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Glucocorticoids (GCs) are commonly used to treat autoimmune and inflammatory diseases, but their clinical effects and long-term use can lead to serious side effects. New drugs that can replace GCs are needed. Glucocorticoid-induced leucine zipper (GILZ) is induced by GCs and mediates many of their anti-inflammatory effects, such as inhibiting the pro-inflammatory molecule NF-κB. The GILZ C-terminal domain (PER region) is responsible for GILZ/p65NF-κB interaction and consequent inhibition of its transcriptional activity. A set of five short peptides spanning different parts of the PER region of GILZ protein was designed, and their anti-inflammatory activity was tested, both in vitro and in vivo. We tested the biological activity of GILZ peptides in human lymphocytic and monocytic cell lines to evaluate their inhibitory effect on the NF-κB-dependent expression of pro-inflammatory cytokines. Among the tested peptides, the peptide named PEP-1 demonstrated the highest efficacy in inhibiting cell activation in vitro. Subsequently, PEP-1 was further evaluated in two in vivo experimental colitis models (chemically induced by DNBS administration and spontaneous colitis induced in IL-10 knock-out (KO) mice (to assess its effectiveness in counteracting inflammation. Results show that PEP-1 reduced disease severity in both colitis models associated with reduced NF-κB pro-inflammatory activity in colon lamina propria lymphocytes. This study explored GILZ-based 'small peptides' potential efficacy in decreasing lymphocyte activation and inflammation associated with experimental inflammatory bowel diseases (IBDs). Small peptides have several advantages over the entire protein, including higher selectivity, better stability, and bioavailability profile, and are easy to synthesize and cost-effective. Thus, identifying active GILZ peptides could represent a new class of drugs for treating IBD patients. [ABSTRACT FROM AUTHOR]

Since their discovery, glucocorticoids (GCs) have been used to treat almost all autoimmune and chronic inflammatory diseases, as well as allergies and some forms of malignancies, because of their immunosuppressive and anti-inflammatory effects. Although GCs provide only symptomatic relief and do not eliminate the cause of the pathology, in the majority of treatments, GCs frequently cannot be replaced by other classes of drugs. Consequently, long-term treatments cause adverse effects that may, in turn, lead to new pathologies that sometimes require the withdrawal of GC therapy. Therefore, thus far, researchers have focused their efforts on molecules that have the same efficacy as that of GCs but cause fewer adverse effects. To this end, some GC-induced proteins, such as glucocorticoid-induced leucine zipper (GILZ), have been used as drugs in mouse models of inflammatory pathologies. In this review, we focus on some important but rare autoimmune and chronic inflammatory diseases for which the biomedical research investment in new therapies is less likely. Additionally, we critically evaluate the possibility of treating such diseases with other drugs, either GC-related or unrelated. [ABSTRACT FROM AUTHOR]

Poor prognosis in heart failure and the lack of real breakthrough strategies validate targeting myocardial remodelling and the intracellular signalling involved in this process. So far, there are no effective strategies to counteract hypertrophy, an independent predictor of heart failure progression and death. Glucocorticoid‐induced leucine zipper (GILZ) is involved in inflammatory signalling, but its role in cardiac biology is unknown. Using GILZ‐knockout (KO) mice and an experimental model of hypertrophy and diastolic dysfunction, we addressed the role of GILZ in adverse myocardial remodelling. Infusion of angiotensin II (Ang II) resulted in myocardial dysfunction, inflammation, apoptosis, fibrosis, capillary rarefaction and hypertrophy. Interestingly, GILZ‐KO showed more evident diastolic dysfunction and aggravated hypertrophic response compared with WT after Ang II administration. Both cardiomyocyte and left ventricular hypertrophy were more pronounced in GILZ‐KO mice. On the other hand, Ang II–induced inflammatory and fibrotic phenomena, cell death and reduction in microvascular density, remained invariant between the WT and KO groups. The analysis of regulators of hypertrophic response, GATA4 and FoxP3, demonstrated an up‐regulation in WT mice infused with Ang II; conversely, such an increase did not occur in GILZ‐KO hearts. These data on myocardial response to Ang II in mice lacking GILZ indicate that this protein is a new element that can be mechanistically involved in cardiovascular pathology. [ABSTRACT FROM AUTHOR]

