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Next, we identified DEGs by comparing 4 h and 12 h timing for each treatment, 18 10 up regulated and 8 down regulated , up-regulated and down-regulated and 81 up-regulated and down-regulated DEGs were found in control, RNase3, and RNase3-H15A treatment as a function of time, respectively. The small amount of DEGs between 4 and 12 h found in control samples indicated that the biology status of the cells has been stable during all the experiment.

A direct comparison between RNase3 and RNase3-H15A-treated cells found 30 8 up-regulated and 22 down-regulated and 10 up-regulated and down-regulated DEGs at 4 h and 12 h post of treatment, respectively. All the identified DEGs are listed in Additional file 2. Core response genes not related to the protein ribonucleolytic activity are listed in Additional file 3 and are up-regulated and down-regulated, respectively; see Fig.

To examine more closely the correlations between the genes of the core response to RNase3 and RNase3-H15A treatment, we incorporated a network-based approach using the NetworkAnalyst3. EGFR activation positively regulates transcription factors that mediate the inflammatory response: STAT1, responding to cell stress injuries [ 39 ]; NF-kB, which controls pro-inflammatory cytokine production and cell survival [ 40 ], SRC, a regulatory protein kinase [ 41 ] and EGR1, activated during tissue remodelling.

The interaction between each protein pair is indicated with lines, the circle size is proportional to interaction degree. Only the genes with more than 25 interactions were shown. The colour bar indicated the log2FC of each gene. Down-regulated pathways include cell cycle, DNA replication, homologous recombination and mismatch repair among others, which are indicators of a cellular growth inhibition and duplication arrest response.

Following, we studied the macrophage cell response upon protein exposure as a function of time. Considering that cytokine—cytokine receptor interaction is within the top common enriched pathway upon wt RNase3 and RNase3-H15A treatment, with more than 78 genes significantly up-regulated Fig.

According to the timeline, the gene counts of most pro-inflammatory genes decreased by 12 h in comparison to a 4 h Fig. Overall, the observed immune-metabolic response is characteristic of a M1 macrophage activation type [ 44 ]. Heat-map of DEGs enriched in cytokine—cytokine receptor interaction pathways.

Colour bar indicates the Log2FC for each gene. Red stars indicate correlation with the protein catalytic activity. Moreover, we evaluated the inhibitory action of a monoclonal antibody anti-EGFR Cetuximab on the gene expression levels. The antibody recognizes the extracellular domain of the receptor and blocks its activation. Following treatment of THP1-derived macrophages with Cetuximab, we confirmed the inhibition of the protein effect Figure S3. Although RNase3 and RNase3-H15A elicited an overall similar transcriptional response of macrophage, a direct comparison between macrophage cells treated with the wt and mutant proteins identified significant changes related to the RNase catalytic activity.

Within the DEGs paired set, we found only six genes activated at both 4 and 12 h time points Fig. Furthermore, SOCS3 activation is usually observed following infection and plays a tissue protecting role against inflammation side effects [ 48 ]. On the other hand, when all the DEGs were analysed by connectivity and betweenness centrality, we observed 31 seed genes connected by 44 edges.

ISG15 expression is regulated by STAT1 transcription factor, which is characteristic of the macrophage activation by the interferon pathway and adaptation to an antiviral state. In contrast, no significant pathway was found when applying the DEGs at 4 h. In addition, the comparative heatmap of DEGs highlighted that significant changes of wt versus H15A mutant are mostly prominent at 12 h.

Overall, the results indicate that the late immune-regulatory action 12 h induced by RNase3 dependent on the protein catalytic activity would complement the first short-term response 4 h , mostly mediated by EGFR activation. Results suggest that both RNase3-activated pathways, catalytic-dependent and independent, might work by complementing each other. To corroborate our transcriptomic results and evaluate the potential contribution in situ of the macrophage endogenous RNase3, we decided to obtain a THP1 cell line that overexpresses the protein.

Therefore, the overexpression of RNase3 in THP1 cells could serve to analyse the protein action within the macrophage-derived cells. An empty lentiviral plasmid was used for the control reference cells.

The best performance cell line OX5 was selected for final characterization. Besides, we confirmed that overexpression of RNase3 did not alter the macrophage cell viability, as evaluated by the MTT assay. However, the overexpression of RNase3 is significantly slowing down the duplication rate. A total of 18 genes were selected among the top DEGs from libraries of wild-type versus control and catalytically defective mutant versus wild-type for further qPCR analysis.

The results confirmed similar expression levels induced by both endogenous and recombinant proteins Fig. Considering that our whole transcriptome analysis pointed out to a direct activation of EGFR by RNase3, we decided to explore further the protein-receptor-binding process. In particular, the QYRD sequence was spotted to participate in EGF interaction with the receptor by site-directed mutagenesis [ 49 ] and molecular dynamics [ 50 ].

Considering that the region of RNase5 involved in the interaction with EGFR is mostly conserved in RNase3, we decided to explore the potential protein interaction to the receptor. Notably, we obtained for RNase3 similar or even slightly higher binding energies, as calculated for EGF.

