SESLA suppresses the activation of macrophages and dendritic cells after Gram-positive bacterial challenge

Abstract Background Secoeudesma sesquiterpenes lactone A (SESLA) is a sesquiterpene derived from Inula japonica Thunb. and is known to possess many pharmacological properties, e.g. anti-tumor and anti-inflammatory activities. However, the immunomodulatory role of SESLA in gram-positive (G+) bacterial infection is not clear. Materials and methods To set up a G+ bacterial infection model in vitro, we carried out a bacterial mimic (PGN or Pam3CSK4) or Methicillin-resistant Staphylococcus aureus (MRSA) stimulated experiment using macrophages or dendritic cells (DCs). ELISA and qPCR were performed to measure the expression of inflammatory cytokines. Flow cytometry was used to detect the expression of MHC II and co-stimulatory molecules on the surface of DCs. The network pharmacology was used to identify the molecular mechanism and potential targets of SESLA that are predicted to be involved in the MRSA-elicited inflammation. Western blot and dual luciferase reporter assay were adopted to certify possible molecular mechanism of SESLA. Results This study demonstrated that SESLA treatment significantly reduced the levels of inflammatory cytokines stimulated by PGN, Pam3CSK4 or even MRSA in vitro, and it also reduced PGN-induced expression of MHC II and co-stimulatory molecules on the surface of DCs. Mechanistically, the inhibition of IκBα phosphorylation and the suppression of T cells activation could account for its anti-inflammatory activity. Conclusion The present study validated the notable anti-inflammatory activity of SESLA and discovered its previously uncharacterized immunoregulatory role and the underlying mechanism in G+ bacterial infections. Overall, SESLA has a potential to be an antibiotic adjuvant for the treatment of G+ bacterial infections.


Introduction
Bacterial infections are a major threat to human health and commonly associate with poor clinical outcomes (e.g. sepsis), especially with the emergence of antibiotic-resistant bacteria in recent years [1,2]. Antibiotics are the indispensable antibacterial drugs in the clinic, however, their clinical application is severely limited due to the antibiotic resistance and side effects. Therefore, development of novel therapeutic options for controlling bacterial infections is becoming increasingly urgent.
During infection, innate immune cells, such as macrophages and dendritic cells (DCs), are typically the first responders to clear the bacteria and trigger antibacterial immunity. By secreting chemokine monocyte chemoattractant protein 1 (MCP-1) etc., they can recruit more immune cells (e.g. peripheral monocytes) to the site of inflammation and resist infection [3][4][5]. Beyond this, as the most professional antigen presenting cells (APCs), DCs could also present bacterial antigens to T cells to evoke an antigen-specific adaptive immune response [6]. However, these over-activation of innate immune cells and the excessive proliferation/ activation of adaptive T cells, instead, could lead to a hyperinflammatory response, becoming the main etiology of adverse outcome of bacterial infections [7]. This unbridled inflammation is characterized by a high level of different cytokines and amplified cytokine cascade (termed as 'cytokine storm') and commonly tightly linked to widespread tissue damage, multi-organ failure, and even death [8].
Host-directed therapy (HDT) is a new alternative antiinfective strategy that was proposed recently [9]. Owing to its advantages in targeting the reprograming of the host immune system other than the pathogen itself during infection, it appears to be a promising treatment in combination with antibiotics for bacterial infections, particularly drugresistant bacterial infections.
Traditional Chinese Medicine (TCM) possesses abundant active pharmaceutical ingredients with a wide range of pharmacological activities. Consistently, it has been considered as an essential source for the discovery and development of new anti-inflammatory agents that can be used for HDT-based treatments of infectious diseases, such as sepsis [10,11]. Inula japonica Thunb. (I. japonica), known as xuanfuhua in China, is a member of the Compositae (Asteraceae) family and broadly distributes in East Asian countries. It has been widely used in TCM formulae (e.g. xuanfuhua decoction and 'Huang-Lian-Shang-Qing' pills) for years, and are commonly used to treat digestive disorders, infectious diseases, and various inflammatory diseases clinically [12,13]. Secoeudesma sesquiterpene lactone A (SESLA) is a sesquiterpene compound, an intermediate product generated during the synthesis of ivangustin in Inula japonica Thunb. [14]. Our previous study demonstrated that SESLA could inhibit inflammatory responses induced by lipopolysaccharide (LPS), the main component of gram-negative (G -) bacterial cell wall, or carbapenem-resistant Klebsiella pneumoniae (CRKP), thereby exhibiting combined protective effects in CRKP-infected septic mice [15]. However, whether SESLA has an immunomodulatory role in gram-positive (G þ ) bacterial infections (including antibiotic resistant bacteria) remains unclear.
As a common G þ bacterium, methicillin-resistant Staphylococcus aureus (MRSA) has been deemed as a highpriority pathogen by the World Health Organization due to its extensive drug resistance and high mortality [16]. Therefore, in this study, we aim to explore whether and how SESLA regulates the activation of macrophages and DCs and DC-mediated activation of T cells upon stimulations by the mimics of G þ bacterial cell wall components (peptidoglycan (PGN) and Pam3CSK4) and even resistant bacteria MRSA.  [14]. In this experiment, SESLA was dissolved in DMSO (Cat: D2650, Sigma-Aldrich) to produce a stock solution (40 mM) and froze at À20 C. The stock solution was diluted with medium to the experimental concentration before use. Peptidoglycan (PGN) from Staphylococcus aureus (S. aureus) was purchased from Sigma (St. Louis, MO). Pam3CSK4 (Cat: tlrl-pms) was purchased from InvivoGen (San Diego, CA, USA). Recombinant mouse GM-CSF (Cat. 415-ML) and IL-4 (Cat. 404-ML) were purchased from R&D Systems (Minneapolis, MN).

