When an animal is infected, its innate immune response needs to be tightly regulated across tissues and coordinated with other aspects of organismal physiology. Previous studies with Caenorhabditis elegans have demonstrated that insulin-like peptide genes are differentially expressed in response to different pathogens. They represent prime candidates for conveying signals between tissues upon infection. Here, we focused on one such gene, ins-11 and its potential role in mediating cross-tissue regulation of innate immune genes. While diverse bacterial intestinal infections can trigger the up-regulation of ins-11 in the intestine, we show that epidermal infection with the fungus Drechmeria coniospora triggers an upregulation of ins-11 in the epidermis.
Using the Shigella virulence factor OpsF, a MAP kinase inhibitor, we found that in both cases, ins-11 expression is controlled cell autonomously by p38 MAPK, but via distinct transcription factors, STA-2/STAT in the epidermis and HLH-30/TFEB in the intestine. We established that ins-11, and the insulin signaling pathway more generally, are not involved in the regulation of antimicrobial peptide gene expression in the epidermis. The up-regulation of ins-11 in the epidermis does, however, affect intestinal gene expression in a complex manner, and has a deleterious effect on longevity. These results support a model in which insulin signaling, via ins-11, contributes to the coordination of the organismal response to infection, influencing the allocation of resources in an infected animal.
Infection of Caenorhabditis elegans induces broad changes in gene expression (reviewed in [1,2]). These are characterized by pathogen-specific responses and more generic changes. We previously noted among the genes induced by infection with the fungi Drechmeria coniospora an overrepresentation of genes repressed after infection by the bacterial pathogens Serratia marcescens, Enterococcus faecalis or Photorhabdus luminescens. Conversely, there was an overrepresentation of genes up-regulated upon infection by each of these bacterial pathogens among the genes down-regulated upon infection with D. coniospora.
Since D. coniospora infects via the epidermis, while the bacterial pathogens colonize the nematode intestine, one possible interpretation of these results is that there is a communication between tissues. Thus, when immune defenses are activated in the epidermis, they would be repressed in the intestine and vice versa. In a parsimonious model, fungal infection of the epidermis or bacterial infection of the intestine would provoke an increase in the expression of a signaling molecule that would then modulate gene Life Science M expression in other tissues.
There are countless instances of such non-cell autonomous control of gene expression across all multicellular species. A number have been characterized in the context of the response of C. elegans to biotic and abiotic insults (reviewed in).
To give one recent example, activation of a conserved p38 MAPK pathway in the intestine by rotenone is associated with protection from neurodegeneration [6], conceivably because induction of the mitochondrial unfolded protein response in the intestine represses the expression of the antimicrobial peptide gene nlp-29 [7], which regulates dendrite degeneration in aging and infection [8]. But, as is commonly the case, the molecular nature of the signal mediating this cross-tissue control has not yet been established.
One prominent exception to this is the complex network of insulin signaling. With 40 genes encoding insulin-like peptides, acting through a common conserved pathway to regulate the activity of the FOXO transcription factor DAF-16 and thereby the expression of hundreds of target genes across all tissues [10], insulin signaling influences many if not all aspects of C. elegans physiology, including diapause, longevity, stress resistance and fertility [9]. It has been demonstrated to play a role in innate immunity too. Specifically, the insulin-like peptide INS-7 from neurons has been shown to inhibit DAF-16 signaling in the intestine and thereby negatively-regulate pathogen resistance. Interestingly, infection with Pseudomonas aeruginosa provokes an increase in neuronal ins-7 expression, leading to the down-regulation of putative immune defense genes in the intestine. And recently, a role in learning aversive behavior has been proposed for insulin-like peptide INS-11 upon infection by P. aeruginosa. In this study, we investigated the induction of ins-11 caused by different infections and propose a non-cell autonomous role for ins-11 in cross-tissue communication.
Expression of insulin peptides upon infection
Given their known roles in cross-tissue communication and in innate immune regulation, insulin-like peptides are prime candidates for mediating the reciprocal control of epidermal and intestinal gene expression seen following infection of C. elegans. We therefore first reviewed data from our previous studies to identify insulin-like genes that are induced both by fungal and bacterial pathogens. Infection of C. elegans by D. coniospora provokes a marked increase in the expression of 4 insulin-like genes, ins-7, ins-11, ins-23 and ins-36.
Among them, although ins-7 expression is induced by P. aeruginosa, it did not figure among the list of genes differentially regulated upon infection by the bacterial pathogens S. marcescens, E. faecalis or P. luminescens. And while the expression of ins-23 and ins-36 was decreased upon infection with S. marcescens, for ins-11 it was increased, as it was also following infection with E. faecalis and P. luminescens [4]. Further, other investigators have reported an induction of ins-11 expression after infection with Bacillus thuringiensis [14], P. aeruginosa [13,15,16], Staphylococcus aureus [17] and the microsporidian species Nematocida parisii [18]. We therefore focused our attention on ins-11 and its potential role in mediating a cross-tissue regulation of innate immune genes.
Dependent upregulation of ins-11 in the intestine upon bacterial infection
As a first step in the characterization of ins-11, we verified its expression upon infection by S. marcescens. By qRT-PCR we observed a clear induction of ins-11 gene expression, as we did for the C-type lectin gene clec-67, a putative defense gene that is expressed in the intestine and is up-regulated by infection by diverse bacteria [3] (Fig. 1A). It has previously been shown that following S. aureus or P. aeruginosa infection, ins-11 expression requires the basic helix-loop-helix transcription factor HLH-30 [13,17]. In the case of infection by S. marcescens, ins-11 expression was also dependent upon hlh-30.
As previously reported, a strain carrying an ins-11 transcriptional reporter construct (ins-11p::GFP) exhibited only a low constitutive fluorescence in adults, in the nerve ring, labial neurons and most obviously in the intestine (Fig. 1B). When this strain was infected by S. marcescens, a clear increase in GFP expression was observed in the intestine (Fig. 1C). Interestingly, the same type of induction in the intestine was also recently reported upon infection by P. aeruginosa [13]. This suggests that diverse bacterial intestinal infections can trigger the up-regulation of ins-11 in the intestine.