Fibrosis is a major cause of mortality and morbidity worldwide with limited therapeutic options. Our understanding of fibrosis has significantly improved and led to the identification of “core” fibrogenic mechanisms that fuel a self-sustaining vicious cycle following the initial insult. The fibrotic niche is the result of complex cellular and molecular interactions that need to be disrupted to achieve transformational therapies. In this Review, we describe the current understanding of fibrogenic mechanisms, the progress and limitations of omics approaches in the identification of novel fibrotic pathways, and advances in therapeutic modalities that all together have the potential to unleash innovative cross-organ antifibrotic therapies.

Idiopathic pulmonary fibrosis (IPF) is a progressive and chronic interstitial lung disease with significant unmet need, and only a few therapeutic options slow decline rather than improve function. An expanding fibrotic niche is one hallmark of this disease, comprising a multicellular environment characterized by epithelial dysfunction and death, infiltration of peripheral immune cells, differentiation of fibroblasts and immune cells, extracellular matrix turnover and deposition of fibrillar collagen. Inflammation is a master regulator spanning all aspects of this fibrotic niche through cellular players and signaling mediators that can be split into three primary types. In many immune responses, the inflammatory milieu is dominated by one type of inflammation at a time, but recent findings suggest there might be more spatial and kinetic overlap present in fibrotic diseases, including IPF, than previously appreciated. In this Review, we summarize some of the key cellular players and soluble mediators involved in IPF progression and how distinct types of inflammation jointly regulate disease progression in concert rather than focus on individual cytokines or immune cells.

Immune checkpoint inhibitors improve outcomes for many patients with cancer, but resistance remains a major clinical challenge. Type 2 cytokines, including IL-4, IL-13, and thymic stromal lymphopoietin (TSLP), have been implicated in suppressing anti-tumor immunity. Here, we investigated whether combined blockade of IL-4, IL-13, and TSLP enhances anti-tumor responses alone and in combination with PD-1 inhibition. IL-4 impaired T cell-mediated tumor control and reduced expression of immune-stimulatory molecules by monocyte-derived dendritic cells in vitro. In CT26 and KP787 tumor models, combined blockade of IL-4, IL-13, TSLP, and PD-1 improved tumor growth inhibition and was associated with reprogramming of T cells, monocytes, and dendritic cells toward anti-tumor phenotypes. Across multiple human cancer datasets, transcriptional signatures associated with IL-4/IL-13 and TSLP activity correlated with poorer survival. These findings identify type 2 cytokine signaling as a suppressive pathway in anti-tumor immunity and support its therapeutic targeting to enhance checkpoint inhibitor efficacy.

IntroductionThe tumor necrosis factor (TNF) and TNF receptor (TNFR) superfamilies comprise 47 proteins that regulate immune signaling and T cell costimulation. While TNF inhibitors are established therapies for immune-mediated inflammatory diseases (IMIDs), their efficacy is limited by primary non-response and secondary loss of efficacy. Preclinical data suggest that the TNF/TNFR members exhibit redundant and synergistic signaling, motivating combination targeting strategies.MethodsWe have systematically evaluated TNF/TNFR combinations as potential immunotolerance targets using integrated computational and experimental approaches. We applied a gene prioritization framework incorporating transcriptomics, genetics, druggability, and pathway regulation data to derive disease association scores for the TNF/TNFR genes in rheumatoid arthritis and inflammatory bowel disease. Based on these scores and T cell expression profiling, ten targets were prioritized for a combinatorial screen using clinical-stage and preclinical pharmacological inhibitors in a mixed lymphocyte reaction (MLR) assay. The effects of the most promising combinations were further characterized by RNA sequencing.ResultsThe combinatorial screen identified four drug combinations that significantly suppressed T cell production of IL-2 and IFN-γ: TNF+CD40L, TNF+OX40L, CD40L+OX40L, and CD40L+LTβ/LIGHT. Transcriptional profiling revealed that these combinations broadly downregulated genes associated with T cell activation, proliferation, differentiation, and cytokine production that were induced during the allogeneic response. Notably, the co-inhibition of TNF and CD40L (Adalimumab+Dapirolizumab) produced the most robust suppression, uniquely downregulating 337 genes enriched for key T cell activation pathways, including NF-κB and ERK1/2.DiscussionThese findings demonstrate that the combinatorial antagonism of TNF/TNFR superfamily members can potently suppress allogeneic T cell responses, with the TNF+CD40L combination showing particularly strong and broad effects. The results support the continued preclinical evaluation of combinatorial TNF/TNFR inhibition as a potential tolerance-inducing therapeutic strategy for patients with refractory IMIDs.

