Mechanism of Innate Immune Response Induced by Albizia julibrissin Saponin Active Fraction Using C2C12 Myoblasts

Albizia julibrissin saponin active fraction (AJSAF), is a prospective adjuvant with dual Th1/Th2 and Tc1/Tc2 potentiating activity. Its adjuvant activity has previously been proven to be strictly dependent on its spatial co-localization with antigens, highlighting the role of local innate immunity in its mechanisms. However, its potential targets and pathways remain unclear. Here, its intracellular molecular mechanisms of innate immune response were explored using mouse C2C12 myoblast by integrative analysis of the in vivo and in vitro transcriptome in combination with experimental validations. AJSAF elicited a temporary cytotoxicity and inflammation towards C2C12 cells. Gene set enrichment analysis demonstrated that AJSAF regulated similar cell death- and inflammatory response-related genes in vitro and in vivo through activating second messenger–MAPK–CREB pathways. AJSAF markedly enhanced the Ca2+, cAMP, and reactive oxygen species levels and accelerated MAPK and CREB phosphorylation in C2C12 cells. Furthermore, Ca2+ chelator, CREB inhibitor, and MAPK inhibitors dramatically blocked the up-regulation of IL-6, CXCL1, and COX2 in AJSAF-treated C2C12 cells. Collectively, these results demonstrated that AJSAF induced innate immunity via Ca2+–MAPK–CREB pathways. This study is beneficial for insights into the molecular mechanisms of saponin adjuvants.


Introduction
Vaccine development is facing serious challenges due to the emergence of new infectious diseases and the spread of cancer [1].Adjuvants should be capable of eliciting appropriate T-cell-mediated and/or humoral immunities targeting specific pathogens [2].However, up to now, few adjuvants have been licensed for human use.In addition to safety issues and lower potency, limited understanding of the mechanism and drug targets is also a key factor behind the lack of effective adjuvants [3].
Saponin adjuvants achieve adaptive immunity by reconstructing the local immune microenvironment.Although considered as an undesired side effect, early inflammatory response is crucial for adjuvants to elicit adaptive immune responses [4].Most vaccines are administered intramuscularly [5].However, the muscle tissues contain only a few resident immune cells, whose abundance and type are altered by most adjuvants [6].Adjuvant-induced acute inflammatory responses in muscle tissues promote the recruitment of immune cells to improve antigen uptake and expedite the migration of antigen-loaded immune cells to the lymph nodes, leading to the efficient activation of naive T cells and the establishment of adaptive immunity [7][8][9].Mouse C2C12 myoblasts are commonly used to study the development and regeneration of human skeletal muscle cells [10].In our previous works, it was found that C2C12 myoblasts could be applied as an in vitro model in understanding the mechanisms of saponin adjuvants [11].
Albizia julibrissin saponin active fraction (AJSAF) is a prospective adjuvant with dual Th1/Th2 and Tc1/Tc2 potentiating activity on OVA and several commercial livestock and poultry vaccines [12].It has been previously reported that AJSAF induced the protein expression of cytokines and chemokines at the site of injection and its adjuvant activity was strictly dependent on its spatial co-localization with antigens [4].Although the potential targets and pathways of the AJSAF-induced local innate immunity in mice has been also studied, the results need to be verified [13].Here, its intracellular molecular mechanisms of innate immune response were explored using mouse C2C12 myoblast through integrative analysis of the in vitro and in vivo transcriptome in combination with experimental validations.
AJSAF was prepared as previously described [12] and was identified to contain 29 saponins including 10 new compounds by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry based on accurate mass database [14].The endotoxin level was 0.253 ± 0.004 EU/mL in AJSAF solution (2 mg/mL), being excluded from endotoxin contamination.

Cell Culture and Stimulation
Mouse C2C12 myoblast cell line (ATCC CRL-1772) was purchased from the cell bank of the Shanghai Branch of the Chinese Academy of Sciences, Shanghai, China, and cultured in DMEM complete medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin in a 37 • C and 5% CO 2 atmosphere.After 24 h of adhesion culture, the cells were treated with AJSAF at the designated concentration from 100 µg/mL to 300 µg/mL, and then the pelleted cells and culture supernatants were collected at the indicated time, respectively.

Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)
Total RNA isolated with TRIzol reagent was subjected to reverse transcription.PCR was performed using the specific primers (Table S1) and FastStart Universal SYBR Green Master (Rox) according to the MMIQE guidelines [15].The relative expression level to Gapdh was calculated using the 2 −∆∆Ct method [11].

