Injury-Induced Innate Immune Response During Segment Regeneration of the Earthworm, Eisenia andrei

Abstract

Regeneration of body parts and their interaction with the immune response is a poorly understood aspect of earthworm biology. Consequently, we aimed to study the mechanisms of innate immunity during regeneration in Eisenia andrei earthworms. In the course of anterior and posterior regeneration, we documented the kinetical aspects of segment restoration by histochemistry. Cell proliferation peaked at two weeks and remitted by four weeks in regenerating earthworms. Apoptotic cells were present throughout the cell renewal period. Distinct immune cell (e.g., coelomocyte) subsets were accumulated in the newly-formed blastema in the close proximity of the apoptotic area. Regenerating earthworms have decreased pattern recognition receptors (PRRs) (e.g., TLR, except for scavenger receptor) and antimicrobial peptides (AMPs) (e.g., lysenin) mRNA patterns compared to intact earthworms. In contrast, at the protein level, mirroring regulation of lysenins became evident. Experimental coelomocyte depletion caused significantly impaired cell divisions and blastema formation during anterior and posterior regeneration. These obtained novel data allow us to gain insight into the intricate interactions of regeneration and invertebrate innate immunity.

Keywords: apoptosis; cell proliferation; coelomocytes; earthworm; gene expression; innate immunity; regeneration.

Figure 1

Histochemistry of intact anterior segments (a,f,k,p) and regenerating blastema at various time points: 4 days (b,g,l,q); 1 week (c,h,m,r); 2 weeks (d,i,n,s) 4 weeks (e,j,o,t). Haematoxylin-eosin (H&E) (a–e), Periodic acid and Schiff reaction (PAS) (f–j), acid phosphatase (ACP) (k–o) and alkaline phosphatase (ALP) (p–t) stainings were performed. Representative images were presented from five independent experiments. Arrows point to ACP (m–o) positive cells. Asterisks mark ALP (q–r) expressing cells. The level of amputation is indicated by a dashed line. Scale bars: 200 μm.

Figure 2

Histochemistry of intact posterior segments (a,f,k,p) and regenerating blastema at various time points such as 4 days (b,g,l,q); 1 week (c,h,m,r); 2 weeks (d,i,n,s); 4 weeks (e,j,o,t). Standard histochemical staining methods were performed: (a–e) H&E, (f–j) PAS, (k–o) ACP and (p–t) ALP. In H&E images arrowheads point to coelomocytes (c–e) in the blastema. Representative images were selected from five independent experiments. Arrows point to ACP (l–n) expressing cells. Asterisks represent cells with ALP (q–t) expression. The level of amputation is denoted by a dashed line. Scale bars: 200 μm.

Figure 3

Cell division and tissue re-organization in intact earthworms and in 2- and 4-week anterior blastema. The blastema is marked by a dashed line. Representative images were chosen from five independent experiments. Arrows point to the EdU-positive cells (green). Asterisks mark the reorganized actin filaments (red) in the blastema. The level of amputation is indicated by a dashed line Scale bars: 200 μm.

Figure 4

Cell division and tissue re-organization in intact earthworms and in 2- and 4-week posterior blastema of regenerating earthworms. Representative images were chosen from five independent experiments. Arrows point to EdU-positive cells (green) and asterisks remark the actin filaments (red) in the blastema. The level of amputation is denoted by a dashed line. Scale bars: 200 μm.

Figure 5

Detection of cell division and localization of coelomocytes by specific mAbs in intact earthworms and 2- and 4-week anterior blastema: (a) granular amoebocytes (EFCC4-positive cells) and (b) eleocytes (EFCC5-positive cells). Arrows point to proliferating EdU-positive cells (green). (a) Number signs mark granular amoebocytes (red) and (b) asterisks denote eleocytes (red). Representative images were selected from five independent experiments. BW—body wall; CC—coelomic cavity; G—gut. Scale bars: 100 μm.

