Tonsillar Microbiome‐Derived Lantibiotics Induce Structural Changes of IL‐6 and IL‐21 Receptors and Modulate Host Immunity

Abstract Emerging evidence emphasizes the functional impacts of host microbiome on the etiopathogenesis of autoimmune diseases, including rheumatoid arthritis (RA). However, there are limited mechanistic insights into the contribution of microbial biomolecules especially microbial peptides toward modulating immune homeostasis. Here, by mining the metagenomics data of tonsillar microbiome, a deficiency of the encoding genes of lantibiotic peptides salivaricins in RA patients is identified, which shows strong correlation with circulating immune cells. Evidence is provided that the salivaricins exert immunomodulatory effects in inhibiting T follicular helper (Tfh) cell differentiation and interleukin‐21 (IL‐21) production. Mechanically, salivaricins directly bind to and induce conformational changes of IL‐6 and IL‐21 receptors, thereby inhibiting the bindings of IL‐6 and IL‐21 to their receptors and suppressing the downstream signaling pathway. Finally, salivaricin administration exerts both prophylactic and therapeutic effects against experimental arthritis in a murine model of RA. Together, these results provide a mechanism link of microbial peptides‐mediated immunomodulation.


Introduction
The human microbiomes are well appreciated for their indispensable role in immune development. The immune system and the microbiota are two components that influence one another to orchestrate host homeostasis as well as to maintain a stable microbial community. [1] Given the wellrecognized roles of microbiome in setting the systemic immune tone, perturbation of the healthy microbial community, termed dysbiosis, has been linked to the pathophysiology of numerous autoimmune diseases, including Type 1 diabetes, multiple sclerosis, and rheumatoid arthritis (RA). [1a,2] Some autoimmune conditions like RA have required the presence of microbes for disease onset. [3] RA is a systemic autoimmune disease characterized by the invasion of joints T (Treg) and/or T helper 17 (Th17) cells. [7,8] However, there are relatively few mechanistic insights into the contribution of microbial macro-molecules such as microbial peptides toward modulating immune homeostasis.
Previously, by profiling the metabolic potential of the tonsillar microbiome, we found that the biosynthesis and transport of lantibiotics were deficient in RA tonsils. [9] Lantibiotics are polycyclic antimicrobial peptides containing lanthionine and/or -methyllanthionine residues produced by bacteria including species of Streptococcus and Lactobacillus. [10] They are among the most promising candidates for future antimicrobials due to their capacity to inhibit the growth of clinically significant pathogens including multidrug-resistant Staphylococci, Streptococci, Enterococci, and Clostridia. [10] The diversity and potency of lantibiotics make them attractive candidates for translational application, and several are already in clinical trials. [10] Besides the classical antibiotic use, they are now receiving increased attention as possible immune-modulating agents. [10,11] The lantibiotic nisin Z is able to modulate host immune responses and mediate protective host immunity through similar mechanisms as natural host defense peptides, engaging multiple signal transduction pathways and growth factor receptors. [12] Another lantibiotic peptide lancovutide (Moli1901) was shown to be a safe and effective therapy for the treatment of cystic fibrosis in a phase II clinical trial. [13] These results suggest that the lantibiotic peptides may represent a new class of secreted bacterial molecules with immunomodulatory activities.
In the present study, we identified deficiency for lantibiotic peptides salivaricins in the tonsillar microbiome of RA patients. More importantly, we demonstrated the immunomodulatory effects of salivaricins, and determined the key receptors that mediated this process. Finally, animal experiments were performed to investigate the potential clinical applications of salivaricins in autoimmune diseases.

