Gene expression profiling of RIP2-knockdown in HD11 macrophages — elucidation of potential pathways (gene network) when challenged with avian pathogenic E.coli (APEC)

Receptor interacting serine/threonine kinase 2 (RIP2), ubiquitous in many tissue/cell types, is the key regulator of immune and inflammatory responses for many diseases, including avian pathogenic E. coli (APEC), which causes a wide variety of localized or systemic infections. However, the molecular mechanisms by which RIP2 drives its transcriptional program to affect immune and inflammatory response upon APEC infection remains poorly understood. In this study, RNA-seq and bioinformatics analyses were used to detect gene expression and new direct/indirect RIP2 targets in the treatments of wild type HD11 cells (WT), RIP2 knockdown cells (shRIP2), APEC stimulation cells (APEC), and RIP2 knockdown cells combined with APEC infection (shRIP2 + APEC). The results revealed that a total of 4691 and 2605 differentially expressed genes (DEGs) were screened in shRIP2 + APEC vs. APEC and shRIP2 vs. WT, respectively. Functional annotation analysis showed that apoptosis, MAPK, p53, Toll-like receptor, and Nod-like receptor signaling pathways were involved in APEC-induced RIP2 knockdown HD11 cells. By analyzing the enriched pathway and gene networks, we identified that several DEGs, including HSP90AB1, BID, and CASP9 were targeted by RIP2 upon APEC infection. As a whole, this study can not only provide data support for constructing gene networks of RIP2 knockdown with APEC challenge but also provide new ideas for improving the immune and inflammatory response.

of antimicrobial resistance among APEC [4,5]. The pathogenesis of APEC is still not well understood but appears to influence apoptosis of immune cells and tissues injury [6][7][8]. Moreover, it has been demonstrated that APEC shared the similar genomic sequences with uropathogenic E. coli responsible for human urinary tract infections [9,10], indicating the horizontal gene transfer ability and zoonotic potential of APEC. Therefore, it is critical to study the mechanism of the immune response and effectively prevent the excessive inflammation at genomic level.
Receptor interacting serine/threonine kinase 2 (RIP2) is the key molecule in the regulation of immune responses, inflammation, and cell death against viral and bacterial infections [11][12][13][14]. The N-terminal CARD of RIP2 can bind to the CARD domain of procaspase-1 to activate NFκB signal cascade and initiate the pro-inflammatory pathways [15][16][17]. Moreover, it has been demonstrated that cells deficient of RIP2 could decrease the activation of NFκB, resulting in impaired expression of IL6, TNFα, IP10 and reduced neutrophil infiltration, which alleviate the excessive inflammatory response [18][19][20]. The research of Homer et al. [21] discovered that RIP2 tyrosine kinase has a dual function in NOD2-dependent autophagy, that is RIP2 both sends a positive autophagy signal through the activation of p38 MAPK and relieves repression of autophagy mediated by the phosphatase PP2A. Currently, RIP2 has been found to play an important role in the response of the avian host to an APEC infection [7]. Although it is considered the master regulator of immune and inflammatory response, it is not clear how the ubiquitous RIP2 can direct the immune or inflammation-specific process.
In this study we attempted to unravel the gene network underlying RIP2 regulation associated with APEC infection via RNA-seq analysis, using the chicken HD11 macrophage cell line. We studied the expression profiles of complete and knockdown of RIP2 HD11 cells with or without 24 h APEC postinfection. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) mechanistic analyses were performed taking into consideration the function of mRNAs, which changed their expression significantly in RNA-seq analysis. Altogether, our results suggest that HSP90AB1, BID, and CASP9 were the new targets downstream of RIP2, whose depletion directly or indirectly caused the suppression of those genes. Thus, HSP90AB1, BID, and CASP9 were involved in the immune and inflammatory response, while precisely RIP2 could down-regulate those genes during APEC stimulation.

RIP2 inhibition level in chicken HD11 macrophages with or without APEC challenge
To identify the inhibition level of RIP2, the protein and mRNA levels were first measured in wild type HD11 cells (WT), HD11 cells with knockdown of RIP2 (shRIP2), APEC-challenged HD11 cells (APEC), and APEC-challenged HD11 cells with knockdown of RIP2 (shRIP2 + APEC). As shown in Fig. 1A and B, a significantly decreased mRNA and protein expression level of RIP2 were observed in the RIP2 knockdown group in comparison to WT (p < 0.05), indicating the expression of RIP2 was indeed inhibited. Also, APEC challenge resulted in the significantly increased mRNA and protein abundance of RIP2 compared with WT (p < 0.05). However, the mRNA and protein level of RIP2 were found to be significantly decreased in the RIP2 knockdown HD11 cells with APEC challenge in comparison to wild type HD11 cells or APEC challenge group ( Fig. 1A-C).