A close inspection of the predicted complex revealed that the interaction of RNase3 with EGFR might be mediated by residues regions 1—4, 19—28, 33—35, 86—87 and 94— In particular, we can identify the key role of N95, R97, Y98 and D of RNase3, which can interact within the — region of the EGF receptor see Additional file 8 for a full list of predicted protein—protein interactions. The molecular modelling also indicates that the RNase active site residues H15, K38 and H would not be involved in the receptor binding, in agreement with our transcriptomic results.

In particular, Hung and co-workers confirmed that residues Q93 and Y94 were essential for the receptor binding to RNase5. Here, in our model for RNase3, we observed the putative binding of R97 to the receptor extracellular domain. The common interacting region is underlined. EGFR is coloured in grey, the protein ligands were coloured in pink, the interacting residues from EGFR and the ligands were coloured in blue and red, respectively.

Next, we evaluated the efficacy of the macrophage endogenous RNase3 against infection. We selected two models of macrophage intracellular infection M. Both intracellular pathogens have been previously proven to be effectively eradicated within the macrophages by recombinant RNase3 addition [ 9 , 14 ]. Intracellular infection was quantified by Colony Forming Unit counting CFUs in lysed cells after removal of extracellular mycobacteria.

No significant differences in M. However, M. On the contrary, mycobacteria cannot proliferate in the RNase3-overexpression cell line. A significant growth inhibition of M. Noteworthy, the growth inhibition was even more prominent as the infection progressed 48 h and 72 h. The present data corroborate our previous results showing that recombinant RNase3 can efficiently mediate the eradication of macrophage intracellular infection by mycobacteria [ 14 ].

RNase3 inhibits bacterial and viral infection within macrophages. Overexpression of RNase3 in macrophage inhibits M. Intracellular and extracellular RSV reached a maximum peak at 24 h and 48 h post of infection, respectively. Importantly, we found that overexpression of RNase3 in macrophage can significantly inhibit the RSV duplication at 24—72 h poi Fig. In addition, overexpression of RNase3 reduced the macrophage cell death rate caused by M.

Moreover, the transcriptional expression levels of RNase3 were quantified in both cell lines WT and OX in the absence and presence of M. In addition, we explored whether the cell infection by M. The results indicate that short-term M. The data corroborate down- and up-regulation profile previously reported upon mycobacteria infection [ 14 ]. Erlotinib inhibits EGFR activation by blocking the receptor tyrosine kinase [ 52 ]. The RNase3 overexpression macrophages were treated with Erlotinib for 24 h before infection of either M.

As illustrated in Fig. Moreover, we quantified and compared the fold change of a set of 18 selected genes in RNase3 overexpressed cell line in the presence and absence of Erlotinib treatment. The selected genes had previously been quantified as DEGs by both recombinant RNase3 addition and endogenous protein overexpression see Fig.

Besides, by the side-by-side comparison of the overall transcriptome between wild-type and catalytic defective RNase3, we identified the DEGs dependent or independent of the protein catalytic activity see Additional file 1. Here, following Erlotinib treatment we observed how gene expression is uniquely altered for the genes not dependent on the protein catalytic activity Fig. The results corroborated the presence of two distinct type of pathways that are regulated by RNase3 and are associated to either catalytic or non-catalytic-dependent mechanisms of action see Fig.

At the indicated post-infection time, the intracellular M. Schematic illustration of the proposed molecular mechanism of RNase3 modulation in human macrophage. The genes associated to RNase catalytic activity were labelled in red. Antimicrobial peptides AMPs are important components of natural defence against a wide range of pathogens [ 53 ].

AMPs, originally reported to work by a direct action at the microbial cell wall, were later ascribed a diversity of immune modulatory properties that can contribute to the infection eradication [ 54 , 55 ]. Human antimicrobial RNases from the RNaseA superfamily can be regarded as multifaceted AMPs that combine a microbial membrane destabilization action with immune regulatory properties [ 4 , 56 , 57 ]. In our recent work, we have reported how RNase3 induction of macrophage autophagy mediated the eradication of M.

The comparative transcriptomic profile of macrophages treated with wild-type and catalytic-defective RNase3 has enabled us to identify the regulation pathways related and unrelated to the protein ribonucleolytic activity. The shared transcriptome profile pattern outlined an up-regulation core response characteristic of a macrophage pro-inflammation condition [ 44 ].

Furthermore, downregulation core response indicated that the protein addition promoted the cell growth arrest and duplication inhibition Fig. Furthermore, inspection of central hub down-regulated genes also revealed an association to EGFR receptor activation.

Interestingly, EGFR, the epidermal growth factor receptor, is not only a key membrane receptor involved in cell survival and tissue remodelling [ 58 , 59 ], but it can also mediate the macrophage activation during bacterial [ 60 ] and virus infection [ 61 ]. Specifically, EGFR signalling is critical for pro-inflammatory cytokine and chemokine production [ 60 , 62 , 63 ]. Therefore, our present results suggest that RNase3 mostly modulates the macrophage response via direct EGFR activation.

Taking into consideration the very recent report of human RNase5 direct binding to EGFR [ 64 ], we envisaged here a structural analysis of the putative interaction of RNase3 to the receptor. Molecular modelling suggested no involvement of the protein catalytic site in the receptor interaction, as indicated by our transcriptome results. Interestingly, we found a high sequence homology between both RNases at the conserved key target sequence previously identified at RNase5 and EGF C-terminus [ 18 ] Fig.