Tonsillar microbiome-Derived Lantibiotic Peptides Salivaricins are Correlated to Circulating Immune Cells
Previously, by profiling the tonsillar microbiome of RA patients and healthy controls using a standard whole-metagenome shotgun sequencing technology (Table S1, Supporting Information), we detected that the biosynthesis and transport of lantibiotics were deficient in RA tonsils. [9] Here, we profiled these metagenomic sequencing data in-depth to determine the genomic capacity of human tonsil-associated microbes to produce lantibiotic peptides. Five lantibiotic-encoding genes were identified from our metagenomic data by searching the antimicrobial peptide database (APD3: the antimicrobial peptide database as a tool for research and education), [14] including salivaricin A2, [15] B, [15a,16] E, [17] and G32, [18] commonly synthesized by S. salivarius, and suicin 65, [19] commonly synthesized by S. suis (Table S2, Supporting Information). Abundance of salivaricin A2 coding gene was significantly reduced in the tonsillar microbiomes of RA patients compared to that of healthy controls (Wilcoxon rank sum test, P = 0.03), while salivaricin B, E, G32, and suicin 65 were also less abundant in RA patients but not significant ( Figure 1A). Additionally, we analyzed the coding capacity of these lantibiotic showing the relative abundances of five lantibiotic genes (salivaricin A2, B, E, G32, and suicin 65) in the tonsillar microbiomes of rheumatoid arthritis (RA) patients and healthy controls. Boxes represented the interquartile range between the first and third quartiles and median (internal line). Whiskers denoted the lowest and highest values within 1.5 times the range of the first and third quartiles, respectively; and dots represented outlier samples beyond the whiskers. RPKM, reads per kilobase per million mapped reads. n = 32 for RA patients and n = 30 for healthy controls, Wilcoxon rank sum test, *P < 0.05. B) Heatmaps displaying the associations of lantibiotic-encoding gene abundances from the tonsillar microbiome with RA-related clinical and immunological indexes (left panel) and with circulating immune cell subsets (right panel). Spearman's rank correlation test, *P < 0.05. peptides in the saliva, dental plaque, and gut microbiomes of RA patients. [6c] Similarly, we found that salivaricin B from the saliva microbiome showed a downtrend (Wilcoxon rank sum test, P = 0.38) in naïve-treated RA patients while became partial recovery to the healthy state in treated patients, whereas salivaricin A2, E2, and G32 were rarely detected in these niches ( Figure S1, Supporting Information).
Subsequently, we explored correlations of lantibiotic peptides with RA clinical and immunological indicators. Despite that a limited number of associations were observed between lantibiotics and RA clinical indexes, we did detect strong associations of these lantibiotic-encoding genes with host circulating immune cell subsets ( Figure 1B). Specifically, the lack of salivaricin A2 was correlated with the increased proportions of precursor follicular helper T (pTfh), dendritic cells, and plasma cells ( Figure 1B), all well-known immune mediators leading to overproduction of pathogenic autoantibodies in RA. [20] Collectively, these results support the notion that salivaricin peptides produced by tonsillar microbiome may function in immunomodulatory roles in RA.

Salivaricins Downregulate IL-21 Production in Human PBMCs
To support investigations of the potential immunomodulatory effects of salivaricins, we chemically synthesized salivaricin A2 and B ( Figure S2, Supporting Information) and performed in vitro experiments with peripheral blood mononuclear cells (PBMCs) isolated from both RA patients and healthy individuals. Treatment of cultured human PBMCs stimulated with CD3 and CD28 antibodies revealed that the salivaricin A2 or B peptides could significantly reduce the level of the immune regulatory cytokine interleukin-21 (IL-21), and the IL-21 reducing effects of both peptides were dose-dependent (Figure 2A,B, and Figure S3, Supporting Information). No salivaricin-related differences were observed in the levels of other cytokines including IL-17A, IL-10, IFN-, TNF-, or IL-6 ( Figure S3, Supporting Information). IL-21 is mainly produced by Tfh and Th17 cells, and this cytokine is known to regulate both germinal center (GC) B cell survival and plasma cell differentiation. [21] Importantly, these in vitro results showing salivaricin A2-and B-mediated reductions in the IL-21 level of PBMCs are consistent with the negative correlations we detected in our RA patient clinical data between the capacity of the tonsillar microbiome to produce salivaricins and the proportions of circulating precursor Tfh and plasma cells.
