNK mitogen-activated protein kinase (MAPK) signaling, but not p38 or ERK MAPK signaling. We further show that LL-37 specifically enhances the activity of Cdc42 Rho GTPase, and that the knockdown of Cdc42 suppresses LL-37-induced production of chemokines without altering the peptide's ability to induce IL-1RA. This is the first study to demonstrate the role of Rho GTPases in LL-37-mediated responses. We demonstrate that LL-37 facilitates chemokine production and leukocyte recruitment engaging Cdc42/Rac1 Rho GTPase via GPCR and the JNK MAPK pathway. In contrast, LL-37-mediated anti-inflammatory cytokine IL-1RA production is independent of either Rho or Ras GTPase. The results of this study suggest that Cdc42 Rho GTPase may be the molecular switch that controls the opposing functions of LL-37 in the process of inflammation.

Keywords: Cdc42 RhoGTPase; JNK mitogen-activated protein kinase; LL-37; cathelicidin; cell migration; host defense peptide; inflammation.

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Figures

Figure 1
Figure 1
Cdc42/Rac1 GTPase controls LL-37-induced chemokine secretion. Macrophage-like THP-1 cells were pre-incubated with Cdc42/Rac1 inhibitor ML141 (10 µM) for 1 h, prior to stimulation with either LL-37 or sLL-37 (5 µM each), lipopolysaccharide (LPS) (10 ng/ml), or 0.01% DMSO as vehicle control (VC). (A) Tissue culture supernatants were monitored by ELISA for the production of chemokines GRO-α and IL-8 after 24 h. Results shown as mean ± SE of eight independent experiments (n = 8). (B) mRNA expressions were evaluated by quantitative real-time PCR for chemokines (GRO-α and IL-8) and anti-inflammatory cytokine IL-1RA, after 4 h. Relative fold changes were calculated compared to the expression in unstimulated cells, using the ΔΔCt method, after normalization with 18sRNA expression. Results shown as mean ± SE of three independent experiments. (C) Macrophage-like THP-1 cells were pre-incubated with the Ras inhibitor FTS (10 µM) for 1 h, prior to stimulation with either LL-37 or sLL-37 (5 µM each), or LPS (10 ng/ml). TC supernatants were monitored by ELISA for the production of chemokines GRO-α and IL-8 after 24 h. Results shown as mean ± SE of eight independent experiments (n = 8). Analysis of variance with Bonferroni’s post hoc test was used for statistical analyses (***p < 0.0005).
Figure 2
Figure 2
LL-37-induced anti-inflammatory cytokine IL-1RA production is independent of Rho and Ras GTPase activity. Macrophage-like THP-1 cells were pre-incubated with either (A) the Cdc42/Rac1 inhibitor ML141 (10 µM) or (B) Ras inhibitor FTS (10 µM), for 1 h prior to stimulation with either LL-37 (5 µM), sLL-37 (5 µM), lipopolysaccharide (10 ng/ml), or 0.01% DMSO as vehicle control (VC). Tissue culture supernatants were monitored by ELISA for the production of the anti-inflammatory cytokine IL-1RA after 48 h. Results shown as mean ± SE of eight independent experiments (n = 8). Analysis of variance with Bonferroni’s post hoc test was used for statistical analyses (***p < 0.0005).
Figure 3
Figure 3
LL-37-mediated JNK mitogen-activated protein kinase (MAPK) phosphorylation is dependent on Cdc42/Rac1 RhoGTPase. Macrophage-like THP-1 cells were pre-incubated with Cdc42/Rac1 inhibitor ML141 (10 µM) for 1 h prior to stimulation with either LL-37 (5 µM), sLL-37 (5 µM), or recombinant human IL-32γ (20 ng/ml), for 15 min. (A) Cell lysates (each containing 20 µg total protein each) were probed with phospho-JNK (T183/Y185) antibody by western blots. Antibodies against total-JNK and β-actin were used as loading controls. Densitometry analyses for western blots represent relative fold change compared to unstimulated cells normalized to 1. Results shown as mean ± SE of five independent experiments (n = 5). (B) Cell lysates (40 µg each) were monitored by PathScan® phospho-JNK (Thr183/Tyr185) Sandwich ELISA for relative quantitative estimation of JNK MAPK phosphorylation. Results shown as mean ± SE of four independent experiments (n = 4). Analysis of variance with Bonferroni’s post hoc test was used for statistical analyses (*p < 0.05, **p < 0.005).
Figure 4
Figure 4
Cdc42/Rac1 Rho GTPase controls LL-37-induced chemokine secretion in human peripheral blood-derived mononuclear cells (PBMCs). Human PBMCs were pre-incubated with Cdc42/Rac1 inhibitor ML141 (10 µM) for 1 h prior to stimulation with either LL-37 (5 µM), sLL-37 (5 µM), or lipopolysaccharide (10 ng/ml). Tissue culture supernatants were monitored by ELISA for the production of chemokines (A) GRO-α, (B) IL-8, and (C) monocyte chemotactic protein-1 after 24 h, and (D) the anti-inflammatory cytokine IL-1RA after 48 h. Results shown are from five independent experiments (n = 5) using PBMCs isolated from independent donors, each dot represents an independent donor and the line shown is the median for each condition. Kruskal–Wallis analysis of variance with Dunn’s multiple comparison test was used for statistical analyses (*p < 0.05, **p < 0.005, ***p < 0.0005).
Figure 5
Figure 5