Gene Set Enrichment Analysis (GSEA)
GSEA was performed for the whole genes detected in AJSAF-treated C2C12 cells and mouse muscle tissues (Supplementary Methods) [13] using mouse GSKB [18].Leadingedge gene sets (LEGSs) were identified based on |normalized enrichment score| > 1, p < 0.05, and false discovery rate < 0.25.The genes in the LEGSs with core enrichment being "Yes" were considered as the core genes.The core genes were plotted for the heat maps at https://www.omicstudio.cn/tool(accessed on 19 February 2023), and their GO functions and KEGG pathways were analyzed using Metascape (http://metascape.org/,accessed on 19 February 2023).The network components were predicted using molecular complex detection technology (MCODE) [19,20].

Relevance Analysis of Transcriptome In Vivo and In Vitro
The heatmap and PPI network of the top 20 clusters with GO-and KEGG-enriched terms of both in vivo and in vitro core genes was build using Metascape (http://metascape.org/, accessed on 19 February 2023) [16,17].Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining (TRRUST) database (https://www.grnpedia.org/trrust/, accessed on 19 February 2023) was employed to predict the transcription factors (TFs) [21].The overlap Circos diagrams were plotted for screening the in vivo and in vitro common core genes.The common core genes were subjected to eight algorithms (EPC, MCC, MNC, Betweenness, Closeness, Degree, Radiality, and Stress) in the Cytoscape cytoHubba plug-in (https://cytoscape.org/,accessed on 19, February, 2023).The intersecting genes of the top 10 genes from each approach were identified to be hub genes using UpSet (https://www.omicstudio.cn/tool,accessed on 19 February 2023).GeneMANIA (http://www.genemania.org/,accessed on 19 February 2023) was applied to construct a co-expression network of hub genes [22].

cAMP, Free Ca 2+ and ROS Detection
The levels of intracellular cAMP, free Ca 2+ , and ROS were determined using cAMP assay kit, Fluo-3 AM, and ROS assay kit, respectively [11,23].

Western Blotting
C2C12 cells were lysed with RIPA lysis buffer containing protease inhibitor and phosphatase inhibitor, and then protein contents were detected using BCA assay.The denatured proteins were separated on SDS-PAGE and transferred to PVDF membrane.After incubation with primary antibodies overnight at 4 • C, the membrane was blotted with HRP-conjugated IgG for 1 h.The signals were visualized with ECL on the iBright TM CL1500 Imaging System (Thermo Fisher Scientific, Waltham, MA, USA) [23].

Statistical Analysis
Data were expressed as mean ± SD and statistically analyzed with ANOVA and Student's t-tests using the GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA, USA).p < 0.05 was statistically significant.

AJSAF Elicited a Temporary Cytotoxicity and Inflammation in C2C12 Cells
AJSAF exhibited remarkable toxicities against C2C12 cells at the concentration of >150 µg/mL for 4 h, with the IC 50 value being 210 µg/mL.No cytotoxicity, however, was found in the AJSAF-treated C2C12 cells for 24 h except for 300 µg/mL (Figure 1A).

Western Blotting
C2C12 cells were lysed with RIPA lysis buffer containing protease inhibitor and phosphatase inhibitor, and then protein contents were detected using BCA assay.The denatured proteins were separated on SDS-PAGE and transferred to PVDF membrane.After incubation with primary antibodies overnight at 4 °C, the membrane was blotted with HRP-conjugated IgG for 1 h.The signals were visualized with ECL on the iBright TM CL1500 Imaging System (Thermo Fisher Scientific, Waltham, MA, USA) [23].

Statistical Analysis
Data were expressed as mean ± SD and statistically analyzed with ANOVA and Student's t-tests using the GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA USA).p < 0.05 was statistically significant.

AJSAF Elicited a Temporary Cytotoxicity and Inflammation in C2C12 Cells
AJSAF exhibited remarkable toxicities against C2C12 cells at the concentration of > 150 µg/mL for 4 h, with the IC50 value being 210 µg/mL.No cytotoxicity, however, was found in the AJSAF-treated C2C12 cells for 24 h except for 300 µg/mL (Figure 1A).AJSAF was reported to facilitate gene expression of Il-6, Cxcl1, and Cox2 (Ptgs2) in mouse quadriceps muscles [13].The mRNA expression of these inflammatory factors in C2C12 cells was also significantly up-regulated by AJSAF; it peaked at 4-6 h and then speedily declined (p < 0.001, Figure 1B).Meanwhile, AJSAF markedly and concentrationdependently induced the production of IL-6, CXCL1, and COX2 in C2C12 cells (p < 0.001, (Figures 1C-E and S1).These results indicated that AJSAF elicited a temporary cytotoxicity and inflammation in C2C12 cells.

