Cell Communication and Signaling

Human embryo implantation, occurring approximately one week after fertilization, remains poorly understood due to ethical and technical limitations of in vivo investigation. To overcome these barriers, and model this critical developmental event, encompassing peri- and early post-implantation stages, we used an in vitro embryo attachment model composed of donor-derived endometrial epithelial cells forming an open-faced endometrial layer (OFEL) and human stem cell-derived blastoids recapitulating human day 5 blastocysts in peri-implantation model. Following attachment, developmental progression was further investigated on laminin-coated substrates to capture early post-implantation dynamics. Despite its central role as the primary endocrine signal of early pregnancy, human chorionic gonadotropin (hCG) remains largely uncharacterized in this context. Here, we describe the transcriptomic profile of blastoid-endometrial co-cultures relative to OFEL alone, identifying CGA and CGB3/5/8 as among the most strongly upregulated genes following blastoid attachment to hormonally stimulated OFEL. Consistent with these findings, immunoassays and luteinizing hormone/choriogonadotropin receptor (LHCGR) activation assays of conditioned media confirmed the secretion of heterodimeric, biologically active hCG and its free subunits in co-cultures, but not in endometrial layers alone. Notably, the hyperglycosylated hCG heterodimer was the predominant isoform detected. Co-culture with the endometrial component significantly increased hCG secretion compared with blastoids cultured alone, an effect further enhanced by hormonal priming in the peri-implantation model. Collectively, these findings indicate that a hormonally primed endometrial environment not only promotes blastoid attachment but also amplifies embryonic hCG production and bioactivity, underscoring the importance of maternal endocrine cues in early embryo-endometrium communication. Furthermore, our peri- and early post-implantation models recapitulate key aspects of reciprocal endocrine signaling between embryonic and endometrial tissues, providing a tractable experimental framework to investigate embryo-endometrium crosstalk.

ObjectivesCD4+ T cells play key roles in regulating immune responses during pregnancy, therefore we aimed to understand the CD4+ T cell surface proteome and transcriptome during pregnancy. MethodsCD4+ T cells were analysed in blood and decidua from term-pregnancies (>37 weeks), and non-pregnant blood. >350 surface proteins were screened via flow cytometry, and transcriptomes were analysed using single-cell RNA sequencing with >130 CITE-seq barcoded antibodies. ResultsSurface protein screening identified changes to ILT4/CD85d, CD9, IFN-{gamma} receptor {beta}-chain, CX3CR1 and CCR5 in the pregnant blood and decidual CD4+ T cells. CX3CR1 and CCR5 had the highest expression on the effector-memory T cell (TEM) subset in the blood, with expression consistent across subsets in decidua. CD126/IL-6R was lower in pregnant blood and decidual CD4+ T cells, while scRNAseq identified enrichment in the IL-6R signalling pathway in naive CD4+ T cells in pregnant blood. Both sIL-6R and IL-6 concentrations were increased in plasma during pregnancy, suggesting perturbations to the IL-6/IL-6R signalling axis. Meanwhile, decidual CD4+ T cells had increased expression of transcription factor RUNX3 in the CD69+ tissue-resident-like subset. ConclusionsOur findings demonstrate altered molecular expression in CD4+ T cells during pregnancy. This provides important mechanistic insight of their adaptation and regulation during placental development, which may drive placental dysfunction or pregnancy complications including preeclampsia, fetal growth restriction and stillbirth. These new data may inform future studies that focus on determining the significance of differentially- expressed immune features in pregnancy to identify potential targets for immune modulation to treat pregnancy complications and infections.