Detection of cytokine and quantitative real-time PCR analysis
Cytokine levels in the culture supernatants were measured separately using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN). The method of total RNA extraction and qRT-PCR analysis was performed as described previously [19]. Calculations (2 ÀDDCt method) were carried out using b-Actin as the reference gene in each sample.

Western blot analysis
The cells were lysed in M-PER TM Protein Extraction Reagent (Pierce, Rockford, IL) containing protease inhibitor cocktail (Calbiochem, San Diego, CA). Total protein concentrations in lysates were measured using Pierce TM BCA Protein Assay Kit (Pierce, Rockford, IL). And these samples were adjusted to equal protein concentration for the further detection by Western blot analysis as described previously [15]. All antibodies used were purchased from Cell Signaling Technology (Beverly, MA).

The strains of bacteria and their growth condition
The clinically isolated methicillin-resistant Staphylococcus aureus (MRSA, HS488) was obtained from the Institute of Antibiotics, Huashan Hospital, Fudan University & Key Laboratory of Clinical Pharmacology of Antibiotics (Shanghai, China). Single colony of MRSA (HS488) was picked and grown in Luria-Bertani (1% Tryptone, 0.5% Yeast extract, 1% NaCl) medium and cultured at 37 C with shaking (200 rpm) overnight. Heat-killed MRSA (HK MRSA) was prepared by boiling the bacterial suspension at 90 C for 30 min. Then they were washed by sterile PBS and re-suspended in DMEM before use.

Luciferase reporter assay
The NF-jB luciferase assay was described previously [15,20,21]. Briefly, mouse NF-jB luciferase reporter gene plasmid (100 ng) and pRL-TK-Renilla-luciferase plasmid (20 ng) were co-transfected into Raw264.7 cells using jetPEI TM (Polyplus). Thirty-six hours later, cells were stimulated with PGN and different concentrations of SESLA for 24 h. The luciferase activity was measured, and the results were expressed as fold changes relative to the internal renilla control.

Identification of differently expressed genes (DEGs) and bioinformatic analysis
The gene expression dataset of whole blood leukocytes in MRSA-infected mice model (GSE38531) were downloaded from gene expression omnibus (GEO) database (http://www. ncbi.nlm.nih.gov/geo/). Genes with adjust p value < .05 and jlog 2(fold change) j >1 between uninfected control group (C) and MRSA-infected group (infected 24 h) (T) were considered to be significantly differential expression genes (DGEs). Gene Ontology (GO) analysis and visualization with the biological process (BP), cellular component (CC) and molecular function (MF) were carried out using R3.6.2 software with Bioconductor packages (p value < 0.05). The top 10 GO items of each functional categories were selected to be visualized. Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for pathway analysis by R3.6.2 and the top 20 items with p value < 0.05 were visualized.

