Tofacitinib for the treatment of refractory immune checkpoint inhibitor-associated immune nephritis.

Immune checkpoint inhibitor (ICI)-associated immune nephritis or acute interstitial nephritis (AIN) is one of the rare but known complication of ICI therapy. Guidelines recommend treatment of ICI-associated AIN with steroids, then TNF-alpha inhibitor infliximab. However, some cases are refractory to these therapies, potentially due to insufficient cytokine blockade. This is the first case where a 65-year-old female with metastatic lung adenocarcinoma, requiring high maintenance doses of steroids for immune nephritis was treated with tofacitinib, an oral Janus kinase (JAK) inhibitor. Tofacitinib enabled successful steroid tapering and might be a therapy option for refractory immune nephritis.

Tertiary lymphoid structure signatures are associated with immune checkpoint inhibitor related acute interstitial nephritis.

Tertiary lymphoid structures (TLSs) are associated with anti-tumor response following immune checkpoint inhibitor (ICI) therapy, but a commensurate observation of TLS is absent for immune related adverse events (irAEs) i.e. acute interstitial nephritis (AIN). We hypothesized that TLS-associated inflammatory gene signatures are present in AIN and performed NanoString-based gene expression and multiplex 12-chemokine profiling on paired kidney tissue, urine and plasma specimens of 36 participants who developed acute kidney injury (AKI) on ICI therapy: AIN (18), acute tubular necrosis (9), or HTN nephrosclerosis (9). Increased T and B cell scores, a Th1-CD8+ T cell axis accompanied by interferon-g and TNF superfamily signatures were detected in the ICI-AIN group. TLS signatures were significantly increased in AIN cases and supported by histopathological identification. Furthermore, urinary TLS signature scores correlated with ICI-AIN diagnosis but not paired plasma. Urinary CXCL9 correlated best to tissue CXCL9 expression (rho 0.75, p < 0.001) and the ability to discriminate AIN vs. non-AIN (AUC 0.781, p-value 0.003). For the first time, we report the presence of TLS signatures in irAEs, define distinctive immune signatures, identify chemokine markers distinguishing ICI-AIN from common AKI etiologies and demonstrate that urine chemokine markers may be used as a surrogate for ICI-AIN diagnoses.

Pharmacokinetics and pharmacodynamics of TTI-101, a STAT3 inhibitor that blocks muscle proteolysis in rats with chronic kidney disease.

Loss of muscle proteins increases the morbidity and mortality of patients with chronic kidney disease (CKD), and there are no reliable preventive treatments. We uncovered a STAT3/CCAAT-enhancer-binding protein-δ to myostatin signaling pathway that activates muscle protein degradation in mice with CKD or cancer; we also identified a small-molecule inhibitor of STAT3 (TTI-101) that blocks this pathway. To evaluate TTI-101 as a treatment for CKD-induced cachexia, we measured TTI-101 pharmacokinetics and pharmacodynamics in control and CKD rats that were orally administered TTI-101or its diluent. The following two groups of gavage-fed rats were studied: sham-operated control rats and CKD rats. Plasma was collected serially (0, 0.25, 0.5, 1, 2, 4, 8, and 24 h) following TTI-101 administration (at oral doses of 0, 10, 30, or 100 mg/kg). Plasma levels of TTI-101 were measured by LC-MS/MS, and pharmacokinetic results were analyzed with the PKSolver program. Plasma TTI-101 levels increased linearly with doses; the maximum plasma concentrations and time to maximal plasma levels (~1 h) were similar in sham-operated control rats and CKD rats. Notably, gavage treatment of TTI-101 for 3 days produced TTI-101 muscle levels in sham control rats and CKD rats that were not significantly different. CKD rats that received TTI-101 for 7 days had suppression of activated STAT3 and improved muscle grip strength; there also was a trend for increasing body and muscle weights. TTI-101 was tolerated at doses of 100 mg·kg-1·day-1 for 7 days. These results with TTI-101 in rats warrant its development as a treatment for cachexia in humans.

Inhibition of myostatin in mice improves insulin sensitivity via irisin-mediated cross talk between muscle and adipose tissues.