Background Checkpoint inhibitors improve survival in patients with several types of tumors. However, resistance to checkpoint inhibitors creates an opportunity for patients to benefit from novel immunotherapies. The type 2 cytokines IL-4, IL-13 and TSLP have been implicated in suppressing anti-tumor immune responses through T and myeloid cells. Our current study tested whether combined therapeutic blockade of IL-4, IL-13 and TSLP improved anti-tumor immunity alone and in combination with PD1 antagonism. Methods We used in vitro experiments with primary cells to identify cell types likely to participate in controlling tumors upon IL-4, IL-13, TSLP and PD1 blockade. Therapeutic blockade in the subcutaneous CT26 model tested in vivo tumor growth inhibition and associated immunological changes. Bioinformatic analysis of human tumor bulk RNA sequencing data probed for survival associations with IL-4/IL-13 and TSLP transcriptional responses. Results In vitro , IL-4 suppressed T cell-mediated tumor growth inhibition and reduced monocyte-derived dendritic cell expression of proteins associated with anti-tumor immunity. In vivo , blocking IL-4, IL-13, TSLP and PD1 improved tumor growth inhibition by creating “hotter” tumors. This was associated with repolarization of CD4 and CD8 T cells and shifts in monocyte, conventional type 1 and type 2 (or monocyte-derived) dendritic cell programs. Transcriptional responses to IL-4/IL-13 and TSLP were associated with poor survival outcomes across patients with several types of cancers. Conclusion Therapeutic blockade of IL-4, IL-13 and TSLP may drive immunological tumor growth inhibition in subsets of cancer patients alone and in combination with checkpoint inhibitors. Improved tumor growth inhibition was likely driven through augmented cytotoxic T cell priming in secondary lymphoid organs and improved reactivation by repolarized monocytes and dendritic cells in tumors. ### Competing Interest Statement All co-authors are current or former employees of Pfizer, Inc.; F. J., R. L. G. III and A. M. S. B. are inventors on the patent application US18/175,796 pertaining to this paper.

Neuropod cells are a newly discovered type of enteroendocrine cell (EEC) that connect the gut and brain functionally into one circuit. In the mouse colon, neuropod cells express various peptide hormones, such as Pyy and Glp1, presynaptic proteins, and make synaptic contacts with sensory neurons. While their function is not fully elucidated, they play a significant role in relaying signals to the brainstem upon sensing nutrients and microbial factors in the gut lumen. Their occurrence in the human gastrointestinal tract is currently not established. In this study, we showed that PYY-expressing cells (L-cells) in the human colon exhibit characteristics of neuropod cells. Utilizing advanced histological methods and confocal microscopy we found that L-cells of the healthy human colon possess distinctive morphology, express synaptic proteins, and exist proximal to sensory neurons. This agrees with our meta-analysis of single-cell RNA sequencing (scRNA-Seq) data that showed that human colonic L-cells express pre- and post-synaptic genes. As inflammatory conditions could affect colonic neuropod cells, we aimed to profile the phenotypic and transcriptional changes of neuropod cells both in human and murine colon in Inflammatory Bowel Disease (IBD) and experimental colitis, respectively. In human IBD, the abundance of neuropod cells and spatial proximity to sensory neurons were decreased in the colon of ulcerative colitis (UC) and Crohn’s disease (CD) patients. L-cells in IBD patients display genes related to innate and adaptive immunity, including antigen presentation genes suggesting a role in immune regulation. We further confirmed the effects of intestinal inflammation in neuropod cells by utilizing the DSS mouse model of colitis, where we showed that acute DSS colitis induced spatially distinct effects on the abundance of neuropod cells and impaired the synaptic connection with sensory neurons. Overall, these findings extend early murine characterizations to the human system and highlight the complex interactions between colonic neuropod cells and the enteric nervous and immune systems during inflammatory diseases. ### Competing Interest Statement All authors are current employees of Pfizer, Inc.