Molecular docking studies for IjBa/NF-jB complex
IjBa/NF-jB complex structure file (1ikn.pdb) was download from PDB Database (https://www.rcsb.org/). Molecular docking analysis was carried out using Autodock Vina 1.1.2 software [22] and the most optimal docking result was visualized by PyMOL 2.0. If the affinity À7.0 kcal/mol indicates that the compound has a high affinity with the receptor protein.

Statistical analysis
All statistical analyses were carried out using the software of GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). Data were given as mean ± standard deviation (SD) from three independent experiments. Statistical significance between two groups was calculated with student's t-test. A p-value of <0.05 indicated a statistical significance.

SESLA reduces the transcription of inflammatory genes in PGN or Pam3CSK4-stimulated Raw264.7 cells
As a major component of the bacterial cell wall, especially of G þ bacteria, PGN is a well-known pathogen-associated molecular patterns (PAMPs) and is usually used to stimulate the immune cells to mimic the infection of G þ bacteria [23]. Pam3CSK4 is a synthetic tripalmitoylated lipopeptide that is analogous to the acylated amino terminus of bacterial lipoproteins and is also commonly used as a mimic of G þ bacteria to elicit an inflammatory response similar to PGN in macrophages and DCs [24]. In this study, to evaluate the effects of SESLA on the inflammatory response during G þ bacterial infection, the mRNA expression levels of the above-mentioned prototypical inflammatory factors induced by mimics of bacterial cell wall components (PGN or Pam3CSK4) in macrophages were detected. As shown in Figures 1 and 2, SESLA significantly decreased the transcriptional levels of IL-6, TNF-a, IL-1b, MCP-1 and IL-10 in mouse macrophage cell line Raw264.7.
Therefore, these results showed that SESLA exhibited an anti-inflammatory role elicited by PGN or Pam3CSK4, suggesting that it could suppress the excessive inflammatory reaction caused by G þ bacterial infection.
SESLA decreases the secretion of IL-6 and TNF-a after stimulation with heat-killed MRSA in mouse primary peritoneal macrophages The bacterial plasma membrane contains a diverse range of PAMPs, and the intact membrane can mimic the real bacterial infection more accurately than the mimics of bacterial cell wall components, PGN or Pam3CSK4. Moreover, primary innate immune cells can respond to stimulators better than continuously passaged cells.
In this study, the anti-inflammatory role of SESLA was verified in primary peritoneal macrophages after the addition of heat-killed MRSA (HK MRSA) to the culture medium. As shown in Figure 3, SESLA could significantly decrease the production of IL-6 and TNF-a in the macrophages. Hence, these data suggested that SESLA exhibited excellent antiinflammatory properties during MRSA infections.

SESLA down-regulates the production of inflammatory cytokines in PGN or HK MRSA-induced dendritic cells (DCs)
The exacerbated inflammatory response triggered by PAMPs, and subsequent cytokine storm are the main pathogeneses of sepsis during bacterial or viral infection. Accumulation and over-activation of macrophages and DCs play important roles during this process. As one of the important first-line defenses against bacterial infections, DCs are also a main source of cytokine production.
Herein, the immunoregulatory roles of SESLA on cytokine secretion by DCs were measured after stimulation with PGN. As shown in Figure 4(A-D), SESLA could suppress the production of IL-6, TNF-a, IL-12p70 and IL-10 in a dose-dependent manner. Similarly, the anti-inflammatory role of SESLA was also confirmed in DCs in response to HK MRSA stimuli (Figure 4(E,F)).

SESLA reduces the expression of co-stimulatory molecules on the surface of PGN-activated DCs
In clinical practice, 28-day survival rate is a main outcome measurement of septic patients. The cytokine secretion and immune injury caused by activated T cells play a dominant role in sepsis from one week after onset [25]. DCs are the only cells to activate naive T cells. They phagocytose bacteria, process antigens and present them to T cells together with co-stimulatory signal. Upon recognizing the peptide by MHC class II complex (e.g. Iab), naive T cells become activated with the help of increased expression of co-stimulatory molecules, e.g. CD80, CD86 and CD40 on DCs [26].
In this study, the expression of MHC and co-stimulatory cell surface markers were detected by flow cytometry. As expected, the expression of CD80, CD86, CD40 and Iab was increased with PGN stimulation ( Figure 5). However, SESLA could decrease their expression, which might result in the down-regulation of T cell activation during infection.
In summary, SESLA was found to have anti-inflammatory effects in G þ bacteria-infected macrophages and DCs, and also down-regulated the expression levels of MHC II and costimulatory molecules on the surface of DCs, indicating that it may exert immunomodulatory activity in the formation and exacerbation of cytokine storm from innate immune cells and activated T cells in infectious diseases.