BackgroundInflammatory bowel disease (IBD) is a chronic inflammatory disorder characterized by gut microbial dysbiosis and immune dysregulation. While compositional changes in the microbiome are well studied, the functional mechanisms through which microbes influence host signalling remain poorly understood. PurposeThis study aimed to investigate microbial-host molecular mimicry in IBD and to elucidate its role in modulating immune and neuronal pathways through a newly proposed Microbial Signal Recognition and Neuronal Mimicry (SRNM) axis. MethodsShotgun metagenomic datasets from IBD patients and healthy controls were analyzed using a custom Molecular Mimicry In Silico Pipeline (MMIP). Reads were assembled, annotated, and subjected to protein homology mapping, Gene Ontology enrichment, PFAM domain analysis, and taxonomic profiling to identify microbial proteins mimicking human functional pathways. ResultsIBD-associated microbiomes exhibited significantly higher functional complexity and enrichment of eukaryote-like proteins compared to healthy controls. Microbial proteins mimicking host pathways involved in neuron projection development, signal recognition particle (SRP)-mediated protein targeting, immune signaling, and stress responses were markedly enriched in IBD. Key human-like targets included TRPV1, CAMK2D, SNCA, MTCP1, TCL1B, and PEAK3. PFAM analysis revealed overrepresentation of kinase domains, zinc-finger motifs, ankyrin repeats, and ABC transporters. These signatures were predominantly contributed by IBD-enriched taxa such as Gammaproteobacteria, Fusobacteria, and Betaproteobacteria. ConclusionThis study identifies a previously unrecognized SRNM axis in IBD, revealing how microbial molecular mimicry may influence neuroimmune signaling and disease pathogenesis, and highlight potential targets for microbiome-based therapeutic intervention.

PurposeTo uncover the molecular mechanisms of corneal sensory nerves (CSN)s involvement in the initiation of Pseudomonas aeruginosa (PA) keratitis and the roles of the miR-183/96/182 cluster (miR-183C) in this process. MethodsmiR-183C conventional knockout (KO) or sensory neuron-specific (SNS) conditional (C)KO mice and their age- and sex-matched wild type (WT) controls were used. TG SN were isolated. Neurite growth and branching were analyzed by neurite tracing. Custom-made microfluidic chambers (MFC) were used to separate the neuronal cell bodies in the soma chamber and their neurites/nerve endings in the axon chamber. TG SNs response to lipopolysaccharide (LPS) or PA infection of the neurites/nerve endings was studied by ELISA assays of CX3CL1 and substance P (sP) in the axon chamber. Target luciferase reporter assays were performed to validate key downstream target genes of miR-183C. ResultsThe total neurite length and number of branches per TG SN were decreased in the CKO vs WT mice, and in the male vs female WT mice. PA infection, but not LPS alone, induced the production and secretion of CX3CL1 and sP in WT mice; while TG SN of miR-183C KO mice responded to both LPS and PA and were significantly enhanced when compared to WT mice. Antagonists to TLR4 and/or FPR1 inhibited PA-induced responses. Target luciferase reporter assays confirmed that genes encoding NRP1, TAC1-the precursor gene of sP, CX3CL1 and ADAM10, a metalloproteinase involved in the production of soluble CX3CL1, were direct targets of miR-183C. ConclusionsPA directly activates TG SN and induces chemokine and neuropeptide production/secretion through TLR4 and FPR1 receptors, which may contribute to the initiation of PA keratitis. miR-183C regulates TG SN neurite growth, chemokine and neuropeptide production/secretion and the response to PA infection by targeting a collection of key genes involved in axon guidance/projection-, chemokine and neuropeptide biogenesis- and receptors mediating PA-induced activation.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and lacks effective therapies. The stimulator of interferon genes (STING) has been shown to both suppress and promote migration in various cancer types, but its role in TNBC remains unclear. To investigate this, we established STING-overexpressing murine TNBC cell lines and assessed their migratory and proliferative behavior. STING overexpression significantly suppressed cell migration without affecting cell proliferation. Furthermore, STING overexpression upregulated expression levels of Itgb1 and Itga6 significantly, but not Icam1, Cxcl3, Itgb2, Lama5, and Rhoa. These findings highlight the potential anti-migratory role of STING beyond immunomodulatory functions.

