Distinct transcriptome architectures underlying lupus establishment and exacerbation.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease involving multiple immune cells. To elucidate SLE pathogenesis, it is essential to understand the dysregulated gene expression pattern linked to various clinical statuses with a high cellular resolution. Here, we conducted a large-scale transcriptome study with 6,386 RNA sequencing data covering 27 immune cell types from 136 SLE and 89 healthy donors. We profiled two distinct cell-type-specific transcriptomic signatures: disease-state and disease-activity signatures, reflecting disease establishment and exacerbation, respectively. We then identified candidate biological processes unique to each signature. This study suggested the clinical value of disease-activity signatures, which were associated with organ involvement and therapeutic responses. However, disease-activity signatures were less enriched around SLE risk variants than disease-state signatures, suggesting that current genetic studies may not well capture clinically vital biology. Together, we identified comprehensive gene signatures of SLE, which will provide essential foundations for future genomic and genetic studies.

Dynamic landscape of immune cell-specific gene regulation in immune-mediated diseases.

Genetic studies have revealed many variant loci that are associated with immune-mediated diseases. To elucidate the disease pathogenesis, it is essential to understand the function of these variants, especially under disease-associated conditions. Here, we performed a large-scale immune cell gene-expression analysis, together with whole-genome sequence analysis. Our dataset consists of 28 distinct immune cell subsets from 337 patients diagnosed with 10 categories of immune-mediated diseases and 79 healthy volunteers. Our dataset captured distinctive gene-expression profiles across immune cell types and diseases. Expression quantitative trait loci (eQTL) analysis revealed dynamic variations of eQTL effects in the context of immunological conditions, as well as cell types. These cell-type-specific and context-dependent eQTLs showed significant enrichment in immune disease-associated genetic variants, and they implicated the disease-relevant cell types, genes, and environment. This atlas deepens our understanding of the immunogenetic functions of disease-associated variants under in vivo disease conditions.

Multimodal repertoire analysis unveils B cell biology in immune-mediated diseases.

OBJECTIVES: Despite the involvement of B cells in the pathogenesis of immune-mediated diseases (IMDs), biological mechanisms underlying their function are scarcely understood. To overcome this gap, here we constructed and investigated a large-scale repertoire catalogue of five B cell subsets of patients with IMDs. METHODS: We mapped B cell receptor regions from RNA sequencing data of sorted B cell subsets. Our dataset consisted of 595 donors under IMDs and health. We characterised the repertoire features from various aspects, including their association with immune cell transcriptomes and clinical features and their response to belimumab treatment. RESULTS: Heavy-chain complementarity-determining region 3 (CDR-H3) length among naïve B cells was shortened among autoimmune diseases. Strong negative correlation between interferon signature strength and CDR-H3 length was observed in naïve B cells and suggested the role for interferon in premature B cell development. VDJ gene usage was skewed especially in plasmablasts and unswitched-memory B cells of patients with systemic lupus erythematosus (SLE). We developed a scoring system to quantify this skewing, and it positively correlated with peripheral helper T cell transcriptomic signatures and negatively correlated with the amount of somatic hyper mutations in plasmablasts, suggesting the association of extrafollicular pathway. Further, this skewing led to high usage of IGHV4-34 gene with 9G4 idiotypes in unswitched-memory B cells, which showed a prominent positive correlation with disease activity in SLE. Gene usage skewing in unswitched-memory B cells was ameliorated after belimumab treatment. CONCLUSIONS: Our multimodal repertoire analysis enabled us the system-level understanding of B cell abnormality in diseases.

Immunomics analysis of rheumatoid arthritis identified precursor dendritic cells as a key cell subset of treatment resistance.

