Systemic acquired resistance (SAR) occurs when primary infected leaves produce several SAR-inducing chemical or mobile signals that are transported to uninfected distal parts via apoplastic or symplastic compartments and activate systemic immunity. The transport route of many chemicals associated with SAR is unknown. Recently, it was demonstrated that pathogen-infected cells preferentially transport salicylic acid (SA) through the apoplasts to uninfected areas. The pH gradient and deprotonation of SA may lead to apoplastic accumulation of SA before it accumulates in the cytosol following pathogen infection. Additionally, SA mobility over a long distance is essential for SAR, and transpiration controls the partitioning of SA into apoplasts and cuticles. On the other hand, glycerol-3-phosphate (G3P) and azelaic acid (AzA) travel via the plasmodesmata (PD) channel in the symplastic route. In this review, we discuss the role of SA as a mobile signal and the regulation of SA transport in SAR. Arabidopsis and tobacco plants, which accumulate little or no SA due to the expression of the bacterial nahG gene, which encodes salicylate hydroxylase [Salicylic acid  is an essential defense hormone in plants that accumulates upon a pathogen attack to induce local defense and systemic acquired resistance (SAR) ,2. Genetroxylase . SA biosSAR provides long-lasting protection against secondary pathogen attacks by priming the plant’s defense response. SAR is triggered by an initial infection, and results in the activation of the plant’s defense response throughout the entire plant, even in tissues that are not yet infected. As a result of SAR, the plant is able to coordinate its defense response and respond appropriately to pathogen attacks, even in distant tissues. Although the identity of a specific mobile signal is unknown, numerous SAR-inducing chemicals have been identified, some of which are physically mobile, and some of which are volatile in nature. These include salicylic acid (SA)  and its Arabidopsis, the majority of pathogen-induced SA is derived from the ICS branch by plastid-localized ICS1 ) [Arabidopsis, there are two isochorismate synthase genes, ICS1 and ICS2; however, only ICS1 is induced by pathogens. The ics1 mutants showed 90–95% less SA accumulation after pathogen infection than wild-type plants [ics1/ics2 double mutant strongly affected SA accumulation, indicating that ICS1 generates most of the SA via isochorismate during pathogen defense. [Arabidopsis mutants with altered SA accumulation are known to result from mutations in three genes encoding ICS pathway components. According to recent studies, bacteria have the Isochorismate Pyruvate Lyase (IPL) enzyme, which directly converts isochorismate to SA, whereas Arabidopsis requires both AvrPphB-susceptible 3 (PBS3) and Enhanced Pseudomonas Susceptibility 1 (EPS1) [eds5-3 mutants, co-expression of ICS1 and engineered chloroplast-targeted PBS3 can restore SA biosynthesis, suggesting that EDS5 could participate in isochorismate transport to the cytosol during SA biosynthesis [In higher plants, SA is synthesized from chorismate through two distinct metabolic pathways, each with multiple steps: the isochorismate synthase (ICS)- and phenylalanine ammonia-lyase (PAL)-derived pathways ,29,30. D (SID2)) . In Arabe plants . Only thdefense. ,32. Seve1 (EPS1) ,35,36,371 (EPS1) ,39. Howeics1/ics2 double mutant, which is blocked by the ICS pathway [Arabidopsis contains four PAL genes, and studies with mutants or PAL-specific inhibitors suggest that the PAL pathway is also involved in SA biosynthesis [A small amount of SA accumulation is induced by pathogens in the  pathway . Arabidoynthesis ,40. Neveynthesis   and Calmodulin-Binding Protein 60-Like.g (CBP60g) [ICS1, EDS5, and PBS3 by binding to its promoter with CBP60g, and is homologous to SARD1 [cbp60g sard1 double mutant impairs ICS1, EDS5, and PBS3 induction and, subsequently, SA biosynthesis by bacterial elicitor flg22 or the avirulent bacterial strain Pseudomonas syringae pv maculicola (Psm) ES4326 avrB, resulting in compromised pattern-triggered immunity (PTI)- and effector-triggered immunity (ETI)-induced resistance as well as SAR [EDS5 show impaired SA accumulation and resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 [pbs3 mutants display reduced SA accumulation in response to pathogens, such as plants with defects in ICS1 and EDS5. Additionally, several proteins contribute to SA accumulation and SAR in response to pathogens. These include Enhanced Disease Susceptibility 1 (EDS1), Phytoalexin Deficient 4 (PAD4), and Non-race-specific Disease Resistance 1 (NDR1), which show partial reductions in SA levels, unlike ICS1 and EDS5 [The expression of  reviews ,42, a la(CBP60g) . The tra(CBP60g) . In addito SARD1 . The cbpl as SAR ,47. Muta) DC3000 . Likewisand EDS5 ,57,58,59Arabidopsis thaliana is activated by two receptor classes: Nonexpressor of Pathogenesis-Related Genes (NPR1) and NPR1-Like Protein 3/4 (NPR3/4). This stimulates the expression of defense-related genes and immunity. Neither NPR1 nor NPR3/NPR4 contain DNA-binding domains; they must interact with transcription factors, such as TGA2/5/6, for signal transduction [npr3/npr4 mutants showing enhanced PR gene expression and basal resistance. NPR3 and NPR4 were identified as SA receptors based on their high SA affinities. NPR4 has an SA-binding core domain that refolds with SA to form a helical fold that completely encloses SA within its hydrophobic core. The interaction between NPR1 and NPR4 is disrupted by SA-induced conformational changes in the core of NPR4, which binds to SA. SA binding by NPR1 has also been reported, although its activity varies between studies. Similarly to their negative association with SA signaling, NPR3 and NPR4 are CRL3 substrate receptors for NPR1 polyubiquitination and degradation. SA-mediated gene expression is controlled by NPR3 and NPR4 by controlling NPR1 stability [NIM1-Interacting 1 (NIMIN1), is strongly induced by SA on top of NPR3 and NPR4 [nimin1 mutant resulted in enhanced SA-induced PR1 expression, while NIMIN1 overexpression exhibited phenotypes similar to npr1 mutants, including reduced PR1 expression after SA induction, compromised SAR, and SA intolerance [The SA signaling pathway in sduction ,62,63,64tability . A negatand NPR4 . NIMIN1 olerance ,67. SAR olerance . Arabidopsis and tobacco nahG plants, although the levels were lower than in wild-type plants [14C-SA to distal tissues was confirmed after infiltration in local leaves in wild-type and nahG Arabidopsis plants.Several studies have shown that SA is crucial for SAR, but its role has been largely unclear until recently. Mobile inducers of SAR are produced in the primary infected leaves, and are then translocated to distal uninfected portions to activate SAR ,70,71,72e plants . Additioacp4 and mod1 mutants, which exhibit increased transpiration, tend to have higher levels of SA in their cuticle waxes, suggesting that the transpiration pull regulates its partitioning [ACP4, a component of fatty acid (FA) biosynthesis and lipid synthesis initiation, can reduce wax components  and cutin aliphatic monomers [MOD1 result in a defective enoyl-ACP reductase enzyme that is necessary for the production of FA, resulting in reduced levels of multiple FA species as well as total lipids. Interestingly, in both acp4 and mod1 mutant plants, the transport of SA into petiole exudate (PEX) and apoplasts was impaired, even though the levels of SA in the infected leaves was similar to that in wild-type leaves [acp4 and mod1 mutants had lower water usage efficiency levels (WUE), and did not transport SA efficiently through the apoplast. As a consequence, both acp4 and mod1 mutant plants had low water potential and increased stomatal aperture. These results indicate that increased transpiration decreases apoplastic hydrostatic pressure in these mutants, which allows SA to enter the cuticle [acp4 and mod1 mutants and restore systemic SA transport and SAR. Interestingly, sid2 mutants exhibited reduced water potential, reduced WUE, and increased stomatal aperture. In addition, exogenous SA restored stomatal aperture and water potential in these plants, but not pathogen infection. In contrast, following both exogenous SA and pathogen infection, the acp4 and mod1 mutant plants had reduced stomatal apertures. SA treatment did not induce jasmonic acid (JA) levels or abscisic acid (ABA) levels, which suggests that SA is independent of ABA. Therefore, the function of SA and ABA in relation to stomatal aperture needs to be further investigated. Thus, the balance between cuticular SA levels and intracellular water potential can regulate stomatal opening [Intriguingly, active SA transport is regulated by the plant cuticle. An intact cuticle is also required for systemic SA transport, since a portion of total SA is partitioned into cuticle wax. Mutants with defects in their cuticles, such as the itioning . For examonomers . Moreovee leaves . A previe leaves . Recentl cuticle ,74,75. T opening .Respiratory Burst Oxidase Homologs (RBOH) mutants rbohD and rbohF, indicating that ROS radicals function downstream of NO in SAR [rbohD and rbohF, and mutation in either of them compromises SAR. Nevertheless, exogenous ROS application restored SAR in NO associated protein 1 (noa1)/nitrate reductases 1 (nia1) or the noa1/nia2 double mutants, which were deficient in NO [GLY1  and GLI1 , resulting in G3P accumulation [Colletotrichum higginsianum and induces SAR in Arabidopsis and monocots [ics1(sid2) mutant plants, which have significantly reduced either basal or pathogen-induced SA levels [gly1 and gli1 mutant plants resulted in a compromised SAR phenotype, which could be restored by exogenous G3P application [gly1 and gli1 mutants exhibited levels of SA and AzA similar to those in the wild-type plants. Additionally, exogenous application of G3P alone was not sufficient to induce SA biosynthesis or SAR. These results suggest that, similarly to SA, AzA and G3P may not be sufficient to induce SAR.Pathogen infections result in rapid NO accumulation during SAR via unknown mechanisms. NO application did not confer SAR to O in SAR . As a rent in NO . Plants nt in NO ,26. A pant in NO . In partnt in NO . G3P is nt in NO . Exogenomulation . Exogenomonocots ,79. InteA levels . As a relication ,26. IntrPDLP5 have impaired AzA and G3P transport, as well as compromised SAR. In addition, 35S-PDLP5-expressing plants show reduced transport of DIR1, a lipid-transfer protein involved in SAR [PDLP1 and PDLP5 [pdlp1 and pdlp5 mutant plants, but only pdlp5 plants showed an increase in PD permeability. As AZI1 interacts with both PDLP1 and PDLP5, it is likely to form complexes with both proteins. Loss of PDLP1 or PDLP5 increases the chloroplastic localization of AZI1, although the biological significance of the chloroplastic localization of AZI1 or its intracellular partitioning during SAR is not known [ics1(sid2) mutant plants, which have significantly reduced either basal or pathogen-induced SA levels.In the presence of DIR1, G3P is systemically mobile, but its direct binding has not yet been identified . DIR1 isd in SAR ,24. The nd PDLP5 . Thus, Sot known ,69. TherArabidopsis and other plants which increase the formation of aromatic amino acids, branched-chain amino acids, and lysine [Arabidopsis, pipecolic acid (Pip) is converted into NHP in Arabidopsis by three different enzymes that respond strongly to biotic stresses [P. syringae [FMO1 overexpression conferred resistance to infection by bacterial and oomycete pathogens, whereas defects in SARD4, another Pip biosynthetic gene, eliminated resistance [14-Pip in distal leaves and, similarly, Návarová et al. [ald1 and fmo1 mutants, suggesting