The injury-induced myokine insulin-like 6 is protective in experimental autoimmune myositis
© Zeng et al.; licensee BioMed Central Ltd. 2014
Received: 4 February 2014
Accepted: 11 June 2014
Published: 4 August 2014
The idiopathic inflammatory myopathies represent a group of autoimmune diseases that are characterized by lymphocyte infiltration of muscle and muscle weakness. Insulin-like 6 (Insl6) is a poorly characterized member of the insulin-like/relaxin family of secreted proteins, whose expression is upregulated upon acute muscle injury.
In this study, we employed Insl6 gain or loss of function mice to investigate the role of Insl6 in a T cell-mediated model of experimental autoimmune myositis (EAM). EAM models in rodents have involved immunization with human myosin-binding protein C with complete Freund’s adjuvant (CFA) emulsions and pertussis toxin.
Insl6-deficiency in mice led to a worsened myositis phenotype including increased infiltration of CD4 and CD8 T cells and the elevated expression of inflammatory cytokines. Insl6-deficient mice show significant motor function impairment when tested with treadmill or Rotarod devices. Conversely, muscle-specific overexpression of Insl6 protected against the development of myositis as indicated by reduced lymphocyte infiltration in muscle, diminished inflammatory cytokine expression and improved motor function. The improvement in myositis by Insl6 could also be demonstrated by acute hydrodynamic delivery of a plasmid encoding murine Insl6. In cultured cells, Insl6 inhibits Jurkat cell proliferation and activation in response to phytohemagglutinin/phorbol 12-myristate 13-acetate stimulation. Insl6 transcript expression in muscle was reduced in a cohort of dermatomyositis and polymyositis patients.
These data suggest that Insl6 may have utility for the treatment of myositis, a condition for which few treatment options exist.
KeywordsInsulin-like 6 Myositis Autoimmune T cells Skeletal muscle Myokine
Idiopathic inflammatory myositis is a category of autoimmune muscle disorders that include polymyositis, dermatomyositis and inclusion body myositis. Features common to all of the subtypes include muscle weakness, histological evidence of muscle inflammation and elevated levels of creatine kinase. However, each subset also has unique clinical, immunopathologic and histologic criteria . Dermatomyositis is a vascular endothelial lesion associated with perimysial inflammation and perifascicular muscle fiber atrophy, whereas polymyositis is associated with injury to muscle fibers by aggressive immune cells which predominantly infiltrate the endomysium . Macrophages, dendritic cells, and T cells are prominently present in muscle of the different myositis subgroups. In dermatomyositis, large numbers of helper T cells (CD4 T cells) are found within the perimysial, often perivascular areas. In polymyositis, activated cytotoxic T cells (CD8 T cells) surround and invade non-necrotic muscle fibers, while helper T cells are found at more distant parts of the infiltrates . These inflammatory muscle diseases are rare and there are few effective treatment strategies for these patients .
Immune cells regulate skeletal muscle regeneration after myofiber damage [5, 6]. Muscle damage stimulates massive infiltration of immune cells into the injury site, including neutrophils, macrophages, and T cells. These cells function in the phagocytosis of necrotic muscle fibers, express pro-inflammatory cytokines and participate in satellite cell activation . We have previously demonstrated that Insl6 has muscle regenerative and anti-inflammatory activities in a cardiotoxin-induced model of severe muscle injury when expressed using an engineered adenovirus vector . We therefore sought to extend these studies and explore the utility of Insl6 in the setting of inflammatory muscle injury using a rodent model of myositis.
In the current study, we employed Insl6 gain or loss of function mice to investigate the role of Insl6 in a T cell-mediated model of experimental autoimmune myositis (EAM). EAM models in rodents have involved immunization with native myosin-binding protein C with complete Freund’s adjuvant (CFA) emulsions and pertussis toxin (PT) [9, 10]. In murine EAM, the infiltration of CD4 and CD8 T cells is observed, similar to what is seen in human PM and DM . Of interest, depletion of both CD4 and CD8 T cells suppresses the myositis phenotype in this model . Using this model, we find that Insl6-deficiency led to greater muscle damage, increased inflammation and reduced motor function in the murine model of EAM. Conversely, Insl6 overexpression diminished inflammation and improved motor function.