As an alternative to lifelong insulin supplementation, potentiation of immune tolerance in patients with type 1 diabetes could prevent the autoimmune destruction of pancreatic islet β-cells. This study was aimed to assess whether the G3c monoclonal antibody (mAb), which triggers the glucocorticoid-induced TNFR-related (Gitr) costimulatory receptor, promotes the expansion of regulatory T cells (Tregs) in SV129 (wild-type) and diabetic-prone NOD mice. The delivery of the G3c mAb via G3C hybridoma cells enveloped in alginate-based microcapsules (G3C/cps) for 3 weeks induced Foxp3+ Treg-cell expansion in the spleen of wild-type mice but not in Gitr−/− mice. G3C/cps also induced the expansion of nonconventional Cd4+Cd25−/lowFoxp3lowGitrint/high (GITR single-positive [sp]) Tregs. Both Cd4+Cd25+GitrhighFoxp3+ and GITRsp Tregs (including also antigen-specific cells) were expanded in the spleen and pancreas of G3C/cps-treated NOD mice, and the number of intact islets was higher in G3C/cps-treated than in empty cps-treated and untreated animals. Consequently, all but two G3C/cps-treated mice did not develop diabetes and all but one survived until the end of the 24-week study. In conclusion, long-term Gitr triggering induces Treg expansion, thereby delaying/preventing diabetes development in NOD mice. This therapeutic approach may have promising clinical potential for the treatment of inflammatory and autoimmune diseases. [ABSTRACT FROM AUTHOR]

Glucocorticoid‐induced leucine zipper (GILZ) exerts anti‐inflammatory effects on the immune cells. However, less is known about GILZ function in neutrophils. We aimed to define the specific role of GILZ in basal neutrophil activity during an inflammatory response. GILZ knockdown resulted in a persistent activation state of neutrophils, as evidenced by increased phagocytosis, killing activity, and oxidative burst in GILZ‐knockout (KO) neutrophils. This enhanced response caused severe disease in a dinitrobenzene sulfonic acid (DNBS)‐induced colitis model, where GILZ‐KO mice had prominent granulocytic infiltrate and excessive inflammatory state. We used a Candida albicans intraperitoneal infection model to unravel the intracellular pathways affected by GILZ expression in activated neutrophils. GILZ‐KO neutrophils had stronger ability to clear the infectious agent than the wild‐type (WT) neutrophils, and there was more activation of the NOX2 (NADPH oxidase 2) and p47phox proteins, which are directly involved in oxidative burst. Similarly, the MAPK pathway components, that is, ERK and p38, which are involved in the oxidative burst pathway, were highly phosphorylated in GILZ‐KO neutrophils. Evaluation of GILZ expression kinetics during C. albicans infection revealed down‐regulation that correlated inversely with the state of neutrophil activation, which was evaluated as oxidative burst. Overall, our findings define GILZ as a regulator of neutrophil functions, as its expression contributes to limiting neutrophil activation by reducing the activation of the signaling pathways that control the basal neutrophil functions. Controlling GILZ expression could help regulate a continuous inflammatory state that can result in chronic inflammatory and autoimmune diseases. GILZ expression contributes to the inhibition of neutrophil activation by reducing MAPK pathway protein and NOX2 activity that control basal neutrophil functions. [ABSTRACT FROM AUTHOR]

Background: The standard technique for implanting a subcutaneous implantable cardioverter defibrillator (S-ICD) requires three incisions and the pocket of the device is created in the subcutaneous tissue of the left lateral thoracic wall. However, a two-incision technique may be adopted, in which the cranial parasternal region is avoided and the device is positioned more deeply, completely under the latissimus dorsi muscle. This can also be combined with ultrasound-guided serratus anterior plane block (US-SAPB) for intraoperative anesthesia and perioperative analgesia. We describe our preliminary experience of US-SAPB combined with the two-incision intermuscular technique. Methods: We performed US-SAPB 40 minutes before starting the procedure, while the patient was in the supine position. The devices were implanted under the latissimus dorsi muscle. All patients were followed-up after hospital discharge. Results: Twelve patients (male 50%, 53 ± 16 years, body mass index 23 ± 4) underwent the S-ICD implantation with the combined technique. The mean procedure duration was 47 ± 11 minutes. The procedure was successful and a shock energy of 65 J was successful in converting the induced ventricular fibrillation in all patients. The US-SAPB was successful in 92% of cases and only one patient required convertion into general anesthesia due to pain during the procedure. In the postoperative period, patients did not report major discomfort and analgesics were not required. During a median follow-up of 12 months, no complications were reported. Conclusions: Serratus anterior plane block combined with the intermuscular and two-incision technique proved to be safe and effective during the S-ICD implantation procedure. [ABSTRACT FROM AUTHOR]

