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Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by fibrosis and vasculopathy. CXCL4 represents an early serum biomarker of severe SSc and likely contributes to inflammation via chemokine signaling pathways, but the exact role of CXCL4 in SSc pathogenesis is unclear. Here, we elucidate an unanticipated mechanism for CXCL4-mediated immune amplification in SSc, in which CXCL4 organizes “self” and microbial DNA into liquid crystalline immune complexes that amplify TLR9-mediated plasmacytoid dendritic cell (pDC)-hyperactivation and interferon-α production. Surprisingly, this activity does not require CXCR3, the CXCL4 receptor. Importantly, we find that CXCL4-DNA complexes are present in vivo and correlate with type I interferon (IFN-I) in SSc blood, and that CXCL4-positive skin pDCs coexpress IFN-I-related genes. Thus, we establish a direct link between CXCL4 overexpression and the IFN-I-gene signature in SSc and outline a paradigm in which chemokines can drastically modulate innate immune receptors without being direct agonists. CXCL4 is an inflammatory chemokine signaling through CXCR3 receptor. Here the authors show a CXCR3-independent function of CXCL4: it forms liquid crystals with DNA, potentiating mammalian and bacterial DNA recognition by TLR9, thereby amplifying interferon-a production in systemic sclerosis.

Cystic fibrosis (CF) is caused by defects of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR-modulating drugs may overcome specific defects, such as the case of Trikafta, which is a clinically approved triple combination of Elexacaftor, Tezacaftor and Ivacaftor (ETI) that exhibited a strong ability to rescue the function of the most frequent F508del pathogenic variant even in genotypes with the mutated allele in single copy. Nevertheless, most rare genotypes lacking the F508del allele are still not eligible for targeted therapies. Via the innovative approach of using nasal conditionally reprogrammed cell (CRC) cell-based models that mimic patient disease in vitro , which are obtainable from each patient due to the 100% efficiency of the cell culture establishment, we theratyped orphan CFTR mutation L1077P. Protein studies, Forskolin-induced organoid swelling, and Ussing chamber assays congruently proved the L1077P variant function rescue by ETI. Notably, this rescue takes place even in the context of a single-copy L1077P allele, which appears to enhance its expression. Thus, the possibility of single-allele treatment also arises for rare genotypes, with an allele-specific modulation as part of the mechanism. Of note, besides providing indication of drug efficacy with respect to specific CFTR pathogenic variants or genotypes, this approach allows the evaluation of the response of single-patient cells within their genetic background. In this view, our studies support in vitro guided personalized CF therapies also for rare patients who are nearly excluded from clinical trials.

Psoriatic arthritis (PsA) is a chronic inflammatory arthritis associated with psoriasis. A third of psoriatic patients develop PsA unknown mechanisms. No reliable diagnostic markers are available for PsA, or prognostic biomarkers for PsA development in psoriasis. We previously uncovered a pro-inflammatory role for cathelicidin LL37 in lesional psoriasis skin. LL37 binds nucleic acids and stimulates plasmacytoid/myeloid dendritic cells (pDC, mDCs) to secrete type I interferon (IFN-I) and pro-inflammatory factors. LL37 becomes an autoantigen for psoriatic Th1-Th17/CD8 T cells. Anti-LL37 antibodies were detected in systemic lupus erythematosus, an autoimmune disease characterized by neutrophil-extracellular-traps release (NETosis) in target organs. LL37 can be substrate of irreversible post-translational modifications, citrullination or carbamylation, linked to neutrophil activity. Here we analyzed inflammatory factors, included LL37, in PsA and psoriasis plasma and PsA synovial fluids (SF)/biopsies. We show that LL37 (as a product of infiltrating neutrophils) and autoantibodies to LL37 are elevated in PsA, but not OA SF. Anti-LL37 antibodies correlate with clinical inflammatory markers. Anti-carbamylated/citrullinated-LL37 antibodies are present in PsA SF/plasma and, at lower extent, in psoriasis plasma, but not in controls. Plasma anti-carbamylated-LL37 antibodies correlate with PsA (DAS44) but not psoriasis (PASI) disease activity. Ectopic lymphoid structures, and deposition of immunoglobulin-(Ig)G-complexes (IC) co-localizing with infiltrating neutrophils, are observed in PsA and not OA synovial tissues (ST). Activated complement (C5a, C9), GM-CSF and IFN-I are up-regulated in PsA and not OA synovia and in PsA and psoriasis plasma but not in HD. C9 and GM-CSF levels in PsA SF correlate with clinical inflammatory markers and DAS44 (C9) and with anti-carbamylated/citrullinated-LL37 antibodies (GM-CSF and IFN-I). Thus, we uncover a role for LL37 as a novel PsA autoantibody target and correlation studies suggest participation of anti-LL37 antibodies to PsA pathogenesis. Notably, plasma antibodies to carbamylated-LL37, which correlate with DAS44, suggest their use as new disease activity markers. GM-CSF and complement C5a and C9 elevation may be responsible for autoantigens release by neutrophils and their modification, fueling inflammation and autoreactivity establishment. Finally, targeting GM-CSF, C5a, C9 can be beneficial in PsA.