OBJECTIVES: Little is known about the immunology underlying variable treatment response in rheumatoid arthritis (RA). We performed large-scale transcriptome analyses of peripheral blood immune cell subsets to identify immune cells that predict treatment resistance. METHODS: We isolated 18 peripheral blood immune cell subsets of 55 patients with RA requiring addition of new treatment and 39 healthy controls, and performed RNA sequencing. Transcriptome changes in RA and treatment effects were systematically characterised. Association between immune cell gene modules and treatment resistance was evaluated. We validated predictive value of identified parameters for treatment resistance using quantitative PCR (qPCR) and mass cytometric analysis cohorts. We also characterised the identified population by synovial single cell RNA-sequencing analysis. RESULTS: Immune cells of patients with RA were characterised by enhanced interferon and IL6-JAK-STAT3 signalling that demonstrate partial normalisation after treatment. A gene expression module of plasmacytoid dendritic cells (pDC) reflecting the expansion of dendritic cell precursors (pre-DC) exhibited strongest association with treatment resistance. Type I interferon signalling was negatively correlated to pre-DC gene expression. qPCR and mass cytometric analysis in independent cohorts validated that the pre-DC associated gene expression and the proportion of pre-DC were significantly higher before treatment in treatment-resistant patients. A cluster of synovial DCs showed both features of pre-DC and pro-inflammatory conventional DC2s. CONCLUSIONS: An increase in pre-DC in peripheral blood predicted RA treatment resistance. Pre-DC could have pathophysiological relevance to RA treatment response.

Control of naive and effector CD4 T cell receptor repertoires by rheumatoid-arthritis-risk HLA alleles.

OBJECTIVE: Human Leukocyte Antigen (HLA) alleles regulate susceptibility to rheumatoid arthritis (RA) and immune-mediated diseases. This study aims to elucidate the impact of HLA alleles to T cell subsets. METHODS: We performed genome-wide and HLA allele association analysis for T cell receptor (TCR) beta chain repertoire in 13 purified T cell subsets from the ImmuNexUT database, consisting of 407 donors with ten immune-mediated diseases and healthy controls. RESULTS: HLA class II alleles were associated with TRBV gene usage and the public clones of CD4 T cells, while HLA class I alleles were associated with CD8 T cells. RA-risk and immune-mediated diseases-risk HLA alleles were associated with TRBV gene usage of naive and effector CD4 T cell subsets and public clones accumulating in Th17. Clonal diversity was independent of HLA alleles and was correlated with transcriptome changes that reflect TCR signaling. CONCLUSION: This study revealed in vivo evidence that both HLA alleles and environmental factors shape naive and effector TCR repertoires in RA and immune-mediated diseases patients.

Dysregulation of the gene signature of effector regulatory T cells in the early phase of systemic sclerosis.

OBJECTIVES: We evaluated flow-cytometric and transcriptome features of peripheral blood immune cells from early-phase (disease duration <5 years) SSc in comparison with late-phase SSc. METHODS: Fifty Japanese patients with SSc (12 early SSc cases and 38 late SSc cases) and 50 age- and sex-matched healthy controls were enrolled. A comparison of flow-cytometric subset proportions and RNA-sequencing of 24 peripheral blood immune cell subsets was performed. We evaluated differentially expressed genes (DEGs), characterized the co-expressed gene modules, and estimated the composition of subpopulations by deconvolution based on single-cell RNA-sequencing data. As a disease control, idiopathic inflammatory myositis (IIM) patients were also evaluated. RESULTS: Analysing the data from early and late SSc, fraction II effector regulatory T cell (Fr. II eTreg) genes showed a remarkable differential gene expression, enriched for genes related to oxidative phosphorylation. Although the flow-cytometric proportion of Fr. II eTregs was not changed in early SSc, deconvolution indicated expansion of the activated subpopulation. Co-expressed gene modules of Fr. II eTregs demonstrated enrichment of the DEGs of early SSc and correlation with the proportion of the activated subpopulation. These results suggested that DEGs in Fr. II eTregs from patients with early SSc were closely associated with the increased proportion of the activated subpopulation. Similar dysregulation of Fr. II eTregs was also observed in data from patients with early IIM. CONCLUSIONS: RNA-seq of immune cells indicated the dysregulation of Fr. II eTregs in early SSc with increased proportion of the activated subpopulation.

Integrated bulk and single-cell RNA-sequencing identified disease-relevant monocytes and a gene network module underlying systemic sclerosis.

