Targeted proteomic approach for quantification of collagen type I and type III in formalin-fixed paraffin-embedded tissue.

Collagen is the most abundant protein in mammals and a major structural component of the extracellular matrix (ECM). Changes to ECM composition occur as a result of numerous physiological and pathophysiological causes, and a common means to evaluate these changes is the collagen 3 (Col3) to collagen 1 (Col1) ratio. Current methods to measure the Col3/1 ratio suffer from a lack of specificity and often under- or over-estimate collagen composition and quantity. This manuscript presents a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of Col3 and Col1 in FFPE tissues. Using surrogate peptides to generate calibration curves, Col3 and Col1 are readily quantified in FFPE tissue sections with high accuracy and precision. The method is applied to several tissue types from both human and reindeer sources, demonstrating its generalizability. In addition, the targeted LC-MS/MS method permits quantitation of the hydroxyprolinated form of Col3, which has significant implications for understanding not only the quantity of Col3 in tissue, but also understanding of the pathophysiology underlying many causes of ECM changes. This manuscript presents a straightforward, accurate, precise, and generalizable method for quantifying the Col3/1 ratio in a variety of tissue types and organisms.

Primary cilia signaling in astrocytes mediates development and regional-specific functional specification.

Astrocyte diversity is greatly influenced by local environmental modulation. Here we report that the majority of astrocytes across the mouse brain possess a singular primary cilium localized to the cell soma. Comparative single-cell transcriptomics reveals that primary cilia mediate canonical SHH signaling to modulate astrocyte subtype-specific core features in synaptic regulation, intracellular transport, energy and metabolism. Independent of canonical SHH signaling, primary cilia are important regulators of astrocyte morphology and intracellular signaling balance. Dendritic spine analysis and transcriptomics reveal that perturbation of astrocytic cilia leads to disruption of neuronal development and global intercellular connectomes in the brain. Mice with primary ciliary-deficient astrocytes show behavioral deficits in sensorimotor function, sociability, learning and memory. Our results uncover a critical role for primary cilia in transmitting local cues that drive the region-specific diversification of astrocytes within the developing brain.

The development of brain pericytes requires expression of the transcription factor nkx3.1 in intermediate precursors.

Brain pericytes are one of the critical cell types that regulate endothelial barrier function and activity, thus ensuring adequate blood flow to the brain. The genetic pathways guiding undifferentiated cells into mature pericytes are not well understood. We show here that pericyte precursor populations from both neural crest and head mesoderm of zebrafish express the transcription factor nkx3.1 develop into brain pericytes. We identify the gene signature of these precursors and show that an nkx3.1-, foxf2a-, and cxcl12b-expressing pericyte precursor population is present around the basilar artery prior to artery formation and pericyte recruitment. The precursors later spread throughout the brain and differentiate to express canonical pericyte markers. Cxcl12b-Cxcr4 signaling is required for pericyte attachment and differentiation. Further, both nkx3.1 and cxcl12b are necessary and sufficient in regulating pericyte number as loss inhibits and gain increases pericyte number. Through genetic experiments, we have defined a precursor population for brain pericytes and identified genes critical for their differentiation.

Novel AAV variants with improved tropism for human Schwann cells.

Gene therapies and associated technologies are transforming biomedical research and enabling novel therapeutic options for patients living with debilitating and incurable genetic disorders. The vector system based on recombinant adeno-associated viral vectors (AAVs) has shown great promise in recent clinical trials for genetic diseases of multiple organs, such as the liver and the nervous system. Despite recent successes toward the development of novel bioengineered AAV variants for improved transduction of primary human tissues and cells, vectors that can efficiently transduce human Schwann cells (hSCs) have yet to be identified. Here, we report the application of the functional transduction-RNA selection method in primary hSCs for the development of AAV variants for specific and efficient transgene delivery to hSCs. The two identified capsid variants, Pep2hSC1 and Pep2hSC2, show conserved potency for delivery across various in vitro, in vivo, and ex vivo models of hSCs. These novel AAV capsids will serve as valuable research tools, forming the basis for therapeutic solutions for both SC-related disorders or peripheral nervous system injury.

