Modulation of In ammatory Cytokines Secretion Response from Stimulated Human PBMCs Following Application of Recombinant Human Cytokines IL-37b and IL-38 Produced in Plants


 Affordable therapeutics are vitally needed for humans worldwide. Plant-based production of recombinant proteins can potentially enhance, back-up, or even substitute for the manufacturing capacity of the conventional, fermenter-based technologies. We plastome-engineered a tobacco cultivar to express high levels of two “plantakines” - recombinant human cytokines, interleukins IL-37b and IL-38, and confirmed their native conformation and folding. Assessment of their biological functionality was performed ex vivo by analyzing the effects exerted by the plantakines on levels of 11 cytokines secreted from human Peripheral Blood Mononuclear Cells (PBMCs) challenged with an inflammatory agent. Application of the plant-produced IL-37b and IL-38 in PBMCs stimulated with Lipopolysaccharide or Phytohaemagglutinin resulted in significant, dose-dependent modulation of pro-inflammatory cytokines secretion and attenuation of levels of several cytokines involved in inflammatory response. Our results demonstrate feasibility of manufacturing functional recombinant human proteins using scalable, cost-effective and eco-friendly plant-based bioreactors.


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Introduction 2 3 Plants make a lot of sense as production platforms for all kinds of biologics. Photosynthetic capacity 4 allowing autotrophic growth renders plants the most energy-efficient and cost-effective platform for 5 manufacturing of various recombinant proteins, secondary metabolites and other assorted small 6 molecules, as plants require only three abundantly available raw input ingredients for biosynthesis -7 carbon dioxide, water and sunlight. Hence, the initial part of the manufacturing process, the "upstream 8 production" that generates the biomass accumulating the desired product ensues significant costs savings, 9 eliminating the need for construction, maintenance and operation of fermenter facilities 1 2 3 . Benefits to 10 "downstream production" steps of the process, where the desired product is extracted and purified are also 11 recognized for plant-based systems, with some of the bottlenecks being addressed in recent studies 4 5 6 . 12 Additional advantages of exploiting plants as single-use, clean and biodegradable bioreactors for 13 production of recombinant proteins include inherent safety due to inability of mammalian pathogens to 14 propagate in plant tissue and virtually unlimited scalability of plant-based production 7 8 . 15 Since the emergence of the first reports of successful genetic transformation of plants and the expression 16 of recombinant heterologous proteins of human origin in transgenic plants, tremendous technological 17 advances were achieved in the "molecular pharming" field, with the first FDA-approved pharmaceutical 18 for human use in 2012, taliglucerase alfa, produced in carrot cells 9 . Today several biopharmaceuticals on 19 the market are sourced from plants and a few biotechnology companies around the world use plant-based 20 production platforms in their manufacturing processes 10 3 11 . Plant-based bioreactors could facilitate 21 making more affordable many biologic drugs in use today and provide a source of therapeutics supplied 22 locally, which can be very beneficial in the context of developing nations, or when global supply chains 23 are disrupted 11 12 . 24 Among the methodologies used for plant-based recombinant protein manufacturing, plastome-engineered 25 plants possess several advantageous features as a platform, simply generating extraction-ready biomass 26 from seed. Plastome-engineered plants can express and accumulate very high yields of the desirable 27 product and, thus, can represent the most cost-effective production route 13 14 15 16 . We set to demonstrate 28 the feasibility of plastome-engineered plant bioreactor platform for production of biologically active 29 recombinant human cytokines. Based on our preliminary screens searching for valuable proteins with a 30 potential for prolific expression in plastids, we engineered the plastome of a low-alkaloid tobacco cultivar 31 to produce "bioreactor lines" expressing mature forms of two "plantakines" -human interleukins IL-37 32 (isoform b, IL-37b) and IL-38, both characterized as anti-inflammatory cytokines 17 18 . IL-37b and IL-38 33 belong to the IL-1 family of 11 interleukins, 7 of which are pro-inflammatory 19 . Both IL-37b and IL-38 34 function in regulation/mitigation of human inflammatory responses; a plethora of studies demonstrated 35 central involvement for IL-37b and IL-38 in immunity and disease and, therefore, as potential candidates 36 for development as therapeutic agents 20 21 . The created plastome-engineered bioreactor lines produced up 37 to ~1 gram of the recombinant protein per 1 kg of fresh leaf biomass. After confirmation of their correct 38 folding, we assessed the biological activity of the plant-produced IL-37b and IL-38 in ex vivo experiments 39 by monitoring the response to inflammatory agents (IAs) in freshly isolated cultured human Peripheral 40 Blood Mononuclear Cells (PBMCs), manifested in the levels of secreted inflammatory cytokines.