Figure 6

Detection of cell proliferation and localization of coelomocytes by specific mAbs in intact segments, and 2- and 4-week posterior blastema: (a) granular amoebocytes (EFCC4-positive cells) and (b) eleocytes (EFCC5-positive cells). Arrows point to dividing EdU-positive cells (green). (a) Number signs mark granular amoebocytes (red) and (b) asterisks indicate eleocytes (red). Representative images were selected from five independent experiments. BW—body wall; CC—coelomic cavity; G—gut. Scale bars: 100 μm.

Figure 7

Detection of apoptosis and localization of EFCC4-positive granular amoebocytes in the 4-week anterior (a) and posterior (b) blastema. The TUNEL positive cells were overwhelmingly increased after 4 weeks and granular amoebocytes (red) were also present in the close vicinity of apoptotic cells (green). CC-coelomic cavity. Arrows point to TUNEL positive cells. Representative images were presented from five independent experiments. Scale bars: 100 μm.

Figure 8

Expression patterns of PRR genes (CCF: (a,b); LBP/BPI: (c,d); TLR: (e,f); SR: (g,h)) during the anterior (a,c,e,g) and posterior (b,d,f,h) regeneration from 1 to 4 weeks. Gene expressions in the regenerating blastema were compared to intact segments. The boxes represent the interquartile ranges (IQR), whiskers denote lowest and highest values, horizontal lines indicate the median of four independent (n = 4) experiments that were carried out in duplicates. The significance of the data was evaluated by one-way ANOVA with Dunnett’s post-hoc test using GraphPad Prism software (* p < 0.05, ** p < 0.01). Results were normalized to RPL17 mRNA level. A.U. arbitrary unit.

Figure 9

Expression patterns of AMP genes (lysozyme: (a,b); Lumbr: (c,d); LuRP: (e,f); lysenin: (g,h)) during anterior (a,c,e,g) and posterior (b,d,f,h) restoration from 1 to 4 weeks. Gene expressions of regenerating blastema were compared to intact ends. The boxes represent interquartile ranges (IQR), whiskers signify lowest and highest values, horizontal lines designate the median of four independent (n = 4) trials that were executed in duplicates. The significance of the data was estimated by one-way ANOVA with Dunnett’s post-hoc test with GraphPad Prism software (* p < 0.05, ** p < 0.01). RPL17 mRNA level used for normalization. A.U. arbitrary unit.

Figure 10

The protein profile of lysenins was studied by Western-blot during (a) anterior and (b) posterior restoration in intact earthworms and in 2 and 4 weeks regenerating blastema. Upper bands supposedly correspond to lysenin-related protein 2 (~40 kDa) and lower bands to lysenin (~37–38 kDa). Three independent experiments are presented (n = 3). The graphs below indicate the band intensity normalized to the corresponding protein band of α-tubulin as reference protein. The values illustrate mean ± SEM.

Figure 11

Detection of cell proliferation in the 2-week (a) anterior or (b) posterior blastema without (left side) or after coelomocyte depletion (right side). Representative images were selected from seven independent experiments of 2-week regenerating earthworms. The level of amputation is indicated by a dashed line. Proliferating cells (green) were enumerated and illustrated on the graphs adjacent to EdU-stained images. Intact earthworms (-) without or (+) with coelomocyte depletion for corresponding intervals used as controls. The amount of proliferating cells (-) without or (+) with coelomocyte ablation, after 2 and 4 weeks, was assessed in the regenerative blastema. Results are depicted on the same graph next to the counted cells of intact segments. The boxes mean interquartile ranges (IQR), whiskers represent lowest and highest values, horizontal lines label median of seven independent (n = 7) repetitions. The significance of the data was interpreted by one-way ANOVA with Dunnett’s post-hoc test with GraphPad Prism software (* p < 0.05, ** p < 0.01, *** p < 0.01). Scale bars: 200 μm.