The glucocorticoid-induced leucine zipper (GILZ) gene is a pivotal mediator of the anti-inflammatory effects of glucocorticoids (GCs) that are known to regulate the function of both adaptive and innate immunity cells. Our aim was to investigate the role of GILZ in GC-induced inhibition of neutrophil migration, as this role has not been investigated before. We found that GILZ expression was induced by dexamethasone (DEX), a synthetic GC, in neutrophils, and that it regulated migration of these cells into inflamed tissues under DEX treatment. Of note, inhibition of neutrophil migration was not observed in GILZ-knockout mice with peritonitis that were treated by DEX. This was because DEX was unable to up-regulate annexin A1 (Anxa1) expression in the absence of GILZ. Furthermore, we showed that GILZ mediates Anxa1 induction by GCs by transactivating Anxa1 expression at the promoter level via binding with the transcription factor, PU.1. The present findings shed light on the role of GILZ in the mechanism of induction of Anxa1 by GCs. As Anxa1 is an important protein for the resolution of inflammatory response, GILZ may represent a new pharmacologic target for treatment of inflammatory diseases. [ABSTRACT FROM AUTHOR]

Glucocorticoid-induced tumour necrosis factor receptor-related protein (GITR, TNFRSF18, and CD357) is expressed at high levels in activated T cells and regulatory T cells (Tregs). In this review, we present data from mouse and human studies suggesting that GITR is a crucial player in the differentiation of thymic Tregs (tTregs), and expansion of both tTregs and peripheral Tregs (pTregs). The role of GITR in Treg expansion is confirmed by the association of GITR expression with markers of memory T cells. In this context, it is not surprising that GITR appears to be a marker of active Tregs, as suggested by the association of GITR expression with other markers of Treg activation or cytokines with suppressive activity (e.g., IL-10 and TGF-β), the presence of GITR+ cells in tissues where Tregs are active (e.g., solid tumours), or functional studies on Tregs. Furthermore, some Treg subsets including Tr1 cells express either low or no classical Treg markers (e.g., FoxP3 and CD25) and do express GITR. Therefore, when evaluating changes in the number of Tregs in human diseases, GITR expression must be evaluated. Moreover, GITR should be considered as a marker for isolating Tregs. [ABSTRACT FROM AUTHOR]

Glucocorticoids are the most powerful anti-inflammatory and immunosuppressive pharmacological drugs available, despite their adverse effects. Glucocorticoid-induced leucine zipper (GILZ) is a glucocorticoid-induced gene that shares several anti-inflammatory properties with glucocorticoids. Although immunosuppressive effects of glucocorticoids on neutrophils remain poorly understood, we previously demonstrated that GILZ suppresses neutrophil activation under glucocorticoid treatment. Here, we sought to explore the regulation of Toll-like receptor 2 (TLR2) by the synthetic glucocorticoid dexamethasone (DEX) on neutrophils and the associated GILZ involvement. Peripheral blood neutrophils were isolated from wild type and GILZ-knock-out (KO) mice. TLR2 was found to be downregulated by the in vivo administration of glucocorticoids in wild type but not in GILZ-KO neutrophils, suggesting the involvement of GILZ in TLR2 downregulation. Accordingly, the TLR2-associated anti-fungal activity of neutrophils was reduced by DEX treatment in wild type but not GILZ-KO neutrophils. Furthermore, GILZ did not interact with NF-κB but was found to bind with STAT5, a pivotal factor in the regulation of TLR2 expression. A similar modulation of TLR2 expression, impaired phagocytosis, and killing activity was observed in circulating human neutrophils treated in vitro with DEX. These results demonstrate that glucocorticoids reduce the ability of neutrophils to respond to infections by downregulating TLR2 via GILZ, thereby reducing critical functions. [ABSTRACT FROM AUTHOR]

