Alzheimer-like behavior and synaptic dysfunction in 3 × Tg-AD mice are reversed with calcineurin inhibition.

Alzheimer's disease is a progressive neurodegenerative disorder characterized by impairments in synaptic plasticity and cognitive performance. Current treatments are unable to achieve satisfactory therapeutic effects or reverse the progression of the disease. Calcineurin has been implicated as part of a critical signaling pathway for learning and memory, and neuronal calcineurin may be hyperactivated in AD. To investigate the effects and underlying mechanisms of FK506, a calcineurin inhibitor, on Alzheimer-like behavior and synaptic dysfunction in the 3 × Tg-AD transgenic mouse model of Alzheimer's disease, we investigated the effect of FK506 on cognitive function and synaptic plasticity in the 3 × Tg-AD transgenic mouse model of Alzheimer's disease. The results showed that FK506 treatment ameliorated cognitive deficits, as indicated by the decreased latency in the water maze, and attenuated tau hyperphosphorylation in 3 × Tg-AD mice. Treatment with FK506 also reduced the levels of certain markers of postsynaptic deficits, including PSD-95 and NR2B, and reversed the long-term potentiation deficiency and dendritic spine impairments in 3 × Tg-AD mice. These findings suggest that treatment with calcineurin inhibitors such as FK506 can be an effective therapeutic strategy to rescue synaptic deficit and cognitive impairment in familial Alzheimer's disease and related tauopathies.

Calcineurin regulates synaptic Ca2+-permeable AMPA receptors in hypothalamic presympathetic neurons via α2δ-1-mediated GluA1/GluA2 assembly.

Hypertension is a major adverse effect of calcineurin inhibitors, such as tacrolimus (FK506) and cyclosporine, used clinically as immunosuppressants. Calcineurin inhibitor-induced hypertension (CIH) is linked to augmented sympathetic output from the hypothalamic paraventricular nucleus (PVN). GluA2-lacking, Ca2+-permeable AMPA receptors (CP-AMPARs) are a key feature of glutamatergic synaptic plasticity, yet their role in CIH remains elusive. Here, we found that systemic administration of FK506 in rats significantly increased serine phosphorylation of GluA1 and GluA2 in PVN synaptosomes. Strikingly, FK506 treatment reduced GluA1/GluA2 heteromers in both synaptosomes and endoplasmic reticulum-enriched fractions from the PVN. Blocking CP-AMPARs with IEM-1460 induced a larger reduction of AMPAR-mediated excitatory postsynaptic current (AMPAR-EPSC) amplitudes in retrogradely labelled, spinally projecting PVN neurons in FK506-treated rats than in vehicle-treated rats. Furthermore, FK506 treatment shifted the current-voltage relationship of AMPAR-EPSCs from linear to inward rectification in labelled PVN neurons. FK506 treatment profoundly enhanced physical interactions of α2δ-1 with GluA1 and GluA2 in the PVN. Inhibiting α2δ-1 with gabapentin, α2δ-1 genetic knockout, or disrupting α2δ-1-AMPAR interactions with an α2δ-1 C terminus peptide restored GluA1/GluA2 heteromers in the PVN and diminished inward rectification of AMPAR-EPSCs in labelled PVN neurons induced by FK506 treatment. Additionally, microinjection of IEM-1460 or α2δ-1 C terminus peptide into the PVN reduced renal sympathetic nerve discharges and arterial blood pressure elevated in FK506-treated rats but not in vehicle-treated rats. Thus, calcineurin in the hypothalamus constitutively regulates AMPAR subunit composition and phenotypes by controlling GluA1/GluA2 interactions with α2δ-1. Synaptic CP-AMPARs in PVN presympathetic neurons contribute to augmented sympathetic outflow in CIH. KEY POINTS: Systemic treatment with the calcineurin inhibitor increases serine phosphorylation of synaptic GluA1 and GluA2 in the PVN. Calcineurin inhibition enhances the prevalence of postsynaptic Ca2+-permeable AMPARs in PVN presympathetic neurons. Calcineurin inhibition potentiates α2δ-1 interactions with GluA1 and GluA2, disrupting intracellular assembly of GluA1/GluA2 heterotetramers in the PVN. Blocking Ca2+-permeable AMPARs or α2δ-1-AMPAR interactions in the PVN attenuates sympathetic outflow augmented by the calcineurin inhibitor.

Concomitant use of interleukin-2 and tacrolimus suppresses follicular helper T cell proportion and exerts therapeutic effect against lupus nephritis in systemic lupus erythematosus-like chronic graft versus host disease.