OBJECTIVE: Immunological disturbances have been reported in systemic sclerosis (SSc). This study assessed the transcriptome disturbances in immune cell subsets in SSc and characterized a disease-related gene network module and immune cell cluster at single cell resolution. METHODS: Twenty-one Japanese SSc patients were enrolled and compared with 13 age- and sex-matched healthy controls (HC). Nineteen peripheral blood immune cell subsets were sorted by flow cytometry and bulk RNA-seq analysis was performed for each. Differential expression and pathway analyses were conducted. Iterative weighted gene correlation network analysis (iWGCNA) of each subset revealed clustered co-expressed gene network modules. Random forest analysis prioritized a disease-related gene module. Single cell RNA-seq analysis of 878 monocytes was integrated with bulk RNA-seq analysis and with a public database for single cell RNA-seq analysis of SSc patients. RESULTS: Inflammatory pathway genes were differentially expressed in widespread immune cell subsets of SSc. An inflammatory gene module from CD16+ monocytes, which included KLF10, PLAUR, JUNB and JUND, showed the greatest discrimination between SSc and HC. One of the clusters of SSc monocytes identified by single-cell RNA-seq analysis characteristically expressed these inflammatory co-expressed genes and was similar to lung infiltrating FCN1hi monocytes expressing IL1B. CONCLUSIONS: Our integrated analysis of bulk and single cell RNA-seq analysis identified an inflammatory gene module and a cluster of monocytes that are relevant to SSc pathophysiology. They could serve as candidate novel therapeutic targets in SSc.

Identifying the most influential gene expression profile in distinguishing ANCA-associated vasculitis from healthy controls.

OBJECTIVE: Previous gene expression analyses seeking genes specific to antineutrophil cytoplasmic antibody-associated vasculitis (AAV) have been limited due to crude cell separation and the use of microarrays. This study aims to identify AAV-specific gene expression profiles in a way that overcomes those limitations. METHODS: Blood samples were collected from 26 AAV patients and 28 healthy controls (HCs). Neutrophils were isolated by negative selection, whereas 19 subsets of peripheral blood mononuclear cells were sorted by fluorescence assisted cell sorting. RNA-sequencing was then conducted for each sample, and iterative weighted gene correlation network analysis (iterativeWGCNA) and random forest were consecutively applied to identify the most influential gene module in distinguishing AAV from HCs. Correlations of the identified module with clinical parameters were evaluated, and the biological role was assessed with hub gene identification and pathway analysis. Particularly, the module's association with neutrophil extracellular trap formation, NETosis, was analyzed. Finally, the module's overlap with GWAS-identified autoimmune disease genes (GADGs) was assessed for validation. RESULTS: A neutrophil module (Neu_M20) was ranked top in the random forest analysis among 255 modules created by iterativeWGCNA. Neu_M20 correlated with disease activity and neutrophil counts but not with the presence of antineutrophil cytoplasmic antibody. The module comprised pro-inflammatory genes, including those related to NETosis, supported by experimental evidence. The genes in the module significantly overlapped GADGs. CONCLUSION: We identified the distinct group of pro-inflammatory genes in neutrophils, which characterize AAV. Further investigations are warranted to confirm our findings as they could serve as novel therapeutic targets.

Internalization Mechanisms of Pyridinium Sulfobetaine Polymers Evaluated by Induced Protic Perturbations on Cell Surfaces.

Understanding the interactions of soft nanomatters with cell membranes is particularly important for research into nanocarrier-based drug delivery systems, cell engineering, and subcellular imaging. Most nanoparticles, vesicles, micelles, and polymeric aggregates are internalized into endosomes and, eventually, lysosomes in the cytosol because of energy-dependent endocytic processes. Endocytic uptake substantially limits the access to the cytoplasm where a cargo agent acts. Bypassing the endocytic pathways by direct penetration into plasma membrane barriers would enhance the efficacy of nanomedicines. Some zwitterionic polymer nanoaggregates have been shown to permeate into the cell interior in an energy-independent manner. We have elucidated this phenomenon by observing changes in the biomembrane barrier functions against protons as the smallest indicator and have used these results to further design and develop poly(betaines). In this work, we investigated the translocation mechanisms for a series of zwitterionic poly(methacrylamide) and poly(methacrylate) species bearing a pyridinium propane sulfonate moiety in the monomers. Minor differences in the monomer structures and functional groups were observed to have dramatic effects on the interaction with plasma membranes during translocation. The ability to cross the plasma membrane involves a balance among the betaine dipole-dipole interaction, NH-π interaction, π-π interaction, cation-π interaction, and amide hydrogen bonding. We found that the cell-penetrating polysulfobetaines had limited or no detrimental effect on cell proliferation. Our findings enhance the opportunity to design and synthesize soft nanomatters for cell manipulations by passing across biomembrane partitions.

[A Case of a Huge, Mixed-Type IPMC with Suspected Rupture of the Omental Bursa That Was Effectively Treated with Gemcitabine plus Nab-Paclitaxel].

