{"paper_id":"0b533189-6b5f-4009-bb70-fed53175ae2d","body_text":"Gingipain regulates isoform switches of PD-L1 in macrophages infected with Porphyromonas gingivalis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Gingipain regulates isoform switches of PD-L1 in macrophages infected with Porphyromonas gingivalis Yilin Zheng, Ziyi Wang, Yao Weng, Heriati Sitosari, Yuhan He, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5411219/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Periodontal pathogen Porphyromonas gingivalis ( Pg ) is believed to possess immune evasion capabilities, but it remains unclear whether this immune evasion is related to host gene alternative splicing (AS). In this study, RNA-sequencing (RNA-seq) revealed significant changes in both AS landscape and transcriptomic profile of macrophages following Pg infection with/without knockout of gingipain (a unique toxic protease of Pg) . Pg infection increased the programmed death ligand 1 (PD-L1) transcripts expression and selectively upregulated a specific coding isoform that more effectively binds to programmed cell death protein 1 (PD-1) receptors on T cells, thereby inhibiting immune function. Biological experiments confirmed these results and demonstrated that the AS switch of PD-L1 was gingipain-dependent. AlphaFold 3 predictions indicated that the protein docking compatibility between PD-1 and Pg -upregulated PD-L1 isoform was over 80% higher than another coding isoform. These findings suggest that Pg employs gingipain to modulate the AS of PD-L1, facilitating immune evasion. Biological sciences/Immunology Biological sciences/Microbiology Porphyromonas gingivalis gingipain macrophage alternative splicing PD-L1 immune evasion Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Porphyromonas gingivalis ( P. gingivalis or Pg ) is considered the primary pathogen responsible for periodontal disease 1 . P. gingivalis promotes the development of the disease by producing multiple proteolytic enzymes that degrade periodontal tissues 2 . Within these proteases, gingipain is considered a major contributor to the proteolytic activity of P. gingivalis 3 . Previously, we found that P. gingivalis infection of macrophages leads to histone leakage 4 . Histone modifications are known to affect alternative splicing (AS) of mRNA 5, 6 . The capacity to generate various transcripts (gene isoforms) through AS significantly contributes to the complexity of proteome in higher vertebrates. A vast majority of human genes utilize alternative isoforms: around 95% of multi-exon genes exhibit evidence of AS, and about 60% of genes have at least one alternative transcription start site 7 . This splicing diversity plays important biological roles in organisms, including increasing protein functional diversity, regulating gene expression levels, and participating in processes such as cell development and tissue specialization. The changes in AS of the transcriptome in infected macrophages are crucial in regulating the immune response 8 . However, the role of mRNA AS in macrophages infected with P. gingivalis has not been elucidated. P. gingivalis has been shown to induce the upregulation of the immune checkpoint protein, programmed death ligand 1 (PD-L1), in periodontal tissue cells 9, 10 . PD-L1, encoded by the CD274 gene, is the ligand for the co-inhibitory receptor programmed cell death protein 1 (PD-1) 11 . Under physiological conditions, PD-L1 is expressed in various immune-privileged organs. Additionally, many immune-related cells express PD-L1, especially activated T and B lymphocytes, dendritic cells (DCs), monocytes, mesenchymal stem cells (MSCs), and bone marrow-derived mast cells. The interaction of PD-1 with PD-L1 provides immune escape for immune-privileged organs or tumor cells by inhibiting T-cell activation and cytokine production. This leads to the attenuation of T cells responses by blocking proliferation, inducing apoptosis, and promoting the differentiation of regulatory T cells 12 . AS in cancer generates neoantigens that can evade immune detection, affecting immunotherapy outcomes. AS in PD-L1 induces resistance to PD-L1 blockade in non-small cell lung cancer 9, 10 . PD-L1 protein is a transmembrane glycoprotein with a substantial extracellular region that includes Immunoglobulin (Ig)-like domains, a hydrophobic transmembrane domain, and a 30-amino acid cytoplasmic tail that does not have canonical signaling motifs 13 . The Ig-like domains can be divided into two types: one is the Ig Variable (IgV)-like domain, and the other is the Ig Constant (IgC)-like domain. According to the Ensembl database, two alternative coding transcripts of PD-L1 are encoded by the CD274 gene 11, 14 . The longer coding transcript (PD-L1 IgV+ ), contains all exons from 1 to 7 and encodes a 290 amino acid protein (33 kDa). The second transcript (PD-L1 IgV− ), produced by alternative splicing, lacks exon 3, resulting in a shorter 160 amino acid isoform without an IgV-like domain. Given that the IgV-like domain is crucial for the interaction of PD-1 with PD-L1 15, 16 , therefore, different PD-L1 isoforms may have different effects on inhibiting T-cell function. However, the AS of PD-L1 has not been reported in the Pg -infected macrophages. In this study, we explored the landscape of isoform switches in macrophages infected with P. gingivalis using high-throughput RNA sequencing (RNA-seq). Our bioinformatics analysis revealed that PD-L1 plays a crucial role in regulating the immune response in macrophages exposed to P. gingivalis through gingipain release. We not only identified switches in AS and the transcriptome related to the immune response in Pg -infected macrophages but also validated the AS events of PD-L1 through biochemical experiments. These findings suggest a novel mechanism by which P. gingivalis may facilitate immune evasion in chronic periodontitis. Results Landscape changes of alternative splicing events in Pg -inf macrophages with/without gingipain The schedules of collecting RNA from untreated macrophages (No-inf), macrophages treated with P. gingivalis ( Pg -inf), and macrophages treated with knockout gingipain P. gingivalis ( ΔKDP -inf) for RNA sequencing are shown in Fig. S1 . The landscape of the gene comparisons, highlighting different isoform switch cases from the RNA sequencing results, is illustrated in Fig. 1 A and Fig. 1 B. In the pairwise comparisons of genes across the No-inf, Pg -inf, and ΔKDP -inf groups, we identified different AS events with variations in the types of AS detected. This funding suggests that P. gingivalis infection influences the original AS in macrophages, with gingipains playing a significant role in this process. Bioinformatics analysis indicated that the alternative splicing of PD-L1 (CD274) in macrophages was correlated with P. gingivalis infection The Venn diagram (Fig. 2 A) illustrates the screening process for genes closely associated with gingipains from previous RNA sequencing data during P. gingivalis infection. The overlapping section of Set 1 and Set 2 revealed that AS events in 32 genes were associated with gingipains, while Set 3 further identified that AS events in 26 of these 32 genes occurred specifically during the infection process. In the functional enrichment analysis of these 26 overlapped genes, “GO:0050870_positive regulation of T cell activation” was found in the top 20 categories (Fig. 2 B). PD-L1 (CD274), as the most significantly upregulated gene, was enriched in this category (Fig. 2 C). To further investigate the specific AS events of PD-L1 after P. gingivalis infection, we conducted isoform switch analysis in “No-inf vs Pg -inf” (Fig. 2 D-G). In this analysis, two types of PD-L1 isoforms, PD-L1 IgV− and PD-L1 IgV+ were identified (Fig. 2 D). P. gingivalis infection resulted in an overall increase in PD-L1 gene expression ( Fig. 2 E). However, the expression trends of PD-L1 IgV− and PD-L1 IgV+ are differed. The increase in PD-L1 IgV+ was more pronounced than that of PD-L1 IgV− , leading to a shift towards greater usage of PD-L1 IgV+ and decreased usage of PD-L1 IgV− (Fig. 2 F-G). This suggests that macrophages infected with P. gingivalis may not only upregulate PD-L1 protein expression but primarily enhance the expression of the PD-L1 IgV+ isoform. P. gingivalis infection upregulated the expression of PD-L1 in macrophages in a gingipain-dependent manner To evaluate the effects of gingipain on PD-L1 protein expression in macrophages, a Western blot was performed to assess the PD-L1 protein level in macrophages treated as described previously (Fig. 3 A). The grayscale analysis of Western blot results is shown in Fig. 3 B. In No-inf macrophages, PD-L1 expression was negligible. However, P. gingivalis infection induced a 17-fold increase in PD-L1 expression. In contrast, infection with ΔKDP resulted in only a 5-fold increase in PD-L1 expression, representing a 12-fold decrease compared to the Pg -inf macrophages. These findings suggested that P. gingivalis infection enhances PD-L1 expression in macrophage in a gingipain-dependent manner. Gingipain facilitated P. gingivalis in selectively increasing the expression of PD-L1 IgV+ during P. gingivalis infection. Because the PD-L1 antibody recognizes all isoforms of PD-L1 proteins, we performed RT-PCR and RT-qPCR to investigate the AS of PD-L1, as confirmed in Fig. 2 D-G. We designed a primer spanning Exon3 (IgV-like domain) and Exon4 (IgC-like domain) of PD-L1 IgV+ isoform ( right part in Fig. 4 A). The RT-PCR products of total RNA from No-inf macrophages using this primer are displayed in the left part of Fig. 4 A. Three distinct bands detected, corresponding to the locations of the three reported PD-L1 isoforms on Ensembl. These bands represented CD274-202, PD-L1 IgV+ isoform (805 bp); CD274-205, Non-coding isoform (632 bp); and CD274-201, PD-L1 IgV− isoform (463 bp). These results indicate multiple isoforms are present in normal macrophages. To further investigate the AS of PD-L1 in Pg -inf and ΔKDP -inf macrophages, we designed specific primers targeting the PD-L1 IgV− and PD-L1 IgV+ isoforms for RT-qPCR analysis. Both P. gingivalis or ΔKDP infections caused a 2-fold increase in PD-L1 IgV− expression compared to No-inf macrophages (Fig. 4 Β ). There was no significant difference in the relative expression level of PD-L1 IgV− isoform between Pg -inf and ΔKDP -inf macrophages. However, compared to No-inf macrophages, P. gingivalis infection led to a 14-fold increase in the relative expression level of the PD-L1 IgV+ isoform (Fig. 4 C). In contrast, ΔKDP -inf results in only a 2-fold increase in PD-L1 IgV+ expression, similar to that observed for PD-L1 IgV− in No-inf macrophages. We also compared the relative expression levels of PD-L1 IgV− and PD-L1 IgV+ isoforms within each group (No-inf, Pg -inf, and ΔKDP -inf macrophages) using their 2 −ΔCt ( Fig. S2 ). In both No-inf and ΔKDP -inf macrophages, there was no significant difference between the expression of PD-L1 IgV− and PD-L1 IgV+ isoforms. However, in the Pg -inf macrophages, the relative expression level of PD-L1 IgV+ was 19-fold higher than that of PD-L1 IgV− ( Fig. S2 ). These findings demonstrate that the selective increase in PD-L1 IgV+ observed during P. gingivalis infection in a gingipain-dependent manner. The exon-inclusion ratio, also known as percent spliced in (PSI), is a widely used statistic for quantifying AS events 17 . To examine the modulation of PD-L1 AS by gingipain, we utilized the data from Fig. S2 to calculate the PSI within No-inf, Pg-inf , and ΔKDP -inf macrophages (Fig. 4 D). Our analysis revealed that PD-L1 IgV+ accounted for 95% of the total coding sequence in Pg -inf macrophages. In No-inf macrophages, PD-L1 IgV+ usage was 79%, while in ΔKDP -inf macrophages, the usage dropped to 69% (Fig. 4 D). These findings suggest that gingipain plays a critical role in modulating the AS of PD-L1, increasing the prevalence of PD-L1 IgV+ isoform, consistent with our previous RNA sequencing results. AlphaFold 3 predictive modeling demonstrated that PD-L1 IgV+ binds more readily to PD-1, forming a stable protein complex compared to PD-L1 IgV- . Since no crystal structure of human PD-L1 IgV− -PD-1 complex reported in the