Insect cytokine paralytic peptide (PP) induces cellular and humoral immune responses in the silkworm Bombyx mori

Abstract

In the blood (hemolymph) of the silkworm Bombyx mori, the insect cytokine paralytic peptide (PP) is converted from an inactive precursor to an active form in response to the cell wall components of microorganisms and contributes to silkworm resistance to infection. To investigate the molecular mechanism underlying the up-regulation of host resistance induced by PP, we performed an oligonucleotide microarray analysis on RNA of blood cells (hemocytes) and fat body tissues of silkworm larvae injected with active PP. Expression levels of a large number of immune-related genes increased rapidly within 3 h after injecting active PP, including phagocytosis-related genes such as tetraspanin E, actin A1, and ced-6 in hemocytes, and antimicrobial peptide genes cecropin A and moricin in the fat body. Active PP promoted in vitro and in vivo phagocytosis of Staphyloccocus aureus by the hemocytes. Moreover, active PP induced in vivo phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) in the fat body. Pretreatment of silkworm larvae with ML3403, a pharmacologic p38 MAPK inhibitor, suppressed the PP-dependent induction of cecropin A and moricin genes in the fat body. Injection of active PP delayed the killing of silkworm larvae by S. aureus, whereas its effect was abolished by preinjection of the p38 MAPK inhibitor, suggesting that p38 MAPK activation is required for PP-dependent defensive responses. These findings suggest that PP acts on multiple tissues in silkworm larvae and acutely activates cellular and humoral immune responses, leading to host protection against infection.

FIGURE 1.

Induction of immune-related genes by BSA or active PP. Silkworm larvae were injected with 50 μl/larva of saline, BSA (66 μg/ml, 1 μm), or active PP (4 μg/ml, 1 μm), and after 3 h RNAs were extracted from hemocytes and fat body tissues. mRNA amounts of immune-related genes in hemocytes (A) and fat body (B) were analyzed by quantitative RT-PCR. mRNA quantity of each gene was normalized to that of a control gene, elongation factor 2, whose expression is not affected by injection of the samples. Furthermore, fold changes in mRNA quantities of BSA- or PP-injected groups relative to saline-injected groups were estimated and are shown on the longitudinal axis. Data represent mean ± S.D. (error bars) of four experiments. Statistical significance was determined by Student's t test (*, p < 0.05; **, p < 0.01).

FIGURE 2.

Time course of induction of phagocytosis-related genes in hemocytes by active PP. mRNA amounts of phagocytosis-related genes (A, tetraspanin E; B, actin A1; C, ced-6) in hemocytes after injection of saline (open circles) or active PP (filled circles) were analyzed by quantitative RT-PCR. mRNA quantity of each gene was normalized to that of a control gene, elongation factor 2, whose expression is not affected by injection of the samples. Furthermore, fold changes in mRNA quantities relative to time 0 (no injection) were estimated and are shown on the longitudinal axis. Data represent mean ± S.D. (error bars) of four or five experiments. Statistical significance was determined by Student's t test (*, p < 0.05).

FIGURE 3.

Time course of induction of antimicrobial peptide genes in the fat body by active PP. mRNA amounts of antimicrobial peptide genes (A, cecropin A; B, moricin) in the fat body after injection of saline (open circles) or active PP (filled circles) were analyzed by quantitative RT-PCR. mRNA quantity of each gene was normalized to that of a control gene, elongation factor 2, whose expression is not affected by injection of the samples. Furthermore, fold changes in mRNA quantities relative to time 0 (no injection) were estimated and are shown on the longitudinal axis. Data represent mean ± S.D. (error bars) of four or five experiments. Statistical significance was determined by Student's t test (*, p < 0.05).

FIGURE 4.

Induction of immune-related genes by peptidoglycan or active PP. Silkworm larvae were injected with saline, peptidoglycan, or active PP, and RNAs were extracted from hemocytes and fat body tissues 3 h later. mRNA amounts of Toll and Imd pathway genes in hemocytes (upper) and fat body (lower) were analyzed by quantitative RT-PCR. The mRNA quantity of each gene was normalized to that of a control gene, elongation factor 2. Furthermore, fold changes in mRNA quantities of peptidoglycan- or PP-injected groups relative to saline-injected groups were estimated and are shown on the longitudinal axis. Data represent mean ± S.D. (error bars) of three to five experiments. Statistical significance was determined by Student's t test (*, p < 0.05; **, p < 0.01).

FIGURE 5.

Effect of active PP on phagocytosis of bacteria by silkworm hemocytes. A, in vitro phagocytosis assay. Hemocytes supplied with the indicated amounts of active PP were incubated with S. aureus for 3 h. After lysing the hemocytes, cfu of S. aureus were determined and normalized to the number of hemocytes. Fold changes in cfu relative to the sample incubated without PP (0 ng/ml) are shown. Data represent mean ± S.D. (error bars) of five experiments. Statistical significance was determined by Student's t test (*, p < 0.05). B, microscope image of fluorescent (Alexa Fluor 594)-labeled S. aureus colocalized with silkworm hemocytes. Hemocytes were collected and fixed 3 h after injection of labeled bacteria. C, in vivo phagocytosis of fluorescence-labeled bacteria. Silkworms were injected with fluorescence-labeled S. aureus. After 3 h, cells were collected, and the numbers of fluorescent particles colocalized with hemocytes were counted. Values were normalized to the number of hemocytes. Statistical significance was determined by Student's t test (*, p < 0.05). D, in vivo phagocytosis of live bacteria. Silkworms were injected with the indicated numbers of live S. aureus cells. After 3 h, hemocytes were collected and lysed to determine the number of internalized bacteria; cfu normalized to the number of hemocytes are shown. Data represent mean ± S.D. of four larvae. Statistical significance was determined by Student's t test (*, p < 0.05). E, in vitro binding assay of active PP to bacteria. S. aureus was incubated with active PP in PBS for 3 h, and samples were separated into supernatant fractions (S) and precipitate fractions (P). The samples were analyzed by immunoblotting.

FIGURE 6.

Effect of active PP on the in vivo activation of p38 MAPK in silkworm fat body. A, phosphorylation of p38 MAPK in silkworm fat body induced by active PP. Eight larvae/group were injected with saline or active PP, and after 20 or 60 min the fat body tissues were collected. Tissue samples were analyzed by immunoblot. B, effect of BSA on p38 MAPK phosphorylation in the silkworm fat body. Five larvae/group were injected with 50 μl of BSA (66 μg/ml, 1 μm) or active PP (4 μg/ml, 1 μm), and the fat body tissues were collected 30 min later. Tissue samples were analyzed by immunoblot. C, effect of a pharmacologic p38 inhibitor on the induction of AMP gene expression by active PP. Ten larvae/group were treated with 10% DMSO (a solvent) or ML3403 prior to the injection of saline or active PP, and the fat body tissues were dissected 3 h later. The mRNA amounts of cecropin A and moricin were evaluated by quantitative RT-PCR, and the values were normalized to the mRNA amounts of elongation factor 2. In each pretreatment condition (DMSO or ML3403), PP-dependent fold-changes relative to saline-injected groups in the cecropin A and moricin mRNA are shown. Data represent mean ± S.D. (error bars) of four experiments. Statistical significance was determined by Student's t test (*, p < 0.05). D, effect of ML3403, a pharmacologic p38 inhibitor, on PP-dependent up-regulation of host resistance to bacterial infection. Silkworm larvae were treated with 10% DMSO or ML3403 prior to injection of saline or active PP mixed with a suspension of S. aureus.

FIGURE 7.

Regulation of cellular and humoral immune responses by insect cytokine PP.