Biology subjects

Pilling, D.

Publications and source records attributed to Pilling, D..

6 recordsLinked to original sources

bioRxiv2025

An extracellular matrix coating similar to articular cartilage inhibits the differentiation of myofibroblasts and limits the development of implant-induced fibrosis

Fibrosis is involved in 45% of deaths in the United States, with no treatment options to reverse progression of the disease. Implantable devices (such as joint replacements, chips, stents, artificial organs, biosensors, catheters, heart valves, scaffolds for tissue engineering, etc.) can trigger a foreign body response, in which fibrotic tissue covers the implant and impedes its function. Myofibroblast are a key cellular component of scar tissue. To explore the relationship between extracellular matrix-based coatings and fibrosis, we coated tissue-culture surfaces using a library of extracellular matrix (ECM) proteins and then performed an in-vitro screen for myofibroblast differentiation on the coated surfaces as an indicator of fibrotic potential. The protein and proteoglycan components of cartilage (collagen II, biglycan, decorin, and chondroitin sulfate) were individually anti-fibrotic. Further, mixtures of collagen II, biglycan, decorin, and chondroitin sulfate inhibited myofibroblast differentiation to a greater degree than collagen II, biglycan, decorin, or chondroitin sulfate as individual coatings. Next, we performed an in-vivo model of a foreign-body response. Implanting an uncoated implant subcutaneously into mice resulted in a thicker layer of fibrotic scar tissue than implants coated with a cartilage-like ECM mixture. Our results indicate that the ECM microenvironment is key to the initiation, progression, and maintenance of fibrosis.

immunology↗

bioRxiv2025

Differences between human male and female neutrophils with respect to which RNAs are present in polysomes

BackgroundHuman males and females show differences in the incidence of neutrophil-associated diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis, and differences in neutrophil physiological responses such as responses to infection, tissue damage, and chemotactic factors. However, little is known about the basis of sex-based differences in human neutrophils. MethodsStarting with human neutrophils from healthy donors, we used RNA-seq to examine total RNA profiles, RNAs not associated with ribosomes and thus not being translated, RNAs in monosomes, and RNAs in polysomes and thus heavily translated. ResultsThere were sex-based differences in the levels of RNAs across free RNA, monosome, and polysome fractions. Male neutrophils had increased levels of mRNAs encoding mitochondrial proteins in the free RNA fractions, indicating low levels of translation. The polysomes of male neutrophils were enriched for mRNAs encoding cytoskeletal organization, cell motility, and cell activation. The polysomes of female neutrophils were enriched for mRNAs associated the regulation of metabolic processes, cytokine responses, and mitochondrial proteins. ConclusionsThese data indicate that male and female neutrophils have different expression patterns and different translation efficiency of some mRNAs. This may contribute to the observed sex-based differences in neutrophil behavior and neutrophil-associated disease incidence and severity.

immunology↗

bioRxiv2022

Inhibiting a mRNA motif binding protein that mediates TGF-β1 upregulation of translation attenuates pulmonary fibrosis in mice

In human lung cells, the profibrotic cytokine TGF-{beta}1 increases sialidase 3 (NEU3) protein by increasing NEU3 translation without increasing levels of NEU3 mRNA. To elucidate how TGF-{beta}1 regulates translation, we treated human lung fibroblasts (HLF) with TGF-{beta}1 and used proteomics and RNA-seq to determine the effect of TGF-{beta}1 on proteins, mRNAs, and mRNA polysome/monosome ratios. We identified 181 mRNAs where TGF-{beta}1 also increases translation to increase protein levels without significantly affecting mRNA levels. These mRNAs share a common 20 nucleotide motif. Deletion or insertion of this motif in mRNAs eliminates or induces the TGF-{beta}1 regulation of translation. At least 5 RNA-binding proteins including DDX3 bind the RNA motif, and TGF-{beta}1 regulates their protein levels and/or binding to the motif. Inhibiting DDX3, either by siRNA or small molecule inhibitors, reduced TGF-{beta}1 induced NEU3 levels. In the mouse bleomycin model of pulmonary fibrosis, injections of the DDX3 inhibitor RK-33 starting 10 days after bleomycin potentiated survival and reduced lung inflammation, fibrosis, and lung tissue levels of DDX3, TGF-{beta}1, and NEU3. Together, these results suggest that TGF-{beta}1 regulates RNA-binding proteins that interact with a mRNA motif that is necessary and sufficient for TGF-{beta}1 to regulate mRNA translation, and that blocking this effect can reduce fibrosis.

