INTRODUCTION: Transplant glomerulitis is a morphological lesion seen in kidney allograft rejection that is associated with poor outcomes; however, little is known about how immune cells infiltrate and organize specifically within glomeruli.METHODS: We used Co-Detection by Indexing (CODEX) multifluorescent imaging to measure 52 protein markers in a retrospective cohort of 41 human allograft nephrectomies (ANs) and evaluated the immunological landscape of transplant glomerulitis.RESULTS: Characterization of 18 cell types identified diverse immune cells within inflamed glomeruli, with unique phenotypes and compositions compared with the extraglomerular microenvironment. Immunological phenotypes were conserved across glomeruli within individuals and associated with the general state of injury, with M1 macrophages and effector CD8 T cells associated with mild inflammation. Distance-based spatial analysis further revealed a profibrotic community composed of M2 macrophages, memory CD8 T cells and exhausted CD8 T cells surrounding endothelial cell hubs. These interaction networks were associated with regions of adverse glomerular remodeling, expression of profibrotic proteins, and were more prevalent in individuals with C4d-positive rejection.CONCLUSION: These results implicate distinct cell-cell interactions as hallmarks of alloimmune injury and chronic remodeling during transplant glomerulitis and may give rise to new tools for histological risk assessment of clinical rejection syndromes.

In eukaryotic cells, reactive oxygen species (ROS) serve as crucial signaling components. ROS are potentially toxic, so constant adjustments are needed to maintain cellular health. Here we describe a single-cell, mass cytometry-based method that we call signaling network under redox stress profiling (SN-ROP) to monitor dynamic changes in redox-related pathways during redox stress. SN-ROP quantifies ROS transporters, enzymes, oxidative stress products and associated signaling pathways to provide information on cellular redox regulation. Applied to diverse cell types and conditions, SN-ROP reveals unique redox patterns and dynamics including coordinated shifts in CD8+ T cells upon antigen stimulation as well as variations in CAR-T cell persistence. Furthermore, SN-ROP analysis uncovers environmental factors such as hypoxia and T cell exhaustion for influencing redox balance, and also reveals distinct features in patients on hemodialysis. Our findings thus support the use of SN-ROP to elucidate intricate redox networks and their implications in immune cell function and disease.

COVID-19 remains a global pandemic of an unprecedented magnitude with millions of people now developing "COVID lung fibrosis." Single-cell transcriptomics of lungs of patients with long COVID revealed a unique immune signature demonstrating the upregulation of key proinflammatory and innate immune effector genes CD47, IL-6, and JUN. We modeled the transition to lung fibrosis after COVID and profiled the immune response with single-cell mass cytometry in JUN mice. These studies revealed that COVID mediated chronic immune activation reminiscent to long COVID in humans. It was characterized by increased CD47, IL-6, and phospho-JUN (pJUN) expression which correlated with disease severity and pathogenic fibroblast populations. When we subsequently treated a humanized COVID lung fibrosis model by combined blockade of inflammation and fibrosis, we not only ameliorated fibrosis but also restored innate immune equilibrium indicating possible implications for clinical management of COVID lung fibrosis in patients.

Patients with severe COVID-19 often suffer from lymphopenia, which is linked to T cell sequestration, cytokine storm and mortality. However, it remains largely unknown how SARS-CoV-2 induces lymphopenia. Here, we studied the transcriptomic profile and epigenomic alterations involved in cytokine production by SARS-CoV-2-infected cells. We adopted a reverse time-order gene coexpression network (TO-GCN) approach to analyze time-series RNA-sequencing data, revealing epigenetic modifications at the late stage of viral egress. Furthermore, we identified SARS-CoV-2-activated NF-κB and IRF1 pathways contributing to viral infection and COVID-19 severity through epigenetic analysis of H3K4me3 ChIP-sequencing. Cross-referencing our transcriptomic and epigenomic datasets revealed that coupling NF-κB and IRF1 pathways mediate PD-L1 immunosuppressive programs. Interestingly, we observed higher PD-L1 expression in Omicron-infected cells than SARS-CoV-2 infected cells. Blocking PD-L1 at an early stage of virally-infected AAV-hACE2 mice significantly recovered lymphocyte counts and lowered inflammatory cytokine levels. Our findings indicate that targeting the SARS-CoV-2-mediated NF-κB and IRF1-PD-L1 axis may represent an alternative strategy to reduce COVID-19 severity. This article is protected by copyright. All rights reserved.