Introduction: Defective interleukin-2 (IL-2) production contributes to immune system imbalance in patients with systemic erythematosus lupus (SLE). Recent clinical studies suggested that low-dose IL-2 treatment is beneficial for SLE and the therapeutic effect is associated with regulatory T cell (Treg) expansion. Pharmacological calcineurin inhibition induces a reduction in the number of Tregs because they require stimulation of T cell receptor signaling and IL-2 for optimal proliferation. However, the activation of T cell receptor signaling is partially dispensable for the expansion of Tregs, but not for that of conventional T cells if IL-2 is present. Aim: We examined whether addition of IL-2 restores the Treg proportion even with concurrent use of a calcineurin inhibitor and if the follicular helper T cell (Tfh) proportion is reduced in an SLE-like murine chronic graft versus host disease model. Methods: Using a parent-into-F1 model, we investigated the effect of IL-2 plus tacrolimus on Treg and Tfh proportions and the therapeutic effect. Results: Treatment with a combination of IL-2 and tacrolimus significantly delayed the initiation of proteinuria and decreased the urinary protein concentration, whereas tacrolimus or IL-2 monotherapy did not significantly attenuate proteinuria. Phosphorylation of signal transducer and activator of transcription 3, a positive regulator of Tfh differentiation, was reduced by combination treatment, whereas phosphorylation of signal transducer and activator of transcription 5, a negative regulator, was not reduced. Conclusion: Addition of calcineurin inhibitors as adjunct agents may be beneficial for IL-2-based treatment of lupus nephritis.

Nsp1 facilitates SARS-CoV-2 replication through calcineurin-NFAT signaling.

SARS-CoV-2, the causative agent of COVID-19, has been intensely studied in search of effective antiviral treatments. The immunosuppressant cyclosporine A (CsA) has been suggested to be a pan-coronavirus inhibitor, yet its underlying mechanism remained largely unknown. Here, we found that non-structural protein 1 (Nsp1) of SARS-CoV-2 usurped CsA-suppressed nuclear factor of activated T cells (NFAT) signaling to drive the expression of cellular DEAD-box helicase 5 (DDX5), which facilitates viral replication. Nsp1 interacted with calcineurin A (CnA) to displace the regulatory protein regulator of calcineurin 3 (RCAN3) of CnA for NFAT activation. The influence of NFAT activation on SARS-CoV-2 replication was also validated by using the Nsp1-deficient mutant virus. Calcineurin inhibitors, such as CsA and VIVIT, inhibited SARS-CoV-2 replication and exhibited synergistic antiviral effects when used in combination with nirmatrelvir. Our study delineated the molecular mechanism of CsA-mediated inhibition of SARS-CoV-2 replication and the anti-SARS-CoV-2 action of calcineurin inhibitors. IMPORTANCE: Cyclosporine A (CsA), commonly used to inhibit immune responses, is also known to have anti-SARS-CoV-2 activity, but its mode of action remains elusive. Here, we provide a model to explain how CsA antagonizes SARS-CoV-2 through three critical proteins: DDX5, NFAT1, and Nsp1. DDX5 is a cellular facilitator of SARS-CoV-2 replication, and NFAT1 controls the production of DDX5. Nsp1 is a viral protein absent from the mature viral particle and capable of activating the function of NFAT1 and DDX5. CsA and similar agents suppress Nsp1, NFAT1, and DDX5 to exert their anti-SARS-CoV-2 activity either alone or in combination with Paxlovid.

Severity and organ distribution of chronic graft-versus-host disease with posttransplant cyclophosphamide-based versus methotrexate/calcineurin inhibitor-based allogeneic hematopoietic cell transplantation.

The reduced risk of chronic graft-versus-host-disease (GVHD) with posttransplant cyclophosphamide (ptCy) in the setting of haploidentical related donor and more recently, with HLA-matched related and matched and mismatched unrelated donor allogeneic transplantation has been established. There is, however, paucity of data to show if ptCy impacts chronic GVHD pathogenesis, its phenotype and evolution after HCT regardless of the donor status. We examined the differences in chronic GVHD incidence and presentation in 314 consecutive patients after receiving their first allogeneic transplantation (HCT) using ptCy-based GVHD prophylaxis (ptCy-HCT; n = 120; including 95 with haploidentical related donor) versus conventional calcineurin inhibitor-based prophylaxis (CNI-MUD; n = 194) between 2012 and 2019. The 1-year cumulative incidence of all-grade chronic GVHD and moderate/severe chronic GVHD was 24% and 12%, respectively, after ptCy-HCT and 40% and 23% in the CNI-MUD recipients (p = 0.0003 and 0.007). Multivariable analysis confirmed that use of CNI-based GVHD prophylaxis and peripheral blood stem cell graft as the risk factors for chronic GVHD. The cumulative incidence of visceral (involving ≥1 of the following organs: liver, lungs, gastrointestinal tract, serous membranes) chronic GVHD was significantly higher with CNI-MUD vs. ptCy-HCT (27% vs. 15% at 1 year, p = 0.009). The incidence of moderate/severe visceral chronic GVHD was 20% in CNI-MUD group vs. 7.7% in the ptCy-HCT group at 1 year (p = 0.002). In addition, significantly fewer ptCy-HCT recipients developed severe chronic GVHD in ≥3 organs (0.8%) vs. 8.8% in the CNI-MUD group at 1-year posttransplant (p = 0.004). There was no significant different in relapse, non-relapse mortality, and relapse-free and overall survival between the two groups. Further investigation is needed to confirm that reduced risk and severity of chronic GVHD, less visceral organ distribution with ptCy-HCT leads to improved quality of life.

