Science Translational Medicine

Individuals with fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder caused by mutations in the bone morphogenetic protein receptor ACVR1 (also known as ALK2), experience progressive and severely debilitating endochondral heterotopic ossification (HO). Previous studies have identified fibro-adipogenic progenitors (FAPs) as a major cellular source of HO in FOP mouse models. Here, we used mRNA sequencing of FAPs isolated from injured muscle of FOP mice to capture the earliest gene expression changes associated with the transformation of muscle tissues into bone. Whereas the transcriptomes of mutant and wild-type FAPs were indistinguishable prior to injury and at 1 day post injury, dramatic dysregulation of gene expression was observed at 3 and 5 days after injury. Surprisingly, gene expression changes suggested early specification of osteochondral lineages by 3 days post injury, approximately 3 to 4 days before the appearance of histologically identifiable cartilage. We identified dysregulation of genes encoding components of major signaling pathways, cell cycle regulators, extracellular matrix components, and hypoxia response mediators, and showed that these gene expression changes are temporally associated with abnormal population growth of mutant FAPs. We further demonstrated that treatment of FOP mice with either an anti-activin A monoclonal antibody or the ACVR1 kinase inhibitor BLU-782 (IPN60130; fidrisertib) normalized the expression of the majority of dysregulated FAP genes. However, the relative efficacy of the drugs differed, and distinct sets of overlapping genes were normalized by the treatments. The substantial transcriptional changes caused by these drugs in FAPs from normally regenerating muscle were also enumerated and described. These findings define early transcriptional events associated with pathogenic FAP reprogramming and provide a molecular framework for understanding the shared and distinct effects of activin A and ACVR1 inhibition.

Microbiome-based therapeutics to treat enteric bacterial infections requires stable engraftment of transplanted beneficial bacteria that target and eliminate the pathogen. The host factors that determine whether a microbiome transplant engrafts remain largely unresolved. Here we demonstrate that Interleukin-10 (IL-10) signaling deficiency leads to impaired fecal microbiota transplantation (FMT) engraftment and failure to resolve Clostridioides difficile infection in mice. In the absence of IL-10-mediated immunoregulation provided by Foxp3+ Treg cells, increased IFN-{gamma} signaling in the intestine leads to elevated production of reactive oxygen/nitrogen species (ROS/RNS) by neutrophils and epithelial cells that supports the growth of inflammation-tolerant microbes, inhibits FMT engraftment, and impairs resolution of C. difficile. FMT treatment success can be restored by antibody-mediated blockade of IFN-{gamma} signaling, neutrophil depletion or inhibition of ROS/RNS production. Lastly, we developed an Il10-mRNA-LNP immunotherapy to boost IL-10 signaling in FMT Non-Responsive mice and demonstrate that Il10-mRNA-LNP administration is sufficient to restore FMT engraftment and subsequent resolution of C. difficile. Combined, these data support a mechanism by which IL-10 released by Treg cells limits IFN-{gamma} mediated intestinal inflammation thereby promoting an intestinal microenvironment receptive to FMT engraftment and resolution of C. difficile. These data demonstrate that the host's immune status can be therapeutically modulated to improve microbiome-based approaches to treat infection.

Sepsis is life-threatening, yet diagnosis remains a major global challenge because existing approaches lack speed and accuracy. Here we show that label-free imaging cytometry of 10 L whole-blood samples by Remoscope rapidly distinguishes patients with sepsis from those with non-infectious critical illness and healthy controls. In a prospective study of 29 critically ill adults, multi-physician adjudication identified sepsis (n=13), non-infectious critical illness (n=12), and indeterminate status (n=4); these patients were compared to healthy controls (n=22). A multiple instance learning (MIL) model pooled unbiased features from thousands of blood images per patient. Optimized for rule-out, the three-class model detected sepsis with 100% sensitivity and 85.3% specificity. Class activation heatmaps, unsupervised single cell embeddings, and morphometrics converged on abnormal leukocyte and erythrocyte morphology, including immature neutrophil-like cells. Label-free whole-blood imaging with MIL detects sepsis-associated hematologic signatures without stains or cold chain, establishing a foundation for rapid critical care diagnostics.