Cell viability in knockdown of RIP2 HD11 macrophages with or without APEC challenge
Morphological changes of APEC-challenged HD11 cells with or without knockdown of RIP2 were observed. As displayed in Fig. 1D, there was no effect of RIP2 knockdown on HD11 cells growth and proliferation. After challenge with APEC at 10 8 cfu/mL for 24 h, cytopathic effects appeared in HD11 cells when compared with the WT, while knockdown of RIP2 gene could significantly alleviate the APEC-challenged cytopathy. Moreover, the viability of chicken HD11 cells toward APEC challenge with or without knockdown of RIP2 was measured using an CCK-8 assay. There was no difference between WT and shRIP2 group (p > 0.05; Fig. 1E), which was consistent with the results of cells morphology. The APEC-challenged chicken HD11 cells had a significantly lower cell survival rate than those in the RIP2 knockdown group at 24 h post-infection (p < 0.05; Fig. 1E), indicating knockdown of RIP2 can effectively inhibit the APEC-challenged cell apoptosis in HD11 cells.

Nitric oxide (NO) production in knockdown of RIP2 HD11 macrophages with or without APEC challenge
Considering the function of chicken HD11 macrophages, NO production in the cell supernatant from the four different groups (WT, shRIP2, APEC, and shRIP2 + APEC) was determined using the Griess reagent kit. As shown in Fig. 1F, there was no significant difference between WT and RIP2 knockdown group for NO production. However, APEC challenge and RIP2 knockdown combined with APEC challenge groups induced significant NO production in HD11 macrophages, with APEC challenged group giving higher levels of NO compared to RIP2 knockdown combined with APEC challenged group (p < 0.05, Fig. 1F). These results suggested that knockdown of RIP2 had the ability to effectively reduce the APEC-induced NO production, although the level was still significantly higher than WT (p < 0.05).

Overview of RNA sequencing data
A total of twelve cDNA libraries were constructed, respectively, from four groups (WT, shRIP2, APEC, and shRIP2 + APEC) with three biological replicates for each. A total of 71,438,338-95,374,638 sequence reads were obtained, and each sample yielded approximately 80,223,040 clean reads (range from 69,393,540 to 92,952,070, Table S3). The percentages of Q30 for the clean reads was more than 99%, and the average of GC content of clean reads was 52.34% (Table S3). Moreover, 92.81-94.10% of the clean reads were found to successfully map to the chicken reference genome, of which 89.16-91.25% were uniquely mapped to genome (Table  S4). Additionally, Table 1 shows the information on the fraction of reads mapping to features for each sample, i.e. any expressed parts of the genome. Interestingly, the percentage of reads mapping to coding sequence (CDS) Fig. 1 Knockdown of RIP2 affects chicken HD11 macrophages with or without APEC challenge. A The relative RIP2 mRNA expression level in the group of WT, shRIP2, APEC, and APEC+shRIP2 (data are shown as mean ± SD; n = 4 independent experiments; *** p < 0.001; **** p < 0.0001). B The RIP2 protein expression level in the group of WT, shRIP2, APEC, and APEC+shRIP2 was analyzed by western blot. C Image J software was used for RIP2 gray-level analysis of western blot results (data are shown as mean ± SD; n = 3 independent experiments; ** p < 0.01; **** p < 0.0001). D The morphology of chicken HD11 macrophages in the group of WT, shRIP2, APEC, and APEC+shRIP2. E The cell viability of chicken HD11 macrophages in the group of WT, shRIP2, APEC, and APEC+shRIP2 (data are shown as mean ± SD; n = 4 independent experiments; *** p < 0.001; **** p < 0.0001; NS, not significant). F The nitric oxide (NO) production of chicken HD11 macrophages in the group of WT, shRIP2, APEC, and APEC+shRIP2 (data are shown as mean ± SD; n = 4 independent experiments; ** p < 0.01; **** p < 0.0001; NS, not significant). Abbreviations: WT, wild type HD11 cells; shRIP2, RIP2 knockdown HD11 cells; APEC, avian pathogenic E.coli challenge group in WT, shRIP2, and shRIP2 + APEC were 59.78, 59.91, and 60.06%, respectively, while that in APEC was 56.95% (Table 1). Also, sequencing homogeneity was assessed to evaluate the bias of RNA-seq data. Results showed reads were evenly distributed on the genes without 5′ or 3′ bias ( Fig. 2A). Then, a multidimensional scaling analysis was performed on the count data, revealing the biological replicates were relatively concentrated in each of the four groups (Fig. 2B). The WT and shRIP2 HD11 macrophages with or without APEC infection were clearly separated (Fig. 2B). Furthermore, the heatmap of sample correlation showed the shRIP2 + APEC group clustered with the WT, and then the shRIP2, finally the APEC group (Fig. 2D), which were consistent with the results of reads distribution to CDS in Table 1. Then, the dynamic range of the expression values was estimated and exhibited as a box plot of logarithmic transformed RPKM values for each sample separately (Fig. 2C), and the RPKM density distribution is presented in Fig. 2E.