[Evidence-based recommendations for the treatment of rheumatic and immunologic diseases with calcineurin inhibitors: a consensus statement].

Calcineurin inhibitors (CNI), including oral cyclosporin A and tacrolimus, are intensive immunosuppressants that are extensively used in the treatment of rheumatic and immunologic diseases in China. CNI selectively inhibit the activation and proliferation of T lymphocytes and the transcription of cytokines [such as tumor necrosis factor-α, interleukin (IL)-6, and IL-17] through inhibiting the activation of calcineurin in cells and reducing the release of IL-2. To standardize the use of CNI in the field of rheumatic and immunologic diseases, this consensus statement was developed by the National Clinical Research Center for Dermatologic and Immunologic Diseases (Peking Union Medical College Hospital), in conjunction with the Chinese Association of Rheumatology and Immunology Physicians, the Chinese Research Hospital Association, the Rheumatology and Immunology Professional Committee, and the Chinese Association of Rehabilitation Medicine. The 2011 Oxford Centre for Evidence-Based Medicine Levels of Evidence was used to rate the quality of the evidence and the strength of the recommendations, and the RIGHT (Reporting Items for practice Guidelines in HealThcare) checklist was followed to report the consensus. The consensus offers recommendations addressing nine clinical challenges to Chinese clinicians. The primary objective of this consensus is to deliver scientific and detailed guidance on CNI for Chinese clinicians, and to improve the quality of patient-centered medical services.

The long-term effects of multidrug immunosuppressive protocols based on calcineurin inhibitors and conversion to rapamycin on the morphology, apoptosis, and proliferation of rat salivary glands.

BACKGROUND: The effect of multidrug immunosuppressive protocols on the salivary glands is still unknown. This study aimed to determine the influence of immunosuppressive regimens based on calcineurin inhibitors (CNIs) and conversion to rapamycin on the morphology, apoptosis, and proliferation of rat salivary glands. METHODS: Male rats received cyclosporin A (CsA), tacrolimus (FK-506), mycophenolate mofetil (MMF), rapamycin (Rapa), and prednisone (Pre) according to three-drug protocols: CMP (CsA, MMF, and Pre), CMP/R (CsA, MMF, and Pre with conversion to Rapa), TMP (FK-506, MMF, and Pre), and TMP/R (FK-506, MMF, and Pre with conversion to Rapa). Morphological and immunohistochemical and quantitative analyses of the salivary glands were performed. RESULTS: Structural changes in salivary glands were observed in all experimental groups, especially in the submandibular gland. In the salivary glands, the percentages of collagen fibers and TUNEL-, Ki67- and PCNA-positive cells were higher in the experimental groups vs. the control but were lower in the CMP/R and TMP/R groups vs. the CMP and TMP groups, with the exception of collagen fibers in the parotid gland in the TMP/R group vs. the TMP group. CONCLUSIONS: Long-term administration of CNIs in triple regimens and after conversion to rapamycin monotherapy, causes morphological changes in the salivary glands of rats. Immunosuppressive treatment based on CNIs is associated with an increase in collagen accumulation. The effects of the conversion of treatment with CNIs to rapamycin in immunosuppressive protocols in rat salivary glands lead to decreased fibrosis, apoptosis, and proliferation. These changes may possibly prevent abnormalities resulting from the application of CNIs.

Brain α2δ-1-Bound NMDA Receptors Drive Calcineurin Inhibitor-Induced Hypertension.