Then we tested the activation of signal transducer and activator of transcription 3 (STAT3), a key molecule downstream of IL-6 and/or IL-21 signaling and also the most important signal transducer in promoting murine Tfh differentiation. [20a] As expected, salivaricin A2 and B largely blocked IL-6-mediated STAT3 phosphorylation in Tfh-like cells ( Figure 3F and Figure S5A Collectively, these results demonstrated that salivaricins could inhibit IL-6R/IL-21R-STAT3 signaling pathway, thus rationalizing the observed effects of salivaricin on both Tfh cell differentiation and IL-21 production.

Salivaricins Directly Bind to IL-6 and IL-21 Receptors
Given the regulatory capacities of salivaricins in IL-6R/IL-21R signaling, we used cell-free assays to test whether salivaricin A2 and B may interact with IL-6R and/or IL-21R. Pull-down assays showed that salivaricin A2 and B, but not similarly sized negative control peptides, bound directly to murine IL-6R subunit alpha (IL-6R ); and only salivaricin B but not A2 bound directly to IL-21R (Table S3, Supporting Information). Additional validation of direct bindings of salivaricins to murine IL-6R was performed using surface plasmon resonance (SPR), and the results demonstrated that salivaricins-IL-6R interactions were concentrationdependent ( Figure 4A). Plus, while pull-down assay did not detect interaction between salivaricin A2 and IL-21R, SPR experiments demonstrated that salivaricin A2 also interacts with murine IL-21R ( Figure 4B). It should be noted that, however, the detected binding affinities of the salivaricins to immobilized IL-6R and IL-21R were 10 3 -10 5 magnitude lower than the binding affinities of IL-6 and IL-21 to their receptors ( Figure S6 and Table S4, Supporting Information). Despite having lower binding affinity to the respective receptors, the competitive inhibition experiments showed that the binding of IL-6 to IL-6R or IL-21 to IL-21R could be partially or completely suppressed in the presence of salivaricin A2 or B, in a dose-dependent manner ( Figure 4C,D).
These results indicated that salivaricins could inhibit the interactions between IL-6 and IL-21 with their receptors, therefore suppressing the IL-6R/IL-21R signaling.
Next, we set to identify the key amino acid sites of salivaricins that mediate the bindings to IL-6R and/or IL-21R. First, computational approach was used to predict the potential amino acid sites on the peptides (Figure5A,B). We then constructed five mutants according to the results generated by PISA (Proteins, Interfaces, Structures, and Assemblies) and validated their binding affinities by SPR. Results showed that salivaricin A2 peptides with mutations at the 2nd arginine residue (salivaricin A2-1), at the 12nd and 13th asparagine residues (salivaricin A2-2), and at the 21st and 22nd cysteine residues (salivaricin A2-3) all had impaired binding affinities to IL-6R ( Figure 5C), yet remained similar binding affinities to IL-21R ( Figure 5D). In addition, these mutants had similar secondary structures with salivaricins A2 (Figure S7, Supporting Information), indicating that these sites were not critical for the conformation of salivaricin A2, but responsible for its associations with IL-6R. However, the predicted sites of salivaricin B showed little effect in the binding affinities to IL-6R or IL-21R ( Figure S8, Supporting Information), indicating a gap in predicting and understanding of functionality of salivaricin B, which would only be addressed in future studies.

