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We have previously reported that TLR4 signaling is increased in lipopolysaccharide (LPS) -stimulated Cystic Fibrosis (CF) macrophages (MΦs), contributing to the robust production of pro-inflammatory cytokines. The heme oxygenase (HO-1)/carbon monoxide (CO) pathway modulates cellular redox status, inflammatory responses, and cell survival. The HO-1 enzyme, together with the scaffold protein caveolin 1 (CAV-1), also acts as a negative regulator of TLR4 signaling in MΦs. Here, we demonstrate that in LPS-challenged CF MΦs, HO-1 does not compartmentalize normally to the cell surface and instead accumulates intracellularly. The abnormal HO-1 localization in CF MΦs in response to LPS is due to decreased CAV-1 expression, which is controlled by the cellular oxidative state, and is required for HO-1 delivery to the cell surface. Overexpression of HO-1 or stimulating the pathway with CO-releasing molecules (CORM2)enhancesCAV-1 expression in CF MΦs, suggesting a positive-feed forward loop between HO-1/CO induction and CAV-1 expression. These manipulations reestablished HO-1 and CAV-1 cell surface localization in CF MΦ's. Consistent with restoration of HO-1/CAV-1 negative regulation of TLR4 signaling, genetic or pharmacological (CORM2)-induced enhancement of this pathway decreased the inflammatory response of CF MΦs and CF mice treated with LPS. In conclusion, our results demonstrate that the counter-regulatory HO-1/CO pathway, which is critical in balancing and limiting the inflammatory response, is defective in CF MΦs through a CAV-1-dependent mechanism, exacerbating the CF MΦ's response to LPS. This pathway could be a potential target for therapeutic intervention for CF lung disease.

Cell migration is a very complex mechanism linked to the inflammatory response as well as to several processes of cell biology and development. The movement of cells in response to a chemokine gradient is the most popular form of interaction between cell environment and surface and specific surface receptor triggers an intracellular signaling pathway. This phenomenon is known as chemotaxis and is characteristic of neutrophil movements in response to the chemokine Interleuchin-8 (IL8) in a 3D space. Another paradigm of dynamic interaction between cells is the mechanism underlying the confluency of cells cultured in a Petri dish. This process is particularly studied in growing epithelial cell lines, and is influenced by still undefined external stimuli as well as by cell proliferation and cytoskeleton reorganization, that allow the contact and interaction between cells in 2D space. Chronic inflammation is an ideal condition for the study of cell migration and its de-regulation; understanding the mechanisms involved in cell motility and their putative modulation, is the first step in setting new appropriate models of study. During PhD project, different cellular models have been used: IB3 cell lines belonging to patients with Cystic Fibrosis (caused by mutations of cystic fibrosis transmembrane regulator (CFTR)), and T84 cell lines, a popular model of study of Coeliac Disease, a common intolerance to proteins of wheat. Both disease are characterized by a pro-inflammatory milieu with high level of tissue Transglutaminase (TG2), a multi-functions enzyme with a defined role in several human pathologies. Cystic Fibrosis is the prototype of diseases in which an uncontrolled production of IL8 leads to a dysregulated neutrophil recruitment. The IB3 cell lines have been used to understand a molecular mechanisms of enhanced IL8 production and their modulation. The results evidence i) the role of TG2 as a new pathogenic factor in Cystic Fibrosis; ii) the role of post-translational modifications of TG2 as a link between genetic defect of CFTR ad inflammation iii) the mechanisms of autophagy inhibition via ROS-TG2 axis, in epithelial airway cell line and mice model of CF. The consequence is a fine modulation of IL8 production. The system allows the design of a 3D model to study cell migration under IL8 diffusive flux in qualitative and quantitative ways. Another important pathological system with chronic inflammation is celiac disease and the analysis of interactions of alimentary peptides with the frontline gut epithelium is a useful model of study. The epithelial cells are pivot in the innate immune activation in CD and cytoskeleton rearrangement is the earliest event in such a response to gliadin peptides. The data in T84 cell model show a gliadin peptide-driven pro-inflammatory environment as a consequence of its impaired lysosomal degradation. Moreover, alterations of motility and cytoskeletal reorganization emphasize the “toxic” effect of peptide. Therefore, CD offers an ideal opportunity to set up a 2D model of study of cell motility. Understanding the mechanisms of migration and the identification of appropriate target of modulation of cell recruitment could allow wide industrial applications in biotechnology and will be successful to provide a useful tool to test potential therapeuthic molecules.