RCSB PDB database ( http://www.rcsb.org ), and the available crystal structure of PD-L1 IgV+ -PD-1 complex includes only the IgV-like domain, we utilized AlphaFold 3 to predict the binding of PD-L1 IgV+ and PD-L1 IgV− with PD-1. First, we retrieved the reported human PD-L1 and PD-1 crystal structures from the PDB database and generated AlphaFold 3 models (as described in 2.9 ). We compared the predicted models to the RCSB PDB structures using PyMOL (Fig. 5 A-B) and found RMSD values of 1.344 for PD-L1 and 0.403 for PD-1 when comparing AlphaFold 3 predictions to the RCSB PDB structures. Additionally, we used AlphaFold 3 to predict a protein structure model based on the sequence corresponding to PDB entry named 3BIK (human PD-L1_mouse PD-1) from the RCSB PDB database. The comparison between the predicted structure and the original 3BIK protein structure showed high similarity (RMSD = 1.718) ( Fig. S3 ). These results indicate that the AlphaFold 3-predicted structures of PD-L1 and PD-1, as well as their docking complex, closely resemble the reported RCSB PDB structures (RMSD < 2) 18, 19 . Subsequently, we used AlphaFold 3 to predict the PD-L1 IgV+ -PD-1 and PD-L1 IgV− -PD-1 complexes (Fig. 5 C) and conducted a comparative assessment. Chain_pair_ inter-chain predicted TM-score (iptm) refers to the accuracy of predicted interactions between protein chains, with higher scores indicating more reliable predictions, commonly used in AlphaFold-Multimer analysis 20 . We found that the chain_pair_iptm of PD-L1 IgV+ -PD-1 was 5-fold that of PD-L1 IgV− -PD-1 (Fig. 5 D). This suggests that PD-L1 IgV+ binds to PD-1 more effectively than PD-L1 IgV− . Additionally, this difference in binding is supported by the hydrogen bonds formed between the amino acid residues of the two PD-L1 isoforms and PD-1 (Fig. 5 E-F). PD-L1 IgV+ forms 18 hydrogen bonds with PD-1, while PD-L1 IgV− forms only 10 hydrogen bonds with PD-1. These results further confirm that during P. gingivalis infection, the selective upregulation of PD-L1 IgV+ by gingipain facilitates more effective binding to PD-1. Discussion The present study provides evidence that gingipain is involved in AS of PD-L1, an inhibitory immune checkpoint protein, during P. gingivalis infecting macrophage. PD-L1, as a well-known transmembrane protein, has been extensively studied in the field of cancer due to its ability to suppress immune responses 21–23 . Some studies have shown that infection with P. gingivalis upregulates the expression of PD-L1 in host cells, thereby exacerbating cancer progression 24–26 . In this study, we also found the upregulation of PD-L1 in Pg -inf macrophages (Fig. 3 A-B). We further showed that P. gingivalis not only upregulates the overall expression of PD-L1 through gingipain but also regulates AS of PD-L1, selectively increasing the PD-L1 IgV+ isoform. Evidence indicates that the IgV-like domain of PD-L1 is the primary region responsible for binding to PD-1, and this interaction has been demonstrated through various biochemical experiments, including structural studies 27, 28 , binding assays 29 , and the use of inhibitors or antibodies targeting the IgV-like domain 30–32 . We also used AlphaFold 3 to predict that the deletion of the IgV-like domain would lead to a significant decrease in the interacting protein docking (Fig. 5 D). This suggests that gingipain can utilize its unique gingipain to influence the AS of PD-L1 in macrophages. By increasing the expression of the IgV-like domain, it enhances the interaction between PD-L1 and PD-1, leading to T cells suppression. This mechanism allows gingipain to promote immune evasion, supporting its survival. Macrophages undergo numerous AS changes during their differentiation and activation in vitro, influencing their roles in detection, phagocytosis, and cytokine secretion 33 . The occurrence of these AS events is mainly attributed to two factors. First, host cells produce splice variants to inhibit the invasion and replication of microbes 34, 35 . Second, microbial infections result in the production of aberrant isoforms of antiviral genes to facilitate the invasion and replication of pathogens 36 . Through comparative analysis of sequence results of No-inf, Pg -inf, and ΔKDP -inf, we had observed a lot of alternative splicing events in macrophages that occurd following infection (Fig. 1 A). These alternative splicing events were categorized into 18 types based on the different kinds of isoform switches (Fig. 1 B), which can be further classified into four major categories: 1. Isoform switching events that remain unchanged during infection, including “IDR with binding region loss or gain”; 2. Isoform switching events that change during infection but are unrelated to gingipain, including “3’UTR is shorter or longer”, “Complete ORF loss or gain”, “IDR loss or gain”, “Length loss or gain”, and “Signal peptide loss or gain”; 3. Isoform switching events that change during infection and are directly influenced by gingipain, including “Domain loss or gain”, “Exon loss or gain”, “IDR length loss or gain”, “Transcript is Noncoding of Coding”, and “TSS more upstream or downstream”; 4. Isoform switching events that occur only in the absence of gingipain during infection, or in other words, gingipain can inhibit the occurrence of these isoform switching events, including “5’UTR is shorter or longer”, “Domain length loss or gain”, “Intron retention (IR) loss or gain”, “Last exon more upstream or downstream”, “NMD sensitive or insensitive”, and “TTS more upstream or downstream”. Hereafter, we focused more on the latter two categories of alternative splicing changes related to gingipain. In the category of alternative splicing regulated by gingipain, we found that gingipain influences AS in macrophages by affecting exon inclusion or exclusion in transcripts and even causing the acquisition or loss of entire protein domains. These changes may directly impact immune cell function. For example, the integrity of the IFNα/β receptor protein structure is closely related to many human immune-related diseases 37 ; the loss of certain N-terminal domains of short FOXP1 (FOXP1 S ) can affect its interaction with downstream proteins or molecules, thereby influencing B cell maturation and function 38 ; the generation of NIRP3 isoforms lacking exon 5 leads to a loss of interaction with NIMA related Kinase 7 (NEK7) and its activity, which in turn affects the innate immune response in vertebrates 39 . Similarly, in our study, we found that the loss of the IgV-like domain of PD-L1 due to P. gingivalis infection, which affects its efficiency in binding to PD-1, also falls into this category. Additionally, gingipain can also regulate macrophages’ AS by increasing or decreasing the number of translatable transcripts, which also impact the function of immune cells. For example, after Mycobacterium tuberculosis ( Mtb ) infection in macrophages, the number of non-translatable isoforms increases, which correlates with the reduction of many cellular protein levels and affects macrophage maturation 40 . Meanwhile, we found that gingipain can inhibit certain AS events in macrophages, which are closely related to immune cell functions during the inflammation. For instance, under the stimulation of the inflammatory cytokine LPS, human Interferon regulatory factor-5 (IRF-5) transcribes five types of longer 5’UTR that are involved in subsequent normal inflammation 41 ; the Nonsense-mediated mRNA decay (NMD) mechanism plays a crucial role in degrading abnormal immunoglobulin and T Cell Receptor (TCR) transcripts in lymphocytes 42 ; the dynamic balance of splicing factors and the level of IR is closely related to B cell differentiation 43 . However, these AS changes, which are essential during inflammation, may potentially be inhibited by gingipain. The elevation of surface PD-L1 protein expression by P. gingivalis via gingipain has previously been confirmed in dendritic cells 26 . Both macrophages and dendritic cells are capable of presenting antigens to T cells, though macrophages possess stronger phagocytic abilities. Typically, monocytes, which differentiate into macrophages at infection sites, use macropinocytosis to ingest nutrients, pathogens, soluble antigens, and large extracellular molecules 44 . However, P. gingivalis can survive for extended periods after being phagocytosed by macrophages 45 . Our previous work demonstrated the presence of live P. gingivalis within macrophages 24 to 48 hours after post-infection 4 , suggesting that phagocytosed P. gingivalis can persist and influence intracellular biochemical processes, such as DNA replication, RNA transcription, and protein translation or modification. P. gingivalis is a major pathogen of periodontitis 46 , which has been linked to various systemic conditions such as diabetes mellitus 47, 48 , rheumatoid arthritis 49 , and Alzheimer’s disease 50 . The dissemination of P. gingivalis to various systemic organs is facilitated by its secretion of outer membrane vesicles (OMVs) 51, 52 , which carry various virulent factors throughout the body via blood circulation 53, 54 . Gingipains help Pg evade the immune system by degrading junctional adhesion molecule (JAM1) on gingival epithelial cells, disrupting the epithelial barrier 55 , and degrading key innate immune components such as defensins and complement proteins 54, 56, 57 . In this study, by analyzing changes in AS of certain genes in macrophages after P. gingivalis infection, we identified a subset of genes associated with gingipains. Among these genes, we found a significant increase in the functional isoform of PD-L1, which is closely related to the inhibition of T cell function. This provides a theoretical basis for gingipains affecting the host’s adaptive immunity by inhibiting T cell activation to help P. gingivalis achieve immune evasion. The mechanism by which gingipain, a unique toxic protease from P. gingivalis , participates in the nucleic acid-driven process remains unclear. Alternative splicing, a process transitioning pre-mRNA to mature mRNA, requires the participation of multiple proteins, either directly or indirectly. Central to this process are small nuclear ribonucleoproteins (snRNPs), which form essential components of the spliceosome 58 . Additionally, RNA-binding proteins (RBPs) indirectly regulate alternative splicing 59 . Although our sequencing data did not show significant changes in snRNP expression in macrophages following P. gingivalis infection, we observed a marked reduction in the expression of RNA Binding Protein with Multiple Splicing (RBPMS) as shown in our sequencing results ( https://d3dcaz4rv8jgb4.cloudfront.net/ ). In Pg -inf macrophages, RBPMS expression dropped to 11.2% of that in No-inf macrophages and 17.6% of that in ΔKDP -infected macrophages. This suggests that P. gingivalis infection may lead to a decrease in RBPMS in macrophages, and this phenomenon may be related to gingipain. Previous studies have demonstrated that RBPMS is closely linked to exon skipping during alternative splicing 60–62 . This aligns with our observation that P. gingivalis infection leads to a decrease in the exon 3 (IgV-like domain) skipping during the AS of PD-L1. However, the relationship between gingipain and RBPMS still requires further validation. Additionally, whether the decrease in RBPMS is related to the increase in the IgV-like domain of PD-L1 also needs to be further investigated. Overall, this study demonstrated that gingipains selectively increase the expression of PD-L1 IgV+ in macrophages by regulating the alternative splicing during P. gingivalis infection, providing a potential mechanism for immune evasion by P. gingivalis in the host. These findings could be important for understanding the mechanisms of P. gingivalis immune evasion and will be beneficial for establishing pharmacological therapies for the infection of P. gingivalis . Declarations Funding This study was funded by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (23K18431, 22H03511, 21K19644, HO; 22H06790, MI). Acknowledgments The authors are grateful to Professor Mariko Naito and Assistant Professor Miko Shoji (Department of Molecular Microbiology and Immunology, Nagasaki University) for