Salivaricins Induce Conformational Changes of IL-6 and IL-21 Receptors
As the salivaricins had much lower binding affinities to the respective receptors, we then hypothesized that the peptides did not directly compete for binding with IL-6 or IL-21 but rather induce changes in the functions of receptors. Given that the receptor structure is critical to the ligand-receptor engagement, we detected the secondary structures of IL-6R and IL-21R in the presence of salivaricins by using circular dichroism (CD) spectroscopy. As shown in Figure 6, the CD spectrum and the statistical proportions of different secondary structures (e.g., alpha-helix, beta-barrels, and random coils) of IL-6R or IL-21R remained unchanged following IL-6 or IL-21 binding (

Salivaricins Exert Anti-Arthritic Effects in Experimental Arthritis
Interventions with agents to modulate IL-6/IL-6R or IL-21/IL-21R have been shown to confer clinical benefit in patients with autoimmune diseases including RA. [21,24] To determine if salivaricins can suppress immune-mediated pathological processes in vivo, we explored both prophylactic and therapeutic applications of salivaricins in collagen-induced arthritis (CIA) model mice, a well-characterized murine arthritis model. Prophylactic  administration with salivaricin A2 or B before arthritis onset (see Figure 7A for the experimental design) significantly decreased the incidence and severity of arthritis in the CIA mice, without affecting body weight ( Figure 7B-D and Figure S10A-G, Supporting Information). Note that similar protective effects were observed for all three of the salivaricin administration routes (intra-orally, intra-peritonelly, or intra-gastrically). We also administered salivaricins as a potential treatment after CIA model induction, and found that both salivaricin A2 and B significantly relieved disease severity and reduced inflammatory cell infiltration in the joints compared to the vehicle controls ( Figure 7E-G). Thus, salivaricins have potential clinical applicability, both for protecting against RA development and for treating RA.
We also explored which immune effector molecules may contribute to the observed anti-arthritic efficacy of salivaricins by profiling serum cytokine levels and lymphocyte subsets in draining lymph nodes (DLN) and spleens of the animals at the end of the prophylactic regime experiment. Salivaricins markedly decreased the proportions of Tfh cells in both DLNs and spleens (Figure 8A,B). We also detected a significant reduction of Tfh cells expressing the aforementioned Tfh polarizing transcription factor Bcl6 ( Figure 8C,D). In addition, the frequencies of Th17, plasmablast, and GCB cells also showed trends of decrease in the salivaricins-treated mice, while the proportions of Th1 and Treg cells remained unchanged ( Figure 8E,F). Similar to Tfh cells, Tfr cells were also reduced in the salivaricin-treated mice ( Figure  S10H,I, Supporting Information), while the ratio of Tfr to Tfh cells showed increased tendency ( Figure 8E,F). Consistently, salivaricins-treated mice showed significantly reduced titers of serum autoantibody anti-collagen type II (CII) and significantly increased serum IL-10 levels ( Figure 8G,H). There was no change in the IL-6 level ( Figure S10J, Supporting Information). However, serum IL-21 in CIA mice was overall very low, which may underlie the fact that we found trend of decrease but no statistical significance in IL-21 level in salivaricin-treated mice (data not shown). Together, these data suggest that salivaricin may act as a promis- Figure 5. Identification of binding sites of salivaricin A2 to receptors. A,B) Computational approach is used to predict the potential amino acid sites of salivaricin A2 binding to mouse IL-6R and IL-21R, identifying five residues according to the results generated by PISA (Proteins, Interfaces, Structures and Assemblies). C) SPR sensorgram for the bindings of salivaricin A2 mutants (12.5-800 μM) to the immobilized murine IL-6R (20 nM). The KD values of salivaricin A2 is 55.1 μM, while salivaricin A2-1 (mutation at the 2nd arginine residue), salivaricin A2-2 (mutations at the 12th and 13th asparagine residues), and A2-3 (mutations at 21st and 22nd cysteine residues) showed not-available KD values to murine IL-6R . D) SPR sensorgram for the bindings of salivaricin A2 mutants (12.5-800 μM) to the immobilized murine IL-21R (20 nM), identifying KD values of 29.4 μM for salivaricin A2, 13.4 μM for salivaricin A2-1, 21.1 μM for salivaricin A2-2 and 16.8 μM for salivaricin A2-3 to murine IL-21R.
ing target in the management of overproduction of pathogenic autoantibodies in autoimmune diseases including RA.

Discussion
In this study, we revealed an immunomodulatory role of lantibiotic peptides salivaricins in autoimmune disease, demonstrating that salivaricins beneficially modulate host immunity by directly binding to and inhibiting IL-6 and IL-21 receptors.
