Anti-tumour effects of antimicrobial peptides, components of the innate immune system, against haematopoietic tumours in Drosophila mxc mutants

Abstract

The innate immune response is the first line of defence against microbial infections. In Drosophila, two major pathways of the innate immune system (the Toll- and Imd-mediated pathways) induce the synthesis of antimicrobial peptides (AMPs) within the fat body. Recently, it has been reported that certain cationic AMPs exhibit selective cytotoxicity against human cancer cells; however, little is known about their anti-tumour effects. Drosophila mxc^mbn1 mutants exhibit malignant hyperplasia in a larval haematopoietic organ called the lymph gland (LG). Here, using RNA-seq analysis, we found many immunoresponsive genes, including those encoding AMPs, to be upregulated in these mutants. Downregulation of these pathways by either a Toll or imd mutation enhanced the tumour phenotype of the mxc mutants. Conversely, ectopic expression of each of five different AMPs in the fat body significantly suppressed the LG hyperplasia phenotype in the mutants. Thus, we propose that the Drosophila innate immune system can suppress the progression of haematopoietic tumours by inducing AMP gene expression. Overexpression of any one of the five AMPs studied resulted in enhanced apoptosis in mutant LGs, whereas no apoptotic signals were detected in controls. We observed that two AMPs, Drosomycin and Defensin, were taken up by circulating haemocyte-like cells, which were associated with the LG regions and showed reduced cell-to-cell adhesion in the mutants. By contrast, the AMP Diptericin was directly localised at the tumour site without intermediating haemocytes. These results suggest that AMPs have a specific cytotoxic effect that enhances apoptosis exclusively in the tumour cells.

Keywords: AMPs; Cytotoxicity; Drosophila; Fat body; Innate immunity; Lymph gland.

Fig. 1.

Hyperplasia of the larval LG in mxc^mbn1 larvae. (A) Schematic of the LG in Drosophila 3rd instar larvae. The lobes are defined as clusters of haematopoietic cells arranged in a hemispheric pattern aligned segmentally in pairs along the A-P axis of the gland and comprise a larger primary (1st) lobe, a secondary (2nd) lobe and a tertiary (3rd) lobe. The 1st lobe consists of a cortical zone (CZ), medullary zone (MZ) and posterior signalling centre (PSC). At the posterior end of these lobes, a cluster of connected pericardial cells (PCs) are lined in a row. (B) DAPI-stained whole LG isolated from normal control. Normal control LG is arranged bilaterally so that it flanks the dorsal vessel at the midline. (C) DAPI-stained whole lobes with part of the PC row isolated from hemizygous larvae (mxc^mbn1/Y) for mxc^mbn1. LG tumour overgrowth is visible in the mxc^mbn1 mutant larva. The anterior lobes are more prominently enlarged. (D) Quantification of tumour size. The right or left halves of whole LG regions of the mutant LGs (mean=0.266 mm², n=35) are five times larger than those of normal control (mean=0.047 mm², n=21) (Welch's t-test, ****P<0.0001). Error bars represent s.e.m.; red horizontal lines represent the mean. (E-H) Hyperplasia and abnormal distribution of differentiated cells labelled by Hml>GFP (E,F) and undifferentiated haemocyte precursors labelled by upd3>GFP (G,H). GFP fluorescence images of LGs prepared from normal control (E,G) and mxc^mbn1 mutant larvae (F,H) at mature 3rd instar stage. Magenta, DAPI staining; green, GFP fluorescence. Scale bars: 100 μm.

Fig. 2.

Activation of two innate immune pathways in the mxc^mbn1 mutant. The relative mRNA levels of the target genes of innate immune pathways in control (w/Y) larvae, tumour-harbouring larvae hemizygous for mxc^mbn1 and larvae hemizygous for non-tumourous mxc^G43are shown as quantified by qRT-PCR. The mRNA levels for samples were normalised to control values. Error bars represent s.e.m. Drs and Def are controlled by the Toll pathway. Dpt is regulated by the Imd pathway. Mtk, AttA and CecA2 are controlled by both the Toll and Imd pathways. Notably, all six AMP genes are specifically upregulated in the mxc^mbn1 mutant larvae.