Single-cell analysis reveals distinct fibroblast plasticity during tenocyte regeneration in zebrafish.

Despite their importance in tissue maintenance and repair, fibroblast diversity and plasticity remain poorly understood. Using single-cell RNA sequencing, we uncover distinct sclerotome-derived fibroblast populations in zebrafish, including progenitor-like perivascular/interstitial fibroblasts, and specialized fibroblasts such as tenocytes. To determine fibroblast plasticity in vivo, we develop a laser-induced tendon ablation and regeneration model. Lineage tracing reveals that laser-ablated tenocytes are quickly regenerated by preexisting fibroblasts. By combining single-cell clonal analysis and live imaging, we demonstrate that perivascular/interstitial fibroblasts actively migrate to the injury site, where they proliferate and give rise to new tenocytes. By contrast, perivascular fibroblast-derived pericytes or specialized fibroblasts, including tenocytes, exhibit no regenerative plasticity. Active Hedgehog (Hh) signaling is required for the proliferation of activated fibroblasts to ensure efficient tenocyte regeneration. Together, our work highlights the functional diversity of fibroblasts and establishes perivascular/interstitial fibroblasts as tenocyte progenitors that promote tendon regeneration in a Hh signaling-dependent manner.

Cystatin C is glucocorticoid responsive, directs recruitment of Trem2+ macrophages, and predicts failure of cancer immunotherapy.

Cystatin C (CyC), a secreted cysteine protease inhibitor, has unclear biological functions. Many patients exhibit elevated plasma CyC levels, particularly during glucocorticoid (GC) treatment. This study links GCs with CyC's systemic regulation by utilizing genome-wide association and structural equation modeling to determine CyC production genetics in the UK Biobank. Both CyC production and a polygenic score (PGS) capturing predisposition to CyC production were associated with increased all-cause and cancer-specific mortality. We found that the GC receptor directly targets CyC, leading to GC-responsive CyC secretion in macrophages and cancer cells. CyC-knockout tumors displayed significantly reduced growth and diminished recruitment of TREM2+ macrophages, which have been connected to cancer immunotherapy failure. Furthermore, the CyC-production PGS predicted checkpoint immunotherapy failure in 685 patients with metastatic cancer from combined clinical trial cohorts. In conclusion, CyC may act as a GC effector pathway via TREM2+ macrophage recruitment and may be a potential target for combination cancer immunotherapy.

Case report: Immune profiling links neutrophil and plasmablast dysregulation to microvascular damage in post-COVID-19 Multisystem Inflammatory Syndrome in Adults (MIS-A).

Despite surviving a SARS-CoV-2 infection, some individuals experience an intense post-infectious Multisystem Inflammatory Syndrome (MIS) of uncertain etiology. Children with this syndrome (MIS-C) can experience a Kawasaki-like disease, but mechanisms in adults (MIS-A) are not clearly defined. Here we utilize a deep phenotyping approach to examine immunologic responses in an individual with MIS-A. Results are contextualized to healthy, convalescent, and acute COVID-19 patients. The findings reveal systemic inflammatory changes involving novel neutrophil and B-cell subsets, autoantibodies, complement, and hypercoagulability that are linked to systemic vascular dysfunction. This deep patient profiling generates new mechanistic insight into this rare clinical entity and provides potential insight into other post-infectious syndromes.

A protocol for single nucleus RNA-seq from frozen skeletal muscle.

Single-cell technologies are a method of choice to obtain vast amounts of cell-specific transcriptional information under physiological and diseased states. Myogenic cells are resistant to single-cell RNA sequencing because of their large, multinucleated nature. Here, we report a novel, reliable, and cost-effective method to analyze frozen human skeletal muscle by single-nucleus RNA sequencing. This method yields all expected cell types for human skeletal muscle and works on tissue frozen for long periods of time and with significant pathological changes. Our method is ideal for studying banked samples with the intention of studying human muscle disease.

A B1a-natural IgG-neutrophil axis is impaired in viral- and steroid-associated aspergillosis.