providing Δ KDP strains in the study. Althor contributions Yilin Zheng: Conceptualization, Methodology, Investigation, Data curation, Visualization, Formal analysis, Resources, Writing- Original draft preparation. Ziyi Wang: Software, Conceptualization, Methodology, Data curation, Formal analysis, Visualization, Writing - Reviewing and Editing, Resources. Yao Weng: Formal analysis, Resources, Writing - Reviewing and Editing. Heriati Sitosari : Formal analysis, Visualization, Writing - Reviewing and Editing. Yuhan He: Formal analysis, Visualization, Writing - Reviewing and Editing. Xiu Zhang: Investigation, Visualization, Writing - Reviewing and Editing. Noriko Shiotsu: Resources. Yoko Fukuhara: Formal Analysis, Resources, Writing - Reviewing and Editing. Mika Ikegame: Project administration, Resources, Funding acquisition, Writing- Reviewing and Editing. Hirohiko Okamura: Methodology, Supervision, Project administration, Resources, Funding acquisition, Writing- Reviewing and Editing. Competing interests The authors declare no competing interests. Data availability The sequencing data generated during this study are available in the NCBI BioProject repository under the accession number PRJNA1163056. The associated metadata can be accessed via the following link: https://dataview.ncbi.nlm.nih.gov/object/PRJNA1163056?reviewer=clcri79f3dn6h4oicc0g5lhlsi . The data analysis results for this study are presented at the following link: https://d3dcaz4rv8jgb4.cloudfront.net/. Methods Bacterial culture Normal (strain ATCC33277) and gingipain knockout P. gingivalis (strain ΔKDP ) (Nakayama University, Japan) 63 were cultured anaerobically at 37°C in Brain Heart Infusion medium (237500, Becton Dickinson, USA) supplemented with 1 μg/mL L-Cysteine (033-20655, Wako, Japan), 10 μg/mL hemin (5180-1G, Sigma-Aldrich, Japan), 1 μg/mL 2-methyl-1,4-naphthoquinone (vitamin K3) (M9A1503, Nacalai Tesque, Japan). Cell culture The THP-1 human monocytic cell line was seeded at a concentration of 30,000 cells/mL and cultured in RPMI medium 1640 supplemented with 10% FBS at 37°C in 5% CO 2 . Bacterial infection At 24 hours post-seeding, THP-1 cells were differentiated into macrophages by treatment with 100 nM phorbol myristate acetate (PMA) for 48 hours. The differentiated THP-1 cells were then exposed to P. gingivalis (MOI = 100) or ΔKDP (MOI = 100) for 4 hours in RPMI 1640 medium supplemented with 10% FBS. After incubation, unbound P. gingivalis and ΔKDP were removed by washing with PBS. The Pg -infected ( Pg -inf) and ΔKDP -infected ( ΔKDP -inf) macrophages were then incubated for 48 hours in RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin. The harvested macrophages were subsequently used for RNA and protein extraction. Libraries Preparation and Sequencing RNA extraction from each sample was performed using the RNeasy Mini Kit (QIAGEN). The concentration of the obtained total RNA was measured using NanoDrop ONE, resulting in total yields of 11.3 µg for No-inf, 4.4 µg for Pg -inf, and 4.5 µg for ΔKDP -inf. Total RNA (50 ng) was subjected to poly(A) RNA extraction and fragmentation using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) and the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB), following the protocol outlined in the NEBNext Ultra II RNA Library Prep Kit for Illumina Instruction Manual. Fragmentation was achieved by adding NEBNext First Strand Synthesis Reaction Buffer and NEBNext Random Primers to the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB), followed by incubation at 94°C for 15 minutes. The fragmented poly(A) RNA was subjected to reverse transcription using the NEBNext First Strand Synthesis Enzyme Mix from the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB), followed by the addition of NEBNext Adaptor (NEB) to generate cDNA. The prepared cDNA was amplified by PCR to generate libraries. To identify the samples, barcode sequences No-inf (10 µg/uL), Pg -inf (15.2 µg/uL) and ΔKDP -inf (10.3 µg/uL) were added using NEBNext Multiplex Oligos for Illumina. Furthermore, analysis was conducted using the Bioanalyzer to confirm the distribution of library lengths. The analysis results indicated that libraries of the desired lengths were prepared, and sufficient concentration and purity were observed, thus proceeding with next-generation sequencing analysis. Bioinformatic Analysis of Bulk RNA-Seq Data After quality trimming with Trim Galore v0.6.10, the reads were aligned to the GRCm38.108 reference genome (Ensembl Release 108) using the STAR aligner v2.7.10b. De novo transcript assembly and annotation were performed using StringTie v2.2.1 and SQANTI3 v5.0. Transcript-level quantification was performed for all samples using Salmon v1.9.0, followed by further quality filtering with isoformSwitchAnalyzeR v1.17.05, based on Salmon’s mapping results. Differentially expressed genes (DEGs) were identified using DESeq2 v1.31.16, with DEGs defined by a False Discovery Rate (FDR) < 0.05 and |log2Fold Change| > 1. Additionally, Gene Ontology (GO) enrichment analysis was performed using clusterProfiler v4.6.0.ss. SDS-PAGE and western blot analysis The No-inf, Pg -inf, and Δ KDP -inf macrophage cells were harvested into lysis buffer, subjected to SDS-PAGE, and then transferred onto PVDF membranes (Merck, Darmstadt, Germany). Primary antibodies included a PD-L1 mouse polyclonal antibody (1:500, 405.9A11, Cell Signaling Technology, USA) and β-actin (1:1000, 3700S, Cell Signaling Technology, USA). Following incubation with primary antibodies overnight, membranes were washed with TBST for 30 minutes at room temperature and subsequently incubated with an anti-mouse IgG and HRP-linked secondary antibody (1:10000, #7076, Cell Signaling Technology) for 45 minutes at room temperature. Finally, signals were detected using Western Blot Chemiluminescence HRP Substrate (WBLUF0100; Millipore, Burlington, MA, USA). Densitometric analysis of the bands was performed using ImageJ (National Institutes of Health, Bethesda, MD, USA). Reverse transcription polymerase chain reaction (RT-PCR) RNA was extracted using Trizol reagent (Invitrogen). The PrimeScript Reverse Transcription kit (Takara) was used for reverse transcription of the total RNA at a concentration of 500 ng/μl. The cDNA served as a template for RT-PCR using GoTaq ® Green Master Mix (LOT 0000471784, Promega, USA). For amplification of PD-L1, RT-PCR was performed with an initial denaturation for 2 min at 95 °C, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min, and a final extension for 10 min at 72 °C. The primers for different lengths of PD-L1 isoforms were shown as follows: PD-L1-forward, 5’- CCT ACT GGC ATT TGC TGA ACG -3’; PD-L1-reverse, 5’- GAG TTT GTA TCT TGG ATG CCA CAT T-3’. All primers were designed by NIH Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) cDNA products were the same with 2.7 . The resulting cDNA products were diluted twofold with pure water, and 2 μl of the diluted cDNA served as a template for quantifying the relative RNA content via qRT-PCR using Luna Universal qPCR Master Mix (#M3003E, New England Biolabs Inc., MA). Relative levels of PCR products were determined using a LightCycler System (Roche Diagnostics, Mannheim, Germany), with the threshold cycle (Ct) automatically determined using default settings on the LightCycler 96 software (version 1.1; Roche Diagnostics, Mannheim, Germany). The primers for each target isoform were shown as follows: β-actin-forward, 5’-TGG CAC CCA GCA CAA TGA A-3’; β-actin-reverse, 5’-CTA AGT CAT AGT CCG CCT AGA AGC-3’; PD-L1 IgV- -forward, 5’-CAT TTG CTG AAC GCC CCA TAC-3’; PD-L1 IgV- -reverse, 5’-TGC TTG TCC AGA TGA CTT CGG-3’; PD-L1 IgV+ -forward, 5’-TAC TGT CAC GGT TCC CAA GGA-3’; PD-L1 IgV+ -reverse, 5’-TGA TTC TCA GTG TGC TGG TCA-3’. All primers were designed as before in 2.7 . Differences in gene expression levels were calculated using the 2 -ΔCt and 2 -ΔΔCt methods after normalization of target gene expression levels within each sample against the expression levels of the reference gene (β-actin). AlphaFold 3 predication We used the protein prediction software AlphaFold 3 to predict the protein complexes formed by PD-L1 IgV- and PD-L1 IgV+ with PD-1, respectively. Specifically, we used the AlphaFold 3 server (https://alphafoldserver.com/) 64 and input the sequences for human PD-L1 IgV-/IgV+ and PD-1 from the RCSB PDB database (https://www.rcsb.org) as follows: IgV like domain is from 4ZQK (AA18-132); PD-L1 IgV- is from 3BIK (AA133-229); PD-L1 IgV+ is from 3BIK (AA18-229); and PD-1 is from 3RRQ (AA31-149). The accuracy of the AlphaFold 3 model was evaluated by Root Mean Square Deviation (RMSD) values between the protein structures predicted by AlphaFold 3 and the structures downloaded from the RCSB PDB database 65 . By analyzing the chain_pair_iptm scores of the 15 PD-L1 and PD-1 complexes generated from three independent AlphaFold 3 predictions (5 models per prediction), we evaluated the confidence of the predicted protein-protein interfaces. All modifications and comparisons of the protein structures in this study were performed using PyMOL (3.0.3). Hydrogen bonds formed in protein docking were predicted using the PDBePISA tool from EMBL-EBI (https://www.ebi.ac.uk/pdbe/pisa/). Statistical analysis All statistical analyses were performed using GraphPad Prism 9 for Mac. The Western blot result and RT-qPCR result were analyzed by A one-way analysis of variance (ANOVA). Tukey's multiple comparisons test or Šídák's multiple comparisons test were performed for these results. In all the above statistics, comparisons with p -value < 0.05 were considered significant. References Socransky, S.S., et al., Microbial complexes in subgingival plaque. J Clin Periodontol, 1998. 25 (2): p. 134-44. Lamont, R.J. and H.F. Jenkinson, Life below the gum line: pathogenic mechanisms of Porphyromonas gingivalis. Microbiol Mol Biol Rev, 1998. 62 (4): p. 1244-63. Potempa, J., R. Pike, and J. 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Kumar, Alternate splicing of transcripts shape macrophage response to Mycobacterium tuberculosis infection. PLoS Pathog, 2017. 13 (3): p. e1006236. Mancl, M.E., et al., Two discrete promoters regulate the alternatively spliced human interferon regulatory factor-5 isoforms. Multiple isoforms with distinct cell type-specific expression, localization, regulation, and function. J Biol Chem, 2005. 280 (22): p. 21078-90. Lambert, J.M., et al., Mechanisms and Regulation of Nonsense-Mediated mRNA Decay and Nonsense-Associated Altered Splicing in Lymphocytes. Int J Mol Sci, 2020. 21 (4). Ullrich, S. and R. Guigó, Dynamic changes in intron retention are tightly associated with regulation of splicing factors and proliferative activity during B-cell development. Nucleic Acids Res, 2020. 48 (3): p. 1327-1340. Kay, R.R., Macropinocytosis: Biology and mechanisms. Cells Dev, 2021. 168 : p. 203713. Sundqvist, G., et al., Phagocytosis and virulence of different strains of Porphyromonas gingivalis. Scand J Dent Res, 1991. 99 (2): p. 117-29. de Pablo, P., et al., Periodontitis in systemic rheumatic diseases. Nat Rev Rheumatol, 2009. 5 (4): p. 218-24. Takamura, H., et al., Porphyromonas gingivalis attenuates the insulin-induced phosphorylation and translocation of forkhead box protein O1 in human hepatocytes. Arch Oral Biol, 2016. 69 : p. 19-24. Ishikawa, M., et al., Oral Porphyromonas gingivalis translocates to the liver and regulates hepatic glycogen synthesis through the Akt/GSK-3β signaling pathway. Biochim Biophys Acta, 2013. 1832 (12): p. 2035-43. Olsen, I., S.K. Singhrao, and J. Potempa, Citrullination as a plausible link to periodontitis, rheumatoid arthritis, atherosclerosis and Alzheimer's disease. J Oral Microbiol, 2018. 10 (1): p. 1487742. Dominy, S.S., et al., Porphyromonas gingivalis in Alzheimer's disease brains: Evidence for disease causation and treatment with small-molecule inhibitors. Sci Adv, 2019. 5 (1): p. eaau3333. Tanai, A. and H. Okamura, The role of extracellular vesicles throughout normal pregnancy and in relation to oral bacteria. J Oral Biosci, 2021. 63 (1): p. 14-22. Okamura, H., et al., Outer membrane vesicles of Porphyromonas gingivalis: Novel communication tool and strategy. Jpn Dent Sci Rev, 2021. 