In particular, structural alignment of RNase5 with the EGF molecule in complex with the extracellular domain of the receptor [ 65 ] highlighted four conserved residues: two cysteines C81 and C92 , a glutamine Q93 and a tyrosine Y94 [ 64 ]. C81 and C92 are conserved in most of the RNaseA family members and participate in the disulphide bonding that connects the protein N and C ends and stabilizes the overall three-dimensional structure.

Pairwise sequence alignment indicated that Y94 is conserved in all RNase members. On the other hand, we found partially conserved substitutions for RNase5 Q In particular, in RNase3 the glutamine is substituted by a threonine T In particular, R97 shows an enhanced cytotoxicity on several tested eukaryote cell lines [ 67 ].

On the contrary, the presence of a T at this position creates an N-glycosylation site and native N-glycosylated forms display a reduced cytotoxicity and antimicrobial activity [ 17 , 68 , 69 ]. Presence of an R at 97 position has been linked to an enhanced severity of malaria and schistosomiasis side-effects [ 71 , 72 ], such as hepatic fibrosis or neurological disorders, that might be associated to an overabundance of the secreted RNase3 at the infectious focus [ 73 , 74 ].

The protein overproduction at the infected tissue could be detrimental to the host health, as also observed in allergic asthma and other chronic inflammatory diseases. Evolutionary studies indicated that R is the amino acid present in RNase3 lineage ancestor and is shared by most of the other primate homologues [ 75 ].

Overall, we observe a similar expression profile for all recombinant proteins. However, we identified a significant increase in the expression levels of the pro-inflammatory IL1A and NFKB1A markers as a function of the protein glycosylation degree, from the non-glycosylated form expressed in E.

In addition, we also confirmed that pre-treatment of macrophage cells with the anti-EGFR Ab inhibits the induced gene expression profile for all the tested recombinant proteins Figure S8. Therefore, the qPCR data also suggest that the protein region around residue 97 is involved in the activation of EGFR related pathways. However, further studies will be required for a deep understanding of the structural underling basis of the distinct biological properties of the natural protein variants.

On the other hand, the analysis of the CQYRD region within the overall vertebrate family context reveals a significant variability at position The importance of this residue was highlighted by Hung and co-workers, who demonstrated that human RNase1 homologue, where position 97 is occupied by an Ala, does not activate the EGFR [ 18 ].

Notably, A99 in human RNase3 is conserved in all primates [ 75 ]. In addition, our modelling study identified another residue key for RNase3 interaction to the receptor, D The residue is conserved in all RNase3 primates but absent in RNase2, its closest homologue counterpart.

Therefore, structural analysis revealed subtle differences that might significantly alter the receptor recognition pattern. We can speculate that the observed variability within the interacting sequence is indicating the targeting of distinct receptor subtypes within the EGFR family. In any case, our results are in agreement with the characterization of RNase5-EGFR interaction, that discarded the contribution of the protein catalytic activity in the receptor activation [ 64 ].

In addition, the present results highlight the induction by both wild-type RNase3 and RNase3-H15A of pro-inflammatory cytokines. The present data would reinforce the RNase3 role in tissue remodelling processes reported by in vitro wound healing models [ 2 , 16 , 77 — 79 ].

Importantly, not only viral RNA ligands but also processed self RNA can be detected by RLRs in the cytoplasm to trigger innate immunity and inflammation and induce gene expression against infection [ 84 , 85 ]. Complementarily, the RNA products can work as intercellular signalling molecules to mediate a prompt host response to infection [ 86 ].

A complex interplay between the host ncRNA and the RNA virus might determine the final outcome during infection [ 87 ]. We can speculate that RNase3 antiviral immune response may be mediated by the generation of specific RNA cleavage products. Our transcriptome results also highlighted that most of the significant DEGs related to RNase3 catalytic activity were visualized at the late 12 h exposure time rather than at the early 4 h time point Figs.

Moreover, among the activated pathways associated to the protein ribonucleolytic we found the activation of endosomal receptors, such as the TLRs 7—9, which are associated to endosomal detection of both foreign and host signalling nucleic acid molecules [ 86 ]. We find in the literature reference to other members of the RNaseA superfamily related to interferon-mediated response and endosomal TLR activation [ 89 — 91 ].

RNase7 was recently reported to activate the TLR9 signalling pathway [ 92 , 93 ]. The present results suggest that both protein families might cooperate to fight infection by shared convergent mechanisms of RNA processing. Indeed, recent work highlighted the importance of coordinated signalling pathways that mediate the macrophage defence mechanism against infection [ 97 , 98 ]. To complement our comparative transcriptome analysis, we decided to analyse in situ the potential role of the RNase3 endogenously expressed by macrophages.

Characterization of a THP1-macrophage-derived cell line that overexpresses RNase3 corroborated the transcriptomic results. The observed gene expression pattern induced by RNase3 is characteristic of both EGFR- and IFN-associated pathways, which can participate in the macrophage response to bacterial and viral infection, respectively [ 60 , 98 , ]. Moreover, our results revealed that native RNase3 expressed within the macrophages can mediate the eradication of M.

