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• W4313487834 abstract "Article4 January 2023Open Access Source DataTransparent process Bacterial expression of a designed single-chain IL-10 prevents severe lung inflammation Ariadna Montero-Blay Corresponding Author Ariadna Montero-Blay ariadna.montero@crg.eu orcid.org/0000-0002-2357-722X Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Conceptualization, Validation, Writing - original draft Search for more papers by this author Javier Delgado Blanco Javier Delgado Blanco Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Software, Methodology Search for more papers by this author Irene Rodriguez-Arce Irene Rodriguez-Arce Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Validation, Methodology, Writing - review & editing Search for more papers by this author Claire Lastrucci Claire Lastrucci Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Methodology Search for more papers by this author Carlos Piñero-Lambea Carlos Piñero-Lambea orcid.org/0000-0002-5263-3702 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Resources Search for more papers by this author Maria Lluch-Senar Maria Lluch-Senar orcid.org/0000-0001-7568-4353 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Resources, Writing - review & editing Search for more papers by this author Luis Serrano Corresponding Author Luis Serrano luis.serrano@crg.eu orcid.org/0000-0002-5276-1392 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain ICREA, Barcelona, Spain Contribution: Conceptualization, Supervision, Writing - original draft Search for more papers by this author Ariadna Montero-Blay Corresponding Author Ariadna Montero-Blay ariadna.montero@crg.eu orcid.org/0000-0002-2357-722X Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Conceptualization, Validation, Writing - original draft Search for more papers by this author Javier Delgado Blanco Javier Delgado Blanco Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Software, Methodology Search for more papers by this author Irene Rodriguez-Arce Irene Rodriguez-Arce Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Validation, Methodology, Writing - review & editing Search for more papers by this author Claire Lastrucci Claire Lastrucci Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Methodology Search for more papers by this author Carlos Piñero-Lambea Carlos Piñero-Lambea orcid.org/0000-0002-5263-3702 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Resources Search for more papers by this author Maria Lluch-Senar Maria Lluch-Senar orcid.org/0000-0001-7568-4353 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Resources, Writing - review & editing Search for more papers by this author Luis Serrano Corresponding Author Luis Serrano luis.serrano@crg.eu orcid.org/0000-0002-5276-1392 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain ICREA, Barcelona, Spain Contribution: Conceptualization, Supervision, Writing - original draft Search for more papers by this author Author Information Ariadna Montero-Blay *,1,†, Javier Delgado Blanco1,†, Irene Rodriguez-Arce1,†, Claire Lastrucci1, Carlos Piñero-Lambea1, Maria Lluch-Senar1 and Luis Serrano *,1,2,3 1Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain 2Universitat Pompeu Fabra (UPF), Barcelona, Spain 3ICREA, Barcelona, Spain † These authors contributed equally to this work *Corresponding author. Tel: +34 933160101; E-mail: ariadna.montero@crg.euCorresponding author. Tel: +34 933160101; E-mail: luis.serrano@crg.eu Molecular Systems Biology (2023)19:e11037https://doi.org/10.15252/msb.202211037 AbstractSynopsis Introduction Results Discussion Materials and Methods Data availability Acknowledgements Author contributions Disclosure and competing interests statementSupporting InformationReferencesPDFDownload PDF of article text and main figures.PDF PLUSDownload PDF of article text, main figures, expanded view figures and appendix. