Graphical abstract graphic file with name fx1.jpg [33]Open in a new tab Highlights * • Identified a cluster of genes as biomarkers to monitor ALY688 activity in blood * • Validate the reliability of proposed biomarkers in human PBMC * • Discovered ALY688 conferred anti-inflammatory effects in LPS-induced sepsis model __________________________________________________________________ Human metabolism; Genetics; Cell biology Introduction Adiponectin is a protein synthesized by adipocytes, exhibiting robust effects on lipid metabolism, insulin sensitivity, and cardiovascular regulation.[34]^1^,[35]^2^,[36]^3 Subsequently, it was discovered that adiponectin is expressed not only by adipocytes but also by skeletal muscle cells, cardiac myocytes, and endothelial cells. Adiponectin exists in two forms: the full-length protein and a proteolytic cleavage fragment. This fragment comprises the globular C-terminal domain, commonly referred to as globular adiponectin. The cleavage process believed to generate the globular fragment of adiponectin is thought to be mediated by leukocyte elastase, which is secreted by activated monocytes and/or neutrophils. Adiponectin has gained a lot of interest due to its insulin sensitizing, anti-inflammatory, and antiapoptotic properties.[37]^4^,[38]^5 Adiponectin serves as a pivotal regulator of the innate immune system and plays a significant role in the development of inflammation and metabolic disorders.[39]^6^,[40]^7 Clinical studies have revealed an independent and inverse correlation between circulating adiponectin levels and various components of the metabolic syndrome. This includes insulin resistance, body weight, blood pressure, and serum lipids. Furthermore, adiponectin has been found to be negatively associated with cardiovascular diseases such as atherosclerosis and myocardial infarction.[41]^8^,[42]^9^,[43]^10^,[44]^11 After more than a decade of research, the functions of adiponectin and its signaling pathways have been largely identified, primarily through two specific adiponectin receptors (AdipoR1 and AdipoR2).[45]^12^,[46]^13 The molecular actions of adiponectin imply that the molecule itself or agonists targeting its receptors hold potential for the treatment of obesity and its associated comorbidities.[47]^12^,[48]^13 Indeed, in rodent models, the administration of adiponectin has shown to enhance insulin sensitivity and glucose metabolism, elevate insulin secretion, and decrease body weight,[49]^14^,[50]^15 as well as demonstrated the role of preventing cardiac remodeling and improving cardiac function after myocardial infarction.[51]^16^,[52]^17 Targeting its receptors, a recent demonstration revealed that AdipoRon, an orally active synthetic small-molecule adiponectin receptor agonist, markedly enhanced insulin sensitivity and glucose tolerance in mice,[53]^18 and demonstrated cardioprotective effect in the ischemic mouse model.[54]^19 Conversely, the inhibition of AdipoR1 activation, coupled with pharmacological administration of adiponectin, demonstrated the reversal of post-myocardial infarction remodeling and mitigation of heart failure progression.[55]^20 Consequently, there is substantial pharmaceutical interest in developing compounds that target the adiponectin receptor to mimic adiponectin actions. One such compound is ALY688, a 10-amino acid peptide spanning the active site of the adiponectin globular domain. Studies have demonstrated its ability to bind to AdipoR1 and AdipoR2 and induce adiponectin-like effects in several cellular and animal models.[56]^21^,[57]^22^,[58]^23^,[59]^24 As therapeutic interventions mimicking adiponectin progress toward the initiation of clinical trials, the establishment of a dependable target engagement marker becomes imperative for monitoring pharmacodynamics in patients. Such an assay would also have significant value in research studies of animal models. The objective of this study was to develop a bioassay suitable for both clinical and experimental applications, based on a small blood sample. The implementation of such an assay stands to facilitate the non-intrusive, temporal monitoring of adiponectin-like effects. In this study, our goal was to develop such an assay via identifying and validating a cluster of genes that were significantly altered by ALY688. This was achieved via whole-genome RNA sequencing, followed by validation in human