We proposed and demonstrated that myelogenous leukemia has a preleukemic phase. In the premalignant phase, normal hematopoietic stem cells (HSCs) gradually accumulate mutations leading to HSC clonal expansion, resulting in the emergence of leukemic stem cells (LSCs). Here, we show that preleukemic HSCs are the basis of clonal hematopoiesis, as well as late-onset blood diseases (chronic-phase chronic myeloid leukemia, myeloproliferative neoplasms, and myelodysplastic disease). The clones at some point each trigger surface expression of "eat me" signals for macrophages, and in the clones and their LSC progeny, this is countered by upregulation of "don't eat me" signals for macrophages such as CD47,opening the possibility of CD47-based therapies. We include evidence that similar processes result in fibroblast expansion in a variety of fibrotic diseases, and arterial smooth muscle clonal expansion is a basis of atherosclerosis, including upregulation of both "eat me" and "don't eat me" molecules on the pathogenic cells.

Pathologic skin scarring presents a vast economic and medical burden. Unfortunately, the molecular mechanisms underlying scar formation remain to be elucidated. We used a hypertrophic scarring (HTS) mouse model in which Jun is overexpressed globally or specifically in α-smooth muscle or collagen type I–expressing cells to cause excessive extracellular matrix deposition by skin fibroblasts in the skin after wounding. Jun overexpression triggered dermal fibrosis by modulating distinct fibroblast subpopulations within the wound, enhancing reticular fibroblast numbers, and decreasing lipofibroblasts. Analysis of human scars further revealed that JUN is highly expressed across the wide spectrum of scars, including HTS and keloids. CRISPR-Cas9–mediated JUN deletion in human HTS fibroblasts combined with epigenomic and transcriptomic analysis of both human and mouse HTS fibroblasts revealed that JUN initiates fibrosis by regulating CD36. Blocking CD36 with salvianolic acid B or CD36 knockout model counteracted JUN-mediated fibrosis efficacy in both human fibroblasts and mouse wounds. In summary, JUN is a critical regulator of pathological skin scarring, and targeting its downstream effector CD36 may represent a therapeutic strategy against scarring.

Thrombopoietin (TPO) and the TPO-receptor (TPO-R, or c-MPL) are essential for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Agents that can modulate TPO-R signaling are highly desirable for both basic research and clinical utility. We developed a series of surrogate protein ligands for TPO-R, in the form of diabodies (DBs), that homodimerize TPO-R on the cell surface in geometries that are dictated by the DB receptor binding epitope, in effect "tuning" downstream signaling responses. These surrogate ligands exhibit diverse pharmacological properties, inducing graded signaling outputs, from full to partial TPO agonism, thus decoupling the dual functions of TPO/TPO-R. Using single-cell RNA sequencing and HSC self-renewal assays we find that partial agonistic diabodies preserved the stem-like properties of cultured HSCs, but also blocked oncogenic colony formation in essential thrombocythemia (ET) through inverse agonism. Our data suggest that dampening downstream TPO signaling is a powerful approach not only for HSC preservation in culture, but also for inhibiting oncogenic signaling through the TPO-R.

To explore how the immune system controls clearance of SARS-CoV-2, we used a single-cell, mass cytometry-based proteomics platform to profile the immune systems of 21 patients who had recovered from SARS-CoV-2 infection without need for admission to an intensive care unit or for mechanical ventilation. We focused on receptors involved in interactions between immune cells and virus-infected cells. We found that the diversity of receptor repertoires on natural killer (NK) cells was negatively correlated with the viral clearance rate. In addition, NK subsets expressing the receptor DNAM1 were increased in patients who more rapidly recovered from infection. Ex vivo functional studies revealed that NK subpopulations with high DNAM1 expression had cytolytic activities in response to target cell stimulation. We also found that SARS-CoV-2 infection induced the expression of CD155 and nectin-4, ligands of DNAM1 and its paired coinhibitory receptor TIGIT, which counterbalanced the cytolytic activities of NK cells. Collectively, our results link the cytolytic immune responses of NK cells to the clearance of SARS-CoV-2 and show that the DNAM1 pathway modulates host-pathogen interactions during SARS-CoV-2 infection.