Cdc42/Rac1 Rho GTPase controls LL-37-induced chemokine secretion and JNK mitogen-activated protein kinase (MAPK) phosphorylation in human monocyte-derived macrophages (MDM). Human MDMs were pre-incubated with Cdc42/Rac1 inhibitor ML141 (10 µM) for 1 h prior to stimulation with either LL-37 (5 µM), sLL-37 (5 µM), or lipopolysaccharide (10 ng/ml). Tissue culture supernatants were monitored by ELISA for the production of chemokines (A) GRO-α and (B) IL-8 after 24 h, and (C) IL-1RA after 48 h. (D) Human MDMs were pre-incubated with Cdc42/Rac1 inhibitor ML141 (10 µM) for 1 h prior to stimulation with either LL-37 (5 µM), sLL-37 (5 µM), or recombinant human IL-32γ (20 ng/ml), for 15 min. Cell lysates (40 µg each) were monitored by PathScan® phospho-JNK (Thr183/Tyr185) Sandwich ELISA for relative quantitative estimation of JNK MAPK phosphorylation. Results shown are from three independent experiments (n = 3) using MDMs isolated from independent donors. Each dot represents an independent donor and the line shown is the median for each condition. Kruskal–Wallis analysis of variance with Dunn’s multiple comparison test was used for statistical analyses (*p < 0.05).
Figure 6
Figure 6
Cdc42/Rac1 Rho GTPases controls LL-37-mediated leukocyte migration. Human peripheral blood-derived mononuclear cells (PBMCs) were pre-incubated with the Cdc42/Rac1 inhibitor ML141 (10 µM) for 1 h prior to stimulation with either LL-37 or sLL-37 (5 µM each). PBMC tissue culture supernatants collected after 24 h were used in transwell cell migration assays to monitor the migration of (A) THP-1 monocytic cells and (B) human neutrophils. RPMI medium spiked with chemokines monocyte chemotactic protein-1, IL-8, and GRO-α (30 ng/ml each) were used as experimental controls as indicated. Results shown are from seven independent experiments using PBMCs isolated from independent donors (n-7). Each dot represents an independent donor and the median line is shown for each condition. Kruskal–Wallis analysis of variance with Dunn’s multiple comparison test was used for statistical analyses (*p < 0.05, **p < 0.005).
Figure 7
Figure 7
LL-37 activates Cdc42 Rho GTPase. Macrophage-like THP-1 cells were serum starved for 1 h prior to stimulation with either LL-37 (5 µM), sLL-37 (5 µM each), or lipopolysaccharide (10 ng/ml), and cell lysates collected after 5 min. The activities of (A) Cdc42 and (B) Rac1 Rho GTPases were determined using respective G-LISA assays. Positive control refers to active Cdc42 or Rac 1 protein provided in the G-LISA kit by the manufacturer. Results shown as mean ± SE of six independent experiments (n = 6). Analysis of variance with Bonferroni’s post hoc test was used for statistical analyses (*p < 0.05, **p < 0.005).
Figure 8
Figure 8
Knockdown of Cdc42 Rho GTPase suppresses LL-37-induced chemokine production, but not anti-inflammatory cytokine IL-1RA. Human monocytic THP-1 cells were treated with either human Cdc42 Accell SiRNA smartpool (1 µM) or non-target control (NTC) Accell SiRNA smartpool (1 µM) for 96 h. The efficiency of knockdown was determined by western blots probing with Cdc42 antibody and protein loading using actin antibody, after (A) 96 h of SiRNA delivery, and subsequently following cell differentiation to plastic-adherent, macrophage-like THP-1 cells after additional (B) 24 and (C) 48 h. Macrophage-like THP-1 cells, either wild type, knockdown (KD), or cells treated with 50 µl SiRNA buffer (BC), were stimulated with LL-37 (5 µM), sLL-37 (5 µM each) or lipopolysaccharide (10 ng/ml). Tissue culture supernatants were monitored by ELISA for the production of chemokines (D) GRO-α and (E) IL-8 after 24 h, and for (F) anti-inflammatory cytokine IL-1RA after 48 h. Results shown as mean ± SE of three independent experiments (n = 3). Analysis of variance with Bonferroni’s post hoc test was used for statistical analyses (**p < 0.005, ***p < 0.0005).
Figure 9
Figure 9
LL-37-induced activation of Cdc42/Rac1 Rho GTPase and chemokine secretion is mediated through G protein-coupled receptor (GPCR). Macrophage-like THP-1 cells were treated 100 ng/ml of the GPCR inhibitor pertussis toxin for 1 h, prior to stimulation with either LL-37 (5 µM), sLL-37 (5 µM each), or lipopolysaccharide (10 ng/ml). (A) Cells lysates were monitored for Cdc42 activation after 5 min of stimulation. Results shown as mean ± SE of five independent experiments (n = 5). Tissue culture supernatants were monitored by ELISA for the production of chemokines (B) GRO-α and (C) IL-8 after 24 h, and (D) anti-inflammatory cytokine IL-1RA after 48 h. ELISA results shown are mean ± SE of six independent experiments (n = 6). Analysis of variance with Bonferroni’s post hoc test was used for statistical analyses (***p < 0.0005).
Figure 10
Figure 10
Model describing mechanism of LL-37-mediated chemokine secretion and leukocyte recruitment. Based on results from this study and previous studies, we propose that LL-37 can interact with G protein-coupled receptor (GPCR) to further activate (→) Cdc42 Rho GTPase (8, 11), resulting in enhanced phosphorylation of JNK mitogen-activated protein kinase (MAPK) (T183/Y185). The LL-37-GPCR-Cdc42 RhoGTPase axis signals via the JNK MAPK pathway to induce the production of chemokines and promote leukocyte migration. Alternatively, intracellular uptake of LL-37 may be mediated by other receptors (37) or using mechanisms similar to cell-penetrating peptides involving the cytoskeletal machinery (62). LL-37-induced anti-inflammatory cytokine IL-1RA production is independent of both GPCR and Cdc42/Rac1 Rho GTPases.
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