peripheral blood mononuclear cells (PBMC). Our investigation also encompassed examining the impact of ALY688 on inflammatory responses in mice subjected to the potent pro-inflammatory endotoxin, lipopolysaccharide (LPS). Results Identification of potential biomarkers of ALY688 administration in mouse peripheral blood In order to determine whether expression of certain genes can be used as a blood-based biomarker for ALY688 administration in vivo, whole blood was collected from mice 24 h post ALY688 (15 mg/kg) or vehicle (PBS) followed by RNA sequencing analysis ([60]Figure 1A). Differentially expressed genes (DEGs) analysis was performed by screening read counts mean value more than 10, adjusted p ≤ 0.05, fold change ≥2, including a total of 170 DEGs, 13 were downregulated and 157 were upregulated in ALY688 treated mice compared to vehicle (PBS) group ([61]Figures 1B and 1C). Hierarchical clustering showed that the gene expression profile of ALY688 was distinct from PBS ([62]Figure 1D). We then ranked genes by the expression to identify the top 20 ALY688-induced genes, with an adjusted p ≤ 0.05 ([63]Table 1; [64]Figure 1E), and top 10 ALY688-induced genes with a higher significance (adjusted p ≤ 0.01) ([65]Table 2). Remarkably, Gene Ontology (GO) term analysis illustrated that these ALY688-induced genes are associated with processes involved in the regulation of T cell differentiation, regulation of inflammatory responses, regulation of polyunsaturated fatty acid metabolic process, DNA damage, collagen formation, copper-dependent protein binding, and ubiquitin-proteasome dependent proteolysis ([66]Figures 1F and 1G). Among the top upregulated genes, Rorc, Myb enriched adaptive immune response and T cell differentiation, lymphocyte differentiation pathways, C1qc enriched immunoglobulin-mediated immune response pathway, and Jag2 was found in the T cell differentiation pathway ([67]Table S3). In essence, these genes can serve as biomarkers for ALY688 activity. Figure 1. [68]Figure 1 [69]Open in a new tab Identification of differentially expressed genes upon ALY688 treatment enriched in inflammatory processes (A) Study design: mice were subjected to 15 mg/kg ALY688 or PBS via sq. injection. After 24 h, cardiac puncture was performed for blood collection for RNA sequencing analysis. (B) Pie chart summarizes the number of genes upregulated and downregulated when comparing ALY688 with PBS. (C) Volcano plot of gene expression changes in gene expression in the ALY688 versus PBS. The volcano plot represents the estimated log2 fold change as a function of the mean of normalized counts. Significant genes were selected when adjusted p value lower than 0.05 and fold-change ≥2. (D) Z score heatmap of RNA-seq transcriptome analysis composed of the 170 differentially expressed genes identified. (E) Expression of top 20 upregulated DEGs sorted by highest fold change with adjusted p value ≤ 0.05. (F) Functional enrichment analysis of robust DEGs. Gene ontology (GO) enrichment analysis of robust DEGs is visualized in a bar plot (p < 0.05). BP: Biological Process; CC: Cellular Component; MF: Molecular Function. (G) An overview chart with functional ontologies and pathways enriched from the differentially expressed genes in ALY688 treated animal. n = 9 per group. Table 1. Top 20 upregulated DEGs sorted by highest fold changes (adjusted p value ≤ 0.05, mean value of raw count ≥10) Gene Official name Mean of count (PBS) Mean of count (ALY) Fold-changes Tgfb2 transforming growth factor β 2 0.33 14.67 18.05 Rorc RAR related orphan receptor C 3.89 20.44 10.05 Siglech CD33 9.00 49.56 7.47 Prg4 proteoglycan 4 8.89 76.89 6.13 C1qc complement C1q C chain 4.11 15.44 5.64 Dntt DNA nucleotidylexotransferase 4.22 34.67 5.50 Endou endonuclease, poly(U) specific 7.22 24.22 4.87 Alox15 arachidonate 15-lipoxygenase 35.56 97.78 3.92 Fgfr1 Fibroblast Growth Factor Receptor 1 7.89 35.67 3.69 Pard6g par-6 family cell polarity regulator gamma 4.67 11.44 3.54 Il17rb interleukin 17 receptor B 5.44 11.56 3.37 Lzts1 leucine zipper tumor suppressor 1 3.44 8.33 3.34 Jchain Joining chain of multimeric IgA and IgM 14.44 57.89 3.31 Spo11 DNA topoisomerase 8.78 17.89 3.16 Jag2 jagged canonical Notch ligand 2 6.00 31.33 3.03 Cd163L1 CD163 molecule like 1 5.44 11.22 2.96 Lyrm1 LYR Motif Containing 1 4.33 18.89 2.96 Rab7b Ras-Related Protein Rab-7b 5.00 19.00 2.96 Emp1 Epithelial Membrane Protein 1 