The lung naturally resists Aspergillus fumigatus (Af) in healthy individuals, but multiple conditions can disrupt this resistance, leading to lethal invasive infections. Core processes of natural resistance and its breakdown are undefined. We investigated three distinct conditions predisposing to lethal aspergillosis-severe SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection, influenza A viral pneumonia, and systemic corticosteroid use-in human patients and murine models. We found a conserved and essential coupling of innate B1a lymphocytes, Af-binding natural immunoglobulin G antibodies, and lung neutrophils. Failure of this axis concealed Af from neutrophils, allowing rapid fungal invasion and disease. Reconstituting the axis with immunoglobulin therapy reestablished resistance, thus representing a realistic pathway to repurpose currently available therapies. Together, we report a vital host resistance pathway that is responsible for protecting against life-threatening aspergillosis in the context of distinct susceptibilities.

Fibroblast inflammatory priming determines regenerative versus fibrotic skin repair in reindeer.

Adult mammalian skin wounds heal by forming fibrotic scars. We report that full-thickness injuries of reindeer antler skin (velvet) regenerate, whereas back skin forms fibrotic scar. Single-cell multi-omics reveal that uninjured velvet fibroblasts resemble human fetal fibroblasts, whereas back skin fibroblasts express inflammatory mediators mimicking pro-fibrotic adult human and rodent fibroblasts. Consequently, injury elicits site-specific immune responses: back skin fibroblasts amplify myeloid infiltration and maturation during repair, whereas velvet fibroblasts adopt an immunosuppressive phenotype that restricts leukocyte recruitment and hastens immune resolution. Ectopic transplantation of velvet to scar-forming back skin is initially regenerative, but progressively transitions to a fibrotic phenotype akin to the scarless fetal-to-scar-forming transition reported in humans. Skin regeneration is diminished by intensifying, or enhanced by neutralizing, these pathologic fibroblast-immune interactions. Reindeer represent a powerful comparative model for interrogating divergent wound healing outcomes, and our results nominate decoupling of fibroblast-immune interactions as a promising approach to mitigate scar.

Application of an instructive hydrogel accelerates re-epithelialization of xenografted human skin wounds.

Poor quality (eg. excessive scarring) or delayed closure of skin wounds can have profound physical and pyschosocial effects on patients as well as pose an enormous economic burden on the healthcare system. An effective means of improving both the rate and quality of wound healing is needed for all patients suffering from skin injury. Despite wound care being a multi-billion-dollar industry, effective treatments aimed at rapidly restoring the skin barrier function or mitigating the severity of fibrotic scar remain elusive. Previously, a hydrogel conjugated angiopoietin-1 derived peptide (QHREDGS; Q-peptide) was shown to increase keratinocyte migration and improve wound healing in diabetic mice. Here, we evaluated the effect of this Q-Peptide Hydrogel on human skin wound healing using a mouse xenograft model. First, we confirmed that the Q-Peptide Hydrogel promoted the migration of adult human keratinocytes and modulated their cytokine profile in vitro. Next, utilizing our human to mouse split-thickness skin xenograft model, we found improved healing of wounded human epidermis following Q-Peptide Hydrogel treatment. Importantly, Q-Peptide Hydrogel treatment enhanced this wound re-epithelialization via increased keratinocyte migration and survival, rather than a sustained increase in proliferation. Overall, these data provide strong evidence that topical application of QHREDGS peptide-modified hydrogels results in accelerated wound closure that may lead to improved outcomes for patients.

Comparison of human skin- and nerve-derived Schwann cells reveals many similarities and subtle genomic and functional differences.