Functions and Pathways of AJSAF-Induced DEGs in C2C12 Cells
To characterize the transcriptional profiling, C2C12 cells treated with AJSAF were subjected to SurePrint G3 microarray (Figure 2).AJSAF resulted in 738 up-regulated and 700 down-regulated DEGs in C2C12 cells (Figure 3A).AJSAF-induced mRNA expression levels of four putative up-regulated (Rgs, Thbd, Hmox1, and Il33) and four putative downregulated (Rnd3, Tgfb3, Wnt4, and Fas) genes by RT-qPCR coincided with microarray data (Figure 3B and Table S2).AJSAF was reported to facilitate gene expression of Il-6, Cxcl1, and Cox2 (Ptgs2) in mouse quadriceps muscles [13].The mRNA expression of these inflammatory factors in C2C12 cells was also significantly up-regulated by AJSAF; it peaked at 4-6 h and then speedily declined (p < 0.001, Figure 1B).Meanwhile, AJSAF markedly and concentrationdependently induced the production of IL-6, CXCL1, and COX2 in C2C12 cells (p < 0.001, Figure 1C-E and Figure S1).These results indicated that AJSAF elicited a temporary cytotoxicity and inflammation in C2C12 cells.

Functions and Hub Genes of AJSAF-Induced Common Core Genes In Vitro and In Vivo
Both the top 20 enriched GO and KEGG terms of the in vitro and in vivo core genes were concerned with cell activation, cell death, granulocyte chemotaxis, inflammatory response, MAPK cascade pathway, and second-messenger-mediated signaling pathway (Figure 6A).These top 20 clusters were correlative and constituted a network centered around granulocyte chemotaxis, inflammatory response, cell activation, cell death, and positive regulation of locomotion (Figures 6B and S2).Meanwhile, the top 20 TFs of the in vitro and in vivo core genes induced by AJSAF were predicted using the TRRUST database, respectively.Among the top 20 TFs, Crebbp, Nfe2l2, Ppara, Stat1, Stat3, Stat5a, Ep300, Sp1, Ets1, Fos, Egr1, Rela, Cebpb, Trp53, Ikbkb, Jun, and Nfkb1 were the shared TFs, except Usf2, Sp3, and EIk1 for C2C12 cells (Figure 6C).

Functions and Hub Genes of AJSAF-Induced Common Core Genes In Vitro and In Vivo
Both the top 20 enriched GO and KEGG terms of the in vitro and in vivo core genes were concerned with cell activation, cell death, granulocyte chemotaxis, inflammatory response, MAPK cascade pathway, and second-messenger-mediated signaling pathway (Figure 6A).These top 20 clusters were correlative and constituted a network centered around granulocyte chemotaxis, inflammatory response, cell activation, cell death, and Furthermore, Il6, Csf2, Cxcl1, Il1b, Ptgs2 (Cox2), and Stat3 were identified to be the hub genes of the common core genes using upset plot (Figure 6F and Table S5).Six hub genes formed a PPI network with co-expression of 89.51, prediction of 8.88, and other of 1.61 (Figure 6G).The functions of the six hub genes mainly included the regulation of ERK1 and ERK2 cascade, cytokine-mediated signaling, cell chemotaxis, acute inflammatory response, ROS metabolic process, chemokine production, and cell activation (Figure 6G).The above results suggested that AJSAF regulated the cell death-and inflammatory responserelated genes in vitro and in vivo through the second-messenger-MAPK pathway.

AJSAF Induced the Inflammation in C2C12 Cells through Ca 2+ -MAPK-CREB Pathway
The microarray analysis revealed that AJSAF potentially activated the secondmessenger-mediated signaling (Figure 6A,B) and regulated the ROS metabolic process (Figure 6G).Therefore, the second messenger components and ROS generation in C2C12 cells were examined.AJSAF significantly and time-dependently increased the cAMP con-tents in C2C12 cells, which ascended at 0.5 h and peaked at 1 h (Figure 7A).AJSAF also time-and concentration-dependently induced a significant Ca 2+ influx and ROS production in C2C12 cells (Figure 7B,C).The transcriptome correlation analysis revealed that AJSAF regulated the MAPK cascade and protein phosphorylation in vitro and in vivo (Figure 6A,B).Meanwhile, six hub genes were identified to be correlative with the ERK1 and ERK2 cascade.TRRUST predicted that the TFs such as CREB-binding protein (Crebbp) and Nfkb1 regulated AJSAFinduced core genes.Therefore, MAPK, NF-κB, and CREB phosphorylation in C2C12 cells was detected using Western blotting.AJSAF markedly promoted JNK, ERK1/2, p38 MAPK, and CREB phosphorylation in C2C12 cells from 15 min to 2 h.However, AJSAF did not affect NF-κB p65 phosphorylation in C2C12 cells (Figures 7D,E and S3).These results suggested that AJSAF activated second messenger-MAPK-CREB pathways.