A 53-year-old woman was referred to our hospital because of upper abdominal pain and expansion of the pancreatic main duct. Enhanced computed tomography revealed expansion of the main pancreatic duct from the head to the tail; in addition, a 30 mm cystic tumor was observed in the pancreatic head and a 56 mm tumor was observed in the ventral side of the pancreatic body. Endoscopy revealed fistula formation in the duodenum of the Vater papilla on the oral side. The patient was diagnosed with an intraductal papillary mucinous carcinoma(IPMC). In addition, PET-CT revealed accumulation of FDG in the ventral side of the pancreatic body, and a disseminated nodule in the omental bursa was suspected. We administered 6 courses of gemcitabine plus nab-paclitaxel therapy, after which, the tumor in the ventral side of the pancreatic body disappeared. We then performed sub-stomach-preserving pancreatoduodenectomy. The results of abdominal cavity washing cytology were negative, and there were no disseminated nodules in the omental bursa. Therefore, we could perform R0 excision.

Induced Proton Perturbation for Sensitive and Selective Detection of Tight Junction Breakdown.

Tight junctions (TJs) in the epithelial cell gap play primary roles in body defense and nutrient absorption in multicellular organisms. Standard in vitro assays lack sensitivity, selectivity, temporal resolution, and throughput for bioengineering applications. Prompted by the rigorous barrier functions of TJ, we developed a TJ assay that senses proton leaks in the cell gap using ion-sensitive field-effect transistors. Upon exposure of Madin-Darby canine kidney (MDCK) cells cultured on a gate dielectric to a calcium-chelator EGTA, ammonia-assisted pH perturbation was enhanced solely in TJ-forming cells. This was supported by simulations with increased ion permeability in the paracellular pathway. Following administration of Clostridium perfringens enterotoxin as a specific TJ inhibitor to the MDCK cells, our method detected TJ breakdown at a 13× lower concentration than a conventional trans-epithelial electrical resistance assay. Thus, the semiconductor-based active pH sensing could offer an alternative to current in vitro assays for TJs in pathological, toxicological, and pharmaceutical studies.

Translocation Mechanisms of Cell-Penetrating Polymers Identified by Induced Proton Dynamics.

Unlike the majority of nanomaterials designed for cellular uptake via endocytic pathways, some of the functional nanoparticles and nanospheres directly enter the cytoplasm without overt biomembrane injuries. Previously, we have shown that a water-soluble nanoaggregate composed of amphiphilic random copolymer of 2-methacryloyloxyethyl phosphorylcholine (MPC) and n-butyl methacrylate (BMA), poly(MPC- random-BMA) (PMB), passes live cell membranes in an endocytosis-free manner. Yet, details in its translocation mechanism remain elusive due to the lack of proper analytical methods. To understand this phenomenon experimentally, we elaborated the original pH perturbation assay that is extremely sensitive to the pore formation on cell membranes. The ultimate sensitivity originates from the detection of the smallest indicator H+ (H3O+) passed through the molecularly sized transmembrane pores upon challenge by exogenous reagents. We revealed that water-soluble PMB at the 30 mol % MPC unit (i.e., PMB30W) penetrated into the cytosol of model mammalian cells without any proton leaks, in contrast to conventional cell-penetrating peptides, TAT and R8 as well as the surfactant, Triton X-100. While exposure of PMB30W permeabilized cytoplasmic lactate dehydrogenase out of the cells, indicating the alteration of cell membrane polarity by partitioning of amphiphilic PMB30W into the lipid bilayers. Nevertheless, the biomembrane alterations by PMB30W did not exhibit cytotoxicity. In summary, elucidating translocation mechanisms by proton dynamics will guide the design of nanomaterials with controlled permeabilization to cell membranes for bioengineering applications.

Replacement of sensor cables for reducing effects on articulation in the Northern Digital Incorporated's Wave electromagnetic articulography system.

This study attempted to improve the five-degrees-of-freedom sensors of the Northern Digital Incorporated's Wave electromagnetic articulography system by replacing their cables with thinner and more flexible cables to reduce interference in articulation. Measurement errors and data loss rates were compared between the original and the proposed sensors. The proposed sensors showed twofold tracking accuracy and data loss rates compared to the original sensors in an experiment using a crank-rocker mechanism. Data loss rates of the proposed sensors increased in articulatory data collection from four speakers. The proposed sensors have been made available commercially.