that NHP may act downstream of Pip [The inoculation of pathogens results in massive metabolic changes in d lysine . A recenstresses . Lysine stresses . Then, 2stresses . The finstresses ,82. Exogsyringae ,81,83. Fsistance ,81,82. Wsistance  detectedá et al. ,85. Pathm of Pip ,81,82. Tm of Pip ,81,82. NPR1 and NPR3/NPR4, suggesting that NHP may be involved in both regulating SA signaling output and promoting SA biosynthesis [UGT76B1, free NHP and SA levels are reduced, and the response to SARs is diminished [SARD1 and CBP60g target not only SA biosynthesis genes, but also genes involved in NHP biosynthesis [SARD1 and CBP60g during SAR is mediated by NHP, how NHP activates SARD1 and CBP60g transcription remains unelucidated. In addition, NADP and NHP induce SA marker gene expression, but the specificity of the response is unclear. It remains unclear how SA and NHP biosynthesis is regulated by various transcription factors upstream of the defense signal pathway. It is necessary to conduct more research to understand how SA and NHP activate SAR.Interestingly, NHP treatment significantly induces the expression of ynthesis . Consideynthesis . In addiynthesis . It has minished . Recent ynthesis . AlthougRecent research has shown that the cuticle regulates SA transport during SAR, and SAR-inducing chemicals can be transported by either apoplastic or symplastic compartments. After pathogen infection, SA levels increase in the apoplastic compartment, suggesting that SA movement into the apoplastic compartment is critical to SAR. The partitioning of SA in the cuticle wax is increased in mutants with defective cuticles, which exhibit increased transpiration. Moreover, defective SA transport in defective cuticle mutants were due to significantly reduced SA levels in their PEX and apoplastic compartments after pathogen infection. This suggests that transpiration pull plays an important role in SA transport. However, the mechanisms involved in SAR and the role of chemical or mobile signals that induce SAR in plants remain unclear. The understanding of how SAR works can lead to a better understanding of how plants interact with their environments, leading to an improved understanding of plant disease management. SAR can be applied in agriculture for disease management by inducing resistance in crops in order to protect against pests and diseases. Furthermore, recent studies indicate that SA improves plants’ tolerance to abiotic stress by influencing several biochemical and physiological changes . As an e


FOLR2+ macrophages. In light of these findings, FRAbs may be a critical factor in pathological pregnancy, and deserve careful consideration in therapies involving folic acid supplementation for pregnancy complications.Though firstly identified in cerebral folate deficiency, autoantibodies against folate receptors (FRAbs) have been implicated in pregnancy complications such as miscarriage; however, the underlying mechanism needs to be further elaborated. FRAbs can be produced via sensitization mediated by folate-binding protein as well as gene mutation, aberrant modulation, or degradation of folate receptors (FRs). FRAbs may interfere with folate internalization and metabolism through blocking or binding with FRs. Interestingly, different types of FRs are expressed on trophoblast cells, decidual epithelium or stroma, and macrophages at the maternal-fetal interface, implying FRAbs may be involved in the critical events necessary for a successful pregnancy. Thus, we propose that FRAbs may disturb pregnancy establishment and maintenance by modulating trophoblastic biofunctions, placental development, decidualization, and decidua homeostasis as well as the functions of  Unlike dulation . Functiodulation , immune dulation ,5, and rdulation ,7.Since the 1960s, folate deficiency has been regarded as the major cause of neural tube defects (NTDs). Well-designed studies have proven that folate supplementation could prevent fetal NTDs and ameliorate maternal anemia ,9. ConseFolate status can be affected by various factors, including dietary intake, gene polymorphisms, and post-translational modifications of folate transporters and metabolic enzymes. Notably, accumulated evidence has shown that autoantibodies against folate receptors (FRAbs) can significantly disrupt folate internalization by interacting with folate receptors (FRs). FRAbs were first identified in cases of infant cerebral folate deficiency (CFD), in which folate transport from cerebrospinal fluid to the central nervous system is blocked, resulting in relative cerebral folate deficiency, even when peripheral folate status is normal . In addiFOLR1, FOLR2, FOLR3, and FOLR4, respectively. FRs are the most important transporters at neutral pH, with high affinity binding to folates in comparison with the RFC and PCFT [The abundance of intracellular folate is determined by intake and exportation mediated by corresponding transporters, a process which depends on the pH of the internal environment and the specificity of the substrate . Folate and PCFT ,30. The and PCFT ,31,32. Fand PCFT ,34.Dietary folate is mainly absorbed in the upper small intestine via the PCFT on enterocytes and, then, it is metabolized to 5-MTHF, which is then released into circulation by MRP3. Serum 5-MTHF is transported into cells through their membrane folate transporters, where it participates in FOCM mediated by a series of enzymes ,36. Duri12 acting as a co-factor. THF is then recycled to 5-MTHF in the form of 5,10-methylenetetrahydrofolate  by methylenetetrahydrofolate reductase (MTHFR). There is also a functional cycle among THF, 5,10-methylene THF and dihydrofolate (DHF), which involves two key events: (1) de novo purine biosynthesis, which is mediated by phosphoribosylglycinamide formyltransferase (GART); (2) de novo dTMP biosynthesis, which is assisted by thymidylate synthetase (TS). In the Met cycle, Met is synthesized from homocysteine (Hcy) via MS then, upon the methyl transfer and modification, Met is converted to Hcy [In the folate cycle, 5-MTHF is initially catalyzed to tetrahydrofolate (THF) via methionine synthase (MS), with vitamin Bd to Hcy ,37,38. Id to Hcy ,40.In general, there have been several classical theories for autoantibodies formation, such as molecular mimicry, newly exposed epitope, aberrant antigen presentation, dysregulated immune network, and so on ,42. InteFOLRs may alter their antigenicity and have been reported to coexist with blocking FRAb in CFD patients [FOLR1 was identified in a CFD patient, but the authors did not investigate the FRAb status further [FOLR1 and FOLR2 in the coding region were found to be correlated with CFD [FOLRs.Receptor mutations are a common cause of autoantibody formation in autoimmune diseases. Mutations and variants of patients . In one  further ; howeverwith CFD . NeverthFOLR1 in an epigenetic way. Interestingly, most participants (5 out of 6 subjects enrolled) who presented missense variants of KDM6B were also FRAb-positive in their serum samples [Epigenetic modification may also contribute to the development of FRAbs. Han et al. reported that missense variants of lysine demethylase 6B (KDM6B) resulted in the decreased expression of KDM6B protein and, thus, dysregulated methylation by upregulating H3K27me2 and downregulating H3K27Ac. This could further inhibit the transcriptional activity of  samples ; moreove samples ,48,49, i12 deficiency inhibits the remethylation or trans-sulfuration pathways, Hcy is more likely to form an active Hcy thiolactone (HTL), which can homocysteinylate proteins by interacting with lysine residues and cause structural abnormalities and loss of protein function [Epidemiological evidence suggests that the level of FRAbs is positively associated with folate status but negatively associated with Hcy . Dependifunction . Animal function ; moreovefunction .It is interesting to note that FRs contain high levels of lysine residues, which suggests the possibility that they could be modulated by HTL and potentially misidentified as autoantigens . This coHomocysteinylation mediated by HTL has also been observed in other proteins related to FRAb function. For example, Tang et al. demonstrated that excessive Hcy induced by folate deficiency may lead to increased homocysteinylation of heterogeneous nuclear ribonucleoprotein E1(hnRNP-E1), which interacts with its mRNA cis-element and forms a positive feedback loop, eventually resulting in accumulation of hnRNP-E1. hnRNP-E1 has a high affinity to an 18-nucleotide cis-element in the 5′-untranslated region of FRα mRNA, which triggers proportionate translational upregulation of FRs . This reAs a soluble form of FR, FBP is primarily present in human or bovine milk and shares approximately 80–90% homology with the amino acid sequence of membrane-anchored FRα and FRβ in the human placenta ,69. The + T cells, accompanied by increased levels of certain antibodies against FRα [Certain membrane-anchored receptors such as FRs, the follicle-stimulating hormone receptor, and the thyrotropin receptor, exert their effects via internalization through a process named receptor-mediated endocytosis ,75. Uponinst FRα . These rMTHFR , DNMT3A (rs7560488 C → T), and MTHFD2 , and elevated plasma levels in FRAbs [It is important, however, to note that other factors, such as genetics or immune activation, may also induce the production of FRAbs. For example, studies have reported an association between genetic variations in FOCM-related enzymes, including in FRAbs ; neverthin FRAbs . Further3H-folic acid and the ELISA method can be used to distinguish blocking and binding FRAbs, respectively, according to their properties [FRAbs can be classified into blocking and binding subtypes based on their function. Blocking FRAbs refer to autoantibodies that share the same binding sites as folates and inhibit the combination of folates with FRs. Conversely, binding FRAbs recognize different binding sites from folates and, therefore, do not compete with folates but, instead, inactivate FRs by triggering an immune response and inflammation through antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) ,84,85. Toperties . The funoperties . Frye etoperties . Besidesoperties . These fMinor differences in the heavy chain of immunoglobulin distinguish the IgG and IgM isoforms of FRAbs in different situations. For instance, IgG1 and IgG2 predominated in FRAbs found in pregnant women with NTDs, while IgG1 and IgG4 isotypes constituted the majority of serum FRAbs in CFD children, with an average biological half-life of 21 days ,88. CabrFOLR1 (encoding FRα) and SLC19A1 (encoding RFC) have been found to be associated with an increased risk of recurrent miscarriage [In addition to limited folate intake and elevated endogenous Hcy ,91,92,93carriage ,98,99.However, the association between miscarriage and the mutation of enzymes or folate transporters at different loci are inconsistently demonstrated in previous studies ,102,103.The maternal-fetal interface is primarily composed of the maternal decidua and the maternal side of the placenta; it plays a critical role in promoting proper fetal growth and development while maintaining a healthy pregnancy. As the target of FRAbs, FRs present a specific expressional profile at the maternal-fetal interface .FOLR1, is a high-affinity FR anchored to the cell membrane via GPI. FRα exhibits a restricted distribution pattern in normal tissues [FRα, encoded by  tissues , with ex tissues ,120,121. tissues ,107,108. tissues ,123. Fun tissues ,105. In  tissues , indicat tissues ,110, andFOLR2. It has been reported to primarily express on activated macrophages and dendritic cells, while resting macrophages are devoid of FRβ expression under physiological