RPMI-1640 medium, DMEM, FBS, penicillin-streptomycin mixture, and Alexafluor 488® goat anti-rabbit IgG antibody were obtained from Invitrogen (Carlsbad, CA). Anti-CD4 and anti-CD8 antibodies conjugated with eFluor® 650NC were purchased from eBioscience. Fluorescein isothiocyanate (FITC)-conjugated anti-CD4 antibody was obtained from BD Biosciences (San Jose, CA). Freund’s complete adjuvant (CFA), PT, phytohemagglutinin (PHA), phorbol 12-myristate 13-acetate (PMA), protease inhibitor cocktail, RIPA buffer, and anti-laminin antibody were purchased from Sigma (St. Louis, MO). Tubulin-α antibody and horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies were purchased from Cell Signaling (Danvers, MA). 3H-thymidine was purchased from PerkinElmer (Waltham, MA).
MCK-Insl6-TG mice in a C57BL/6 background were generated as previously described . These mice express murine Insl6 cDNA from a 4.8 kb pair fragment of the murine creatine kinase M promoter. Insl6-deficient mice were described previously . In Insl6-deficient mice, exon 1 of Insl6 genomic DNA is replaced by the PGK-neomycin(Neo) selection cassette. Insl6+/- heterozygous female and male pairs were used for breeding, and the offspring littermates (Insl6+/+ and Insl6-/-) were used for experiments. In some instances, C57BL/6 mice were purchased from Charles River Laboratories (Cambridge, MA). Mice were housed on a fixed 12-hour light/dark cycle and fed a normal chow diet (Teklad Global 18% protein rodent diet, 2018, Harlan Teklad). Study protocols were approved by the Institutional Animal Care and Use Committee at Boston University.
Fc tagged Insl6 plasmid
The human IgG1 Fc was fused at the N-terminus of full-length mouse Insl6. In brief, mouse Insl6 cDNA was PCR amplified and cloned into pLEV113 mammalian expression vector (LakePharma, Belmont, CA) downstream of an in-frame human IgG1 Fc domain and a secretion signal peptide. This plasmid was used to perform hydrodynamic injection.
Recombinant human skeletal muscle myosin-binding C protein
The recombinant skeletal muscle myosin-binding C protein was prepared using a prokaryotic expression procedure. A cDNA fragment of human fast-type skeletal muscle C protein was amplified from human skeletal muscle cDNA using PCR. Primers sequences were previously described by Sugihara et al. . The cDNA fragment was sub-cloned into Qiagen pQE30 expression vector and the plasmid was maintained and amplified in E. coli XL-1 Blue strain. E. coli M15 (pREP4) contained in the QIAexpressionist™ was used to prepare the recombinant C protein according to the manufacturer’s protocol (Qiagen, Valencia, CA). The soluble recombinant protein was dialyzed against 0.5 M arginine, 2 mM reduced glutathione, and 0.2 mM oxidized glutathione in PBS, pH 7.4. Endotoxin removal was performed with Detoxi-Gel. The molecular weight of C protein is approximately 35 kDa.
Chronic inflammatory myositis model
Female mice, eight to ten weeks old, were immunized intradermally with 600 μg of the C protein emulsified in CFA containing 100 μg of heat-killed Mycobacterium butyricum (Sigma, St. Louis, MO). The immunogens were injected subcutaneously at multiple sites around the torso or on the feet, and 2 μg of PT in PBS was injected intraperitoneally at the same time.
Cardiotoxin-induced muscle injury model
A 10 μM solution of cardiotoxin (CTX) or equal volume of PBS was injected into the tibialis anterior (TA) muscle at 2 μl/g body weight using an insulin syringe as described previously . The needle was inserted parallel to the muscle fiber longitude until reaching the tendon which inserts into the knee and then slowly withdrawn while simultaneously injecting the CTX solution in its path.
Hydrodynamic delivery of plasmid DNA
Mice were pre-warmed by heat lamp for 20 minutes, and a plastic restrainer was used to keep mice in position during injection. Ten micrograms of Insl6-Fc or control vectors were dissolved in a large volume of saline (80 μl/g body weight) and injected quickly within 8 seconds via the tail vein .
