Administration Of FK506 Research Articles

Effects of immunosuppression after limb fracture in mice on nociceptive, cognitive, and anxiety-related outcomes.

Chronic pain is a common and problematic consequence of injuries with few proven methods for prevention or treatment. In addition to pain, functional limitations and neuropsychiatric changes such as cognitive impairment and anxiety worsen outcomes. To determine whether inhibiting activation of the adaptive immune response after limb fracture would reduce pain, functional loss, memory changes, and anxiety. These experiments used a murine tibial fracture/cast immobilization model that develops these adverse outcomes. Adaptive immunity was blocked using the immunosuppressant FK506 beginning at the time of fracture. The administration of FK506 reduced mechanical allodynia and hind limb unweighting for weeks after cast removal as well as nonevoked pain measures. Fracture was associated with working memory loss in the Y-maze assay in vehicle- but not FK506-treated mice. Object recognition memory was not improved with FK506 after fracture. Also, vehicle- but not FK506-treated mice developed an anxiety phenotype. Impaired running wheel performance after cast removal over the following 2 weeks was not improved with FK506 administration. In addition, FK506 treatment blocked Immunoglobulin M (IgM) accumulation in the skin of the fractured limbs, and hippocampal enhancement of matrix metalloproteinase-8 expression, a metalloproteinase associated with neuroplastic changes after injuries, was completely blocked. Taken together, our results show that blocking the adaptive immune response after limb trauma reduces the severity of nociceptive and biological changes. The same treatment may reduce the adverse consequences of anxiety and memory deficits using some measures, but other measures of memory are not affected, and activity is not enhanced.

Relationship Between Dysbiotic Wound Microbiota and Critical Colonization: Involvement of FOXP3-Positive Cells in Rats.

Detection of critical colonization is gaining importance in wound management, but its pathophysiology remains unclear. We previously clarified that a dysbiotic wound microbiota differing from skin commensal microbiota may be involved in critical colonization and that such wounds contain fewer Forkhead box protein P3 (FOXP3)-positive cells in the tissue. However, it is not clear whether FOXP3-positive cells contribute to the development of critical colonization. Here, we examined whether inhibition of FOXP3-positive cell could induce critical colonization when the commensal microbiota was present in the wounds. Sprague-Dawley rats were administered FK506 or vehicle to inhibit differentiation into FOXP3-positive cells. Full-thickness wounds were made on the dorsal skin and inoculated with bacterial solution (dysbiosis group) or Luria-Bertani medium (commensal group). A bacterial solution was prepared by anaerobically culturing bacteria from the skin of donor rats on an artificial dermis in Luria-Bertani medium for 72 hours. Tissues were collected on day 4 postwounding for histological evaluation. After microbiota transplantation, excessive inflammation occurred in the FK506 + commensal group. In contrast, wounds with transplanted dysbiotic microbiota showed the same level of neutrophil infiltration, regardless of FK506 administration. Furthermore, the wound area was larger in the FK506 + commensal group than in the vehicle + commensal group on day 4 postwounding (P = 0.01). This area was also significantly larger in both the vehicle + dysbiosis (P = 0.01) and FK506 + dysbiosis groups (P = 0.03) than in the vehicle + commensal group. This study has shown that dysbiosis may be at least related to developing critical colonization, and the results suggest that FOXP3-positive cells are involved in this process. Our study may contribute to establishing new interventions that prevent critical colonization by correcting wound microbiota.

NFAT3-FasL axis synchronously regulates apoptosis and necroptosis in murine cochlear outer hair cells after noise trauma.