BACKGROUND/OBJECTIVE: In mice, a high-fat diet (HFD) induces obesity, insulin resistance and myostatin production. We tested whether inhibition of myostatin in mice can reverse these HFD-induced abnormalities. SUBJECTS/METHODS: C57BL/6 mice were fed a HFD for 16 weeks including the final 4 weeks some mice were treated with an anti-myostatin peptibody. Body composition, the respiratory exchange ratio plus glucose and insulin tolerance tests were examined. Myostatin knock down in C2C12 cells was performed using small hairpin RNA lentivirus. Adipose tissue-derived stem cells were cultured to measure their responses to conditioned media from C2C12 cells lacking myostatin, or to recombinant myostatin or irisin. Isolated peritoneal macrophages were treated with myostatin or irisin to determine whether myostatin or irisin induce inflammatory mechanisms. RESULTS: In HFD-fed mice, peptibody treatment stimulated muscle growth and improved insulin resistance. The improved glucose and insulin tolerances were confirmed when we found increased muscle expression of p-Akt and the glucose transporter, Glut4. In HFD-fed mice, the peptibody suppressed macrophage infiltration and the expression of proinflammatory cytokines in both the muscle and adipocytes. Inhibition of myostatin caused the conversion of white (WAT) to brown adipose tissue, whereas stimulating fatty acid oxidation and increasing energy expenditure. The related mechanism is a muscle-to-fat cross talk mediated by irisin. Myostatin inhibition increased peroxisome proliferator-activated receptor gamma, coactivator 1α expression and irisin production in the muscle. Irisin then stimulated WAT browning. Irisin also suppresses inflammation and stimulates macrophage polarization from M1 to M2 types. CONCLUSIONS: These results uncover a metabolic pathway from an increase in myostatin that suppresses irisin leading to the activation of inflammatory cytokines and insulin resistance. Thus, myostatin is a potential therapeutic target to treat insulin resistance of type II diabetes as well as the shortage of brown/beige fat in obesity.

Inhibition of Stat3 activation suppresses caspase-3 and the ubiquitin-proteasome system, leading to preservation of muscle mass in cancer cachexia.

Cachexia occurs in patients with advanced cancers. Despite the adverse clinical impact of cancer-induced muscle wasting, pathways causing cachexia are controversial, and clinically reliable therapies are not available. A trigger of muscle protein loss is the Jak/Stat pathway, and indeed, we found that conditioned medium from C26 colon carcinoma (C26) or Lewis lung carcinoma cells activates Stat3 (p-Stat3) in C2C12 myotubes. We identified two proteolytic pathways that are activated in muscle by p-Stat3; one is activation of caspase-3, and the other is p-Stat3 to myostatin, MAFbx/Atrogin-1, and MuRF-1 via CAAT/enhancer-binding protein δ (C/EBPδ). Using sequential deletions of the caspase-3 promoter and CHIP assays, we determined that Stat3 activation increases caspase-3 expression in C2C12 cells. Caspase-3 expression and proteolytic activity were stimulated by p-Stat3 in muscles of tumor-bearing mice. In mice with cachexia caused by Lewis lung carcinoma or C26 tumors, knock-out of p-Stat3 in muscle or with a small chemical inhibitor of p-Stat3 suppressed muscle mass losses, improved protein synthesis and degradation in muscle, and increased body weight and grip strength. Activation of p-Stat3 stimulates a pathway from C/EBPδ to myostatin and expression of MAFbx/Atrogin-1 and increases the ubiquitin-proteasome system. Indeed, C/EBPδ KO decreases the expression of MAFbx/Atrogin-1 and myostatin, while increasing muscle mass and grip strength. In conclusion, cancer stimulates p-Stat3 in muscle, activating protein loss by stimulating caspase-3, myostatin, and the ubiquitin-proteasome system. These results could lead to novel strategies for preventing cancer-induced muscle wasting.

Glucocorticoids increase adipocytes in muscle by affecting IL-4 regulated FAP activity.

An increase in intramuscular adipocyte tissue (IMAT) is associated with glucose dysregulation, decreased muscle strength, and increased risk of disability. Unfortunately, the mechanisms stimulating intramuscular adipogenesis remain unclear. We found that dexamethasone (Dex) administration to mice with injured muscles stimulates the accumulation of IMAT. To identify precursors of these adipocytes, we isolated satellite cells and fibro/adipogenic progenitors (FAPs) from muscle; satellite cells did not differentiate into adipocytes even following Dex treatment. In contrast, Dex stimulated FAP differentiation into adipocytes. In vivo, we transplanted purified FAPs from transgenic, EGFP mice into the injured muscles of C57/BL6 mice and found that Dex administration stimulated adipogenesis from FAP-EGFP. The increase in adipogenesis depended on Dex-induced inhibition of interleukin-4 (IL-4). In the injured muscle of IL-4-knockout mice, the levels of adipocytes were increased, while in the injured muscles of Dex-treated mice with IL-4 injections, adipogenesis was suppressed. In cultured FAPs, IL-4 inhibited Dex-induced conversion of FAPs into adipocytes; this did not occur in FAPs expressing knockdown of the IL-4 receptor. Thus, we concluded that glucocorticoids stimulate FAPs to differentiate into adipocytes in injured muscles. This process is blocked by IL-4, suggesting that interfering with IL-4 signaling could prevent adipogenesis in muscle.

