Proinflammatory Signaling Research Articles

Sodium-Glucose Co-Transporter-2 Inhibitor Empagliflozin Attenuates Sorafenib-Induced Myocardial Inflammation and Toxicity.

Environmental antineoplastics such as sorafenib may pose a risk to humans through water recycling, and the increased risk of cardiotoxicity is a clinical issue in sorafenib users. Thus, developing strategies to prevent sorafenib cardiotoxicity is an urgent work. Empagliflozin, as a sodium-glucose co-transporter-2 (SGLT2) inhibitor for type 2 diabetes control, has been approved for heart failure therapy. Still, its cardioprotective effect in the experimental model of sorafenib cardiotoxicity has not yet been reported. Real-time quantitative RT-PCR (qRT-PCR), immunoblot, and immunohistochemical analyses were applied to study the effect of sorafenib exposure on cardiac SGLT2 expression. The impact of empagliflozin on cell viability was investigated in the sorafenib-treated cardiomyocytes using Alamar blue assay. Immunoblot analysis was employed to delineate the effect of sorafenib and empagliflozin on ferroptosis/proinflammatory signaling in cardiomyocytes. Ferroptosis/DNA damage/fibrosis/inflammation of myocardial tissues was studied in mice with a 28-day sorafenib ± empagliflozin treatment using histological analyses. Sorafenib exposure significantly promoted SGLT2 upregulation in cardiomyocytes and mouse hearts. Empagliflozin treatment significantly attenuated the sorafenib-induced cytotoxicity/DNA damage/fibrosis in cardiomyocytes and mouse hearts. Moreover, GPX4/xCT-dependent ferroptosis as an inducer for releasing high mobility group box 1 (HMGB1) was also blocked by empagliflozin administration in the sorafenib-treated cardiomyocytes and myocardial tissues. Furthermore, empagliflozin treatment significantly inhibited the sorafenib-promoted NFκB/HMGB1 axis in cardiomyocytes and myocardial tissues, and sorafenib-stimulated proinflammatory signaling (TNF-α/IL-1β/IL-6) was repressed by empagliflozin administration. Finally, empagliflozin treatment significantly attenuated the sorafenib-promoted macrophage recruitments in mouse hearts. In conclusion, empagliflozin may act as a cardioprotective agent for humans under sorafenib exposure by modulating ferroptosis/DNA damage/fibrosis/inflammation. However, further clinical evidence is required to support this preclinical finding.

Regulation of STAT1 Signaling in Human Pancreatic β-Cells by the Lysine Deacetylase HDAC6: A New Therapeutic Opportunity in Type 1 Diabetes?

Type 1 diabetes arises from the selective destruction of pancreatic β-cells by autoimmune mechanisms and intracellular pathways driven by Janus (JAK)-kinase mediated STAT isoforms (especially STAT1 & STAT2) are implicated as mediators of β-cell demise. Despite this, the molecular mechanisms that regulate JAK-STAT signalling in β-cells during the autoimmune attack remain only partially disclosed and the factors acting to antagonise pro-inflammatory STAT1 signalling are uncertain. We have recently implicated Signal Regulatory Protein (SIRP)-α in promoting β-cell viability in the face of ongoing islet autoimmunity and now reveal that this protein controls the availability of a cytosolic lysine deacetylase, HDAC6, whose activity regulates the phosphorylation and activation of STAT1. We provide evidence that STAT1 serves as a substrate for HDAC6 in β-cells and that sequestration of HDAC6 by SIRPα in response to anti-inflammatory cytokines (such as interleukin-13) leads to increased STAT1 acetylation. This then impairs the ability of STAT1 to promote gene transcription in response to pro-inflammatory cytokines including interferon-gamma (IFNγ). We further find that SIRPα is lost from the β-cells of subjects with recent-onset type 1 diabetes under conditions when HDAC6 is retained and STAT1 levels are increased. On this basis, we report a previously unrecognised role for cytokine-induced regulation of STAT1 acetylation in the control of β- cell viability and propose that targeted inhibition of HDAC6 activity may represent a novel therapeutic modality to promote β-cell viability in the face of active islet autoimmunity.

Epidermal growth factor receptor/mitogen-activated kinase inhibitor treatment induces a distinct inflammatory hair follicle response that includes collapse of immune privilege.

