Human cathelicidin LL-37 and its derivative IG-19 regulate interleukin-32-induced inflammation

Abstract

Human cathelicidin LL-37 protects against infections and endotoxin-induced inflammation. In a recent study we have shown that IG-19, an LL-37-derived peptide, protects in a murine model of arthritis. Cytokine interleukin-32 (IL-32) is elevated and directly associated with the disease severity of inflammatory arthritis. Therefore, in this study we examined the effects of LL-37 and IG-19 on IL-32-induced responses in human peripheral blood-derived mononuclear cells (PBMC) and macrophages. We showed that CD14(+) monocytes are the primary cells that produce pro-inflammatory tumour necrosis factor-α (TNF-α) following stimulation of PBMC with IL-32. We demonstrated that LL-37 and IG-19 significantly suppress IL-32-induced production of pro-inflammatory cytokines, e.g. TNF-α and IL-1β, without altering chemokine production. In contrast, LL-37 and IG-19 enhance the production of the anti-inflammatory cytokine IL-1RA. Further mechanistic studies revealed that LL-37 and IG-19 suppress IL-32-mediated phosphorylation of Fyn (Y420) Src kinase. In contrast, IL-32-mediated phosphorylation of AKT-1 (T308) and MKP-1 (S359) is not suppressed by the peptides. LL-37 and IG-19 alone induce the phosphorylation of MKP-1 (S359), which is a known negative regulator of inflammation. Furthermore, the peptides induce the activity of p44/42 mitogen-activated protein kinase, which is known to phosphorylate MKP-1 (S359). This is the first study to demonstrate the regulation of IL-32-induced inflammation by LL-37 and its derivative peptide IG-19. The mechanistic results from this study suggest that regulation of immune-mediated inflammation by these peptides may be controlled by the dual phosphatase MKP-1. We speculate that LL-37 and its derivatives may contribute to the control of immune-mediated inflammatory diseases.

Keywords: LL-37; cathelicidin; host defence peptides; inflammation; interleukin-32.

Figure 1

Production of pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated with interleukin-32γ (IL-32γ; 20 ng/ml) in the presence and absence of 5 μm of LL-37, IG-19 or sLL-37, for 24 hr. Tissue culture supernatants were monitored for production of (a) tumour necrosis factor-α (TNF-α), (b) IL-1β and (c) IL-6 by ELISA. Results shown are an average of six independent biological experiments performed with PBMC isolated from different donors ± standard error. The P-values are calculated using Student's paired t-test.

Figure 2

Production of anti-inflammatory cytokines in human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated with interleukin-32γ (IL-32γ; 20 ng/ml) in the presence and absence of 5 μm of either LL-37 or IG-19. Tissue culture supernatants were monitored for the production of cytokines (a) IL-1RA (n = 9) and (b) IL-10 (n = 11), after 48 hr by ELISA. Results shown are an average of independent biological experiments performed with PBMC isolated from different donors ± standard error. The P-values are calculated using Student's paired t-test (NS = non-significant).

Figure 3

Flow cytometry analysis of interleukin-32γ (IL-32γ)-induced tumour necrosis factor-α (TNF-α) production in human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated with IL-32γ (20 ng/ml) in the presence and absence of LL-37, IG-19 or sLL-37 (5 μm each). The cells were stimulated with bacterial lipopolysaccharide (LPS) as a positive control. After 18 hr of incubation at 37°, cells were stained with antibodies for specific surface markers, fixed and permeabilized, followed by staining with anti-TNF-α antibody. (a) TNF-α⁺ cells were gated, and (b) CD14⁺ cells were gated.