BACKGROUND: Calcineurin is highly enriched in immune T cells and the nervous system. Calcineurin inhibitors, including cyclosporine and tacrolimus (FK506), are the cornerstone of immunosuppressive regimens for preserving transplanted organs and tissues. However, these drugs often cause persistent hypertension owing to excess sympathetic outflow, which is maintained by N-methyl-D-aspartate receptor (NMDAR)-mediated excitatory input to the hypothalamic paraventricular nucleus (PVN). It is unclear how calcineurin inhibitors increase NMDAR activity in the PVN to augment sympathetic vasomotor activity. α2δ-1 (encoded by the Cacna2d1 gene), known colloquially as a calcium channel subunit, is a newly discovered NMDAR-interacting protein. In this study, we determined whether α2δ-1 plays a role in calcineurin inhibitor-induced synaptic NMDAR hyperactivity in the PVN and hypertension development. METHODS: Immunoblotting and coimmunoprecipitation assays were used to quantify synaptic protein levels and the physical interaction between GluN1 (the obligatory NMDAR subunit) and α2δ-1. Whole-cell patch-clamp recordings of retrogradely labeled, spinally projecting PVN were conducted in perfused brain slices to measure presynaptic and postsynaptic NMDAR activity. Radio-telemetry was implanted in rodents to continuously record arterial blood pressure in conscious states. RESULTS: Prolonged treatment with FK506 in rats significantly increased protein levels of α2δ-1, GluN1, and the α2δ-1-GluN1 complex in PVN synaptosomes. These effects were blocked by inhibiting α2δ-1 with gabapentin or interrupting the α2δ-1-NMDAR interaction with an α2δ-1 C-terminus peptide. Treatment with FK506 potentiated the activity of presynaptic and postsynaptic NMDARs in spinally projecting PVN neurons; such effects were abolished by gabapentin, Cacna2d1 knockout, or α2δ-1 C-terminus peptide. Furthermore, microinjection of α2δ-1 C-terminus peptide into the PVN diminished renal sympathetic nerve discharges and arterial blood pressure that had been increased by FK506 treatment. Remarkably, concurrent administration of gabapentin prevented the development of FK506-induced hypertension in rats. Additionally, FK506 treatment induced sustained hypertension in wild-type mice but not in Cacna2d1 knockout mice. CONCLUSIONS: α2δ-1 is essential for calcineurin inhibitor-induced increases in synaptic NMDAR activity in PVN presympathetic neurons and sympathetic outflow. Thus, α2δ-1 and α2δ-1-bound NMDARs represent new targets for treating calcineurin inhibitor-induced hypertension. Gabapentinoids (gabapentin and pregabalin) could be repurposed for treating calcineurin inhibitor-induced neurogenic hypertension.

Calcineurin inhibitor inhibits tolerance induction by suppressing terminal exhaustion of donor T cells after allo-HCT.

Calcineurin inhibitor-based graft-versus-host disease (GVHD) prophylaxis is standard in allogeneic hematopoietic stem cell transplantation (HCT) but fails to induce long-term tolerance without chronic GVHD (cGVHD) in a considerable number of patients. In this study, we addressed this long-standing question in mouse models of HCT. After HCT, alloreactive donor T cells rapidly differentiated into PD-1+ TIGIT+ terminally exhausted T cells (terminal Tex). GVHD prophylaxis with cyclosporine (CSP) suppressed donor T-cell expression of TOX, a master regulator to promote differentiation of transitory exhausted T cells (transitory Tex), expressing both inhibitory receptors and effector molecules, into terminal Tex, and inhibited tolerance induction. Adoptive transfer of transitory Tex, but not terminal Tex, into secondary recipients developed cGVHD. Transitory Tex maintained alloreactivity and thus PD-1 blockade restored graft-versus-leukemia (GVL) activity of transitory Tex and not terminal Tex. In conclusion, CSP inhibits tolerance induction by suppressing the terminal exhaustion of donor T cells, while maintaining GVL effects to suppress leukemia relapse.

Preclinical safety evaluation of calcineurin inhibitors delivered through an intraductal route to prevent post-ERCP pancreatitis demonstrates endocrine and systemic safety.

OBJECTIVE: There is an urgent need for safe and targeted interventions to mitigate post-ERCP pancreatitis (PEP). Calcineurin inhibitors (CnIs) offer therapeutic promise as calcineurin signaling within acinar cells is a key initiating event in PEP. In previous proof-of-concept studies using experimental models, we showed that concurrent intra-pancreatic ductal administration of the CnIs, tacrolimus (Tac) or cyclosporine A (CsA) with the ERCP radiocontrast agent (RC) prevented PEP. To translate this finding clinically, we investigated potential toxic effects of intraductal delivery of a single-dose RC-CnI formulation on endocrine pancreas function and systemic toxicities in a preclinical PEP model. METHODS: C57BL/6J mice underwent ductal cannulation and received a single, intra-pancreatic ductal infusion of RC or RC with Tac or CsA (treatment groups) or underwent ductal cannulation without infusion ('sham' group). To assess endocrine function, intraperitoneal glucose tolerance test (IPGTT) was performed at two days before infusion and on day 2 and 14 post-surgery. To evaluate off-target tissue toxicities, renal and hepatic function-related parameters including blood urea nitrogen, plasma creatinine, potassium, aspartate aminotransferase, alanine aminotransferase, and total bilirubin were measured at the same time-points as IPGTT. Histological and biochemical indicators of pancreas injury and inflammation were also evaluated. RESULTS: No abnormalities in glucose metabolism, hepatic or renal function were observed on day 2 or 14 in mice administered with intraductal RC or RC with Tac or CsA. CONCLUSION: Intraductal delivery of RC-CnI formulation was safe and well-tolerated with no significant acute or subacute endocrine or systemic toxicities, underscoring its clinical utility to prevent PEP.