Skin is an easily accessible tissue and a rich source of Schwann cells (SCs). Toward potential clinical application of autologous SC therapies, we aim to improve the reliability and specificity of our protocol to obtain SCs from small skin samples. As well, to explore potential functional distinctions between skin-derived SCs (Sk-SCs) and nerve-derived SCs (N-SCs), we used single-cell RNA-sequencing and a series of in vitro and in vivo assays. Our results showed that Sk-SCs expressed typical SC markers. Single-cell sequencing of Sk- and N-SCs revealed an overwhelming overlap in gene expression with the exception of HLA genes which were preferentially up-regulated in Sk-SCs. In vitro, both cell types exhibited similar levels of proliferation, migration, uptake of myelin debris and readily associated with neurites when co-cultured with human iPSC-induced motor neurons. Both exhibited ensheathment of multiple neurites and early phase of myelination, especially in N-SCs. Interestingly, dorsal root ganglion (DRG) neurite outgrowth assay showed substantially more complexed neurite outgrowth in DRGs exposed to Sk-SC conditioned media compared to those from N-SCs. Multiplex ELISA array revealed shared growth factor profiles, but Sk-SCs expressed a higher level of VEGF. Transplantation of Sk- and N-SCs into injured peripheral nerve in nude rats and NOD-SCID mice showed close association of both SCs to regenerating axons. Myelination of rodent axons was observed infrequently by N-SCs, but absent in Sk-SC xenografts. Overall, our results showed that Sk-SCs share near-identical properties to N-SCs but with subtle differences that could potentially enhance their therapeutic utility.

Dexamethasone modulates immature neutrophils and interferon programming in severe COVID-19.

Although critical for host defense, innate immune cells are also pathologic drivers of acute respiratory distress syndrome (ARDS). Innate immune dynamics during Coronavirus Disease 2019 (COVID-19) ARDS, compared to ARDS from other respiratory pathogens, is unclear. Moreover, mechanisms underlying the beneficial effects of dexamethasone during severe COVID-19 remain elusive. Using single-cell RNA sequencing and plasma proteomics, we discovered that, compared to bacterial ARDS, COVID-19 was associated with expansion of distinct neutrophil states characterized by interferon (IFN) and prostaglandin signaling. Dexamethasone during severe COVID-19 affected circulating neutrophils, altered IFNactive neutrophils, downregulated interferon-stimulated genes and activated IL-1R2+ neutrophils. Dexamethasone also expanded immunosuppressive immature neutrophils and remodeled cellular interactions by changing neutrophils from information receivers into information providers. Male patients had higher proportions of IFNactive neutrophils and preferential steroid-induced immature neutrophil expansion, potentially affecting outcomes. Our single-cell atlas (see 'Data availability' section) defines COVID-19-enriched neutrophil states and molecular mechanisms of dexamethasone action to develop targeted immunotherapies for severe COVID-19.

Marginating transitional B cells modulate neutrophils in the lung during inflammation and pneumonia.

Pulmonary innate immunity is required for host defense; however, excessive neutrophil inflammation can cause life-threatening acute lung injury. B lymphocytes can be regulatory, yet little is known about peripheral transitional IgM+ B cells in terms of regulatory properties. Using single-cell RNA sequencing, we discovered eight IgM+ B cell subsets with unique gene regulatory networks in the lung circulation dominated by transitional type 1 B and type 2 B (T2B) cells. Lung intravital confocal microscopy revealed that T2B cells marginate in the pulmonary capillaries via CD49e and require CXCL13 and CXCR5. During lung inflammation, marginated T2B cells dampened excessive neutrophil vascular inflammation via the specialized proresolving molecule lipoxin A4 (LXA4). Exogenous CXCL13 dampened excessive neutrophilic inflammation by increasing marginated B cells, and LXA4 recapitulated neutrophil regulation in B cell-deficient mice during inflammation and fungal pneumonia. Thus, the lung microvasculature is enriched in multiple IgM+ B cell subsets with marginating capillary T2B cells that dampen neutrophil responses.

A Primary Care Provider's Guide to Prevention and Management of Pressure Injury and Skin Breakdown in People With Spinal Cord Injury.

Skin breakdown, including burns and pressure injuries (PrIs), is a devastating complication of spinal cord injury (SCI). Chronic wounds place the person with SCI at high risk of infections, sepsis, and death. Skin health and breakdown is individual and multifactorial, thus prevention requires individualized education focused on patient preferences and goals. Assessment requires an accurate description of wound type/PrI stage, location, size, wound bed, wound margin, epithelialization, exudate, and peri-wound condition. PrIs should be staged using the National Pressure Injury Advisory Panel (NPIAP) staging system. Successful treatment requires optimal wound bed preparation, pressure off-loading, and access to surgical specialists if needed. Mattress and seating systems, pressure relief, skin microclimate, nutrition, and home supports should be optimized. To promote wound healing and aid prevention, identifiable causes need to be removed, risk factors improved, and wound care provided. Infection should be treated with input from infectious disease specialists. Consideration for specialized surgical management including flaps and primary closures should be coordinated with the interdisciplinary team to optimize outcomes. If comorbid conditions promote wound chronicity, a palliative rather than curative treatment plan may be needed.