Inhibitors of epidermal growth factor receptor (EGFRi) or mitogen-activated kinase (MEKi) induce a folliculitis in 75-90% of patients, the pathobiology of which remains insufficiently understood. To characterize changes in the skin immune status and global transcriptional profile of patients treated with EGFRi; to investigate whether EGFRi affects the hair follicle's (HF) immune privilege (IP); and to identify early proinflammatory signals induced by EGFRi/MEKi in human scalp HFs ex vivo. Scalp biopsies were taken from patients exhibiting folliculitis treated long term with EGFRi ('chronic EGFRi' group, n = 9) vs. healthy scalp skin (n = 9) and patients prior to commencing EGFRi treatment and after 2 weeks of EGFRi therapy ('acute EGFRi' group, n = 5). Healthy organ-cultured scalp HFs were exposed to an EGFRi (erlotinib, n = 5) or a MEKi (cobimetinib, n = 5). Samples were assessed by quantitative immunohistomorphometry, RNA sequencing (RNAseq) and in situ hybridization. The 'chronic EGFRi' group showed CD8+ T-cell infiltration of the bulge alongside a partial collapse of the HF's IP, evidenced by upregulated major histocompatibility complex (MHC) class I, β2-microglobulin (B2 M) and MHC class II, and decreased transforming growth factor-β1 protein expression. Healthy HFs treated with EGFRi/MEKi ex vivo also showed partial HF IP collapse and increased transcription of human leucocyte antigen (HLA)-A, HLA-DR and B2 M transcripts. RNAseq analysis showed increased transcription of chemokines (CXCL1, CXCL13, CCL18, CCL3, CCL7) and interleukin (IL)-26 in biopsies from the 'chronic EGFRi' cohort, as well as increased IL-33 and decreased IL-37 expression in HF biopsies from the 'acute EGFRi' group and in organ-cultured HFs. The data show that EGFRi/MEKi compromise the physiological IP of human scalp HFs and suggest that future clinical management of EGFRi/MEKi-induced folliculitis requires HF IP protection and inhibition of IL-33.

Effect of Clonal Hematopoiesis Mutations and Canakinumab Treatment on Incidence of Solid Tumors in the CANTOS Randomized Clinical Trial.

Clonal hematopoiesis (CH) is more common in older persons and has been associated with an increased risk of hematological cancers and cardiovascular diseases. The most common CH mutations occur in the DNMT3A and TET2 genes and result in increased proinflammatory signaling. The Canakinumab Anti-inflammatory Thrombosis Outcome Study (NCT01327846) evaluated the neutralizing anti-IL1β antibody canakinumab in 10,061 randomized patients with a history of myocardial infarction and persistent inflammation; DNA samples were available from 3,923 patients for targeted genomic sequencing. We examined the incidence of non-hematological malignancy by treatment assignment and CH mutations and estimated the cumulative incidence of malignancy events during trial follow-up. Patients with TET2 mutations treated with canakinumab had the lowest incidence of non-hematological malignancy across cancer types. The cumulative incidence of at least one reported malignancy was lower for patients with TET2 mutations treated with canakinumab versus those treated with placebo. These findings support a potential role for canakinumab in cancer prevention and provide evidence of IL1β blockade cooperating with CH mutations to modify the disease course. Prevention Relevance: We reveal that administering canakinumab is associated with a decrease in non-hematological malignancies among patients with clonal hematopoiesis (CH) mutations. These findings underscore canakinumab's potential in preventing cancer and provide proof of IL1β blockade collaborating with CH mutations to enhance its clinical benefits. See related Spotlight, p. 399.

Photobiomodulation in the infrared spectrum reverses the expansion of circulating natural killer cells and brain microglial activation in Sanfilippo mice.

Sanfilippo syndrome results from inherited mutations in genes encoding lysosomal enzymes that catabolise heparan sulfate (HS), leading to early childhood-onset neurodegeneration. This study explores the therapeutic potential of photobiomodulation (PBM), which is neuroprotective and anti-inflammatory in several neurodegenerative diseases; it is also safe and PBM devices are readily available. We investigated the effects of 10-14 days transcranial PBM at 670 nm (2 or 4 J/cm2/day) or 904 nm (4 J/cm2/day) in young (3 weeks) and older (15 weeks) Sanfilippo or mucopolysaccharidosis type IIIA (MPS IIIA) mice. Although we found no PBM-induced changes in HS accumulation, astrocyte activation, CD206 (an anti-inflammatory marker) and BDNF expression in the brains of Sanfilippo mice, there was a near-normalisation of microglial activation in older MPS IIIA mice by 904 nm PBM, with decreased IBA1 expression and a return of their morphology towards a resting state. Immune cell immunophenotyping of peripheral blood with mass cytometry revealed increased pro-inflammatory signalling through pSTAT1 and p-p38 in NK and T cells in young but not older MPS IIIA mice (5 weeks of age), and expansion of NK, B and CD8+ T cells in older affected mice (17 weeks of age), highlighting the importance of innate and adaptive lymphocytes in Sanfilippo syndrome. Notably, 670 and 904 nm PBM both reversed the Sanfilippo-induced increase in pSTAT1 and p-p38 expression in multiple leukocyte populations in young mice, while 904 nm reversed the increase in NK cells in older mice. In conclusion, this is the first study to demonstrate the beneficial effects of PBM in Sanfilippo mice. The distinct reduction in microglial activation and NK cell pro-inflammatory signalling and number suggests PBM may alleviate neuroinflammation and lymphocyte activation, encouraging further investigation of PBM as a standalone, or complementary therapy in Sanfilippo syndrome.