conditions [FRβ, which shares 70% identity with FRα in homology, is encoded by nditions ,126,127.nditions  and locanditions .FOLR3, is a soluble protein that lacks a GPI anchor domain and originates from the secretory granules of neutrophil granulocytes. It has a much lower affinity for folate than FRα and FRβ [FRγ, encoded by  and FRβ . Some st and FRβ , inflamm and FRβ , and ant and FRβ . The rol and FRβ ,129. In  and FRβ . More reFOLR4, is a pseudo folate receptor that cannot transport folates. It was initially identified in sperm cells and is responsible for fertilization via interaction with its ligand, Izumo1, on oocytes of several mammalian species, including humans and mice [FRδ, also named Izumo1 receptor (Izumo1R) and encoded by and mice . Besidesand mice ,116,117;FRAbs target the placenta and embryos, and have been shown to inhibit folate transport from rats to their fetuses, leading to folate deficiency during pregnancy ,132. Da Initially, epidemiological evidence revealed a link between miscarriage and FRAb-related birth defects, like NTDs, in human pregnancy ,134. WomDNMT3A (rs7560488 C → T) and the elevated plasma levels of FRAbs [DNMT1 and DNMT3 [Based on the hypotheses we discussed above, several risk factors have been implicated in facilitating FRAb production in miscarriage. These include aberrant expression, the modulation or degradation of FRs, homocysteinylation mediated by Hcy thiolactone (HTL), and FBP-induced sensitization. In complicated cases of miscarriage, limiting FBP intake through a milk-free diet has shown to have an adjuvant therapeutic effect on reducing FRAb titers ,136. Intof FRAbs . In factnd DNMT3 ,92,93, iOther factors that could lead to the unexpected exposure of FR epitopes at the maternal-fetal interface may also contribute to the production of FRAbs. Rothenberg et al. proposed that clinically unrecognized pregnancies, known as biochemical pregnancies, could partially explain the high incidence of spontaneous miscarriage concurrent with the presence of FRAbs in apparently healthy individuals . The risTrophoblast cells, which originate from the outer layer of blastocysts, play a vital role in placentation. They differentiate into villous trophoblasts (VTs) and extravillous trophoblasts (EVTs); however, impaired trophoblastic biofunctions and placentation can lead to pregnancy complications such as miscarriage. Since FRα is responsible for over 60% of folate transport in trophoblast cells, and plays a crucial role in determining intracellular folate status ,105, it −6 M culture condition of folate, but inhibited in 10−8 M and 10−10 M [Several studies have confirmed the role of FRAbs in inhibiting cellular folate uptake and interfering with FOCM, particularly in cases of CFD or NTDs . These c 10−10 M , suggest 10−10 M ,143, whi 10−10 M . It woulFolic acid plays a key role in the early stages of placental development, including EVT invasion, angiogenesis, and secretion of the matrix metalloproteinases (MMPs) . PericonFRAbs might disturb placentation in a metabolic-dependent way. Firstly, by inhibiting intracellular folate bioavailability and reducing MMP production or by inducing oxidative stress, folate deficiency caused by FRAbs has been shown to dysregulate trophoblastic migration and invasion ,146,147.The process of decidualization involves the transformation of secretory-phase endometrium into decidua, which is stimulated by estradiol (E2) and progesterone (P4). It prepares the uterus for successful implantation, embryonic development, and pregnancy maintenance ,153. DurIt is believed that FRαAb blocks or inhibits the uptake of folate by EEC, as FRs mainly express on EECs in decidua. A deficiency of folate in mice has been shown to result in the defective progression of DSC decidualization, probably through the modulation of genomic methylation or a decrease in E2 and P4 hormones ,92; howeTrp53), differentiation , and angiogenesis (Ereg), were hypermethylated and therefore downregulated in the decidua [VEGFA, VEGFR2, and PLGF, resulting in a poorly vascularized decidua [Folate has also been shown to attenuate LPS-induced increases in COX-2 in mouse placentae . As COX- decidua . Further decidua . These sIn contrast to E2 and P4 or a combination of the two, the cAMP-induced decidualization of DSCs in vitro can produce soluble pro-apoptotic factors that might be critical for decidual remodeling and involution during pregnancy ; howeverAutophagy, a highly conserved cellular process responding to survival stress, has recently been found to regulate menstruation, placentation, decidualization, and maternal immune tolerance during pregnancy. Both in vivo and in vitro studies have demonstrated that autophagy is suppressed under folate-deficient conditions by aberrantly activating AMPK/mTOR, which results in impaired decidualization ,169. IntFOLR2 expression accounts for the maturation and enrichment of macrophages, and that FOLR2+ macrophages may be involved in regulating cancer progression, immune response, and angiogenesis (Recent studies have found that FRβ is expressed in inflammatory or autoimmune diseases and tumor-associated macrophages (TAMs), and it may be regulated by its positive transcriptional factor PU.1 . FRβ expogenesis . This pr+ stem cells in situ, and tissue-resident macrophages prior to pregnancy, could also be potential origins [+ cells during cell differentiation, particularly among subgroups of monocyte–macrophage precursors such as CD34+CD15+ and CD34+CD38+ cells [FOLR2+ macrophages have been reported to secrete a series of inflammatory cytokines and chemokines, such as IL-8, CCL2, CCL3, and CCL4 [FOLR2+ macrophages and newly recruited ones. It would be fascinating to investigate whether and how FRβ and its autoantibodies regulate dMφ populations by regulating cell recruitment.According to classical viewpoints, dMφ are derived from peripheral monocytes that originate in bone marrow; however, recent hypotheses propose that the embryonic precursors of macrophages, pluripotent CD34 origins . Interes8+ cells . Polyclo8+ cells . These fand CCL4 . Among tand CCL4 ,174,175,+CD86+CD11c+) and M2 (CD163+CD206+) macrophages. Resting macrophages are polarized to M1 phenotype macrophages when stimulated by lipopolysaccharide (LPS), IFN-γ, or a granulocyte macrophage colony-stimulating factor (GM-CSF). M1 macrophages, on the one hand, produce pro-inflammatory cytokines like IL-12 and TNF-α. M2 macrophages, on the other hand, can be induced by IL-4, IL-10, or M-CSF, and produce anti-inflammatory factors IL-10 and TGF-β [Macrophages can be categorized into two main phenotypes based on their functional characteristics: M1 , in the human placental stroma during the first trimester. HBCs secrete VEGFA, osteopontin (OPN), the tissue inhibitor of metalloproteinase 1 (TIMP-1), and MMP-9 to promote angiogenesis and vascular remodeling in a paracrine manner [FOLR2+ macrophages in regulating decidual angiogenesis and vascular remodeling; however, the regulatory mechanisms of FOLR2 that facilitate angiogenesis and vascular remodeling are still unclear. Further studies that intervene with FOLR2 expression and investigate correlated signaling pathways could assist in understanding the mechanisms involved.r stroma  and secrr stroma . Thomas e manner . Notablye manner . These rFOLR2 has long been recognized as a marker for the immunosuppressive phenotype of tissue-resident macrophages. In human hepatocellular carcinoma, the overexpression of CD68 and CD163 in FOLR2+ TAMs have been associated with a worse prognosis [FOLR2+ TAMs mediate immunotolerance in hepatocellular carcinoma by interacting with Foxp3+ Treg cells through CD86/CTLA4 interaction and crosstalk with endothelial cells mediated by NOTCH signaling [FOLR2+ TAMs can maintain immune tolerance in situ by secreting immunosuppressive cytokines such as IL-10 [FOLR2+ macrophages co-express CD39 and CD73, which boosts immunosuppression by activating the purinergic pathway, releasing extracellular ATP and converting it to immunosuppressive adenosine, thereby inhibiting the activity of CD4+ T cells [FOLR2 and its downstream adenosine play a role in mediating mutual immune regulation among macrophages, NK cells, and CD4+ T cells.rognosis . To reveignaling . Additioas IL-10 ,186. In  T cells . The bal T cells . These fFOLR2 may impair immune tolerance. Treating mouse peritoneal macrophages with CpG-DNA, combined with an anti-IL-10 receptor (IL-10R) antibody, can upregulate MHCII, IL-2, IL-6, and IL-13 molecular expression while suppressing FOLR2, MMP-9, and VEGF, and can reverse M2 polarization to M1. Additionally, research has demonstrated a significant reduction in detectable lung-metastasis foci in vivo, suggesting that targeting IL-10R could alter the immune tolerance established by FOLR2 [+ TAMs, an enrichment of pro-inflammatory monocytes, an influx of cytotoxic CD8+ T cells, the inhibition of tumor progression, and better prognosis in a mouse model [FOLR2 M2-like macrophages can increase the proportions of M1 macrophages and CD3+ T cells; however, it has no effect on CD4+ and CD8+ T cells [FOLR2+ macrophages play a major role in mediating immune tolerance.On the other hand, artificially inhibiting by FOLR2 . Specifise model . Therape T cells ,192. OveFOLR2+ macrophage in immune regulation. For instance, although fetal macrophages in placental stroma are confirmed to be FOLR2-positive and highly express CD206, they also demonstrate an active response to Toll-like receptor stimulation and possess active microbicidal capacity [FOLR2+ macrophages in the immune microenvironment of breast cancer that was colocalized with CD8+ cytotoxic T lymphocytes (CTLs) adjacent to lymphoid tissue. Surprisingly, they found that +FOLR2 TAMs in breast cancer were MHCII-positive and expressed the gene signatures of both M1 and M2 simultaneously. These macrophages were capable of activating CTLs via long-lasting interaction, contributing to the antitumor immune response [FOLR2 expression on macrophages regulates the intricate immune landscapes of the microenvironment through crosstalk with other lymphocytes in situ.Some studies, however, have challenged this point of view and added complexity to the roles of the capacity . Nalio eresponse . FurtherFRAbs were initially discovered in birth defects such as CFD, NTDs, and fetal malformation, which could partially explain why normal folate status aberrantly came alongside relative local folate deficiency in these cases. Recently, the roles of FRAbs in early pregnancy and miscarriage have been reported; however, since preliminary studies were only conducted using polyclonal antiserum against placental FRs (mainly consisting of FRα and FRβ) or FBP, the pathogenesis of miscarriage associated with FRAbs has not been deeply explored. In this review, we summarized the existing classical viewpoints of FRAbs and inferred several hypotheses regarding the generation of FRAbs during the periconceptional period. As emerging research on the expressional profile of FRs at the maternal-fetal interface accumulates, we attempt to discuss the potential roles of FRAbs, with regard to each specific FR isoform, in pregnancy maintenance and miscarriage. FRα mainly localizes in trophoblast cells, epithelia, and the stroma of decidua at the maternal-fetal interface; as such, FRαAb might interfere with trophoblastic biofunctions, placental development, decidualization, and decidual homeostasis. FRβ is a key signature of dMφ. Autoantibodies against FRβ might correlate with the origin of dMφ and angiogenesis, vascular remodeling, and immune regulation mediated by dMφ . FRγ andFRAbs could potentially present a new strategy for predicting pregnancy outcomes. The efficacy of FRAbs in predicting subfertility has already been verified by preliminary studies . Further