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PBMCs are the central and crucial components of the immune system that brings forth a response to 42 intruder pathogens, as well as identifies and fights own body cells that have undergone malignant 43 transformation (cancer). PBMCs are an assorted mixture of highly specialized immune cells, PBMCs 44 population is comprised of a multitude of immune cell types including lymphocytes (~85%), monocytes 1 (~15%) and dendritic cells (<1%) 22 . In vitro and ex vivo human PBMCs studies are ubiquitous in cell 2 biology and immunology research and an important biotechnological tool in developing new therapeutics 3 and diagnostics 23 24 25 26 27 . We hypothesized that by monitoring the inflammatory response in IA-4 stimulated PBMCs we could study the effects of the plantakines (and their active concentrations) exerted 5 on the levels of specific inflammatory markers. 6 We report significant modulation of inflammation responses from PBMCs stimulated with different IAs 7 as a result of treatments with the plant-produced IL-37b and IL-38. We observed attenuation of levels of 8 several secreted inflammatory cytokines, generally consistent with the previous reports characterizing the 9 biological activity of IL-37b and IL-38 as anti-inflammatory. We engineered the plastome transformation constructs to produce IL-37b and IL-38 as mature peptides 22 (V46 -D218 for IL-37b, C2-W152 for IL-38), optimizing the expression by selecting suitable cis-acting 23 regulatory genetic elements and using plastid-preferable codons (data not shown). Screening for prolific 24 producer lines of IL-37b and IL-38 identified the best configurations of plastid expression cassettes by 25 examining their crude leaf tissue extracts with Western blots (Figure 1a). Two bioreactor lines were 26 selected and grown in greenhouse to maturity, expressing the recombinant human IL-37b and IL-38 at ~1 27 g and 0.75 g, respectively, per 1 kg of fresh leaf tissue. Interestingly, prevalent amounts of both 28 plantakines were found to accumulate in older leaves, demonstrating significant stability of these 29 recombinant proteins in the chloroplasts (Figure 1b). We observed large amounts of the monomeric 30 forms, as well as the dimerized and multimerized forms of the cytokines IL-37b and IL-38 in the crude 31 leaf extracts and in samples after purification; the dimers (and higher molecular weight multimers) were 32 very stable and detectable in SDS-PAGE analyses gels even after harsh denaturing conditions of the 33 sample preparation. That observation was in stark contrast to the bacteria-produced recombinant IL-37b 34 and IL-38 counterparts available commercially, that predominantly presented the monomeric forms of the 35 cytokines when used as controls in Western blot experiments using specific antibodies (Figure 1c, d).