Repair of the Infarcted Heart: Cellular Effectors, Molecular Mechanisms and Therapeutic Opportunities.

The adult mammalian heart has limited endogenous regenerative capacity and heals through the activation of inflammatory and fibrogenic cascades that ultimately result in the formation of a scar. After infarction, massive cardiomyocyte death releases a broad range of damage-associated molecular patterns that initiate both myocardial and systemic inflammatory responses. TLRs (toll-like receptors) and NLRs (NOD-like receptors) recognize damage-associated molecular patterns (DAMPs) and transduce downstream proinflammatory signals, leading to upregulation of cytokines (such as interleukin-1, TNF-α [tumor necrosis factor-α], and interleukin-6) and chemokines (such as CCL2 [CC chemokine ligand 2]) and recruitment of neutrophils, monocytes, and lymphocytes. Expansion and diversification of cardiac macrophages in the infarcted heart play a major role in the clearance of the infarct from dead cells and the subsequent stimulation of reparative pathways. Efferocytosis triggers the induction and release of anti-inflammatory mediators that restrain the inflammatory reaction and set the stage for the activation of reparative fibroblasts and vascular cells. Growth factor-mediated pathways, neurohumoral cascades, and matricellular proteins deposited in the provisional matrix stimulate fibroblast activation and proliferation and myofibroblast conversion. Deposition of a well-organized collagen-based extracellular matrix network protects the heart from catastrophic rupture and attenuates ventricular dilation. Scar maturation requires stimulation of endogenous signals that inhibit fibroblast activity and prevent excessive fibrosis. Moreover, in the mature scar, infarct neovessels acquire a mural cell coat that contributes to the stabilization of the microvascular network. Excessive, prolonged, or dysregulated inflammatory or fibrogenic cascades accentuate adverse remodeling and dysfunction. Moreover, inflammatory leukocytes and fibroblasts can contribute to arrhythmogenesis. Inflammatory and fibrogenic pathways may be promising therapeutic targets to attenuate heart failure progression and inhibit arrhythmia generation in patients surviving myocardial infarction.

Advanced nanomedicines and immunotherapeutics to treat respiratory diseases especially COVID-19 induced thrombosis

Immunotherapy and associated immune regulation strategies gained huge attraction in order to be utilized for treatment and prevention of respiratory diseases. Engineering specifically nanomedicines can be used to regulate host immunity in lungs in the case of respiratory diseases including coronavirus disease 2019 (COVID-19) infection. COVID-19 causes pulmonary embolisms, thus new therapeutic options are required to target thrombosis, as conventional treatment options are either not effective due to the complexity of the immune-thrombosis pathophysiology. In this review, we discuss regulation of immune response in respiratory diseases especially COVID-19. We further discuss thrombosis and provide an overview of some antithrombotic nanoparticles, which can be used to develop nanomedicine against thrombo-inflammation induced by COVID-19 and other respiratory infectious diseases. We also elaborate the importance of immunomodulatory nanomedicines that can block pro-inflammatory signalling pathways, and thus can be recommended to treat respiratory infectious diseases.

Differential Responses to Aging Among the Transcriptome and Proteome of Mesenchymal Progenitor Populations.

The biological aging of stem cells (exhaustion) is proposed to contribute to the development of a variety of age-related conditions. Despite this, little is understood about the specific mechanisms which drive this process. In this study, we assess the transcriptomic and proteomic changes in 3 different populations of mesenchymal progenitor cells from older (50-70 years) and younger (20-40 years) individuals to uncover potential mechanisms driving stem cell exhaustion in mesenchymal tissues. To do this, we harvested primary bone marrow mesenchymal stem and progenitor cells (MPCs), circulating osteoprogenitors (COP), and adipose-derived stem cells (ADSCs) from younger and older donors, with an equal number of samples from men and women. These samples underwent RNA sequencing and label-free proteomic analysis, comparing the younger samples to the older ones. There was a distinct transcriptomic phenotype in the analysis of pooled older stem cells, suggestive of suppressed proliferation and differentiation; however, these changes were not reflected in the proteome of the cells. Analyzed independently, older MPCs had a distinct phenotype in both the transcriptome and proteome consistent with altered differentiation and proliferation with a proinflammatory immune shift in older adults. COP cells showed a transcriptomic shift to proinflammatory signaling but no consistent proteomic phenotype. Similarly, ADSCs displayed transcriptomic shifts in physiologies associated with cell migration, adherence, and immune activation but no proteomic change with age. These results show that there are underlying transcriptomic changes with stem cell aging that may contribute to a decline in tissue regeneration. However, the proteome of the cells was inconsistently regulated.

Granzyme K mediates IL-23-dependent inflammation and keratinocyte proliferation in psoriasis.