Epigenetic targets of Janus kinase inhibitors are linked to genetic risks of rheumatoid arthritis.

BACKGROUND: Current strategies that target cytokines (e.g., tumor necrosis factor (TNF)-α), or signaling molecules (e.g., Janus kinase (JAK)) have advanced the management for allergies and autoimmune diseases. Nevertheless, the molecular mechanism that underpins its clinical efficacy have largely remained elusive, especially in the local tissue environment. Here, we aimed to identify the genetic, epigenetic, and immunological targets of JAK inhibitors (JAKis), focusing on their effects on synovial fibroblasts (SFs), the major local effectors associated with destructive joint inflammation in rheumatoid arthritis (RA). METHODS: SFs were activated by cytokines related to inflammation in RA, and were treated with three types of JAKis or a TNF-α inhibitor (TNFi). Dynamic changes in transcriptome and chromatin accessibility were profiled across samples to identify drug targets. Furthermore, the putative targets were validated using luciferase assays and clustered regularly interspaced short palindromic repeat (CRISPR)-based genome editing. RESULTS: We found that both JAKis and the TNFi targeted the inflammatory module including IL6. Conversely, specific gene signatures that were preferentially inhibited by either of the drug classes were identified. Strikingly, RA risk enhancers for CD40 and TRAF1 were distinctively regulated by JAKis and the TNFi. We performed luciferase assays and CRISPR-based genome editing, and successfully fine-mapped the single causal variants in these loci, rs6074022-CD40 and rs7021049-TRAF1. CONCLUSIONS: JAKis and the TNFi had a direct impact on different RA risk enhancers, and we identified nucleotide-resolution targets for both drugs. Distinctive targets of clinically effective drugs could be useful for tailoring the application of these drugs and future design of more efficient treatment strategies.

Long-read sequencing for 29 immune cell subsets reveals disease-linked isoforms.

Alternative splicing events are a major causal mechanism for complex traits, but they have been understudied due to the limitation of short-read sequencing. Here, we generate a full-length isoform annotation of human immune cells from an individual by long-read sequencing for 29 cell subsets. This contains a number of unannotated transcripts and isoforms such as a read-through transcript of TOMM40-APOE in the Alzheimer's disease locus. We profile characteristics of isoforms and show that repetitive elements significantly explain the diversity of unannotated isoforms, providing insight into the human genome evolution. In addition, some of the isoforms are expressed in a cell-type specific manner, whose alternative 3'-UTRs usage contributes to their specificity. Further, we identify disease-associated isoforms by isoform switch analysis and by integration of several quantitative trait loci analyses with genome-wide association study data. Our findings will promote the elucidation of the mechanism of complex diseases via alternative splicing.

Age-associated CD4+ T cells with B cell-promoting functions are regulated by ZEB2 in autoimmunity.

Aging is a significant risk factor for autoimmunity, and many autoimmune diseases tend to onset during adulthood. We conducted an extensive analysis of CD4+ T cell subsets from 354 patients with autoimmune disease and healthy controls via flow cytometry and bulk RNA sequencing. As a result, we identified a distinct CXCR3midCD4+ effector memory T cell subset that expands with age, which we designated "age-associated T helper (THA) cells." THA cells exhibited both a cytotoxic phenotype and B cell helper functions, and these features were regulated by the transcription factor ZEB2. Consistent with the highly skewed T cell receptor usage of THA cells, gene expression in THA cells from patients with systemic lupus erythematosus reflected disease activity and was affected by treatment with a calcineurin inhibitor. Moreover, analysis of single-cell RNA sequencing data revealed that THA cells infiltrate damaged organs in patients with autoimmune diseases. Together, our characterization of THA cells may facilitate improved understanding of the relationship between aging and autoimmune diseases.

Functional Genetics to Understand the Etiology of Autoimmunity.

Common variants strongly influence the risk of human autoimmunity. Two categories of variants contribute substantially to the risk: (i) coding variants of HLA genes and (ii) non-coding variants at the non-HLA loci. We recently developed a novel analytic pipeline of T cell receptor (TCR) repertoire to understand how HLA coding variants influence the risk. We identified that the risk variants increase the frequency of auto-reactive T cells. In addition, to understand how non-coding variants contribute to the risk, the researchers conducted integrative analyses using expression quantitative trait loci (eQTL) and splicing quantitative trait loci (sQTL) and demonstrated that the risk non-coding variants dysregulate specific genes' expression and splicing. These studies provided novel insight into the immunological consequences of two major genetic risks, and we will introduce these research achievements in detail in this review.