Lantibiotic peptides are the most extensively studied antimicrobial peptides that are derived from bacteria. They are now receiving increased attention as potential clinical antimicrobials and as possible immune-modulating agents. The identification of lantibiotics with immunomodulatory activity other than antimicrobial effects will enlarge their possibilities for applications. Salivaricins are produced by certain strains of Streptococcus salivarius, which almost exclusively reside in the human oral cavity. Salivaricins have been useful in the development of novel antibacterial therapies as an alternative to conventional antibiotics. [10] Here, we investigated in-depth the immunomodulatory effect of salivaricins, and demonstrated that salivaricin A2 and B beneficially modulate host immunity by inhibiting Tfh cell differentiation and IL-21 production. Consistent with the dominant roles of Tfh cells and IL-21 in promoting GCB cell survival, plasma cell differentiation, and antibody production, [20,21] we detected reduction of plasmablast, GCB cells, and autoantibody levels along with the downregulated Tfh cells in salivaricin-treated CIA mice. Therefore, we speculate that the deficiencies of salivaricins in RA tonsils reduce the capacity for proper modulation of local immune responses and autoantibody production, by which they are able to regulate autoimmune responses at distal sites involving the synovial joints. The present study, together with previous reports showing that gut microbiota can remotely regulate experimental arthritis by driving the induction and egress of gut Tfh cells, [5] suggests that the disease-relevant T cell subsets or autoantibodies in RA may be initially generated (and proliferated) outside of the joints. Nevertheless, we do not have direct evidence to support the notion that pathological T cells primed at the oral cavity migrate into the joints.
The cytokine milieu is critical for Tfh cell differentiation. IL-6/IL-6R and/or IL-21/IL-21R are indispensable regulators that affect the differentiation and function of Tfh cells. [25] Previous studies have reported that Tfh cell induction was impaired in IL-6 −/− or IL-6R −/− mice. [26] Similarly, there was a defect in the generation of Tfh cells in IL-21 −/− or IL-21R −/− mice. [27] In addition, IL-21 or IL-21R deficient naïve CD4 + T cells showed significantly reduced Bcl6 expression in the presence of IL-6. [23a] IL-21 is induced by IL-6 in activated T cells, and can in turn signal to reinforce its own production. [28] In the present study, we demonstrate that salivaricin A2 and B can directly bind to IL-6R and IL-21R. Although salivaricin A2 or B bind to IL-6R or IL-21R with much lower affinities than that of IL-6 or IL-21, they are able to change the structures of IL-6R and IL-21R. It is likely that the altered conformations of IL-6R and IL-21R in response to salivaricin-binding may be responsible for the salivaricins' competition with interleukins and the reduced interactions of IL-6/IL-6R and IL-21/IL-21R. Consequently, salivaricins can inhibit the IL-6R/IL-21R-STAT3 signaling, thereby suppressing Tfh cell differentiation and IL-21 production. Although both salivaricin A2 and B can bind to and inhibit IL-6R, it is noted that salivaricin A2 had slightly higher affinity to IL-6R than salivacicin B, which may be responsible for the better inhibition in IL-21 production. Moreover, the abundance of salivaricin A2 coding gene was more abundant and frequent than salivaricin B in healthy individuals, making it more suitable for health concerns in the future.
Furthermore, IL-6/IL-6R and IL-21/IL-21R are important contributors to the development of multiple autoimmune disorders characterized by overproduction of pathogenic autoantibodies. [21a,24] Studies have reported that knockout of IL-6 or IL-21R protected mice from experimental arthritis in various models. [24,29] In light of these discoveries, numerous agents which target components of these two signaling pathways have drawn much biomedical attention. [21b,24] For instance, monoclonal antibodies against IL-6R (e.g., Tocilizumab) have been developed for the treatment of RA. [24] Here, we confirmed the ability of salivaricin A2 and B to modulate host immunity in vivo, as evidenced by the reduced autoantibody production and Tfh cells of murine RA model, and detected both prophylactic and therapeutic efficacy against experimental arthritis. Therefore, establishing strategies for manipulating oral microbiome with salivaricins or related probiotics (e.g, S. salivarius) may represent a useful strategy for developing therapeutics against overproduction of pathogenic autoantibodies in autoimmune diseases including RA. Our study paves the way for novel regime of treatment for RA and perhaps other autoimmune disorders.