Psoriasis is an inflammatory disease with systemic manifestations that most commonly presents as itchy, erythematous, scaly plaques on extensor surfaces. Activation of the IL-23/IL-17 pro-inflammatory signaling pathway is a hallmark of psoriasis and its inhibition is key to clinical management. Granzyme K (GzmK) is an immune cell-secreted serine protease elevated in inflammatory and proliferative skin conditions. In the present study, human psoriasis lesions exhibited elevated GzmK levels compared to non-lesional psoriasis and healthy control skin. In an established murine model of imiquimod (IMQ)-induced psoriasis, genetic loss of GzmK significantly reduced disease severity, as determined by delayed plaque formation, decreased erythema and desquamation, reduced epidermal thickness, and inflammatory infiltrate. Molecular characterization in vitro revealed that GzmK contributed to macrophage secretion of IL-23 as well as PAR-1-dependent keratinocyte proliferation. These findings demonstrate that GzmK enhances IL-23-driven inflammation as well as keratinocyte proliferation to exacerbate psoriasis severity.

Therapeutic potential of the topical recombinant human interleukin-1 receptor antagonist in guinea pigs with allergic rhinitis

BackgroundRecombinant human Interleukin receptor antagonist (rhIL-Ra) can bind to the IL-1 receptor on the cell membrane and reversibly blocks the proinflammatory signaling pathway. However, its effect on allergic rhinitis (AR) and the underlying mechanism remains unknown. This study aims to investigate the efficacy of recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) on AR guinea pigs.MethodsGuinea pigs were systemically sensitized by intraperitoneal injection and topical intranasal instillation with ovalbumin within 21 days. Animals administrated with saline served as the normal control. The AR animals were randomly divided into the model group and distinct concentrations of rhIL-1Ra and budesonide treatment groups. IL-1β and ovalbumin specific IgE levels were detected by ELISA kits. Nasal mucosa tissues were stained with hematoxylin & eosin (HE) for histological examination.ResultsIt was found that the numbers of sneezing and nose rubbing were remarkably reduced in rhIL-1Ra and budesonide-treated guinea pigs. Besides, rhIL-1Ra distinctly alleviated IgE levels in serum and IL-1β levels in nasal mucus, together with decreased exfoliation of epithelial cells, eosinophilic infiltration, tissue edema and vascular dilatation.ConclusionsrhIL-1Ra is effective in AR guinea pigs and may provide a novel potential choice for AR treatments.

Modulation of Redox and Inflammatory Signaling in Human Skin Cells Using Phytocannabinoids Applied after UVA Irradiation: In Vitro Studies.

UVA exposure disturbs the metabolism of skin cells, often inducing oxidative stress and inflammation. Therefore, there is a need for bioactive compounds that limit such consequences without causing undesirable side effects. The aim of this study was to analyse in vitro the effects of the phytocannabinoids cannabigerol (CBG) and cannabidiol (CBD), which differ in terms of biological effects. Furthermore, the combined use of both compounds (CBG+CBD) has been analysed in order to increase their effectiveness in human skin fibroblasts and keratinocytes protection against UVA-induced alternation. The results obtained indicate that the effects of CBG and CBD on the redox balance might indeed be enhanced when both phytocannabinoids are applied concurrently. Those effects include a reduction in NOX activity, ROS levels, and a modification of thioredoxin-dependent antioxidant systems. The reduction in the UVA-induced lipid peroxidation and protein modification has been confirmed through lower levels of 4-HNE-protein adducts and protein carbonyl groups as well as through the recovery of collagen expression. Modification of antioxidant signalling (Nrf2/HO-1) through the administration of CBG+CBD has been proven to be associated with reduced proinflammatory signalling (NFκB/TNFα). Differential metabolic responses of keratinocytes and fibroblasts to the effects of the UVA and phytocannabinoids have indicated possible beneficial protective and regenerative effects of the phytocannabinoids, suggesting their possible application for the purpose of limiting the harmful impact of the UVA on skin cells.

The effects of HIV-1-proteins and antiretroviral therapy on aortic endothelial cells (AECs). A mechanistic in vitro approach