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. MetricsMetricsTotal downloads1,312Last 6 Months1,312View all metrics ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Interleukin-10 (IL-10) is an anti-inflammatory cytokine that is active as a swapped domain dimer and is used in bacterial therapy of gut inflammation. IL-10 can be used as treatment of a wide range of pulmonary diseases. Here we have developed a non-pathogenic chassis (CV8) of the human lung bacterium Mycoplasma pneumoniae (MPN) to treat lung diseases. We find that IL-10 expression by MPN has a limited impact on the lung inflammatory response in mice. To solve these issues, we rationally designed a single-chain IL-10 (SC-IL10) with or without surface mutations, using our protein design software (ModelX and FoldX). As compared to the IL-10 WT, the designed SC-IL10 molecules increase the effective expression in MPN four-fold, and the activity in mouse and human cell lines between 10 and 60 times, depending on the cell line. The SC-IL10 molecules expressed in the mouse lung by CV8 in vivo have a powerful anti-inflammatory effect on Pseudomonas aeruginosa lung infection. This rational design strategy could be used to other molecules with immunomodulatory properties used in bacterial therapy. Synopsis Computer-designed single-chain IL-10 molecules expressed in the mouse lung by an engineered attenuated Mycoplasma pneumoniae in vivo have a powerful anti-inflammatory effect on Pseudomonas aeruginosa lung infection. A high-affinity single-chain IL-10 is engineered using protein design software. Mycoplasma pneumoniae (MPN) can express active IL-10. MPN-expressing engineered IL-10 suppresses mouse lung inflammatory response caused by Pseudomonas aeruginosa. Introduction The IL-10 cytokine has potent anti-inflammatory properties and plays a central role in limiting the host immune response, thereby preventing damage to the host and maintaining normal tissue homeostasis. IL-10 has potential applications as an immunotherapeutic agent in tumours, as it can boost CD8+ tissue T-cell infiltration and increase IFN-γ production, thereby stimulating the T-cell memory response (Fillatreau & O'Garra, 2014). To date, however, clinical trials using IL-10 have failed, in part due to the short half-life of IL-10 in the bloodstream, as well as its proinflammatory effects when applied systemically at high concentrations (van Deventer et al, 1997; Saxena et al, 2015). To improve its pharmacodynamics, IL-10 has been fused to an IgG-Fc region (Terai et al, 2009) or to polyethylene glycol (Duncan et al, 2019), and to dampen its pro-inflammatory effects, novel systemically engineered variants have been generated that modulate its binding to the low-affinity receptor 2 (R2; Gorby et al, 2020) or that do not activate the proinflammatory cascade (Saxton et al, 2021). The use of bacteria as delivery vectors or live biotherapeutic product (LBP) has the advantage of being able to generate the therapeutic agent locally, which lowers the required administered dose, thereby reducing adverse effects and production costs. Recently, IL-10 expressed by Lactococcus lactis has been shown to be effective in preventing and treating colitis in a preclinical mice model (Steidler et al, 2000; Cardoso et al, 2018) as well as in phase I clinical trials of Crohn's disease (Braat et al, 2006). In the lung, IL-10 can also be used to prevent tissue damage in transplanted organs (Cypel et al, 2009) or as treatment of a wide range of pulmonary diseases, such as fibrosis (Shamskhou et al, 2019), asthma or acute respiratory distress syndrome (ARDS; Ouyang & O'Garra, 2019; Wang et al, 2019). Ideally, a bacterium used for therapy should be administered to the organ where it is naturally present, to ensure the survival and adaptation to the host niche. We have previously demonstrated the potential application of Mycoplasma pneumoniae (MPN) as an engineered vector for the treatment of Staphylococcus aureus biofilm in vivo (Garrido et al, 2021). Although MPN is a mild pathogen that causes pneumonia in humans, it has some advantages as a LBP, mainly related to the absence of a cell wall, which allows the secretion of molecules directly into the medium. Further, MPN also has