Type 2 diabetes mellitus (T2DM) is a persistent metabolic condition that contributes to the development of cardiovascular diseases. Numerous studies have provided evidence that individuals with T2DM are at a greater risk of developing cardiovascular diseases, typically two to four times more likely than those without T2DM, mainly due to an increased risk of atherosclerosis. The rupture of an atherosclerotic plaque leading to pathological thrombosis is commonly recognized as a significant factor in advancing cardiovascular diseases caused by TD2M, with platelets inducing the impact of plaque rupture in established atherosclerosis and predisposing to the primary expansion of atherosclerosis. Studies suggest that individuals with T2DM have platelets that display higher baseline activation and reactivity than those without the condition. The expression enhancement of several platelet receptors is known to regulate platelet activation signaling, including platelet glycoprotein-Ib (GPIb). Furthermore, the high expression of platelet GP1b has been reported to increase the risk of platelet adhesion, platelet-leucocyte interaction, and thrombo-inflammatory pathology. However, the study exploring the role of GP1b in promoting platelet activation-induced cardiovascular diseases in T2DM patients is still limited. Therefore, we summarize the important findings regarding pathophysiological continuity between T2DM, platelet GPIb, and atherosclerosis and highlight the potential therapy targeting GPIb as a novel antiplatelet agent for preventing further cardiovascular incidents in TD2M patients. For the past decades, type 2 diabetes mellitus (T2DM) cases have sharply increased in many countries of all income levels ,2. It isAtherosclerosis is a long-term inflammatory condition characterized by a disturbed interplay between inflammation and the hemostatic system, primarily involving the platelets, and can lead to serious cardiovascular events ,10. In tDysregulation of glucose metabolism in T2DM patients promotes an inflammatory environment, resulting in atherosclerosis development ,4,5,9,10While these agents have been successful in reducing cardiovascular events in diabetic patients, there are reported limitations associated with the current treatment strategies ,17. ThesThe platelet receptor glycoprotein (GP) Ib is a member of the family of leucine-rich repeat (LRR) protein kinases and has been recognized as transmembrane receptor type 1 ,22. It i9/L [Platelets are non-nucleated blood components identified over 130 years ago ,30. It i9/L ,30. Plat9/L ,30. Plat9/L . The pro9/L . After t9/L . Platele9/L . Further9/L .The platelet’s cytoplasm contains three types of granules ,33. The Hemostasis, thrombosis, and wound healing are regarded as the platelet’s fundamental functions and are achieved through a complicated activation process, resulting in plug development at the injury site, including vascular injury ,35. To aThe hemostatic cascade signaling pathway starts with platelet receptors and some ligand interactions expressed on the surface of endothelial cells, in the subendothelial matrix, and as soluble proteins in the bloodstream ,25,36. TIndividuals with TD2M are at great risk of developing cardiovascular disease, which can lead to severe illness and death due to pathological thrombosis resulting from the rupture of atherosclerotic plaque ,10,27. PStudies have shown that metabolic changes in T2DM can increase platelet activation, which is mostly associated with endothelial cell dysfunction ,41,42,43T2DM is associated with accelerated atherosclerosis due to reactive oxygen species (ROS) production, causing mitochondrial impairment, increased activation of protein kinase C (PKC), and advanced glycation end-products (AGEs) ,46. ROS Hyperglycemic conditions have also been associated with decreased antioxidant production, such as glutathione, associated with increased TXA2 production, leading to increased platelet activation ,38. MoreThus, platelets become more responsive to thrombin and aggregate more, increasing platelet adhesion and sensitivity ,50. SomeDuring the pathological process, platelets play an important role in vascular interactions through their receptors ,52. PlatPlatelet GPIb/IX/V’s structure has been well-reviewed elsewhere ,56. BrieThe activation, adhesion, and aggregation of platelets are crucial processes significantly contributing to advanced atherosclerosis-induced cardiovascular disease, leading to serious clinical events of CAD such as myocardial infarction or heart attack ,9,10. ThT2DM has been marked as a chronic disease with prothrombotic status because it has shown platelet changes and coagulation characteristics ,61,62. IThe platelet GPIb/IX receptor complex is essential in regulating normal and pathological platelet processes . For exaIn addition, the link between GP1b and vWF induces thromboxane A2 production, resulting in ADP secretion and fibrinogen receptor activation, the key steps in platelet activation ,72. The The interaction between the platelet receptor GPIbα and the leukocyte integrin macrophage-1 antigen (Mac-1) also plays a role in thrombosis . This inSince patients with T2DM have demonstrated higher atherogenesis and atherothrombotic complications, antithrombotic drugs are needed as management therapy for these individuals ,18,44,77Collectively, it underscores the need for newer targeted antiplatelet treatment approaches in diabetic patients. This may involve utilizing more potent medications or combining different antiplatelet drugs to enhance effectiveness ,16,17,18Researchers have conducted several studies to develop drugs targeting the GPIb receptor, subsequently decreasing platelet adhesion and preventing thrombosis ,87,89,90Another interesting study involved compounds derived from snake venom called C-type lectins, which have generated mixed results in various experimental settings ,87,94,95Moreover, the study revealed a promising safety profile for anfibatide, providing a foundation for advancing to subsequent clinical trial phases: Phases Ib-IIa and Phase II . AnfibatPlatelet adhesion and aggregation are important in preventing excessive bleeding during tissue injury, including vascular injury. These processes are particularly significant in vascular thrombosis, such as in cerebral arteries or atherosclerotic coronary vessels, which can result in stroke, heart attack, and lower extremity PAD. Numerous studies have reported that individuals with T2DM face a high risk of atherosclerotic cardiovascular disease. To manage cardiovascular thrombotic disorders in patients with T2DM, antiplatelet agents such as aspirin, GPIIb/IIIa inhibitors, and clopidogrel are currently used as treatment options. However, recent studies have highlighted the limitations of these therapies, including resistance issues and severe side effects, indicating the need for newer antiplatelet agents. Studies have shown that individuals with T2DM often experience platelet hyperreactivity and a prothrombotic state linked to increased expression of various platelet glycoproteins, including GPIb. When exposed to strong shear forces, GPIb facilitates platelet adhesion by interacting with vWF. Subsequently, the adhered platelets become activated and aggregate, forming a hemostatic plug or an occlusive thrombus. Collectively, these findings highlight the crucial role of GPIb as a bridge connecting T2DM and atherosclerosis . Therefo


Beta cells couple stimulation by glucose with insulin secretion and impairments in this coupling play a central role in diabetes mellitus. Cyclic adenosine monophosphate (cAMP) amplifies stimulus-secretion coupling via protein kinase A and guanine nucleotide exchange protein 2 (Epac2A). With the present research, we aimed to clarify the influence of cAMP-elevating diterpene forskolin on cytoplasmic calcium dynamics and intercellular network activity, which are two of the crucial elements of normal beta cell stimulus-secretion coupling, and the role of Epac2A under normal and stimulated conditions. To this end, we performed functional multicellular calcium imaging of beta cells in mouse pancreas tissue slices after stimulation with glucose and forskolin in wild-type and Epac2A knock-out mice. Forskolin evoked calcium signals in otherwise substimulatory glucose and beta cells from Epac2A knock-out mice displayed a faster activation. During the plateau phase, beta cells from Epac2A knock-out mice displayed a slightly higher active time in response to glucose compared with wild-type littermates, and stimulation with forskolin increased the active time via an increase in oscillation frequency and a decrease in oscillation duration in both Epac2A knock-out and wild-type mice. Functional network properties during stimulation with glucose did not differ in Epac2A knock-out mice, but the presence of Epac2A was crucial for the protective effect of stimulation with forskolin in preventing a decline in beta cell functional connectivity with time. Finally, stimulation with forskolin prolonged beta cell activity during deactivation, especially in Epac2A knock-out mice. Insulin serves as an important regulator of nutrient homeostasis and a lack of its effects, either due to beta cell failure or resistance in peripheral tissues, or both, results in diabetes mellitus, which affects more than 537 million people worldwide . 90% of ATP channels. The subsequent decrease in potassium efflux depolarizes the cell membrane, leading to an increase in permeability of VDCC allowing Ca2+ ions influx into the cytoplasm -dependent pathway and activation of the guanine nucleotide exchange protein 2 (Epac2A) (The triggering pathway is augmented and fine-tuned by amplifying pathways which start with either (i) an increase in [Ca2+]IC , (ii) GP2+]IC , or (iii2+]IC . The res(Epac2A) –13, whosATP channel activity in an ADP-dependent manner  and complied with all national and European recommendations relating to care and work with experimental animals to minimize animal discomfort.tm1.1Sse) were generated by Professor Susumu Seino of Kobe University as described previously  kept at 40°C dissolved in extracellular solution  125 NaCl, 26 NaHCO3, 6 glucose, 6 lactic acid, 3 myo-inositol, 2.5 KCl, 2 Na-pyruvate, 2 CaCl2, 1.25 NaH2PO4, 1 MgCl2, 0.5 ascorbic acid) with access through the proximal common bile duct, which was clamped distally at the major duodenal papilla. Following injection, the pancreas was cooled with ice-cold ECS and extracted from the abdominal cavity. A vibratome  was used to create 140 µm thick tissue slices, which were collected at room temperature in HEPES-buffered saline  150 NaCl, 10 HEPES, 6 glucose, 5 KCl, 2 CaCl2, 1 MgCl2; titrated to pH=7.4 using 1 M NaOH). The slices were incubated in the dye-loading solution , 0.03% Pluronic F-127 (w/v), and 0.12% dimethylsulphoxide (v/v) dissolved in HBS) for 50 minutes at RT. All chemicals were obtained from Sigma-Aldrich  unless otherwise specified.Acute pancreas tissue slices were obtained from 12-24 weeks old Epac2A KO mice and their WT littermates of either sex were kept in individually ventilated cages  with a 12:12 hours light: dark schedule, as described previously , 80. In 2+]IC oscillations in the phases of sustained activity, a resolution of 10 Hz at 256 x 256 pixels was maintained throughout the whole stimulation protocol. The laser power was adapted to avoid photobleaching and prolong the maximum time of recording, while maintaining a satisfactory signal strength. Cells were imaged at the approximate depth of 15 µm or more to avoid recording from cells at the surface. The optical section thickness was kept at about 4 µm to assure recordings from a