Background & Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a leading cause of liver fibrosis, morbidity, and mortality. It is characterized by the accumulation of fat in hepatocytes causing cell death followed by stromal cell activation and scar deposition at later disease stages. While a marked accumulation of cytotoxic CD8 T cells is observed in MASH in humans and mice, the role of adaptive immune cells in fibrosis progression remains debated. Methods: Transcriptional data were curated from human datasets and preclinical mouse models, including mice deficient in TCRαβ T cells ( Tcrb-/- ), which lack conventional CD4 helper and CD8 cytotoxic T cells, in diet-induced fibrosing MASH for single-cell RNA-sequencing. Specific human and mouse liver transcriptomic immune signatures were corroborated by flow cytometry and immunofluorescence. Results: MASH was associated with the expansion of lymphocytes and myeloid cells. Auto-aggressive CXCR6+PD1+FASLG+ CD8 T cells were enriched in livers of high-fat-diet-fed mice and correlated with MASH and fibrosis. Notably, Tcrb -deficient mice in a chemical and dietary preclinical MASH model developed fibrosis to the same extent as wild-type mice and exhibited exacerbated pro-fibrotic type 3 inflammation. Loss of conventional CD8 T cells neither impacted myeloid cell number nor phenotype within the fibrotic niche. Neutrophil- and non-conventional lymphocyte-derived GM-CSF and IL-17A were central drivers in the fibrotic niche composed of pathogenic macrophages and activated myofibroblasts. Furthermore, myeloid cells were identified as the main source of CXCL16 in diseased livers, retaining auto-aggressive CD8 T cells to the scar and contributing to their accumulation. Thus, non-adaptive cells, including scar-associated macrophages (SAMs) and myofibroblasts, are sufficient for MASH-driven fibrosis in this model. Conclusions: Targeting myeloid cells and fibroblasts should be prioritized as anti-fibrotic therapies for MASH. ### Competing Interest Statement C.L, K.L.D., S.M.C., M.H.W., K.B., S.D., T.A.W., K.M.H., T.F. are or J.M., F.S., C.W., X.C., A.M.S.B. were employees of Pfizer Inc.

Dysregulated signaling from TNF and TNFR proteins is implicated in several immune-mediated inflammatory diseases (IMIDs). This review centers around seven IMIDs (rheumatoid arthritis, systemic lupus erythematosus, Crohn’s disease, ulcerative colitis, psoriasis, atopic dermatitis, and asthma) with substantial unmet medical needs and sheds light on the signaling mechanisms, disease relevance, and evolving drug development activities for five TNF/TNFR signaling axes that garner substantial drug development interest in these focus conditions. The review also explores the current landscape of therapeutics, emphasizing the limitations of the approved biologics, and the opportunities presented by small-molecule inhibitors and combination antagonists of TNF/TNFR signaling.

Background:Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding it's pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. Methods:We performed scRNAseq of 13 fresh full thickness CD resections containing non-involved, inflamed non-strictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next generation sequencing, proteomics and animal models. Results:Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and upregulated, and its pro-fibrotic function was validated by NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and two animal models of experimental colitis. Conclusion:A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and opens potential therapeutic developments.

Allergic diseases are a global health challenge. Individuals harboring loss-of-function variants in transforming growth factor–β receptor (TGFβR) genes have an increased prevalence of allergic disorders, including eosinophilic esophagitis. Allergic diseases typically localize to mucosal barriers, implicating epithelial dysfunction as a cardinal feature of allergic disease. Here, we describe an essential role for TGFβ in the control of tissue-specific immune homeostasis that provides mechanistic insight into these clinical associations. Mice expressing a TGFβR1 loss-of-function variant identified in atopic patients spontaneously develop disease that clinically, immunologically, histologically, and transcriptionally recapitulates eosinophilic esophagitis. In vivo and in vitro, TGFβR1 variant–expressing epithelial cells are hyperproliferative, fail to differentiate properly, and overexpress innate proinflammatory mediators, which persist in the absence of lymphocytes or external allergens. Together, our results support the concept that TGFβ plays a fundamental, nonredundant, epithelial cell–intrinsic role in controlling tissue-specific allergic inflammation that is independent of its role in adaptive immunity.