Discussion
Although saponin adjuvants have been widely investigated for their use in vaccines, their mechanisms of action are poorly understood [24].A high concentration of adjuvant is generated at the local injection site after intramuscular vaccination, and the dominant cell population in contact with adjuvants is muscle cells.AJSAF was found to up-regulate both neutrophil-active (CCL3, CCL7, CXCL1, and CXCL5) and neutrophil-derived genes (CCL2, CCL3, and CCL4) in mouse quadricep muscles [13].In fact, many different chemoattractants with similar functions are usually present at sites of inflammation [25].In this study, AJSAF elicited a temporary cytotoxicity and inflammation in C2C12 cells (Figure 1).RNA-seq analysis showed that AJSAF induced 1438 DEGs in C2C12 cells (Figure 3A).These DEGs were involved in cell proliferation, differentiation, migration, and death, as well as response to wounding and external stimulus (Figure 3D).The core genes in C2C12 cells induced by AJSAF involved the cell chemotaxis, inflammatory response, cell death, cell migration, apoptotic process, cytokine activity, chemokine activity, and growth factor activity and were correlated with TNF signaling, PI3K-Akt signaling, MAPK signaling, and cAMP signaling (Figure 4C).Similarly, AJSAF-induced core genes in mouse quadricep muscles were relative with the GO functions including inflammatory response, cell chemotaxis, cell migration, cell death, apoptotic process, chemokine activity, cytokine activity, and growth factor activity, as well as KEGG pathways such as TNF signaling, JAK-STAT signaling, MAPK signaling, and cAMP signaling (Figure 5C).Moreover, both top 20 enriched terms of AJSAF-induced core genes in vitro and in vivo were co-regulated and associated with cell activation, cell death, granulocyte chemotaxis, inflammatory response, MAPK pathway, and second-messenger-mediated signaling (Figure 6A,B).These results suggested that AJSAF induced similar in vitro and in vivo functions and pathways.
The transcriptomic analysis revealed that AJSAF potentially activated the second messenger-MAPK-CREB pathway in vitro and in vivo.Actually, a very early response was observed showing that the intracellular Ca 2+ , cAMP, and ROS levels peaked in AJSAFstimulated C2C12 cells within 2 h (Figure 7A-C).These three second messengers could promote the phosphorylation of MAPK and activate the inflammasome [26][27][28].In this study, AJSAF significantly and rapidly induced the MAPK and CREB phosphorylation in C2C12 cells, especially ERK1/2 and CREB, reaching almost their peak at 0.25 h after stimulation.CREB is an important TF for mediating the immune-related genes containing a cAMP-responsive element, including Il2, Il6, Il10, Tnfα, and Cox2 [29].Resident macrophages in healthy skeletal muscles regulate tissue homeostasis.CREB-C/EBPβ cascade induces the expression of M2 genes and promotes muscle injury repair [30].A previous study showed that AJSAF activated RAW264.7 macrophages to secrete IL-1β, TNF-α, CCL2, CCL22, and CXCL2 [23].In addition, CREB phosphorylation directly inhibited NF-κB activation [31], which might explain why AJSAF did not affect NF-κB p65 phosphorylation in C2C12 cells.Furthermore, the inhibition assay revealed that all Ca 2+ , ERK1/2, CREB, JNK, and p38 MAPK inhibitors could reverse the up-regulation of IL-6, CXCL1, and COX2 in the AJSAF-treated C2C12 cells (Figure 7F-H).These findings confirmed that the Ca 2+ -MAPK-CREB pathway was involved in the AJSAF-induced inflammation in C2C12 cells.However, how AJSAF affects the intracellular Ca 2+ , cAMP, and ROS levels in C2C12 cells and the role of these second messengers in mediating the adjuvant activity of AJSAF are an issue that warrants further evaluation.
AJSAF induced the lysis of C2C12 cells at 250 µg/mL from 2 h to 4 h.However, the clearance of cell debris and maintenance of homeostasis were found in AJSAF-treated C2C12 cells at the same concentration for 24 h.The dying and/or dead cells released danger-associated molecular patterns (DAMPs), which were sensed by immune and nonimmune cells.DAMPs were reported to activate the ERK1/2-CREB pathway to induce an inflammatory response and adaptive immunity [32][33][34][35].Host DNA released from alumtreated cells influenced its adjuvanticity [36].AJSAF was observed to induce DAMPs with adjuvant activities including S100A8, S100A9, and IL-33 in mouse quadricep muscles [13].Therefore, which DAMPs released by muscle cells are essential to the adjuvant activity of AJSAF also remains to be further elucidated.
In conclusion, this study demonstrated that AJSAF elicited a temporary cytotoxicity and inflammation in C2C12 cells through the Ca 2+ -MAPK-CREB pathway (Figure 8).AJSAF might exert adjuvant activity by eliciting the inflammatory cytokines, chemokines, and DAMPs at the injection site.This study is beneficial for understanding the molecular mechanism of action of saponin adjuvants.AJSAF induced the lysis of C2C12 cells at 250 µg/mL from 2 h to 4 h.However, the clearance of cell debris and maintenance of homeostasis were found in AJSAF-treated C2C12 cells at the same concentration for 24 h.The dying and/or dead cells released danger-associated molecular patterns (DAMPs), which were sensed by immune and non-immune cells.DAMPs were reported to activate the ERK1/2-CREB pathway to induce an inflammatory response and adaptive immunity [32][33][34][35].Host DNA released from alumtreated cells influenced its adjuvanticity [36].AJSAF was observed to induce DAMPs with adjuvant activities including S100A8, S100A9, and IL-33 in mouse quadricep muscles [13].Therefore, which DAMPs released by muscle cells are essential to the adjuvant activity of AJSAF also remains to be further elucidated.
In conclusion, this study demonstrated that AJSAF elicited a temporary cytotoxicity and inflammation in C2C12 cells through the Ca 2+ -MAPK-CREB pathway (Figure 8).AJSAF might exert adjuvant activity by eliciting the inflammatory cytokines, chemokines, and DAMPs at the injection site.This study is beneficial for understanding the molecular mechanism of action of saponin adjuvants.