Muscle function was evaluated with a Rotarod device. The test was performed on each mouse by measuring the running time until the mouse fell off the rod. Mice were initially trained to acclimate them to the task, and then tested two days thereafter. The turning speed was set at 10 revolutions/minute for knockout (KO) mice and their littermate controls, and at 20 revolutions/minute for transgenic (TG) mice and controls, to better distinguish the differences between the genetically manipulated KO and TG strains of mice and control littermates. The protocol was modified from published work . Each mouse was scored according to its time on the rotating rod: score 1 corresponding to 0 to 30 seconds, score 2 for 31 to 60 seconds, score 3 for 61 to 90 seconds, and so on. A maximum score of 20 was recorded for mice running more than 10 minutes.
The treadmill test employs a moving belt (Columbus Instruments, Columbus, OH). All mice were subjected to a 30 minute run on a horizontal treadmill at 12 m/minute, twice per week before the experimental analysis . To assess running performance, the treadmill was initiated with a start speed of six m/minute, and speed was increased by two m/minute every five minutes for KO mice and their littermate control mice. For TG mice and littermate controls, a starting speed was ten m/minute, and speed was increased by five m/minute every five minutes. Different belt speeds were chosen to better distinguish between the running performance of test and control sets of mice when KO and TG groups were analyzed. Electrical stimulation was applied to encourage the mice to run on the belt. The total running distance was recorded after mice elected not to run for a period of 30 seconds. Study protocols were approved by the Institutional Animal Care and Use Committee at Boston University.
Histological analysis of murine samples
Skeletal muscle tissues were embedded in OCT compound and snap-frozen in liquid nitrogen. Serial cryostat sections (8 μm) were fixed for 10 minutes in 10% formalin, and were stained with H&E (Fisher Scientific, Pittsburgh, PA) for histological analysis. The inflammatory lesion was visualized using a × 40 objective. For immunofluorescence staining, serial cryostat sections (8 μm) were fixed in cold acetone and stained with eFluor® 650NC conjugated anti-CD4 or CD8 antibody (1:100 dilution), and anti-laminin primary antibodies (1:25 dilution). Alexafluor 488® goat anti-rabbit IgG secondary antibody (1:200 dilution) was used to visualize laminin expression. Fifteen randomly chosen microscopic fields from three different sections in each tissue block were examined for the presence of antigen expressing cells.
Insl6 antibody production
Insl6 rabbit polyclonal antibody against peptides 1) VPAGVSQKKGTHT, 2) QLQKKSTNKMNTF and 3) TKEEMAVACLPFVDF was custom ordered from Thermo Fisher Inc. (Pittsburgh, PA). The antibody was purified from antiserum by antigen specific affinity purification. Antiserum was passed through a column containing resin beads conjugated to the immunogenic Insl6 peptides. Antibodies were eluted using a pH gradient, collected in a neutralizing buffer and concentrated before use.
Skeletal muscle tissues were homogenized in radioimmunoprecipitation assay (RIPA) buffer which was supplemented with protease inhibitor cocktail. Equal amount of proteins were loaded on 10% NuPAGE Gels (Invitrogen, Carlsbad, CA) and run as instructed by the manufacturer. The transfer to PVDF membranes was done under wet conditions. Membranes were blocked in 5% skimmed milk or BSA solution, and then incubated with primary antibodies at 4°C overnight. Membranes were washed several times in Tris-buffered saline with Tween (TBST) while agitating, and then incubated with HRP-conjugated anti-rabbit secondary antibodies at room temperature for one hour. Chemiluminescent antigen expression was captured on film by electrochemiluminescence (ECL) using a Western Blotting Detection kit (GE Healthcare, Buckinghamshire, UK.