Our previous work using recombinant RNases highlighted the protein antimicrobial activity against both extracellular and macrophage intracellular dwelling mycobacteria [ 6 , 14 ]. The present results indicate that overexpression of endogenous RNase3 within macrophages can inhibit both M.

In addition, we observed how the mycobacteria infection is modulating the expression of RNase3, as reported for other host defence peptides [ — ]. The receptor blockage only altered the expression profile of genes unrelated to RNase3 catalytic activity Fig. More importantly, the experimental results indicated that the receptor is required for RNase3 antibacterial but not for antiviral activity. The present data are in agreement with previous reports on RNase3 catalytic activity direct contribution on the protein antiviral [ 9 ] but not antibacterial action [ 14 , 37 , 38 ].

RNase3 has been reported to contribute against persistent intracellular pathogens, such as the tuberculosis bacilli or the HIV virus, that frequently coexist and threaten immune-depressed patients [ 98 , ]. Therefore, it is crucial to understand the protein mechanism of action against macrophage intracellular infections. Unfortunately, RNase3 pro-inflammatory action following infection might also have a detrimental effect on the host tissues. Nevertheless, the protein action at the infection focus might turn out beneficial by promoting the tissue remodelling and healing [ 4 , 16 , 78 ].

Besides, RNase3 induction of leukocyte recruitment should reinforce the role of other blood cell type. In particular, RNase3 is abundantly secreted by eosinophils during inflammation and infection. Upon eosinophil degranulation, pro-inflammatory cytokines and chemokines are released to the infected tissue.

On its turn, the eosinophil secretory proteins can be engulfed by macrophages and participate in the eradication of intracellular infection. Therefore, the protein would mediate a positive feedback and will ensure an efficient host response. Further work is in progress to fully comprehend RNase3 signalling role.

A better understanding of the regulatory pathways that mediate the host response processes induced by RNase3 should facilitate the design of alternative anti-infective drugs. The present work underlines once again the therapeutic potentiality of our own defence molecules. Comparative transcriptome profile analysis of macrophages treated with wild-type RNase3 and the catalytic-defective mutant RNase3-H15A revealed that the protein triggers an early pro-inflammatory response in a ribonuclease-independent manner.

Moreover, protein—protein network analysis of comparative gene expression profiles indicated that the overall cell response is triggered by a direct activation of the EGFR. By structural analysis, we have identified the protein region potentially involved in the receptor binding. Complementarily, comparative transcriptome analysis suggested that RNase3 catalytic activity would participate in the activation of specific pathways associated to antiviral host defence.

In addition, the specific blockage of EGFR by Erlotinib indicates that the receptor-associated pathways participate in the protein antibacterial but not antiviral actions. Last, we demonstrated that endogenous overexpression of RNase3 in macrophages can inhibit M. LL and EB designed the experimental work.

All authors approved the final version of the manuscript. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Cellular and Molecular Life Sciences.

Cell Mol Life Sci. Published online Nov Author information Article notes Copyright and License information Disclaimer. Ester Boix, Email: se. Corresponding author. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

This article has been cited by other articles in PMC. Figure S2. The relative transcriptional expression level was calculated by comparison with the housekeeping gene GAPDH. Figure S3. The relative transcriptional expression level was calculated by comparing with the housekeeping gene GAPDH. Figure S4. Figure S5. Figure S6. A RSV stock with known concentration was serially diluted and used to build the standard curve.

Figure S7. Figure S8. Comparison of gene expression profiles induced by non-glycosylated recombinant RNase3 expressed in E. Non-coding genes and genes with less than 10 reads sum were filtered out. Additional file 2: Differential expressed genes for each paired wise comparison.

Supplementary file3 Table S1. Total RNA quality. Supplementary file4 Table S2. Supplementary file5 Table S3. Basic information on sequencing output and processing DOCX 14 kb. Electronic supplementary material The online version of this article Recombinant protein expression and purification RNase3 and RNase3-H15A recombinant proteins were produced as previously reported [ 8 ]. Transcriptome analysis FastQC was used to carry out the quality assessment of reads, assessing the distribution of phred quality scores and mean percentage GC content across each read.

Macrophage infection by mycobacteria and CFU assay Infection of THP1 cells derived to macrophage by mycobacteria was performed as previously described [ 14 ]. Cell viability assay Cell viability was measured using MTT assay as previously reported [ 35 ]. Results Comparative transcriptome analysis of THP1-derived macrophages treated with wild-type RNase3 and catalytic-defective RNase3-H15A mutant To characterize the immunomodulatory properties of RNase3 on macrophages we incubated THP1-derived cells in the presence of the protein and analysed the cell transcriptome at 4 h and 12 h incubation time.

Open in a separate window. RNase3 modulates the macrophage antiviral pathway in a catalytic-dependent manner Although RNase3 and RNase3-H15A elicited an overall similar transcriptional response of macrophage, a direct comparison between macrophage cells treated with the wt and mutant proteins identified significant changes related to the RNase catalytic activity.