Clinical translation of mesenchymal stromal cell (MSC) therapies derived from primary tissue sources is limited by manufacturing inconsistencies driven by donor-to-donor variability, which reduces predictability of clinical response. Induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) offer a promising donor-independent, clonal alternative strategy that is also more amenable to engineering, but whether engineered iMSCs reproduce the potency attributes of primary MSCs remains unclear. Here, we benchmarked a doxycycline-inducible, hTERT-immortalized iMSC line engineered to overexpress TGFB1 (TGFB1-iMSCs), a key regulator of macrophage polarization, against multiple adipose tissue-derived MSC (MSC(AT)) donors across predefined immunomodulatory and angiogenic potency attributes. TGFB1-iMSCs were smaller and more circular with comparable or higher proliferative rates than primary MSC(AT) donors. Interestingly, they had a distinct angiogenic signature (EDIL3, EDN1, PDGFA), and nine differentially expressed microRNAs were predicted to target secretory trafficking and growth factor receptor signalling. Functionally, however, TGFB1-iMSCs secreted less VEGF and showed intermediate HUVEC tube formation, but matched or exceeded all primary MSC(AT) donors in a monocyte-macrophage transwell immunomodulatory assay. Notably, in a murine destabilization of the medial meniscus (DMM) model of post-traumatic osteoarthritis, a single intra-articular injection of TGFB1-iMSCs, but not MSC(AT), reduced total synovial macrophage numbers and lowered the MHCII/CD206 ratio at eight weeks, although it did not reduce cartilage degeneration, synovitis, or fibrosis. Together, these findings show that TGFB1-engineered iMSCs have a macrophage-directed potency profile distinct from that of primary MSC(AT). Immortalized iMSCs therefore provide a reproducible platform for mechanistically dissecting engineered MSC potency and its effects on the immune environment.

Antiretroviral therapy suppresses HIV-1 replication but cannot eliminate the latent viral reservoir persisting in memory CD4 T cells. Existing latency reversal candidates have yet to achieve robust viral reactivation at tolerable doses. We engineered T4IL15{triangleup}, a bispecific biologic that selectively delivers IL-15 signaling to memory CD4 T cells, with minimal off-target stimulation. T4IL15{triangleup} reactivated latent virus in 94% (48/51) of clinical samples ex vivo, 75% (15/20) of administrations in HIV-1-infected and ART-suppressed humanized mice, and 100% (6/6) of SIV-infected and ART-suppressed rhesus macaques at doses well tolerated. We also found that off-target stimulation of non-CD4 cells hindered HIV-1 reactivation ex vivo. T4IL15{triangleup} provides a robust reactivation platform that could complement other reservoir-elimination strategies toward an HIV-1 cure.

NOS1AP is a genetically validated driver of tau pathology in Alzheimer's disease (AD), yet the NOS1AP-tau interaction itself has remained pharmacologically untargeted. We report CAPTOR-1, a first-in-class small molecule that disrupts this interaction by binding a pocket in the N-terminal region of NOS1AP (residues 1-126). A drug-resistant NOS1AP Y16A/F42A variant that retained tau binding but exhibited a 78-fold reduction in CAPTOR-1 affinity provides genetic evidence supporting an on-target mechanism. In human iPSC-derived neurons, CAPTOR-1 dose-dependently reduces endogenous NOS1AP-tau association, tau seeding activity, and pathogenic tau phosphorylation, phenocopying genetic NOS1AP knockdown; these effects extend to neurons from patients with sporadic AD. Orally dosed CAPTOR-1 crosses the blood-brain barrier and engages NOS1AP in brain dose-dependently. Chronic oral dosing rescues pathology, synaptic function, and memory in a tauopathy model (PS19) and an amyloidosis model (APP/PS1), sparing wild-type littermates in both. This work establishes pharmacological disruption of the NOS1AP-tau interaction as a viable, disease-modifying strategy for AD and positions CAPTOR-1 as a biomarker-ready chemical probe (pTau217, neurofilament light) for translational studies.