Toll-like receptors (TLRs) are vital components of the innate immune system, recognizing both exogenous pathogens signals (PAMPs) and internal stress signals (DAMPs). TLR2 is unique among the human (Homo sapiens) TLR family members, as it contains a large cavity for binding hydrophobic ligands, such as lipoteichoic acid (LTA) and di/triacyl lipopeptides (Pam2/3CSK4). This study analyzed the structural phylogeny of cavity presence in the TLR2 lineage in vertebrates (vTLR) enabled by AI protein structure predictions and explored the potential convergent evolution of similar features in invertebrates (iTLRs). Analysis of AI models of TLR2s shows that this cavity is consistently present in TRL2 orthologs across jawed vertebrates (Gnathostomata). In jawless vertebrates (Cyclostomatha), these cavities were found in lamprey (Petromyzon marinus) TLR2 model, but only in some extant hagfish (Myxini), suggesting an ancestral origin in basal vertebrates followed by lineage-specific losses. TLR2 paralogs were found in several species, with a similar central cavity but potentially different ligand specificities. In silico ligand docking showed Pam2CSK4 binds to this cavity in all TLRs and paralogs consistently, demonstrating the conserved function of the ligand-binding pocket in gram-positive bacteria recognition across TLR2 branches. Changes in the TLR2 cavity size and shape in some vertebrate groups show the evolution of this DAMP recognition mechanism adapted to its respective pathogens. iTLRs form a separate phylogenetic branch with distinct structural features, but in literature some are considered to be TLR2 orthologs. Indeed, TLRs from some species of Helobdella and Ciona, contain a cavity with some similarity to that in the vTLR2 lineage. However, detailed structural comparisons of their location in the LRR domain and the structural details of the models suggest that their cavities have developed independently from that in TLR2s. Smaller cavities are present in other branches of the LRR family, but show different locations, shapes, and features, indicating that the binding of small ligands in the internal cavities within the LRR domains evolved multiple times in the LRR domain family history.

Objective: To investigate, in the context of endometriosis management, the perceptions of patients and healthcare professionals regarding hormonal treatment options. Design: Qualitative study using semi-structured focus group methodology. Setting: University hospitals and academic research center. Subject(s): Patients with endometriosis (n=20) and healthcare professionals (n=13) involved in their care. Intervention(s): Not applicable Main Outcome Measure(s): Focus group topics investigated representations on the concept of treatment effectiveness, emotion associated to this medical management and the perceived impact of these therapies on patient-professional and patient-environment relationship. Result(s): We highlighted a discrepancy between patients and doctors regarding the concept of efficacy of hormonal therapies. Long-term amenorrhea is the main priority for healthcare professionals, whereas pain reduction remains the immediate wait for patients. Interviewed patients reported a lack of listening and empathy, a shared-information deficit as regards treatment options and side-effects and a need to involved partner and family in care. These factors contribute to communication issues between patients and doctors and appear to contribute to significant mental burden on both sides. Among healthcare professionals, mental burden appears to arise primarily from the resource-intensive demands of endometriosis management, whereas among patients it is driven more by the need to take an active role in their own care to compensate for insufficient information provided by physicians. Conclusion: In this study, we highlighted the ambiguities surrounding the concept of therapeutic efficacy of hormonal therapies and collected several factors to try to improve shared-decision-making process in the management of endometriosis. This is designed to help us create a shared decision-making tool in the near future.

Macrophages/osteoclasts are highly fusogenic cells that interact closely with bone-metastatic breast cancer cells. These cancer cells adapt to bone microenvironments by undergoing osteomimicry, acquiring bone-like phenotypes. Exploration using human breast cancer-bone metastases dataset revealed that a small population of epithelial breast cancer cells express osteoclast-like and osteomimicry genes at the single-cell level. Cell fusion and cell-in-cell (CIC) processes are two uncommon yet prognostically significant mechanisms in cancer. We showed that co-culture between murine breast cancer cells and osteoclasts yielded a unique osteoclast phenotype through dynamic cell-in-cell (CIC) interactions and fusion-like behaviours between pre-osteoclasts/mature osteoclasts and breast tumor cells, resulting in osteoclast-tumor hybrid-like cells. These tumor cell interactions characterized by membrane retention and nuclear adjacency to host nuclei were consistently observed throughout osteoclast differentiation. Single-cell sequencing analysis and interpretative assays on hybrid-like cells revealed altered extracellular matrix (ECM) modification processes, immunoregulatory, and cancer-associated pathways compared to unfused osteoclasts. Tumor cells co-cultured with osteoclasts expressed hematopoietic and osteoclast-lineage factors more strongly than tumor cells cultured alone with their effects amplified under direct cell-cell contact. The presence of these hybrid-like cells was validated in human breast cancer-bone metastases. We propose that disseminated bone-tropic breast cancer cells were stimulated by osteoclasts to undergo a non-canonical, dynamic osteoclast differentiation and CIC formation to form hybrid-like cells that may facilitate bone metastatic lesions.