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Placement of the HIS-tag at the N-terminal had no effect on the formation of dimers/multimers for both 37 expressed cytokines (data not shown). clones generated for expression of plantakines IL-37b and IL-38 tagged with a HIS-tag at the C-terminus. 5 Numbers 1 -5 for each IL-37b and IL-38 represent extracts (~100 µg fresh leaf tissue) from different 6 clones; clones 1, 2 and 5 for IL-38 show no expression. VGFP (EGEH 28 ) is a HIS-tagged GFP variant 7 used as quantifiable control protein; lanes 1, 2 and 3 represent 12.5, 25 and 50 ng, respectively. All blots 8 probed with the same anti-His tag antibody. Molecular weight marker (MW) ladder is in kiloDaltons. Single black triangle arrows depict the 7 monomers of the plantakines of the predicted molecular sizes (20.3 kDa and 18.3 kDa for IL-37b and IL-8 38, respectively), double arrows depict the dimers. Higher molecular weight multimeric structures are 9 also detectable. 10 11 12 13 Plantakines bioactivity assessment -experimental design 14 The bioactivity of our plantakines IL-37b and IL-38 was assessed by monitoring the secretion of 11 15 cytokines, generally regarded as inflammatory markers, from PBMCs stimulated with an IA. Different IAs bring about different magnitude of inflammatory responses from PBMCs 35 In order to validate the obtained data, as well as to gain insights into the quantitative and qualitative 36 differences in the PBMCs' inflammatory responses between the two IAs tested, we first compared the 37 mean values for each monitored secreted cytokine elicited by either LPS or PHA. The magnitude of the 38 general responses from the stimulated PBMCs, manifested in levels of the secreted pro-inflammatory 39 cytokines was found significantly different between the two IAs. PHA elicited stronger response in 7 out 40 of 11 pro-inflammatory cytokines monitored; namely, IL-17, IFNγ, TNFα, IL-12, IL-22, IL-10 and IL-6 41 displayed, respectively, 2930%, 1240%, 55.7%, 118%, 21.5%, 86.4% and 10.1% higher levels, compared 42 with the LPS-elicited levels (p < 0.001p < 0.05, Figure 2). The levels of GM-CSF, IL-8, IL-1α and IL-43 1β showed no statistically significant difference between the IAs in our experiments. Legend: 10 n/sno significant difference; 11 *significant difference, p < 0.05; 12 ***significant difference, p < 0.001. 13 14 15 16 17

Plant-produced IL-37b and IL-38 modulate inflammatory responses from IA-stimulated PBMCs 18
To gain insight into the bioactivity of the plantakines IL-37b and IL-38 exerted on IA-stimulated PBMCs 19 we compiled the data generated from treatments with observed modulatory effects on secreted 20 inflammatory markers. GEE analysis was performed four times, for each combination of the IA and its 21 concentration, for each secreted cytokine monitored, generating statistically significant (p<0.05) level 22 modulations displayed in 118 treatments out of the total 286 treatment combinations assessed. Plantakines 23 exerted statistically significant modulatory effects on the levels of secreted inflammatory cytokines in 67 24 and 51 treatments that occurred in LPS-and PHA-stimulated PBMCs, respectively (Table I). 25 Collectively, treatments with plantakines IL-37b and IL-38 resulted in more profound anti-inflammatory 26 activity in LPS-stimulated PBMCs rather than PBMCs stimulated with PHA, as only 10 treatments 27 resulted in increased secretion of inflammatory cytokines in LPS-stimulated PBMCs, while decreased 28 secretion was observed in 57 treatments. In contrast, secretion of inflammatory markers in PHA-29 stimulated PBMCs was suppressed in 17 treatments with plantakines and increased in 34. Notably, all the 1 treatments with the simultaneous application of both IL-37b and IL-38 brought about increases in 2 secretion of inflammatory cytokines under stimulations with either IA, while separate applications of the 3 plant-produced IL-37b or IL-38 suppressed secretion of inflammatory cytokines in 44 and 30 treatments, 4 and increased it in 12 and 22, respectively. Fewer treatments with plantakines caused suppression of 5 inflammatory cytokines secretion and the numbers of treatments where inflammatory cytokines secretion 6 increased grew in association with a higher concentration of either IA used to stimulate the PBMCs 7 (Table I) We further analysed the changes in levels of the secreted inflammatory cytokines from the stimulated 23 PBMCs resulting from treatments with different doses of the plantakines IL-37b and IL-38. For each 24 inflammatory marker monitored, the outcomes of