Figure 4

Production of pro-inflammatory cytokines in macrophages. (a) Differentiated THP-1 macrophages and (b) human monocyte-derived macrophages (MDM) were stimulated with interleukin-32γ (IL-32γ; 20 ng/ml) in the presence and absence of 5 μm of LL-37, IG-19, or sLL-37, for 24 hr. Tissue culture supernatants were monitored for production of tumour necrosis factor-α (TNF-α), IL-1β and IL-6 by ELISA. Results shown are an average of eight independent experiments with THP-1 cells, and at least six independent biological experiments performed with MDM isolated from different donors, ± standard error. The P-values are calculated using Student's paired t-test (NS = non-significant).

Figure 5

Production of anti-inflammatory interleukin-1 receptor agonist (IL-1RA) in macrophages. (a) Differentiated THP-1 macrophages and (b) human monocyte-derived macrophages (MDM) were stimulated with IL-32γ (20 ng/ml) in the presence and absence of 5 μm of either LL-37 or IG-19. Tissue culture supernatants were monitored for production of IL-1RA after 48 hr by ELISA. Results shown are an average of five independent experiments with THP-1 cells, and six independent biological experiments performed with MDM isolated from different donors, ± standard error. The P-values are calculated using Student's paired t-test (NS = non-significant).

Figure 6

Western blot analysis of protein phosphorylation in response to interleukin-32γ (IL-32γ). Differentiated THP-1 macrophages were stimulated with IL-32γ (20 ng/ml) in the presence or absence of either LL-37 or IG-19 (5 μm each) for 15 min. Cell lysates were probed in Western blots with specific antibodies to human (a) phospho-Fyn (Y420), (b) phospho-MKP-1 (S359) and (c) phospho-AKT-1 (T308). Antibodies to the non-phosphorylated forms of Fyn and AKT-1, and β-actin were used as paired loading control. The immunoblots shown are representative of at least three independent experiments. (d) Densitometry; ratio of band density of the phosphorylated proteins over unstimulated cells (y-axis), was calculated after normalization to band density of loading paired controls of the respective proteins for each sample. Results represent an average of at least three independent experiments. The P-values are calculated using Student's paired t-test (*P < 0·05, **P < 0·01, NS = non-significant, compared with unstimulated control cells).

Figure 7

Activation of p44/42 mitogen-activated protein kinase (MAPK). Differentiated THP-1 macrophages were stimulated with interleukin-32γ (IL-32γ) (20 ng/ml) in the presence or absence of either LL-37 or IG-19 (5 μm each). Equal amounts of total protein (150 μg) were immunoprecipitated with a monoclonal antibody to phospho-p44/42 MAPK (Thr 202/Tyr 204) for all cell lysates. The immunoprecipitated eluates were incubated with p44/42 MAPK substrate Elk-1 in the presence of ATP, and the activity of p44/42 MAPK was evaluated by monitoring the phosphorylation of the substrate Elk-1 using an anti-phospho-Elk-1 (Ser383) specific antibody. Total cell lysates (5 μg) were probed with β-actin-specific antibody as a measure of input protein. (a) Kinetics of IL-32γ-mediated p44/42 MAPK activity, and (b) effect of the peptides LL-37 and IG-19 on IL-32γ-mediated p44/42 MAPK activity. Results shown are representative of at least three independent experiments.

Figure 8

MKP-1 may be a critical mediator of the regulatory function of LL-37 and derivatives. Human cathelicidin LL-37 and its derivative peptide IG-19 promotes (↑) the activation of p44/42 mitogen-activated protein kinase (MAPK) and subsequent phosphorylation of dual phosphatase MKP-1 (S359). Phospho-MKP-1 (S359) inhibits (|) interleukin-32γ (IL-32γ)-induced activation of p38 and Jun-N-terminal kinase (JNK) MAPK, and nuclear factor-κB (NF-κB) signalling, resulting in the suppression of production of pro-inflammatory cytokines such as tumour necrosis factor (TNF-α). Hence, the dual phosphatase MKP-1 may be a key player in the regulatory role of cathelicidin LL-37 and its derivatives, resulting in the control of IL-32γ-induced inflammation.