Bundibugyo ebolavirus (BDBV) is causing an ongoing outbreak in the Democratic Republic of Congo (DRC) with no licensed vaccine available. Recent reports have shown the presence of BDBV-cross-reactive antibody responses in humans vaccinated against the related Ebola virus (EBOV). Here, we used an IFN-{gamma} ELISpot assay to assess T cell responses against the EBOV, BDBV and SUDV glycoproteins in clinical trial participants vaccinated with the licensed VSV-EBOV vaccine. We could show that BDBV-cross-reactive T cell responses are detectable in the majority of individuals and that conserved regions within the glycoprotein are targeted. In the absence of a BDBV-specific vaccine, these data support efficacy trials of the VSV-EBOV vaccine during the current outbreak.

Fibrous dysplasia (FD) arises from activating GNAS mutations that increase cAMP signaling in skeletal stem/progenitor cells (SSPCs), but the underlying mechanisms and whether the resulting fate imbalance can be reversed remain unclear. Using controlled GNAS induction with transcriptomic, pharmacologic, and cell-fate analyses, we identify a PKA-dependent checkpoint governing SSPC lineage commitment. GNASR201C redirects SSPCs toward osteoclastogenesis while suppressing osteoblast maturation; genetic or pharmacologic PKA inhibition reverses both effects, revealing this fate decision is dynamically reversible. Mechanistically, PKA phosphorylates RUNX2, the master regulator of osteogenesis, enforcing its cytoplasmic retention and blocking osteogenic transcription; a non-phosphorylatable RUNX2 mutant restores nuclear localization and osteoblast differentiation, defining RUNX2 phosphorylation as a disease-initiating molecular switch. Critically, PKA inhibition in vivo reduces fibrosis and osteoclast burden in established bone lesions, demonstrating reversibility even in advanced FD. These findings define a druggable GNAS-PKA-RUNX2 checkpoint and identify PKA inhibition as a strategy to reverse existing disease in FD.

West Nile virus (WNV) is one of the leading causes of arthropod-transmitted encephalitis in the world, yet its effects on neurons, the principal target of WNV in the brain, remain poorly defined. Here we establish and use Ngn2-induced human iPSC-derived glutamatergic neurons to define the neuronal response to WNV through integrated temporal proteomics and transcriptomics. Temporal multi-omic profiling revealed that infection activated innate immunity and the Unfolded Protein Response while suppressing axonogenesis and synaptic programs. WNV infection also disrupted calcium homeostasis, reduced STMN2 abundance, and promoted cytoplasmic redistribution of TDP-43. Applying this neuronal model to characterize contemporary California isolates, we identified LA24, a lineage 1 strain that replicates efficiently in non-neuronal cell lines but is selectively attenuated in neurons, with reduced viral burden in the brain and attenuated pathogenicity in mice. Together, these findings uncover neuron-specific signatures of WNV pathogenesis and demonstrate that neuronal replication phenotypes can reveal strain-dependent virulence differences not captured by conventional cell lines.

Mosquito-transmitted alphaviruses, including chikungunya (CHIKV), Mayaro, Eastern equine encephalitis, and Venezuelan equine encephalitis viruses, cause severe arthritic or neurologic disease in humans. There are currently no approved antiviral treatments against alphavirus infection. Here, we report that the purine ribonucleoside EIDD-3525 is a potent inhibitor of alphavirus replication in vitro with broad spectrum antiviral activity against the Alphavirus genus. Using a trans-replicase assay, we find that EIDD-3525 blocks alphavirus RNA replication. Mutational profiling suggests that EIDD-3525 targets the alphavirus nsP4 RNA-dependent RNA polymerase that is conserved across medically important alphaviruses. In a pre-clinical mouse model of CHIKV infection and disease, oral treatment with EIDD-3525 reduced viral tissue burdens and the severity of virus-induced disease. Collectively, our findings support the continued development of the purine ribonucleoside EIDD-3525 as an orally available antiviral therapeutic for use against a broad group of alphaviruses.