B7-H3 (CD276) is an immune checkpoint molecule frequently overexpressed in hepatocellular carcinoma (HCC) and represents a promising therapeutic target. However, its roles in tumor cell adhesion, metastatic behavior and immune evasion--particularly in interactions with natural killer (NK) cells--remain incompletely understood. In the present study, B7-H3 was depleted using small interfering RNA and CRISPR/Cas9 in epithelial (Huh7 and HepG2) and mesenchymal (SNU449) HCC cell lines. Tumor cell survival, apoptosis, adhesion, migration and invasion were evaluated using functional assays. Expression of adhesion molecules and immune checkpoint proteins was assessed by flow cytometry and western blotting. Oncogenic signaling pathways were analyzed by examining phosphorylation of Akt, ERK, FAK and STAT3. NK cell-mediated cytotoxicity was assessed using primary human NK cells. B7-H3 depletion reduced clonogenic survival and increased apoptosis in mesenchymal HCC cells under anchorage-independent conditions. Loss of B7-H3 impaired cell adhesion, migration and invasion, accompanied by downregulation of PTGFRN, E-cadherin, integrin 3 and integrin V, and reduced cell-to-cell aggregation under anchorage-independent conditions. B7-H3 depletion also decreased surface expression of PD-L1, PD-L2 and CD47. Notably, B7-H3-deficient cells exhibited enhanced susceptibility to primary NK cell-mediated cytotoxicity. Mechanistically, B7-H3 promoted tumorigenic signaling through Akt/S6, MVP/ERK and FAK/Src pathways in epithelial cells, and through FAK/Src and JAK2/STAT3 pathways in mesenchymal cells. Together, these findings reveal previously unrecognized roles for B7-H3 in coordinating adhesion and NK immune evasion in HCC, and support its therapeutic targeting for next-generation immunotherapies.

Background: Endometriosis affects approximately 10% of women of reproductive age and is associated with pelvic pain, infertility, and reduced quality of life. Dienogest is widely used for medical management. This study evaluated the effects of dienogest on endometrioma size, serum CA-125 levels, and pelvic pain. Methods: In this retrospective study, medical records of 45 women aged 18-49 years who received oral dienogest (2 mg/day) for at least six months were reviewed. Endometrioma size was assessed by ultrasound, pelvic pain using the Visual Analog Scale (VAS), and serum CA-125 levels from laboratory records. Baseline and six-month values were compared using the Wilcoxon test and correlations were analyzed using Spearman's test. Results: After six months of treatment, significant reductions were observed in endometrioma size and VAS scores (p<0.001) and CA-125 levels (p<0.001) compared with baseline. No significant correlation was found between endometrioma size and VAS scores or CA-125 levels either before or after treatment (p>0.05). A significant negative correlation was identified between patient age and post-treatment endometrioma size (r = -0.320, p<0.05). Conclusion: Six months of dienogest therapy was associated with significant improvements in lesion size, pain, and biochemical markers. Dienogest may represent an effective medical treatment option for symptomatic patients, particularly for those seeking to avoid surgery and preserve ovarian reserve.