Fig. 3.

Enhancement of AMP expression in the mxc^mbn1 mutant. (A,B,E,F) Bright-field images of control (w/Y) (A,E) and mxc^mbn1 (B,F) whole larvae. (C,D,G,H,I,J,K,L) Bright-field images of the larval fat bodies around the salivary glands from the control (C,G,I,K) and mxc^mbn1 (D,H,J,L) larvae, raised on normal diet (A-H) or on a diet containing antibiotics (I-L). (A′,B′) Fluorescence images of whole larvae carrying Drs-YFP in control (A′) and mxc^mbn1 (B′). (C′,D′) Fluorescence images of larval fat bodies carrying Drs-YFP from control (C′) and mxc^mbn1 larvae (D′). Notably, Drs-YFP was induced in fat bodies of the mxc^mbn1 mutant, but not in the control. (E′,F′) Fluorescence images of the whole larvae carrying Dpt-YFP in control (E′) and mxc^mbn1(F′). (G′,H′) Fluorescence images of larval fat bodies caring Dpt-YFP from control (G′) and mxc^mbn1 larvae (H′). Notably, Dpt-YFP was also induced in fat bodies of the mxc^mbn1 mutant, but not in control. (I′-L′) Fluorescence images of larval fat bodies carrying Drs-YFP (I′,J′) or Dpt-YFP (K′,L′) from control (I′,K′) and mxc^mbn1 larvae (J′,L′). Notably, Drs-YFP and Dpt-YFP were also induced in fat bodies of the mxc^mbn1 larvae raised under aseptic conditions. Scale bars: 500 μm.

Fig. 4.

Overexpression of innate immune pathway components suppresses LG tumour overgrowth. (A-G) LGs stained with DAPI from mature larvae at 3rd instar stage showing a control larva (w/Y) (A), an mxc^mbn1 larva (B), LGs from mxc^mbn1 larvae overexpressing GFP (C) or Dl (D) by Lsp2-Gal4, or LGs from mxc^mbn1 larvae overexpressing GFP (E), Tl^10B (constitutively active form of Toll) (F) or Rel.68 (constitutively active form of Rel) (G). (H) Quantification of tumour size (Welch's t-test, n.s. not significant, ****P<0.0001). Error bars represent s.e.m. The horizontal red lines represent the mean. Scale bars: 100 μm.

Fig. 5.

Fat body-specific induction of AMP genes that are targets of innate immunity pathways in mxc^mbn1 larvae. (A-H) LGs stained with DAPI from a control (r4-Gal4/+) larva (A), from mxc^mbn1 mutant larvae (B) or from mxc^mbn1 larvae expressing GFP (mxc^mbn1/Y; r4>GFP) (C), Drs (mxc^mbn1/Y; r4>Drs) (D), Def (mxc^mbn1/Y; r4>Def) (E), Dpt (mxc^mbn1/Y; r4>Dpt) (F), Mtk (mxc^mbn1/Y; r4>Mtk) (G) and AttA (mxc^mbn1/Y; r4>AttA) (H). (I) Quantification of tumour size (Welch's t-test, n.s. not significant, **P<0.01, ****P<0.0001). The error bars represent s.e.m. The horizontal red lines represent the mean. Scale bars: 100 μm.

Fig. 6.

Observation of LG cells undergoing apoptosis in mxc^mbn1 larvae overexpressing AMPs in the fat bodies. (A-G,I-N) Cell Event Caspase-3/7 Detection Reagent assays in LGs (A-G), wing and haltere discs (I-K), and eye discs (L-N). Red signals in A-G and I-N, and white signals in A′-G′ and I′-N′, represent cells containing activated caspases. Blue, DAPI staining. (A-G) Fluorescence of LG. (A-C,A′-C′) Control LG expressing Drs (r4>Drs) (A,A′), Def (r4>Def) (B,B′) and Dpt (r4>Dpt) (C,C′). Notably, no apoptotic signals are evident. LGs of mxc^mbn1 larvae expressing GFP (D,D′), Drs (E,E′), Def (F,F′) and Dpt (G,G′) in their fat bodies. The LG of mxc^mbn1 contains many apoptotic signals (B,B′). LGs of mxc^mbn1 larvae overexpressing Drs (C,C′), Def (D,D′) and Dpt (E,E′) show more apoptotic signals than mxc^mbn1 larvae. (H) Proportion of apoptotic area in total LG area (Welch's t-test, **P<0.01, ***P<0.001). The error bars represent s.e.m. Red horizontal lines represent the mean. (I-K) Fluorescence of wing and haltere discs. (L-N) Fluorescence of eye discs. No apoptotic signals can be seen in the wing and haltere discs or eye discs in the control larvae overexpressing Drs or Dpt genes in their fat bodies. Scale bars: 100 μm.