SESLA suppresses the production of inflammatory cytokines in a MAPK-independent manner
Given the significant anti-inflammatory activity of SESLA during G þ bacterial infections (e.g. MRSA), we further investigated its mechanism of action (MOA). According to the gene expression dataset GSE38531, a total of 734 differentially expressed genes (DEGs) were identified in the blood between uninfected control mice (C) and MRSA-infected mice model (T) (Supplementary Table 1). The heat map and the volcano plot of DEGs (adjusted p < 0.05) are shown in Supplementary Fig. 2. To analyze the function of these DEGs, they were further analyzed by Gene Ontology (GO) analysis consisting of three categories: biological process (BP), cellular component (CC) and molecular function (MF), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway functional enrichment analysis. As shown in Supplementary Table 2, 1214 terms in BP category, 50 terms in CC category, and 63 terms in MF category (p < 0.05) were enriched. Among them, the most significant enrichment items included T cell activation, positive regulation of cytokine production, T cell receptor complex, cytokine receptor activity, Toll-like receptor binding, etc. (Figure 6(A)), suggesting that MRSA infection mainly triggered the activation of immune cells (e.g. T cells) and the occurrence and development of inflammatory cytokines.
Additionally, the KEGG pathway analysis of DEGs showed that 53 pathways were significantly enriched (p < 0.05) (Supplementary Table 3). The top 20 KEGG pathways were shown in Figure 6(B). As one of the PRRs expressed on the cell membrane, Toll-like receptor 2 (TLR2) can form a heterodimer with a similarly shaped partner TLR6. During S. aureus infection, TLR2/6 heterodimer and other intracellular PRRs (e.g. NOD2 or cryopyrin) activate immune cells upon stimulation by the components of bacteria, e.g. PGN or bacterial lipoproteins, which are considered as the main PAMPs to trigger overwhelming inflammation harmful to the host. The extracellular signal regulated kinase 1/2 (ERK1/2), c-Jun-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) pathways and nuclear factor jB (NF-jB) pathways are common intracellular signaling pathways that are activated and lead to subsequent production of IL-6, TNF-a, IL-1b, IL-12, chemokines and IL-10, etc. [27][28][29]. PGN is a wellknown PAMP of G þ bacteria that is mainly recognized by TLR2, NOD2 or cryopyrin in various immune cells, such as monocytes, macrophages and DCs. Therefore, to investigate the molecular regulatory mechanism by which SESLA suppresses the inflammatory response in macrophages during G þ bacterial infections, we examined the role of SESLA in the modulation of MAPK activation after PGN stimulation. As shown in Figure 6(C), the phosphorylated status of ERK, JNK and p38 was measured in primary peritoneal macrophages, and the results showed that SESLA did not affect the activation of these signaling pathways.
SESLA attenuates inflammatory response via downregulating the activation of NF-jB signaling pathway in mouse primary macrophages As shown in Figure 6(B), as a common downstream signaling pathway of inflammatory response, NF-jB is one of the main signaling pathways during MRSA infection. Besides ERK and p38 MAPK, NF-jB pathway is also a crucial signaling pathway in the induction of IL-10 expression stimulated by lipopeptides in macrophages and DCs [30]. Therefore, in order to further explore the regulatory role of SESLA in NF-jB signaling pathway, molecular docking was performed to evaluate the binding potential between SESLA and the key proteins in NF-jB pathway. Molecular docking is an important aspect of computational biology, which is widely applied to predict the modes and affinities of non-covalent binding between small molecule compounds with the active sites of a protein whose three-dimensional structure is established [31]. According to computational biology, affinity itself can represent the binding ability of a compound to a specific protein. The generally accepted standard is the free energy ˂À5.0 kcal/mol, which means they can bind because the value is the energy required for a high energy bond to form, and when affinity ˂ -7.0 kcal/mol, it means they have strong binding ability. Hence, the greater the absolute value of the affinity, the greater is the binding force.
The docking results showed that SESLA had high affinity for IjBa/NF-jB complex (-7.7 kcal/mol) (Figure 6(D)). The affinity of BAY11-7085 (NF-jB inhibitor) and IjBa/NF-jB complex was À7.4 kcal/mol ( Supplementary Fig. 3B), suggesting that the binding capacity of SESLA and IjBa/NF-jB complex was stronger than that of BAY11-7085 and the complex. To confirm this, the phosphorylated status of important components in NF-jB signaling pathway was detected. As shown in Figure 6(E), SESLA suppressed the phosphorylation of I-jBa upon PGN stimulation in mouse peritoneal macrophages. As a common method to elucidate the transcriptional activity of promoter, luciferase reporter gene assay was used in this study. Results showed that NF-jB transcriptional activity was down-regulated in PGNstimulated Raw264.7 cells by SESLA (Figure 6(F)), consistent with the decreased expression of phosphorylated I-jBa, suggesting that SESLA suppressed the activation of I-jBa and thereby inhibited the transcriptional activity of NF-jB, which may account for the decrease in IL-6, IL-1b, TNF-a, IL-10, etc.