Colorectal cancer across sub-Saharan Africa presents a growing global health burden, with increasing cases and mortality linked to late diagnosis, limited healthcare access and lack of effective treatments. African patients typically present with aggressive disease marked by distinct genomic signatures, indicating the need for targeted treatment approaches. We integrated genetic modelling, phenotypic scoring, imaging and biochemical analysis to explore how mutations found in individual Nigerian colorectal cancer patients influence drug responsiveness. We used the data from Cancer Genome Atlas to identify mutation profiles specific to Nigerian patients. We then generated ten stable Drosophila melanogaster personalised patient avatar lines designed to model patient genomic profiles. This study focused on three lines; each line included oncogenic RAS plus targeting patient-specific variants. These models exhibited various phenotypes including altered larval size, gut size and reduced survival. Two of the three avatar lines showed improved survival, reduced hindgut proliferation zone expansion and restored redox balance after treatment with regorafenib and trametinib. Mirroring clinical patient responses, we found that response to therapy is dependent on the specific genetic profile of the tumour. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/714433v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@110518aorg.highwire.dtl.DTLVardef@5965a0org.highwire.dtl.DTLVardef@11f16a3org.highwire.dtl.DTLVardef@744a1_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIAfrican colorectal cancer showed distinct mutation patterns that contribute to tumour heterogeneity. C_LIO_LIPatient-derived Drosophila avatars were engineered using tumour-specific genetic mutations with key features of human colorectal cancer. C_LIO_LITreatment with targeted therapies showed responses patterned by tumour genotype. C_LIO_LIResponse patterns indicated the need for personalised for colorectal cancer therapies among diverse populations. C_LI

Biofilm formation is a major cause of chronic implant-related bone infections and is associated with impaired immune responses. In a previous study, we identified the cGAS-STING pathway as a potential therapeutic target, as its activation--observed in response to planktonic Staphylococcus aureus (SA)--was absent in the corresponding biofilm setting. The present study aimed to identify potential mechanisms underlying the lack of cGAS activation in the biofilm environment. As biofilm-derived nucleases might degrade cGAS ligands, we assessed presence and activity of micrococcal nuclease in conditioned media from planktonic and biofilm-grown SA and evaluated the impact of extracellular DNases on cGAS pathway activation in macrophages. In addition, we examined altered cGAS expression, the requirement for continuous biofilm exposure and potential downstream inhibition resulting from degradation of the cGAS product. Biofilm formation was associated with dynamic nuclease expression, and exposure to the biofilm environment led to reduced cGAS levels in macrophages, accompanied by a lack of interferon response. Exogenous cGAS activation by G3-YSD failed to restore signaling, independent of nuclease activity or continuous biofilm exposure. In contrast, supplementation with the cGAS product and STING ligand 2'3'-cGAMP fully restored interferon responses and enhanced macrophage activation, indicating that increased degradation of the second messenger in the biofilm environment is not responsible for impaired pathway activation. Similar effects observed with Staphylococcus epidermidis and primary macrophages suggest a broader mechanism that is not SA- or cell line-specific. In conclusion, our data provide novel mechanistic insight into biofilm-mediated impairment of cGAS-STING signaling, revealing a previously unrecognized mechanism of immune evasion in staphylococcal biofilms. These findings extend our previous work and support the therapeutic potential of targeting STING as promising strategy to restore immune responses in chronic implant-related bone infections. HighlightsO_LIBiofilm-derived factors impair cGAS-STING pathway activation and suppress interferon responses in macrophages. C_LIO_LIImpaired signaling is not primarily explained by extracellular micrococcal nuclease-mediated degradation of potential cGAS ligands. C_LIO_LIBiofilm exposure reduces cGAS expression levels and inhibits exogenous cGAS activation independently of continuous presence. C_LIO_LIExogenous 2'3'-cGAMP fully restores interferon responses, indicating that impaired signaling is not due to degradation of the cGAS product. C_LIO_LIDirect activation of STING broadly enhances macrophage activation and by this could amplify overall immune responses. C_LIO_LIBypassing cGAS via direct STING targeting represents a potential therapeutic strategy to overcome immune evasion in chronic implant-related bone infections. C_LI