Existing studies have indicated that noise induces apoptosis and necroptosis in cochlear outer hair cells (OHCs). However, the role of the extrinsic cell death pathway, initiated by death ligands in the cochlea, remains unknown. In this study, we hypothesized that noise could induce the NFAT3/FasL axis-mediated extrinsic death pathway in the cochlea. We found that NFAT3/FasL signaling was silent in normal OHCs. Noise exposure induced apoptosis and necroptosis in OHCs with specifically high FasL expression. Multiplex immunofluorescence staining revealed that NFAT3 nuclear translocation and FasL upregulation were colocalized in the apoptotic and necroptotic OHCs following noise trauma. Administration of FK506 or 11R-vivit (an specific NFAT inhibitor) blocked NFAT3 nuclear translocation, inhibited FasL expression, mitigated apoptosis and necroptosis, and protected against noise-induced hearing loss (NIHL). Finally, FasL knockdown by delivering siRNA intratympanically attenuated apoptosis and necroptosis in OHCs and alleviated NIHL, confirming the role of FasL in OHC death. Collectively, our study demonstrates that the NFAT3/FasL axis mediates noise-induced extrinsic death pathway in OHCs, leading to their apoptosis and necroptosis.

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/GluA2heteromers 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/GluA2heteromers 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/GluA2heterotetramers in the PVN. Blocking Ca2+-permeable AMPARs or α2δ-1-AMPAR interactions in the PVN attenuates sympathetic outflow augmented by the calcineurin inhibitor.

Nerve Wrap for Local Delivery of FK506/Tacrolimus Accelerates Nerve Regeneration.

Peripheral nerve injuries (PNIs) occur frequently and can lead to devastating and permanent sensory and motor function disabilities. Systemic tacrolimus (FK506) administration has been shown to hasten recovery and improve functional outcomes after PNI repair. Unfortunately, high systemic levels of FK506 can result in adverse side effects. The localized administration of FK506 could provide the neuroregenerative benefits of FK506 while avoiding systemic, off-target side effects. This study investigates the utility of a novel FK506-impregnated polyester urethane urea (PEUU) nerve wrap to treat PNI in a previously validated rat infraorbital nerve (ION) transection and repair model. ION function was assessed by microelectrode recordings of trigeminal ganglion cells responding to controlled vibrissae deflections in ION-transected and -repaired animals, with and without the nerve wrap. Peristimulus time histograms (PSTHs) having 1 ms bins were constructed from spike times of individual single units. Responses to stimulus onsets (ON responses) were calculated during a 20 ms period beginning 1 ms after deflection onset; this epoch captures the initial, transient phase of the whisker-evoked response. Compared to no-wrap controls, rats with PEUU-FK506 wraps functionally recovered earlier, displaying larger response magnitudes. With nerve wrap treatment, FK506 blood levels up to six weeks were measured nearly at the limit of quantification (LOQ ≥ 2.0 ng/mL); whereas the drug concentrations within the ION and muscle were much higher, demonstrating the local delivery of FK506 to treat PNI. An immunohistological assessment of ION showed increased myelin expression for animals assigned to neurorrhaphy with PEUU-FK506 treatment compared to untreated or systemic-FK506-treated animals, suggesting that improved PNI outcomes using PEUU-FK506 is mediated by the modulation of Schwann cell activity.

Harnessing IGF-1 and IL-2 as biomarkers for calcineurin activity to tailor optimal FK506 dosage in α-synucleinopathies.

Introduction: Rise in Calcium (Ca2+) and hyperactive Ca2+-dependent phosphatase calcineurin represent two key determinants of a-synuclein (a-syn) pathobiology implicated in Parkinson's Disease (PD) and other neurodegenerative diseases. Calcineurin activity can be inhibited with FK506, a Food and Drug Administration (FDA)-approved compound. Our previous work demonstrated a protective effect of low doses of FK506 against a-syn pathology in various models of a-syn related pathobiology. Methods: Control and a-syn-expressing mice (12-18 months old) were injected with vehicle or two single doses of FK506 administered 4 days apart. Cerebral cortex and serum from these mice were collected and assayed using a meso scale discovery quickplex SQ 120 for cytokines and Enzyme-linked immunosorbent assay for IGF-1. Results: In this study we present evidence that reducing calcineurin activity with FK506 in a-syn transgenic mice increased insulin growth factor (IGF-1), while simultaneously decreasing IL-2 levels in both cerebral cortex and serum. Discussion: The highly conserved Ca2+/calcineurin signaling pathway is known to be affected in a-syn-dependent human disease. FK506, an already approved drug for other uses, exhibits high brain penetrance and a proven safety profile. IL-2 and IGF-1 are produced throughout life and can be measured using standard clinical methods. Our findings provide two potential biomarkers that could guide a clinical trial of FK506 in PD patients, without posing significant logistical or regulatory challenges.