Discussion
Excessive release of inflammatory cytokines induced by microbial infection is one of the primary causes of homeostatic imbalance of the host. As a primary nosocomial Grampositive (G þ ) bacterium, methicillin-resistant Staphylococcus aureus (MRSA), can induce severe infectious disease (e.g. sepsis), which is associated with high mortality in the ICU due to the excessive and fatal systemic hyper-inflammatory response [21,32]. Host-directed therapy (HDT), aimed to recover immune homeostasis, is a promising strategy to combat severe infectious diseases. We previously demonstrated that the synergistic of SESLA and ineffective antibiotic, meropenem, could provide protective effects against carbapenem-resistant Klebsiella pneumoniae (CRKP)-induced septic mice through alleviating excessive inflammatory response. Unique to this study, we further confirmed that SESLA could suppress overwhelming inflammation by decreasing the expression of inflammatory cytokines (such as IL-6 and TNF-a) and inhibiting the activation of naive T cells. The suppression of NF-jB signaling pathway and down-regulating the expression of MHC II as well as co-stimulatory molecules on the surface of DCs by SESLA could account for its anti-inflammatory activity.
In the initial stage of infection, immune cells (e.g. monocytes/macrophages and DCs) can recognize bacterial components and subsequently elicit the secretion of inflammatory cytokines and chemokines [33]. Generally speaking, chemokines are beneficial for the recruitment of monocytes and neutrophils to the sites of infection in order to control the infection in the early stage [34]. However, excessive production of inflammatory cytokines from these cells contributes to the formation of cytokine storm and its associated pathological damage. For example, as a multifunctional cytokine, IL-6 is strongly related to the pathogenesis of severe sepsis [35]. Extremely high level of IL-6 in septic patients is associated with high mortality. As important pro-inflammatory cytokines, both of IL-1b and TNF-a can cause excessive inflammation in a positive feedback loop in innate immune cells [8]. Their combination results in the cascade amplification of cytokines [36], acting as key facilitators of systemic inflammation or multiple organ dysfunction (MODS) [37]. In this study, we found that SESLA could inhibit the production of IL-6, TNF-a, IL-1b and IL-12 in PGN or Pam3CSK4-stimulated macrophages and DCs. Even upon the stimulation of HK MRSA, SESLA could also suppress the production of typical pro-inflammatory cytokines IL-6 and TNF-a dramatically. These results might imply that SESLA displays anti-inflammatory effects in immune cells.
Furthermore, along with pro-inflammatory cytokines, chemokines can also be secreted by epithelial cells, etc. in infectious diseases. They can recruit various immune cells specifically to the foci of infection, resulting in exacerbated inflammation. JC/MCP-1 is a well-known chemokine that recruits monocytes [8]. It was shown that MCP-1 could be down-regulated by SESLA with the stimulation of PGN or Pam3CSK4 in macrophages, hinting that SESLA could inhibit the recruitment of monocytes to the sites of infection and thus prevent the amplifying cascade of inflammatory response.
As a classical anti-inflammatory cytokine, IL-10 is associated with the inhibition of inflammation and maintaining homeostasis during infection [38]. However, high level of serum IL-10 in septic patients is usually associated with high mortality [39]. We found that SESLA could also inhibit the production of IL-10 upon PGN-or Pam3CSK4-stimulation in macrophages or DCs. Therefore, all the above-mentioned results suggest that SESLA might suppress the excessive inflammatory response in response to G þ bacterial infection, potentially contributing to the in vivo protective effect.