The human neonatal immune system is developmentally specialized to balance the unique requirements of perinatal transition. Disruption of this finely tuned balance, as in preterm birth, may have profound consequences for immunity and overall health. However, the impact of prematurity on immune composition and functional responsiveness across gestational ages (GA) remains incompletely understood. Single-cell profiling has advanced our understanding of neonatal immunity, yet most studies were limited to unimodal readouts, narrow GA windows, or baseline function. Here, we present a comprehensive human neonatal CITE-seq atlas (82 samples from 25 neonates and 10 adults as controls) at the first days of life covering a wide GA range and integrating baseline and stimulated conditions. Most notably, we identify a GA-dependent immune transition point centered around 32 weeks of GA, which discriminates extremely and very preterm neonates (GA <32wks) from those of higher GA ([&ge;]32wks). In particular, early-life immunity in extremely and very preterm infants showed CD15+ granulocytic myeloid derived suppressor cell-like predominance, whereas more mature neonates exhibited interferon-primed transcriptional profiles. This resulted in divergent myeloid-to-lymphocyte signaling networks and qualitatively distinct NK- and T-cell bystander responses upon activation. Together, these findings show that intrauterine development imprints GA-specific immune programs. By defining a developmental transition around a GA of 32 weeks that regulates baseline and induced responses of neonatal immune cells, our atlas provides a framework for understanding the vulnerability of preterm infants and thus may pave the way for developing GA-adapted immunomodulatory strategies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/715643v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1db4534org.highwire.dtl.DTLVardef@9c9665org.highwire.dtl.DTLVardef@55f063org.highwire.dtl.DTLVardef@190a52_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundThis study aims to examine the independent relationships between individual components of metabolic syndrome (MetS) and two key clinical outcomes in patients with Crohns disease (CD): disease activity, as quantified by the Crohns Disease Activity Index (CDAI), and the occurrence of complications. MethodsThis retrospective cross-sectional study included 376 adults with newly diagnosed Crohns disease. Multiple linear regression was used to examine associations between metabolic parameters and CDAI scores, while multivariate logistic regression assessed links to complications. Analyses were also based on clinical CDAI cut-offs. Predictive nomograms were developed and internally validated via bootstrap resampling. ResultsMultiple linear regression indicated that higher CDAI scores were independently associated with lower BMI (B = -5.866, P < 0.001), lower HDL-C levels (B = -81.770, P < 0.001), higher triglycerides (B = 15.618, P = 0.001), and lower ESR (B = -0.375, P = 0.03). Multivariate logistic regression established low HDL-C (OR = 0.042, P < 0.001), low BMI (OR = 0.915, P = 0.034), and high triglycerides (OR = 1.792, P = 0.007) as significant independent risk factors for complications. The developed nomograms demonstrated strong predictive performance, with an adjusted R2 of 0.207 for the CDAI model and an AUC of 0.765 for the complication model. For both predictive tasks, the model incorporating separate TG and HDL-C measurements significantly outperformed the TG/HDL-C ratio model. ConclusionMetabolic disturbances demonstrate a significant association with increased disease severity and a higher risk of complication development in Crohns disease. Core tipO_LIDual-outcome study reveals HDL-C and TG differentially link to CD inflammation and complications, pointing to distinct mechanisms. C_LIO_LILow HDL-C is the strongest independent predictor for CD complications, underscoring its protective role beyond cholesterol transport. C_LIO_LIIndividual TG and HDL-C metrics outperform their ratio in prediction, challenging its use and suggesting independent pathways in CD. C_LIO_LILow BMI independently associates with both adverse outcomes, refining the "obesity paradox" and highlighting malnutritions key role. C_LIO_LIA practical, validated nomogram (AUC=0.765) integrates HDL-C, TG, and BMI to stratify complication risk, aiding clinical decision-making. C_LI

RNF43 is best known for removing the Wnt-receptor complex from the cell surface, thereby maintaining Wnt-signaling at minimal essential levels. Recent studies reported that RNF43-mutant colorectal cancers carrying the common BRAFV600E mutation, respond more effectively to combined BRAF/EGFR inhibition. To determine whether RNF43 directly regulates EGFR or BRAF protein abundance, multiple pancreatic and colorectal cancer cell line models were generated in which RNF43 was knocked out, repaired, or stably overexpressed. Total and cell surface EGFR levels, as well as endogenous BRAF expression, were quantified. Across all models, no consistent evidence emerges that RNF43 modulates endogenous EGFR or BRAF levels. R-spondins likewise fail to alter EGFR levels or internalization. Notably, elevated EGFR expression observed in a subset of RNF43 knockout clones is induced by unintended CRISPR/Cas9 vector integration rather than the absence of RNF43 itself, highlighting a previously underappreciated artefact that can confound interpretations of EGFR regulation in genome edited lines. Overall, the data argue against a direct and general role for RNF43 in controlling EGFR or BRAF protein abundance, contradicting recent reports that propose degradation of these targets. Further studies are required to resolve these discrepancies and clarify the mechanistic basis underlying these conflicting observations.