57 : p. 138-146. Xie, H., Biogenesis and function of Porphyromonas gingivalis outer membrane vesicles. Future Microbiol, 2015. 10 (9): p. 1517-27. Lunar Silva, I. and E. Cascales, Molecular Strategies Underlying Porphyromonas gingivalis Virulence. J Mol Biol, 2021. 433 (7): p. 166836. Takeuchi, H., et al., Porphyromonas gingivalis induces penetration of lipopolysaccharide and peptidoglycan through the gingival epithelium via degradation of junctional adhesion molecule 1. PLoS Pathog, 2019. 15 (11): p. e1008124. Carlisle, M.D., R.N. Srikantha, and K.A. Brogden, Degradation of human alpha- and beta-defensins by culture supernatants of Porphyromonas gingivalis strain 381. J Innate Immun, 2009. 1 (2): p. 118-22. Wilensky, A., et al., Porphyromonas gingivalis gingipains selectively reduce CD14 expression, leading to macrophage hyporesponsiveness to bacterial infection. J Innate Immun, 2015. 7 (2): p. 127-135. Patel, S.B. and M. Bellini, The assembly of a spliceosomal small nuclear ribonucleoprotein particle. Nucleic Acids Res, 2008. 36 (20): p. 6482-93. Saltzman, A.L., Q. Pan, and B.J. Blencowe, Regulation of alternative splicing by the core spliceosomal machinery. Genes Dev, 2011. 25 (4): p. 373-84. Nakagaki-Silva, E.E., et al., Identification of RBPMS as a mammalian smooth muscle master splicing regulator via proximity of its gene with super-enhancers. Elife, 2019. 8 . Barnhart, M.D., et al., Phosphorylation of the smooth muscle master splicing regulator RBPMS regulates its splicing activity. Nucleic Acids Res, 2022. 50 (20): p. 11895-11915. Yang, Y., et al., Cell-type specific regulator RBPMS switches alternative splicing via higher-order oligomerization and heterotypic interactions with other splicing regulators. Nucleic Acids Res, 2023. 51 (18): p. 9961-9982. Shi, Y., et al., Genetic analyses of proteolysis, hemoglobin binding, and hemagglutination of Porphyromonas gingivalis. Construction of mutants with a combination of rgpA, rgpB, kgp, and hagA. J Biol Chem, 1999. 274 (25): p. 17955-60. Abramson, J., et al., Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 2024. 630 (8016): p. 493-500. Reva, B.A., A.V. Finkelstein, and J. Skolnick, What is the probability of a chance prediction of a protein structure with an rmsd of 6 A? Fold Des, 1998. 3 (2): p. 141-7. Additional Declarations No competing interests reported. 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\\u003cem\\u003ePg\\u003c/em\\u003e-inf, \\u003cem\\u003eΔKDP\\u003c/em\\u003e-inf macrophages.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A) \\u003c/strong\\u003eLandscape volcano plots showing differential alternative splicing events in No-inf, \\u003cem\\u003ePg\\u003c/em\\u003e-inf, and \\u003cem\\u003eΔKDP\\u003c/em\\u003e-inf macrophages. \\u003cstrong\\u003e(B) \\u003c/strong\\u003eThe landscape of different types of alternative splicing change in No-inf, \\u003cem\\u003ePg\\u003c/em\\u003e-inf, and \\u003cem\\u003eΔKDP\\u003c/em\\u003e-inf macrophages. UTR, Untranslated Region. ORF, Open Reading Frame. IDR, Intrinsically Disordered Region. NMD, Nonsense-Mediated mRNA Decay. Tss, Transcription Start Site. Tts, Transcription Termination Site.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5411219/v1/7933423a36c393187a7fb3ca.png\"},{\"id\":70201015,\"identity\":\"558cfb98-1589-4b78-8c85-d4531d409592\",\"added_by\":\"auto\",\"created_at\":\"2024-11-29 12:37:31\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":222487,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePD-L1 was targeted due to isoform switch after \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infection.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A) \\u003c/strong\\u003e26 genes were targeted due to high relation with gigipain during \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infecting macrophage. (q \\u0026lt; 0.05 and dIF \\u0026gt; 0.05). \\u003cstrong\\u003e(B) \\u003c/strong\\u003eDotplot showed the top 20 related cell signaling pathways about 26 selected genes by Functional enrichment analysis. \\u003cstrong\\u003e(C) \\u003c/strong\\u003eNetplot presented the relation among the top 20 cell signaling pathways with the related genes within 26 selected genes. \\u003cstrong\\u003e(D-G) \\u003c/strong\\u003eIsoform switch analysis of PD-L1 within Set1: No-inf vs \\u003cem\\u003ePg\\u003c/em\\u003e-inf. *, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05，****, \\u003cem\\u003ep \\u003c/em\\u003e\\u0026lt; 0.0001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5411219/v1/69b8494deacc8c09a3803fe6.png\"},{\"id\":70201012,\"identity\":\"c736d3ae-6c5f-44f5-a0ab-4ab708f33c1a\",\"added_by\":\"auto\",\"created_at\":\"2024-11-29 12:37:30\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":88550,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003eP. gingivalis\\u003c/em\\u003einfection upregulated the expression of PD-L1 in macrophage in a gingipain dependent manner.\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Western blots showing the expression of PD-L1 (~35-55 kDa) and β-actin (~45 kDa) on macrophages infected with \\u003cem\\u003eP. gingivalis\\u003c/em\\u003eor \\u003cem\\u003eΔKDP\\u003c/em\\u003e. Both proteins were detected using the same sample (4 µg per lane), analyzed on two separate gels. (B) The densitometric analysis results of A with an Ordinary one-way ANOVA. *, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05; **, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.01.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5411219/v1/f33e0c2040af127d5a03eab1.png\"},{\"id\":70201013,\"identity\":\"b9247f8c-049f-48b3-9be0-bfeba3a60304\",\"added_by\":\"auto\",\"created_at\":\"2024-11-29 12:37:31\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":133840,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eGingipain mainly upregulated the Functional PD-L1 (with IgV-like domain) during \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infection.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A) \\u003c/strong\\u003eRT-PCR products with exons are depicted at the right. Lane 1, 100 bp DNA ladder: lane 2, RT-PCR product of total RNA from No-inf macrophage. \\u003cstrong\\u003e(B-C) \\u003c/strong\\u003ePD-L1\\u003csup\\u003eIgV-\\u003c/sup\\u003e and PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e RNA expression level of PD-L1 in No-inf, \\u003cem\\u003ePg\\u003c/em\\u003e-inf and \\u003cem\\u003eΔKDP-\\u003c/em\\u003einf macrophages.\\u003cstrong\\u003e (D) \\u003c/strong\\u003eThe PSI of PD-L1\\u003csup\\u003eIgV-\\u003c/sup\\u003e and PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e isoform in\\u003cstrong\\u003e \\u003c/strong\\u003eNo-inf, \\u003cem\\u003ePg\\u003c/em\\u003e-inf and \\u003cem\\u003eΔKDP-\\u003c/em\\u003einf macrophage. *, \\u003cem\\u003ep \\u003c/em\\u003e\\u0026lt; 0.05; ****, \\u003cem\\u003ep \\u003c/em\\u003e\\u0026lt; 0.001.\\u003cbr\\u003e\\n\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5411219/v1/f356372f6065b225a56e3edf.png\"},{\"id\":70201010,\"identity\":\"9f5f3fe7-3cf3-4ee0-b0d0-0d6e33fe8e7f\",\"added_by\":\"auto\",\"created_at\":\"2024-11-29 12:37:30\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":282380,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAlphafold 3 predictive model showed PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e more readily binds to PD-1 to form a stable protein complex compared to PD-L1\\u003csup\\u003eIgV-\\u003c/sup\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A) \\u003c/strong\\u003eThe align model of PD-L1 protein structure RCSB PDB database (green) and corresponding AlphaFold predicting protein structure (blue) is showed. RMSD = 1.34. The yellow-highlighted regions within\\u0026nbsp; PD-L1 protein structure predicted by AlphaFold 3 indicate the parts with less than 2Å deviation compared to the PD-L1 protein structure from the RCSB PDB database. \\u003cstrong\\u003e(B) \\u003c/strong\\u003eThe align model of PD-1 protein structure from PDB database (red) and corresponding AlphaFold predicting protein structure (brightorange) is showed. RMSD = 0.403. The yellow-highlighted regions within PD-1 protein structure predicted by AlphaFold 3 indicate the parts with less than 2Å deviation compared to the PD-1 protein structure from the PDB database.\\u003cstrong\\u003e (C) \\u003c/strong\\u003eAlphaFold 3 predicted model of PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e-PD-1 (Left) and PD-L1\\u003csup\\u003eIgV-\\u003c/sup\\u003e-PD-1. \\u003cstrong\\u003e(D) \\u003c/strong\\u003eThe chain_pair_iptm of AlphaFold 3 predicted models of PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e-PD-1 (n = 15) and PD-L1\\u003csup\\u003eIgV-\\u003c/sup\\u003e-PD-1 (n = 15). \\u003cstrong\\u003e(E) \\u003c/strong\\u003eZoomed in cartoon view of the PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e- PD-1 interface with Hydrogen bonds (green).\\u003cstrong\\u003e (F) \\u003c/strong\\u003eZoomed in cartoon view of the PD-L1\\u003csup\\u003eIgV-\\u003c/sup\\u003e- PD-1 interface with Hydrogen bonds (green).****, \\u003cem\\u003ep \\u003c/em\\u003e\\u0026lt; 0.001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5411219/v1/1ec554ff20410881567210e0.png\"},{\"id\":79604711,\"identity\":\"8bdb1c7b-b858-485f-bd52-8e8cbd61c9c9\",\"added_by\":\"auto\",\"created_at\":\"2025-03-31 16:00:21\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1867899,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5411219/v1/d2abc4ec-222c-4a70-bfc8-e876260d9b20.pdf\"},{\"id\":70201016,\"identity\":\"f8b2cb5e-26d1-49ce-b2af-99b8516c5eec\",\"added_by\":\"auto\",\"created_at\":\"2024-11-29 12:37:31\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":2637435,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementoryInfo.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5411219/v1/4ab4995a1ea2695d272f24ab.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Gingipain regulates isoform switches of PD-L1 in macrophages infected with Porphyromonas gingivalis\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003e \\u003cem\\u003ePorphyromonas gingivalis\\u003c/em\\u003e (\\u003cem\\u003eP. gingivalis\\u003c/em\\u003e or \\u003cem\\u003ePg\\u003c/em\\u003e) is considered the primary pathogen responsible for periodontal disease \\u003csup\\u003e1\\u003c/sup\\u003e. \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e promotes the development of the disease by producing multiple proteolytic enzymes that degrade periodontal tissues \\u003csup\\u003e2\\u003c/sup\\u003e. Within these proteases, gingipain is considered a major contributor to the proteolytic activity of \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e \\u003csup\\u003e3\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003ePreviously, we found that \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infection of macrophages leads to histone leakage \\u003csup\\u003e4\\u003c/sup\\u003e. Histone modifications are known to affect alternative splicing (AS) of mRNA \\u003csup\\u003e5, 6\\u003c/sup\\u003e. The capacity to generate various transcripts (gene isoforms) through AS significantly contributes to the complexity of proteome in higher vertebrates. A vast majority of human genes utilize alternative isoforms: around 95% of multi-exon genes exhibit evidence of AS, and about 60% of genes have at least one alternative transcription start site \\u003csup\\u003e7\\u003c/sup\\u003e. This splicing diversity plays important biological roles in organisms, including increasing protein functional diversity, regulating gene expression levels, and participating in processes such as cell development and tissue specialization. The changes in AS of the transcriptome in infected macrophages are crucial in regulating the immune response \\u003csup\\u003e8\\u003c/sup\\u003e. However, the role of mRNA AS in macrophages infected with \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e has not been elucidated.