Identification of differentially expressed genes (DEGs)
In order to investigate the specific mechanism that RIP2 affected cell immune and inflammatory response upon APEC infection, DEGs were identified from WT and shRIP2 HD11 cells with or without APEC challenge. Pairwise comparisons were performed as follows: shRIP2 (knockdown of RIP2 HD11 cells) vs. WT (complete HD11 macrophages) and shRIP2 + APEC (knockdown of RIP2 HD11 cells combined with avian pathogenic E. coli infection) vs. APEC (avian pathogenic E. coli infection HD11 cells). A total of 2605 DEGs were obtained when adjusted p-value ≤0.05 and |logFC| ≥ 0.58 were set as the cut-off limits (Fig. 3A, B) in shRIP2 vs. WT. Among them, 1133 down-regulated DEGs and 1472 up-regulated DEGs were identified (Fig. 3C). In shRIP2 + APEC vs. APEC, a total of 4691 DEGs were identified (Fig. 3D, E), of which 2340 DEGs were down-regulated and 2351 DEGs were upregulated (Fig. 3F).

Functional annotation of DEGs in different comparisons
The gene ontology (GO) classification system was used to classify the possible functions of DEGs in different comparisons. A total of 734 genes (28.18%) and 1476 (31.46%) were successfully assigned to at least one GO term annotation in shRIP2 vs. WT (Table S5) and shRIP2 + APEC vs. APEC (Table S6), respectively. The significantly enriched GO terms in shRIP2 vs. WT and shRIP2 + APEC vs. APEC were similar (Fig. 4). For the molecular function category, the top two largest categories were "binding" and "catalytic activity". According to biological process, the top categories were "cellular process", "biological regulation", and "metabolic process". More remarkable, the immune related GO terms were also identified, including "cell communication", "immune response", "signal transduction", and "response to stimulus". These results show that knockdown of RIP2 significantly affected immune system processes in chicken HD11 macrophages with or without APEC challenge.
Then, Kyoto Encyclopedia of Genes and Genomes (KEGG) classification system was also performed to identify the possible functions of DEGs. A total of 15 and 55 significantly changed pathways were detected in shRIP2 vs. WT (Table S7) and shRIP2 + APEC vs. APEC (Table S8), respectively, with an adjusted p-value ≤0.05. The significantly enriched pathways were primarily involved in immune system for the two comparisons, which included "Phagosome", "Lysosome", "MAPK signaling pathway" (Fig. 5). In addition, the pathways related to cell growth, differentiation, survival, signal transduction were also significantly enriched in the two comparisons, including "Apoptosis", "ECMreceptor interaction", "VEGF signaling pathway", and "Focal adhesion" (Fig. 5). However, the "p53 signaling pathway", "cell cycle", "Toll-like receptor signaling pathway", and "NOD-like receptor signaling pathway" were uniquely identified to be significantly enriched in shRIP2 + APEC vs. APEC (Fig. 5B).