single cell only. OGB-1 was excited by an argon 488 nm laser line and emitted fluorescence was detected by Leica HyD hybrid detector in the range of 500-700 nm , as described previously  and a Leica TCS SP5 AOBS Tandem II upright confocal system . Acquisition frequency was set to 1-2 Hz at 512 x 512 pixels. For a more precise quantification of IC oscillations  after prolonged stimulation with 12 mM glucose only. In the stimulation protocol with forskolin, the node degree in the second part of the plateau phase was on average higher in both types of mice when compared to stimulation with glucose only, with median values k≈10 and k≈5 for the islets from WT and KO mice, respectively. However, the difference between the control and the forskolin protocol was significant only for WT mice.To gain a more general and quantitative insight, we show in Next, we show the average correlation coefficients  and rat protein ). Experi protein , 125 and protein . While t protein . Based orrelated , 128. Werrelated , 23–25. Forskolin was able to weakly activate beta cells exposed to substimulatory glucose and beta cells from Epac2A KO mice responded faster than beta cells from WT littermates to both high glucose and to forskolin added to low glucose. During the plateau phase, forskolin added to high glucose resulted in increased oscillation frequency and relative active time, with well-coordinated activity among beta cells and this response did not critically depend on Epac2A. Prolonged exposure to glucose caused Epac2A-independent beta cell desynchronization, lower network integrity, and higher segregation, while activation of the neurohormonal amplifying pathway prevented this decline in network function in an at least partly Epac2A-dependent manner. In the end, following stimulation with high glucose and forskolin, beta cells deactivated more slowly and the prolongation of activity into the otherwise already silent period was especially well pronounced in beta cells from Epac2A KO mice. Taken together, our results suggest that especially under conditions of stimulated cAMP production, Epac2A may play a role in coordinating beta cell collective activity, with its absence resulting in earlier activation, weaker functional coupling during activity, and later deactivation.https://doi.org/10.6084/m9.figshare.23723700.v2.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The animal study was approved by Administration for Food Safety, Veterinary Sector and Plant Protection of the Republic of Slovenia. The study was conducted in accordance with the local legislation and institutional requirements.Conceptualization, MSK, AS, and MSR; methodology, MSK, JD, MG and AS; software, JD and MG; validation, MSK and AS; formal analysis, MSK, JD and MG; investigation, MSK, JD, LK and VP; resources, MSK, JD and MG; data curation, MSK, JD and MG; writing—original draft preparation. MSK, JD, LK, VP, MG and AS; writing—review and editing, AS and MSK; visualization, MSK and MG; supervision, AS and MSR; project administration, MSK, AS and MSR; funding acquisition, AS and MSR. All authors have read and agreed to the published version of the manuscript.


Immunoglobulin G4 (IgG4) is a member of the human immunoglobulin G (IgG) subclass, a protein involved in immunity to pathogens and the body’s resistance system. IgG4-related diseases (IgG4-RD) are intractable diseases in which IgG4 levels in the blood are elevated, causing inflammation in organs such as the liver, pancreas, and salivary glands. IgG4-RD are known to be more prevalent in males than in females, but the etiology remains to be elucidated. This study was conducted to investigate the relationship between gut microbiota (GM) and serum IgG4 levels in the general population.In this study, the relationship between IgG4 levels and GM evaluated in male and female groups of the general population using causal inference. The study included 191 men and 207 women aged 40 years or older from Shika-machi, Ishikawa. GM DNA was analyzed for the 16S rRNA gene sequence using next-generation sequencing. Participants were bifurcated into high and low IgG4 groups, depending on median serum IgG4 levels.Anaerostipes, Lachnospiraceae, Megasphaera, and [Eubacterium] hallii group were associated with IgG4 levels in women, while Megasphaera, [Eubacterium] hallii group, Faecalibacterium, Ruminococcus.1, and Romboutsia were associated with IgG4 levels in men. Linear non-Gaussian acyclic model indicated three genera, Megasphaera, [Eubacterium] hallii group, and Anaerostipes, and showed a presumed causal association with IgG4 levels in women.ANCOVA, Tukey’s HSD, linear discriminant analysis effect size, least absolute shrinkage and selection operator logistic regression model, and correlation analysis revealed that This differential impact of the GM on IgG4 levels based on sex is a novel and intriguing finding. AlthougIn recent years, there has been increasing attention in medical research towards the gut microbiota (GM). It has been found that the GM plays a crucial role in maintaining human health and influencing the development of diseases . AdditioTo date, no studies have examined the causal relationship between GM and serum IgG4 levels in the general population. The GM varies widely according to sex . IgG4-RD22.1The participants were 398 residents (191 men and 207 women) aged 40 years or older, of Shika-machi, Hakui-gun, Ishikawa Prefecture, Japan, whose fecal samples were collected in 2019. The following five conditions were excluded from the analysis. 1) participants without measured serum IgG4 levels, 2) patients taking immunosuppressive drugs as described below; Methotrexate and Enbrel, 3) patients taking medications that significantly affect GM as described below; antibiotics, steroids, bowel regulators and antibacterial agents, proton pump inhibitor (PPI), 4) patients suspected cancer and IgG4-related disease, 5) individuals with missing data, 6) patients with inflammatory bowel disease (IBD).2.2Data from the Shika-machi Super Preventive Health Examination, a population survey aimed at establishing preventive methods for lifestyle-related diseases, were used. The survey was conducted between 2019. The four model districts selected from the Shika area were Horimatsu, Higashimasuho, Tsuchida, and Higashiki .2). Venous blood was collected early in the morning after a 12-hour fast. The 24-hour urinary sodium excretion was calculated based on the 24-hour urinary creatinine and sodium excretion values  was calculated by dividing the current weight (kg) by the square of the height ; Immunoglobulin G (IgG) ; Immunoglobulin E (IgE) ; 50% hemolytic unit of complement (CH50) ; Anti-CCP antibody ; antinuclear antibody (ANA) (anti-nuclear antibody (ANA) (FA) [FR], FUJIREBIO INC., Tokyo, Japan); Aniti-SS-A/Ro antibody ; rheumatoid factor (RF) levels .2.3Fecal samples were collected using previously described methods  and stor2.4For microbiome analysis, QIIME2 software was used . Demulti2.5Statistical analysis and machine learning were performed using Python (version 3.10.9)  or R, usth–75th percentile). The significance of differences in clinical information between the groups was assessed using Student’s t-test for normally distributed data and the Wilcoxson rank-sum test for non-normally distributed data.The clinical information of the participants underwent a normality assessment using the Shapiro-Wilk test. Normally distributed data are expressed as mean ± standard deviation, while non-normally distributed data are presented as median  from the “glmnet” package in R  model was built using “LiNGAM” in Python . The bac33.1Data on GM were procured from the fecal specimens of 234 study participants. The study dismissed 138 samples lacking IgG4 quantification, six individuals under immunosuppressive or gut flora-altering medications, one potential cancer case, and one suspected IgG4-RD case. None of the participants had IBD. In total, 88 patients  participated in the analysis. 3.2Anaerostipes were more prevalent in women than men  had VIF <10 in all participants. In LASSO in all participants, none of the 13 bacterial genera had statistically significant odds ratios. The classification prediction model in all participants was the area under the receiver operating characteristic curve showed 0.658, sensitivity 0.750, and specificity 0.568  had VIF <10 in male. Of the 12 bacterial genera, only Romboutsia was statistically significant with an odds ratio of 2.696   was associated with a decrease in serum IgG4 levels. In the robustness analysis of causal results, a causal direction from Megasphaera to IgG4 was detected in 82.6% of cases  and from group C to IgG4 in 78.3% of cases  in all participants. A causal direction from Megasphaera to IgG4 was detected in 81.8% of cases  and from group C to IgG4 in 59.1% of cases  in women. In contrast, no causal relationship between bacterial genus and IgG4 could be inferred in males.4Anaerostipes than men in the general population. Causal inference in women showed that Megasphaera increased IgG4 levels, while the groups including [Eubacterium] hallii group and Anaerostipes decreased IgG4 levels. In men, Megasphaera, [Eubacterium] hallii group, Faecalibacterium, Ruminococcus.1, and Romboutsia were important bacterial genera for classifying high IgG4 groups and low IgG4 groups. No bacterial genera presented a causal relationship with serum IgG4 levels in men. Serum IgG4 levels may be associated with changes in the gut bacterial genera.Women had a significantly higher proportion of Blautia and elevated succinate may be responsible for hepatitis  . IL-4, ILactobacilli and Bifidobacteria are non-spore-forming, gram-positive, lactic acid-producing bacteria. Despite some common properties, Lactobacilli and Bifidobacteria belong to two taxonomically distinct groups: the genus Lactobacillus in the phylum Firmicutes and the genus Bifidobacterium in the phylum Actinobacteria, respectively  the results revealed in the present study, in which the proportion of Anaerostipes present in a suspected causal role in reducing IgG4 was significantly higher in women, and (ii) the previously reported fact that women have a lower incidence of IgG4-RD than men. These results may lead to a new hypothesis that women are less likely to develop IgG4-RD than men due to the abundance of Anaerostipes in the gut. To elucidate the pathogenesis of IgG4-RD of unknown cause, the metabolites derived from gut bacteria that regulate serum IgG4 levels need to be investigated in detail in the future.In conclusion, The raw data of the sequencing was registered at DNA Data Bank of Japan (DDBJ) (Number DRA016467). The studies involving humans were approved by the ethics committee for Human Studies at Kanazawa University Hospital. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.AK: Conceptualization, Data curation, Project administration, Validation, Visualization, Writing – original draft, Writing – review & editing. SK: Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing. KoO: Methodology, Validation, Writing – review & editing, Data curation. YM: Data curation, Investigation, Methodology, Validation, Writing – review & editing. KaO: Methodology, Writing – review & editing. RM: Data curation, Methodology, Validation, Writing – review & editing. YI: Methodology, Writing – review & editing. SH: Investigation, Writing – review & editing. IM: Investigation, Writing – review & editing. HF: Investigation, Writing – review & editing. MK: Investigation, Writing – review & editing. HT: Data curation, Writing – review & editing. AH: Investigation, Writing – review & editing. HN: Writing – review & editing, Investigation. SO: Funding acquisition, Investigation, Supervision, Writing – review & editing.