\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e has been shown to induce the upregulation of the immune checkpoint protein, programmed death ligand 1 (PD-L1), in periodontal tissue cells \\u003csup\\u003e9, 10\\u003c/sup\\u003e. PD-L1, encoded by the CD274 gene, is the ligand for the co-inhibitory receptor programmed cell death protein 1 (PD-1) \\u003csup\\u003e11\\u003c/sup\\u003e. Under physiological conditions, PD-L1 is expressed in various immune-privileged organs. Additionally, many immune-related cells express PD-L1, especially activated T and B lymphocytes, dendritic cells (DCs), monocytes, mesenchymal stem cells (MSCs), and bone marrow-derived mast cells. The interaction of PD-1 with PD-L1 provides immune escape for immune-privileged organs or tumor cells by inhibiting T-cell activation and cytokine production. This leads to the attenuation of T cells responses by blocking proliferation, inducing apoptosis, and promoting the differentiation of regulatory T cells \\u003csup\\u003e12\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eAS in cancer generates neoantigens that can evade immune detection, affecting immunotherapy outcomes. AS in PD-L1 induces resistance to PD-L1 blockade in non-small cell lung cancer \\u003csup\\u003e9, 10\\u003c/sup\\u003e. PD-L1 protein is a transmembrane glycoprotein with a substantial extracellular region that includes Immunoglobulin (Ig)-like domains, a hydrophobic transmembrane domain, and a 30-amino acid cytoplasmic tail that does not have canonical signaling motifs \\u003csup\\u003e13\\u003c/sup\\u003e. The Ig-like domains can be divided into two types: one is the Ig Variable (IgV)-like domain, and the other is the Ig Constant (IgC)-like domain. According to the Ensembl database, two alternative coding transcripts of PD-L1 are encoded by the CD274 gene \\u003csup\\u003e11, 14\\u003c/sup\\u003e. The longer coding transcript (PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e), contains all exons from 1 to 7 and encodes a 290 amino acid protein (33 kDa). The second transcript (PD-L1\\u003csup\\u003eIgV\\u0026minus;\\u003c/sup\\u003e), produced by alternative splicing, lacks exon 3, resulting in a shorter 160 amino acid isoform without an IgV-like domain. Given that the IgV-like domain is crucial for the interaction of PD-1 with PD-L1 \\u003csup\\u003e15, 16\\u003c/sup\\u003e, therefore, different PD-L1 isoforms may have different effects on inhibiting T-cell function. However, the AS of PD-L1 has not been reported in the \\u003cem\\u003ePg\\u003c/em\\u003e-infected macrophages.\\u003c/p\\u003e \\u003cp\\u003eIn this study, we explored the landscape of isoform switches in macrophages infected with \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e using high-throughput RNA sequencing (RNA-seq). Our bioinformatics analysis revealed that PD-L1 plays a crucial role in regulating the immune response in macrophages exposed to \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e through gingipain release. We not only identified switches in AS and the transcriptome related to the immune response in \\u003cem\\u003ePg\\u003c/em\\u003e-infected macrophages but also validated the AS events of PD-L1 through biochemical experiments. These findings suggest a novel mechanism by which \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e may facilitate immune evasion in chronic periodontitis.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e \\u003cb\\u003eLandscape changes of alternative splicing events in\\u003c/b\\u003e \\u003cb\\u003ePg\\u003c/b\\u003e\\u003cb\\u003e-inf macrophages with/without gingipain\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eThe schedules of collecting RNA from untreated macrophages (No-inf), macrophages treated with \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e (\\u003cem\\u003ePg\\u003c/em\\u003e-inf), and macrophages treated with knockout gingipain \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e (\\u003cem\\u003eΔKDP\\u003c/em\\u003e-inf) for RNA sequencing are shown in \\u003cb\\u003eFig. S1\\u003c/b\\u003e. The landscape of the gene comparisons, highlighting different isoform switch cases from the RNA sequencing results, is illustrated in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA and Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB. In the pairwise comparisons of genes across the No-inf, \\u003cem\\u003ePg\\u003c/em\\u003e-inf, and \\u003cem\\u003eΔKDP\\u003c/em\\u003e-inf groups, we identified different AS events with variations in the types of AS detected. This funding suggests that \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infection influences the original AS in macrophages, with gingipains playing a significant role in this process.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eBioinformatics analysis indicated that the alternative splicing of PD-L1 (CD274) in macrophages was correlated with\\u003c/b\\u003e \\u003cb\\u003eP. gingivalis\\u003c/b\\u003e \\u003cb\\u003einfection\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eThe Venn diagram (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA) illustrates the screening process for genes closely associated with gingipains from previous RNA sequencing data during \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infection. The overlapping section of Set 1 and Set 2 revealed that AS events in 32 genes were associated with gingipains, while Set 3 further identified that AS events in 26 of these 32 genes occurred specifically during the infection process. In the functional enrichment analysis of these 26 overlapped genes, \\u0026ldquo;GO:0050870_positive regulation of T cell activation\\u0026rdquo; was found in the top 20 categories (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). PD-L1 (CD274), as the most significantly upregulated gene, was enriched in this category (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eTo further investigate the specific AS events of PD-L1 after \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infection, we conducted isoform switch analysis in \\u0026ldquo;No-inf vs \\u003cem\\u003ePg\\u003c/em\\u003e-inf\\u0026rdquo; (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD-G). In this analysis, two types of PD-L1 isoforms, PD-L1\\u003csup\\u003eIgV\\u0026minus;\\u003c/sup\\u003e and PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e were identified (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD). \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infection resulted in an overall increase in PD-L1 gene expression \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eE). However, the expression trends of PD-L1\\u003csup\\u003eIgV\\u0026minus;\\u003c/sup\\u003e and PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e are differed. The increase in PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e was more pronounced than that of PD-L1\\u003csup\\u003eIgV\\u0026minus;\\u003c/sup\\u003e, leading to a shift towards greater usage of PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e and decreased usage of PD-L1\\u003csup\\u003eIgV\\u0026minus;\\u003c/sup\\u003e(Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eF-G). This suggests that macrophages infected with \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e may not only upregulate PD-L1 protein expression but primarily enhance the expression of the PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e isoform.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eP. gingivalis\\u003c/b\\u003e \\u003cb\\u003einfection upregulated the expression of PD-L1 in macrophages in a gingipain-dependent manner\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eTo evaluate the effects of gingipain on PD-L1 protein expression in macrophages, a Western blot was performed to assess the PD-L1 protein level in macrophages treated as described previously (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA). The grayscale analysis of Western blot results is shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB. In No-inf macrophages, PD-L1 expression was negligible. However, \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infection induced a 17-fold increase in PD-L1 expression. In contrast, infection with \\u003cem\\u003eΔKDP\\u003c/em\\u003e resulted in only a 5-fold increase in PD-L1 expression, representing a 12-fold decrease compared to the \\u003cem\\u003ePg\\u003c/em\\u003e-inf macrophages. These findings suggested that \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infection enhances PD-L1 expression in macrophage in a gingipain-dependent manner.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eGingipain facilitated\\u003c/b\\u003e \\u003cb\\u003eP. gingivalis\\u003c/b\\u003e \\u003cb\\u003ein selectively increasing the expression of PD-L1\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003eIgV+\\u003c/b\\u003e\\u003c/sup\\u003e \\u003cb\\u003eduring\\u003c/b\\u003e \\u003cb\\u003eP. gingivalis\\u003c/b\\u003e \\u003cb\\u003einfection.\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eBecause the PD-L1 antibody recognizes all isoforms of PD-L1 proteins, we performed RT-PCR and RT-qPCR to investigate the AS of PD-L1, as confirmed in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD-G. We designed a primer spanning Exon3 (IgV-like domain) and Exon4 (IgC-like domain) of PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e isoform (\\u003cb\\u003eright part in\\u003c/b\\u003e Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). The RT-PCR products of total RNA from No-inf macrophages using this primer are displayed in \\u003cb\\u003ethe left part of\\u003c/b\\u003e Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA. Three distinct bands detected, corresponding to the locations of the three reported PD-L1 isoforms on Ensembl. These bands represented CD274-202, PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e isoform (805 bp); CD274-205, Non-coding isoform (632 bp); and CD274-201, PD-L1\\u003csup\\u003eIgV\\u0026minus;\\u003c/sup\\u003e isoform (463 bp). These results indicate multiple isoforms are present in normal macrophages.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eTo further investigate the AS of PD-L1 in \\u003cem\\u003ePg\\u003c/em\\u003e-inf and \\u003cem\\u003eΔKDP\\u003c/em\\u003e-inf macrophages, we designed specific primers targeting the PD-L1\\u003csup\\u003eIgV\\u0026minus;\\u003c/sup\\u003e and PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e isoforms for RT-qPCR analysis. Both \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e or \\u003cem\\u003eΔKDP\\u003c/em\\u003e infections caused a 2-fold increase in PD-L1\\u003csup\\u003eIgV\\u0026minus;\\u003c/sup\\u003e expression compared to No-inf macrophages (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e \\u003cb\\u003eΒ\\u003c/b\\u003e). There was no significant difference in the relative expression level of PD-L1\\u003csup\\u003eIgV\\u0026minus;\\u003c/sup\\u003e isoform between \\u003cem\\u003ePg\\u003c/em\\u003e-inf and \\u003cem\\u003eΔKDP\\u003c/em\\u003e-inf macrophages. However, compared to No-inf macrophages, \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infection led to a 14-fold increase in the relative expression level of the PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e isoform (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC). In contrast, \\u003cem\\u003eΔKDP\\u003c/em\\u003e-inf results in only a 2-fold increase in PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e expression, similar to that observed for PD-L1\\u003csup\\u003eIgV\\u0026minus;\\u003c/sup\\u003e in No-inf macrophages.\\u003c/p\\u003e \\u003cp\\u003eWe also compared the relative expression levels of PD-L1\\u003csup\\u003eIgV\\u0026minus;\\u003c/sup\\u003e and PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e isoforms within each group (No-inf, \\u003cem\\u003ePg\\u003c/em\\u003e-inf, and \\u003cem\\u003eΔKDP\\u003c/em\\u003e-inf macrophages) using their 2\\u003csup\\u003e\\u0026minus;ΔCt\\u003c/sup\\u003e (\\u003cb\\u003eFig. S2\\u003c/b\\u003e). In both No-inf and \\u003cem\\u003eΔKDP\\u003c/em\\u003e-inf macrophages, there was no significant difference between the expression of PD-L1\\u003csup\\u003eIgV\\u0026minus;\\u003c/sup\\u003e and PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e isoforms. However, in the \\u003cem\\u003ePg\\u003c/em\\u003e-inf macrophages, the relative expression level of PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e was 19-fold higher than that of PD-L1\\u003csup\\u003eIgV\\u0026minus;\\u003c/sup\\u003e (\\u003cb\\u003eFig. S2\\u003c/b\\u003e). These findings demonstrate that the selective increase in PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e observed during \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infection in a gingipain-dependent manner.