RIP2 is a critical regulator for MAPK signaling and apoptosis pathway in chicken HD11 macrophages with or without APEC challenge
After knockdown of RIP2, the MAPK signaling and apoptosis pathway were significantly activated with or without APEC challenge. A total of 39 and 67 DEGs were involved in "MAPK signaling pathway" in shRIP2 vs. WT and shRIP2 + APEC vs. APEC, respectively, as shown in Tables S9 and S10. Nineteen DEGs were commonly involved in the MAPK signaling pathway in both shRIP2 vs. WT and shRIP2 + APEC vs. APEC. To further corroborate the correlation between RIP2 and MAPK signaling pathway, we used qRT-PCR to detect the expression of the commonly identified DEGs-IL1β, JUN, and CASP3 in the two comparisons, as well as the uniquely expressed DEGs-MAPK9 and FOS in shRIP2 + APEC vs. APEC. The qRT-PCR results were in agreement with their transcript abundance changes determined by RNA-seq (Fig. 6). Moreover, a total of 25 and 48 DEGs were enriched in "apoptosis pathway" in shRIP2 vs. WT (Table S11) and shRIP2 + APEC vs. APEC (Table S12), respectively. Twelve DEGs were enriched in apoptosis pathway in the two comparisons, 3 (CTSB, CTSC, and PIK3CB) of which were selected and used for qRT-PCR validation. Moreover, the uniquely enriched DEGs-FADD, CFLAR, BID, and CASP9 in the comparison of shRIP2 + APEC vs. APEC were also identified for RNA-seq data reliability. The results showed that knockdown of RIP2 indeed affected the expression of the the selected genes with or without APEC challenge, which presented 100% consistency between RNA-seq and qRT-PCR (Fig. 6). And, more remarkable, three DEGs (JUN, CASP3, and ENSGALG00000031518) were involved in both MAPK signaling and apoptosis pathway in the two comparisons (Fig. S1), suggesting they had important role in APEC challenge and related with RIP2. All these results show that RIP2 is a critical regulator for MAPK signaling and apoptosis pathway with or without APEC infection.

New target genes of RIP2 upon APEC challenge
In order to find the new target of RIP2 toward APEC infection, we collected and analyzed the DEGs enriched in the uniquely significantly changed pathways "p53 signaling pathway", "Toll-like receptor signaling pathway", and "NOD-like receptor signaling pathway" in the comparison of shRIP2 + APEC vs. APEC (Fig. 9). Compared to APEC challenge, RNA-seq data showed that BID, CASP9, and HSP90AB1 were significantly downregulated in RIP2 knockdown HD11 cells toward APEC infection. To evaluate the RIP2 role in those genes, we studied the expression of above genes in RIP2 knockdown HD11 cell following APEC challenge. As shown in Fig. 10, the mRNA and protein levels of HSP90AB1 showed remarkable reduction in RIP2 knockdown HD11 cells (p < 0.001) combined APEC challenge compared to the APEC challenge group. Interestingly, compared to the wild type HD11 cells, the mRNA and protein expression level of HSP90AB1 were significantly decreased in the knockdown of RIP2 HD11 macrophages group (Fig. 10C, D, and G), which is consistent with the results of RNAseq. These results indicate a strong relationship exists between RIP2 and HSP90AB1 with or without APEC challenge. However, interfering RIP2 did not affect the mRNA and protein expression level of BID and CASP9 (Fig. 10A, B, D, E, and F), which is in agreement with the RNA-seq data. APEC infection had the ability to induce a significant difference in the mRNA and protein expression level of BID and CASP9 between RIP2 knockdown HD11 cells and wild type HD11 cells, indicating RIP2 is involved in the regulation of BID and CASP9 in a certain relationship as a result of APEC infection. Taken together, these results showed that the RNA-seq results were considered to be reliable and RIP2 was involved in the regulation of BID, CASP9, and HSP90AB1 in response to APEC infection.