intrinsic properties that limits horizontal gene transfer, such as its use of a UGA triplet as a tryptophan codon rather than a STOP codon (Inamine et al, 1990; Wodke et al, 2013). Further, some strains (i.e., MPN 129) have a weaker recombination capacity (Sluijter et al, 2010). Finally, it divides every 8–12 h, making it more easily controlled. However, this bacterium has limited protein synthesis capacities as compared to Escherichia coli or Bacillus subtilis; for instance, it has 100 to 200 ribosomes per cell (Kühner et al, 2009; Maier et al, 2011) vs. 1 × 105 ribosomes per cell in E. coli in the exponential phase (Scott et al, 2010). Thus, it is critical to optimise the effective concentration and activity of the expressed IL-10 molecule. To address this, we first took into account the molecular characteristics of IL-10 that distinguish it from other interleukins (ILs): it comprises a homodimeric swapped domain (Bennett et al, 1994) in which each monomer is bridged by Cys12-Cys108 and Cys62-Cys114 disulphide bonds (PDB residue numbering), but with no intermonomeric disulphide bonds. An engineered monomeric version created by enlarging a loop by inserting six amino acids (aa; e.g., GGGSGG) is significantly less active (Josephson et al, 2000), underscoring the importance of the dimeric folding of IL-10 for its activity. However, this requirement for a dimeric state reduces the effectiveness of IL-10 expressed and secreted by a bacterial chassis in a human organ, as dimerisation is hindered in large spaces, leaving the proteins more prone to dissociation at low concentrations and/or low pH (Syto et al, 1998), which could lead to degradation, aggregation and extensive multimerisation (Westerhof et al, 2012). This is especially important in the lung, which has a limited microbiome capacity as compared to the intestine (Huffnagle et al, 2017), preventing large amounts of bacteria from being administered. Here, we (i) designed mutations in IL-10 that increase its affinity for the R1 and R2 receptors (Gorby et al, 2020) and (ii) engineered a single-chain (SC) variant composed of two IL-10 monomers, using two protein design softwares: FoldX (Schymkowitz et al, 2005; Delgado et al, 2019) and ModelX (Blanco et al, 2018; Delgado Blanco et al, 2019; Cianferoni et al, 2020). MPN was able to express active IL-10 and the new IL-10 variants that exhibited an increased affinity as compared to the wild-type (WT) IL-10 (IL-10 ORF) in vitro and had a powerful antiinflammatory effect in an acute lung infection model induced by Pseudomonas aeruginosa. This strategy could reduce production costs and be used in other therapeutic bacteria, as a more active product secreted by the bacterial chassis leads to a lower required bacterial dose and hence a reduction in any potential pathogenicity. Results M. pneumoniae secretes functional IL-10 dimers with disulphide bridges We first tested whether MPN can actively express a complex molecule, such as the human IL-10-swapped dimer with two disulphide bridges. For this purpose, we cloned the human IL-10 fused to the mpn142 secretion signal previously described for MPN (Garrido et al, 2021). We showed by ELISA that the MPN WT strain secreted IL-10 in the supernatant (Table EV1). We confirmed by mass spectroscopy (MS) that both disulphide bridges were generated in the expressed IL-10 (Dataset EV1), indicating that the dimer was correctly folded. We next tested the functionality of the produced IL-10 (IL-10 ORF) for its ability to modulate the primary anti-inflammatory response activation program of human macrophages (see Materials and Methods). For this, MPN expressing IL-10 was grown for 2 days, and the supernatant was added to a cell culture containing approximately one million circulating macrophages (see Materials and Methods). As a positive control, the human IL-10 recombinant protein (hIL-10r) was added. Commercial hIL-10r treatment of macrophages enhanced the expression of anti-inflammatory markers (Fig 1A) and decreased the levels of pro-inflammatory ones (see Fig 1B) as compared to non-treated