Transcriptome Profiling of Immune Cell Types in Peripheral Blood Reveals Common and Specific Pathways Involved in the Pathogenesis of Myositis-Specific Antibody-Positive Inflammatory Myopathies.

OBJECTIVE: Idiopathic inflammatory myopathies (IIM) demonstrate characteristic clinical phenotypes depending on the myositis-specific antibody (MSAs) present. We aimed to identify common or MSA-specific immunological pathways in different immune cell types from peripheral blood by transcriptome analysis. METHODS: We recruited 33 patients with IIM who were separated into the following groups: 15 patients with active disease at onset and 18 with inactive disease under treatment. All patients were positive for MSAs: anti-melanoma differentiation-associated gene 5 (MDA5) antibody (Ab) in 10 patients, anti-Mi-2 Ab in 7, and anti-aminoacyl-transfer RNA synthetase (ARS) Ab in 16. The patients were compared with 33 healthy controls. Twenty-four immune cell types sorted from peripheral blood were analyzed by flow cytometry, RNA sequencing, and differentially expressed gene analysis combined with pathway analysis. RESULTS: The frequencies of memory B cell types were significantly decreased in active patients, and the frequency of plasmablasts was prominently increased in active patients with anti-MDA5 Ab in comparison with healthy controls. The expression of type I interferon (IFN)-stimulated genes of all immune cell types was increased in the active, but not inactive, patients. Endoplasmic reticulum stress-related genes in all IIM memory B cells and oxidative phosphorylation-related genes in inactive IIM double negative B cells were also increased, suggesting prominent B cell activation in IIM. Furthermore, active patients with anti-MDA5 Ab, anti-Mi-2 Ab, or anti-ARS Ab were distinguished by IFN-stimulated and oxidative phosphorylation-related gene expression in plasmablasts. CONCLUSION: Unique gene expression patterns in patients with IIM with different disease activity levels and MSA types suggest different pathophysiologies. Especially, B cells may contribute to common and MSA-specific immunological pathways in IIM.

Transepithelial delivery of insulin conjugated with phospholipid-mimicking polymers via biomembrane fusion-mediated transcellular pathways.

Epithelial barriers that seal cell gaps by forming tight junctions to prevent the free permeation of nutrients, electrolytes, and drugs, are essential for maintaining homeostasis in multicellular organisms. The development of nanocarriers that can permeate epithelial tissues without compromising barrier function is key for establishing a safe and efficient drug delivery system (DDS). Previously, we have demonstrated that a water-soluble phospholipid-mimicking random copolymer, poly(2-methacryloyloxyethyl phosphorylcholine30-random-n­butyl methacrylate70) (PMB30W), enters the cytoplasm of live cells by passive diffusion manners, without damaging the cell membranes. The internalization mechanism was confirmed to be amphiphilicity-induced membrane fusion. In the present study, we demonstrated energy-independent permeation of PMB30W through the model epithelial barriers of Madin-Darby canine kidney (MDCK) cell monolayers in vitro. The polymer penetrated epithelial MDCK monolayers via transcellular pathways without breaching the barrier functions. This was confirmed by our unique assay that can monitor the leakage of the proton as the smallest indicator across the epithelial barriers. Moreover, energy-independent transepithelial permeation was achieved when insulin was chemically conjugated with the phospholipid-mimicking nanocarrier. The bioactivity of insulin as a growth factor was found to be maintained even after translocation. These fundamental findings may aid the establishment of transepithelial DDS with advanced drug efficiency and safety. STATEMENT OF SIGNIFICANCE: A nanocarrier that can freely permeate epithelial tissues without compromising barrier function is key for successful DDS. Existing strategies mainly rely on paracellular transport associated with tight junction breakdown or transcellular transport via transporter recognition-mediated active uptake. These approaches raise concerns about efficiency and safety. In this study, we performed non-endocytic permeation of phospholipid-mimicking polymers through the model epithelial barriers in vitro. The polymer penetrated via transcytotic pathways without breaching the barriers of biomembrane and tight junction. Moreover, transepithelial permeation occurred when insulin was covalently attached to the nanocarrier. The bioactivity of insulin was maintained even after translocation. The biomimetic design of nanocarrier may realize safe and efficient transepithelial DDS.