Discussion
Although RIP2 has been cloned in chicken [22], the function of RIP2 was only focused on the activation of NFκB signal pathway [23]. This study first demonstrated the essential genes or pathways, as well as the downstream targets regulated by RIP2 with or without APEC challenge through transcriptome analysis. We compared the transcriptomes of APEC-induced chicken HD11 cells with knockdown or complete expression of RIP2. It was found that in RIP2 knockdown HD11 cells, many identified DEGs were grouped into the unique functional categories, p53 (p = 1.67 × 10 − 3 ), TLRs (p = 4.14 × 10 − 3 ), and NLRs (p = 2.54 × 10 − 2 ) signaling, toward APEC infection, as expected in cases of impairment of the key regulator for HD11 cells transcriptional program. p53 signaling pathway is involved in the regulation of a serials normal life activities of cells, such as apoptosis and senescence [24][25][26]. Normally, p53 has the ability to prevent DNA replication in order to provide time for DNA repair, otherwise it triggers apoptosis, suggesting that p53 plays an important role in monitoring and identifying DNA damage points [27]. Earlier studies have shown that the p53 signaling pathway was significantly suppressed in the comparison of susceptible vs. non-challenged birds at 5 day APEC post-infection in bone marrow and thymus [6,7], indicating APEC might tend to promote host tumorigenesis. Also, compared to the control group, this pathway was significantly downregulated in the APEC-induced HD11 cells in the current study, which was consistent with previous studies. However, p53 signaling pathway was significantly up-regulated in the knockdown of RIP2 HD11 cells combined with APEC infection in comparison to APEC infection in the present study (Table S13). Then, we focused on the p53 related DEGs, since the identification of these genes could help to elucidate the molecular mechanism that underlies the function of RIP2 in response to APEC infection. Take SIAH1 as an example, it was up-regulated (Fold changes = 2.3) in shRIP2 + APEC vs. APEC, while down-regulated (Fold changes = − 1.96) in APEC vs. WT. This result was expected, since SIAH1 was a tumor suppressor and played essential role in regulating cell apoptosis [28]. Another two genes of interest that changed in shRIP2 + APEC vs. APEC were PMAIP1 and MDM4. It has been demonstrated that PMAIP1 belonged to the pro-apoptotic subfamily and could determine whether a cell commits to apoptosis [29]. Also, this study demonstrated that MDM4 had the ability to promote cell apoptosis upon genotoxic stress [30]. These two genes were up-regulated in shRIP2 + APEC vs. APEC, indicating knockdown of RIP2 triggered the activation of p53 signaling pathway and further promote the apoptosis upon APEC challenge. These results coincide with the expectations, since RIP2 activity correlated with tumor and metastasis et al. [31][32][33].
Another interesting gene HSP90AB1 is downregulated in the comparisons of shRIP2 vs. WT and shRIP2 + APEC vs. APEC. We validated these results finding decreased expression levels of both mRNA and protein in RIP2 knockdown HD11 cells in any conditions compared to wild type HD11 cells (Fig. 10C, D, and G), suggesting the existence of a positive and cross-regulation between RIP2 and HSP90AB1 in chicken HD11 cells with or without APEC challenge. Then, bioinformatics analyses showed that HSP90AB1 was involved to the GO group of "response to stimulus" and, most importantly, to the KEGG group of "NLR signaling", closely related to the immune activity of RIP2. HSP90AB1 is a member of the large family of HSPs and function as molecular chaperones, which is important to signal transduction, protein folding, apoptosis, inflammation, and cell survival [34,35]. It has been demonstrated that the expression of HSP90AB1 mRNA was often expressed on the tissues of heart, liver, brain, and spleen, regulating the heat stress in chickens [36]. In fact, HSP90AB1 was involved in antiapoptotic and immunostimulatory effects of CpG in both mouse and chicken macrophages [37,38]. Our transcriptome data showed that the apoptosis and immune related pathways were significantly changed in RIP2 knockdown HD11 cells with or without APEC challenge, which included the gene of interest HSP90AB1. Herein, this study strongly suggests that HSP90AB1 is a downstream target of RIP2 and positively modulated by RIP2. Moreover, BID and CASP9 were another two important genes that were down-regulated in shRIP2 + APEC vs. APEC, whereas these genes were up-regulated when APEC vs. WT were compared. BID is a member of the proapoptotic BCL2 family, which could stimulate mitochondrial outer membrane permeabilization to induce the release of regulators or other cell-death mediators [39]. CASP9 is the member of cysteine aspartate-specific proteases, serving as an intrinsic initiator of apoptosis [40,41]. Data obtained by qRT-PCR and western blot showed that the mRNA and protein of BID and CASP9 were both significantly decreased when shRIP2+ APEC vs APEC were compared (Fig. 10A, B, E and F). It has been demonstrated that BID and CASP9 were both involved in the apoptosis pathway that was downstream of RIP2 [42,43]. Accordingly, our data point to BID and CASP9 as the targets of RIP2, since RIP2-depletion inhibits the expression of BID and CASP9 upon APEC infection. Apoptosis is a safety and important process to remove the damaged DNA, abnormal-proliferated or dedifferentiated cells, which is indispensable for maintaining cellular homeostasis, and normal regulation of the immune system [44,45]. In the present study, the apoptosis was significantly activated in shRIP2 + APEC vs. APEC, indicating silence of RIP2 could induce apoptosis to inhibit tumorgenesis. These results were reasonable and expected, since knockdown of RIP2 could avoid the excessive tissues/cells injury and inflammatory response [42,46]. In summary, our data suggest that BID and CASP9 could be the two important factors that determine immune and inflammatory response towards HD11 macrophages in the absence of RIP2 upon APEC challenge, even if the cellular fate in the presence of BID and CASP9 down-regulation is presently unknown. The specific regulation mechanism between RIP2 and BID/CASP9 involved in response to APEC challenge warrants further study.