Mucosal niches including the gut and the oral are viewed as likely extra-articular origins for RA development. [3,30] Multiple studies have proposed that interactions between the gut and oral mucosal immune system and an aberrant local microbiome might have a causal role in the development of RA. [5] The tonsillar microbiome is an example of a lymphoid organ-attached community. [31] The close contacts of such communities with mucosa enable their direct interaction with the immune system. [31,32] Focal infections in the tonsils have long been implicated in RA pathogenesis, albeit without well-defined mechanisms. [30b,33] Supporting this idea, our data identified that lantibiotic peptides salivaricins were deficient in the tonsillar microbiome of RA patients. However, the expression of salivaricin in healthy subjects was too low for liquid chromatography-mass spectrometry detection. Previous study has detected the presence of salivaricin peptides in human saliva using the induction assay, [34] but only in subjects in which significant levels of salivaricin-producing strain (eg., S. salivarius K12) colonization were achieved. In their study, the lowest concentration of S. salivarius to detect salivaricin appeared to be 8*10 5 CFU per mL. [34] By contrast, the S. salivarius levels were only 2.6*10 5 CFU per mL in healthy subjects, [34] thus making it hard to detect the presence of salivaricin without supplementation of salivaricin-producing strain. In addition, currently there are no ELISA-kits or antibodies available, we, therefore, have to rely on metagenomic data for inferring abundances of bacterial peptides, a common practice in microbiome research.
More importantly, we observed that the lack of salivaricin A2 at tonsils was correlated with the increased proportions of pTfh, dendritic cells, and plasma cells in the circulating, which is consistent with the immunomodulatory effects of salivaricin A2 in inhibiting Tfh cell differentiation and function. In addition, salivaricin B also showed negative correlations with pTfh cells but did not reach significance, the trend is consistent with the negative regulation of salivaricin B in Tfh cells. Presumably, the relatively small sample size and/or low expression of salivaricin B in human might be a contributing factor. Moreover, salivaricins www.advancedsciencenews.com www.advancedscience.com are known to exert antimicrobial activities selectively against oral and upper-respiratory-tract pathogenic bacteria including S. pyogenes and S. dysgalactiae, [10a,15a,35] which are expectedly enriched in RA tonsillar microbiome [9] and known to trigger autoimmune responses in the joints and the heart. [36] Therefore, we speculate that the potential therapeutic advantage of salivaricins may stem from its dual action involving an immunoregulatory mechanism and an antimicrobial activity. In addition, although salivaricin A2 was positively correlated with lung disease, whether salivaricin A2 is a risky factor to lung health or causally implicated in lung diseases needs to be studied further.
In summary, the major findings of the present study are the identification of immunomodulatory roles of lantibiotics salivaricins in modulating host autoimmunity and the recovery of key receptors that mediate this process.

Experimental Section
Participant Enrollment: Adult patients (n = 32) diagnosed with RA according to the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) 2010 classification criteria [37] were recruited from Peking University People's Hospital, Beijing, China. In the RA group, 37.5% (12/32) patients were treatment-naïve. Healthy volunteers (n = 30) with no history of inflammatory arthritis and rheumatic diseases were enrolled. All the participants had not taken antibiotic treatment or probiotic supplements in the three months prior to sample collection. Informed consent was obtained from all subjects. This study was approved by Peking University People's Hospital Ethics Committee (2101000499). Detailed information of the cohort was given in Tables S1.