cells. The results using IL-10 ORF were comparable to those using hIL-10r. We also confirmed that human primary blood CD14+ macrophages become activated upon hIL-10r or IL-10 ORF stimulation by phosphorylation of Tyr705 of STAT3 (p-STAT3; Yu et al, 2009; Fig EV1A). Additionally, p-STAT3 was activated in HAFTL murine B-cell line (Fig EV1B), indicating that both the human and murine cell lines were activated by the human IL-10. Figure 1. Effects of human IL-10 recombinant protein (hIL-10r) or IL-10 secreted by M. pneumoniae on macrophage primary cells on anti-inflammatory (A) or pro-inflammatory biomarkers (B) A, B. Data were normalised using the control group and are represented as fold-change (FC) of the mean fluorescence intensity (MFI). Macrophage cells were obtained from human donors (n = 4 biological replicas) and incubated with medium (CON) or recombinant human IL-10 (hIL-10r), MPN wild-type (WT) or MPN expressing IL-10 ORF (WT_IL10). Data are represented as mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way ANOVA + post hoc Tukey's multiple comparison test (*P < 0.05; **P < 0.001, ***P < 0.0001). Source data are available online for this figure. Source Data for Figure 1 [msb202211037-sup-0010-SDataFig1.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Analysis of SC mutants designed in this work A, B. Detection of phosphorylated Tyr 705 of STAT3 (p-STAT3) and unphosphorylated STAT3 by Western blot (see Materials and Methods). (A) Data from macrophages isolated from two independent donors, showing unstimulated cells (control, CON) or cells incubated for 24 h with human IL-10 recombinant protein (hIL-10r), supernatant of MPN WT (WT) or of MPN expressing IL-10 (WT_IL-10 ORF). (B) Data from HAFTL murine B-cell line after exposure for 20 min to the supernatant of MPN WT (WT) or MPN expressing IL-10 (WT_IL-10 ORF). C. HEK-Blue™ reporter cell activation dose–response analysis by MutSC1 (linker NGGLD) and MutSC1_Gly (linker GGGGG) supernatants. The x-axis shows the range of IL-10 concentration analysed (Molar, M), and the y-axis represents the mean ± SD of the absorbance at 630 nm. Data were generated in three independent assays with two technical replicas (n > 6). D. Western blot of p-STAT3 activation after 20 min of induction with a fixed IL-10 concentration (20 ng/ml) of supernatants from MPN WT (WT) or MPN expressing IL-10 WT (WT_IL10 ORF), MutSC1 (WT_MutSC1), MutSC2 (WT_MutSC2) in two different cell lines: THP-1 (human monocyte) and HAFTL (murine pre-B cell line). Download figure Download PowerPoint In summary, these results demonstrated that MPN expresses functional human IL-10 that is capable of activating phosphorylation of STAT3 in human primary blood CD14+ macrophages and in a murine cell line. Engineering IL-10 for increased receptor affinity To overcome the limitations of a low protein production capacity in MPN, and to decrease the number of bacterial cells administered to a patient, one strategy is to engineer mutations in IL-10 with increased binding affinity to the high- and low-affinity receptors R1 and R2, respectively. To generate IL-10 versions with an increased affinity towards R1, we used the hIL-10 molecules crystallised in complex with R1 with the best crystal resolution (PDB 1y6k; 2.5 Å). We then performed an in silico mutagenesis scanning of the complex interface residues using the PositionScan FoldX command, which individually mutates all R1-contacting IL-10 aa to the other 19 natural aa (Schymkowitz et al, 2005). Based on this analysis and on sequence conservation across different species (Fig EV2), we identified a set of non-conserved residues that, when mutated with FoldX, improved binding to R1 without compromising protein stability (Dataset EV2). Using the selected mutations, we modelled two multiple mutants, termed Mut1 and Mut2, that contain D28E, S31K/R, N45S and T155M (note that they differ at position 31, with the WT Ser31 mutated to Lys in Mut1 and Arg in Mut2; Fig 2). We verified that the differences in stability and binding energies were not the result of a particular conformation