Quantitative real-time PCR
cDNA was produced from total RNA using ThermoScript RT-PCR Systems (Invitrogen, Carlsbad, CA). Transcript levels were determined relative to the signal from 36B4, and normalized to the mean value of samples from control mice. Primers were purchased from Integrated DNA Technology (Coralville, IA). The following primer sequences were used; mouse Insl6 exon1 (F: 5′-GCAGCTGTGCTGTTCTTGTCTGTT-3′, R: 5′-AGGACTTTGCTCCTCCATCTCGAA-3′); Neo (F: 5′-TGTCGATCAGGATGATCTGG-3′, R: 5′-CCACCATGATATTCGGCAAG-3′); mouse CD8 (F: 5′-CTGTTTTCTGCCATGAGGGACACG-3′, R: GTTCACTTTCTGAAGGACTGGCACG-3′); mouse CD4 (F: 5′-GCTGTCACAACTCCTAGCTGTCAC-3′, F: 5′-CCTCTAATTAATACACCTTTGCCATGC-3′); mouse TNF-α (F: 5′-ACAGAAAGCATGATCCGGGA-3′, R: 5′-TCTGGGCCATAGAACTGATG-3′); mouse IFN-γ (F: 5′-CACGGCACAGTCATTGAAAG-3′, R: 5′-CATCCTTTTGCCAGTTCCTC-3′); mouse 36B4 (F: 5′-GCTCCAAGGAGATGCAGCA-3′, R: CCGGATGTGAGGCAGCAG-3′).
Jurkat T cell proliferation assay
Jurkat cells obtained from the American Type Culture Collection (Manassas, VA) were maintained in growth medium (RPMI supplemented with 10% FBS). Cells were transiently transfected with β-galactosidase (β-gal) or murine Insl6 expression plasmids (pcDNA3, Invitrogen, Carlsbad, CA) that employed the cytomegalovirus promoter. Electroporation by Gene Pulser™ (BioRad, Hercules, CA) was conducted at 250 V and 960 μF with time constant of 24 to 30 seconds. Cell proliferation was assessed by 3H-thymidine incorporation. Forty-eight hours after transfection, cells were incubated with 3H-thymidine (2.0 μCi/107 cells) for four hours. Cells were washed twice with ice cold PBS and lysed with 10% sodium hydroxide. Cell lysates were suspended in liquid scintillation buffer. The incorporation of radioactivity was determined by liquid scintillation analyzer (TRI-CARB2900TR, PerkinElmer, Waltham, MA). In related experiments, C2C12 cells were incubated with Ad-Insl6 and Ad-β-gal virus vectors at a multiplicity of infection (MOI) of 250 to 500 in culture medium for 16 hours. At this time, the virus was removed by media replacement. Cell culture media was collected 48 hours after the transfection and 3H-thymidine (1.0 μCi/ml medium) was added. Jurkat cells were incubated in this conditioned medium for 48 hours. The incorporation of 3H-thymidine was measured as described above.
IL-2 ELISA assay
PHA and PMA activate T-lymphocytes and stimulate production of IL-2 [15, 16]. Jurkat cells were transiently transfected with β-gal or murine Insl6 expression plasmids (pcDNA3, Invitrogen, Carlsbad, CA) as described above. PHA and PMA were added into culture medium 24 hours after transfection. Supernatant was collected 48 hours post-transfection and IL-2 concentration was measured by human IL-2 ELISA (Quantikine ELISA, R&D Systems, Minneapolis, MN).
Analysis in human myositis muscle biopsy samples
Skeletal muscle samples of polymyositis and dermatomyositis patients were collected by muscle biopsy at New England Medical Center, St. Elizabeth Hospital and Tufts University. The procedures were performed after obtaining informed consent from all study subjects. The mean time in storage at -80°C for control samples was 8.8 ± 0.9 years, compared to 8.1 ± 4.0 years for the polymyositis/dermatomyositis samples. Insl6 mRNA levels were determined by real-time RT-PCR using an Applied Biosystems (Woburn, MA) Model 7300 thermal cycler. Total RNA was isolated from human fresh frozen skeletal muscles (30 μg) using RNeasy mini kit or Trizol, following the manufacturers’ instructions (Qiagen, Germantown, MD or Invitrogen, Carlsbad, CA). RNA was reverse transcribed using oligo dT and primers (50 μM each) and reverse transcriptase (RT) (Multiscribe, 250 U) in the presence of 10 mM dNTP mixture in a total volume of 100 μl. First strand cDNA and PCR reactions were generated with equal amounts of starting material using the Applied Biosystems (Woburn, MA) TaqMan Kit. Following denaturation at 95°C for 10 minutes, real-time PCR was undertaken for 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. GAPDH was used as an internal control for quantification of relative expression of specific transcripts. The sequences were as follows: human Insl6 (F: 5′-GCGGCAGGTACTTGGTGAAAGAAA-3′, R: 5′-TTTGCGGGCTTTCGAACTGGTATG-3′); GAPDH (F: 5′-GAGAGATGATGACCCTTTTGGC-3′, R: 5′-CCATCACCATCTTCCAGGAGCG-3′). Histological sections of these muscle biopsy specimens from the polymyositis and dermatomyositis patients were evaluated for the degree of atrophy by a neuropathologist who was blinded to the the level of Insl6 in the tissue. The 5, 10, 20, 25 values refer the percentage of myofibers that appeared atrophic.