the treatments were calculated as percentages of 25 secretion modulation with its probability value in comparison with the positive controls at the 26 corresponding IAs concentrations (Table II). The modulatory effects of the plantakines IL-37b and IL-38 1 could be observed on the levels of most of the monitored secreted inflammatory cytokines elicited with 2 either LPS or PHA, showing a general tendency of attenuation. IL-37b attenuated levels of IFNγ, IL-1α, 3 IL-1β, IL-22, IL-17 and TNFα in LPS-stimulated PBMCs, the effect could be seen at all the 4 concentrations examined, levels of IFNγ and IL-22 were also reduced by IL-37b in PHA-stimulated 5 PBMCs (Table II). Unexpectedly, IL-37b at all 3 concentrations brought about an increase in IL-17 6 secreted from PBMCs stimulated with PHA at 10 µg/mL, similar increases were observed for IL-1α and 7 GM-CSF levels with IL-37b at 100 ng/mL. Modulation of GM-CSF levels by both IL-37b and IL-38 8 displayed dose-dependent character: at low concentrations (1 ng/mL) both plantakines attenuated GM-9 CSF levels by more than 50% in PBMCs stimulated with 150 pg/mL LPS, while 100 ng/mL plantakines 10 concentration increased the levels of GM-CSF, those increases observed more profoundly at LPS 300 11 pg/mL concentration. plantakines IL-37b and IL-38 were applied at the lowest concentration (1 ng/mL), statistically significant 23 attenuation (-9.5% for IL-6 elicited at 150 pg/mL LPS, p=0.012, and -28.5% for IL-8 elicited at 300 24 pg/mL LPS, p=0.032) was detected, aligned with the anti-inflammatory functions expected from IL-37b 25 and IL-38 (Table II) In the present study we engineered the tobacco plant plastome to produce green bioreactors capable of 7 manufacturing profuse amounts of two functional recombinant human cytokines, IL-37b and IL-38, 8 mainly known as anti-inflammatory modulators of immune responses 17 29 . To our best knowledge, this is 9 the first report describing such a prolific expression and production of both recombinant human cytokines 10 in their active forms in plants, a previous successful attempt to produce IL-37b in tobacco via nuclear 11 genome transformation reported much lower yields, while there are no reports on IL-38 production in 12 plants hitherto 30 . Very interesting is the fact that the penultimate amino acid (the second amino acid in the 13 peptide chain after the initiating Methionine) of the IL-38 peptide is Cysteine, which was underlined, 14 along with Histidine, as the strongest instability-conferring penultimate amino acid for protein expression 15 and accumulation in plastids, leading the researchers to propose existence of an N-terminus-dependent 16 protein degradation pathway in plastids 31 . Our bioreactor lines produced IL-38 peptide with the 17 penultimate Cysteine at 8% -10% of the total soluble protein in the leaf tissue, contradicting the 18 proposed model. Further, the same transformation construct expressing an IL-38 peptide variant with an 19 added penultimate Serine, which was reported as a stabilizing penultimate amino acid, reached similar 20 levels of IL-38 accumulation (data not shown), suggesting that the proposed N-terminus-dependent 1 protein degradation pathway in chloroplasts is either limited in its processing capabilities, or involves 2 additional unknown regulatory factors that specifically direct degradation of select proteins 31 . 3 The accumulated dimerized (and multimerized) forms of the plantakines IL-37b and IL-38 in the crude 4 leaf extracts and in purified samples constituted ~50% of the entire recombinant protein yields, 5 contrasting the results of successful recombinant production studies of IL-37b and IL-38 proteins in 6 bacteria, which never reported dimerization of the purified cytokines in their SDS-PAGE analyses, even 7 when the purified IL-37b was concentrated by ultrafiltration 32 33 34 . This observation suggests that the 8 chloroplast stroma compartment, accumulating the synthesized recombinant proteins, provides a 9 beneficial milieu of internal conditions/chaperones/scaffolds assisting the folding of these cytokines and 10 promoting their further dimerization and multimerization. It is also reasonable to assume the remarkable 11 stability of those dimer/multimeric forms of the cytokines in plastids, since the highest levels of 12 accumulation were observed in older leaves. Bacteria-produced IL-37b was shown to form dimers at 13 nanomolar concentrations and tetramers at higher concentrations, which greatly diminished its bioactivity, 14 suggesting a mechanism of activity regulation through