Conclusion
In summary, this study has provided an analysis of the genetic landscape associated with RIP2 knockdown in HD11 macrophages in the presence or absence of an APEC challenge. In total, 4691 and 2605 DEGs were screened in shRIP2 + APEC vs. APEC and shRIP2 vs. WT, respectively. Functional annotation analysis showed that apoptosis, MAPK, p53, Toll-like receptor, and Nod-like receptor signaling pathways were involved in APEC-induced RIP2 knockdown HD11 cells. By analyzing the enriched pathway and gene networks, we identified that several key DEGs, like HSP90AB1, BID, and CASP9 were targeted by RIP2 upon APEC infection. As a whole, this study can not only provide data support for constructing gene networks of RIP2 knockdown associated with APEC challenge but also provide new ideas for improving the immune and inflammatory response.

Cell culture
Chicken HD11 macrophage, an immortalized cell line, was selected as experimental materials as it is similar morphology and function to the in vivo macrophages and helpful to understand the host immune response in the early infection during APEC infection. The chicken HD11 cell line was kindly provided by Dr. Xuming Hu (Yangzhou University). The chicken macrophage-like cell line HD11 was maintained in RPMI1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) in a humidified incubator with 5% CO 2 at 37 °C, and cells were passaged before 80-90% confluence.

Cell transfection
Small hairpin RIP2 (shRIP2) plasmid (Table S1) were synthesized by GenePharma (Shanghai, China). The detailed information for the establishment of shRIP2 HD11 cells can be found in the study of Sun et al. [47]. The Lipofectamine ™ 2000 reagent (Invitrogen, Carlsbad, CA, USA) was used for the cell transfection according to the manufacturer's instructions. After transfection with shRIP2 for 48 h, cells were challenged with or without APEC for 24 h, and collected for further study.

Quantitative real time PCR (qRT-PCR) analysis
Total RNA was isolated from cells of wild type group (WT), RIP2 knockdown group (shRIP2), APEC challenge group (APEC), and RIP2 knockdown with APEC challenge group (shRIP2 + APEC) using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Then the RNA was reverse transcribed into cDNA using a Reverse Transcription Kit

Western blotting
Cells from each of the four groups were lysed on ice using 200 μL RIPA buffer (Beyotime Biotechnology, Shanghai, China) for 30 min. Next, the lysis mixtures were centrifuged and the supernatants were collected. BCA ™ Protein Assay Kit (Pierce, Appleton, WI, USA) was used for quantification of proteins. Then, the isolated proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and electrophoretically transferred to PVDF membranes. Afterwards, membranes were blocked in 5% BSA for 2 h at room temperature and then probed with the primary antibodies at 4 °C overnight. The primery antibodies included anti-GAPDH (ab181602, Abcam, MA, USA), anti-RIP2 (70R-10,459, Fitzgerald, MA, USA),

Nitric oxide (NO) production assay
Chicken HD11 cells from different groups (WT, shRIP2, APEC, and shRIP2 + APEC) were incubated for 24 h, then NO production in the cell supernatant was determined using the Griess reagent kit (Molecular Probes, Carlsbad, CA, USA). The cell supernatant was mixed with Griess reagents and incubated for 30 minutes in the dark, and then measured at 540 nm on a spectrophotometer. The absorbance values were compared to the sodium nitrite standard curve to determine nitrite concentrations (μM).

Cell viability assay
Cell counting kit-8 (CCK-8) was utilized to determine the viability of cells from different groups (WT, shRIP2, APEC, and shRIP2 + APEC). Cells from each of the four groups were placed in three replicates at a density of 1 × 10 5 cells per well in a 96-well plate with 100 μL of medium and incubated for 48 h. Then, the cells were incubated for 2 h in 10 μL of CCK-8 solution. Absorbance (optical density, OD) was assessed at 450 nm using a spectrophotometer. The experiment was performed in triplicate.