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Research Foundation Introduction Endothelial dysfunction (ED) is an early precursor of cardiovascular disease characterized by decreased nitric oxide (NO) and increased oxidative stress, creating a pro-inflammatory environment. Increased ED and cardiovascular risk have been observed in HIV-1 infection and antiretroviral therapy (ART). Purpose The United Nations AIDS update indicates that in 2015 17.0 million people worldwide were living with HIV and receiving antiretroviral therapy. Considering the extent of this global pandemic and its comorbidity with CVD, zooming in on HIV’s intersection with antiretroviral therapy and cardiovascular health is of paramount importance. Yet, many of the mechanisms involved are still poorly understood. This knowledge can ultimately assist with the careful selection of antiretroviral-agents in patients with underlying cardiovascular risk factors. Objectives Establishing a simulated model of in vitro HIV-1-infection in aortic endothelial cells and determining the effects of non/nucleoside reverse transcriptase inhibitors (NRTI/NNRTIs) and protease inhibitors (PIs) on markers of endothelial function and the expression/activation of vascular signalling proteins. Methods A simulated HIV-1-model was established by adding recombinant HIV-1 proteins to growth medium in a single cocktail containing Tat, Gp160 and Nef. ART1 (NRTI/NNRTIs) were administered as a single cocktail to growth medium, containing efavirenz (EFV), emtricitabine (FTC) and tenofovir (TDF). ART2 (PIs) were administered as a single cocktail to growth medium, containing lopinavir (LPV) and ritonavir (RTV). All the aforementioned treatments were subject to dose-response investigations and treatment lasted 24 hours. Cell viability, NO-production and oxidative stress were measured using fluorescent probes. Selected dosages from above studies were further used and important vascular signalling proteins in HIV-1 exposed AECs were investigated by western blotting. Results HIV-1 (100ng/ml, 24h) resulted in ↓NO-levels (DAF-fluorescence intensity: 100±1.25% vs. 76.00±3.67%; p<0.05) with no oxidative stress. HIV-1+PI treatment lowered NO levels (DAF-fluorescence: 100±0.72% vs. 94.47±1.91%; p<0.05). HIV-1+ NRTI/NNRTI treatment had no effects. HIV-1+PI treatment ↓IκBα expression (inhibitory signalling protein in pro-inflammatory NF-κB pathway) (1.0 ± 0.15 vs 0.37 ± 0.03; p<0.05) and ↓eNOS expression (1.0 ± 0.21 vs. 0.28 ± 0.04; p<0.05). The same was observed for PKB/Akt (1.0 ± 0.03 vs. 0.68 ± 0.02; p<0.05). No effects on cell viability and oxidative stress were observed. Conclusion Exposure of AECs to combined HIV-1-protein and PI treatment lowered NO-production, which may be linked to underlying PKB/Akt and eNOS downregulation. HIV-1+PI treatment was associated with increased pro-inflammatory NF-κB signalling. Overall, our results suggest that a dysfunctional state was induced in AECs exposed to HIV-1 proteins and PI treatment.

Limosilactobacillus reuteri DSM 17938 relieves inflammation, endoplasmic reticulum stress, and autophagy in hippocampus of western diet-fed rats by modulation of systemic inflammation.

The consumption of western diets, high in fats and sugars, is a crucial contributor to brain molecular alterations, cognitive dysfunction and neurodegenerative diseases. Therefore, a mandatory challenge is the individuation of strategies capable of preventing diet-induced impairment of brain physiology. A promising strategy might consist in the administration of probiotics that are known to influence brain function via the gut-brain axis. In this study, we explored whether Limosilactobacillus reuteri DSM 17938 (L. reuteri)-based approach can counteract diet-induced neuroinflammation, endoplasmic reticulum stress (ERS), and autophagy in hippocampus, an area involved in learning and memory, in rat fed a high fat and fructose diet. The western diet induced a microbiota reshaping, but L. reuteri neither modulated this change, nor the plasma levels of short-chain fatty acids. Interestingly, pro-inflammatory signaling pathway activation (increased NFkB phosphorylation, raised amounts of toll-like receptor-4, tumor necrosis factor-alpha, interleukin-6, GFAP, and Haptoglobin), as well as activation of ERS (increased PERK and eif2α phosphorylation, higher C/EBP-homologous protein amounts) and autophagy (increased beclin, P62-sequestosome-1, and LC3 II) was revealed in hippocampus of western diet fed rats. All these hippocampal alterations were prevented by L. reuteri administration, showing for the first time a neuroprotective role of this specific probiotic strain, mainly attributable to its ability to regulate western diet-induced metabolic endotoxemia and systemic inflammation, as decreased levels of lipopolysaccharide, plasma cytokines, and adipokines were also found. Therapeutic strategies based on the use of L. reuteri DSM17938 could be beneficial in reversing metabolic syndrome-mediated brain dysfunction and cognitive decline.

Multi-omic integration reveals alterations in nasal mucosal biology that mediate air pollutant effects on allergic rhinitis.

Allergic rhinitis is a common inflammatory condition of the nasal mucosa that imposes a considerable health burden. Air pollution has been observed to increase the risk of developing allergic rhinitis. We addressed the hypotheses that early life exposure to air toxics is associated with developing allergic rhinitis, and that these effects are mediated by DNA methylation and gene expression in the nasal mucosa. In a case-control cohort of 505 participants, we geocoded participants' early life exposure to air toxics using data from the US Environmental Protection Agency, assessed physician diagnosis of allergic rhinitis by questionnaire, and collected nasal brushings for whole-genome DNA methylation and transcriptome profiling. We then performed a series of analyses including differential expression, Mendelian randomization, and causal mediation analyses to characterize relationships between early life air toxics, nasal DNA methylation, nasal gene expression, and allergic rhinitis. Among the 505 participants, 275 had allergic rhinitis. The mean age of the participants was 16.4 years (standard deviation = 9.5 years). Early life exposure to air toxics such as acrylic acid, phosphine, antimony compounds, and benzyl chloride was associated with developing allergic rhinitis. These air toxics exerted their effects by altering the nasal DNA methylation and nasal gene expression levels of genes involved in respiratory ciliary function, mast cell activation, pro-inflammatory TGF-β1 signaling, and the regulation of myeloid immune cell function. Our results expand the range of air pollutants implicated in allergic rhinitis and shed light on their underlying biological mechanisms in nasal mucosa.