Background The invasive and metastatic spread of serous ovarian cancer (SOC) results from the cooperative interactions between cancer and stroma, which include extracellular matrix (ECM) and cellular components, including cancer-associated fibroblasts (CAFs). Soluble factors secreted by cancer and stromal cells contribute to stroma remodeling through the secretion of ECM proteins, providing a favorable environment for cancer cell dissemination. The peptide endothelin-1 (ET-1), through two G protein-coupled receptors (GPCR), endothelin receptor type A (ET A R) and B (ET B R), acts on both cancer and stromal cells, engaging the protein β-arrestin1 (β-arr1), to bolster SOC progression. However, its role in the regulation of the ECM proteins by ovarian fibroblasts is not understood. This study delves into the role of ET-1 as a regulator of type I collagen (Col1) and fibronectin (FN). Methods We used human primary ovarian fibroblasts (HOFs) and CAFs. The expression of Col1 ( COL1A1 ) and FN ( FN1 ) were detected by western blotting (WB), quantitative real time-polymerase chain reaction (qRT-PCR), immunofluorescence (IF), and confocal laser scanning microscopy (CLSM) in cells and tumor tissue sections from mice xenografts, while the transcription of COL1A1 was detected by luciferase reporter gene assay. The nuclear function of β-arr1 was evaluated by silencing and rescue expression with wild-type (WT) and nuclear mutant plasmid constructs, RNA seq and differential gene expression and gene sets enrichment analyses. The prognostic role of COL1A1 , FN1 , EDN1 (ET-1) and ARRB1 (β-arr1) gene expression was evaluated using the Kaplan–Meier plotter database and clinical ovarian cancer tissue samples. Results We demonstrated that ET-1 boosts Col1 and FN expression in HOFs, akin to ovarian CAF levels. Both receptors are implicated, evident from inhibitory effects after ET A R or ET B R antagonist treatments and notably with bosentan, a dual antagonist, in vitro and in vivo. At the molecular level, ET-1 triggers the activation of COL1A1 promoter activity and its enhanced expression via β-arr1 nuclear function. Transcriptome analysis of β-arr1-silenced HOFs confirms the nuclear role of β-arr1 in collagen and ECM remodeling-related protein transcriptional regulation. Accordingly, a high level of EDN1/ARRB1 expression in combination with either COL1A1 or FN1 is associated with the poor prognosis of SOC patients. Conclusions These findings hint at ET-1 involvement in ECM remodeling and early SOC stages by modulating the expression of Col1 and FN. Targeting ET-1 signaling with ET A R/ET B R antagonists might interfere with the ability of CAFs to produce key ECM proteins in this tumor.