Skin scarring, the end result of adult wound healing, is detrimental to tissue form and function. Engrailed-1 lineage-positive fibroblasts (EPFs) are known to function in scarring, but Engrailed-1 lineage-negative fibroblasts (ENFs) remain poorly characterized. Using cell transplantation and transgenic mouse models, we identified a dermal ENF subpopulation that gives rise to postnatally derived EPFs by activating Engrailed-1 expression during adult wound healing. By studying ENF responses to substrate mechanics, we found that mechanical tension drives Engrailed-1 activation via canonical mechanotransduction signaling. Finally, we showed that blocking mechanotransduction signaling with either verteporfin, an inhibitor of Yes-associated protein (YAP), or fibroblast-specific transgenic YAP knockout prevents Engrailed-1 activation and promotes wound regeneration by ENFs, with recovery of skin appendages, ultrastructure, and mechanical strength. This finding suggests that there are two possible outcomes to postnatal wound healing: a fibrotic response (EPF-mediated) and a regenerative response (ENF-mediated).

In the skin, tissue injury results in fibrosis in the form of scars composed of dense extracellular matrix deposited by fibroblasts. The therapeutic goal of regenerative wound healing has remained elusive, in part because principles of fibroblast programming and adaptive response to injury remain incompletely understood. Here, we present a multimodal -omics platform for the comprehensive study of cell populations in complex tissue, which has allowed us to characterize the cells involved in wound healing across both time and space. We employ a stented wound model that recapitulates human tissue repair kinetics and multiple Rainbow transgenic lines to precisely track fibroblast fate during the physiologic response to skin injury. Through integrated analysis of single cell chromatin landscapes and gene expression states, coupled with spatial transcriptomic profiling, we are able to impute fibroblast epigenomes with temporospatial resolution. This has allowed us to reveal potential mechanisms controlling fibroblast fate during migration, proliferation, and differentiation following skin injury, and thereby reexamine the canonical phases of wound healing. These findings have broad implications for the study of tissue repair in complex organ systems.

Osteoporosis and osteoporotic fractures lead to decreased life quality and high healthcare costs. Current treatments prevent losses in bone mass and fractures to some extent but have side effects. Therefore, better therapies are needed. This study investigated whether the transcription factor Jun has a specific pro-osteogenic potency and whether modulating Jun could serve as a novel treatment for osteoporosis-associated fractures. We demonstrate that ectopically transplanted whole bones and distinct osteoprogenitors increase bone formation. Perinatal Jun induction disturbs growth plate architecture, causing a striking phenotype with shortened and thickened bones. Molecularly, Jun induces hedgehog signaling in skeletal stem cells. Therapeutically, Jun accelerates bone growth and healing in a drilling-defect model. Altogether, these results demonstrate that Jun drives bone formation by expanding osteoprogenitor populations and forcing them into the bone fate, providing a rationale for future clinical applications.

Adhesions are fibrotic scars that form between abdominal organs following surgery or infection, and may cause bowel obstruction, chronic pain, or infertility. Our understanding of adhesion biology is limited, which explains the paucity of anti-adhesion treatments. Here we present a systematic analysis of mouse and human adhesion tissues. First, we show that adhesions derive primarily from the visceral peritoneum, consistent with our clinical experience that adhesions form primarily following laparotomy rather than laparoscopy. Second, adhesions are formed by poly-clonal proliferating tissue-resident fibroblasts. Third, using single cell RNA-sequencing, we identify heterogeneity among adhesion fibroblasts, which is more pronounced at early timepoints. Fourth, JUN promotes adhesion formation and results in upregulation of PDGFRA expression. With JUN suppression, adhesion formation is diminished. Our findings support JUN as a therapeutic target to prevent adhesions. An anti-JUN therapy that could be applied intra-operatively to prevent adhesion formation could dramatically improve the lives of surgical patients.

Scleroderma is a devastating fibrotic autoimmune disease. Current treatments are partly effective in preventing disease progression but do not remove fibrotic tissue. Here, we evaluated whether scleroderma fibroblasts take advantage of the "don't-eat-me-signal" CD47 and whether blocking CD47 enables the body's immune system to get rid of diseased fibroblasts. To test this approach, we used a Jun-inducible scleroderma model. We first demonstrated in patient samples that scleroderma upregulated transcription factor JUN and increased promoter accessibilities of both JUN and CD47. Next, we established our scleroderma model, demonstrating that Jun mediated skin fibrosis through the hedgehog-dependent expansion of CD26+Sca1- fibroblasts in mice. In a niche-independent adaptive transfer model, JUN steered graft survival and conferred increased self-renewal to fibroblasts. In vivo, JUN enhanced the expression of CD47, and inhibiting CD47 eliminated an ectopic fibroblast graft and increased in vitro phagocytosis. In the syngeneic mouse, depleting macrophages ameliorated skin fibrosis. Therapeutically, combined CD47 and IL-6 blockade reversed skin fibrosis in mice and led to the rapid elimination of ectopically transplanted scleroderma cells. Altogether, our study demonstrates the efficiency of combining different immunotherapies in treating scleroderma and provides a rationale for combining CD47 and IL-6 inhibition in clinical trials.