BackgroundPolycystic ovary syndrome (PCOS) is a prevalent endocrine disorder with complex pathophysiology and limited therapeutic options. Identifying key molecular drivers and potential drug candidates is critical for improving clinical outcomes. MethodsWe integrated multi-cohort transcriptomics (GSE155489, GSE138518, GSE226146) with weighted gene co-expression network analysis (WGCNA), protein-protein interaction (PPI) network analysis, and drug repurposing. Differential expression analysis identified 1,039 DEGs, and WGCNA identified 10 PCOS-associated modules. Intersection of DEGs with module genes yielded 498 core candidate genes, which were subjected to functional enrichment, PPI network analysis, and connectivity map-based drug repurposing (CLUE/LINCS). Candidate drugs were further evaluated by molecular docking and ADMET prediction using a triple intersection strategy (hub genes, high differential expression, drug-target evidence). ResultsFunctional enrichment revealed significant enrichment in cell adhesion and TGF-beta signaling. PPI network analysis identified CD44 as the top hub gene (degree=42). Drug repurposing identified 106 candidate drugs, including troglitazone and enzalutamide. Using the triple intersection strategy, five genes (ID2, NR4A1, GJA5, ID1, MYH11) were prioritized for molecular docking. GJA5 showed strong predicted binding affinity with flufenamic acid (-7.88 kcal/mol), and cytosporone B exhibited favorable druglikeness (0 Lipinski violations). ConclusionThis study systematically characterizes PCOS-associated gene networks and provides a prioritized set of candidate targets and drugs through a purely computational framework. CD44 emerges as a key network node with potential relevance in PCOS pathophysiology. These findings offer testable hypotheses for future mechanistic studies and drug discovery efforts in PCOS.

Alternative pathways of antigen presentation are crucial in different immunological contexts such as autoimmunity and anti-microbial defense. Among these pathways, autophagy has a central role in delivering cytosolic substrates to the MHC class II compartment. However, its contribution to endogenous MHC class II presentation was only demonstrated for a few antigens. Here we focused our study on the contribution of autophagy to the peptidome of one major allele of the HLA-DR shared epitope, HLA DRB1*04:01 conferring the greatest risk factor for the development of rheumatoid arthritis (RA). We provide an extensive qualitative and quantitative mass spectrometry analysis of the autophagy related MHC class II peptide repertoire of the human DRB1*04:01 allele. A fraction of peptides representing 30% of the repertoire differ profoundly between autophagy sufficient and deficient cells. Our analysis demonstrates that autophagy contributes to MHC class II presentation of peptides from seven described RA autoantigens, the majority of them being related to the ER folding and stress response (calreticulin, calnexin, the 78 kDa glucose-regulated protein (GRP78)-also known as binding immunoglobulin protein (BiP) and several protein from the heat-shock-protein 70 family). Our results correlate with an increased activation of autophagy, in situ, in synovial biopsies and synovial fibroblast (SF) of RA patients. We could further show that SF upregulate MHC class II and present peptides from autophagy related auto-antigens to CD4 T cells from RA patients. Our finding identifies autophagy as a potential process that could contribute to the break of peripheral tolerance during RA.