Fig. 7.

Anti-PH3 immunostaining of LGs from mature larvae to visualise mitotic cells. (A-C′) LGs from 3rd instar larvae at a mature stage. A whole LG from normal control larva (A), whole LG lobes from r4>Def (B) and whole LG lobes from r4>Dpt (C). (D-F) Anterior lobes of LGs from mxc^mbn1 larvae (D), mxc^mbn1 overexpressing Def (E) and mxc^mbn1 overexpressing Dpt (F) at mature 3rd instar stage. Magenta, DAPI staining; green, anti-PH3 (Ser10) immunostaining. (G) Percentage of the area occupied by PH3-positive cells in LG lobes within the total area of LG lobes (except pericardial cells). The error bars represent s.e.m. Red horizontal lines represent the mean. (Welch's t-test; n.s., not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Scale bar: 100 μm.

Fig. 8.

Visualisation of AMP on LGs and in circulating haemocytes from normal control and mxc^mbn1 larvae overexpressing AMPs. (A-L″) Localisation on LGs of AMPs fused with HA-tag showing control LG (r4>Drs) (A,B), LGs from mxc^mbn1 overexpressing Drs (mxc^mbn1/Y; r4>Drs) (C,D), control LG (r4>Def) (E,F), LGs from mxc^mbn1 overexpressing Def (mxc^mbn1/Y; r4>Def) (G,H), control LG (r4>Dpt) (I,J) and LGs from mxc^mbn1 overexpressing Dpt (mxc^mbn1/Y; r4>Dpt) (K,L). (A,C,E,G,I,K) Red, DE-cadherin (Cad); green, AMP (Drosomycin, Defensin or Diptericin); blue (G), DAPI staining. (B,D,F,H,J,L) High-magnification views (of boxed areas in A,C,E,G,I,K, respectively), showing the DNA (magenta) and AMP (green) in the pericardial cells. AMPs are often localised on regions exhibiting reduced DE-cadherin immunostaining signals. Drosomycin and Defensin appear to be taken into haemocyte-like cells (D,H), whereas Diptericin is associated with the LG in a fibrous shape (H). Scale bars: 50 μm.

Fig. 9.

Accumulation of AMPs in circulating haemocytes from normal control and mxc^mbn1 larvae overexpressing AMPs in their fat bodies. (A-L‴) The cellular localisation of AMPs fused with a HA-tag in circulating haemocytes immunostained with anti-P1 antibody. Two typical examples of the haemocytes are shown for each genotype. (A-D) Circulating haemocytes from larvae overexpressing HA-tagged Def in their fat bodies, control (r4>Def) (A,B) and mxc^mbn1/Y; r4>Def (C,D). (E-H) Circulating haemocytes from larvae overexpressing HA-tagged Drs in their fat bodies, control larvae (r4>Drs) (E,F) and mxc^mbn1/Y; r4>Drs (G,H). (I-L) Circulating haemocytes from larvae overexpressing HA-tagged Dpt in their fat bodies, control (r4>Dpt) (I,J) and mxc^mbn1/Y; r4>Dpt (K,L). Red, anti-HA immunostaining to recognise each AMP; green, anti-P1 immunostaining to recognise circulation plasmatocytes; blue, DAPI staining. (M) Quantification of the fluorescence intensity of anti-HA immunostaining images from larvae overexpressing Defensin, Drosomycin and Diptericin. Student's t-test, *P<0.05, ***P<0.001, ****P<0.0001. The error bars represent s.e.m. The horizontal red lines represent the mean. Scale bars: 10 μm.