Glucocorticoids are hormones that regulate several functions in living organisms and synthetic glucocorticoids are the most powerful anti-inflammatory pharmacological tool that is currently available. Although glucocorticoids have an immunosuppressive effect on immune cells, they exert multiple and sometimes contradictory effects on neutrophils. From being extremely sensitive to the anti-inflammatory effects of glucocorticoids to resisting glucocorticoid-induced apoptosis, neutrophils are proving to be more complex than they were earlier thought to be. The aim of this review is to explain these complex pathways by which neutrophils respond to endogenous or to exogenous glucocorticoids, both under physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Cannabinoids are known to possess anti-inflammatory and immunomodulatory properties, but the mechanisms involved are not fully understood. CB2 is the cannabinoid receptor that is expressed primarily on hematopoietic cells and mediates the immunoregulatory functions of cannabinoids. In order to study the effect of JTE907, a selective/inverse agonist of CB2 with anti-inflammatory properties, on the differentiation of T cell subtypes, we used an in vitro system of Th lineage-specific differentiation of naïve CD4+ T lymphocytes isolated from the mouse spleen. The results indicate that JTE907 was able to induce the differentiation of Th0 cells into the Treg cell phenotype, which was characterized by the expression of FoxP3, TGF-β and IL-10. P38 phosphorylation and STAT5A activation were found to mediate the signaling pathway triggered by JTE907 via the CB2 receptor in Th0 lymphocytes. In mice with DNBS-induced colitis, JTE907 treatment was able to induce an increase in the number of CD4+CD25+FoxP3+ cells in the lamina propria after 24 h of disease onset and reduce disease severity after 48 h. Further, longer JTE907 treatment resulted in less severe colitis even when administered orally, resulting in less body weight loss, reduction of the disease score, prevention of NF-κB activation, and reduction of the expression of adhesion molecules. Collectively, the results of this study indicate that specific signals delivered through the CB2 receptor can drive the immune response towards the Treg cell phenotype. Thus, ligands such as JTE907 may have use as potential therapeutic agents in autoimmune and inflammatory diseases. [ABSTRACT FROM AUTHOR]

Autoimmune diseases decrease life expectancy and quality of life for millions of women and men. Although treatments can slow disease progression and improve quality of life, all currently available drugs have adverse effects and none of them are curative; therefore, requiring patients to take immunosuppressive drugs for the remainder of their lives. A curative therapy that is safe and effective is urgently needed. We believe that therapies promoting the in vivo expansion of regulatory T cells (Tregs) or injection of in vitro expanded autologous/heterologous Tregs (cellular therapy) can alter the natural history of autoimmune diseases. In this review, we present data from murine and human studies suggesting that 1) glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) plays a crucial role in thymic Treg (tTreg) differentiation and expansion; 2) GITR plays a crucial role in peripheral Treg (pTreg) expansion; 3) in patients with Sjögren syndrome and systemic lupus erythematosus, CD4 + GITR + pTregs are expanded in patients with milder forms of the disease; and 4) GITR is superior to other cell surface markers to differentiate Tregs from other CD4 + T cells. In this context, we consider two potential new approaches for treating autoimmune diseases consisting of the in vivo expansion of GITR + Tregs by GITR-triggering drugs and in vitro expansion of autologous or heterologous GITR + Tregs to be infused in patients. Advantages of such an approach, technical problems, and safety issues are discussed. [ABSTRACT FROM AUTHOR]