of the crystal structure that we used by computing the in silico variation of free energies of the multiple mutations of IL-10 in Mut1 and Mut2 using other experimental structures (IL-10 apo form: PDB 2ilk, 1.6 Å; IL-10 holo form with R1: PDB 1j7v, 2.9 Å; IL-10 holo form with R1&R2: PDB 6x93, 3.5 Å). We looked for changes in energy that affected IL-10 stability, interactions with R1 and R2 (when present in the PDB structure) and stability of IL-10 receptor complex (Dataset EV2). Figure 2. Schematics of all mutations generated in this work to increase IL-10/R1/R2 receptor affinityDetails for each of the positions mutated to improve interactions with R1 are shown in the individual panels. Note that when aspartic acid-28 is mutated to glutamate in IL-10, an electrostatic displacement occurs over the arginine-24, allowing it to form an H-bond with glutamate-145 from the receptor 1 (yellow double arrow in the subfigure indicates D28E). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Multiple sequence alignment of IL-10 from different vertebrate species performed with the ClustalX algorithmThe first residue corresponds to the first residue of IL-10 in the crystal structure 1y6k. Download figure Download PowerPoint At the start of this work, no structure was available for IL-10 bound to R2. Therefore, we included mutations in Mut2 that had been previously reported to potentially enhance the affinity of the interactions between IL-10 and R2 (e.g., N18I, N92I and K99N; Gorby et al, 2020), resulting in Mut3. When modelling these three mutations in X-ray IL-10 structures in a complex with the R1 but without the R2 receptor (1y6k, 2ilk), we found that they decreased the stability of IL-10 (Dataset EV2). This apparent decrease in stability when using the 1y6k and 2ilk structures is due to a conformational change in the regions where the mutations are introduced upon binding to the R2 receptor (see the X-ray; 1y6k, 2ilk) superimposed on a cryo-EM structure (6x93; 3.5 Å) of the complex between IL-10 and R1/R2 (Saxton et al, 2021; Fig EV3). In fact, two of these previously described mutations (N18I and N92I) modelled on the 6x93 improved stability and binding to R2 (Dataset EV2). Click here to expand this figure. Figure EV3. Superimposition of the crystal structures of IL-10 with R1 (1yl6k) and of IL-10 with both R1 and R2 (6x93)Unstructured regions adopted a different conformation when interacting with R2. Gorby (9)-mutated positions (N19, N92, K99) are zoomed-in on the right side in the crystallographic superimposition with cryo-EM (9), showing an appreciable backbone shifting upon binding to R2 in some of the positions. Download figure Download PowerPoint Measurement of EC-50 and relative affinity ratios for the designed multiple mutants To assess the relative affinity of the different designed IL-10 variants, we used a reporter cell line (HEK-Blue™) that contains a secreted embryonic alkaline phosphatase (SEAP) reporter. Briefly, when IL-10 is bound to R1 and R2, the SEAP reporter triggers a JAK1/STAT3-mediated response resulting in SEAP expression, which can be measured by absorbance at 630 nm (see Materials and Methods). The intensity of the signal before saturation is proportional to the activation of STAT3, and a kinetic model (Heck, 1971) can be fitted to the data to obtain EC-50 values (see Materials and Methods), which could be used to determine relative activity differences between WT and mutants (Gorby et al, 2020). Mutants Mut1 and Mut2 (designed for improved binding to R1) had similar EC-50 values (1.45 e−10 M ± 6.59 e−11 and 6.45 e−11 ± 1.73 e−11 M, respectively), but were better than IL-10 ORF (1.98 e−10 ± 9.2 e−11 M; 1.4-fold and 3.0-fold enhancement in relative affinity, respectively). Mut3, based on Mut2, incorporates new mutations previously described to improve R2 binding (namely, N18I, N92I and K99N; Gorby et al, 2020). Inclusion of these three mutants resulted in an improved EC-50 value (2.61 e−11 ± 1.49 e−11 M), with 8-fold enhancement in relative affinity as compared to the IL10 ORF (Fig 4A). This increased