Salvage therapies of autoimmune hepatitis limit proinflammatory immune cells while sparing regulatory T cells.

BACKGROUND: Autoimmune hepatitis (AIH) can be clinically controlled by first-line immunosuppressive therapy in the majority of patients. However, a selective decrease in intrahepatic regulatory T cells (Treg) was observed with immunosuppressive therapy, which was even more pronounced in patients with incomplete responses than in patients who achieved biochemical remission. The effects of salvage therapies on the number of intrahepatic T and B cells, including Treg, are unclear. The hypothesis was that calcineurin inhibitors would further decrease intrahepatic Treg numbers, and the mammalian target of rapamycin inhibitors would increase intrahepatic Treg numbers. METHODS: In this retrospective study at 2 centers, CD4+, CD8+ and CD4+FOXP3+ T cells, and CD79a+ B cells were quantified in surveillance biopsies under non-standard-of-care treatment [non-SOC: calcineurin inhibitor (n=10), second-line antimetabolites (n=9), mammalian target of rapamycin inhibitors (n=4)] compared with patients under the standard-of-care treatment (SOC). RESULTS: Intrahepatic T-cell and B-cell counts were not significantly different between patients with biochemical remission under SOC and non-SOC. However, patients with incomplete response under non-SOC had significantly lower liver infiltration with T and B cells, whereas Treg were not reduced compared with SOC. This resulted in an even higher ratio of Treg to T and B cells in non-SOC compared with SOC when biochemical remission was not achieved. The different non-SOC regimens showed no significant difference in liver infiltration with T cells, including Treg and B cells. CONCLUSIONS: Non-SOC in AIH partially controls intrahepatic inflammation by limiting the hepatic infiltration of total T and B cells as the main drivers of inflammation without further decreasing intrahepatic Treg. A negative effect of calcineurin inhibitor and a positive effect of mammalian target of rapamycin inhibitors on the number of intrahepatic Treg was not observed.

Tacrolimus induces fibroblast-to-myofibroblast transition via a TGF-ß-dependent mechanism to contribute to renal fibrosis.

Use of immunosuppressant calcineurin inhibitors (CNIs) is limited by irreversible kidney damage, hallmarked by renal fibrosis. CNIs directly damage many renal cell types. Given the diverse renal cell populations, additional targeted cell types and signaling mechanisms warrant further investigation. We hypothesized that fibroblasts contribute to CNI-induced renal fibrosis and propagate profibrotic effects via the transforming growth factor-ß (TGF-ß)/Smad signaling axis. To test this, kidney damage-resistant mice (C57BL/6) received tacrolimus (10 mg/kg) or vehicle for 21 days. Renal damage markers and signaling mediators were assessed. To investigate their role in renal damage, mouse renal fibroblasts were exposed to tacrolimus (1 nM) or vehicle for 24 h. Morphological and functional changes in addition to downstream signaling events were assessed. Tacrolimus-treated kidneys displayed evidence of renal fibrosis. Moreover, α-smooth muscle actin expression was significantly increased, suggesting the presence of fibroblast activation. TGF-ß receptor activation and downstream Smad2/3 signaling were also upregulated. Consistent with in vivo findings, tacrolimus-treated renal fibroblasts displayed a phenotypic switch known as fibroblast-to-myofibroblast transition (FMT), as α-smooth muscle actin, actin stress fibers, cell motility, and collagen type IV expression were significantly increased. These findings were accompanied by concomitant induction of TGF-ß signaling. Pharmacological inhibition of the downstream TGF-ß effector Smad3 attenuated tacrolimus-induced phenotypic changes. Collectively, these findings suggest that 1) tacrolimus inhibits the calcineurin/nuclear factor of activated T cells axis while inducing TGF-ß1 ligand secretion and receptor activation in renal fibroblasts; 2) aberrant TGF-ß receptor activation stimulates Smad-mediated production of myofibroblast markers, notable features of FMT; and 3) FMT contributes to extracellular matrix expansion in tacrolimus-induced renal fibrosis. These results incorporate renal fibroblasts into the growing list of CNI-targeted cell types and identify renal FMT as a process mediated via a TGF-ß-dependent mechanism.NEW & NOTEWORTHY Renal fibrosis, a detrimental feature of irreversible kidney damage, remains a sinister consequence of long-term calcineurin inhibitor (CNI) immunosuppressive therapy. Our study not only incorporates renal fibroblasts into the growing list of cell types negatively impacted by CNIs but also identifies renal fibroblast-to-myofibroblast transition as a process mediated via a TGF-ß-dependent mechanism. This insight will direct future studies investigating the feasibility of inhibiting TGF-ß signaling to maintain CNI-mediated immunosuppression while ultimately preserving kidney health.

