Interplay between trauma and Pseudomonas entomophila infection in flies: a central role of the JNK pathway and of CrebA

In mammals, both sterile wounding and infection induce inflammation and activate the innate immune system, and the combination of both challenges may lead to severe health defects, revealing the importance of the balance between the intensity and resolution of the inflammatory response for the organism’s fitness. Underlying mechanisms remain however elusive. Using Drosophila, we show that, upon infection with the entomopathogenic bacterium Pseudomonas entomophila (Pe), a sterile wounding induces a reduced resistance and increased host mortality. To identify the molecular mechanisms underlying the susceptibility of wounded flies to bacterial infection, we analyzed the very first steps of the process by comparing the transcriptome landscape of infected (simple hit flies, SH), wounded and infected (double hit flies, DH) and wounded (control) flies. We observed that overexpressed genes in DH flies compared to SH ones are significantly enriched in genes related to stress, including members of the JNK pathway. We demonstrated that the JNK pathway plays a central role in the DH phenotype by manipulating the Jra/dJun activity. Moreover, the CrebA/Creb3-like transcription factor (TF) and its targets were up-regulated in SH flies and we show that CrebA is required for mounting an appropriate immune response. Drosophila thus appears as a relevant model to investigate interactions between trauma and infection and allows to unravel key pathways involved.


Immediate early response to trauma and infection:
The 645 genes differentially expressed between SH and DH flies at 30 minutes, corresponding to genes of the 'immediate early' response to the challenges, were partitioned into 4 clusters.
-Cluster 1 contained genes highly expressed in DH flies compared to both SH and control ones (Fig.   2a). This gene set was enriched in GO annotation associated with 'response to stress' (P = 2.10e -3 ) ( Table 1).
As detailed in the main text, several are known actors of the JNK signaling pathway (Gadd45, Hsromega and Cabut) while a number of genes participating in the heat shock response were also retrieved (Hsp70Ab ,   Hsp70Bb, Hsp70Aa, Hsp70Ba, Hsp70Bc, Hsp70Bbb).
-Cluster 2, which corresponded to genes showing higher expression in SH fly population compared to DH and control, was enriched in genes involved in the anti-bacterial humoral response (P = 2.79e -5 ) ( Table 1, see main text for detailed description of this cluster).
-Cluster 3 was composed of 84 genes down-regulated in DH flies compared to SH and control populations. This cluster was enriched in genes annotated as participating in eggshell formation (P = 10 -7 ) ( Table 1), what may support a stress induced down-regulation of the reproductive status of DH females compared to both SH and control ones. Noticeably, i-cisTarget analysis indicated that non-coding sequences of genes in cluster 3 were enriched for both Tata Binding Protein (TBP) and TBP associated factor 1 ( Taf1) binding motifs, which are hallmarks of actively transcribed genes. This suggests that this cluster was comprised of genes that are usually actively transcribed by RNA polymerase II and may therefore be actively down-regulated upon combined trauma and infection.
The 147 genes of cluster 4 had a reduced expression in SH flies compared mainly to control ones and were enriched in GO annotation corresponding to 'tissue development' (P = 0.03) ( Table 1). As in cluster 1, i-cisTarget method retrieved an enrichment for Heat shock factor (Hsf) protein binding motif in the non-coding region of this gene set suggesting that a part of them are transcribed downstream this stress pathway. In addition, binding motif for the zinc finger TF ken and barbie (Ken) -a potential modulator of the JAK/STAT pathway 1 -was enriched in the non-coding sequences of cluster 4 genes. Finally, i-cisTarget also retrieved the Blimp-1 protein binding motif. Blimp-1 is a transcriptional repressor recently identified as involved in precise timing of gene expression during Drosophila pupation 2 . It could therefore also be implicated in the controlled expression of these immediate early genes, which displayed dynamic expression within the first 30 minutes of the experiment.

Early response to trauma and infection:
At 3h, 786 genes were found to be differentially expressed and their expression profiles are divided into 3 clusters (Fig. 2b).
-Cluster 5 was composed of 355 genes down-regulated in SH flies compared to both DH and control ones. The GO enrichment analysis revealed the term 'response to stress' as being enriched (P = 0.02) ( Table   1). A detailed description of this cluster is provided in the main text.
-Cluster 6 consisted of 416 up-regulated genes in the SH group, for which the GO enriched terms were 'egg coat formation' (P = 4.56e -7 ), 'transmembrane transport' (P = 2.41e -6 ) and 'immune response' (P = 0.03) ( Table 1). Genes involved in the formation of protective coverings of the egg thus appeared to be downregulated in DH and control compared to SH flies. This may highlight a down-regulation of egg laying in response to trauma in DH flies, a (persistent) effect that has already been found after 30 minutes (cluster 3, Table 1). It is interesting to note that expression of innate immune response genes was maintained at higher levels in SH flies compared to DH ones, similarly to what was observed at 30 minutes (cluster 2, Table 1, see also main text). Importantly, a significant proportion of cluster 6 genes were identified as potential transcriptional targets of the CrebA/Creb3-like protein (hypergeometric test; P = 4.90e -13 ) (see supplementary   Table S5) and relaxing the stringency of the differential expression analysis by considering genes identified only by EdgeR, pointed out the CrebA gene as down-regulated in DH flies compared to SH ones (see Supplementary Table S2 and main text).
-Cluster 7 was composed of 15 genes up-regulated in DH flies, which did not exhibit statistically significant GO term enrichment.

Late phase of the early response to trauma and infection :
Finally, at 6h, 449 genes were found to be differentially expressed between the three conditions and were partitioned into 3 clusters (Fig. 2c).
-Cluster 8 was composed of 160 genes down-regulated in SH condition compared to both DH and controls. The GO enrichment analysis revealed the term 'defense response' (P = 2.27e -4 ) suggesting that at 6 hours, genes linked to the innate immune response start to be activated in DH flies. This hypothesis was further supported by i-cisTarget analysis, which highlighted the Relish binding motif enrichment in the potential regulatory regions of this gene set. Since this cluster comprised genes that are up-regulated in DH flies but also in control flies that were not subjected to bacterial challenge, this may reflect a fly response to inflammation. The 158 genes forming the cluster 9 were up-regulated in SH condition. Similarly to what was observed at 3 hours (cluster 6), the GO enrichment analysis pointed out the term 'egg coat formation' (P = 4.05e -13 ). Cluster 10, which were composed of 131 up-regulated genes in both SH and DH flies compared to control alone, were significantly enriched in GO term associated to process of cell-cell signaling (P = 7.82e -7 ).