affinity is to be expected, as it contains mutations that enhance both R1 and R2 binding, while Mut2 only has mutations to enhance R1 binding. We showed that the EC-50 of the monomeric IL-10 (MutM) is on average lower (although not significant statistically; P > 0.05) than that of the IL-10 ORF (2.56 e−10 ± 8.77 e−11), as previously described (Josephson et al, 2000; Fig 4A and Dataset EV3). The expression level in MPN of the best mutant (Mut3) was comparable to that of the IL-10 ORF (Fig 4B). Thus, we observed that mutations incorporated in Mut3 enhanced the relative activity by about 8-fold as compared to that of the IL-10 ORF. Single-chain IL-10 rational design Active IL-10 is a swapped dimer, with no intramonomeric disulphide bonds, composed of IL-10L and IL-10M monomers (Fig 3A). However, reaching an equilibrium between the unfolded monomer and the folded dimer decreases effective concentration and could result in potential degradation, misfolding and/or extensive multimerisation (Westerhof et al, 2012). To solve this problem, we decided to create a SC protein by linking the N-terminal region of monomer M with the C-terminal region of the monomer L in the swapped dimer (MutSC1; Fig 3A). To design the linker, we used the ModelX software (Bridging command) developed by our group to screen for linkers in silico between Asn18' from monomer M and Lys157 from monomer L as anchoring points (see Materials and Methods). Bridging connected the anchoring points with all geometrically compatible peptide fragments from PepXDB (see Materials and Methods). Fragments were retrieved with their corresponding side chains, allowing us to explore both conformational and sequence space at a time. We obtained 541 models that were side chain-repaired and ranked by energy using the FoldX v5 (Delgado et al, 2019) RepairPDB and Stability commands, respectively (see Materials and Methods). We determined that the best linker sequence was N157-FGGLD-Y18', whereby Asn18' was replaced by Tyr and Lys157 by Asn, and the FGGLD sequence was inserted. By introducing this linker, we deleted the N-terminal residues of M in the crystal structure (residues 12′ to 17′; CTHFPG). Afterwards, we mutated the Phe after N157 to Asn to improve binding to R1 (this results in NGGLD as the inserted sequence; Fig 3B); this improved the FoldX predicted overall stability of the SC molecule by creating an intramolecular HBond with G160 that rigidified the inserted loop as well as its binding to R1 by stabilising the bound state. Afterwards, we renumbered the sequence with the inserted connecting loop (see Materials and Methods). Note that we deleted the N-terminal sequence of one of the monomers containing Cys12', which makes a disulphide bridge with Cys108' in the original IL-10 ORF sequence (new residue 259); therefore, we mutated Cys108' to Asn, to prevent spurious disulphide bridges. The original Cys12 and Cys108 remained in the structural equivalent of the other monomer of the swapped dimer. Thus, MutSC1 has three of the four original disulphide bridges. MutSC2 was designed by grafting the Mut3 mutations into MutSC1 (Fig 3B). We verified by MS that both MutSC1 and MutSC2 generated the two different disulphide bridges present in the original monomers of the swapped structure (Dataset EV1). Figure 3. Schematic depiction of the design of single-chain (SC) IL-10sIL-10L and IL-10M monomers are respectively shown in blue and magenta. To avoid confusion, IL-10M residue numbers are denoted by adding the prime (′) character. Steps to generate two different sewing patterns: (1) deletion of fringe residues; (2) rewiring schema; (3) structural rearrangement after rewiring the corresponding regions; (4) final numbering in the SC, including numerical gaps long enough to host peptide bridges with different lengths (up to 20) during linker search or bridging. Monomer 2 (IL-10M, in magenta) residue numbers are marked by '. MutSC1 and MutSC2 built from the IL-10 sewing pattern 1. Linker sequences are shown as red labels; post-rewiring mutations are included for MutSC2 using monomeric IL-10 