Low-dose Pimecrolimus, an FDA-approved Calcineurin Inhibitor, Sensitizes Drug-resistant Cancer Cells via Strong P-gp Inhibition.

BACKGROUND/AIM: Co-treatment with calcineurin inhibitors, such as tacrolimus and cyclosporin A, can sensitize chemotherapy-resistant cancer cells with P-glycoprotein (P-gp)-over-expression. Pimecrolimus (PIME) is a clinically available calcineurin inhibitor with a structure similar to that of tacrolimus. Whether PIME can sensitize P-gp-over-expressing resistant cancer cells remains unclear. MATERIALS AND METHODS: Cell viability assay, annexin V analyses, cellular morphology and density observation with a microscope, western-blotting, fluorescence-activated cell sorting (FACS), and analysis for P-gp inhibitory activity were performed to investigate the mechanism of action. RESULTS: PIME exhibited strong cytotoxicity to vincristine (VIC)-treated drug-resistant cell lines (KBV20C and MCF-7/ADR) over-expressing P-gp. Co-treatment with VIC and PIME increased apoptosis and down-regulated the ERK signaling pathway, resulting in G2 arrest. PIME could be co-administered with vinorelbine or eribulin to sensitize resistant KBV20C or MCF-7/ADR cancer cells. Moreover, PIME strongly inhibited the efflux of both rhodamine 123 and calcein-AM substrates through P-gp after 4 h of treatment, indicating that VIC+PIME sensitized cancer cells by inhibiting VIC efflux via direct PIME binding to P-gp. Low doses of PIME, tacrolimus, and cyclosporin A showed similar sensitizing efficiencies in resistant KBV20C cells. These drugs showed similar P-gp inhibitory activities using both rhodamine 123 and calcein-AM substrates, suggesting that calcineurin inhibitors generally have strong P-gp inhibitory activities that sensitize drug-resistant cancer cells with P-gp over-expression. CONCLUSION: PIME, currently used in clinics, can be repositioned for treating patients with P-gp-over-expressing resistant cancer (stem) cells.

Calcineurin inhibitors stimulate Kir4.1/Kir5.1 of the distal convoluted tubule to increase NaCl cotransporter.

We examine whether calcineurin or protein phosphatase 2B (PP2B) regulates the basolateral inwardly rectifying potassium channel Kir4.1/Kir5.1 in the distal convoluted tubule (DCT). Application of tacrolimus (FK506) or cyclosporine A (CsA) increased whole-cell Kir4.1/Kir5.1-mediated K+ currents and hyperpolarized the DCT membrane. Moreover, FK506-induced stimulation of Kir4.1/Kir5.1 was absent in kidney tubule-specific 12 kDa FK506-binding protein-knockout mice (Ks-FKBP-12-KO). In contrast, CsA stimulated Kir4.1/Kir5.1 of the DCT in Ks-FKBP-12-KO mice, suggesting that FK506-induced stimulation of Kir4.1/Kir5.1 was due to inhibiting PP2B. Single-channel patch-clamp experiments demonstrated that FK506 or CsA stimulated the basolateral Kir4.1/Kir5.1 activity of the DCT, defined by NPo (a product of channel number and open probability). However, this effect was absent in the DCT treated with Src family protein tyrosine kinase (SFK) inhibitor or hydroxyl peroxide. Fluorescence imaging demonstrated that CsA treatment increased membrane staining intensity of Kir4.1 in the DCT of Kcnj10fl/fl mice. Moreover, CsA treatment had no obvious effect on phosphorylated NaCl cotransporter (pNCC) expression in Ks-Kir4.1-KO mice. Immunoblotting showed acute FK506 treatment increased pNCC expression in Kcnj10fl/fl mice, but this effect was attenuated in Ks-Kir4.1-KO mice. In vivo measurement of thiazide-induced renal Na+ excretion demonstrated that FK506 enhanced thiazide-induced natriuresis. This effect was absent in Ks-FKBP-12-KO mice and blunted in Ks-Kir4.1-KO mice. We conclude that inhibition of PP2B stimulates Kir4.1/Kir5.1 of the DCT and NCC and that PP2B inhibition-induced stimulation of NCC is partially achieved by stimulation of the basolateral Kir4.1/Kir5.1.