Characterization of an RNase3 overexpression THP1 cell line To corroborate our transcriptomic results and evaluate the potential contribution in situ of the macrophage endogenous RNase3, we decided to obtain a THP1 cell line that overexpresses the protein. Analysis of the putative RNase3-EGFR interaction by molecular modelling Considering that our whole transcriptome analysis pointed out to a direct activation of EGFR by RNase3, we decided to explore further the protein-receptor-binding process.

Overexpression of endogenous RNase3 inhibits the macrophage intracellular infection by both M. Discussion Antimicrobial peptides AMPs are important components of natural defence against a wide range of pathogens [ 53 ]. Conclusions Comparative transcriptome profile analysis of macrophages treated with wild-type RNase3 and the catalytic-defective mutant RNase3-H15A revealed that the protein triggers an early pro-inflammatory response in a ribonuclease-independent manner. Electronic supplementary material Below is the link to the electronic supplementary material.

Supplementary file1 Figure S1. Author contribution LL and EB designed the experimental work. Compliance with ethical standards Conflict of interest The authors declare no conflicts of interest. Footnotes Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References 1. Eosinophil granule proteins: form and function.

J Biol Chem. Eosinophil cationic protein ECP : molecular and biological properties and the use of ECP as a marker of eosinophil activation in disease. Clin Exp Allergy. Analysing the eosinophil cationic protein—a clue to the function of the eosinophil granulocyte.

Respir Res. Immune modulation by human secreted RNases at the extracellular space. Front Immunol. Eosinophil cationic protein induces insulin-like growth factor I receptor expression on bronchial epithelial cells.

Int Arch Allergy Immunol. Two human host defense ribonucleases against mycobacteria, the eosinophil cationic protein RNase 3 and RNase 7. Antimicrob Agents Chemother. Comparison of human RNase 3 and RNase 7 bactericidal action at the Gram-negative and Gram-positive bacterial cell wall. On the contrary, mycobacteria cannot proliferate in the RNase3-overexpression cell line. A significant growth inhibition of M. Noteworthy, the growth inhibition was even more prominent as the infection progressed 48 h and 72 h.

The present data corroborate our previous results showing that recombinant RNase3 can efficiently mediate the eradication of macrophage intracellular infection by mycobacteria [ 14 ]. RNase3 inhibits bacterial and viral infection within macrophages. Overexpression of RNase3 in macrophage inhibits M. Intracellular and extracellular RSV reached a maximum peak at 24 h and 48 h post of infection, respectively.

Importantly, we found that overexpression of RNase3 in macrophage can significantly inhibit the RSV duplication at 24—72 h poi Fig. In addition, overexpression of RNase3 reduced the macrophage cell death rate caused by M. Moreover, the transcriptional expression levels of RNase3 were quantified in both cell lines WT and OX in the absence and presence of M. In addition, we explored whether the cell infection by M. The results indicate that short-term M. The data corroborate down- and up-regulation profile previously reported upon mycobacteria infection [ 14 ].

Erlotinib inhibits EGFR activation by blocking the receptor tyrosine kinase [ 52 ]. The RNase3 overexpression macrophages were treated with Erlotinib for 24 h before infection of either M. As illustrated in Fig. Moreover, we quantified and compared the fold change of a set of 18 selected genes in RNase3 overexpressed cell line in the presence and absence of Erlotinib treatment.

The selected genes had previously been quantified as DEGs by both recombinant RNase3 addition and endogenous protein overexpression see Fig. Besides, by the side-by-side comparison of the overall transcriptome between wild-type and catalytic defective RNase3, we identified the DEGs dependent or independent of the protein catalytic activity see Additional file 1. Here, following Erlotinib treatment we observed how gene expression is uniquely altered for the genes not dependent on the protein catalytic activity Fig.

The results corroborated the presence of two distinct type of pathways that are regulated by RNase3 and are associated to either catalytic or non-catalytic-dependent mechanisms of action see Fig. At the indicated post-infection time, the intracellular M.

Schematic illustration of the proposed molecular mechanism of RNase3 modulation in human macrophage. The genes associated to RNase catalytic activity were labelled in red. Antimicrobial peptides AMPs are important components of natural defence against a wide range of pathogens [ 53 ]. AMPs, originally reported to work by a direct action at the microbial cell wall, were later ascribed a diversity of immune modulatory properties that can contribute to the infection eradication [ 54 , 55 ].

Human antimicrobial RNases from the RNaseA superfamily can be regarded as multifaceted AMPs that combine a microbial membrane destabilization action with immune regulatory properties [ 4 , 56 , 57 ]. In our recent work, we have reported how RNase3 induction of macrophage autophagy mediated the eradication of M.

The comparative transcriptomic profile of macrophages treated with wild-type and catalytic-defective RNase3 has enabled us to identify the regulation pathways related and unrelated to the protein ribonucleolytic activity. The shared transcriptome profile pattern outlined an up-regulation core response characteristic of a macrophage pro-inflammation condition [ 44 ].

Furthermore, downregulation core response indicated that the protein addition promoted the cell growth arrest and duplication inhibition Fig. Furthermore, inspection of central hub down-regulated genes also revealed an association to EGFR receptor activation.