Inhibition of Adipocyte Necroptosis Alleviates Fat Necrosis and Fibrosis After Grafting in a Murine Model.

Because of the delicate structure of the adipose tissue, fat necrosis accounts for 43.7% of all complications after autologous fat grafting; however, its regulation remains unclear. The purpose of this study was to examine the role of necroptosis in fat graft remodeling after grafting. Clinical fat graft necrosis samples were collected, and the expression levels of the necroptosis marker phosphorylated(p)-MLKL were analyzed. Transcriptome analysis was performed on fat grafts before and 1 week after transplantation in C57BL/6 mouse fat grafting models. Additionally, the in vivo effects of RIPK1 inhibitor Nec-1s or RIPK3 inhibitor GSK'872 on the fat grafting complications, including fat necrosis and fibrosis, were investigated. Necroptosis markers were observed and associated with higher occurrence of fibrosis in clinical fat graft necrosis samples compared to normal fat tissue. Amplification and RNA-Seq were conducted on RNA isolated from fat grafts before and after grafting. MLKL, RIPK1, and RIPK3's expression levels were significantly upregulated in comparison to controls. Higher expression levels of necroptotic RNAs were associated with higher levels of DAMPs, including Cxcl2, HMGB1, S100a8, S100a9, Nlrp3, and IL33, and activated proinflammatory signaling pathways, including the TNF, NF-kappa B, and chemokine signaling pathways. Necroptotic inhibitor Nec-1s and GSK'872 robustly suppressed the p-MLKL expression level and significantly inhibited necroptotic cell death, especially in adipocytes. Moreover, administration of Nec-1s and GSK'872 significantly alleviated fat necrosis and subsequent fibrosis in fat grafts. Collectively, our study findings highlight the potential therapeutic applications of necroptosis inhibitors in preventing fat necrosis and fibrosis after grafting.

Polo-like kinase 1 (PLK1) is a novel CARD14-binding protein in keratinocytes

Caspase recruitment domain (CARD)-containing protein 14 (CARD14) is an intracellular protein that mediates nuclear factor-kappa B (NF-ĸB) signaling and proinflammatory gene expression in skin keratinocytes. Several hyperactivating CARD14 mutations have been associated with psoriasis and other inflammatory skin diseases. CARD14-induced NF-ĸB signaling is dependent on the formation of a CARD14-BCL10-MALT1 (CBM) signaling complex, but upstream receptors and molecular mechanisms that activate and regulate CARD14 signaling are still largely unclear. Using unbiased affinity purification and mass spectrometry (AP-MS) screening, we discover polo-like kinase 1 (PLK1) as a novel CARD14-binding protein. CARD14-PLK1 binding is independent of the CARD14 CARD domain but involves a consensus phospho-dependent PLK1-binding motif in the CARD14 linker region (LR). Expression of the psoriasis-associated CARD14(E138A) variant in human keratinocytes induces the recruitment of PLK1 to CARD14-containing signalosomes in interphase cells, but does not affect the specific location of PLK1 in mitotic cells. Finally, disruption of the PLK1-binding motif in CARD14(E138A) increases CARD14-induced proinflammatory signaling and gene expression. Together, our data identify PLK1 as a novel CARD14-binding protein and indicate a negative regulatory role for PLK1 in CARD14 signaling.

#1284 Proteinuria causes excessive proliferation and senescence in proximal tubular cells via receptor-mediated endocytosis