Microneedle-mediated transdermal delivery of FK506 for treatment of acute cardiac allograft rejection

FK506 has been widely used to prevent acute rejection in heart transplantation (HT). However, its strong hydrophobicity, poor bioavailability, high pharmacokinetic variability, and narrow therapeutic window are obstacles to its application in HT. It has been demonstrated that localized and sustained immunosuppressant delivery may be a suitable route for FK506 administration. Hence, the objective of this study was to evaluate the efficacy of a FK506-loaded microneedle (FK506 MN) in the treatment of acute rejection in HT. The physicochemical properties of FK506 MN were evaluated, and it exhibited constant release over the course of at least 9 days with a 94.6 % cumulative release rate. Also, the FK506 MN treatment could successfully alleviate acute rejection and prolong allograft survival compared with the untreated group (mean survival time, 12.33 versus 7.25 days). Furthermore, T cell and macrophage infiltration as well as secretion of IL-2, IL-6 and IFN-γ were dramatically reduced in the FK506 MN group. As expected, no hepatoxicity and nephrotoxicity were observed after the application of FK506 MN. Taken together, FK506 MN based drug delivery system provides a novelty strategy to deliver FK506 in heart transplantation, which may be also suitable for other organ transplantations in the future.

Therapeutic strategies for peripheral nerve injuries: FK506 and electrostimulation

Efficacious therapeutics for peripheral nerve injuries remain incompletely described in the literature. However, over the last several decades, delivery of FK506 (Tacrolimus) and electrostimulation have demonstrated great promise for supplementing surgical advances in treating peripheral nerve injuries. This review describes the discovery, mechanistic investigations, and clinical translation of these strategies to promote functional recovery. FK506 has demonstrated the ability to increase the regeneration rate after nerve injury by a variety of hypothesized mechanisms, yet clinical utility remains limited due to systemic immunosuppression. Local administration of FK506 continues to be an active area of inquiry for minimizing side effects while maintaining its neuroregenerative effects. Electrostimulation of a nerve proximal to the site of surgical nerve repair has demonstrated increased axonal regeneration and accelerated recovery of both motor and sensory nerves. In addition, electrostimulation also appears to improve axon matching during reinnervation from motor to motor and sensory to sensory pathways and is used clinically in our surgeries. However, the specific parameters to best incorporate electrostimulation into the operating theater are still evolving. Utilizing translational rodent and murine models, surgical techniques and these therapeutic strategies have gradually become more viable as safety profiles and mechanisms are gradually understood. This review presents the state of the field for these therapeutic avenues and discusses further areas of research.

Therapeutic strategies for peripheral nerve injuries: FK506 and electrostimulation

Efficacious therapeutics for peripheral nerve injuries remain incompletely described in the literature. However, over the last several decades, delivery of FK506 (Tacrolimus) and electrostimulation have demonstrated great promise for supplementing surgical advances in treating peripheral nerve injuries. This review describes the discovery, mechanistic investigations, and clinical translation of these strategies to promote functional recovery. FK506 has demonstrated the ability to increase the regeneration rate after nerve injury by a variety of hypothesized mechanisms, yet clinical utility remains limited due to systemic immunosuppression. Local administration of FK506 continues to be an active area of inquiry for minimizing side effects while maintaining its neuroregenerative effects. Electrostimulation of a nerve proximal to the site of surgical nerve repair has demonstrated increased axonal regeneration and accelerated recovery of both motor and sensory nerves. In addition, electrostimulation also appears to improve axon matching during reinnervation from motor to motor and sensory to sensory pathways and is used clinically in our surgeries. However, the specific parameters to best incorporate electrostimulation into the operating theater are still evolving. Utilizing translational rodent and murine models, surgical techniques and these therapeutic strategies have gradually become more viable as safety profiles and mechanisms are gradually understood. This review presents the state of the field for these therapeutic avenues and discusses further areas of research.