All values are presented as mean ± SEM. Student’s t-tests were performed to assess the statistical significance of two-way analyses. For multiple comparisons, analysis of variance (ANOVA) was employed. P-values of less than 0.05 were considered statistically significant.
Insl6 ablation impairs muscle regeneration
Insl6-deficiency promotes experimental autoimmune myositis severity
Infiltration of CD4 and CD8 T cells in muscle is a hallmark of the CIM model . Thus, CD4 and CD8 T cell infiltration was assessed in multiple lower limb muscles, including TA, GA, and quadriceps. No appreciable T cell infiltration was observed in wild-type or Insl6-KO mice at baseline (data not shown). The infiltration of CD4 and CD8 T cells was observed in most of the limb muscles of the CIM mice. In Figure 3b, a representative image of TA muscle section stained with CD4/CD8 and laminin is shown. Quantitative analyses revealed more CD4 and CD8 T cells present in the Insl6-KO muscle, compared with wild-type controls. T cell infiltration was observed in the perimysial and perivascular areas. No obvious endomysial CD8 T cell infiltration was observed in any experimental condition, in contrast with a prior report . Analysis of histological sections of muscle with the macrophage marker F4/80 showed a trend toward greater infiltration of macrophages in the Insl6-KO mice under CIM conditions, but this difference was not statistically significant (Additional file 2: Figure S3).Using RNA isolated from lower limb muscles, qRT-PCR analyses were performed to measure the expression of inflammatory marker genes. Consistent with the histological analyses in Figure 3b, CD4 and CD8 transcript levels were elevated in Insl6-KO mice relative to wild-type in the CIM model (Figure 3c). Transcript levels of inflammatory cytokines TNF-α and IFN-γ were also significantly increased in Insl6-KO versus wild-type mice in the CIM condition (Figure 3d). At baseline, transcript levels of CD8, CD4, TNF-α, and IFN-γ are very low in both Insl6-KO and wild-type mice, and there was no difference between groups.
Transgenic Insl6 overexpression ameliorates experimental autoimmune myositis
Analyses of limb muscle revealed that transgenic overexpression of Insl6 diminished inflammation in the CIM model. Muscle transcript levels of CD4, CD8, TNF-α and IFN-γ increased in both wild-type and TG mice in the CIM model, but the induction of each of these transcripts was significantly less in Insl6-overexpressing mice (Figure 4b, c). However, analysis of histological sections of muscle with the marker F4/80 did not show a difference in macrophage infiltration between wild-type and TG mice (Additional file 2: Figure S3).
Hydrodynamic Insl6 gene delivery ameliorates experimental autoimmune myositis
Insl6 inhibits Jurkat T cell activation
IL-2 signaling promotes proliferation, survival and cytokine production in T cells . Little or no IL-2 is produced from Jurkat cells at baseline, but PHA- and PMA-induced protein kinase C activation can robustly stimulate IL-2 production . As shown in Figure 6a, Insl6 overexpression reduced the PMA/PHA-induced IL-2 secretion by 40%. Since Insl6 is a secreted protein [8, 20], we also tested the ability of culture medium containing Insl6 to inhibit Jurkat cell proliferation. Myogenic C2C12 cells were transduced with adenoviral vectors expressing murine Insl6  or a control vector expressing β-gal. Cell culture medium was collected 48 hours post-transduction. mRNA was extracted from C2C12 cell lysates and Insl6 transcript was measured by quantitative RT-PCR to confirm the Insl6 overexpression (data not shown). Jurkat cells were incubated with Insl6 conditioned medium for 24 hours, and cell proliferation was measured by the amount of incorporated 3H-thymidine. As shown in Figure 6b, incubation of cells with Insl6-conditioned media led to a significant reduction in proliferation.