Obesity induces pathological remodeling of white adipose tissue (WAT), including chronic inflammation and fibrosis, which drive metabolic dysfunction. Although weight loss (WL) is the cornerstone of obesity treatment and improves systemic metabolic health, its impact on WAT remodeling remains incompletely understood. Using a mouse model of diet-induced obesity followed by nutritional weight loss, we show that visceral WAT (vWAT) fails to fully recover despite normalization of body weight and marked metabolic improvment. Notably, vWAT fibrosis persists and continues to progress after WL. This occurs while extracellular matrix (ECM) production by adipose progenitor cells is markedly reduced following WL, explaining why pharmacological inhibition of ECM synthesis fails to prevent fibrosis during WL. Instead, we observed impaired collagen degradation by both progenitor cells and macrophages, along with lysosomal dysfunction, during WL. This suggests that defective ECM degradation, rather than continued ECM production, underlies fibrosis persistence and aggravation during WL. Together, these findings identify impaired collagenolysis as a critical barrier to adipose tissue recovery after WL and suggest that enhancing ECM clearance, rather than inhibiting ECM synthesis, may represent a more effective therapeutic strategy to restore adipose tissue function.

Mechanical stability critically influences bone fracture healing, yet how the early mechanical environment directs the transition from inflammation to regeneration remains unclear. Using a murine femoral osteotomy model, we compared rigid, semirigid, and dynamically adjusted semirigid-to-rigid fixation. Semirigid fixation delayed healing on day 21, whereas a strategy of early compliance followed by increased stiffness after 7 days restored bridging and improved bone regeneration beyond constant rigid fixation, pointing to a critical early mechanosensitive window after fracture. Single-cell RNA sequencing across the first week, comparing semirigid with rigid fixation, showed that fixation stiffness altered intercellular signaling within one day of injury. Signaling among myeloid populations was more broadly increased under semirigid fixation, with monocytes the single exception, and signaling associated with resolution of inflammation was reduced. From day 5, periosteal and skeletal progenitor populations increasingly directed cartilage-associated matrix and growth factor signaling toward macrophages and chondrocytes, accompanied by increased Sox9 regulon activity and increased COL10+ hypertrophic cartilage matrix by day 7. Reduced fixation stiffness therefore promotes a chondrogenic trajectory of repair, but increasing stiffness is required to redirect this response toward bone formation. Piezo1 was expressed across the responding populations and shifted between compartments as healing progressed. PIEZO1 activation with Yoda1 under rigid fixation enhanced bone formation but provided no additional benefit under semirigid or dynamically adjusted fixation, whereas inhibition of mechanosensitive signaling with GsMTx4 impaired repair across all conditions. Together, these findings establish the early mechanical environment as a determinant of fracture-healing trajectory, shaping the transition from inflammation to regeneration, and show that successful repair depends not on maximal stability but on when stiffness is applied. Increasing either early mechanical stimulation or cellular mechanosensitivity enhanced regeneration, supporting mechanosensitive signaling as a key component of the early healing response.