monomer/dimer equilibrium and leading to an 15 engineered monomeric IL-37b variants with much stronger biological activity 35 36 . Those monomeric 16 variants, however, along with the natural mature recombinant IL-37b peptide showed appearance of 17 minor bands that corresponded to the dimer size in SDS-PAGE analyses and further investigation of these 18 protein structures is needed 35 . Also, intriguing is the question whether the formation of dimers/multimers 19 contributes to the overall stability of those cytokines against proteolysis, thus enabling the proposed 20 mechanism of self-regulation and in situ preservation in a stable inactive form in the intercellular space, 21 where the local IL-37b concentration reached the dimerization constant values 35 . A mechanism of 22 bioactivity regulation through the dimer formation, similar to that of IL-37b, was proposed for IL-38 in a 23 recent review 29 ; however, no scientific reports are available addressing this subject. Further structural 24 studies will answer the question whether IL-38 can be engineered into a stable, bioactive monomer, 25 similarly to IL-37b.

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Bodily inflammatory processes are a part of innate immune responses, promoted by pro-inflammatory 27 cytokines released from the cells of the immune system as a reaction to the presence of an inflammatory 28 agent or stimuli. Secreted levels of 11 well-characterized inflammatory cytokines, namely GM-SCF, 29 IFNγ, TNFα, IL-1α, IL-1β, IL-6, IL-8, IL-22, IL12, IL-17 and IL-10 were monitored in our experiments 30 with freshly isolated PBMCs subjected to treatments with two different IAs in combination with two 31 plant-produced anti-inflammatory cytokines IL-37b and IL-38 at different concentrations. This 32 experimental setup allowed for focusing on the biological effects exerted by the plantakines IL-37b and 33 IL-38 via a direct comparison of the levels of pro-inflammatory cytokines secreted from IAs-stimulated 34 PBMCs with or without the plantakines treatments at corresponding concentrations (Tables I, II).

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Although PHA and LPS bind to completely different sets of receptors, both IAs seem to trigger varying 36 signal transduction cascades, particularly leading to the activation of NF-κB, which plays an essential role 37 in regulating the expression of genes linked to innate immunity and inflammatory responses 37 38 . Seven 38 out of 11 inflammatory cytokines monitored in our study displayed significantly higher secretion from 39 PBMCs stimulated with PHA rather than LPS, IL-17 secretion varied more than 30-fold ( Figure 1). 40 Remarkably, our findings strongly corroborate a recent report stating that after PHA-stimulation human 41 PBMCs secreted significantly higher levels of inflammatory cytokines, compared to stimulation with 42 LPS, which apparently failed to notably induce IL-17, TNFα and IL-12 39 . 43 The modulatory effects exerted on levels of the inflammatory markers secreted from IA-stimulated 1 PBMCs confirmed the biological activity of plant-produced IL-37b and IL-38. Statistically significant 2 modulations occurred in both LPS-and PHA-stimulated PBMCs due to treatments with the plantakines 3 (Table I). For quenching inflammation, treatments with plantakines appeared to be more effective in LPS-4 stimulated PBMCs, where 85% of all treatments resulted in attenuations of the levels of secreted 5 inflammatory markers, while only 30% of treatments attenuated inflammatory markers in PHA-stimulated 6 PBMCs, implying varying efficacy and specificity of the anti-inflammatory action under different stimuli. 7 Attenuated, rather than increased secretion of inflammatory cytokines occurred 3.5 and 1.7 times more 8 frequently in treatments with IL-37b and IL-38, respectively, in accord with the proposed role for IL-37b 9 as a primary and fundamental inhibitor of inflammation 17 40 . Treatments combining both plantakines, 10 however, resulted in increased secretion of inflammatory markers under either IA; also, with higher 11 concentrations of either IA applied for PBMCs stimulation, treatments with plantakines suppressive of 12 secretion of inflammatory cytokines became scarcer, while more treatments caused increased 13 inflammatory secretion (Table I). These observed phenomena are inexplicable at this point and require 14 further scientific exploration. 