Abstract Background and Aims Proteinuria induces proximal tubule (PT) injury, oxidative stress, lysosomal dysfunction, and inflammation, which contribute to the progression of CKD. Cellular senescence and the associated secretory phenotype, where cells secrete proinflammatory and profibrotic mediators, have been linked to renal interstitial inflammation and fibrosis. Premature senescence is induced by excessive proliferation, oxidative stress, or DNA injury. In kidneys, hyperglycemia or hypertension have been reported to elicit this response. However, the direct association between proteinuria-induced PT protein overload and cellular senescence is unknown. We aimed to clarify the effect of protein overload on cellular proliferation and senescence in proteinuric mice with or without knockout (KO) of the receptors responsible for protein endocytosis, megalin and cubilin. Method Podocin-KO (proteinuric mouse model) and megalin-cubilin-podocin triple-KO mice were used in study. Kidneys from these mice were analyzed using immunohistochemical and immunofluorescent staining and the mice were injected with EdU to investigate renal proliferation. mRNA expression of different markers in renal cortex were evaluated with quantitative rt-PCR. To investigate albumin induced cellular proliferation and senescence, immortalized proximal tubular cells (RPTEC/TERT1, ATCC) were treated with human serum albumin (HSA; Sigma), fatty acid-free HSA (Sigma), and transferrin (Sigma). Western blotting (WB) and quantitative rt-PCR, of different markers were conducted and staining for senescence-associated beta-galactosidase (SA-β-gal) was performed. Results PCNA, a proliferation marker, was detected at a higher level in PTs of podocin-KO mice compared to wild-type (WT) mice, and triple-KO mice. Furthermore, we evaluated EdU DNA incorporation, in mosaic megalin and cubilin-expressing triple-KO mice. Coimmunostaining of megalin (Fig. 1-a) or cubilin (Fig. 1-b) with incorporated EdU revealed that most of the EdU-positive nuclei was observed in megalin or cubilin-positive tubules (white arrowheads). A few EdU positive nuclei was detected in megalin or cubilin-negative tubules or the interstitium (yellow arrowheads). The mRNA expression of PDGF- β, a growth factor, was more upregulated in the cortex of podocin-KO mice than in triple-KO mice. These data indicate that protein uptake via endocytic receptors induces secretion of PDGF-β from PT cells, acting in a paracrine manner potentially inducing proliferation. Additionally, immunofluorescent staining revealed that senescence markers, p21 and γ-H2AX, were detected in PTs of podocin-KO mice at a higher level than in triple-KO mice. The mRNA expression of TGF-β (profibrotic) and MCP-1 (proinflammatory) were higher in podocin-KO, than in triple-KO mice in accordance with more senescent cells in podocin KO mice. Upregulation of p21, p16, and γ-H2AX as well as positive staining for SA-β-gal in RPTEC/TERT1 cells, were only detected in HSA (containing free fatty acids)-treated cells in a dose dependent manner and not in cells treated with HSA depleted for free fatty acids or transferrin. Our results show that excessive protein reabsorption generates senescent cells in proximal tubules potentially through increased proliferation elicited by paracrine signaling. The increase in senescent cells was associated to profibrotic and proinflammatory signaling in line with a senescent secretory phenotype. Our studies in PRTEC/TERT1 confirmed our animal experiments and showed that senescence was induced only by fatty acid bound albumin. Conclusion The present study showed that proteinuria directly induced cellular senescence in PTECs via megalin/cubilin endocytosis of filtered protein. Fatty acid bound-albumin is suggested to cause the cellular senescence.

#2027 Activation of histone deacetylase-1 in fibroblasts and pericytes induces renal interstitial fibrosis after ischemia-reperfusion injury

Abstract Background and Aims Following an acute kidney ischemic event the chromatin remodelling enzyme, histone deacetylase-1 (HDAC1), is activated in many cell types of the kidney including fibroblasts and pericytes. During kidney injury, the fibroblasts/pericytes differentiate into the myofibroblasts and produce extracellular matrix that can lead to a permanent decline in kidney function. Pharmacological inhibition of HDACs can attenuate ischemia-reperfusion-injury (IRI) mediated interstitial fibrosis. The hypothesis of this study was that fibroblast/pericyte HDAC1 activation promotes myofibroblast differentiation and interstitial fibrosis. Method Tamoxifen inducible, fibroblast/pericyte HDAC1 knockout (KO) mice (HDAC1Fl/Fl; Col1a2-CreEr) and littermate controls (HDAC1Fl/Fl) were used. Male and female mice were given tamoxifen by ip at 8-10 weeks of age and IRI or sham surgery completed after a 2-week tamoxifen washout period. A mild, 18 min, bilateral, warm IRI model was used, and samples collected over 4 weeks. Glomerular filtration rate (GFR) was assessed using transdermal FITC-Sinistrin clearance. In vitro experiments with kidney fibroblast cells (NRK49F) overexpressing empty vector or HDAC1 were used for RNA-sequencing to determine the HDAC1-dependent myofibroblast transcriptome. Results We confirmed that HDAC1 was knockdown in myofibroblast cells from KO mice with co-localization of HDAC1 and PDGFRβ or α-smooth muscle actin in the kidneys of IRI mice. Twenty-four hours post-surgery, all IRI mice exhibited 2-3 times increase in serum creatinine and a ∼50% reduction in GFR compared to sham mice (control IRI = 513 ± 129 μl /min/100 g body mass, control sham = 1010 ± 218, n = 4-5, p = 0.02; KO IRI = 529 ± 116, KO sham = 1187 ± 59 μl/min/100 g body mass, n = 4-5, p = 0.004). Subsequent GFR measurements over the next 4 weeks showed a gradual recovery of renal function of IRI mice starting at week 2 and fully restored to sham values by week 4. However, the male control IRI mice had significant focal interstitial fibrosis 2- and 4-weeks post injury (4-week data: sham = 0.3 ± 0.09 % of cortical area, IRI = 1.2 ± 0.2%, n = 6/group, p = 0.0011) but this was attenuated in the KO male IRI mice (sham = 0.4 ± 0.07% n = 4, IRI = 0.5 ± 0.1%, n = 7, p = 0.93). The female mice, regardless of genotype, had very little kidney damage or interstitial fibrosis at 4 weeks. Transcriptomes of NRK49F cells overexpressing HDAC1 had an upregulation of 55 genes related to pro-inflammatory cytokines and tumour necrosis factor signalling and downregulation of 367 genes related to cell junctions, cytoskeleton proteins, and cell adhesion compared to the empty vector control cells. Fibroblast proliferation rates were not statistically different among the groups, suggesting HDAC1 does not affect myofibroblast proliferation but rather pro-inflammatory gene expression. Conclusion In male mice, fibroblast/pericyte activation of HDAC1 leads to the production of pro-inflammatory cytokines that we predict will lead to immune cell infiltration and crosstalk that leads to the excessive production of interstitial fibrosis following IRI.