Shedding light on key pharmacological knowledge and strategies for pediatric atopic dermatitis.

INTRODUCTION: Atopic dermatitis (AD) is an inflammatory disease affecting over 20% of the pediatric population, with 85% of cases presenting before the age of five. Recently, therapeutic options in pediatric patients have evolved rapidly, following extensive development in adult treatments. AREAS COVERED: This review will encompass relevant molecular drivers, along with an overlook on treatment modalities in pediatric AD, as well as a summary of pipeline treatments in clinical trials for pediatric patients from PubMed, Google Scholar, and Clinicaltrials.gov up to July 2022. Topical corticosteroids are the mainstay for AD flares in adults and children. Topical approved agents in pediatric AD are calcineurin inhibitors, crisaborolecrisaborole, and ruxolitinib. Dupilumab is the only FDA approved biologic for patients with AD from six months of age. A Janus kinase inhibitor, upadacitinib, is a systemic treatment approved for pediatric AD patients (age >12 years). Systemic immunosuppressants used in pediatric AD include methotrexate, azathioprine, cyclosporinecyclosporine, and mycophenolate mofetil. EXPERT OPINION: Data regarding disease prevention are conflicting, however, an abundance of research has transpired regarding amelioration of symptoms and induction of disease clearance by targeting numerous pathological mechanisms. Understanding the pediatric AD phenotype will further advance the field and the development of improved therapeutics.

Tacrolimus inhibits stress responses and hyphal formation via the calcineurin signaling pathway in Trichosporon asahii.

The pathogenic fungus Trichosporon asahii causes fatal deep-seated mycosis in immunocompromised patients. Calcineurin, which is widely conserved in eukaryotes, regulates cell growth and various stress responses in fungi. Tacrolimus (FK506), a calcineurin inhibitor, induces sensitivity to compounds that cause stress on the cell membrane and cell wall integrity. In this study, we demonstrated that FK506 affects stress responses and hyphal formation in T. asahii. In silico structural analysis revealed that amino acid residues in the binding site of the calcineurin-FKBP12 complex that interact with FK506 are conserved in T. asahii. The growth of T. asahii was delayed by FK506 in the presence of SDS or Congo red but not in the presence of calcium chloride. FK506 also inhibited hyphal formation in T. asahii. A mutant deficient of the cnb gene, which encodes the regulatory subunit B of calcineurin, exhibited stress sensitivities on exposure to SDS and Congo red and reduced the hyphal forming ability of T. asahii. In the cnb-deficient mutant, FK506 did not increase the stress sensitivity or reduce hyphal forming ability. These results suggest that FK506 affects stress responses and hyphal formation in T. asahii via the calcineurin signaling pathway.

Pharmacodynamic Monitoring of Ciclosporin and Tacrolimus: Insights From Nuclear Factor of Activated T-Cell-Regulated Gene Expression in Healthy Volunteers.

BACKGROUND: Although therapeutic drug monitoring of calcineurin inhibitor (CNI) concentrations is performed routinely in clinical practice, an identical concentration may lead to different effects in different patients. Although the quantification of nuclear factor of activated T-cell-regulated gene expression (NFAT-RGE) is a promising method for measuring individual CNI effects, CNI pharmacodynamics are as of yet incompletely understood. METHODS: CNI concentrations and NFAT-RGEs were quantified in 24 healthy volunteers receiving either ciclosporin or tacrolimus in 2 clinical trials. NFAT-RGE was measured using quantitative reverse transcription polymerase chain reaction tests of whole-blood samples. Pharmacokinetics and pharmacodynamics were analyzed using compartmental modeling and simulation. In addition, NFAT-RGE data from renal transplant patients were analyzed. RESULTS: The average NFAT-RGE during a dose interval was reduced to approximately 50% with ciclosporin, considering circadian changes. The different effect-time course with ciclosporin and tacrolimus could be explained by differences in potency (IC 50 204 ± 41 versus 15.1 ± 3.2 mcg/L, P < 0.001) and pharmacokinetics. Residual NFAT-RGE at the time of maximum concentration (RGE tmax ) of 15% when using ciclosporin and of 30% when using tacrolimus was associated with similar average NFAT-RGEs during a dose interval. Renal transplant patients had similar but slightly stronger effects compared with healthy volunteers. CONCLUSIONS: Ciclosporin and tacrolimus led to similar average suppression of NFAT-RGE in a dose interval, despite considerably different RGE tmax . Pharmacodynamic monitoring of average NFAT-RGE should be considered. When using NFAT-RGE at specific time points, the different effect-time courses and circadian changes of NFAT-RGEs should be considered.