Macrophages are central orchestrators of the tissue response to injury, with distinct macrophage activation states playing key roles in fibrosis progression and resolution. Identifying key macrophage populations found in human fibrotic tissues could lead to new treatments for fibrosis. Here, we used human liver and lung single-cell RNA sequencing datasets to identify a subset of CD9 + TREM2 + macrophages that express SPP1 , GPNMB , FABP5 , and CD63 . In both human and murine hepatic and pulmonary fibrosis, these macrophages were enriched at the outside edges of scarring and adjacent to activated mesenchymal cells. Neutrophils expressing MMP9, which participates in the activation of TGF-β1, and the type 3 cytokines GM-CSF and IL-17A coclustered with these macrophages. In vitro, GM-CSF, IL-17A, and TGF-β1 drive the differentiation of human monocytes into macrophages expressing scar-associated markers. Such differentiated cells could degrade collagen IV but not collagen I and promote TGF-β1–induced collagen I deposition by activated mesenchymal cells. In murine models blocking GM-CSF, IL-17A or TGF-β1 reduced scar-associated macrophage expansion and hepatic or pulmonary fibrosis. Our work identifies a highly specific macrophage population to which we assign a profibrotic role across species and tissues. It further provides a strategy for unbiased discovery, triage, and preclinical validation of therapeutic targets based on this fibrogenic macrophage population.

Crohn’s disease (CD) is a chronic transmural inflammation of intestinal segments caused by dysregulated interaction between microbiome and gut immune system. Recurrent/relapsing CD and resistance to medical treatments result in complications requiring surgery. High-dimensional single-cell profiling approaches, such as scRNA-seq and mass cytometry, have been recently performed on intestinal specimens from patients with IBD and controls. However, most of these studies have analyzed whole mucosal biopsies or the lamina propria (LP) compartment, while few have addressed the intraepithelial lymphocytes (IEL) compartment. Here, we profiled T cells purified from the IEL and LP from terminal ileum resections of adult severe CD cases by single cell technologies. Our study defined a vast heterogeneity of T cell lineages in the IEL compartment. IEL included, among others, unique γδT cell subsets: NKp30+γδ T cells expressing RORγt, which produced IL-26 upon NKp30 engagement and a subset expressing PDGFD and CSF1, which may act on epithelial cells, IEL ILC1s, and macrophages, respectively. We have also observed long-lived memory TCF7+CD8+ T cells expressing DC chemoattractants and TFH subsets that may respond to distinct glutathione-conjugated lipids. CD IEL showed a significant increase of activated TH17, coupled with decreased CD8+ T cells, γδT cells, TFH, and Treg. Conversely, the LP showed increased CD8+ T cells and reduced CD4+ T cells with a relative increase of TH17 over Treg/TFH. Results provide an unbiased view of diversity of cell lineages and their functional states in the intestinal mucosa of controls and CD and identify an altered spatial distribution of T cell subsets between the IEL and the LP compartments.

BACKGROUND AND AIMS:Organoids provide a powerful system to study epithelia in vitro. Recently, this approach was applied successfully to the biliary tree, a series of ductular tissues responsible for the drainage of bile and pancreatic secretions. More precisely, organoids have been derived from ductal tissue located outside (extrahepatic bile ducts; EHBDs) or inside the liver (intrahepatic bile ducts; IHBDs). These organoids share many characteristics, including expression of cholangiocyte markers such as keratin (KRT) 19. However, the relationship between these organoids and their tissues of origin, and to each other, is largely unknown. APPROACH AND RESULTS:Organoids were derived from human gallbladder, common bile duct, pancreatic duct, and IHBDs using culture conditions promoting WNT signaling. The resulting IHBD and EHBD organoids expressed stem/progenitor markers leucine-rich repeat-containing G-protein-coupled receptor 5/prominin 1 and ductal markers KRT19/KRT7. However, RNA sequencing revealed that organoids conserve only a limited number of regional-specific markers corresponding to their location of origin. Of particular interest, down-regulation of biliary markers and up-regulation of cell-cycle genes were observed in organoids. IHBD and EHBD organoids diverged in their response to WNT signaling, and only IHBDs were able to express a low level of hepatocyte markers under differentiation conditions. CONCLUSIONS:Taken together, our results demonstrate that differences exist not only between extrahepatic biliary organoids and their tissue of origin, but also between IHBD and EHBD organoids. This information may help to understand the tissue specificity of cholangiopathies and also to identify targets for therapeutic development.

