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, 18 (7), 481-9

Distinct Innate Immune Responses to Infection and Wounding in the C. Elegans Epidermis


Distinct Innate Immune Responses to Infection and Wounding in the C. Elegans Epidermis

Nathalie Pujol et al. Curr Biol.


Background: In many animals, the epidermis is in permanent contact with the environment and represents a first line of defense against pathogens and injury. Infection of the nematode Caenorhabditis elegans by the natural fungal pathogen Drechmeria coniospora induces the expression in the epidermis of antimicrobial peptide (AMP) genes such as nlp-29. Here, we tested the hypothesis that injury might also alter AMP gene expression and sought to characterize the mechanisms that regulate the innate immune response.

Results: Injury induces a wound-healing response in C. elegans that includes induction of nlp-29 in the epidermis. We find that a conserved p38-MAP kinase cascade is required in the epidermis for the response to both infection and wounding. Through a forward genetic screen, we isolated mutants that failed to induce nlp-29 expression after D. coniospora infection. We identify a kinase, NIPI-3, related to human Tribbles homolog 1, that is likely to act upstream of the MAPKK SEK-1. We find NIPI-3 is required only for nlp-29 induction after infection and not after wounding.

Conclusions: Our results show that the C. elegans epidermis actively responds to wounding and infection via distinct pathways that converge on a conserved signaling cassette that controls the expression of the AMP gene nlp-29. A comparison between these results and MAP kinase signaling in yeast gives insights into the possible origin and evolution of innate immunity.