numbering. Download figure Download PowerPoint Characterisation of the engineered IL-10 mutants in vitro in human and murine cell lines We next addressed whether expressing a SC-IL10 rather than the swapped dimer increased the amount of functional protein expressed by MPN. We quantified the IL-10 protein concentration in the cell culture supernatant by ELISA and determined the number of cells by counting colony-forming units (CFU; see Materials and Methods). We found that the supernatant with MutSC1 contained up to 4-fold more secreted IL-10 protein than the supernatant with IL-10 ORF or Mut3 (Fig 4B). We then assessed the capacity of each mutant to activate the IL-10 receptors in HEK-Blue™ cells. As compared to IL-10 ORF (EC-50 1.98 e−10 ± 9.24 e−11 M), the SC mutants MutSC1 (1.90 e−11 ± 5.12 e−12 M) and MutSC2 (1.20 e−11 ± 2.8 e−12 M) had better EC-50 values than the IL-10 ORF, with an improved relative activity of 10.4- and 16.5-fold, respectively. Importantly, MutSC2 had a better EC-50 than the best multiple mutant in the swapped dimer (Mut3). To determine the importance of the linker sequence in the activity of MutSC1 and MutSC2, we replaced the linker sequence N-NGGLD-Y with N-GGGGG-Y (MutSC1_Gly). The new control mutant MutSC1_Gly had a very significant decrease in activity, indicating that it is important for a short linker to have a specific sequence (Fig EV1C). To address whether this improvement in the relative activities of MutSC1 and MutSC2 was specific to the species cell type used in the assay, we assessed STAT3 phosphorylation (p-STAT3) upon ligand stimulation by FACS in two different cell lines: murine HAFTL (a mouse pre-B cell line) and human BLaER1 (a B-cell precursor leukaemia cell line). As compared to IL-10 ORF, both the MutSC1 and MutSC2 mutants were significantly superior in activating p-STAT3 (enhanced relative activity: 29.0 ± 9.5-fold and 57.1 ± 13-fold in BLaER1; 11.2 ± 1.8-fold and 18.6 ± 1.9-fold in HAFTL, for MutSC1 and MutSC2, respectively; Fig 4C and D, and Dataset EV4). Altogether, the engineered mutants MutSC1 and MutSC2 resulted in enhanced relative activity in different cell types of human or mouse origin. As observed for the HEK-Blue™ cells, MutSC2 was around 100% better than MutSC1. Similar qualitative results were found by Western blot in murine HAFTL and in the human monocyte cell line THP-1 (Fig EV1D). Figure 4. Expression levels and apparent dissociation constant of selected IL-10 variants expressed by M. pneumoniaeEach point in the figure is a biological replica. A. EC-50 (molar, M) for human IL-10 recombinant protein (hIL-10r) and IL-10 WT (IL-10 ORF) and different variants (MutM, hIL-10r, Mut1, Mut2, Mut3, MutSC1 and MutSC2) expressed by M. pneumoniae. Data are represented as mean ± SD. Statistical comparison was done by one-way ANOVA + post hoc Bonferroni multiple comparison test, using the IL-10 ORF condition as a reference (*P < 0.05). B. Fold-change (FC) in expression levels (fg/CFU) of IL-10 variants Mut3 and MutSC1 secreted to the medium normalised by expression level of IL-10 ORF (ORF). Statistical comparison of mean ± SD was performed by one-way ANOVA + Tukey's post hoc test (*P < 0.05). C, D. Average and SD values for the FC in the relative EC-50 values determined by flow cytometry analysis of phosphorylated STAT3 after a 20-min exposure of the BlaER1 (C) or HAFTL (D) cell lines to IL-10 ORF (reference) or the mutant MutSC1 or MutSC2 (see Materials and Methods). Numbers indicate the average FC ± SD. Source data are available online for this figure. Source Data for Figure 4 [msb202211037-sup-0011-SDataFig4.zip] Download figure Download PowerPoint Overall, these results support the idea that the IL-" @default.
• W4313487834 created "2023-01-06" @default.
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• W4313487834 date "2023-01-01" @default.
• W4313487834 modified "2023-10-14" @default.
• W4313487834 title "Bacterial expression of a designed single‐chain <scp>IL</scp> ‐10 prevents severe lung inflammation" @default.
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