As an alternative to lifelong insulin supplementation, potentiation of immune tolerance in patients with type 1 diabetes could prevent the autoimmune destruction of pancreatic islet β-cells. This study was aimed to assess whether the G3c monoclonal antibody (mAb), which triggers the glucocorticoid-induced TNFR-related (Gitr) costimulatory receptor, promotes the expansion of regulatory T cells (Tregs) in SV129 (wild-type) and diabetic-prone NOD mice. The delivery of the G3c mAb via G3C hybridoma cells enveloped in alginate-based microcapsules (G3C/cps) for 3 weeks induced Foxp3 + Treg-cell expansion in the spleen of wild-type mice but not in Gitr -/- mice. G3C/cps also induced the expansion of nonconventional Cd4 + Cd25 -/low Foxp3 low Gitr int/high (GITR single-positive [sp]) Tregs. Both Cd4 + Cd25 + Gitr high Foxp3 + and GITRsp Tregs (including also antigen-specific cells) were expanded in the spleen and pancreas of G3C/cps-treated NOD mice, and the number of intact islets was higher in G3C/cps-treated than in empty cps-treated and untreated animals. Consequently, all but two G3C/cps-treated mice did not develop diabetes and all but one survived until the end of the 24-week study. In conclusion, long-term Gitr triggering induces Treg expansion, thereby delaying/preventing diabetes development in NOD mice. This therapeutic approach may have promising clinical potential for the treatment of inflammatory and autoimmune diseases., (© 2020 by the American Diabetes Association.)

Glucocorticoid-Induced Leucine Zipper (GILZ) is a glucocorticoid-inducible gene that mediates glucocorticoid anti-inflammatory effects. GILZ and the isoform L-GILZ are expressed in a variety of cell types, especially of hematopoietic origin, including macrophages, lymphocytes and epithelial cells, and strongly upregulated upon glucocorticoid treatment. A quantitative analysis of GILZ expression in mouse tissues is technically difficult to perform because of the presence of a pseudogene and the high homology of GILZ gene with other genes of TSC22 family. We here propose specific primer pairs to be used in Real Time PCR to avoid unwanted amplification of GILZ pseudogene and TSC-22 family member d1iso3. These primer pairs were used to determine GILZ and L-GILZ expression, in either untreated or in vivo and in vitro dexamethasone-treated tissues. Results indicate that GILZ and L-GILZ are upregulated by glucocorticoids, being GILZ more sensitive to glucocorticoid induction than L-GILZ, but they are differently expressed in all examined tissues, confirming a different role in specific cells. An inappropriate primer pair amplified also GILZ pseudogene and TSC22d1iso3, thus producing misleading results. This quantitative evaluation may be used to better characterize the role of GILZ and L-GILZ in mice and may be translated to humans.

Background: Among the various factors influencing the success rate, onset time, and duration of peripheral nerve blocks, the role of local anesthetics concentration remains uncertain. In this prospective, randomized, single-blinded study, we evaluated whether varying the dilution of a fixed dose of mepivacaine solution influenced onset time and duration of sciatic nerve block., Methods: Ninety ASA physical status I to II patients scheduled for foot surgery were randomly allocated to receive a double-injection Labat sciatic nerve block with 12 mL mepivacaine 2% (group concentration I = 45 patients) or 24 mL of mepivacaine 1% (group volume II = 45 patients). The nerve stimulator was initially set at 2 Hz, 0.1 millisecond, 1 mA. The total amount of local anesthetic (240 mg) was kept constant and equally divided between the peroneal and tibial nerves. All patients also received an ultrasound-guided popliteal sciatic nerve catheter for postoperative analgesia. Times to readiness for surgery, performance, and offset of local anesthetic were recorded. Our primary end point was to determine a possible difference in offset time between groups. Continuous variables were expressed as median (IQR) and compared with the Wilcoxon-Mann-Whitney U test; WMWodds are reported together with their 95% confidence interval., Results: The overall success rate of sciatic nerve block was 99%. Time of performance was shorter in group I, 120 seconds (90-150 seconds), than that in group II, 150 seconds (120-180 seconds) (P = 0.0048; WMWodds 2.26 [1.35-4.34]). The onset time of sensory and motor sciatic nerve block was 4 minutes (2-9 minutes) in group I and 6 minutes (4-10 minutes) in group II (P = 0.41; WMWodds 1.21 [0.77-1.95]), while the duration of sensory block was 235 minutes (203-250 minutes) in group I, and 240 minutes (218-247 minutes) in group II respectively (P = 0.51; WMWodds 1.20 [0.69-2.16])., Conclusions: We found no evidence that varying volume and concentration while maintaining a fixed total dose of mepivacaine alters the onset time and duration of double-injection sciatic nerve block. Considering our WMWodds results, possible differences in onset time and duration comparable to differences in the performance time between groups cannot be excluded.