#6635 ARNT AND ALK3 MEDIATE RENO PROTECTION AFTER ISCHEMIC PRECONDITIONING

Abstract Background and Aims Ischemia and following hypoxia are a common cause of acute kidney injury (AKI). Strategies for prevention and treatment of AKI are limited. In several animal models, ischemic preconditioning (IPC) has been established as an effective intervention to prevent ischemic organ injury. An IPC protocol of ischemia and reperfusion mediates organ protection for a few hours. This first phase of organ protection is followed by a second, protective phase occurring after 24–48 h. The second phase, named second window of protection (SWOP), persists for 3–4 d. Mechanisms and transcriptional mediators during SWOP are mostly unknown. The aim of the underlying study was to investigate effectors and target genes during SWOP and to identify a potential pharmacological therapeutic target. Method 6–8-week-old C57BL/6 mice were undergoing an IPC intervention; five minutes of renal sinistra artery ischemia was followed by five minutes of reperfusion. This cycle was performed five times. Mice were sacrificed 2, 4, 8, 12, 25, 48, 72 h after IPC. Two additional groups of mice were pharmacologically preconditioned for 48 h with low dose FK506 and small molecule GPI1046. Analysis of murine kidneys were performed by SDS-Page and Western blot. Results The first phase after IPC (2–4 h) was characterized by an increasing expression of hypoxia inducible factor-1α (HIF1α); 48 h after intervention a significant increase of expression of aryl hydrocarbon receptor nuclear translocator (ARNT) and consecutive activin receptor-like kinase 3 (ALK3) was observed. Comparable induction of ARNT and ALK3 expression was obtained by pharmacological preconditioning with FK506 or GPI1046. Conclusion Affected by hypoxia, HIF1α activates transcriptional upregulation of ARNT 48 h after IPC. The increasing expression of ARNT, follows an increased ARNT homodimerization. ARNT homodimer binds to the palindromic E-box 5′-CACGTG-3′ of the proximal ALK3 promoter. By this it induces the expression of the renoprotective ALK3 during SWOP. The organ-protective effects of constitutively expressed ARNT during SWOP can be mimicked pharmacologically by administration of FK506 or GPI1046 and may be used as a future therapeutic target.

Drug suspending during wound healing effectively weakens immunosuppression-related complications by preserving CD8+ T cell function.

Immunosuppressive medications, which interfere with the activation and proliferation of T and B cells, increase the risk of wound healing complications. To address it, this study aimed to validate the feasibility of drug suspending during wound healing, whilst exploring the mechanisms exerted by T cells, which are important in the wound healing process. For this, a mouse skin wound model was set up. Tacrolimus (FK506) and fingolimod (FTY720) were both administered intraperitoneally prior to wounding to inhibit the T cell activation and migration, respectively. Flow-cytometric analysis subsequently revealed the functional T cell subtypes detected during the healing process. A CD8a antibody was also administered to deplete CD8+ T cells in vivo to verify their specific function. It was found that FK506 or FTY720 administration delayed the early phase of wound healing by reducing collagen production, which was also supported by the downregulation of col1a1, col3a1 and tgfb1. However, there was no significant difference in the total healing period. Both spleen- and skin-derived CD8+ T cells were proliferated and activated after injury without intervention, whereas CD4+ T cells showed no significant changes. Furthermore, selectively depleting CD8+ T cells retarded the healing process by downregulating collagen production-associated genes (col1a1, col3a1, tgfβ1 and en1) and proteins (collagen type 1 and 3). In addition, the CD8a antibody decreased the expression of genes lta, tnfa, il13 and il13ra, and protein interleukin-13Rα. In conclusion, suspending immunosuppressive drugs during wound healing was shown to be feasible through restraining the migration of activated T cells. CD8+ T cells represented the primary functional subtype positively associated with wound healing.