The transcription factor JUN is highly expressed in pulmonary fibrosis. Its induction in mice drives lung fibrosis, which is abrogated by administration of anti-CD47. Here, we use high-dimensional mass cytometry to profile protein expression and secretome of cells from patients with pulmonary fibrosis. We show that JUN is activated in fibrotic fibroblasts that expressed increased CD47 and PD-L1. Using ATAC-seq and ChIP-seq, we found that activation of JUN rendered promoters and enhancers of CD47 and PD-L1 accessible. We further detect increased IL-6 that amplified JUN-mediated CD47 enhancer activity and protein expression. Using an in vivo mouse model of fibrosis, we found two distinct mechanisms by which blocking IL-6, CD47 and PD-L1 reversed fibrosis, by increasing phagocytosis of profibrotic fibroblasts and by eliminating suppressive effects on adaptive immunity. Our results identify specific immune mechanisms that promote fibrosis and suggest a therapeutic approach that could be used alongside conventional anti-fibrotics for pulmonary fibrosis.

Graft-vs-host disease (GVHD) remains a major complication of allogeneic hematopoietic cell transplantation. Acute GVHD results from direct damage by donor T cells, while the biology of chronic GVHD (cGVHD) with its autoimmune-like manifestations remains poorly understood - mainly due to the paucity of representative preclinical models. We examined over an extended time period seven MHC-matched, minor antigen mismatched mouse models for development of cGVHD. Development and manifestations of cGVHD were determined by a combination of MHC-allele type and recipient strain, with BALB recipients being the most susceptible. The C57BL/6 into BALB.B combination most closely modeled the human syndrome. In this strain combination moderate acute GVHD was observed and BALB.B survivors developed overt cGVHD at 6-12 months affecting eyes, skin and liver. Naïve CD4+ cells caused this syndrome as no significant pathology was induced by grafts composed of purified hematopoietic stem cells (HSC) or HSC plus memory CD4+ or CD8+ cells. Furthermore, co-transferred naïve and memory CD4+ T cells demonstrated differential homing patterns and locations of persistence. No clear association with donor TH17 cells and the phenotype of acute or chronic GVHD was observed in this model. Donor CD4+ cells caused injury to medullary thymic epithelial cells, a key population responsible for negative T-cell selection, suggesting that impaired thymic selection was an underlying cause of the cGVHD syndrome. In conclusion, we report for the first time that the C57BL/6 into BALB.B combination is a representative model of cGVHD that evolves from immunological events during the early post-transplant period.

Hematopoietic stem cell transplantation (HSCT) is a curative therapy for blood and immune diseases with potential for many settings beyond current standard-of-care. Broad HSCT application is currently precluded largely due to morbidity and mortality associated with genotoxic irradiation or chemotherapy conditioning. Here we show that a single dose of a CD117-antibody-drug-conjugate (CD117-ADC) to saporin leads to > 99% depletion of host HSCs, enabling rapid and efficient donor hematopoietic cell engraftment. Importantly, CD117-ADC selectively targets hematopoietic stem cells yet does not cause clinically significant side-effects. Blood counts and immune cell function are preserved following CD117-ADC treatment, with effective responses by recipients to both viral and fungal challenges. These results suggest that CD117-ADC-mediated HSCT pre-treatment could serve as a non-myeloablative conditioning strategy for the treatment of a wide range of non-malignant and malignant diseases, and might be especially suited to gene therapy and gene editing settings in which preservation of immunity is desired.

Peritoneal adhesions are fibrous tissues that tether organs to one another or to the peritoneal wall and are a major cause of postsurgical and infectious morbidity. The primary molecular chain of events leading to the initiation of adhesions has been elusive, chiefly due to the lack of an identifiable cell of origin. Using clonal analysis and lineage tracing, we have identified injured surface mesothelium expressing podoplanin (PDPN) and mesothelin (MSLN) as a primary instigator of peritoneal adhesions after surgery in mice. We demonstrate that an anti-MSLN antibody diminished adhesion formation in a mouse model where adhesions were induced by surgical ligation to form ischemic buttons and subsequent surgical abrasion of the peritoneum. RNA sequencing and bioinformatics analyses of mouse mesothelial cells from injured mesothelium revealed aspects of the pathological mechanism of adhesion development and yielded several potential regulators of this process. Specifically, we show that PDPN+MSLN+ mesothelium responded to hypoxia by early up-regulation of hypoxia-inducible factor 1 alpha (HIF1α) that preceded adhesion development. Inhibition of HIF1α with small molecules ameliorated the injury program in damaged mesothelium and was sufficient to diminish adhesion severity in a mouse model. Analyses of human adhesion tissue suggested that similar surface markers and signaling pathways may contribute to surgical adhesions in human patients.