8.11 16.89 2.87 [70]Open in a new tab Table 2. Top 10 upregulated DEGs sorted by highest fold changes (adjusted p value ≤ 0.01, mean value of raw count ≥10) Gene Official name Mean of count (PBS) Mean of count (ALY) Fold-changes Siglech CD33 8.67 93.56 7.49 Prg4 Proteoglycan 4 8.89 76.89 6.13 Alox15 Arachidonate 15-lipoxygenase 29.00 139.67 3.92 Fgfr1 Fibroblast Growth Factor Receptor 1 7.89 35.67 3.69 Jchain Joining chain of multimeric IgA and IgM 14.44 57.89 3.31 Igha Immunoglobulin heavy constant alpha 103.78 403.56 3.08 Jag2 Jagged canonical Notch ligand 2 6.00 31.33 3.03 Zc3h6 Zinc finger CCCH-type containing 6 20.67 73.89 3.46 Igkj5 Immunoglobulin Kappa Joining 5 25.22 82.33 2.65 Myb MYB proto-oncogene 37.44 117.56 2.54 [71]Open in a new tab Validation of the potential biomarker in human PBMCs and mouse peripheral blood To determine whether the biomarker candidates identified in mice are clinically relevant, healthy human PBMCs were treated with ALY688 ex vivo ([72]Figure 2A) to examine gene expression of the top DEGs identified in our RNA sequencing analysis in mice. Remarkably, expression of 16 out of 20 genes that were examined were significantly increased in the ALY688 treated PBMCs compared to those treated with PBS ([73]Figures 2B–2Q). Aclam, Igha, Msmo1, and Zbtb20 were unchanged following ALY688 treatment ([74]Figures S1A–S1D). Then PLS-DA (Partial Least Squares Discriminant Analysis) was performed, showing the degree of separation between clusters of different treatment, PBS and ALY688 ([75]Figure 2R). Based on the proximity within the genes altered by ALY688, which suggesting the high intra-group similarity, we then proposed the group of genes, Tgfb2, Rorc, Myb, Prg4, and Il17rb, as a candidate panel of biomarkers to assess the action of ALY688 in circulation ([76]Table 3). To further verify the reliability of these 6 genes expression upon ALY688 stimulation, blood from mice subjected to PBS or ALY688 injection was collected. Expression of the proposed biomarkers were then measured and suggested that Tgfb2, Rorc, Prg4, and Il17rb demonstrated significant increase upon ALY688, and there is a trend of increase in Myb (p = 0.0576), together intensifying the feasibility of this assay ([77]Figure 3). Figure 2. [78]Figure 2 [79]Open in a new tab Verification of RNA sequencing findings in human PBMC (A) Study design: DEGs selected by RNA sequencing analysis were further validated in human PMBC treated with or without ALY688 by qPCR. Gene expressions of (B) Tgfb2, (C) Rorc, (D) Myb, (E) Prg4, (F) Jag2, (G) Il17rb (H) Cox15, (I) Tomm40, (J) Alox15, (K) Fgfr1, (L) Acaca, (M) Ldlr, (N) Zc3h6, (O) Cox17, (P) Jchain, and (Q) Siglech in PBMC treated with or without 300 nM ALY688 for 24 h. (R) PLSDA analysis. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 compare with PBS, n = 10 per group. Data are presented as fold change to PBS. Ordinary One-way ANOVA with Tukey’s multiple comparison was run for statistical analysis. Table 3. Proposed panel of genes as biomarkers of ALY688 action Gene Official name Tgfb2 Transforming growth factor β 2 Myb MYB proto-oncogene Rorc RAR related orphan receptor C Jag2 Jagged canonical Notch ligand 2 Prg4 Proteoglycan 4 Il17rb Interleukin 17 receptor B [80]Open in a new tab Figure 3. [81]Figure 3 [82]Open in a new tab Biomarkers validation in mouse blood (A) Study design: Proposed biomarkers were validated in whole blood cells isolated from mice subjected to PBS or ALY688 injection. Gene expressions of (B) Tgfb2, (C) Rorc, (D) Myb, (E) Prg4, (F) Jag2, (G) Il17rb. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compare with PBS, n = 7–8 per group. Data are presented as fold change to PBS. Ordinary One-way ANOVA with Tukey’s multiple comparison was run for statistical analysis. ALY688 administration inhibits LPS-induced inflammatory gene expression in mice whole blood To assess potential anti-inflammatory effects of ALY688, we injected mice with a sublethal dose of LPS[83]^25 ([84]Figure 4A). RNA sequencing analysis from whole blood samples demonstrated that 2994 genes were differentially expressed in LPS treated mice compared to control (PBS) ([85]Figure 4B). DEGs highlighted a strong distinction between the two groups, as evidenced by the heatmap ([86]Figure 4C). Besides, among the DEGs, it’s suggested that 1536 genes were upregulated by the stimulation of LPS, while 1,458 genes showed downregulation ([87]Figures 4B and 4D). Among DEGs, significantly increased the output of pro-inflammatory cytokines (Il2ra, Il-1β, Il15ra), Tnfα, chemokines interferon was observed, and together contributes to the pathophysiology of septic shock. The analysis of GO terms through binary cut clustering provided additional confirmation of the validity of our LPS-induced sepsis mouse model. This analysis revealed a significant enrichment in pathways associated with lymphocyte cell adaptive responses and immune activation ([88]Figure S2A), further validating the reliability of our RNA sequencing methodology. Figure 4. [89]Figure 4 [90]Open in a new tab Altered gene expressions uncovered from ALY688 treatment upon LPS stimulation versus LPS (A) Schematic diagram. Scheme 1: Mice were subjected to 15 mg/kg ALY688 vis sq. 15 min prior to 10 mg/kg LPS or PBS i.p. injection. Scheme 2: Mice were subjected to 10 mg/kg LPS or PBS i.p. injection. After 24 h, cardiac puncture was performed for blood collection for RNA sequencing analysis. (B) Pie chart summarizes the number of genes upregulated and downregulated when comparing LPS with PBS. (C) Z score heatmap of RNA-seq transcriptome analysis composed of the 2994 differentially expressed genes identified. (D) Volcano plot of gene expression changes in gene expression in the LPS versus PBS. The volcano plot represents the estimated log2 fold change as a function of the mean of normalized counts. Significant genes were selected when adjusted p value lower than 0.05 and fold-change ≥2. (E) Pie chart summarizes the number of genes upregulated and downregulated when comparing ALY688+LPS versus LPS. (F) Z score heatmap of RNA-seq transcriptome analysis composed of the 76 differentially expressed genes identified. (G) Volcano plot of gene expression changes in gene expression in the ALY688+LPS versus LPS. The volcano plot represents the estimated log2 fold change as a function of the mean of normalized counts. Significant genes were selected when adjusted p value lower than 0.01 and fold-change ≥1.5. n = 6 per group. Next, to determine the biological impact of administering adiponectin on inflammation, mice were injected with 15 mg/kg ALY688 or vehicle (PBS) 15 min prior to LPS injection ([91]Figure 4A). Whole blood was collected after 24 h and RNA sequencing analysis was conducted to assess alterations in whole blood. Using similar analytical approaches to those in [92]Figure 1, we identified a total of 76 genes that were differentially expressed between LPS injected mice with or without ALY688 treatment ([93]Figures 4E and 4F). Among the 76 DEGs, 56 were upregulated and 20 were downregulated in the ALY+LPS group compared to the PBS+ LPS group ([94]Figures 4E and 4G). Specifically, the increase of pro-inflammatory cytokines Il27, Traip (TRAF-interacting protein), Tnfrsf9 (tumor necrosis factor receptor superfamily, member 9) and Irf2bp1 (Interferon regulatory factor 2 binding protein 1) induced by LPS was reversed by ALY688 treatment ([95]Figures 3A–3D). Taken together, our data suggested that ALY688 administration reversed the LPS-induced genes profile in whole blood cells. Discussion Adiponectin, a multifunctional protein with a multitude of beneficial metabolic and anti-inflammatory properties, has garnered attention for its therapeutic potential in addressing cardiometabolic disorders.[96]^5^,[97]^26^,[98]^27 In this study, we used the adiponectin receptor agonist ALY688 which will soon enter phase I clinical trials. We first demonstrated the adiponectin-mimetic action of ALY688 via an anti-inflammatory effect in a mouse model treated with LPS. Our RNA sequencing analysis of whole blood in a sublethal LPS mouse model pretreated with or without ALY688 demonstrated that the adiponectin receptor agonist elicited a substantial impact on pro-inflammatory gene expression. In the LPS injected group, whole blood cell RNA sequencing revealed the significant increase of Tnf, Il-1β, and interferon, which confirmed the phenotype of septic model.[99]^28 The decrease in pro-inflammatory genes such as Il27, and Traip observed with ALY688 in this LPS model suggests that ALY688 may mitigate the deleterious impacts of inflammation during acute inflammatory conditions, which is in line with previously reported adiponectin anti-inflammatory effect in various diseases model[100]^29^,[101]^30^,[102]^31^,[103]^32 The inflammatory cytokines, including interferon-γ, and tumor necrosis factor found suppressed by adiponectin signaling in the disease model like, obese, atherosclerosis, and NASH were also observed in the sequencing analysis of ALY+LPS versus LPS.