Chronic FK506 treatment reduces amyloid β and tau levels in the hippocampus and cortex of 3xTg‐AD mice

AbstractBackgroundAlzheimer’s disease (AD) is the most common form of neurodegenerative dementia. While major histopathological hallmarks of AD are amyloid‐β (Aβ) plaques and neurofibrillary tau tangles, soluble oligomeric forms of Aβ and tau (TauO) have been identified as the most neurotoxic species involved in synaptic degeneration. Previous studies associate alterations of the phosphatase calcineurin (CaN) to AβO‐driven toxicity. We previously have shown that CaN mediates both the neurotoxic and cognitive effects of AβO and elevated CaN levels have been also shown in AD patients, suggesting a central role of CaN in AD onset and/or clinical progression. Our previous studies in mouse models demonstrated that an acute treatment with the CaN inhibitor FK506/Tacrolimus in Tg2576 acutely injected intracerebroventricularly with AβO restores memory function in these cognitively impaired animals. Moreover, we reported absence of AD in aging human chronically treated with KF506 following solid organ transplant. To deeper investigate the involvement of CaN in AD onset and progression, we used a 3xTgAD mouse model presenting both Aβ and Tau aggregates.Method7 month‐old 3xTg‐AD mice were chronically treated for two weeks with intraperitoneal administration of either FK506 (1mg/kg) or PBS. Using immunofluorescence and western blot analyses we analyzed the levels of CaN, total Tau and Aβ in hippocampal (dentate gyrus‐DG, CA1 and CA3) and cortical areas (frontal and parietal‐occipital cortex) in our experimental groups.ResultWe found significantly decreased levels of CaN in DG, CA1 and CA3 and cortical areas in FK506 treated 3xTg‐AD mice as compared to 3xTg‐AD mice injected with PBS. Furthermore, performing double staining for Aβ and total Tau, FK506 treated mice displayed significant lower levels of Aβ and Tau in all the considered areas as compared to PBS‐treated 3xTg‐AD mice. These data suggest that the treatment with FK506 might trigger some mechanisms involved in the elimination of Tau and Aβ, i.e. autophagy.ConclusionOur data confirm the role of CaN as a key player in AD. The reduced key AD pathology following CaN inhibition with FK506 suggests the use of an FDA approved drug as a potential treatment with rapid clinical translation to slow the progression of the disease.

Electrical stimulation or tacrolimus (FK506) alone enhances nerve regeneration and recovery after nerve surgery, while dual use reduces variance and combines strengths of each in promoting enhanced outcomes.

Repaired nerve injuries can fail to achieve functional recovery. Therapeutic options beyond surgery, such as systemic tacrolimus (FK506) and electrical stimulation (E-stim), can improve recovery. We tested whether dual administration of FK506 and E-stim enhances regeneration and recovery more than either therapeutic alone. Rats were randomized to four groups: E-stim, FK506, FK506 + E-stim, and repair alone. All groups underwent tibial nerve transection and repair. Two sets of animals were created to measure outcomes of early nerve regeneration using nerve histology (n=36) and functional recovery (n=42) (21- and 42-day endpoints, respectively). Functional recovery was measured by behavioral analyses (walking track and grid walk) and, at the endpoint, muscle mass and force. Dual E-stim and FK506 administration produced histomorphometric measurements of nerve regeneration no different than either therapeutic alone. All treatments were superior to repair alone (FK506, P < .0001; E-stim, P < .05; FK506 + E-stim, P < .05). The E-stim and FK506 + E-stim groups had improved behavioral recovery compared with repair alone (at 6 weeks: E-stim, P < .05; FK506 + E-stim, P < .01). The FK506 group had improved recovery based on walking-track analysis (at 6 weeks: P < .001) and muscle force and mass (P < .05). The concurrent use of both therapies ensured earlier functional recovery and decreased variability in functional outcomes compared with either therapy alone, suggesting a moderate benefit. Dual administration of FK506 and E-stim showed minimal additive effects to further improve regeneration or recovery compared with either therapy alone. The data suggest the combination of FK506 and E-stim appears to combine the relative strengths of each therapeutic.