Distinct Regulatory Programs Control the Latent Regenerative Potential of Dermal Fibroblasts during Wound Healing.

Dermal fibroblasts exhibit considerable heterogeneity during homeostasis and in response to injury. Defining lineage origins of reparative fibroblasts and regulatory programs that drive fibrosis or, conversely, promote regeneration will be essential for improving healing outcomes. Using complementary fate-mapping approaches, we show that hair follicle mesenchymal progenitors make limited contributions to wound repair. In contrast, extrafollicular progenitors marked by the quiescence-associated factor Hic1 generated the bulk of reparative fibroblasts and exhibited functional divergence, mediating regeneration in the center of the wound neodermis and scar formation in the periphery. Single-cell RNA-seq revealed unique transcriptional, regulatory, and epithelial-mesenchymal crosstalk signatures that enabled mesenchymal competence for regeneration. Integration with scATAC-seq highlighted changes in chromatin accessibility within regeneration-associated loci. Finally, pharmacological modulation of RUNX1 and retinoic acid signaling or genetic deletion of Hic1 within wound-activated fibroblasts was sufficient to modulate healing outcomes, suggesting that reparative fibroblasts have latent but modifiable regenerative capacity.

Macrophages and Associated Ligands in the Aged Injured Nerve: A Defective Dynamic That Contributes to Reduced Axonal Regrowth.

The regenerative capacity of injured peripheral nerves is diminished with aging. To identify factors that contribute to this impairment, we compared the immune cell response in young vs. aged animals following nerve injury. First, we confirmed that macrophage accumulation is delayed in aged injured nerves which is due to defects in monocyte migration as a result of defects in site-specific recruitment signals in the aged nerve. Interestingly, impairment in both macrophage accumulation and functional recovery could be overcome by transplanting bone marrow from aged animals into young mice. That is, upon exposure to a youthful environment, monocytes/macrophages originating from the aged bone marrow behaved similarly to young cells. Transcriptional profiling of aged macrophages following nerve injury revealed that both pro- and anti-inflammatory genes were largely downregulated in aged compared to young macrophages. One ligand of particular interest was macrophage-associated secreted protein (MCP1), which exhibited a potent role in regulating aged axonal regrowth in vitro. Given that macrophage-derived MCP1 is significantly diminished in the aged injured nerve, our data suggest that age-associated defects in MCP1 signaling could contribute to the regenerative deficits that occur in the aged nervous system.

Dysfunction of Hair Follicle Mesenchymal Progenitors Contributes to Age-Associated Hair Loss.

Skin aging is accompanied by hair loss due to impairments in hair follicle (HF) epithelial progenitor cells and their mesenchymal niche. This inductive mesenchyme, called dermal papilla (DP), undergoes progressive cell loss and eventual miniaturization that contributes to HF pathogenesis. Using laser ablation and fate mapping, we show that HF dermal stem cells (hfDSCs) reconstitute the damaged DP and maintain hair growth, suggesting that hfDSC dysfunction may trigger degeneration of the inductive niche. Fate mapping over 24 months revealed progressive hfDSC depletion, and in vivo clonal analysis of aged hfDSCs showed impaired self-renewal and biased differentiation. Single-cell RNA-seq confirmed hfDSCs as a central precursor, giving rise to divergent mesenchymal trajectories. In aged skin, hfDSCs exhibited senescent-like characteristics, and senescence-associated secretory phenotypes were identified in the aging HF mesenchyme. These results clarify fibroblast dynamics within the HF and suggest that progressive dysfunction within the mesenchymal progenitor pool contributes to age-related hair loss.