Pregnancy is a unique period regarding immune cell regulation. Within the placenta, maternal immune cells play a central role in immune surveillance and tissue remodeling. However, regulatory mechanisms of systemic immunity during pregnancy are less clear. Here, we show that neutrophil function is altered in pregnant mice (E13.5), indicated by increased slow rolling velocity and reduced adhesion. Mechanistically, PreImplantation factor (PIF), a 15 amino acid peptide which is produced by human and murine trophoblast cells of the placenta, is continuously secreted into the maternal circulation and plays a key role in modulating neutrophil function via blocking the voltage-gated potassium channel KV1.3. This resulted in impaired intracellular Ca2+ signaling and subsequently disturbance of neutrophil post-arrest modifications and a higher susceptibility to physiological shear forces in vivo and in vitro. Furthermore, PIF-mediated KV1.3 blockade impaired E-selectin-mediated release of S100A8/A9 and phagocytosis. Taken together, we have identified PIF as an important modulator of neutrophil function during pregnancy suggesting a critical role in regulating innate immune responses throughout gestation.

T cell development in the thymus is a tightly regulated process where epigenetic modifications, such as histone 3 lysine 27 acetylation (H3K27ac), play a crucial role in controlling the activation of genes. The epigenetic regulation of human mucosal-associated invariant T (MAIT) cell development is unknown; we mapped the regulatory chromatin landscape in the three developmental stages of thymic MAIT cells to identify the regulatory elements and enhancer activity involved in thymic maturation and analysed whether these chromatin dynamics are associated with the acquisition of effector programs in developing MAIT cells. Utilising cleavage under target and tagmentation (CUT&Tag), genome-wide H3K27ac profiles were generated and combined with transcriptome data from thymic MAIT cells, which revealed how developmental shifts in enhancer activity correspond to changes in gene expression. In total, 41,958 genomic regions with H3K27ac signal were identified in MAIT cells across the three development stages, of which 1,200 regions showed acetylation changes during differentiation from stage 1 to stage 3. At dynamic regions, the greatest differences were observed between stage 1 and stage 3, highlighting a progressive gain or loss of H3K27ac during MAIT cell development. Overall, MAIT cell maturation was associated with the gradual accumulation of H3K27ac at promoters and enhancers, which closely correlated with gene expression changes during development. Stage-specific enrichment of H3K27ac was observed at key transcription factor gene loci involved in MAIT cell development, including ZBTB16 (PLZF), EOMES, RUNX3, NFATC2, FOXO1, TGIF1, IRF1, and MAF genes. Epigenetic remodelling was also observed at cytokine and cytokine receptors (IL7R, IL18R1, IL23R, IFNG), chemokines and chemokine receptors (CCL4, CCL5, CCR5, CCR9, CXCR4, CXCR6), as well as several surface molecules with known immunological function. Our work reveals a previously uncharacterised epigenetic profile of human MAIT cells that regulates and inuences their development.

BackgroundInterferon-gamma (IFN-{gamma}) is the primary effector cytokine of adaptive anti-tumor immunity, yet it paradoxically induces a potent immunosuppressive tumor microenvironment (TME). The full mechanistic scope of this paradox in head and neck squamous cell carcinoma (HNSC) has not been characterized at the transcriptomic scale. MethodsUsing TCGA HNSC RNA-seq data (n = 522), we applied an integrated computational pipeline: Spearman correlation analysis, principal component analysis (PCA), UMAP, K-means clustering (k = 4), Random Forest regression, deep neural networks, permutation importance, JAK-STAT cascade mapping, and DNN-based transcriptome-wide mediation analysis across 57 IFN-{gamma} pathway and 78 immunosuppressive genes. ResultsIFN-{gamma} pathway activity was universally and positively correlated with six immunosuppressive axes, including checkpoints (CD274; LAG3; IDO1), Tregs, myeloid suppression, and tryptophan catabolism. K-means clustering identified four immunologically distinct tumor subgroups. DNN models predicted suppressive TME. Permutation importance identified IRF8 as the dominant mediator linking IFN-{gamma} signaling to immunosuppression. DNN mediation analysis identified PDCD1LG2 (PD-L2) as the strongest intermediary between IFNG and PD-L1 regulation, followed by JAK2 and GBP5. ConclusionsIFN-{gamma} orchestrates coordinated immunosuppression in HNSC through JAK-STAT-IRF8 signaling. PDCD1LG2 and JAK2 are actionable mediators of this paradox, supporting combination strategies co-targeting IFN-{gamma}-induced checkpoint induction and direct checkpoint blockade in HNSC immunotherapy. GRAPHICAL ABSTRACT