Stat3 activation links a C/EBPδ to myostatin pathway to stimulate loss of muscle mass.

Catabolic conditions like chronic kidney disease (CKD) cause loss of muscle mass by unclear mechanisms. In muscle biopsies from CKD patients, we found activated Stat3 (p-Stat3) and hypothesized that p-Stat3 initiates muscle wasting. We created mice with muscle-specific knockout (KO) that prevents activation of Stat3. In these mice, losses of body and muscle weights were suppressed in models with CKD or acute diabetes. A small-molecule that inhibits Stat3 activation produced similar responses, suggesting a potential for translation strategies. Using CCAAT/enhancer-binding protein δ (C/EBPδ) KO mice and C2C12 myotubes with knockdown of C/EBPδ or myostatin, we determined that p-Stat3 initiates muscle wasting via C/EBPδ, stimulating myostatin, a negative muscle growth regulator. C/EBPδ KO also improved survival of CKD mice. We verified that p-Stat3, C/EBPδ, and myostatin were increased in muscles of CKD patients. The pathway from p-Stat3 to C/EBPδ to myostatin and muscle wasting could identify therapeutic targets that prevent muscle wasting.

Interactions between p-Akt and Smad3 in injured muscles initiate myogenesis or fibrogenesis.

In catabolic conditions such as aging and diabetes, IGF signaling is impaired and fibrosis develops in skeletal muscles. To examine whether impaired IGF signaling initiates muscle fibrosis, we generated IGF-IR(+/-) heterozygous mice by crossing loxP-floxed IGF-IR (exon 3) mice with MyoD-cre mice. IGF-IR(+/-) mice were studied because we were unable to obtain homozygous IGF-IR-KO mice. In IGF-IR(+/-) mice, both growth and expression of myogenic genes (MyoD and myogenin; markers of satellite cell proliferation and differentiation, respectively) were depressed. Likewise, in injured muscles of IGF-IR(+/-) mice, there was impaired regeneration, depressed expression of MyoD and myogenin, and increased expression of TGF-ß1, α-SMA, collagen I, and fibrosis. To uncover mechanisms stimulating fibrosis, we isolated satellite cells from muscles of IGF-IR(+/-) mice and found reduced proliferation and differentiation plus increased TGF-ß1 production. In C2C12 myoblasts (a model of satellite cells), IGF-I treatment inhibited TGF-ß1-stimulated Smad3 phosphorylation, its nuclear translocation, and expression of fibronectin. Using immunoprecipitation assay, we found an interaction between p-Akt or Akt with Smad3 in wild-type mouse muscles and in C2C12 myoblasts; importantly, IGF-I increased p-Akt and Smad3 interaction, whereas TGF-ß1 decreased it. Therefore, in muscles of IGF-IR(+/-) mice, the reduction in IGF-IR reduces p-Akt, allowing for dissociation and nuclear translocation of Smad3 to enhance the TGF-ß1 signaling pathway, leading to fibrosis. Thus, strategies to improve IGF signaling could prevent fibrosis in catabolic conditions with impaired IGF signaling.

Transcription factor FoxO1, the dominant mediator of muscle wasting in chronic kidney disease, is inhibited by microRNA-486.

Chronic kidney disease (CKD) accelerates muscle protein degradation by stimulating the ubiquitin proteasome system through activation of the E3 ligases, Atrogin-1/MAFbx and MuRF-1. Forkhead transcription factors (FoxOs) can control the expression of these E3 ligases, but the contribution of individual FoxOs to muscle wasting is unclear. To study this we created mice with a muscle-specific FoxO1 deletion. The absence of FoxO1 blocked 70% of the increase in E3 ligase induction by CKD as well as the proteolysis and loss of muscle mass. Thus, FoxO1 has a role in controlling ubiquitin proteasome system-related proteolysis. As microRNA (miR)-486 reportedly dampens FoxO1 expression and its activity,we transfected a miR-486 mimic into primary cultures of myotubes and found this blocked dexamethasone-stimulated protein degradation without influencing protein synthesis.It also decreased FoxO1 protein translation and increased FoxO1 phosphorylation by downregulation of PTEN phosphatase, a negative regulator of p-Akt. To test its efficacy in vivo, we electroporated miR-486 into muscles and found that the expression of the E3 ligases was suppressed and muscle mass increased despite CKD. Thus, FoxO1 is a dominant mediator of CKD-induced muscle wasting, and miR-486 coordinately decreases FoxO1 and PTEN to protect against this catabolic response.

Role of integrin-ß3 protein in macrophage polarization and regeneration of injured muscle.