The efficacy and longevity of medical implants and devices is largely determined by the host immune response, which extends along a continuum from pro-inflammatory/pro-fibrotic to anti-inflammatory/pro-regenerative. Using a rat subcutaneous implantation model, along with histological and transcriptomics analyses, we characterized the tissue response to a collagen polymeric scaffold fabricated from polymerizable type I oligomeric collagen (Oligomer) in comparison to commercial synthetic and collagen-based products. In contrast to commercial biomaterials, no evidence of an immune-mediated foreign body reaction, fibrosis, or bioresorption was observed with Oligomer scaffolds for beyond 60 days. Oligomer scaffolds were noninflammatory, eliciting minimal innate inflammation and immune cell accumulation similar to sham surgical controls. Genes associated with Th2 and regulatory T cells were instead upregulated, implying a novel pathway to immune tolerance and regenerative remodeling for biomaterials. Engineered collagen polymeric scaffolds exhibit regenerative remodeling without eliciting a foreign body reaction, fibrosis, or bioresorption. By definition, a biomaterial is any substance (other than a drug), synthetic or natural, that can be used as a system or part of a system that treats, augments, or replaces any tissue, organ, or function of the body.1 This definition immediately brings to light the importance of biomaterial-tissue interactions and associated tissue responses as prioritized considerations when designing biomaterials for various intended uses. Synthetic materials, whether permanent or degradable, continue to be attractive candidates for medical product design, owing to advantages associated with sourcing, batch-to-batch reproducibility, high customizability, physical stability, amenability to scalable manufacturing, and cost. However, increasing concerns have been raised regarding fibrotic encapsulation and associated foreign body reactions (FBRs) elicited by these materials as well as their lack of biological signaling capacity (bioactivity).2,3 Alternative approaches have targeted use or incorporation of biological materials, including collagen, glycosaminoglycans, and other components of the tissue extracellular matrix (ECM). Here, the goal is to engage the body's cells via binding and signaling motifs inherent to these biomolecules and their structural assemblies for improved material biocompatibility and tissue integration.4 While biological materials are routinely used for ound, dental, and various surgical applications, their broader utility is limited by sensitivity to standard manufacturing processes, limited customizability of material format and physical properties, and rapid immune-mediated material degradation (bioresorption).4–6 Consequently, the search continues for advanced engineering biomaterials that are highly customizable and adaptable to patient-specific needs and support improved tissue response outcomes, namely promotion of immune tolerance, tissue integration, and stimulation of scar-free regenerative healing, where compromised tissues are restored to a normal histological and functional state.7–9Biomaterials play an integral role in modern health care, with applications extending from hemostatic and wound dressings to medical implants for tissue reinforcement or replacement , which are often associated with tissue-derived or other biological materials. Implantation of these foreign materials yields well-characterized FBRs, which are initiated by the immune system at the tissue-implant interface.10–14 Mechanistically, FBRs are often described as having multiple sequential but overlapping phases, including protein adsorption, acute inflammation, chronic inflammation, foreign body giant cell (FBGC) formation, and fibrosis/fibrous capsule formation.2,15 This reaction initiates immediately upon tissue contact, with blood-derived and interstitial protein adsorption to the material surface and associated complement activation (the first step of innate immunity). In turn, chemokines and other chemoattractants guide cells of the innate or non-specific immune system, notably neutrophils, mast cells, dendritic cells, monocytes, and macrophages, to accumulate at the implant site. Continued activation of macrophages, as well as their fusion to form multi-nucleated FBGCs, leads to a state of chronic inflammation, with engagement of adaptive immune cell populations , recruitment of additional cell types , and ultimately deposition of dense fibrous connective tissue (fibrotic capsule) that walls off the foreign material. This process is further exacerbated by phagocytosis, where neutrophils, macrophages, and FBGCs work to actively engulf and break down foreign materials, releasing reactive oxygen species and matrix degradative enzymes.It is well established that nature-derived or synthetic materials not inherent to the host are recognized as foreign objects by the immune system. Also recognized as foreign are remnant cellular debris and modified biomolecules  pathways that suppress inflammation and promote immune homeostasis.21 Given that both innate and adaptive immune cells operate along phenotypic continuums extending from pro-inflammatory/pro-fibrotic to anti-inflammatory/pro-regenerative, it is this delicate balance that ultimately determines biomaterial tissue response and healing outcomes.21 To influence this balance, many current biomaterial design strategies focus on modulating the tissue response by manipulating specific physicochemical properties, including size and shape,22 pore size,23 surface properties , mechanical properties,28,29 and degradability.30 To further augment biomaterials and promote favorable host reactions, researchers have also commonly explored controlled release of immunomodulatory agents  and surface functionalization with bioactive coatings . Overall, these contemporary approaches highlight the importance of tissue and immune response considerations in the design and translation of next-generation biomaterials.While FBGCs, fibrosis, and fibrous capsule formation are commonly reported outcomes following biomaterial implantation, immune cell phenotypes and cellular signaling pathways, material degradation or bioresorption, thickness of the fibrous capsule, and consequences of the inflammatory response vary with biomaterial type and implantation microenvironment.via physical, chemical, and/or enzymatic methods) while maintaining the complex molecular composition, architecture, mechanical properties, and bioactivity inherent to the ECM.4 Other collagen-based products, including freeze-dried collagen sponges, are fabricated from more refined starting materials, namely tissue particulate consisting of microfibrillar collagen or collagen hydrolysates, with hydrolysates representing denatured and/or enzymatically treated collagen molecules cleaved into shorter chains of amino acids.4,34 All commercial collagen-based materials, regardless of source material and method of manufacture, engage innate and adaptive immune systems, resulting in a distinct FBR, commonly referenced as constructive remodeling, that features immune-mediated material bioresorption (phagocytosis and proteolytic breakdown) and fibrous tissue formation and remodeling.35–38 Depending on a number of factors , tissue responses can vary between collagen-based products, affecting macrophage polarization, number and distribution of FBGCs, degradation rates, and tissue response outcomes.39 Overall, the complexity of present-day collagen-based biomaterials and their associated design poses ongoing challenges to the elucidation of their mechanism of action and predictive modulation of their immune response.6Given that type I collagen represents the major structural and mechanical framework of tissues, numerous collagen-based biomaterials exist within the market today. Scaffolds fashioned from decellularized animal and human tissues represent a prominent type of collagen-based product. To create these products, tissues are processed to eliminate cellular components  by mixing with a buffer, polymerization is initiated, giving rise to highly-interconnected and physically-stable fibrillar scaffolds, without use of exogenous additives or crosslinking agents.40–42 To date, engineering design tools  and scalable fabrication methods  have been applied for customization of wide variety of collagen polymeric material formats, including in situ scaffold-forming formulations, high-strength thin materials , compression-resistant constructs, and materials with gradient or aligned fibrillar microstructures.42–50In vivo, these biomaterials exhibit a unique noninflammatory, regenerative healing response with no FBR or immune-mediated bioresorption, which has been documented by implanting various material formats in a number of different anatomical locations, including dermis, subcutaneous tissue, skeletal muscle, intraperitoneal cavity, larynx, and breast.44,46–50Our design strategy for next-generation engineered collagen materials employs a unique starting material consisting of a polymerizable type I collagen protein known as oligomeric collagen . Oligomer is readily extracted and purified from porcine dermis, as well as other collagen-containing tissues from animal and human sources, thereby eliminating cellular and other immunogenic components.via immunostaining, and RNA-sequencing (RNA-seq) transcriptomics analysis, which provided expression levels of roughly 12,000 gene products. Gross and histological assessments were also performed 60 days following Oligomer scaffold implantation to define more long-term tissue responses. Collectively, results from this study further demonstrate that engineered collagen polymeric materials fashioned from Oligomer elicit a unique regenerative mechanism of action that benefits from host immune tolerance. Additionally, results suggest new paradigms where inflammation and immune-mediated bioresorption of collagen materials are not required for desirable endogenous tissue regeneration outcomes.To extend this work and continue to foster the bench-to-bedside translation of this biopolymer technology, we conducted a rat subcutaneous implantation study  to syste2.2.13) were obtained from GeniPhys, Inc. . HeliCote, a commercially available collagen wound dressing was purchased from Integra LifeSciences Corporation . Polypropylene surgical mesh (Prolene Mesh) was purchased from Ethicon . Test sample materials were created using a 10 mm biopsy punch . Oligomer scaffold and commercial collagen test samples were created under aseptic conditions; commercial mesh test samples were autoclaved prior to implantation.Custom-fabricated high-density scaffolds prepared from type I oligomeric collagen . At time points representing 3, 7, and 14 days, animals were euthanized, and the dorsum subcutaneous tissue was exposed and photographed. Tissue explants and associated biomaterials were then processed and submitted for immunostaining, histological analysis, and RNA-seq analysis. To evaluate more long-term outcomes, additional animals were implanted with Oligomer scaffold only (n = 8), with gross and histological tissue reaction assessments performed after 60 days. Subcutaneous tissue from normal, untreated rats was used as reference normal tissue.Material biocompatibility and immune response of Oligomer scaffold compared to commercial implant materials was assessed using a rat subcutaneous implant