Targeting inflammation and immune activation to improve CTLA4-Ig-based modulation of transplant rejection.

For the last few decades, Calcineurin inhibitors (CNI)-based therapy has been the pillar of immunosuppression for prevention of organ transplant rejection. However, despite exerting effective control of acute rejection in the first year post-transplant, prolonged CNI use is associated with significant side effects and is not well suited for long term allograft survival. The implementation of Costimulation Blockade (CoB) therapies, based on the interruption of T cell costimulatory signals as strategy to control allo-responses, has proven potential for better management of transplant recipients compared to CNI-based therapies. The use of the biologic cytotoxic T-lymphocyte associated protein 4 (CTLA4)-Ig is the most successful approach to date in this arena. Following evaluation of the BENEFIT trials, Belatacept, a high-affinity version of CTLA4-Ig, has been FDA approved for use in kidney transplant recipients. Despite its benefits, the use of CTLA4-Ig as a monotherapy has proved to be insufficient to induce long-term allograft acceptance in several settings. Multiple studies have demonstrated that events that induce an acute inflammatory response with the consequent release of proinflammatory cytokines, and an abundance of allograft-reactive memory cells in the recipient, can prevent the induction of or break established immunomodulation induced with CoB regimens. This review highlights advances in our understanding of the factors and mechanisms that limit CoB regimens efficacy. We also discuss recent successes in experimentally designing complementary therapies that favor CTLA4-Ig effect, affording a better control of transplant rejection and supporting their clinical applicability.

Sirolimus Attenuates Calcineurin Inhibitor-Induced Epithelial-Mesenchymal Transition in Hepatocellular Carcinoma.

BACKGROUND: Calcineurin inhibitors (CNIs), which are potent immunosuppressants (ISs), increase the risk for hepatocellular carcinoma (HCC) recurrence after liver transplantation (LTx). Epithelial-mesenchymal transition (EMT) is a key process in which epithelial cancer cells lose their polarity, resulting in cancer progression and metastasis. The aim of this study was to evaluate the effect of sirolimus (SRL) individually and in combination with other ISs to reduce EMT. METHODS: HCC SK-Hep1 cells were used and various ISs (SRL, tacrolimus, cyclosporine A, or mycophenolate mofetil) were administered at 2 dosages and in combination therapies. Mice were transplanted with SK-Hep1 cells (in the liver) and were monitored after 2 weeks. RESULTS: The in vitro treatment with SRL showed a dose-dependent attenuation of cell proliferation and migration in case of the individual and IS combination treatments; further, decreased levels of pro-EMT proteins, namely, N-cadherin, transforming growth factor-ß, ZEB1, Slug, and Snail were observed. In contrast, E-cadherin expression was upregulated after both the individual and IS combination treatments. These results were also observed in the samples from mice transplanted with the SK-Hep1 cells. CONCLUSION: The present study demonstrated that SRL reduced HCC metastasis by inhibiting EMT. Thus, our findings provide a rationale for the use of SRL in combination with ISs in HCC LTx patients.

IL-2 receptor engineering enhances regulatory T cell function suppressed by calcineurin inhibitor.

Clinical trials utilizing regulatory T cell (Treg) therapy in organ transplantation have shown promising results, however, the choice of a standard immunosuppressive regimen is still controversial. Calcineurin inhibitors (CNIs) are one of the most common immunosuppressants for organ transplantation, although they may negatively affect Tregs by inhibiting IL-2 production by conventional T cells. As a strategy to replace IL-2 signaling selectively in Tregs, we have introduced an engineered orthogonal IL-2 (ortho IL-2) cytokine/cytokine receptor (R) pair that specifically binds with each other but does not bind with their wild-type counterparts. Murine Tregs were isolated from recipients and retrovirally transduced with ortho IL-2Rß during ex vivo expansion. Transduced Tregs (ortho Tregs) were transferred into recipient mice in a mixed hematopoietic chimerism model with tacrolimus administration. Ortho IL-2 treatment significantly increased the ortho IL-2Rß(+) Treg population in the presence of tacrolimus without stimulating other T cell subsets. All the mice treated with tacrolimus plus ortho IL-2 achieved heart allograft tolerance, even after tacrolimus cessation, whereas those receiving tacrolimus treatment alone did not. These data demonstrate that Treg therapy can be adopted into a CNI-based regimen by utilizing cytokine receptor engineering.