Interestingly, EGFR, the epidermal growth factor receptor, is not only a key membrane receptor involved in cell survival and tissue remodelling [ 58 , 59 ], but it can also mediate the macrophage activation during bacterial [ 60 ] and virus infection [ 61 ]. Specifically, EGFR signalling is critical for pro-inflammatory cytokine and chemokine production [ 60 , 62 , 63 ]. Therefore, our present results suggest that RNase3 mostly modulates the macrophage response via direct EGFR activation.

Taking into consideration the very recent report of human RNase5 direct binding to EGFR [ 64 ], we envisaged here a structural analysis of the putative interaction of RNase3 to the receptor. Molecular modelling suggested no involvement of the protein catalytic site in the receptor interaction, as indicated by our transcriptome results.

Interestingly, we found a high sequence homology between both RNases at the conserved key target sequence previously identified at RNase5 and EGF C-terminus [ 18 ] Fig. In particular, structural alignment of RNase5 with the EGF molecule in complex with the extracellular domain of the receptor [ 65 ] highlighted four conserved residues: two cysteines C81 and C92 , a glutamine Q93 and a tyrosine Y94 [ 64 ].

C81 and C92 are conserved in most of the RNaseA family members and participate in the disulphide bonding that connects the protein N and C ends and stabilizes the overall three-dimensional structure. Pairwise sequence alignment indicated that Y94 is conserved in all RNase members.

On the other hand, we found partially conserved substitutions for RNase5 Q In particular, in RNase3 the glutamine is substituted by a threonine T In particular, R97 shows an enhanced cytotoxicity on several tested eukaryote cell lines [ 67 ]. On the contrary, the presence of a T at this position creates an N-glycosylation site and native N-glycosylated forms display a reduced cytotoxicity and antimicrobial activity [ 17 , 68 , 69 ].

Presence of an R at 97 position has been linked to an enhanced severity of malaria and schistosomiasis side-effects [ 71 , 72 ], such as hepatic fibrosis or neurological disorders, that might be associated to an overabundance of the secreted RNase3 at the infectious focus [ 73 , 74 ].

The protein overproduction at the infected tissue could be detrimental to the host health, as also observed in allergic asthma and other chronic inflammatory diseases. Evolutionary studies indicated that R is the amino acid present in RNase3 lineage ancestor and is shared by most of the other primate homologues [ 75 ].

Overall, we observe a similar expression profile for all recombinant proteins. However, we identified a significant increase in the expression levels of the pro-inflammatory IL1A and NFKB1A markers as a function of the protein glycosylation degree, from the non-glycosylated form expressed in E.

In addition, we also confirmed that pre-treatment of macrophage cells with the anti-EGFR Ab inhibits the induced gene expression profile for all the tested recombinant proteins Figure S8. Therefore, the qPCR data also suggest that the protein region around residue 97 is involved in the activation of EGFR related pathways. However, further studies will be required for a deep understanding of the structural underling basis of the distinct biological properties of the natural protein variants.

On the other hand, the analysis of the CQYRD region within the overall vertebrate family context reveals a significant variability at position The importance of this residue was highlighted by Hung and co-workers, who demonstrated that human RNase1 homologue, where position 97 is occupied by an Ala, does not activate the EGFR [ 18 ]. Notably, A99 in human RNase3 is conserved in all primates [ 75 ]. In addition, our modelling study identified another residue key for RNase3 interaction to the receptor, D The residue is conserved in all RNase3 primates but absent in RNase2, its closest homologue counterpart.

Therefore, structural analysis revealed subtle differences that might significantly alter the receptor recognition pattern. We can speculate that the observed variability within the interacting sequence is indicating the targeting of distinct receptor subtypes within the EGFR family. In any case, our results are in agreement with the characterization of RNase5-EGFR interaction, that discarded the contribution of the protein catalytic activity in the receptor activation [ 64 ].

In addition, the present results highlight the induction by both wild-type RNase3 and RNase3-H15A of pro-inflammatory cytokines. The present data would reinforce the RNase3 role in tissue remodelling processes reported by in vitro wound healing models [ 2 , 16 , 77 — 79 ].

Importantly, not only viral RNA ligands but also processed self RNA can be detected by RLRs in the cytoplasm to trigger innate immunity and inflammation and induce gene expression against infection [ 84 , 85 ]. Complementarily, the RNA products can work as intercellular signalling molecules to mediate a prompt host response to infection [ 86 ].

A complex interplay between the host ncRNA and the RNA virus might determine the final outcome during infection [ 87 ]. We can speculate that RNase3 antiviral immune response may be mediated by the generation of specific RNA cleavage products. Our transcriptome results also highlighted that most of the significant DEGs related to RNase3 catalytic activity were visualized at the late 12 h exposure time rather than at the early 4 h time point Figs.

Moreover, among the activated pathways associated to the protein ribonucleolytic we found the activation of endosomal receptors, such as the TLRs 7—9, which are associated to endosomal detection of both foreign and host signalling nucleic acid molecules [ 86 ]. We find in the literature reference to other members of the RNaseA superfamily related to interferon-mediated response and endosomal TLR activation [ 89 — 91 ].