model as summarized in 2.3Processing for histological evaluation involved excision of material implant and sham sites along with a margin of surrounding normal tissue. Tissue explants were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) or Masson's Trichrome (MTC). For immunohistochemistry, sections were deparaffinized, rehydrated, processed for antigen retrieval, and stained with mouse anti-rat CD68 antibody , rabbit anti-rat CD3 , rabbit anti-rat CD4 , mouse anti-rat CD8a , rabbit anti-rat Foxp3 , or mouse anti-rat IL-17 . Slides were then treated with ImmPRESS® HRP Horse Anti-Mouse IgG Polymer Detection Kit  or ImmPRESS HRP Horse Anti-Rabbit IgG Polymer Detection Kit (Vector Laboratories) and counterstained with hematoxylin. Histological assessments were performed by an independent pathologist in a blinded fashion.2.451 with the following parameter: “–outSAMmapqUnique 60”. To evaluate quality of the RNA-seq data, the number of reads that fell into different annotated regions  of the reference genome was assessed using bamutils (from ngsutils v.0.5.9).52 Uniquely mapped reads were used to quantify the gene level expression employing featureCounts (subread v.1.5.1)53 with the following parameters: “-s 2 -Q 10”. The data was normalized using TMM  method. Differential expression analysis was performed using edgeR (v.3.12.1).54,55 False discovery rate (FDR) was computed from p-values using the Benjamini–Hochberg procedure.Each material implant and sham site, along with a specified margin of surrounding subcutaneous tissue, was carefully dissected away from the rest of the skin layers and immediately snap-frozen in liquid nitrogen. Samples were mechanically homogenized using an Omni tissue homogenizer  and total RNA isolated using TRIzol . Total RNA was further purified with an RNase-Free DNase treatment for 20 minutes , followed by clean-up using the RNeasy MinElute Cleanup kit (Qiagen) according to manufacturer's instructions. Samples were then submitted to the Indiana University School of Medicine Center for Medical Genomics for RNA-seq analysis. Total RNA was first evaluated for its quantity and quality using Agilent Bioanalyzer 2100 . Total RNA (100 ng) was used for library preparation with the KAPA mRNA Hyperprep Kit (KK8581)  following the manufacturer's protocol. Each resulting uniquely dual-indexed library was quantified and quality assessed by Qubit and Agilent Bioanalyzer, and multiple libraries were pooled in equal molarity. The pooled libraries were sequenced with 2×100 bp paired-end configuration on an Illumina NovaSeq 6000 sequencer  using the v1.5 reagent kit. The sequencing reads (25–30 million read pairs per sample) were first quality checked using FastQC  for quality control. The sequence data were then mapped to the rat reference genome rn6 using the RNA-seq aligner STAR (v.2.5)2.5post-hoc analysis (p < 0.05) using JMP Pro 15 .Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was completed using the clusterProfiler package in R, and enriched pathways were displayed using the barplot function. For individual gene results, data was compiled using GraphPad Prism 6 . Statistical significance was determined through ANOVA with Tukey 3.3.156,57 was employed to characterize and compare the local immune and tissue response of Oligomer scaffolds with those for conventional biological and synthetic materials. All animals remained healthy and displayed expected weight gains throughout the study. Qualitative assessment of surgical sites showed no evidence of erythema (redness); however, roughly 50% of commercial mesh sites across all time points showed moderate edema (swelling) and seroma formation. Based on gross and histological assessments of material implant and sham sites, it was apparent that Oligomer scaffolds were highly biocompatible and elicited a distinct short-term (≤14 days) tissue response compared to the commercial materials. To confirm that Oligomer scaffolds did not elicit any long-term deleterious reactions, additional 60-day gross and histological assessments were performed only for this material. Grossly, sham and Oligomer sites showed no obvious tissue reaction or evidence of fibrous tissue overgrowth at both 14-day . As shown in 58Principal component analysis (PCA) at each time point revealed different group profiles, with distinct clusters converging by day 14 Fig. S3. Given tR < 0.01 . For OliR < 0.01 . KEGG ove.g., neutrophil and macrophage) phenotype and activation. As shown in Ccl9, Csf2, Spp1, and Il1rn, with levels decreasing over the 14-day study period. A notable exception was Ccl17 and Ccl22, which for Oligomer scaffold maintained increased expression at 7 and 14 days . Interestingly, these genes encode chemokines known to recruit Tregs.7 Additionally, high and sustained expression of Cxcl6, a chemoattractant of neutrophils and potent angiogenic chemokine,59 was observed for Oligomer scaffold but not sham . Upregulation of macrophage phenotype and function genes was also observed with commercial collagen, including Ccl17, Ccl22, Ccl20, Il1rn, Arg1, Il10, Cxcl13, and Ifng, many of which foster engagement of the adaptive immune system.18,60 The heatmaps for mesh and commercial collagen were most similar with the exceptions that Itgam, Cd86, Ccl3, Ccl17, Ccl22, Ccl20, Il1rn, Arg1, and Cxcl1 showed somewhat lesser or no upregulation, and Ifng was downregulated for the mesh group , Csf2 (log2FC = 6.6 at 14 days), Mmp9 (log2FC = 6.4 at 14 days), and Spp1 (log2FC = 9.7 at 14 days), were amongst the most highly upregulated factors throughout the 14-day study period  and markers associated with FBGC and phagosomes (Cd44 and Itgb2) also showed moderate upregulation across all time points.10,13 Again, mesh exhibited a similar profile to commercial collagen, with the exceptions that lesser upregulation of Mmp9 (log2FC = 3.7 at 14 days) and Itgb2 (log2FC = 2.2 at 14 days) and no upregulation of Syk were observed. These differences are likely associated with divergent pathways and outcomes for FBRs, with commercial collagen and mesh yielding bioresorption and fibrotic encapsulation, respectively.Since FBGCs, which are a hallmark of FBRs, appeared to play a significant role in the tissue responses to commercial collagen and mesh but not Oligomer scaffold, signaling pathways associated with macrophage fusion and FBGC formation were also evaluated in detail. As shown in y period .13,58,61Mmp13 and Mmp9, and the tissue inhibitor of matrix metalloproteinases (Timp1), further confirming the lack of observed bioresorption, material degradation, and fibrotic response (Mmp9 (log2FC = 6.5 at 14 days), Mmp13 (log2FC = 12.6 at 14 days), and Itgb2 (log2FC = 3.5 at 14 days). Upregulation of this gene combination corroborated the active phagocytosis and rapid ECM proteolysis observed histologically.13,58,62 Modest upregulation of genes favoring ECM deposition and maintenance was also observed, including Tgfb1 (log2FC = 1.1 at 14 days), P4ha1 (log2FC = 1.3 at 14 days), Plod2 (log2FC = 4.9 at 14 days), and Timp1 (log2FC = 2.6 at 14 days) , Thbs4 (log2FC = 5.0 at 14 days) as well as Timp1 (log2FC = 3.2 at 14 days) (65 including Col3a1 (log2FC = 1.9 at 14 days), Col5a3 (log2FC = 2.3 at 14 days), Fn1 (log2FC = 1.4 at 14 days), P4ha1 (log2FC = 1.1 at 14 days), Adamts14 (log2FC = 2.0 at 14 days), and Lox (log2FC = 1.8 at 14 days)  . On the 14 days) . This wa14 days) .CD3e), T cell activation and proliferation , and inflammation , especially at later time points  and Th2  pathways . Interestingly, heatmaps also revealed a number Th1/Th17 pathway genes that were only upregulated for commercial collagen (21Il17a (log2FC = 8.3 at 14 days); Irf4 (log2FC = 1.1 at 14 days), a transcription factor implicated in induction, amplification, and stabilization of the Th17 phenotype;66Ccr6 (log2FC = 2.5 at 14 days), a receptor upregulated on Th17 cells; Ccl20 (log2FC = 3.8 at 14 days), a known Th17 and Treg chemokine;67 and Ifng (log2FC = 2.0 at 14 days) and Tgfb1 (log2FC = 1.1 at 14 days). The heatmap for mesh , synthetic polymers , biological materials , and nature-derived materials .17,19,22,24,68 Targeted cell knockout or depletion studies show that macrophages are primary contributors to the fibrotic response,69 and both neutrophils and macrophages participate in degradation of collagen-based materials.71 Consistent with this notion as well as other published preclinical and human clinical studies,72–77 we show that the commercial mesh, when implanted subcutaneously, induced fibrotic capsule formation driven by large numbers of macrophages and FBGCs. The filamentous mesh structure supported rapid cell accumulation around and throughout the mesh as well as an extended inflammatory reaction that progressed outward into the surrounding normal tissue over time. Commercial collagen featured a similar temporal accumulation of macrophages and FBGCs; however, its material composition and porous microstructure led to a different cellular infiltration pattern. In this case, cells penetrated deeper into the material from the tissue interface over time, with progressive material degradation. A distinct response was observed with Oligomer scaffold, where macrophage activation appeared transient, similar to the sham control. Because macrophages did not appear to drive the Oligomer scaffold response, definition of macrophage polarization according to traditional M1/M2 types was considered outside the scope of this work. Broadly, transcriptomics showed common and persistent upregulation of Cxcl6, Cd68, Ccl9, Csf2, and Spp1 for both commercial materials, and increased expression of Cd86, Ccl2, Il1rn, Arg1, Ccl20, Ccl22, Il10, Cxcl13, and Ifng for commercial collagen. Such inflammatory cytokine and cell marker profiles are consistent with mixed or hybrid macrophage phenotypes as suggested by other published work.78,79 For both materials, FBGCs were readily identifiable at 7- and 14-day time points, with corroborating upregulation of macrophage fusion genes  and FBGC surface markers (Cd44). Mmp9, Mmp13, and Itgb2 expression was enhanced only for commercial collagen, which is consistent with the active phagocytosis and proteolysis of collagen materials.58,62Neutrophils and macrophages are dominate innate immune cell players during both early and late phases of FBRs to implantable materials, including metals  are largely driven by immunogenic cell remnants , non-collagenous molecules, as well as leachables  and deleterious effects caused by material processing and formatting .39,84 By contrast, Oligomer materials are fabricated from highly-purified type I collagen protein building blocks, comprising full-length collagen molecules held together by the natural, non-reducible intermolecular crosslink chemistry known as histidinohydroxylysinonorleucine (HHL).41,85 The component collagen polypeptide chains  and intermolecular crosslink chemistry are highly conserved across species,86 with reported values for porcine and human sequence homologies being 97% and 94% for component α1 and α2 chains, respectively.87 Relevant to the present study, sequence homology between porcine and rat collagen is slightly less but still high, with values of 91% for the α1 chain and 90% for α2 chain. Further, the structure of Oligomer scaffolds closely resembles native collagen found within tissue ECMs,42 which likely contributes to its noninflammatory response. The molecular self-assembly (polymerization) of Oligomer occurs at physiologically-relevant