Whole-Metagenome Shotgun Sequencing and Bioinformatic Analyses: The tonsillar microbiota samples were collected by following the procedure of Human Microbiome Project (https://hmpdacc.org/doc/HMP_ MOP_Version12_0_072910). Genomic bacterial DNA was extracted using the MoBio PowerSoil DNA Isolation Kit 12888-100 protocol (MoBio Laboratories). The samples were placed at −80°C less than two months before DNA extraction. The fresh genomics DNA samples (RA = 32 and HC = 30) were mechanically fragmented to ≈400 bp with Bioruptor Pico (Diagenode, Belgium). A magnetic beads-based method was used for DNA fragments selection following a standard protocol (Agencourt AMPure XP). Libraries were prepared by using the NEBnext® Ultra II DNA Library Prep Kit for Illumina® (New England BioLabs). The Illumina HiSeq X platform was then used for 2 × 150 bp paired-end whole-metagenome sequencing. A de novo gene catalogue was constructed based on the metagenomic data from the tonsillar samples of all individuals. A non-redundant gene catalogue was generated after removing the redundancy genes.
The lantibiotics and their encoding genes from the metagenomic data are identified based on integrating the antimicrobial peptide database (APD3) [14] and literature search with a threshold of >95% similarity. For the APD3 database, only lantibiotics that sourced from the organisms of Streptococcus, Lactococcus, and Lactobacillus were selected for further analysis. And a literature search was performed by searching the PubMed database using keywords "lantibiotics", or "salivaricin". This procedure identified 11 additional salivaricins from several strains of Streptococcus spp. and Lactobacillus salivarius. The detailed information on lantibiotics inferred in this study was shown in Table S2, Supporting Information.
The Chemosynthesis of Peptides: Salivaricin A2 (KRGTGWFATITDDCPNSVFVCC), [15b] salivaricin B (GGGVIQTISHECRMNSWQFLFTCCS), [16] negative control peptide A (CCVFVSNPCDDTITAFWGTGRK), and negative control peptide B (SCCTFLFQWSNMRCEHSITQIVGGG) were synthesized by solid-phase techniques on a CS336X Peptide Synthesizer (Cs Bio, USA) in RoYo Biotech Co., Ltd (Shanghai, China). The reverse peptides of salivaricin A2 and B were chosen as their negative control peptides according to previous study. [38] The peptides were purified by high-performance liquid chromatography (Shimadzu Corp., Japan) with a purity of more than 96% and identified by Liquid chromatography-mass spectrometer (Shimadzu Corp., Japan). The endotoxin unit was tested. Detailed information on the two salivaricins was given in Figure S2, Supporting Information.
Real-Time Quantitative PCR (qPCR): For comparison of gene expression, total RNA from in vitro polarized Th0 or Tfh-like cells was extracted with RNAprep Pure Micro Kit (TIANGEN Biotech Co., Ltd) according to the manufacturer's instructions. Oligonucleotide, Revertaid reverse transcriptase, and RiboLock RNase Inhibitor (Invitrogen) were used to generate cDNA. Gene expressions were examined using the AceQ qPCR SYBR Green Master Mix (Vazyme Biotech). For qPCR analysis, the data shown were normalized to the expression of reference gene Actb. The primers were listed in Table S5, Supporting Information.
Western Blot: Cells from in vitro polarized Th0 or Tfh-like cells were lysed using RIPA lysis buffer (Sigma-Aldrich, St Louis, MO, USA) supplemented with 1% protease inhibitor cocktail and 5% phosphatase inhibitor (Roche, Basel, Switzerland. Protein concentrations were determined using BCA protein assays (Pierce, Rockford, IL, USA). Cell lysates were separated by SDS-PAGE gels and electrotransferred onto polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Buckinghamshire, UK), then probed with primary antibodies and subsequently HRP-labeled secondary antibodies. Signals were detected by ChemiDoc MP (BIO RAD). Immunoblotting was performed using standard protocols. Actin was used as an internal control throughout. The antibodies against pSTAT3 (Tyr705) and STAT3 (79D7) were obtained from Cell Signaling Technology.