OBJECTIVE: To investigate the effects of local doxycycline administration on skin scarring.BACKGROUND: Skin scarring represents a major source of morbidity for surgical patients. Doxycycline, a tetracycline antibiotic with off-target effects on the extracellular matrix, has demonstrated antifibrotic effects in multiple organs. However, doxycycline's potential effects on skin scarring have not been explored in vivo.METHODS: Female C57BL/6J mice underwent dorsal wounding following an established splinted excisional skin wounding model. Doxycycline was administered by local injection into the wound base following injury. Wounds were harvested upon complete wound closure (postoperative day 15) for histological examination and biomechanical testing of scar tissue.RESULTS: A one-time dose of 3.90 mM doxycycline (2 mg/mL) within 12 hours of injury was found to significantly reduce scar thickness by 24.8% (P < 0.0001) without compromising tensile strength. The same effect could not be achieved by oral dosing. In doxycycline-treated scar matrices, collagen I content was significantly reduced (P = 0.0317) and fibers were favorably arranged with significantly increased fiber randomness (P = 0.0115). Common culprits of altered wound healing mechanics, including angiogenesis and inflammation, were not impacted by doxycycline treatment. However, engrailed1 profibrotic fibroblasts, responsible for scar extracellular matrix deposition, were significantly reduced with doxycycline treatment (P = 0.0005).CONCLUSIONS: Due to the substantial improvement in skin scarring and well-established clinical safety profile, locally administered doxycycline represents a promising vulnerary agent. As such, we favor rapid translation to human patients as an antiscarring therapy.

BACKGROUND: Animal models of optic nerve injury are often used to study central nervous system (CNS) degeneration and regeneration, and targeting the optic nerve is a powerful approach for axon-protective or remyelination therapy. However, the experimental delivery of drugs or cells to the optic nerve is rarely performed because injections into this structure are difficult in small animals, especially in mice.NEW METHOD: We investigated and developed methods to deliver drugs or cells to the mouse optic nerve through 3 different routes: a) intraorbital, b) through the optic foramen and c) transcranial.RESULTS: The methods targeted different parts of the mouse optic nerve: intraorbital proximal (intraorbital), intracranial middle (optic-foramen) or intracranial distal (transcranial) portion.COMPARISON WITH EXISTING METHODS: Most existing methods target the optic nerve indirectly. For instance, intravitreally delivered cells often cannot cross the inner limiting membrane to reach retinal neurons and optic nerve axons. Systemic delivery, eye drops and intraventricular injections do not always successfully target the optic nerve. Intraorbital and transcranial injections into the optic nerve or chiasm have been performed but these methods have not been well described. We approached the optic nerve with more selective and precise targeting than existing methods.CONCLUSIONS: We successfully targeted the murine optic nerve intraorbitally, through the optic foramen, and transcranially. Of all methods, the injection through the optic foramen is likely the most innovative and fastest. These methods offer additional approaches for therapeutic intervention to be used by those studying white matter damage and axonal regeneration in the CNS.

Fibrotic diseases are not well-understood. They represent a number of different diseases that are characterized by the development of severe organ fibrosis without any obvious cause, such as the devastating diseases idiopathic pulmonary fibrosis (IPF) and scleroderma. These diseases have a poor prognosis comparable with endstage cancer and are uncurable. Given the phenotypic differences, it was assumed that the different fibrotic diseases also have different pathomechanisms. Here, we demonstrate that many endstage fibrotic diseases, including IPF; scleroderma; myelofibrosis; kidney-, pancreas-, and heart-fibrosis; and nonalcoholic steatohepatosis converge in the activation of the AP1 transcription factor c-JUN in the pathologic fibroblasts. Expression of the related AP1 transcription factor FRA2 was restricted to pulmonary artery hypertension. Induction of c-Jun in mice was sufficient to induce severe fibrosis in multiple organs and steatohepatosis, which was dependent on sustained c-Jun expression. Single cell mass cytometry revealed that c-Jun activates multiple signaling pathways in mice, including pAkt and CD47, which were also induced in human disease. αCD47 antibody treatment and VEGF or PI3K inhibition reversed various organ c-Jun-mediated fibroses in vivo. These data suggest that c-JUN is a central molecular mediator of most fibrotic conditions.