Following injury, skeletal muscle achieves repair by a highly coordinated, dynamic process resulting from interplay among numerous inflammatory, growth factors and myogenic regulators. To identify genes involved in muscle regeneration, we used a microarray analysis; there was a significant increase in the expression of a group of integrin genes. To verify these results, we used RT-PCR and Western blotting and found that 12 integrins were up-regulated from 3 h to 15 days following injury. Following muscle injury, integrin-ß3 was initially expressed, mainly in macrophages. In integrin-ß3 global KO mice, the expression of myogenic genes was decreased and muscle regeneration was impaired, whereas fibrosis was enhanced versus events in wild type (WT) mice. The mechanism for these responses in integrin-ß3 KO mice included an infiltration of macrophages that were polarized into the M2 phenotype. These macrophages produced more TGF-ß1 and increased TGF-ß1/Smad signaling. In vitro, we confirmed that M2 macrophages lacking integrin-ß3 produced more TGF-ß1. Furthermore, transplantation of bone marrow cells from integrin-ß3 KO mice into WT mice led to suppression of the infiltration and accumulation of macrophages into injured muscles. There was also impaired muscle regeneration with an increase in muscle fibrosis. Our results demonstrate that integrin-ß3 plays a fundamental role in muscle regeneration through a regulation of macrophage infiltration and polarization leading to suppressed TGF-ß1 production. This promotes efficient muscle regeneration. Thus, an improvement in integrin-ß3 function could stimulate muscle regeneration.

Ginsenoside Rb1 attenuates homocysteine-augmented guidewire injury-induced intimal hyperplasia in mice.

BACKGROUND: Intimal hyperplasia (IH) is the primary cause for post-angioplasty restenosis. The purpose of this study is to investigate the effects of homocysteine (Hcy) and ginsenoside Rb1 (Rb1) on IH using a guidewire injury animal model. METHODS: In 12-wk-old C57BL/6J mice, the left common carotid artery (CCA) was denudated with a guidewire and the right CCA was used as the uninjured control. They were treated with saline (NS), Hcy, Rb1, or Hcy + Rb1 for 4 wk prior to sacrifice. Animals were sacrificed at 4, 6, or 8 wk. Both CCAs were harvested and intimal-medium thickness (IMT) ratios were calculated. Local macrophage distribution was also studied. RESULTS: Histology analyses demonstrated consistent internal elastic lamina disruption and focal IH in the injured CCA segments. The degree of IH correlated to the lengths of time following injury. Hcy treated group had significant increase in IMT compared with the NS group (P < 0.05), while Rb1 group was similar to the NS group. In addition, Hcy + Rb1 group showed significant improvement in IMT compared with Hcy group (P < 0.01). Furthermore, Hcy significantly increased local macrophage content as compared with either lesion alone or Rb1 treated animals. CONCLUSIONS: Our study showed that Hcy increased the degree of IH and macrophage content in the injured CCA and that Rb1 attenuated these adverse effects. These changes might be mediated through antioxidative effects of Rb1. Our data suggests a potential clinical application of ginseng in controlling Hcy-related vascular injuries.

Targeted disruption of luteinizing hormone beta-subunit leads to hypogonadism, defects in gonadal steroidogenesis, and infertility.

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) act on gonadal cells to promote steroidogenesis and gametogenesis. Clarifying the in vivo roles of LH and FSH permits a feasible approach to contraception involving selective blockade of gonadotropin action. One way to address these physiologically important problems is to generate mice with an isolated LH deficiency and compare them with existing FSH loss-of-function mice. To model human reproductive disorders involving loss of LH function and to define LH-responsive genes, we produced knockout mice lacking the hormone-specific LHbeta-subunit. LHbeta-null mice are viable but demonstrate postnatal defects in gonadal growth and function resulting in infertility. Mutant males have decreased testes size, prominent Leydig cell hypoplasia, defects in expression of genes encoding steroid biosynthesis pathway enzymes, and reduced testosterone levels. Furthermore, spermatogenesis is blocked at the round spermatid stage, causing a total absence of the elongated spermatids. Mutant female mice are hypogonadal and demonstrate decreased levels of serum estradiol and progesterone. Ovarian histology demonstrates normal thecal layer, defects in folliculogenesis including many degenerating antral follicles, and absence of corpora lutea. The defects in both sexes are not secondary to aberrant FSH regulation, because FSH levels were unaffected in null mice. Finally, both male and female null mice can be pharmacologically rescued by exogenous human chorionic gonadotropin, indicating that LH-responsiveness of the target cells is not irreversibly lost. Thus, LHbeta null mice represent a model to study the consequences of an isolated deficiency of LH ligand in reproduction, while retaining normal LH-responsiveness in target cells.