Crohn’s disease (CD) is a chronic transmural inflammation of intestinal segments caused by dysregulated interaction between microbiome and gut immune system. Here, we profile, via multiple single-cell technologies, T cells purified from the intestinal epithelium and lamina propria (LP) from terminal ileum resections of adult severe CD cases. We find that intraepithelial lymphocytes (IEL) contain several unique T cell subsets, including NKp30 + γδT cells expressing RORγt and producing IL-26 upon NKp30 engagement. Further analyses comparing tissues from non-inflamed and inflamed regions of patients with CD versus healthy controls show increased activated T H 17 but decreased CD8 + T, γδT, T FH and Treg cells in inflamed tissues. Similar analyses of LP find increased CD8 + , as well as reduced CD4 + T cells with an elevated T H 17 over Treg/T FH ratio. Our analyses of CD tissues thus suggest a potential link, pending additional validations, between transmural inflammation, reduced IEL γδT cells and altered spatial distribution of IEL and LP T cell subsets.

Rationale and Objectives Fibrosis is characterized by progressive replacement of normal tissue by extracellular matrix. Diagnosis relies on biopsies as noninvasive methods for detection and quantification of fibrosis are still limited. This work aimed to address the ability of 2 molecular magnetic resonance (MR) probes, EP-3533 and Gd-Hyd, to identify fibrosis and fibrogenesis, respectively, independently of the presence of underlying inflammation in a mouse model of chronic liver disease caused by infection with Schistosoma mansoni. Methods Three groups of mice that develop either mild type 2 inflammation and fibrosis (wild type), severe fibrosis with exacerbated type 2 inflammation (Il10−/−Il12b−/−Il13ra2−/−), or minimal fibrosis with marked type 1 inflammation (Il4ra∂/∂) after infection with S. mansoni were imaged using both probes for determination of signal enhancement. Schistosoma mansoni–infected wild-type mice developed chronic liver fibrosis. Results The liver MR signal enhancement after either probe administration was significantly higher in S. mansoni–infected wild-type mice compared with naive animals. The S. mansoni–infected Il4ra∂/∂ mice presented with little liver signal enhancement after probe injection despite the presence of substantial inflammation. Schistosoma mansoni–infected Il10−/−Il12b−/−Il13ra2−/− mice presented with marked fibrosis, which correlated to increased signal enhancement after injection of either probe. Conclusions Both MR probes, EP-3533 and Gd-Hyd, were specific for fibrosis in this model of chronic liver disease regardless of the presence or severity of the underlying inflammation. These results, in addition to previous findings, show the potential application of both molecular MR probes for detection and quantification of fibrosis from various etiologies.

Fibrosis can affect any organ and is responsible for up to 45% of all deaths in the industrialized world. It has long been thought to be relentlessly progressive and irreversible, but both preclinical models and clinical trials in various organ systems have shown that fibrosis is a highly dynamic process. This has clear implications for therapeutic interventions that are designed to capitalize on this inherent plasticity. However, despite substantial progress in our understanding of the pathobiology of fibrosis, a translational gap remains between the identification of putative antifibrotic targets and conversion of this knowledge into effective treatments in humans. Here we discuss the transformative experimental strategies that are being leveraged to dissect the key cellular and molecular mechanisms that regulate fibrosis, and the translational approaches that are enabling the emergence of precision medicine-based therapies for patients with fibrosis.