Computational Analysis of Binding Sites of Salivaricins with Receptors: Four complexes (salivaricin A2-mouse IL-6R complex, salivaricin A2mouse IL-21R complex, salivaricin B-mouse IL-6R complex, salivaricin B-mouse IL-21R complex) all used AlphaFold (version 2.1.0) with the parameter "-model_preset = multimer" to predict their structures. Finally, the relaxed model was selected as the final prediction results and the surface state and contact interface of the above-mentioned complexes were analyzed by PISA (version 1.52). Five mutants were constructed according to the results generated by PISA: salivaricin A2-1 (mutation at the 2nd arginine residue), salivaricin A2-2 (mutations at the 12th and 13th asparagine residues), salivaricin A2-3 (mutations at 21st and 22nd cysteine residues), salivaricin B-1 (mutation at the 23rd and 24th cysteine residue), and salivaricin B-2 (mutations at the 11th glutamic acid and 13th arginine residues). CD Spectroscopy: CD experiments were performed at 25°C using a Chirascan V100 (Jasco, Easton, MD, USA). Protein or peptide samples at 0.1-10 mg mL −1 were examined in a 1.0 mm-path length Suprasil (quartz) cell (Hellma UK). CD spectra were collected over a wavelength range of 180-260 nm in 1 nm steps using 1s time per point with a bandwidth of 1 nm. All CD spectra were corrected for the baseline by subtracting the spectra of the corresponding IL-6R or IL-21R solutions and representing the average of three runs. The content of the secondary structures was estimated with the CDNN v2.1 software. [40] CIA Induction and Intervention: Male DBA/1 mice (6-8 weeks old) were purchased from Huafukang Co. Ltd. (Beijing, China) and fed under specific pathogen-free conditions. All experiments were carried out in accordance with guidelines prescribed by the Animal Care and Use Committee of Peking University People's Hospital (2019PHE047). CIA induction was established by following a previously published protocol. [41] Briefly, DBA/1 mice were immunized intradermally at the base of the tail with 200 μg of bovine type II collagen (CII, Chondrex) emulsified in complete Freund's adjuvant (Sigma-Aldrich, St Louis, MO, USA) in equal volumes. Three weeks later, a booster was delivered using 100 μg CII emulsified in Freund's incomplete adjuvant. Mice were monitored for signs of arthritis after the booster immunization. Clinical score was assessed by using the following system as detailed previously: [41] 0, normal; 1, erythema and swelling of one or several digits; 2, erythema and moderate swelling extending from the ankle to the mid-foot (tarsals); 3, erythema and severe swelling extending from the ankle to the metatarsal joints; and 4, complete erythema and swelling encompassing the ankle, foot, and digits, resulting in deformity and/or ankyloses. The scores of all four limbs were summed, yielding total scores of 0-16 per mouse.
We randomized mice into control or treatment groups. Salivaricin A2 (50, 100, or 200 μg per mice) or B (50, 100, or 200 μg per mice) was supplemented intra-orally, intraperitoneally or intragastrically three times a week. The preventive group started on day 1, and the therapeutic groups commenced after the onset of CIA (approximately day 26). At the study endpoints, mice were euthanized and serum samples were collected for cytokine and auto-antibody detection. The spleens and joint DLNs (popliteal and axillary lymph nodes, DLN) were obtained from mice, sieved through a 70 μm cell strainer (Corning) in RPMI 1640 medium with 10% FBS and single-cell suspensions (10 6 cells per 100 μL) were prepared for flow cytometry. 10-23 mice were used in the indicated group, and data were pooled from two independent experiments.
Radiography Evaluations and Histological Analyses: The paws from each mouse were collected and fixed in 4% paraformaldehyde (PFA) for 48 h, then scanned using a Micro-CT scanner (Quantum FX, Caliper, USA). After that, the paws were decalcified in 5% EDTA, paraffin-embedded, sectioned, and stained with hematoxylin and eosin. A microscopic assessment of sagittal sections was performed and histopathological changes were scored based on the following previously reported parameters: [42] 0, normal synovium; 1, synovial membrane hypertrophy and cell infiltrates; 2, pannus and cartilage erosion; 3, major erosion of cartilage and subchondral bone; and 4, loss of joint integrity and ankylosis. The scores of all four limbs were summed and divided by 4, yielding average scores of 0-4 per mouse.