Figure 4 . 4 .
Figure 4. Function of the AJSAF-induced core gens in C2C12 cells.(A) Enrichment plot of the leading-edge gene sets by the gene set enrichment analysis.(B) Heatmap of the core genes.(C) GO Figure 4. Function of the AJSAF-induced core gens in C2C12 cells.(A) Enrichment plot of the leading-edge gene sets by the gene set enrichment analysis.(B) Heatmap of the core genes.(C) GO function and KEGG pathway of the core genes.(D,E) Three densely connective networks (D) and their functional annotation (E) of the core genes by Cytoscape MCODE plug-in.BP: biological process.

Figure 5 .
Figure 5. Function of the AJSAF-induced core gens in mouse quadricep muscles.(A) Enrichment plot of the leading-edge gene sets by the gene set enrichment analysis.(B) Heatmap of the core genes.(C) GO function and KEGG pathway of the core genes.(D,E) Four densely connective networks (D) and their functional annotation (E) of the core genes by Cytoscape MCODE plug-in.BP: biological process, MF: molecular function.

Figure 5 .
Figure 5. Function of the AJSAF-induced core gens in mouse quadricep muscles.(A) Enrichment plot of the leading-edge gene sets by the gene set enrichment analysis.(B) Heatmap of the core genes.(C) GO function and KEGG pathway of the core genes.(D,E) Four densely connective networks (D) and their functional annotation (E) of the core genes by Cytoscape MCODE plug-in.BP: biological process, MF: molecular function.

Figure 6 .
Figure 6.Functions and hub genes of AJSAF-induced common core genes in vitro and in vivo.(A) and (B) Heatmap (A) and network (B) of the Top20 clusters with GO and KEGG enriched terms.(C) and (D) top 20 transcription factors (C) and overlap Circos diagram (D) of the in vitro and in vivo core genes induced by AJSAF.(E) GO function and KEGG pathway of the common core genes.(F,G) Upset plot (F) and protein-protein interaction network (G) of 6 hub genes.

Figure 6 .
Figure 6.Functions and hub genes of AJSAF-induced common core genes in vitro and in vivo.(A) and (B) Heatmap (A) and network (B) of the Top20 clusters with GO and KEGG enriched terms.(C) and (D) top 20 transcription factors (C) and overlap Circos diagram (D) of the in vitro and in vivo core genes induced by AJSAF.(E) GO function and KEGG pathway of the common core genes.(F,G) Upset plot (F) and protein-protein interaction network (G) of 6 hub genes.