immunology↗

bioRxiv2022

Changes in lung sialidases in male and female mice after bleomycin aspiration

Aim of the studySialidases, also called neuraminidases, are enzymes that cleave terminal sialic acids from glycoconjugates. In humans and mice, lung fibrosis is associated with desialylation of glycoconjugates and upregulation of sialidases. There are four mammalian sialidases, and it is unclear when the four mammalian sialidases are elevated over the course of inflammatory and fibrotic responses, whether tissue resident and inflammatory cells express different sialidases, and if sialidases are differentially expressed in male and females. Materials and MethodsTo determine the time course of sialidase expression and the identity of sialidase expressing cells, we used the bleomycin model of pulmonary fibrosis in mice to examine levels of sialidases during inflammation (days 3 - 10) and fibrosis (days 10 - 21). ResultsBleomycin aspiration increased sialidase NEU1 at days 14 and 21 in male mice and day 10 in female mice. NEU2 levels increased at day 7 in male and day 10 in female mice. NEU3 appears to have a biphasic response in male mice with increased levels at day 7 and then at days 14 and 21, whereas in female mice NEU3 levels increased over 21 days. In control mice, the sialidases were mainly expressed by EpCAM positive epithelial cells, but after bleomycin, epithelial cells, CD45 positive immune cells, and alveolar cells expressed NEU1, NEU2, and NEU3. Sialidase expression was higher in male compared to female mice. There was little expression of NEU4 in murine lung tissue. ConclusionsThese results suggest that sialidases are dynamically expressed following bleomycin, that sialidases are differentially expressed in male and females, and that of the four sialidases only NEU3 upregulation is associated with fibrosis in both male and female mice.

immunology↗

bioRxiv2022

The sialidase NEU3 promotes pulmonary fibrosis in mice

Sialic acid is often the distal sugar on glycoconjugates, and sialidases are enzymes that remove this sugar. In fibrotic lesions in human and mouse lungs, there is extensive desialylation of glycoconjugates, and upregulation of sialidases including the extracellular sialidase NEU3. In the bleomycin model of pulmonary fibrosis, mice lacking NEU3 (Neu3-/-) showed strongly attenuated bleomycin-induced weight loss, lung damage, inflammation, upregulation of TGF-{beta}1, and fibrosis. This indicates that NEU3 is necessary for the full spectrum of bleomycin-induced pulmonary fibrosis. To determine if NEU3 is sufficient for fibrosis, mice not treated with bleomycin were treated with recombinant murine NEU3 or inactive NEU3. Aspiration of NEU3 caused inflammation and fibrosis in the lungs, while inactive NEU3 caused inflammation but not fibrosis. Mice were also treated with NEU3 starting 10 days after bleomycin. In male but not female mice, NEU3 increased inflammation and fibrosis. Inactive NEU3 did not enhance bleomycin-induced lung fibrosis. These results suggest that NEU3 is sufficient to induce fibrosis in the lungs, and that this effect is mediated by NEU3s enzymic activity.

immunology↗

bioRxiv2020

Serum Amyloid P inhibits single stranded RNA-induced lung inflammation, lung damage, and cytokine storm in mice

SARS-CoV-2 is a single stranded RNA (ssRNA) virus and contains GU-rich sequences distributed abundantly in the genome. In COVID-19, the infection and immune hyperactivation causes accumulation of inflammatory immune cells, blood clots, and protein aggregates in lung fluid, increased lung alveolar wall thickness, and upregulation of serum cytokine levels. A serum protein called serum amyloid P (SAP) has a calming effect on the innate immune system and shows efficacy as a therapeutic for fibrosis in animal models and clinical trials. In this report, we show that aspiration of the GU-rich ssRNA oligonucleotide ORN06 into mouse lungs induces all of the above COVID-19-like symptoms. Men tend to have more severe COVID-19 symptoms than women, and in the aspirated ORN06 model, male mice tended to have more severe symptoms than female mice. Intraperitoneal injections of SAP starting from day 1 post ORN06 aspiration attenuated the ORN06-induced increase in the number of inflammatory cells and formation of clot-like aggregates in the mouse lung fluid, reduced ORN06-increased alveolar wall thickness and accumulation of exudates in the alveolar airspace, and attenuated an ORN06-induced upregulation of the inflammatory cytokines IL-1{beta}, IL-6, IL-12p70, IL-23, and IL-27 in serum. Together, these results suggest that aspiration of ORN06 is a simple model for both COVID-19 as well as cytokine storm in general, and that SAP is a potential therapeutic for diseases with COVID-19-like symptoms as well as diseases that generate a cytokine storm.

immunology↗