Treatment with hepatocyte transplantation in a novel mouse model of persistent liver failure

Background and AimCell-based transplantation therapy using hepatocytes, hepatic stem cells, hepatocyte-like cells induced from stem cells, etc. is thought to be an attractive alternative to liver transplantation, and have been studied to date. For its clinical application, however, it is extremely important to develop a model that reproduces the pathological conditions with indication for treatment and enables the study for the ideal treatment strategy. MethodsThe transgenic mice which express the thymidine kinase (TK) gene of human herpes simplex virus (HSV) in their hepatocytes with normal immunity has been developed (designated as HSVtk). After ganciclovir (GCV) administration which injure TK-expressing hepatocytes, the primary hepatocytes (PHs) isolated from green fluorescent protein (GFP) transgenic mouse (GFP-tg) were transplanted to HSVtk intrasplenically, and replacement index (RI) with transplanted PHs in the liver, liver histology, and mRNA expressions in the liver were analyzed up to 8 weeks after transplantation. ResultsHSVtk without PH transplantation after GCV administration developed persistent liver failure with degenerated hepatocytes, persistent elevation of ALT and hepatic p16 mRNA levels, suggesting the existence of cellular senescence in the base of the disease. When autologous GFP-PHs were transplanted to HSVtk, the transplanted cells were successfully engrafted in the liver. Eight weeks after transplantation, serum ALT levels and liver histology were almost normalized, while RIs varied from 19.8 to 73.8%. Since the hepatic p16 mRNA levels were decreased significantly in these mice, the senescence of hepatocytes associated with liver injury was thought to be resolved. On the other hand, allogenic GFP-PHs transplanted to HSVtk were eliminated as early as 1 week after transplantation. In these mice, hepatic p16 mRNA levels were significantly increased at 8 weeks after transplantation, suggesting the aggravation of hepatocyte senescence. FK506 administration to HSVtk protected the transplanted hepatocytes with allogenic background from rejection at 2 weeks after transplantation, but the condition of mice and the senescent status in the liver seemed worsened. ConclusionsThe mouse model with HSVtk/GCV system was useful for studying the mechanism of liver regeneration and the immune rejection responses in the hepatocyte transplantation treatment. It may also be utilized to develop the effective remedies to avoid immune rejection.

FK506 increases susceptibility to musculoskeletal infection in a rodent model

BackgroundDelayed fracture healing caused by soft tissue loss can be resolved by the administration of a Th1 immunosuppressant, such as FK506. Additionally, open fractures are at high risk for infection. We hypothesized that the inclusion of an immunosuppressant to a subject at risk for a musculoskeletal infection will increase the likelihood of infection.MethodsA rat model of musculoskeletal infection was used. Sprague Dawley rats received a stabilized femur defect and were inoculated with 104 CFU Staphylococcus aureus via a collagen matrix. Six hours after inoculation, the wounds were debrided of collagen and devitalized tissue and irrigated with sterile saline. The animals were randomized into two groups: carrier control and FK506, which were administered daily for 14 days and were euthanized and the tissues harvested to measure local bioburden.ResultsThe dosing regimen of FK506 that restored bone healing increased the bioburden in the bone and on the fixation implant compared to the carrier control animals. As expected, the administration of FK506 decreased circulating white blood cells, lymphocytes, neutrophils, and monocytes. Additionally, the red blood cell count, hematocrit, and body weight were lower in those animals that received FK506 compared to carrier control.ConclusionsFK506 administration decreased the systemic immune cell counts and increased the bacterial bioburden within a model of musculoskeletal infection. Collectively, these outcomes could be attributed to the overall T cell suppression by FK506 and the altered antimicrobial activity of innate cells, thereby allowing S. aureus to thrive and subsequently leading to infection of severe, musculoskeletal injuries. These observations reveal the crucial continued investigation for the clinical use of FK506, and other immunosuppressant compounds, in trauma patients who are at increased risk of developing infections.