Nemaline myopathy is a severe inherited skeletal muscle disorder characterized by profound muscle weakness, impaired mobility, and early mortality in severe cases. Despite the devastating clinical burden, no disease-modifying therapies are currently available. Nemaline myopathy-causing genes either encode sarcomeric structural proteins or regulators of sarcomeric protein stability, resulting in primary defects in sarcomere structure and function. Recessive mutations in KLHL40 cause one of the most severe forms of nemaline myopathy, frequently resulting in perinatal lethality. Given its loss-of-function mechanism, KLHL40-associated nemaline myopathy is an attractive candidate for gene replacement therapy. We therefore evaluated the therapeutic potential of systemic AAV9-mediated KLHL40 gene replacement in a mouse model of severe nemaline myopathy. A single systemic administration of an AAV9 vector encoding human KLHL40 during early disease produced durable rescue of survival, skeletal muscle pathology, motor performance, and contractile function in KLHL40-deficient mice. Therapeutic benefit was accompanied by restoration of sarcomere organization and normalization of myofiber size without detectable long-term histopathological abnormalities in major organs. Collectively, our findings provide a strong preclinical foundation for developing AAV9-mediated KLHL40 gene replacement as a treatment for severe KLHL40-associated nemaline myopathy.

Oral premalignant lesions (OPLs) are common; however, histopathological grading incompletely identifies lesions destined for cancer. To define tissue ecosystems that precede malignant transformation, we integrated single-cell-resolution Xenium spatial transcriptomics of 20 HPV-negative biospecimens from 16 patients, with independent single-cell RNA sequencing and immunofluorescence data from 38 patients with OPLs after quality control. Progressive OPLs exhibit a coordinated epithelial-immune program comprising MX1/NOTCH3-high KRT14+/KRT15+ basal epithelial states, S100A9-high inflammatory macrophages, TIGIT-high, exhaustion-associated T cells, and altered dendritic cell states. Spatial analyses showed that T cells and dendritic cells were displaced from the basal epithelial interface, and that epithelial neighborhoods were depleted of dendritic cells but enriched for TIGIT-high T cells, revealing immune reorganization not captured by cellular abundance alone. Independent single-cell and protein-level analyses reproduced basal epithelial interferon/stress programs, S100A9-associated myeloid inflammation, TIGIT-high, exhaustion-associated T-cell programs, and dendritic cell redistribution. Ligand-receptor inference suggested convergent myeloid and lymphoid signals linked to epithelial stress, checkpoint regulation, and extracellular matrix remodeling. Functionally, recombinant S100A9 accelerated wound closure in oral epithelial and cancer cells, whereas S100A9 and S100A8/S100A9 enhanced oral cancer cell proliferation. These responses were attenuated by pharmacologic inhibition of TLR4 or RAGE. Together, these data define a spatially organized, myeloid-skewed, checkpoint-enriched ecosystem present before invasion and suggest S100A9-TLR4/RAGE signaling and epithelial-immune geometry as candidate mechanisms and biomarkers for oral cancer interception.

Successful treatment of primary Clostridioides difficile infection (CDI) while preventing its recurrence remains a major clinical challenge, with recurrent CDI (R-CDI) affecting 20-30% of treated patients. Persistence of C. difficile spores within the intestinal mucosa is thought to drive R-CDI following antibiotic treatment. Here, we show that intracellular C. difficile spores remain protected from taurocholate-triggered germination, and that nystatin, an FDA-approved antifungal that blocks spore entry into host cells, reduces this protected intracellular population and redirects spores toward an extracellular, germinating population accessible to antibiotics. In a murine model of R-CDI, oral co-administration of taurocholate with nystatin and antibiotics during primary CDI reduced R-CDI incidence from 100% to 44%. When administered during an active R-CDI episode, the same formulation reduced a second R-CDI from 100% to 62%. To improve bioavailability, we encapsulated nystatin and vancomycin in chitosan nanoparticles. Oral administration of nanoparticle-encapsulated vancomycin, alone or combined with nanoparticle-encapsulated nystatin, reduced R-CDI colonization to 33% and 44%, respectively, compared with 100% in mice receiving free vancomycin. Together, these findings support a therapeutic strategy that repurposes nystatin and taurocholate, in combination with antibiotics and nanoparticle-based delivery, to target intracellular spore persistence and reduce the incidence of C. difficile recurrence..