pH and ionic strength and is driven by hydrophobic and electrostatic interactions, yielding highly interconnected fibrillar collagen materials.41 The component D-banded fibrils, with roughly 67 nm periodicity, are similar to those found in connective tissues,42 facilitating cellular recognition, macromolecular interactions, and mechanochemical signaling. In summary, it is reasonable to suppose that tissue response differences between Oligomer scaffold and commercial collagen are largely driven by differences in both molecular composition and multi-scale structural features.By contrast, Oligomer scaffold showed minimal innate immune cell accumulation and activation, with associated histological findings and heatmaps that largely mirrored the extent and temporal response of the sham surgical group. A notable exception was the early and persistent upregulation of et al., 2021 21 for recent review). In short, stimulation of lymphocyte migration, proliferation, and downstream maturation occurs following innate immunity engagement through antigen-presenting dendritic cells and macrophages, along with secreted cytokines. Integrative signaling between innate and adaptive immune components, in turn, drives biomaterial responses, ranging from tissue regeneration to tissue repair (fibrosis) to chronic inflammation.2,16 In the present study, transcriptomics data indicated adaptive immune system engagement within the first week following Oligomer scaffold implantation, with upregulation of Th2 and Treg pathway genes dominating by 14 days. Specifically, Oligomer induced high levels of Ccr4, which encodes a Treg receptor that binds the recruitment chemokines CCL17 and CCL22.83 Treg participation was further confirmed by the upregulation of Forkhead box P3 (Foxp3), a transcription factor essential for Treg maturation and function, as well as Treg surface receptors Ctla4 (cytotoxic T lymphocyte antigen), Icos (inducible T cell costimulatory), and Il2ra (interleukin 2 receptor α-chain).80,88,89 Previous studies have documented that recruited and resident Treg populations control inflammation after tissue injury and restrain immune responses to self and foreign antigens by modulating neutrophil and macrophage function and suppressing CD4+ and CD8+ T cell-mediated inflammation.90,91 Moreover, it has been suggested that Tregs directly facilitate tissue regeneration via activating local stem and progenitor cell populations.92–94 In addition to promoting Treg activity, Oligomer scaffold implantation led to increased Th2 pathway gene expression, including the key transcription factor GATA3 and cytokine IL-13.95 Th2 cells, in addition to Tregs, are known suppressors of Th1- and Th17-driven inflammation, with many studies suggesting a critical role of IL-13 and IL-4 activated macrophages in the resolution of inflammation and the restoration of immune homeostasis.96,97 Th2 cells have also been identified as a required cell type for the constructive remodeling response observed with decellularized ECM scaffolds.78,98,99 Overall, the promotion of Th2/Treg signaling by Oligomer scaffolds suggests that adaptive immune engagement is likely a key attribute of its unique noninflammatory mechanism of action.While the innate immune system plays a critical role in biomaterial FBRs, more recent investigations have focused on adaptive immune system participation . In the present study, commercial collagen showed rapid bioresorption, which is consistent with modest crosslinking and the 10- to 14-day resorption rating of this product. As with other collagen materials, cell-mediated material phagocytosis and proteolysis was facilitated by neutrophils, macrophages, and FBGCs, as well as associated matrix metalloproteinases MMP-13 and MMP-9, which are known to degrade intact collagen fibrils and collagen fragments, respectively.10,105,106 This degradation process is accompanied by the influx of fibroblasts and other stromal cell populations that deposit ECM components, including collagen, and progressively remodel the fibrous connective tissue. This well-known collagen biomaterial response is referred to as “constructive remodeling”, with tissue response outcomes “culminating in the formation of a tissue that is usually site appropriate, at least partially functional, and devoid of any persistent or chronic inflammatory reaction”.71,107 This remodeling process does not represent tissue morphogenesis or regeneration in the true sense, but rather is best described as a departure from the default wound contraction and scar tissue formation observed with tissue injury.34,107 Interestingly, in most situations when conventional collagen materials are exogenously crosslinked, improved persistence is achieved, but chronic inflammation and a more robust FBR are also observed.39 There is an increased number and prolonged presence of macrophages and FBGCs, enhanced type 1 immune response, and enhanced fibrosis and/or fibrotic encapsulation, ultimately yielding undesirable outcomes.39,84,107 This has led to suppositions within the biomaterials field that inflammation and biological scaffold degradation are requisite processes to achieve favorable constructive remodeling outcomes.107,108Present-day collagen materials, regardless of their method of fabrication, undergo immune-mediated bioresorption with fibrous connective tissue formation and remodeling.via mechanisms that largely involve cell-collagen mechanobiological signaling and collagen metabolism (turnover). It is notable that these mechanisms occur in absence of a material-induced inflammatory response, which is important since inflammatory signals and processes may modulate or even override these normal tissue homeostatic mechanisms.109,110 This and previously published work show that Oligomer materials persist within both non-wound and wound environments, where they maintain their structural and physical integrity, and exhibit physiological collagen turnover and regenerative remodeling, all of which is dependent upon the material format and implant microenvironment.44,47,49,50 The slow turnover observed with Oligomer materials is not surprising, since fibrillar collagen in normal connective tissues is naturally metabolized (turned over) at a very slow and controlled rate via a process that is distinct from immune-mediated bioresorption.111,112 In native tissues, molecules of fibrillar collagens are held together by mature intermolecular crosslink chemistries that influence tissue mechanical properties and collagen metabolism during tissue turnover and remodeling.113,114 As an inherent component of polymerizable oligomeric collagen, these mature, trivalent crosslink chemistries contribute to the distinct suprafibrillar self-assembly observed with these collagen building blocks, which in turn yields scaffolds with improved mechanical integrity and proteolytic resistance compared to those formed by other polymerizable collagens.40,41The immune response and regenerative remodeling observed following Oligomer material implantation is distinct from conventional collagen materials, thereby challenging current paradigms. Unlike commercial collagen scaffolds, Oligomer scaffolds do not undergo degradation (bioresorption) following implantation in the body. Oligomer scaffolds undergo cellular remodeling e.g., volume and shape), fibrillar density and microstructure, implantation microenvironment, and whether there is an associated tissue injury or void. In general, the remodeling rate increases with decreased scaffold volume, increased scaffold surface area, increased scaffold fibrillar density, and presence of tissue void/injury. The present study shows that Oligomer materials fashioned as relatively thin, high-density scaffolds show limited cellular infiltration and slow remodeling over a 60-day period when implanted subcutaneously, where there is limited tissue injury and no tissue void and limited disruption of the tissue mechanobiological continuum. Similar slow remodeling, along with immunoprotective properties, have been reported previously following subcutaneous implantation of islets  encapsulated within polymerized oligomeric collagen.48,49 More specifically, euglycemia was induced for beyond 90 days in streptozotocin-induced type 1 diabetic mice following injection/implantation of 250 islets encapsulated in 500 μL of oligomeric collagen (4.2 mg/cm3 in density) in two separate locations along their dorsum. In this case, the noninflammatory and persistent oligomeric scaffold supported and protected the foreign islets, preventing detection by the host immune system. In a separate study, thin, high-density Oligomer scaffolds, similar to those evaluated in the present study, were applied for reconstruction of a full-thickness mucosal layer defect (∼3–4 cm2) within a porcine hemilaryngectomy model.47 In that study, the thin, high-strength sheet was reported to provide immediate and suitable barrier function, with progressive regenerative remodeling occurring over an 8-week period. The newly formed mucosa was histologically similar to normal mucosa, featuring a stratified squamous epithelium supported by a vascularized and cellularized stromal layer with evidence of gland formation. Similar endogenous regeneration was reported when an Oligomer soft tissue filler formulation was evaluated in a porcine lumpectomy (breast conserving surgery) model.50 In this study, the in situ scaffold-forming collagen  conformed to and filled the complex surgical void, where it prevented contraction and supported breast tissue neogenesis, including adipose tissue and mammary glands and ducts over a 16-week period. Collectively, the regenerative remodeling observed with Oligomer materials to date has many similarities to processes associated with tissue development and morphogenesis, highlighting the importance of minimizing inflammation and maintaining the mechanobiological continuum between the tissue and material.The rate of cellularization and regenerative remodeling observed following implantation of Oligomer materials is dependent upon material geometry  as a next-generation engineering collagen polymer platform that can help address unmet tissue restoration and reconstruction needs and bring the burgeoning field of personalized regenerative medicine to clinical reality.In summary, we have further defined, through histological and tissue-level transcriptomic assessments, the immunological response and mechanism of action of implantable Oligomer materials. To the best of our knowledge, this is the first study that documents that engineered polymeric materials that recapitulate molecular composition and ultrastructural features of stable, mature fibrillar collagen support regenerative remodeling in vivo experiments. R. A. M. and S. B. performed the implantation procedures and post-surgical assessments. T. J. P. prepared the implants. R. A. M. prepared the explants for analysis. H. G. and Y. L. aided in the acquisition and interpretation of transcriptomic analysis. A. C. performed all histopathological analyses. S. V-H. and R. A. M. wrote the manuscript and all authors review, provided comments, and edited the manuscript.R. A. M, S. V.-H. and S. B. designed the S. V.-H. is the Founder and CSO of GeniPhys, a start-up company who licensed polymerizable collagen technology from Purdue Research Foundation for development and commercialization. T. J. P. serves as the GeniPhys Project Development Manager.BM-011-D3BM00091E-s001