Transglutaminase 2 (TG2) is a ubiquitously expressed member of the transglutaminase family with Ca2+-dependent protein crosslinking activity. Its subcellular localization is crucial in determining its function, and indeed, TG2 is found in the extracellular matrix, mitochondria, recycling endosomes, plasma membrane, cytosol, and nucleus because it is associated with cell growth, differentiation, and apoptosis. It is involved in several pathologies, such as celiac disease, cardiovascular, hepatic, renal, and fibrosis diseases, carrying out opposite functions of up and down regulation in the progression of the same pathology. Therefore, this fine regulation requires a very sensitive and specific method of identification of TG2, which is to be detected in very small quantities in a deregulated condition. Here, we demonstrate the possibility of detecting TG2 down to attomolar concentration by using organic electrochemical transistors driven by gold electrodes functionalized with anti-TG2 antibodies. In particular, a direct correlation between the TG2 concentration and the transistor transconductance values, as extracted from typical transfer curves, was found. Overall, our findings highlight the potentialities of this new biosensing approach for the detection of TG2 in the context of pathological diseases, offering a rapid and cost-effective alternative to traditional methods.

Cystic fibrosis (CF), the most common lethal monogenic disease in Caucasians, is characterized by recurrent bacterial infections and colonization, mainly by Pseudomonas aeruginosa, resulting in unresolved airway inflammation. CF is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which functions as a chloride channel in epithelial cells, macrophages, and other cell types. Impaired bacterial handling by macrophages is a feature of CF airways, although it is still debated how defective CFTR impairs bacterial killing. Recent evidence indicates that a defective autophagy in CF macrophages leads to alterations of bacterial clearance upon infection. Here we use bone marrow-derived macrophages from transgenic mice to provide the genetic proof that defective CFTR compromises both uptake and clearance of internalized Pseudomonas aeruginosa. We demonstrate that the proteostasis regulator cysteamine, which rescues the function of the most common F508del-CFTR mutant and hence reduces lung inflammation in CF patients, can also repair the defects of CF macrophages, thus restoring both bacterial internalization and clearance through a process that involves upregulation of the pro-autophagic protein Beclin 1 and re-establishment of the autophagic pathway. Altogether these results indicate that cysteamine restores the function of several distinct cell types, including that of macrophages, which might contribute to its beneficial effects on CF.

Cystic fibrosis (CF) is most frequently due to homozygous ΔF508-CFTR mutation. The ΔF508-CFTR protein is unstable in the plasma membrane (PM), even if it is rescued by pharmacological agents that prevent its intracellular retention and degradation. Restoring defective autophagy in CF airways by proteostasis regulators (such as cystamine and its reduced form, cysteamine) can rescue and stabilize ΔF508-CFTR at the PM, thus enabling the action of CFTR potentiators, which are pharmacological agents that stimulate the function of CFTR as an ion channel. The effects of cystamine extend for days (in vitro) and weeks (in vivo) beyond washout, suggesting that once peripheral proteostasis has been re-established, PM-resident ΔF508-CFTR sustains its own stability. We demonstrated that the pharmacological inhibition of wild-type CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette subfamily C, member 7)], in bronchial epithelial cells decreases the stability of the CFTR protein by inhibiting autophagy, elevating the abundance of SQSTM1/p62 and its interaction with CFTR at the PM, increasing the ubiqutination of CFTR, stimulating the lysosomal degradation of CFTR and avoiding its recycling. All these effects could be inhibited by cystamine. Moreover, CFTR-sufficient epithelia generate permissive conditions for incorporating ΔF508-CFTR into the PM and stabilizing it at this location. These results provide the rationale for a combination therapy of CF in which pretreatment with cystamine or cysteamine enables the later action of CFTR potentiators.