[104]^33^,[105]^34^,[106]^35^,[107]^36^,[108]^37 In conclusion, using this well-established model of acute inflammation we demonstrated that pretreatment of mice with ALY688 successfully attenuated induction of various pro-inflammatory factors. We also performed discovery-based research using RNA sequencing (RNAseq) of whole blood to identify DEGs in wild-type mice treated with ALY688 versus vehicle. The goal of this approach was to develop an assay biomarking adiponectin receptor activation that could be used not only in animal research but would be amenable to implementation in human clinical study sites. Thus, our investigation focused on the identification of potential blood-based biomarkers altered in response to ALY688 treatment. Based on DEG analysis derived from whole blood sample RNA sequencing datasets, we were able to identify 157 genes significantly upregulated by ALY688 treatment. The identified genes, including Tgfb2, Rorc, Myb, Prg4, Jag2, and Il17rb, selected by magnitude of fold change and clustering, were thus selected as promising candidates for clinical target engagement monitoring. Among this selected cluster genes, Tgfb2 belongs to the family of the transforming growth factor β (TGF-β), and is a versatile cytokine that induces responses which have been correlated with an increased risk of developing various hallmarks of metabolic syndrome.[109]^38^,[110]^39 TGF-β can originate from multiple sources, primarily fibroblasts and, to a lesser extent, resident macrophages, contributing to the promotion of fibrosis.[111]^40 TGF-β plays a key role in inhibiting immune responses, either directly by impeding the functions of Th1 and Th2 CD4^+ effector cells and NK cells or indirectly through the promotion and activation of Tregs.[112]^41^,[113]^42 Notably, recent studies have demonstrated that ALY688 increased TGF-β protein following muscle damage in a Duchenne muscular dystrophy model.[114]^43 Our findings suggest TGF-β level is increased by ALY688 and that this could convey an anti-inflammatory outcome, which may explain the reduction of pro-inflammatory cytokines evoked by LPS stimulation, and in line with adiponectin’s anti-inflammatory effect in various models.[115]^29^,[116]^44^,[117]^45^,[118]^46^,[119]^47 RNA sequencing data also identified the receptor of IL-17, Il17rb, as one of the most predominant upregulated genes upon ALY688 administration. IL-17 family cytokines have been shown to play a pivotal role in host defense against extracellular pathogens and inflammatory responses.[120]^48 Among this cytokine family, IL-25 acts as an alarm signal, generated in response to cell injury or tissue damage. IL-25 activates immune cells through its interaction with the IL-17RA and IL-17RB receptors, thereby initiating immune responses.[121]^49 Splenocytes from IL-17RB-KO (Il17rb^−/−) mice ceased to produce numerous inflammatory factors (e.g., IL-5 or IL-13) upon in vitro stimulation with IL-25.[122]^50 Thus, IL-17 stands out as a pivotal cytokine produced by TH17 cells, playing a central role in regulation of gut immunity.[123]^51^,[124]^52 Its significance extends beyond infection control, as TH17 cells actively contribute to the safeguarding of epithelial and mucosal tissues.[125]^53^,[126]^54^,[127]^55 Jag2, a member of the Notch signaling pathway, has been implicated in adipogenesis and adipose tissue inflammation and, interestingly, adiponectin was shown to activate Notch signaling in the hippocampus through upregulating ADAM10 and Notch1.[128]^56 While the direct relationship between Jag2 and adiponectin remains unclear, our data suggest further investigation of this understudied association is warranted. Furthermore, Treg cell expansion required cell-to-cell contact and Notch3 signaling, which was mediated selectively through the Notch ligand Jag2 expression.[129]^57 For example, in experimental autoimmune encephalomyelitis Treg expansion is regulated by signaling through Jag2.[130]^58 Rorc is the lineage-specific transcription factor for Th17 cells whose upregulation in developing Th17 cells is critically regulated by IL-6 and TGF-β.[131]^57^,[132]^59 Mice lacking the transcription factor Myb in Tregs succumbed to a multi-organ inflammatory disease.