RNase7 was recently reported to activate the TLR9 signalling pathway [ 92 , 93 ]. The present results suggest that both protein families might cooperate to fight infection by shared convergent mechanisms of RNA processing. Indeed, recent work highlighted the importance of coordinated signalling pathways that mediate the macrophage defence mechanism against infection [ 97 , 98 ].

To complement our comparative transcriptome analysis, we decided to analyse in situ the potential role of the RNase3 endogenously expressed by macrophages. Characterization of a THP1-macrophage-derived cell line that overexpresses RNase3 corroborated the transcriptomic results.

The observed gene expression pattern induced by RNase3 is characteristic of both EGFR- and IFN-associated pathways, which can participate in the macrophage response to bacterial and viral infection, respectively [ 60 , 98 , ].

Moreover, our results revealed that native RNase3 expressed within the macrophages can mediate the eradication of M. Our previous work using recombinant RNases highlighted the protein antimicrobial activity against both extracellular and macrophage intracellular dwelling mycobacteria [ 6 , 14 ].

The present results indicate that overexpression of endogenous RNase3 within macrophages can inhibit both M. In addition, we observed how the mycobacteria infection is modulating the expression of RNase3, as reported for other host defence peptides [ — ]. The receptor blockage only altered the expression profile of genes unrelated to RNase3 catalytic activity Fig. More importantly, the experimental results indicated that the receptor is required for RNase3 antibacterial but not for antiviral activity.

The present data are in agreement with previous reports on RNase3 catalytic activity direct contribution on the protein antiviral [ 9 ] but not antibacterial action [ 14 , 37 , 38 ]. RNase3 has been reported to contribute against persistent intracellular pathogens, such as the tuberculosis bacilli or the HIV virus, that frequently coexist and threaten immune-depressed patients [ 98 , ].

Therefore, it is crucial to understand the protein mechanism of action against macrophage intracellular infections. Unfortunately, RNase3 pro-inflammatory action following infection might also have a detrimental effect on the host tissues.

Nevertheless, the protein action at the infection focus might turn out beneficial by promoting the tissue remodelling and healing [ 4 , 16 , 78 ]. Besides, RNase3 induction of leukocyte recruitment should reinforce the role of other blood cell type. In particular, RNase3 is abundantly secreted by eosinophils during inflammation and infection.

Upon eosinophil degranulation, pro-inflammatory cytokines and chemokines are released to the infected tissue. On its turn, the eosinophil secretory proteins can be engulfed by macrophages and participate in the eradication of intracellular infection. Therefore, the protein would mediate a positive feedback and will ensure an efficient host response. Further work is in progress to fully comprehend RNase3 signalling role. A better understanding of the regulatory pathways that mediate the host response processes induced by RNase3 should facilitate the design of alternative anti-infective drugs.

The present work underlines once again the therapeutic potentiality of our own defence molecules. Comparative transcriptome profile analysis of macrophages treated with wild-type RNase3 and the catalytic-defective mutant RNase3-H15A revealed that the protein triggers an early pro-inflammatory response in a ribonuclease-independent manner. Moreover, protein—protein network analysis of comparative gene expression profiles indicated that the overall cell response is triggered by a direct activation of the EGFR.

By structural analysis, we have identified the protein region potentially involved in the receptor binding. Complementarily, comparative transcriptome analysis suggested that RNase3 catalytic activity would participate in the activation of specific pathways associated to antiviral host defence. In addition, the specific blockage of EGFR by Erlotinib indicates that the receptor-associated pathways participate in the protein antibacterial but not antiviral actions.

Last, we demonstrated that endogenous overexpression of RNase3 in macrophages can inhibit M. LL and EB designed the experimental work. All authors approved the final version of the manuscript. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Cellular and Molecular Life Sciences. Cell Mol Life Sci. Published online Nov Author information Article notes Copyright and License information Disclaimer. Ester Boix, Email: se. Corresponding author. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.

If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

This article has been cited by other articles in PMC. Figure S2. The relative transcriptional expression level was calculated by comparison with the housekeeping gene GAPDH. Figure S3. The relative transcriptional expression level was calculated by comparing with the housekeeping gene GAPDH.

Figure S4. Figure S5. Figure S6. A RSV stock with known concentration was serially diluted and used to build the standard curve. Figure S7. Figure S8. Comparison of gene expression profiles induced by non-glycosylated recombinant RNase3 expressed in E. Non-coding genes and genes with less than 10 reads sum were filtered out. Additional file 2: Differential expressed genes for each paired wise comparison. Supplementary file3 Table S1. Total RNA quality. Supplementary file4 Table S2.

Supplementary file5 Table S3. Basic information on sequencing output and processing DOCX 14 kb. Electronic supplementary material The online version of this article Recombinant protein expression and purification RNase3 and RNase3-H15A recombinant proteins were produced as previously reported [ 8 ]. Transcriptome analysis FastQC was used to carry out the quality assessment of reads, assessing the distribution of phred quality scores and mean percentage GC content across each read.