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe exon-inclusion ratio, also known as percent spliced in (PSI), is a widely used statistic for quantifying AS events \\u003csup\\u003e17\\u003c/sup\\u003e. To examine the modulation of PD-L1 AS by gingipain, we utilized the data from \\u003cb\\u003eFig. S2\\u003c/b\\u003e to calculate the PSI within No-inf, \\u003cem\\u003ePg-inf\\u003c/em\\u003e, and \\u003cem\\u003eΔKDP\\u003c/em\\u003e-inf macrophages (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD). Our analysis revealed that PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e accounted for 95% of the total coding sequence in \\u003cem\\u003ePg\\u003c/em\\u003e-inf macrophages. In No-inf macrophages, PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e usage was 79%, while in \\u003cem\\u003eΔKDP\\u003c/em\\u003e-inf macrophages, the usage dropped to 69% (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD). These findings suggest that gingipain plays a critical role in modulating the AS of PD-L1, increasing the prevalence of PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e isoform, consistent with our previous RNA sequencing results.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eAlphaFold 3 predictive modeling demonstrated that PD-L1\\u003c/b\\u003e \\u003csup\\u003e \\u003cb\\u003eIgV+\\u003c/b\\u003e \\u003c/sup\\u003e \\u003cb\\u003ebinds more readily to PD-1, forming a stable protein complex compared to PD-L1\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003eIgV-\\u003c/b\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eSince no crystal structure of human PD-L1\\u003csup\\u003eIgV\\u0026minus;\\u003c/sup\\u003e-PD-1 complex reported in the RCSB PDB database (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://www.rcsb.org\\u003c/span\\u003e\\u003cspan address=\\\"http://www.rcsb.org\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e), and the available crystal structure of PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e-PD-1 complex includes only the IgV-like domain, we utilized AlphaFold 3 to predict the binding of PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e and PD-L1\\u003csup\\u003eIgV\\u0026minus;\\u003c/sup\\u003e with PD-1. First, we retrieved the reported human PD-L1 and PD-1 crystal structures from the PDB database and generated AlphaFold 3 models (as described in \\u003cb\\u003e2.9\\u003c/b\\u003e). We compared the predicted models to the RCSB PDB structures using PyMOL (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA-B) and found RMSD values of 1.344 for PD-L1 and 0.403 for PD-1 when comparing AlphaFold 3 predictions to the RCSB PDB structures. Additionally, we used AlphaFold 3 to predict a protein structure model based on the sequence corresponding to PDB entry named 3BIK (human PD-L1_mouse PD-1) from the RCSB PDB database. The comparison between the predicted structure and the original 3BIK protein structure showed high similarity (RMSD\\u0026thinsp;=\\u0026thinsp;1.718) (\\u003cb\\u003eFig. S3\\u003c/b\\u003e). These results indicate that the AlphaFold 3-predicted structures of PD-L1 and PD-1, as well as their docking complex, closely resemble the reported RCSB PDB structures (RMSD\\u0026thinsp;\\u0026lt;\\u0026thinsp;2) \\u003csup\\u003e18, 19\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eSubsequently, we used AlphaFold 3 to predict the PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e-PD-1 and PD-L1\\u003csup\\u003eIgV\\u0026minus;\\u003c/sup\\u003e-PD-1 complexes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC) and conducted a comparative assessment. Chain_pair_ inter-chain predicted TM-score (iptm) refers to the accuracy of predicted interactions between protein chains, with higher scores indicating more reliable predictions, commonly used in AlphaFold-Multimer analysis \\u003csup\\u003e20\\u003c/sup\\u003e. We found that the chain_pair_iptm of PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e-PD-1 was 5-fold that of PD-L1\\u003csup\\u003eIgV\\u0026minus;\\u003c/sup\\u003e-PD-1 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD). This suggests that PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e binds to PD-1 more effectively than PD-L1\\u003csup\\u003eIgV\\u0026minus;\\u003c/sup\\u003e. Additionally, this difference in binding is supported by the hydrogen bonds formed between the amino acid residues of the two PD-L1 isoforms and PD-1 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE-F). PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e forms 18 hydrogen bonds with PD-1, while PD-L1\\u003csup\\u003eIgV\\u0026minus;\\u003c/sup\\u003e forms only 10 hydrogen bonds with PD-1. These results further confirm that during \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infection, the selective upregulation of PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e by gingipain facilitates more effective binding to PD-1.\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eThe present study provides evidence that gingipain is involved in AS of PD-L1, an inhibitory immune checkpoint protein, during \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infecting macrophage. PD-L1, as a well-known transmembrane protein, has been extensively studied in the field of cancer due to its ability to suppress immune responses \\u003csup\\u003e21\\u0026ndash;23\\u003c/sup\\u003e. Some studies have shown that infection with \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e upregulates the expression of PD-L1 in host cells, thereby exacerbating cancer progression \\u003csup\\u003e24\\u0026ndash;26\\u003c/sup\\u003e. In this study, we also found the upregulation of PD-L1 in \\u003cem\\u003ePg\\u003c/em\\u003e-inf macrophages (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA-B). We further showed that \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e not only upregulates the overall expression of PD-L1 through gingipain but also regulates AS of PD-L1, selectively increasing the PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e isoform. Evidence indicates that the IgV-like domain of PD-L1 is the primary region responsible for binding to PD-1, and this interaction has been demonstrated through various biochemical experiments, including structural studies \\u003csup\\u003e27, 28\\u003c/sup\\u003e, binding assays \\u003csup\\u003e29\\u003c/sup\\u003e, and the use of inhibitors or antibodies targeting the IgV-like domain \\u003csup\\u003e30\\u0026ndash;32\\u003c/sup\\u003e. We also used AlphaFold 3 to predict that the deletion of the IgV-like domain would lead to a significant decrease in the interacting protein docking (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD). This suggests that gingipain can utilize its unique gingipain to influence the AS of PD-L1 in macrophages. By increasing the expression of the IgV-like domain, it enhances the interaction between PD-L1 and PD-1, leading to T cells suppression. This mechanism allows gingipain to promote immune evasion, supporting its survival.\\u003c/p\\u003e \\u003cp\\u003eMacrophages undergo numerous AS changes during their differentiation and activation in vitro, influencing their roles in detection, phagocytosis, and cytokine secretion \\u003csup\\u003e33\\u003c/sup\\u003e. The occurrence of these AS events is mainly attributed to two factors. First, host cells produce splice variants to inhibit the invasion and replication of microbes \\u003csup\\u003e34, 35\\u003c/sup\\u003e. Second, microbial infections result in the production of aberrant isoforms of antiviral genes to facilitate the invasion and replication of pathogens \\u003csup\\u003e36\\u003c/sup\\u003e. Through comparative analysis of sequence results of No-inf, \\u003cem\\u003ePg\\u003c/em\\u003e-inf, and \\u003cem\\u003eΔKDP\\u003c/em\\u003e-inf, we had observed a lot of alternative splicing events in macrophages that occurd following infection (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). These alternative splicing events were categorized into 18 types based on the different kinds of isoform switches (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB), which can be further classified into four major categories: 1. Isoform switching events that remain unchanged during infection, including \\u0026ldquo;IDR with binding region loss or gain\\u0026rdquo;; 2. Isoform switching events that change during infection but are unrelated to gingipain, including \\u0026ldquo;3\\u0026rsquo;UTR is shorter or longer\\u0026rdquo;, \\u0026ldquo;Complete ORF loss or gain\\u0026rdquo;, \\u0026ldquo;IDR loss or gain\\u0026rdquo;, \\u0026ldquo;Length loss or gain\\u0026rdquo;, and \\u0026ldquo;Signal peptide loss or gain\\u0026rdquo;; 3. Isoform switching events that change during infection and are directly influenced by gingipain, including \\u0026ldquo;Domain loss or gain\\u0026rdquo;, \\u0026ldquo;Exon loss or gain\\u0026rdquo;, \\u0026ldquo;IDR length loss or gain\\u0026rdquo;, \\u0026ldquo;Transcript is Noncoding of Coding\\u0026rdquo;, and \\u0026ldquo;TSS more upstream or downstream\\u0026rdquo;; 4. Isoform switching events that occur only in the absence of gingipain during infection, or in other words, gingipain can inhibit the occurrence of these isoform switching events, including \\u0026ldquo;5\\u0026rsquo;UTR is shorter or longer\\u0026rdquo;, \\u0026ldquo;Domain length loss or gain\\u0026rdquo;, \\u0026ldquo;Intron retention (IR) loss or gain\\u0026rdquo;, \\u0026ldquo;Last exon more upstream or downstream\\u0026rdquo;, \\u0026ldquo;NMD sensitive or insensitive\\u0026rdquo;, and \\u0026ldquo;TTS more upstream or downstream\\u0026rdquo;.\\u003c/p\\u003e \\u003cp\\u003eHereafter, we focused more on the latter two categories of alternative splicing changes related to gingipain. In the category of alternative splicing regulated by gingipain, we found that gingipain influences AS in macrophages by affecting exon inclusion or exclusion in transcripts and even causing the acquisition or loss of entire protein domains. These changes may directly impact immune cell function. For example, the integrity of the IFNα/β receptor protein structure is closely related to many human immune-related diseases \\u003csup\\u003e37\\u003c/sup\\u003e; the loss of certain N-terminal domains of short FOXP1 (FOXP1\\u003csub\\u003eS\\u003c/sub\\u003e) can affect its interaction with downstream proteins or molecules, thereby influencing B cell maturation and function \\u003csup\\u003e38\\u003c/sup\\u003e; the generation of NIRP3 isoforms lacking exon 5 leads to a loss of interaction with NIMA related Kinase 7 (NEK7) and its activity, which in turn affects the innate immune response in vertebrates \\u003csup\\u003e39\\u003c/sup\\u003e. Similarly, in our study, we found that the loss of the IgV-like domain of PD-L1 due to \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infection, which affects its efficiency in binding to PD-1, also falls into this category.\\u003c/p\\u003e \\u003cp\\u003eAdditionally, gingipain can also regulate macrophages\\u0026rsquo; AS by increasing or decreasing the number of translatable transcripts, which also impact the function of immune cells. For example, after \\u003cem\\u003eMycobacterium tuberculosis\\u003c/em\\u003e (\\u003cem\\u003eMtb\\u003c/em\\u003e) infection in macrophages, the number of non-translatable isoforms increases, which correlates with the reduction of many cellular protein levels and affects macrophage maturation \\u003csup\\u003e40\\u003c/sup\\u003e. Meanwhile, we found that gingipain can inhibit certain AS events in macrophages, which are closely related to immune cell functions during the inflammation. For instance, under the stimulation of the inflammatory cytokine LPS, human Interferon regulatory factor-5 (IRF-5) transcribes five types of longer 5\\u0026rsquo;UTR that are involved in subsequent normal inflammation \\u003csup\\u003e41\\u003c/sup\\u003e; the Nonsense-mediated mRNA decay (NMD) mechanism plays a crucial role in degrading abnormal immunoglobulin and T Cell Receptor (TCR) transcripts in lymphocytes \\u003csup\\u003e42\\u003c/sup\\u003e; the dynamic balance of splicing factors and the level of IR is closely related to B cell differentiation \\u003csup\\u003e43\\u003c/sup\\u003e. However, these AS changes, which are essential during inflammation, may potentially be inhibited by gingipain.