Distinct signalling pathways promote phagocytosis of bacteria, latex beads and lipopolysaccharide in medfly haemocytes

Abstract

In insects, phagocytosis is an important innate immune response against pathogens and parasites, and several signal transduction pathways regulate this process. The focal adhesion kinase (FAK)/Src and mitogen activated protein kinase (MAPK) pathways are of central importance because their activation upon pathogen challenge regulates phagocytosis via haemocyte secretion and activation of the prophenoloxidase (proPO) cascade. The goal of this study was to explore further the mechanisms underlying the process of phagocytosis. In particular, in this report, we used flow cytometry, RNA interference, enzyme-linked immunosorbent assay, Western blot and immunoprecipitation analysis to demonstrate that (1) phagocytosis of bacteria (both Gram-negative and Gram-positive) is dependent on RGD-binding receptors, FAK/Src and MAPKs, (2) latex bead phagocytosis is RGD-binding-receptor-independent and dependent on FAK/Src and MAPKs, (3) lipopolysaccharide internalization is RGD-binding-receptor-independent and FAK/Src-independent but MAPK-dependent and (4) in unchallenged haemocytes in suspension, FAK, Src and extracellular signal-regulated kinase (ERK) signalling molecules participating in phagocytosis show both a functional and a physical association. Overall, this study has furthered knowledge of FAK/Src and MAPK signalling pathways in insect haemocyte immunity and has demonstrated that distinct signalling pathways regulate the phagocytic activity of biotic and abiotic components in insect haemocytes. Evidently, the basic phagocytic signalling pathways among insects and mammals appear to have remained unchanged during evolution.

Figure 1

Bacteria and cell-surface LPS adhered to and were engulfed by medfly haemocytes. (a) Suspended haemocytes (5 × 10⁵ cells/100 ml medium) from wandering larval stage were incubated for 30 min with either FITC-labelled E. coli (i, v) or FITC-labelled S. aureus (ii, vi) or FITC-labelled LPS (iii, vii) or FITC-labelled latex beads (iv, viii) and the uptake of these components by haemocytes was analysed using flow cytometry, either after trypan blue quenching (v–viii), or not (i–iv). (b) and (c) Kinetic analysis of E. coli and S. aureus with flow cytometry. (d) Identification of E. coli phagocytosis by confocal microscopy. Haemocytes were fixed and stained with antibodies against mouse tubulin and propidium iodide. MIF, medium intense fluorescence.

Figure 2

FAK regulates phagocytosis of E. coli/S. aureus/latex beads in medfly haemocytes. Suspended haemocytes (5 × 10⁵ cells/100 ml medium) from wandering larval stage were incubated with either FITC-labelled E. coli (a–c), FITC-labelled S. aureus (d–f), FITC-labelled LPS (g–i), or FITC-labelled latex beads (j–l), after preincubation for 6 hr at 25° with 8 μg FAK dsRNA (b, e, h, k). After trypan blue quenching, the haemocytes were analysed with flow cytometry. Control experiments, after trypan blue quenching, were performed in haemocyte suspensions in plain medium (a, d, g, j) or with 8 μg paxillin dsRNA (c, f, i, l). MIF, medium intense fluorescence.

Figure 3

Pathogens, latex beads, LPS, fibronectin (FN) and RGD phosphorylate FAK at Y397. (a) Haemocyte suspensions stimulated with E. coli (lane 1), S. aureus (lane 2), LPS (lane 3), latex beads (lane 4), FN (lane 5), and RGD (lane 6). A haemocyte suspension that was stimulated with RGE (lane 7) was used as negative control. Haemocytes incubated in Grace's medium (lanes 8 and 9) were used to show any minor activation because of the isolation procedure. Cell lysates were subjected to immunoblotting analysis with anti-p^Y397FAK. Actin was used as a loading control. (b) Phosphorylation of FAK at Y397 was also detected immunocytochemically. Suspended haemocytes were attached to glass slides and were at first incubated with anti-p^Y397FAK (i, iii, iv), followed by a secondary anti-rabbit antibody coupled to a fluorochrome (FITC), as described in the Materials and methods. Samples that were incubated only with secondary rabbit antibody FITC (ii) were used to detect any non-specific binding sites of secondary antibody. Haemocytes incubated in plain medium were used as a control (i).

Figure 4

RGD-binding receptors regulate phagocytosis of E. coli and S. aureus in medfly haemocytes. Suspended haemocytes (5 × 10⁵ cells/100 ml medium) from wandering larval stage were incubated for 30 min with either FITC-labelled E. coli (a–d) or FITC-labelled S. aureus (e–h) or FITC-labelled LPS (i–l) or FITC-labelled latex beads (m–p), after preincubation for 30 min at 25° with 2 mm RGD (b, f, j, n) or 0·01γ/μl FN (c, g, k, o). Control samples were preincubated in plain medium (a, e, i, m) or in 2 mm RGE (d, h, l, p). After trypan blue quenching, haemocytes were analysed with flow cytometry.