Given that G þ and Gbacteria possess fundamentally diverse cell wall architecture, it is believed that different molecular mechanisms should account for the anti-inflammatory properties of SESLA. Our KEGG analysis on normal mice and MRSA-infected mice suggested that MAPK pathway and NF-jB pathway might play major effects in the production of inflammatory cytokines. In this study, we found that the expression of phosphorylated p38, ERK and JNK MAPKs was Representative results were shown from three independent experiments. The bar graphs showed the statistical results for phosphorylation levels of I-jBa. (F) Raw264.7 were seeded (8 Â 10 4 cells/well) in 96-well plates overnight and then co-transfected with NF-jB luciferase reporter plasmid and pRL-TK-Renilla-luciferase plasmid. At one and a half day after transfection, cells were stimulated with PGN (25 mg/mL) with or without SESLA (10 mM) for 24 h. The NF-jB luciferase activities were determined and presented as fold increase. Data were expressed as mean ± SD from three independent experiments; Ã , p < 0.05, ÃÃ , p < 0.01. unaffected by SESLA, suggesting that MAPK pathways were not involved in anti-inflammatory immune regulatory role of SESLA. Therefore, a molecular docking study was performed to analyze the affinity between SESLA and IjBa/NF-jB complex. Results indicated an intense binding force between them which gave a hint that SESLA might be involved in the regulation of NF-jB activation. Data showed that SESLA could inhibit the phosphorylation of IjBa. Moreover, SESLA could also decrease the transcriptional activity of NF-jB. Collectively, the above results suggested that the inhibition of NF-jB activation by SESLA might account for its antiinflammatory activities during G þ bacterial infection.
An uncontrolled cytokine storm produced by over-activated immune cells after bacterial infections is fatal. It can cause the imbalance of homeostasis resulting in severe tissue damage, organ failure and even death. The critical kinase (e.g. IKK) is considered as a therapeutic target for research and development of new anti-inflammatory drugs [40]. In addition to the innate immune response, adaptive immune system also plays a major role in the formation of cytokine storm [25]. Activated T cells are also an important contributor to cytokine storm [41]. The GO analysis revealed that a substantial number of immune cells (e.g. T cells) was also activated significantly in MRSA-infected mouse model. Our study demonstrated that SESLA could down-regulate the expression of MHC II and co-stimulatory molecules (e.g. CD40, CD80 and CD86), giving a hint that SESLA might inhibit T cell activation to alleviate overactivated inflammation.
Given its potential anti-inflammatory activity by inhibiting NF-jB pathway, SESLA could be a promising immunomodulatory drug candidate targeted to maintain the immunological homeostasis in G þ bacterial infection, or even multidrug-resistant MRSA. Recent studies have shown that patients with severe coronavirus disease 2019 (COVID-19) are characterized by massive cytokine secretion, which eventually causes life-threatening acute respiratory distress syndrome (ARDS). As a negative regulator of cytokine storm, SESLA might also be a drug candidate with potential anti-inflammatory role in the treatment of similar bacterial or even viral sepsis. However, the processes and the regulatory network of different cells in vivo are quite complicated. Thus, the exact anti-inflammatory and immune protective role of SESLA during MRSA infection in vivo needs to be further investigated.

Conclusions
In summary, this study further revealed the anti-inflammatory activity of SESLA against G þ bacterial infection. The possible mechanisms might include the down-regulation of inflammatory cytokine secretion in immune cells via inhibition of NF-jB signaling pathway in macrophages and suppression the activation of T cells by inhibiting the expression of MHC II and co-stimulatory molecules on DCs. Therefore, this study might provide a mechanistic insight into the anti-inflammatory role of SESLA, inhibiting the cytokine storm and maintaining the immune homoeostasis upon the infection of G þ bacteria or even drug-resistant bacteria (e.g. MRSA).