Recruitment of fibroblasts to tumors and their activation into cancer-associated fibroblasts (CAFs) is a strategy used by tumor cells to direct extracellular matrix (ECM) remodeling, invasion, and metastasis, highlighting the need to investigate the molecular mechanisms driving CAF function. Endothelin-1 (ET-1) regulates the communication between cancer and stroma and facilitates the progression of serous ovarian cancer (SOC). By binding to Endothelin A (ET A ) and B (ET B ) receptors, ET-1 enables the recruitment of β-arrestin1 (β-arr1) and the formation of signaling complexes that coordinate tumor progression. However, how ET-1 receptors might “educate” human ovarian fibroblasts (HOFs) to produce altered ECM and promote metastasis remains to be elucidated. This study identifies ET-1 as a pivotal factor in the activation of CAFs capable of proteolytic ECM remodeling and the generation of heterotypic spheroids containing cancer cells with a propensity to metastasize. An autocrine/paracrine ET-1/ET A/B R/β-arr1 loop enhances HOF proliferation, upregulates CAF marker expression, secretes pro-inflammatory cytokines, and increases collagen contractility, and cell motility. Furthermore, ET-1 facilitates ECM remodeling by promoting the lytic activity of invadosome and activation of integrin β1. In addition, ET-1 signaling supports the formation of heterotypic HOF/SOC spheroids with enhanced ability to migrate through the mesothelial monolayer, and invade, representing metastatic units. The blockade of ET A/B R or β-arr1 silencing prevents CAF activation, invadosome function, mesothelial clearance, and the invasive ability of heterotypic spheroids. In vivo, therapeutic inhibition of ET A/B R using bosentan (BOS) significantly reduces the metastatic potential of combined HOFs/SOC cells, associated with enhanced apoptotic effects on tumor cells and stromal components. These findings support a model in which ET-1/β-arr1 reinforces tumor/stroma interaction through CAF activation and fosters the survival and metastatic properties of SOC cells, which could be counteracted by ET A/B R antagonists.

Breast cancer is one of the most diffuse cancers in the world and despite the availability of the different drugs employed against it, the need for new and particularly more specific molecules is ever growing. In this framework, natural products are increasingly assuming an important role as new anticancer drugs. Aloe-emodin (AE) is one of the best characterized molecules in this field. The functionalization of bioactive natural products with selected peptide sequences to enhance their bioavailability and specificity of action is a powerful and promising strategy. In this study, we analyzed the cell specificity, cell viability effects, intracellular distribution, and immune cell response of a new peptide conjugate of Aloe-emodin in SKBR3 and A549 cell lines by means of viability tests, flow cytometry, and confocal microscopy. The conjugate proved to be more effective at reducing cell viability than AE in both cell lines. Furthermore, the results showed that it was mainly internalized within the SKBR3 cells, showing a nuclear localization, while A459 cells displayed mainly a cytoplasmic distribution. A preserving effect of the conjugate on NKs’ cell function was also observed. The designed conjugate showed a promising specific activity towards HER2-expressing cells coupled with an enhanced water solubility and a higher cytotoxicity; thus, the resulting proof-of-concept molecule can be further improved as an anticancer compound.

Chronic inflammation of the lung, as a consequence of persistent bacterial infections by several opportunistic pathogens represents the main cause of mortality and morbidity in cystic fibrosis (CF) patients. Mechanisms leading to increased susceptibility to bacterial infections in CF are not completely known, although the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in microbicidal functions of macrophages is emerging. Tissue macrophages differentiate in situ from infiltrating monocytes, additionally, mature macrophages from different tissues, although having a number of common activities, exhibit variation in some molecular and cellular functions. In order to highlight possible intrinsic macrophage defects due to CFTR dysfunction, we have focused our attention on in vitro differentiated macrophages from human peripheral blood monocytes. Here we report on the contribution of CFTR in the bactericidal activity against Pseudomonas aeruginosa of monocyte derived human macrophages. At first, by real time PCR, immunofluorescence and patch clamp recordings we demonstrated that CFTR is expressed and is mainly localized to surface plasma membranes of human monocyte derived macrophages (MDM) where it acts as a cAMP-dependent chloride channel. Next, we evaluated the bactericidal activity of P. aeruginosa infected macrophages from healthy donors and CF patients by antibiotic protection assays. Our results demonstrate that control and CF macrophages do not differ in the phagocytic activity when infected with P. aeruginosa. Rather, although a reduction of intracellular live bacteria was detected in both non-CF and CF cells, the percentage of surviving bacteria was significantly higher in CF cells. These findings further support the role of CFTR in the fundamental functions of innate immune cells including eradication of bacterial infections by macrophages.

Cockayne syndrome group A (CS-A) is a rare recessive progeroid disorder characterized by sun sensitivity and neurodevelopmental abnormalities. Cells derived from CS-A patients present as pathological hallmarks excessive oxidative stress, mitochondrial fragmentation and apoptosis associated with hyperactivation of the mitochondrial fission dynamin related protein 1 (DRP1). In this study, by using human cell models we further investigated the interplay between DRP1 and CSA and we determined whether pharmacological or genetic inhibition of DRP1 affects disease progression. Both reactive oxygen and nitrogen species are in excess in CS-A cells and when the mitochondrial translocation of DRP1 is inhibited a reduction of these species is observed together with a recovery of mitochondrial integrity and a significant decrease of apoptosis. This study indicates that the CSA-driven modulation of DRP1 pathway is key to control mitochondrial homeostasis and apoptosis and suggests DRP1 as a potential target in the treatment of CS patients.