Macrophage infection by mycobacteria and CFU assay Infection of THP1 cells derived to macrophage by mycobacteria was performed as previously described [ 14 ]. Cell viability assay Cell viability was measured using MTT assay as previously reported [ 35 ]. Results Comparative transcriptome analysis of THP1-derived macrophages treated with wild-type RNase3 and catalytic-defective RNase3-H15A mutant To characterize the immunomodulatory properties of RNase3 on macrophages we incubated THP1-derived cells in the presence of the protein and analysed the cell transcriptome at 4 h and 12 h incubation time.

Open in a separate window. RNase3 modulates the macrophage antiviral pathway in a catalytic-dependent manner Although RNase3 and RNase3-H15A elicited an overall similar transcriptional response of macrophage, a direct comparison between macrophage cells treated with the wt and mutant proteins identified significant changes related to the RNase catalytic activity. Characterization of an RNase3 overexpression THP1 cell line To corroborate our transcriptomic results and evaluate the potential contribution in situ of the macrophage endogenous RNase3, we decided to obtain a THP1 cell line that overexpresses the protein.

Analysis of the putative RNase3-EGFR interaction by molecular modelling Considering that our whole transcriptome analysis pointed out to a direct activation of EGFR by RNase3, we decided to explore further the protein-receptor-binding process. Overexpression of endogenous RNase3 inhibits the macrophage intracellular infection by both M. Discussion Antimicrobial peptides AMPs are important components of natural defence against a wide range of pathogens [ 53 ].

Conclusions Comparative transcriptome profile analysis of macrophages treated with wild-type RNase3 and the catalytic-defective mutant RNase3-H15A revealed that the protein triggers an early pro-inflammatory response in a ribonuclease-independent manner. Electronic supplementary material Below is the link to the electronic supplementary material.

Supplementary file1 Figure S1. Author contribution LL and EB designed the experimental work. Compliance with ethical standards Conflict of interest The authors declare no conflicts of interest. Footnotes Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References 1. Eosinophil granule proteins: form and function.

J Biol Chem. Eosinophil cationic protein ECP : molecular and biological properties and the use of ECP as a marker of eosinophil activation in disease. Clin Exp Allergy. Analysing the eosinophil cationic protein—a clue to the function of the eosinophil granulocyte. Respir Res. Immune modulation by human secreted RNases at the extracellular space. Front Immunol. Eosinophil cationic protein induces insulin-like growth factor I receptor expression on bronchial epithelial cells.

Int Arch Allergy Immunol. Two human host defense ribonucleases against mycobacteria, the eosinophil cationic protein RNase 3 and RNase 7. Antimicrob Agents Chemother. Comparison of human RNase 3 and RNase 7 bactericidal action at the Gram-negative and Gram-positive bacterial cell wall. FEBS J. Nucleic Acids Res. Structural determinants of the eosinophil cationic protein antimicrobial activity. Biol Chem. Topography studies on the membrane interaction mechanism of the eosinophil cationic protein.

Both aromatic and cationic residues contribute to the membrane-lytic and bactericidal activity of eosinophil cationic protein. FEBS J, p. Human antimicrobial RNases inhibit intracellular bacterial growth and induce autophagy in mycobacteria-infected macrophages.

Stimulation of basophil and rat mast cell histamine release by eosinophil granule-derived cationic proteins. J Immunol. Eosinophil cationic protein stimulates migration of human lung fibroblasts in vitro. Scand J Immunol.

The functional heterogeneity of eosinophil cationic protein is determined by a gene polymorphism and post-translational modifications. Cancer Cell. Nat Protoc. Genome Biol. NetworkAnalyst 3. J Mol Biol. J Am Chem Soc. Optimized production and concentration of lentiviral vectors containing large inserts. J Gene Med. Production, concentration and titration of pseudotyped HIVbased lentiviral vectors. Genomic identification and expression analysis of the cathelicidin gene family of the forest musk deer.

Animals Basels doi: An in vitro model to study immune responses of human peripheral blood mononuclear cells to human respiratory syncytial virus infection. J Vis Exp. Immunostimulatory defective viral genomes from respiratory syncytial virus promote a strong innate antiviral response during infection in mice and humans.

PLOS Pathog. J Virol Methods. Respiratory syncytial virus infection in mice and detection of viral genomes in the lung using RTqPCR. Bio Protoc. MTT colorimetric assay for testing macrophage cytotoxic activity in vitro. J Immunol Methods. J Infect Dis. Rosenberg HF.

Recombinant human eosinophil cationic protein. Ribonuclease activity is not essential for cytotoxicity. Exploring new biological functions of amyloids: bacteria cell agglutination mediated by host protein aggregation. PLoS Pathog. Mol Carcinog. Ligand-independent EGFR signaling.

Cancer Res. Sci Rep. Tyrosine phosphorylation controls PCNA function through protein stability. Nat Cell Biol. Macrophage activation and polarization: nomenclature and experimental guidelines. Identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays. Fensterl V, Sen GC. Interferon-induced Ifit proteins: their role in viral pathogenesis. J Virol. The association of OASL and type I interferons in the pathogenesis and survival of intracellular replicating bacterial species.

Front Cell Infect Microbiol. Carow B, Rottenberg ME. SOCS3, a major regulator of infection and inflammation. Development of a human epidermal growth factor derivative with EGFR-blocking and depleted biological activities: a comparative in vitro study using EGFR-positive breast cancer cells.

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