Figure 1
Figure 1. The pnlp-29::GFP reporter is induced by Drechmeria coniospora
Control wt;frIs7 worms (A) and worms 24 h after infection (B). Red and green fluorescence is visualized simultaneously. C Quantification of fluorescence of infected (yellow) and non-infected (blue) worms. The mean values for green and red fluorescence (in arbitrary, but constant units for each colour) for the uninfected and infected populations were 59 and 287 (green) and 331.5 and 295.0 (red), respectively. D Continuous distribution of fluorescence levels in the population. The analysis of large numbers of individuals revealed a continuous distribution of fluorescence levels, with the median value for the fluorescence ratio (green/red) being close to the mean (triangles). Although there was an extremely broad range of values for individuals (from 4 to 589, and 11 to 700 in arbitrary units for 1369 non-infected and 1232 infected worms, respectively, in this experiment), leading to high standard deviations (157% and 54% of the mean values, respectively; shown as error bars with the mean at the median position), and noise (variance/mean2, 2.4 and 0.3, respectively). Due to the nature of the distribution, standard deviations are not an informative parameter and are not shown on subsequent figures using the Biosort.
Figure 2
Figure 2. Needle and laser wounding of the epidermis cause scar formation and local cuticle synthesis
A–D Epidermal scars in wild type (N2) 60 h after needle wound (A,B) and immediately after femtosecond laser wound (C,D); DIC image (panels A, C) and epifluorescence (GFP long pass filter, panels B,D). The autofluorescent material accumulates at the wound immediately (see Suppl. Movies). Scale 10 μm. E–H Ultrastructural analysis of scar and cuticle synthesis at epidermal wounds. The lateral epidermis just ventral to the alae was damaged internally by aiming for the PLM axonal process (in the zdIs5 strain) and cutting it with MHz femtosecond laser. F–H wounded and unwounded (E) sides of the same animal 24 h post wounding. The innermost (basal) layer of cuticle (b) is immediately adjacent to the epidermis hyp7 and is approximately 100 nm thick in unwounded epidermis (b, coloured in inset, E). Surrounding the electron dense material of the scar, the basal layer is up to 20 times thicker than in unwounded cuticle, and contains ribosome-sized electron dense particles (inset, H). Images are all from one animal, sections in F, G, and H are 1.3 μm apart (schematic in inset, G). Scale bar 2 μm.
Figure 3
Figure 3. Epidermal damage induce pnlp-29::GFP
An individual wt;frIs7 worm before (A) and 2 h after needle wounding (B). C An individual that has been wounded with a laser 5 h previously is lying above a mock-wounded worm. D–G An individual worm viewed by DIC (D) or epifluorescence (E) microscopy just after needle wounding. The arrowhead marks the wound site. The same worm 3 hours (F) or 24 hours (G) after wounding. H & I Part of the epidermis of an eff-1(hy21);frIs7 worm. One unfused hyp7 cell shows strong GFP fluorescence (I). With the exception of D (DIC) and H (epifluorescence using a red filter to visualize pcol-12:DsRed only), in all images, red and green fluorescence are visualized simultaneously.
Figure 4
Figure 4. nlp-29 induction after infection and injury is dependent on the TIR-1B/p38 pathway
Quantification with the Biosort of the normalized fluorescence ratio of worms carrying the frIs7 transgene. A RNAi was used to target the different tir-1 isoforms, and fluorescence measured 24 h post-infection (yellow) or 6 h after wounding (green) and compared to control worms (blue). B Mutants affecting the long tir-1 isoforms (ok1052) and (gk264) have an essentially wild type phenotype. C–F Different mutants were analyzed after infection and wounding: sek-1(ag1), sek-1(km4), nsy-1(ky397), pmk-1(km25), mek-1(ks54), jkk-1(km2), unc-16(ju146) and unc-43(n498n1186). G Over-expression of sek-1 in the epidermis, under the control of the col-12 promoter provokes high fluorescence ratios in sek-1 mutants. The number of worms in each sample is given in parentheses.
Figure 5
Figure 5. nipi-3 encodes a kinase required for the induction of nlp-29 and nlp-31 after infection but not after wounding A
Quantification with the Biosort of the normalized fluorescence ratio of transgenic worms carrying the frIs7 transgene 24 h post-infection (yellow; left panel) or 6 h after wounding (green; right panels) in wt, nipi-2(fr2) and nipi-3(fr4) mutant worms compared to controls (blue). B Quantification by qRT-PCR of nlp-29 and nlp-31 mRNA in wild-type and nipi-3 mutant worms after infection (yellow and orange, respectively) or wounding (green and lime, respectively). C Quantification with the Biosort of the normalized fluorescence ratio of wt;frIs7 worms 24 h post-infection (yellow) or 6 h after wounding (green) compared to controls (blue), following mock RNAi or RNAi of nipi-3. D Structure of the nipi-3 locus. The location of the fr4 mutation is shown relative to the exon/intron structure of nipi-3; the red shading in 4 exons represents the kinase domain. Grey boxes represent the predicted 5′ and 3′-most exons of the neighbouring upstream and downstream genes, respectively. The first line below the gene indicates the extent of the genomic fragment used to rescue the mutant phenotype; the next line (labelled RNAi), that used for RNAi and the third line, the promoter region used to drive the expression of GFP in the reporter construct. E The nipi-3 gene encodes a protein with similarity to human Tribbles homolog 1 (TRIB1). Part of the predicted amino acid sequence of the NIPI-3 is compared to that of human Tribbles homolog 1 (TRIB1; accession number AAH63292). The N-terminal region outside the kinase domain is underlined; the site corresponding to the nipi-3 mutation is marked by an asterisk. Identical residues are boxed in black, similar residues in grey, using Hofmann and Baron’s Boxshade.
Figure 6
Figure 6
A–D Fluorescence images of pnipi-3::GFP transgenic worms showing expression in the intestine, head neurons and motoneurons in a L1 larva (A), in the epidermis in a 3-fold embryo (B). In the adult head (C) and tail (D), expression is seen in the epidermis (arrow) and intestine (arrowhead), as well as a subset of neurons. Scale bar 10 μm. E, F Quantification with the Biosort of the normalized fluorescence ratio of worms carrying the frIs7 transgene following infection (yellow) compared to controls (blue). wt and nipi-3(fr4) mutants, with or without the rescuing transgene containing nipi-3 under its own promoter (E). wt, nipi-3(fr4) and pmk-1 mutants, with or without the transgene containing sek-1 under the control of the col-12 promoter (F). G–N Images of uninfected (G, H, I, J) or infected (K, L, M, N) worms carrying the frIs7 transgene in the wt (G, K) and nipi-3 background (H–L), and in some cases a second transgene driving expression of nipi-3 in the epidermis (I, M) or intestine (J, N). The worms shown in (M) are representative only of the rescued worms (see Suppl. Figure 6).
Figure 7
Figure 7
Multiple signals converge on a p38 MAPK signaling cassette conserved between C. elegans (left panel) and budding yeast (right). NIPI-3 may function analogously to UNC-43 as an activator of TIR-1, specifically in the response to infection.

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