Inflammatory T-cell infiltration is widely considered a hallmark of cardiac immune-mediated tissue injury, yet whether inflammation alone is sufficient to cause cardiac damage remains unclear. Using a spontaneous genetic model of immune checkpoint inhibitor myocarditis, we show that cardiac immune infiltration and tissue injury are separable processes. CD4+ T-cells promote disease by licensing pathogenic CD8+ T-cell responses through CD40L signaling, whereas perforin-dependent CD8+ cytotoxicity is specifically required to induce cardiomyocyte death and cardiac arrhythmias. Loss of perforin prevented cardiac injury and rescued survival despite persistent myocardial inflammation, demonstrating that inflammatory infiltration is insufficient to produce lethal myocarditis in the absence of a cytotoxic effector program. CD40L blockade similarly attenuated pathogenic CD8+ T-cell activation, myocardial inflammation, and mortality. These findings identify a cooperative CD4-CD8 T-cell circuit that governs autoimmune cardiac injury and establish that acquisition of cytotoxic effector function, rather than inflammation alone, determines the transition from immune infiltration to tissue destruction.

Objectives: To develop and validate pixel data analysis (PDA) for reconstructing pixel-level shear-wave velocity (SWV) from saved Holder-Optimized Elastography (HOE) elastograms and to determine whether whole-field subcutaneous heterogeneity provides a quantitative signature of lymphatic obstruction compared with visual high-velocity-area (HVA) counting and sparse manual region-of-interest (ROI) sampling. Materials and Methods: In this cross-sectional study, 110 women after breast cancer surgery underwent lymphoscintigraphy and HOE, a pressure-minimized acquisition framework that preserves conventionally captured SWV information while additionally revealing spatial abnormalities attenuated by handheld acquisition. Reconstruction validation used 200 paired ROIs from 49 PACS-retrieved images spanning nearly the full displayed SWV range; reproducibility was assessed across 96 matched anatomical sites from 6 participants undergoing independently reacquired HOE examinations. PDA, visual HVA counting, and manual ROI sampling were evaluated on the same HOE-acquired elastograms in task-specific common complete-case cohorts. Results: PDA reconstructed machine-reported SWV with near-zero error (mean difference, +0.0009 m/s; mean absolute error, 0.0075 m/s; 95% limits of agreement, -0.0202 to +0.0221 m/s). For subcutaneous variance, interobserver/intraobserver ICCs were 0.718/0.799 for PDA and 0.161/0.560 for manual ROI sampling. In the primary no-versus-partial comparison (n = 93), forearm-pooled PDA subcutaneous variance ratio yielded the highest observed AUC (0.859; 95% CI, 0.772 - 0.947), compared with 0.820 for visual HVA counting and 0.769 for manual ROI sampling. PDA's subcutaneous variance ratio remained a leading feature across all three comparisons (AUC, 0.859 - 0.878), including partial-versus-total grading, where all three frameworks showed high discrimination. Conclusions: PDA accurately reconstructs pixel-level SWV from saved HOE elastograms and enables reproducible whole-field quantification of superficial SWE heterogeneity. Subcutaneous variance ratio was the leading quantitative PDA feature across lymphatic-obstruction comparisons and was particularly informative in partial obstruction. The same approach also enables retrospective quantitative reanalysis of compatible archived elastograms.