[133]^60^,[134]^61 Overall, the cluster gene of 6 presented robust and consistent upregulation in response to ALY688 treatment in both murine and human samples, as well as strong correlation of adiponectin mimetic effect in immune response, position them as panel of potential non-invasive biomarkers for assessing the pharmacokinetics of ALY688 action. Translating our findings to human relevance, we validated the identified biomarkers based on the highest fold changes in RNA sequencing analysis, in human PBMC. The significant alignment between mouse and human studies, particularly the consistent upregulation of key genes, suggests the potential applicability of these markers across species. Analysis using functional annotation tools revealed enrichment in pathways related to immune response and inflammation, aligning with the anticipated effects of adiponectin-based therapy.[135]^44^,[136]^62^,[137]^63^,[138]^64 To refine our biomarker selection, we scrutinized the most significantly upregulated genes, pinpointing those involved in T cell differentiation, inflammation, fatty acid metabolism, DNA damage, collagen formation, and ubiquitin-proteasome-dependent proteolysis.[139]^65^,[140]^66^,[141]^67 The subsequent GO term analysis emphasized the immune response-related pathways enriched among the top upregulated genes, such as Myb, Rorc, and Il17rb enriched in the pathway regulating CD4, alpha-beta T cell activation, and Th17 cell differential and immune response. Interestingly, it’s well known that Th17 cells secrete IL-17A, IL-17F, and express master transcription factor RORγt, encoded by Rorc, in response to diseases.[142]^68^,[143]^69^,[144]^70 Multiple pre-clinical studies have shown the protective effect of ALY688, featuring its anti-inflammatory property. In a mouse model of metabolic dysfunction-associated steatohepatitis (MASH), ALY688 administration elicited the suppression effect of pro-inflammatory genes including Tnfα, Mcp1, IL-1β.[145]^71 ALY688 also exerted a cardioprotective effect in part by lowering IL-6, TLR4, and IL-1β gene expression in heart from a preclinical mouse model of heart failure with reduced ejection fraction induced by pressure overload.[146]^72 Circulating IL-6 level, as well as the skeletal muscle expression of TNFα and IL-1β which are enhanced by D2.mdx mouse model of Duchenne muscular dystrophy was decreased by ALY688 treatment.[147]^73^,[148]^74 Together, our findings reinforce the regulatory potential of ALY688 upon inflammation. In conclusion, our study demonstrated anti-inflammatory effects of ALY688, and identified a gene signature indicating adiponectin action using whole blood samples, which were validated in human PBMC. The identified biomarker candidates offer a foundation for future clinical applications, enabling the non-intrusive monitoring of adiponectin-like effects. New knowledge from our study and the future use of the bioassay emerging from this work will be valuable in the clinical development of adiponectin-based therapeutic strategies. Limitations of the study We acknowledge a potential limitation of this study in using only lipopolysaccharide (LPS) to induce acute inflammation. Alternative approaches to consider include peritoneal contamination and infection (PCI) or and cecal ligation and puncture (CLP). LPS and PCI models demonstrate similar degrees of immune response, indicated by serum pro-inflammatory cytokines and oxidative stress and apoptosis in spleen, whereas a delayed and a lower degree of response are typically found in CLP. Considering the purpose of this study, the temporal nature and size of response made LPS the most suitable model to mimic the characteristic features of human systemic inflammation. Resource availability Lead contact Further information and reasonable requests for resources and reagents should be directed to the lead contact, Dr. Gary Sweeney (gsweeney@yorku.ca). Material availability This study did not generate any unique materials. The full RNAseq dataset has been deposited at GEO (see information above). Data and code availability RNA-seq data have been deposited to GEO with the identifier [149]GSE275088. This study did not generate any new code. Any additional information required to reanalyze the data reported in this paper is available from the [150]lead contact upon request. Acknowledgments