Figure 5

Src is involved in the phagocytosis process of bacteria and latex beads. (a) Suspended haemocytes (5 × 10⁵ cells/100 ml medium) from wandering larval stage were stimulated with FITC-labelled E. coli (i, ii), FITC-labelled S. aureus (iii, iv), FITC-labelled LPS (v, vi), FITC-labelled latex beads (vii, viii), after preincubation for 30 min at 25° in plain medium (i, iii, v, vii) or in 2 μl PP2 (ii, iv, vi, viii). After trypan blue quenching, haemocytes were analysed with flow cytometry. (b) The involvement of Src in phagocytosis was detected immunocytochemically. Suspended haemocytes were attached on glass slides and were at first incubated with non-p⁵²⁷Src (i, iii, iv), followed by a secondary anti-rabbit antibody coupled to a fluorochrome (FITC), as described in the Materials and methods. Samples that were incubated only with secondary anti-rabbit antibody FITC (ii) were used to detect any non-specific binding sites of secondary antibody. Haemocytes incubated in plain medium were used as a control (i). MIF, medium intense fluorescence.

Figure 6

RGD-binding receptors phosphorylate MAP kinases to the same extent as pathogens, LPS and latex beads. (a) Haemocyte suspensions were stimulated with E. coli (lane 2), S. aureus (lane 3), latex beads (lane 4), LPS (lane 5), FN (lane 6), and RGD (lane 7). Haemocytes incubated in Grace's medium (lane 1) were used to show any minor activation caused by the isolation procedure. Cell lysates were subjected to immunoblotting analysis with anti-pJNK. Membrane was stripped and reprobed with anti-pp38. Actin was used as a loading control. (b) Haemocyte suspensions were stimulated with E. coli (lane 2), S. aureus (lane 3), latex beads (lane 4), LPS (lane 5), FN (lane 6), and RGD (lane 7). Haemocytes incubated in Grace's medium (lane 1) were used to show any minor activation caused by the isolation procedure. Cell lysates were subjected to immunoblotting analysis with anti-pERK. Actin was used as a loading control. (c) Phosphorylation of MAP kinases was detected immunocytochemically. Suspended haemocytes were attached on glass slides and were at first incubated with anti-pp38 (iii), anti-pJNK (iv), anti-pERK (v), followed by a secondary anti-rabbit antibody coupled to a fluorochrome (FITC). Samples that were incubated only with secondary rabbit antibody FITC (ii) were used to detect any non-specific binding sites of secondary antibody. Haemocytes incubated in plain medium were used as a control (i).

Figure 7

Effect of FAK silencing on the phosphorylation of ERK, p38, JNK, MEK and Src. Suspended haemocytes from wandering larval stage were either incubated for 5½ hr in Grace followed by 30 min treatment with E. coli (E. coli) or incubated in the presence of FAK dsRNA for up to 6 hr, but for the last 30 min E. coli was added (FAK dsRNA + E. coli). Haemocytes incubated in plain medium were used as a control (control). Afterwards, the haemocytes were lysed and the lysates were plated in a 96-well assay plate and analysed by ELISA to detect the status of ERK, p38, JNK, MEK and Src phosphorylation using polyclonal antibodies against pERK (a), pp38 (b), pJNK (c), pMEK (d), p^Y416Src (e), and p^Y527Src (f), respectively. Each well contained 4 μg/ml haemocyte lysate.

Figure 8

Src interacts with FAK and MAP kinases act downstream of FAK. Suspended haemocytes were stimulated with E. coli (lanes 2–9) after preincubation for 30 min at 25° with Src inhibitor, 5 μm PP2 (lane 3) or with kinase inhibitors, 0·4 μm SB 203580 (lane 4) and 10 μm SB 202190 (lane 5) for p38, 4 μm SP 600125 (lanes 6 and 7) for JNK and 10 μl U0126 (lane 8) for MEK1/2 and 4 μm PD 098059 (lane 7) for ERK. Haemocytes incubated in Grace's medium (lane 1) were used to show any minor activation as a result of the isolation procedure. Cell lysates were subjected to immunoblotting analysis with anti-pFAK Y397. Actin was used as a loading control.

Figure 9

Co-immunoprecipitation and reciprocal co-immunoprecipitation of FAK, Src and ERK signalling molecules. Suspended haemocytes from wandering larval stages were isolated and then lysed. FAK, Src or ERK were immunoprecipitated from the lysates. When FAK was immunoprecipitated, using FAK polyclonal antibody, the precipitates were then resolved on 10% SDS–PAGE and blotted with polyclonal antibodies against Src and ERK. When Src or ERK were immunoprecipitated using polyclonal antibodies against Src and ERK, the precipitates were then resolved on 10% SDS–PAGE and blotted with FAK polyclonal antibody.

Figure 10

Proposed signalling model for the engulfment of pathogens or latex beads or LPS by medfly haemocytes.