\\u003c/p\\u003e \\u003cp\\u003eThe elevation of surface PD-L1 protein expression by \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e via gingipain has previously been confirmed in dendritic cells \\u003csup\\u003e26\\u003c/sup\\u003e. Both macrophages and dendritic cells are capable of presenting antigens to T cells, though macrophages possess stronger phagocytic abilities. Typically, monocytes, which differentiate into macrophages at infection sites, use macropinocytosis to ingest nutrients, pathogens, soluble antigens, and large extracellular molecules \\u003csup\\u003e44\\u003c/sup\\u003e. However, \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e can survive for extended periods after being phagocytosed by macrophages \\u003csup\\u003e45\\u003c/sup\\u003e. Our previous work demonstrated the presence of live \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e within macrophages 24 to 48 hours after post-infection \\u003csup\\u003e4\\u003c/sup\\u003e, suggesting that phagocytosed \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e can persist and influence intracellular biochemical processes, such as DNA replication, RNA transcription, and protein translation or modification.\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e is a major pathogen of periodontitis \\u003csup\\u003e46\\u003c/sup\\u003e, which has been linked to various systemic conditions such as diabetes mellitus \\u003csup\\u003e47, 48\\u003c/sup\\u003e, rheumatoid arthritis \\u003csup\\u003e49\\u003c/sup\\u003e, and Alzheimer\\u0026rsquo;s disease \\u003csup\\u003e50\\u003c/sup\\u003e. The dissemination of \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e to various systemic organs is facilitated by its secretion of outer membrane vesicles (OMVs) \\u003csup\\u003e51, 52\\u003c/sup\\u003e, which carry various virulent factors throughout the body \\u003cem\\u003evia\\u003c/em\\u003e blood circulation \\u003csup\\u003e53, 54\\u003c/sup\\u003e. Gingipains help \\u003cem\\u003ePg\\u003c/em\\u003e evade the immune system by degrading junctional adhesion molecule (JAM1) on gingival epithelial cells, disrupting the epithelial barrier \\u003csup\\u003e55\\u003c/sup\\u003e, and degrading key innate immune components such as defensins and complement proteins \\u003csup\\u003e54, 56, 57\\u003c/sup\\u003e. In this study, by analyzing changes in AS of certain genes in macrophages after \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infection, we identified a subset of genes associated with gingipains. Among these genes, we found a significant increase in the functional isoform of PD-L1, which is closely related to the inhibition of T cell function. This provides a theoretical basis for gingipains affecting the host\\u0026rsquo;s adaptive immunity by inhibiting T cell activation to help \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e achieve immune evasion.\\u003c/p\\u003e \\u003cp\\u003eThe mechanism by which gingipain, a unique toxic protease from \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e, participates in the nucleic acid-driven process remains unclear. Alternative splicing, a process transitioning pre-mRNA to mature mRNA, requires the participation of multiple proteins, either directly or indirectly. Central to this process are small nuclear ribonucleoproteins (snRNPs), which form essential components of the spliceosome \\u003csup\\u003e58\\u003c/sup\\u003e. Additionally, RNA-binding proteins (RBPs) indirectly regulate alternative splicing \\u003csup\\u003e59\\u003c/sup\\u003e. Although our sequencing data did not show significant changes in snRNP expression in macrophages following \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infection, we observed a marked reduction in the expression of RNA Binding Protein with Multiple Splicing (RBPMS) as shown in our sequencing results (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://d3dcaz4rv8jgb4.cloudfront.net/\\u003c/span\\u003e\\u003cspan address=\\\"https://d3dcaz4rv8jgb4.cloudfront.net/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e). In \\u003cem\\u003ePg\\u003c/em\\u003e-inf macrophages, RBPMS expression dropped to 11.2% of that in No-inf macrophages and 17.6% of that in \\u003cem\\u003eΔKDP\\u003c/em\\u003e-infected macrophages. This suggests that \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infection may lead to a decrease in RBPMS in macrophages, and this phenomenon may be related to gingipain. Previous studies have demonstrated that RBPMS is closely linked to exon skipping during alternative splicing \\u003csup\\u003e60\\u0026ndash;62\\u003c/sup\\u003e. This aligns with our observation that \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infection leads to a decrease in the exon 3 (IgV-like domain) skipping during the AS of PD-L1. However, the relationship between gingipain and RBPMS still requires further validation. Additionally, whether the decrease in RBPMS is related to the increase in the IgV-like domain of PD-L1 also needs to be further investigated.\\u003c/p\\u003e \\u003cp\\u003eOverall, this study demonstrated that gingipains selectively increase the expression of PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e in macrophages by regulating the alternative splicing during \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e infection, providing a potential mechanism for immune evasion by \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e in the host. These findings could be important for understanding the mechanisms of \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e immune evasion and will be beneficial for establishing pharmacological therapies for the infection of \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003eFunding\\u003c/p\\u003e\\n\\u003cp\\u003eThis study was funded by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (23K18431, 22H03511, 21K19644, HO; 22H06790, MI).\\u003c/p\\u003e\\n\\u003cp\\u003eAcknowledgments\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors are grateful to Professor Mariko Naito and Assistant Professor Miko Shoji (Department of Molecular Microbiology and Immunology, Nagasaki University) for providing \\u003cem\\u003e\\u0026Delta;\\u003c/em\\u003e\\u003cem\\u003eKDP\\u003c/em\\u003e strains in the study.\\u003c/p\\u003e\\n\\u003cp\\u003eAlthor contributions\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eYilin Zheng:\\u003c/strong\\u003e Conceptualization, Methodology, Investigation, Data curation, Visualization, Formal analysis, Resources, Writing- Original draft preparation. \\u003cstrong\\u003eZiyi Wang:\\u003c/strong\\u003e Software, Conceptualization, Methodology, Data curation, Formal analysis, Visualization, Writing - Reviewing and Editing, Resources. \\u003cstrong\\u003eYao Weng:\\u003c/strong\\u003e Formal analysis, Resources, Writing - Reviewing and Editing. \\u003cstrong\\u003eHeriati Sitosari\\u003c/strong\\u003e\\u003cstrong\\u003e:\\u003c/strong\\u003e Formal analysis, Visualization, Writing - Reviewing and Editing. \\u003cstrong\\u003eYuhan He: \\u003c/strong\\u003eFormal analysis, Visualization, Writing - Reviewing and Editing. \\u003cstrong\\u003eXiu Zhang: \\u003c/strong\\u003eInvestigation, Visualization, Writing - Reviewing and Editing. \\u003cstrong\\u003eNoriko Shiotsu: \\u003c/strong\\u003eResources. \\u003cstrong\\u003eYoko Fukuhara:\\u003c/strong\\u003e Formal Analysis, Resources, Writing - Reviewing and Editing. \\u003cstrong\\u003eMika Ikegame: \\u003c/strong\\u003eProject administration, Resources, Funding acquisition, Writing- Reviewing and Editing. \\u003cstrong\\u003eHirohiko Okamura:\\u003c/strong\\u003e Methodology, Supervision, Project administration, Resources, Funding acquisition, Writing- Reviewing and Editing.\\u003c/p\\u003e\\n\\u003cp\\u003eCompeting interests\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare no competing interests.\\u003c/p\\u003e\\n\\u003cp\\u003eData availability\\u003c/p\\u003e\\n\\u003cp\\u003eThe sequencing data generated during this study are available in the NCBI BioProject\\u003c/p\\u003e\\n\\u003cp\\u003erepository under the accession number PRJNA1163056. The associated metadata can be\\u003c/p\\u003e\\n\\u003cp\\u003eaccessed via the following link: https://dataview.ncbi.nlm.nih.gov/object/PRJNA1163056?reviewer=clcri79f3dn6h4oicc0g5lhlsi . The data analysis results for this study are presented at\\u003c/p\\u003e\\n\\u003cp\\u003ethe following link: https://d3dcaz4rv8jgb4.cloudfront.net/.\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cp\\u003eBacterial culture\\u003c/p\\u003e\\n\\u003cp\\u003eNormal (strain ATCC33277) and gingipain knockout \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e (strain \\u003cem\\u003e\\u0026Delta;KDP\\u003c/em\\u003e) (Nakayama University, Japan) \\u003csup\\u003e63\\u003c/sup\\u003e were cultured anaerobically at 37\\u0026deg;C in Brain Heart Infusion medium (237500, Becton Dickinson, USA) supplemented with 1 \\u0026mu;g/mL L-Cysteine (033-20655, Wako, Japan), 10 \\u0026mu;g/mL hemin (5180-1G, Sigma-Aldrich, Japan), 1 \\u0026mu;g/mL 2-methyl-1,4-naphthoquinone (vitamin K3) (M9A1503, Nacalai Tesque, Japan).\\u003c/p\\u003e\\n\\u003cp\\u003eCell culture\\u003c/p\\u003e\\n\\u003cp\\u003eThe THP-1 human monocytic cell line was seeded at a concentration of 30,000 cells/mL and cultured in RPMI medium 1640 supplemented with 10% FBS at 37\\u0026deg;C in 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eBacterial infection\\u003c/p\\u003e\\n\\u003cp\\u003eAt 24 hours post-seeding, THP-1 cells were differentiated into macrophages by treatment with 100 nM phorbol myristate acetate (PMA) for 48 hours. The differentiated THP-1 cells were then exposed to \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e (MOI = 100) or \\u003cem\\u003e\\u0026Delta;KDP\\u003c/em\\u003e (MOI = 100) for 4 hours in RPMI 1640 medium supplemented with 10% FBS. After incubation, unbound \\u003cem\\u003eP. gingivalis\\u003c/em\\u003e and \\u003cem\\u003e\\u0026Delta;KDP\\u003c/em\\u003e were removed by washing with PBS. The \\u003cem\\u003ePg\\u003c/em\\u003e-infected (\\u003cem\\u003ePg\\u003c/em\\u003e-inf) and \\u003cem\\u003e\\u0026Delta;KDP\\u003c/em\\u003e-infected (\\u003cem\\u003e\\u0026Delta;KDP\\u003c/em\\u003e-inf) macrophages were then incubated for 48 hours in RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin. The harvested macrophages were subsequently used for RNA and protein extraction.\\u003c/p\\u003e\\n\\u003cp\\u003eLibraries Preparation and Sequencing\\u003c/p\\u003e\\n\\u003cp\\u003eRNA extraction from each sample was performed using the RNeasy Mini Kit (QIAGEN).\\u0026nbsp;The concentration of the obtained total RNA was measured using NanoDrop ONE, resulting in total yields of 11.3 \\u0026micro;g for No-inf, 4.4 \\u0026micro;g for \\u003cem\\u003ePg\\u003c/em\\u003e-inf, and 4.5 \\u0026micro;g for\\u0026nbsp;\\u003cem\\u003e\\u0026Delta;KDP\\u003c/em\\u003e-inf. Total RNA (50 ng) was subjected to poly(A) RNA extraction and fragmentation using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) and the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB), following the protocol outlined in the NEBNext Ultra II RNA Library Prep Kit for Illumina Instruction Manual. Fragmentation was achieved by adding NEBNext First Strand Synthesis Reaction Buffer and NEBNext Random Primers to the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB), followed by incubation at 94\\u0026deg;C for 15 minutes. The fragmented poly(A) RNA was subjected to reverse transcription using the NEBNext First Strand Synthesis Enzyme Mix from the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB), followed by the addition of NEBNext Adaptor (NEB) to generate cDNA. The prepared cDNA was amplified by PCR to generate libraries. To identify the samples, barcode sequences No-inf (10 \\u0026micro;g/uL), \\u003cem\\u003ePg\\u003c/em\\u003e-inf (15.2 \\u0026micro;g/uL) and\\u0026nbsp;\\u003cem\\u003e\\u0026Delta;KDP\\u003c/em\\u003e-inf (10.3 \\u0026micro;g/uL) were added using NEBNext Multiplex Oligos for Illumina. Furthermore, analysis was conducted using the Bioanalyzer to confirm the distribution of library lengths. The analysis results indicated that libraries of the desired lengths were prepared, and sufficient concentration and purity were observed, thus proceeding with next-generation sequencing analysis.