RNA-Seq
Total RNA was extracted from HD11 cells of different groups (WT, shRIP2, APEC, and shRIP2 + APEC) using an RNA isolation kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. The quality of RNA was analyzed by agarose gel electrophoresis and a Nanodrop ™ OneCspectrophotometer (Thermo Fisher Scientific Inc., MA, USA). The RNA Integrity was confirmed by Qseq (Qseq100, Guangding, Taiwan). A total of 2 μg of RNA was used for stranded RNA sequencing library preparation using Ribo-off rRNA depletion kit (Catalog NO. MRZG12324, Illumina, San Diego, CA, USA) following the manufacturer's instruction. The library products corresponding to 200-500 bps were enriched, quantified and finally sequenced on NovaSeq 6000 sequencer (Illumina, San Diego, CA, USA) with PE150 sequencing platform.

Quality control and differentially expressed genes (DEGs) analysis
For quality control, raw data were first filtered by Trimmomatic (version 0.36) (i.e., removing low-quality reads (Q ≤ 10) and repeated and adaptor sequences (10 bp overlap (AGA TCG GAAG)). The obtained clean reads were further treated with in-house scripts to eliminate duplication bias introduced in library preparation and sequencing. Deduplicated reads were mapped to the reference genome of the chicken (Gallus gallus) from Ensembl (https:// asia. ensem bl. org/ info/ data/ ftp/ index. html) using STRA software (version 2.5.3a) with default parameters. Reads mapped to the exon regions of each gene were counted by feature-Counts (Subread-1.5.1; Bioconductor) and then RPKM was calculated. Genes differentially expressed between groups were identified using the edgeR package (version 3.12.1). A p-value cutoff of 0.05 and fold-change cutoff of 1.5 were used to judge the statistical significance of gene expression differences.

GO and KEGG pathway enrichment analyses
GO analysis [48] and KEGG enrichment analysis [49,50] for DEGs were both implemented by KOBAS software (version: 2.1.1) with a p-value cutoff of 0.05 to judge statistically significant enrichment.

Statistical analyses
Statistical analysis was conducted using a one-way ANOVA and Tukey Honestly Significant differences test (HSD; SAS, 2000; Cary, NC) using JMP statistical software (version 15.2.1, SAS Institute). Data are expressed as the mean ± standard error. The statistical significance Fig. 9 Gene network analysis was used to select the target genes of RIP2 upon APEC challenge by String. Colored nodes: query proteins and first shell of interactors; white nodes: second shell of interactors; empty nodes: proteins of unknown 3D structure; filled nodes: some 3D structure is known or predicted. The light blue line and purple line represent known interaction. Other color lines indicate predicted interactions (data are shown as mean ± SD; n = 4 independent experiments; different letters represent a significant difference, p < 0.001; same letters indicate no significance, p > 0.05). C The relative HSP90AB1 mRNA expression level in the group of wild type HD11 cells (WT), knockdown of RIP2 HD11 cells (shRIP2), avian pathogenic E. coli (APEC), and knockdown of RIP2 HD11 cells combined with APEC infection (APEC+shRIP2). (data are shown as mean ± SD; n = 4 independent experiments; different letters represent a significant difference, p < 0.001; same letters indicate no significance, p > 0.05). D The protein level of BID, CASP9, and HSP90AB1 in the group of wild type HD11 cells (WT), knockdown of RIP2 HD11 cells (shRIP2), avian pathogenic E. coli (APEC), and knockdown of RIP2 HD11 cells combined with APEC infection (APEC+shRIP2). E Image J software was used for BID gray-level analysis of western blot results (data are shown as mean ± SD; n = 3 independent experiments; different letters represent a significant difference, p < 0.05; same letters indicate no significance, p > 0.05). F Image J software was used for CASP9 gray-level analysis of western blot results (data are shown as mean ± SD; n = 3 independent experiments; different letters represent a significant difference, p < 0.05; same letters indicate no significance, p > 0.05). G Image J software was used for HSP90AB1 gray-level analysis of western blot results (data are shown as mean ± SD; n = 3 independent experiments; different letters represent a significant difference, p < 0.05; same letters indicate no significance, p > 0.05)