Background Bacterial toxins are emerging as promising hallmarks of colorectal cancer (CRC) pathogenesis. In particular, Cytotoxic Necrotizing Factor 1 (CNF1) from E. coli deserves special consideration due to the significantly higher prevalence of this toxin gene in CRC patients with respect to healthy subjects, and to the numerous tumor-promoting effects that have been ascribed to the toxin in vitro. Despite this evidence, a definitive causal link between CNF1 and CRC was missing. Here we investigated whether CNF1 plays an active role in CRC onset by analyzing pro-carcinogenic key effects specifically induced by the toxin in vitro and in vivo. Methods Viability assays, confocal microscopy of γH2AX and 53BP1 molecules and cytogenetic analysis were carried out to assess CNF1-induced genotoxicity on non-neoplastic intestinal epithelial cells. Caco-2 monolayers and 3D Caco-2 spheroids were used to evaluate permeability alterations specifically induced by CNF1, either in the presence or in the absence of inflammation. In vivo, an inflammatory bowel disease (IBD) model was exploited to evaluate the carcinogenic potential of CNF1. Immunohistochemistry and immunofluorescence stainings of formalin-fixed paraffin-embedded (FFPE) colon tissue were carried out as well as fecal microbiota composition analysis by 16 S rRNA gene sequencing. Results CNF1 induces the release of reactive oxidizing species and chromosomal instability in non-neoplastic intestinal epithelial cells. In addition, CNF1 modifies intestinal permeability by directly altering tight junctions’ distribution in 2D Caco-2 monolayers, and by hindering the differentiation of 3D Caco-2 spheroids with an irregular arrangement of these junctions. In vivo, repeated intrarectal administration of CNF1 induces the formation of dysplastic aberrant crypt foci (ACF), and produces the formation of colorectal adenomas in an IBD model. These effects are accompanied by the increased neutrophilic infiltration in colonic tissue, by a mixed pro-inflammatory and anti-inflammatory cytokine milieu, and by the pro-tumoral modulation of the fecal microbiota. Conclusions Taken together, our results support the hypothesis that the CNF1 toxin from E. coli plays an active role in colorectal carcinogenesis. Altogether, these findings not only add new knowledge to the contribution of bacterial toxins to CRC, but also pave the way to the implementation of current screening programs and preventive strategies.

Dissemination of high-grade serous ovarian cancer (HG-SOC) in the omentum and intercalation into a mesothelial cell (MC) monolayer depends on functional α5β1 integrin (Intα5β1) activity. Although the binding of Intα5β1 to fibronectin drives these processes, other molecular mechanisms linked to integrin inside-out signaling might support metastatic dissemination. Here, we report a novel interactive signaling that contributes to Intα5β1 activation and accelerates tumor cells toward invasive disease, involving the protein β-arrestin1 (β-arr1) and the activation of the endothelin A receptor (ET A R) by endothelin-1 (ET-1). As demonstrated in primary HG-SOC cells and SOC cell lines, ET-1 increased Intβ1 and downstream FAK/paxillin activation. Mechanistically, β-arr1 directly interacts with talin1 and Intβ1, promoting talin1 phosphorylation and its recruitment to Intβ1, thus fueling integrin inside-out activation. In 3D spheroids and organotypic models mimicking the omentum, ET A R/β-arr1-driven Intα5β1 signaling promotes the survival of cell clusters, with mesothelium-intercalation capacity and invasive behavior. The treatment with the antagonist of ET A R, Ambrisentan (AMB), and of Intα5β1, ATN161, inhibits ET-1-driven Intα5β1 activity in vitro, and tumor cell adhesion and spreading to intraperitoneal organs and Intβ1 activity in vivo. As a prognostic factor, high EDNRA/ITGB1 expression correlates with poor HG-SOC clinical outcomes. These findings highlight a new role of ET A R/β-arr1 operating an inside-out integrin activation to modulate the metastatic process and suggest that in the new integrin-targeting programs might be considered that ET A R/β-arr1 regulates Intα5β1 functional pathway.