15 Levels of the inflammatory markers secreted from the stimulated PBMCs displayed distinct and varying 16 patterns of modulation following application of treatments with the plant-produced IL-37b and IL-38 17 ( Table II). The number of treatments that triggered an increased, rather than attenuated secretion, was 18 higher only in 2 among the 11 inflammatory cytokines monitored, namely GM-CSF and IL-12, indicating 19 general anti-inflammatory effects exerted by the treatments with plantakines in IA-stimulated PBMCs. 20 Notable were the differences in the magnitude and the scope of the modulation, reflected in the outcomes 21 of the treatments being either an attenuation or an increase of the inflammatory secretion levels: an 22 attenuation of the secretion was the outcome of 74 treatments, averaging -28% level reduction, while 23 increases in secreted levels of inflammatory cytokines, observed in 44 treatments, displayed 79% on 24 average. Among the 11 cytokines monitored, only the levels of IFNγ exhibited consistent attenuation 25 from treatments with either plantakine in PBMCs stimulated with either IA, displaying also the strongest 26 attenuation observed in our experiments (-63.9%, p<0.001), exerted by application of 1 ng/mL IL-38 in 27 PBMCs stimulated with 150 pg/mL LPS. In contrast, GM-CSF levels were 3 times more frequently 28 increased, rather than attenuated by treatments with the plantakines, with the strongest increase reaching 29 380.5%, p<0.001, upon application of 100 ng/mL IL-38 in PBMCs stimulated with 5 µg/mL PHA (Table  30 II). Strikingly, the plant-produced IL-37b and IL-38 both exerted dose-dependent regulation of GM-CSF 31 secreted levels, bringing about attenuation at low concentrations, while causing increases at high 32 concentrations. Although both IL-37b and IL-38 are generally characterized as anti-inflammatory 33 cytokines active in quenching inflammation 41 18 , studies have reported that recombinant unprocessed IL-34 38 could increase inflammatory cytokine IL-6 production in human macrophages in response to LPS or 35 IL-1β stimuli 42 43 . In addition, IL-37b was reported to increase TNFα production in higher concentrations 36 and Candida-induced IL-17 production was reportedly blocked by low concentrations of IL-38, while 37 higher doses of IL-38 induced more IL-17 production, a pattern which resembled IL-37b bioactivity 44 42 . 38 Both IFNγ and GM-CSF are crucial cytokines for activation/differentiation of myeloid cell populations 45 39 46 and their nuanced regulation by the plant-produced IL-37b and IL-38 may serve as a primer for future 40 studies to discern novel patterns in PBMCs inflammatory responses. Interesting also to note that 41 statistically significant attenuation of IL-6 and IL-8, two profound inflammation markers monitored in our 42 study 47 48 was only detected upon applications of low concentrations of the plantakines, aligned with the 43 anti-inflammatory functions expected from IL-37b and IL-38. 44 In conclusion, we developed plastome-engineered, low-alkaloid tobacco bioreactor lines for cost-efficient 1 and prolific production of two functional human cytokines with profound anti-inflammatory properties, 2 IL-37b and IL-38, which are underlined as prospective therapeutic agents. Our explorative study 3 demonstrated that the plantakines exerted significant modulation of levels of secreted cytokines involved 4 in inflammatory responses monitored in IA-stimulated PBMCs, indicating a dose-dependent mode of 5 action and general attenuation of several secreted inflammation markers. Enhancement of several pro-6 inflammatory cytokines, associated with higher concentrations of the plantakines applied in treatments 7 was also observed, revealing novel patterns of inflammation regulation by IL-37b and IL-38. Different 8 magnitude of responses from PBMCs were seen in levels of secreted cytokines elicited by different IAs, 9 where PHA elicited stronger response than LPS in levels of most secreted cytokines monitored. 10 Cumulatively BioTek Instruments (VT, USA) Epoch Microplate Spectrophotometer was used to acquire numerical 7 ELISA data. 8 9 Experiments with PBMCs -General design and Multiplex cytokine analysis 10 After isolation, the PBMCs from each donor separately were counted, plated in equal numbers per well 11 and stimulated for 24h with the applied treatments. Control wells on the plate contained media only (as 12 basal level controls), and LPS and PHA (as positive controls at their corresponding concentrations). After 13 the 24h treatments stimulation the PBMCs' culture supernatants were used in the multiplex Luminex 14 platform-assisted analysis of the concentrations of the 11 secreted pro-inflammatory cytokines.