Calcineurin Controls Hypothalamic NMDA Receptor Activity and Sympathetic Outflow.

Hypertension is a common and serious adverse effect of calcineurin inhibitors, including cyclosporine and tacrolimus (FK506). Although increased sympathetic nerve discharges are associated with calcineurin inhibitor-induced hypertension, the sources of excess sympathetic outflow and underlying mechanisms remain elusive. Calcineurin (protein phosphatase-2B) is broadly expressed in the brain, including the paraventricular nuclear (PVN) of the hypothalamus, which is critically involved in regulating sympathetic vasomotor tone. We determined whether prolonged treatment with the calcineurin inhibitor causes elevated sympathetic output and persistent hypertension by potentiating synaptic N-methyl-D-aspartate (NMDA) receptor activity in the PVN. Telemetry recordings showed that systemic administration of FK506 (3 mg/kg per day) for 14 days caused a gradual and profound increase in arterial blood pressure in rats, which lasted at least 7 days after discontinuing FK506 treatment. Correspondingly, systemic treatment with FK506 markedly reduced calcineurin activity in the PVN and circumventricular organs, but not rostral ventrolateral medulla, and increased the phosphorylation level and synaptic trafficking of NMDA receptors in the PVN. Immunocytochemistry labeling showed that calcineurin was expressed in presympathetic neurons in the PVN. Whole-cell patch-clamp recordings in brain slices revealed that treatment with FK506 increased baseline firing activity of PVN presympathetic neurons; this increase was blocked by the NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist. Also, treatment with FK506 markedly increased presynaptic and postsynaptic NMDA receptor activity of PVN presympathetic neurons. Furthermore, microinjection of the NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist into the PVN of anesthetized rats preferentially attenuated renal sympathetic nerve discharges and blood pressure elevated by FK506 treatment. In addition, systemic administration of memantine, a clinically used NMDA receptor antagonist, effectively attenuated FK506 treatment-induced hypertension in conscious rats. Our findings reveal that normal calcineurin activity in the PVN constitutively restricts sympathetic vasomotor tone via suppressing NMDA receptor activity, which may be targeted for treating calcineurin inhibitor-induced hypertension.

Targeting calcineurin induces cardiomyocyte proliferation in adult mice.

The mammalian neonatal heart can regenerate for 1 week after birth, after which, the majority of cardiomyocytes exit the cell cycle. Recent studies demonstrated that calcineurin mediates cell-cycle arrest of postnatal cardiomyocytes, partly through induction of nuclear translocation of the transcription factor Hoxb13 (a cofactor of Meis1). Here we show that inducible cardiomyocyte-specific deletion of calcineurin B1 in adult cardiomyocytes markedly decreases cardiomyocyte size and promotes mitotic entry, resulting in increased total cardiomyocyte number and improved left ventricular (LV) systolic function after myocardial infarction (MI). Similarly, pharmacological inhibition of calcineurin activity using FK506 promotes cardiomyocyte proliferation in vivo and increases cardiomyocyte number; however, FK506 administration after MI in mice failed to improve LV systolic function, possibly due to inhibition of vasculogenesis and blunting of the post-MI inflammatory response. Collectively, our results demonstrate that loss of calcineurin activity in adult cardiomyocytes promotes cell cycle entry; however, the effects of the calcineurin inhibitor FK506 on other cell types preclude a significant improvement of LV systolic function after MI.

FK506-loaded PLGA nanoparticles improve long-term survival of a vascularized composite allograft in a murine model.