COVID-19 highlighted the need for antivirals that can be rapidly deployed against both current and future coronavirus threats. CMX990 is a clinical stage coronavirus main protease (Mpro) inhibitor bearing a unique trifluoromethoxymethyl ketone warhead. Here, we broadly evaluate its pan-coronavirus target engagement, antiviral breadth, and pharmacologic differentiation. Across a reporter panel of 24 Mpro enzymes spanning alpha-, beta-, delta-, and gamma-coronaviruses, CMX990 retained robust potent activity, including against nirmatrelvir-resistant SARS-CoV-2 variants. In direct comparison, nirmatrelvir and ensitrelvir showed diminished potency against non-SARS-CoV-2 coronaviruses. Live-virus assays corroborated this breadth, with potent, selective activity against SARS-CoV-2, SARS-CoV-1, MERS-CoV, and additional human coronaviruses. Structural studies revealed a conserved covalent binding mode centered on the catalytic Cys of coronavirus Mpro. In serial-passage studies, resistance emerged only after approximately twice as many serial passages under CMX990 selection as under nirmatrelvir selection, without selecting for the canonical nirmatrelvir-resistance substitution E166V. Extracellular washout experiments revealed superior persistence of antiviral activity relative to nirmatrelvir, consistent with durable target engagement. In a murine SARS-CoV-2 infection model, once-daily CMX990 protected against weight loss and reduced lung viral titers to near baseline levels. Collectively, these findings identify CMX990 as a structurally and functionally validated pan-coronavirus protease inhibitor supporting its potential as a pandemic-ready antiviral candidate.

CD4+ T-cell recovery is widely used to assess immune reconstitution in people with HIV, but its relationship to virus-specific humoral immunity across pathogens remains unclear. We characterized neutralizing activity against SARS-CoV-2, HIV-1, and influenza viruses in ART-treated, virologically suppressed individuals spanning distinct CD4+ T-cell levels. BA.5 was the dominant SARS-CoV-2 neutralization target across CD4 strata, whereas D614G neutralization increased with greater immune reconstitution. This gradient was largely absent for the antigenically divergent NB.1.8.1 and XFG variants. HIV-1 neutralization was inversely associated with CD4+ T-cell count, while influenza showed strain-dependent patterns. Neutralization-to-protection bridging preserved a CD4-associated gradient for D614G but not for highly immune-evasive variants. These findings show that immune reconstitution does not translate uniformly into antiviral humoral function. Instead, antibody profiles reflect host immune status, prior antigenic exposure, and viral antigenic evolution, supporting pathogen-specific functional assessment alongside conventional immune monitoring.

BACKGROUND & OBJECTIVE Tofacitinib, a Janus kinase (JAK) inhibitor, is approved for ulcerative colitis (UC). We investigated baseline and longitudinal transcriptomics and proteomics in intestinal biopsies, peripheral blood, and serum from UC patients starting tofacitinib to characterise treatment-associated effects and identify which molecular compartment harbours the most predictive response-associated biomarkers. METHODS In this prospective, open-label study, patients with moderately to severely active UC started tofacitinib 10 mg BID for eight weeks. Clinical, biochemical, endoscopic, and histological data were collected at baseline and week 8. The primary outcome, histo-endoscopic mucosal improvement (HEMI), was defined as endoscopic Mayo subscore [≤]1 with histologic remission (Robarts Histopathology Index [≤]3, no mucosal neutrophils). Mucosal biopsies and peripheral blood were collected at baseline and week 8 for bulk RNA-sequencing (RNA-seq); biopsies and serum underwent proteomic profiling using Olink Explore 384 (Cardiometabolic, Inflammation, Neurology, Oncology panels). RESULTS In total, 127 paired multi-omic samples were collected longitudinally from 40 UC patients: 15 (38%) achieved HEMI at week 8 (responders), 13 (32.5%) were late responders, and 12 (30%) were non-responders. Pretreatment mucosal biopsies from future responders showed elevated interferon, IL-6-family, IL-2/common-{gamma}-chain, and T-cell-receptor signalling gene expression, which declined on treatment only in responders. A responder-specific JAK-STAT-dependent mechanism spanned interferon, cytokine-receptor, T-cell-receptor, and NF-{kappa}B signalling, with concordant gene- and protein-level reductions. Compared with a public infliximab dataset, JAK-STAT/interferon downregulation was shared and response-independent, while non-canonical NF-{kappa}B downregulation was tofacitinib-specific. In blood, shared pathway labels reflected distinct genes from mucosal effectors. CONCLUSION Response to tofacitinib depends on a JAK-tractable mucosal pathway absent in non-responders, suggesting a pretreatment biomarker for predicting treatment response.