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The whole blood from 5 random human donors was collected in ACD Vacutainer tubes and immediately 16 processed for isolation of PBMCs by gradient density centrifugation using Lympholyte. The freshly 17 isolated PBMCs from each donor separately were cultured in a volume of 200 µL at a concentration of 18 1.25 X 10 6 cells/mL in 96-well plates (~250,000 cells/well) in an incubator set at 37°C, 5% CO2 and 19 >80% humidity. PBMCs were treated with one of two IAs at 2 concentrations each (LPS at 150 and 300 20 pg/mL; PHA at 5 and 10 µg/mL) in combination with two test items (plant-produced IL-37b or IL-38) at 21 three concentrations each (1, 10 and 100 ng/mL), based on the monomeric form amounts estimated with 22 densitometry (ImageJ). Each treatment was tested in triplicates. Control wells contained media only 23 (negative control, basal level of detection) and the examined IAs at both tested concentrations as the 24 positive controls. The PBMCs were incubated for 24 hours after the stimulation for analysis of the levels 25 of the secreted cytokines that were determined in the culture supernatant using a multiplex immunoassay 26 (MAGPix ® , Luminex), analytes were selected from the Milliplex panel HCYTOMAG-60K, sensitivity 27 range of 3.2 to 10,000 pg/mL. All parameters of the Milliplex panel cytokines analysis using Luminex 28 platform were validated (Millipore-Sigma).

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Statistical analysis 31 Non-linear logarithm transformation was performed to address non-normality of distribution of Luminex-32 derived numeric data. Generalized Estimating Equation (GEE) method was used in the analysis of nested 33 (correlated) structure of the data. Secreted inflammatory cytokine values were used as dependent 34 variables and test item (plantakines) with dosage as independent predictors (factors). Separate analyses 35 were conducted for each of the 11 monitored cytokines and each inflammatory agent/concentration 36 combination. Analysis was performed using SPSS software version 27 using the level of significance 0.05 37 (p-values < 0.05 are reported as statistically significant). 38 39 40 to Anton Svendrovski, MBA, M.Sc., for the proficient help with the statistical analyses. Our sincere 1 appreciation goes to Mr. Felipe Campusano, B.Sc., B.Pharm. (Solar Grants Biotechnology Inc.) for his 2 support and guidance throughout the project. 3 We are grateful for the opportunity provided by AAFC London Research and Development Centre and 4 Fanshawe College CARIB lab, London, Ontario, to conduct collaborative projects. 5 6 7 Author contributions 8 9 IK performed all the work regarding creation of the plastome-engineered bioreactor lines expressing the 10 plantakines, purification and the initial molecular characterization of the purified recombinant proteins. 11 Immuni T Inc. performed the study with PBMCs, statistical analyses of the data were carried out by 12 UZIK Consulting Inc. IK wrote the manuscript.

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Competing interests 15 16 This study was funded and conducted by Solar Grants Biotechnology Inc., of which Dr. Igor Kolotilin is 17 the founder and Chief Scientific Officer. 18 19