Physical Activity Decreases Inflammation and Delays the Development of Obesity-Associated Pancreatic Ductal Adenocarcinoma.

Obesity is a risk factor for pancreatic ductal adenocarcinoma (PDAC), a deadly disease with limited preventive strategies. Lifestyle interventions to decrease obesity represent a potential approach to prevent obesity-associated PDAC. In this study, we examined whether decreasing obesity through physical activity (PA) and/or dietary changes could decrease inflammation in humans and prevent obesity-associated PDAC in mice. Comparison of circulating inflammatory-associated cytokines in subjects (overweight and obese) before and after a PA intervention revealed PA lowered systemic inflammatory cytokines. Mice with pancreatic-specific inducible KrasG12D expression were exposed to PA and/or dietary interventions during and after obesity-associated cancer initiation. In mice with concurrent diet-induced obesity and KrasG12D expression, the PA intervention led to lower weight gain, suppressed systemic inflammation, delayed tumor progression, and decreased proinflammatory signals in the adipose tissue. However, these benefits were not as evident when obesity preceded pancreatic KrasG12D expression. Combining PA with diet-induced weight loss (DI-WL) delayed obesity-associated PDAC progression in the genetically engineered mouse model, but neither PA alone nor combined with DI-WL or chemotherapy prevented PDAC tumor growth in orthotopic PDAC models regardless of obesity status. PA led to the upregulation of Il15ra in adipose tissue. Adipose-specific overexpression of Il15 slowed PDAC growth but only in nonobese mice. Overall, our study suggests that PA alone or combined with DI-WL can reduce inflammation and delay obesity-associated PDAC development or progression. Lifestyle interventions that prevent or manage obesity or therapies that target weight loss-related molecular pathways could prevent progression of PDAC. Significance: Physical activity reduces inflammation and induces changes to adipose-related signaling to suppress pancreatic cancer, supporting the potential of obesity management strategies to reduce the risk of developing pancreatic cancer. See related commentary by Sogunro and Muzumdar, p. 2935.

Macrophage proteomic analysis of covalent immobilization of hyaluronic acid and graphene oxide on CoCr alloy in a tribocorrosive environment.

In this work, a sequential covalent immobilization of graphene oxide (GO) and hyaluronic acid (HA) is performed to obtain a biocompatible wear-resistant nanocoating on the surface of the biomedical grade cobalt-chrome (CoCr) alloy. Nanocoated CoCr surfaces were characterized by Raman spectroscopy and electrochemical impedance spectroscopy (EIS) in 3 g/L HA electrolyte. Tribocorrosion tests of the nanocoated CoCr surfaces were carried out in a pin on flat tribometer. The biological response of covalently HA/GO biofunctionalized CoCr surfaces with and without wear-corrosion processes was studied through the analysis of the proteome of macrophages. Raman spectra revealed characteristic bands of GO and HA on the functionalized CoCr surfaces. The electrochemical response by EIS showed a stable and protective behavior over 23 days in the simulated biological environment. HA/GO covalently immobilized on CoCr alloy is able to protect the surface and reduce the wear volume released under tribocorrosion tests. Unsupervised classification analysis of the macrophage proteome via hierarchical clustering and principal component analysis (PCA) revealed that the covalent functionalization on CoCr enhances the macrophage biocompatibility in vitro. On the other hand, disruption of the HA/GO nanocoating by tribocorrosion processes induced a macrophage proteome which was differently clustered and was distantly located in the PCA space. In addition, tribocorrosion induced an increase in the percentage of upregulated and downregulated proteins in the macrophage proteome, revealing that disruption of the covalent nanocoating impacts the macrophage proteome. Although macrophage inflammation induced by tribocorrosion of HA/GO/CoCr surfaces is observed, it is ameliorated by the covalently grafting of HA, which provides immunomodulation by eliciting downregulations in characteristic pro-inflammatory signaling involved in inflammation and aseptic loosening of CoCr joint arthroplasties. Covalent HA/GO nanocoating on CoCr provides potential applications for in vivo joint prostheses led a reduced metal-induced inflammation and degradation by wear-corrosion.