BackgroundThe side effects of life-long administration of FK506 limit the clinical practice of vascularized composite allografts (VCAs). This study aimed to evaluate the feasibility of FK506-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles (FK506 NPs) for prolonging the long-term survival of VCAs and reducing the side effects of FK506.MethodsPLGA nanoparticles loaded with FK506 were prepared by the solvent evaporation method. The characterization of FK506 NPs was evaluated by electron microscopy. To confirm the function and safety of FK506 NPs, these particles were administrated into rats by intraperitoneal injection. The survival time of the allograft, systemic concentration of FK506, anti-rejection activity, and side-effect of FK506 NPs were evaluated in a Brown Norway (BN)-to-Sprague Dawley (SD) epigastric VCA transplantation model.ResultsCompared with the nontreatment, PLGA control and FK506 groups, the median survival times (MST) of the FK506 NP groups were significantly prolonged. The FK506 NPs could maintain therapeutic drug concentration for 60 days. Moreover, cytokine concentrations, flow cytometry of regulatory T cells (Tregs) and histopathology of allografts revealed significantly prolonged immunosuppression by FK506 NPs. FK506 NPs also ameliorated FK506 nephrotoxicity.ConclusionsFK506 NPs prolong the survival time of VCAs in a murine model with minimal nephrotoxicity, and provide a potential clinical strategy for ameliorating long-term side effects of immunosuppressive therapy.

Effect of immunosuppressants on a mouse model of osteogenesis imperfecta type V harboring a heterozygous Ifitm5 c.-14C\u2009>\u2009T mutation

Osteogenesis imperfecta (OI) type V is an autosomal dominant disorder caused by the c.-14C > T mutation in the interferon-induced transmembrane protein 5 gene (IFITM5), however, its onset mechanism remains unclear. In this study, heterozygous c.-14C > T mutant mice were developed to investigate the effect of immunosuppressants (FK506 and rapamycin) on OI type V. Among the mosaic mice generated by Crispr/Cas9-based technology, mice with less than 40% mosaic ratio of c.-14C > T mutation survived, whereas those with more than 48% mosaic ratio exhibited lethal skeletal abnormalities with one exception. All heterozygous mutants obtained by mating mosaic mice with wild-type mice exhibited a perinatal lethal phenotype due to severe skeletal abnormalities. Administration of FK506, a calcineurin inhibitor, in the heterozygous fetuses improved bone mineral content (BMC) of the neonates, although it did not save the neonates from the lethal effects of the mutation, whereas rapamycin, an mTOR inhibitor, reduced BMC, suggesting that mTOR signaling is involved in the bone mineralization of heterozygous mutants. These findings could clarify certain aspects of the onset mechanism of OI type V and enable development of therapeutics for this condition.

Administration of FK506 from Late Stage of Disease Prolongs Survival of Human Prion-Inoculated Mice

Human prion diseases are etiologically categorized into three forms: sporadic, genetic, and infectious. Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common type of human prion disease that manifests as subacute progressive dementia. No effective therapy for sCJD is currently available. Potential therapeutic compounds are frequently tested in rodents infected with mouse-adapted prions that differ from human prions. However, therapeutic effect varies depending on the prion strain, which is one of the reasons why candidate compounds have shown little effect in sCJD patients. We previously reported that intraperitoneal administration of FK506 was able to prolong the survival of mice infected with a mouse-adapted prion by suppressing the accumulation of abnormal prion protein (PrP) and inhibiting the activation of microglia. In this study, we tested oral administration of FK506 in knock-in mice expressing chimeric human prion protein (KiChM) that were infected with sCJD to determine if this compound is also effective against a clinically relevant human prion, i.e., one that has not been adapted to mice. Treatment with FK506, started either just before or just after disease onset, suppressed typical sCJD pathology (gliosis) and slightly but significantly prolonged the survival of sCJD-inoculated mice. It would be worthwhile to conduct a clinical trial using FK506, which has been safety-approved and is widely used as a mild immunosuppressant.