\\u003c/p\\u003e\\n\\u003cp\\u003eBioinformatic Analysis of Bulk RNA-Seq Data\\u003c/p\\u003e\\n\\u003cp\\u003eAfter quality trimming with Trim Galore v0.6.10, the reads were aligned to the GRCm38.108 reference genome (Ensembl Release 108) using the STAR aligner v2.7.10b. De novo transcript assembly and annotation were performed using StringTie v2.2.1 and SQANTI3 v5.0. Transcript-level quantification was performed for all samples using Salmon v1.9.0, followed by further quality filtering with isoformSwitchAnalyzeR v1.17.05, based on Salmon\\u0026rsquo;s mapping results. Differentially expressed genes (DEGs) were identified using DESeq2 v1.31.16, with DEGs defined by a False Discovery Rate (FDR) \\u0026lt; 0.05 and |log2Fold Change| \\u0026gt; 1. Additionally, Gene Ontology (GO) enrichment analysis was performed using clusterProfiler v4.6.0.ss.\\u003c/p\\u003e\\n\\u003cp\\u003eSDS-PAGE and western blot analysis\\u003c/p\\u003e\\n\\u003cp\\u003eThe No-inf, \\u003cem\\u003ePg\\u003c/em\\u003e-inf, and\\u0026nbsp;\\u003cem\\u003e\\u0026Delta;\\u003c/em\\u003e\\u003cem\\u003eKDP\\u003c/em\\u003e-inf macrophage cells were harvested into lysis buffer, subjected to SDS-PAGE, and then transferred onto PVDF membranes (Merck, Darmstadt, Germany). Primary antibodies included a PD-L1 mouse polyclonal antibody (1:500, 405.9A11, Cell Signaling Technology, USA) and \\u0026beta;-actin (1:1000, 3700S, Cell Signaling Technology, USA). Following incubation with primary antibodies overnight, membranes were washed with TBST for 30 minutes at room temperature and subsequently incubated with an anti-mouse IgG and HRP-linked secondary antibody (1:10000, #7076, Cell Signaling Technology) for 45 minutes at room temperature. Finally, signals were detected using Western Blot Chemiluminescence HRP Substrate (WBLUF0100; Millipore, Burlington, MA, USA). Densitometric analysis of the bands was performed using ImageJ (National Institutes of Health, Bethesda, MD, USA).\\u003c/p\\u003e\\n\\u003cp\\u003eReverse transcription polymerase chain reaction (RT-PCR)\\u003c/p\\u003e\\n\\u003cp\\u003eRNA was extracted using Trizol reagent (Invitrogen). The PrimeScript Reverse\\u0026nbsp;Transcription kit (Takara) was used for reverse transcription of the total RNA at a\\u0026nbsp;concentration of 500 ng/\\u0026mu;l.\\u0026nbsp;The\\u0026nbsp;cDNA served as a template for RT-PCR using\\u0026nbsp;GoTaq\\u003csup\\u003e\\u0026reg;\\u003c/sup\\u003e Green Master Mix (LOT 0000471784, Promega, USA). For amplification of PD-L1, RT-PCR was performed with an initial denaturation for 2 min at 95 \\u0026deg;C, followed by 40 cycles of 95 \\u0026deg;C for 30 s, 60 \\u0026deg;C for 30 s, 72 \\u0026deg;C for 1 min, and a final extension for 10 min at 72 \\u0026deg;C. The primers for different lengths of PD-L1 isoforms were shown as follows: PD-L1-forward, 5\\u0026rsquo;- CCT ACT GGC ATT TGC TGA ACG -3\\u0026rsquo;; PD-L1-reverse, 5\\u0026rsquo;- GAG TTT GTA TCT TGG ATG CCA CAT T-3\\u0026rsquo;. All primers were designed by NIH Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/).\\u003c/p\\u003e\\n\\u003cp\\u003eReverse transcription-quantitative polymerase chain reaction (RT-qPCR)\\u003c/p\\u003e\\n\\u003cp\\u003ecDNA products were the same with \\u003cstrong\\u003e2.7\\u003c/strong\\u003e.\\u0026nbsp;The resulting cDNA products were diluted twofold with pure water, and 2 \\u0026mu;l of the diluted cDNA served as a template for quantifying the relative RNA content via qRT-PCR using Luna Universal qPCR Master Mix (#M3003E, New England Biolabs Inc., MA). Relative levels of PCR products were determined using a LightCycler System (Roche Diagnostics, Mannheim, Germany), with the threshold cycle (Ct) automatically determined using default settings on the LightCycler 96 software (version 1.1; Roche Diagnostics, Mannheim, Germany). The primers for each target isoform were shown as follows: \\u0026beta;-actin-forward, 5\\u0026rsquo;-TGG CAC CCA GCA CAA TGA A-3\\u0026rsquo;; \\u0026beta;-actin-reverse, 5\\u0026rsquo;-CTA AGT CAT AGT CCG CCT AGA AGC-3\\u0026rsquo;; PD-L1\\u003csup\\u003eIgV-\\u003c/sup\\u003e-forward, 5\\u0026rsquo;-CAT TTG CTG AAC GCC CCA TAC-3\\u0026rsquo;; PD-L1\\u003csup\\u003eIgV-\\u003c/sup\\u003e-reverse, 5\\u0026rsquo;-TGC TTG TCC AGA TGA CTT CGG-3\\u0026rsquo;; PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e-forward, 5\\u0026rsquo;-TAC TGT CAC GGT TCC CAA GGA-3\\u0026rsquo;; PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e-reverse, 5\\u0026rsquo;-TGA TTC TCA GTG TGC TGG TCA-3\\u0026rsquo;.\\u0026nbsp;All primers were designed as before in\\u0026nbsp;\\u003cstrong\\u003e2.7\\u003c/strong\\u003e.\\u0026nbsp;Differences in gene expression levels were calculated using the 2\\u003csup\\u003e-\\u0026Delta;Ct\\u003c/sup\\u003e and 2\\u003csup\\u003e-\\u0026Delta;\\u0026Delta;Ct\\u003c/sup\\u003e methods after normalization of target gene expression levels within each sample against the expression levels of the reference gene (\\u0026beta;-actin).\\u003c/p\\u003e\\n\\u003cp\\u003eAlphaFold 3 predication\\u003c/p\\u003e\\n\\u003cp\\u003eWe used the protein prediction software AlphaFold 3 to predict the protein complexes formed by PD-L1\\u003csup\\u003eIgV-\\u003c/sup\\u003e and PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e with PD-1, respectively. Specifically, we used the AlphaFold 3 server (https://alphafoldserver.com/) \\u003csup\\u003e64\\u003c/sup\\u003e and input the sequences for human PD-L1\\u003csup\\u003eIgV-/IgV+\\u003c/sup\\u003e and PD-1 from the RCSB PDB database (https://www.rcsb.org) as follows: IgV like domain is from 4ZQK (AA18-132); PD-L1\\u003csup\\u003eIgV-\\u003c/sup\\u003e is from 3BIK (AA133-229); PD-L1\\u003csup\\u003eIgV+\\u003c/sup\\u003e is from 3BIK (AA18-229); and PD-1 is from 3RRQ (AA31-149).\\u0026nbsp;The accuracy of the AlphaFold 3 model was evaluated by Root Mean Square Deviation (RMSD) values between the protein structures predicted by AlphaFold 3 and\\u0026nbsp;the structures downloaded from the RCSB PDB\\u0026nbsp;database \\u003csup\\u003e65\\u003c/sup\\u003e. By analyzing the chain_pair_iptm scores of the 15 PD-L1 and PD-1 complexes generated from three independent AlphaFold 3 predictions (5 models per prediction), we evaluated the confidence of the predicted protein-protein interfaces. All modifications and comparisons of the protein structures in this study were performed using PyMOL (3.0.3).\\u0026nbsp;Hydrogen bonds formed in protein docking were predicted using the PDBePISA tool from EMBL-EBI (https://www.ebi.ac.uk/pdbe/pisa/).\\u003c/p\\u003e\\n\\u003cp\\u003eStatistical analysis\\u003c/p\\u003e\\n\\u003cp\\u003eAll statistical analyses were performed using GraphPad Prism 9 for Mac. The Western blot result and RT-qPCR result were analyzed by A one-way analysis of variance (ANOVA). Tukey\\u0026apos;s multiple comparisons test or \\u0026Scaron;\\u0026iacute;d\\u0026aacute;k\\u0026apos;s multiple comparisons test were performed for these results. 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Construction of mutants with a combination of rgpA, rgpB, kgp, and hagA.\\u003c/em\\u003e J Biol Chem, 1999. \\u003cstrong\\u003e274\\u003c/strong\\u003e(25): p. 17955-60.\\u003c/li\\u003e\\n\\u003cli\\u003eAbramson, J., et al., \\u003cem\\u003eAccurate structure prediction of biomolecular interactions with AlphaFold 3.\\u003c/em\\u003e Nature, 2024. \\u003cstrong\\u003e630\\u003c/strong\\u003e(8016): p. 493-500.\\u003c/li\\u003e\\n\\u003cli\\u003eReva, B.A., A.V. Finkelstein, and J. Skolnick, \\u003cem\\u003eWhat is the probability of a chance prediction of a protein structure with an rmsd of 6 A?\\u003c/em\\u003e Fold Des, 1998. \\u003cstrong\\u003e3\\u003c/strong\\u003e(2): p. 141-7.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Porphyromonas gingivalis, gingipain, macrophage, alternative splicing, PD-L1, immune evasion\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-5411219/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-5411219/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003ePeriodontal pathogen \\u003cem\\u003ePorphyromonas gingivalis\\u003c/em\\u003e(\\u003cem\\u003ePg\\u003c/em\\u003e) is believed to possess immune evasion capabilities, but it remains unclear whether this immune evasion is related to host gene alternative splicing (AS). In this study, RNA-sequencing (RNA-seq) revealed significant changes in both AS landscape and transcriptomic profile of macrophages following \\u003cem\\u003ePg\\u003c/em\\u003e infection with/without knockout of gingipain (a unique toxic protease of \\u003cem\\u003ePg)\\u003c/em\\u003e. \\u003cem\\u003ePg\\u003c/em\\u003e infection increased the programmed death ligand 1 (PD-L1) transcripts expression and selectively upregulated a specific coding isoform that more effectively binds to programmed cell death protein 1 (PD-1) receptors on T cells, thereby inhibiting immune function. Biological experiments confirmed these results and demonstrated that the AS switch of PD-L1 was gingipain-dependent. AlphaFold 3 predictions indicated that the protein docking compatibility between PD-1 and \\u003cem\\u003ePg\\u003c/em\\u003e-upregulated PD-L1 isoform was over 80% higher than another coding isoform. These findings suggest that \\u003cem\\u003ePg \\u003c/em\\u003eemploys gingipain to modulate the AS of PD-L1, facilitating immune evasion.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Gingipain regulates isoform switches of PD-L1 in macrophages infected with Porphyromonas gingivalis\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-11-29 12:37:26\",\"doi\":\"10.21203/rs.3.rs-5411219/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2024-12-17T09:32:59+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-12-05T22:00:19+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-11-25T10:26:50+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"161784581969276110340531815881877528945\",\"date\":\"2024-11-22T00:01:37+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"84466269590597305337979231377876149205\",\"date\":\"2024-11-18T14:10:47+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-11-18T09:25:20+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-11-18T07:31:00+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2024-11-12T13:30:13+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2024-11-11T11:03:34+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Scientific Reports\",\"date\":\"2024-11-07T15:50:02+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"97a0a341-c947-47ef-8cd8-2b5a915ad6e5\",\"owner\":[],\"postedDate\":\"November 29th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[{\"id\":40708909,\"name\":\"Biological sciences/Immunology\"},{\"id\":40708910,\"name\":\"Biological sciences/Microbiology\"}],\"tags\":[],\"updatedAt\":\"2025-03-31T15:58:13+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-5411219\",\"link\":\"https://doi.org/10.1038/s41598-025-94954-7\",\"journal\":{\"identity\":\"scientific-reports\",\"isVorOnly\":false,\"title\":\"Scientific Reports\"},\"publishedOn\":\"2025-03-26 15:56:53\",\"publishedOnDateReadable\":\"March 26th, 2025\"},\"versionCreatedAt\":\"2024-11-29 12:37:26\",\"video\":\"\",\"vorDoi\":\"10.1038/s41598-025-94954-7\",\"vorDoiUrl\":\"https://doi.org/10.1038/s41598-025-94954-7\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-5411219\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-5411219\",\"identity\":\"rs-5411219\",\"version\":[\"v1\"]},\"buildId\":\"7rjqhiLT3MXkJMwkYKINL\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}