Reduced Insl6 expression in clinical inflammatory myositis
Characteristics of patients and control individuals
Control (n = 14)
PM/DM (n = 13)
Age, mean ± SEM
58.8 ± 4.0 (36 to 85)
53.6 ± 5.2 (22 to 78)
Male, % (n)
Female, % (n)
Insl6 relative transcript, mean ± SEM
0.98 ± 0.17
0.50 ± 0.11
Insl6 is upregulated in injured muscle and it has been reported that it has muscle-regenerative and anti-inflammatory properties when overexpressed in the cardiotoxin model of muscle injury . In this report, we demonstrate that mice lacking Insl6 display enhanced muscle inflammation and impaired regeneration following exposure to cardiotoxin. The present study also reports that Insl6 is protective in a murine model resembling human inflammatory myopathy using genetically modified mice either lacking or overexpressing Insl6. Furthermore, it is shown that overexpression of an Fc-Insl6 hybrid protein following hydrodynamic plasmid delivery improves overall muscle pathology in this model based upon functional, histological and biochemical evidence. These data support a therapeutic role for Insl6 in inflammatory myopathies where there is a high unmet need for new therapies.
Experimental myositis has been induced previously in different species with a variety of agents. Here, we employed a model of autoimmune myositis in C57BL/6 mice by administering a single immunization of recombinant human skeletal C-protein fragment emulsified in Freund’s adjuvant with heat-inactivated M. butyricum. The resulting histopathologic abnormalities resembled some features of human polymyositis including CD4+ and CD8+ T cell infiltration. In our analyses, CD4+ and CD8+ T cells were observed in perivascular and perimysial areas, but no pronounced myofiber invasion was observed. While there was no apparent myofiber necrosis, we observed pronounced muscle motor dysfunction in both Rotarod and treadmill within two weeks of immunization. Deficiency of Insl6 led to an increase in CD4 and CD8 T cell infiltration, increased inflammatory cytokine expression and greater motor function impairment in the myositis model. Conversely, overexpression of Insl6, either by chronic overexpression in muscle or acute systemic overexpression, minimized T cell infiltration and cytokine expression in muscle and improved performance in the Rotarod and treadmill tests.
It is widely recognized that subsets of T cells play important roles in the development of the myositis phenotype [3, 25–27]. In our study, Insl6 was shown to inhibit T cell activation and IL-2 cytokine secretion. These effects could be shown either by directly overexpressing Insl6 within Jurkat T cells or by treating these cells with Insl6-conditioned media. In addition, Insl6 negatively regulated the production of inflammatory cytokines in muscle. Previously, we reported that Insl6 can act on myoblasts to promote a regenerative response in muscle . Thus, it appears that Insl6 can act both on inflammatory and muscle cells to control the response to injury in muscle.
Interestingly, Insl6 mRNA transcript levels are down-regulated in human myositis biopsy samples, whereas this factor is upregulated in the murine models of myositis and cardiotoxin injury. We speculate that Insl6 is an endogenous immunosuppressant or immunosurveillance factor. In animal injury models that are relatively short-term, the release of Insl6 may function as a self-protective mechanism that minimizes immune-mediated tissue destruction. However, in chronic human disease, the reduction of Insl6 by an as-yet-unidentified mechanism may contribute to the development of idiopathic inflammatory myopathies or other autoimmune diseases. Consistent with this notion, low levels of Insl6 correlated with disease severity. Thus, reestablishing the normal upregulation of Insl6 in muscle of myositis patients may have therapeutic utility.
Comparative genomic analysis shows that Insl6 is a member of the insulin-like/relaxin family of proteins that have diverse roles in reparative and reproductive processes . Whereas insulin-like growth factor (IGF) and insulin activate tyrosine kinase receptors, most insulin-like family proteins signal through G-protein-coupled-receptors to control cAMP-mediated signaling events . However, the receptor for Insl6 has yet to be identified, and nothing is known about the downstream signaling molecules that respond to this ligand. Related family members, including Insl1 (which is also referred to as relaxin), have been shown to have both muscle- and cardiovascular-protective actions. It has previously been shown that relaxin will promote muscle repair in a laceration model through its ability to diminish fibrosis and promote satellite cell mobilization in both young and aged mice [30, 31]. Human recombinant relaxin-2 protein (RLX030) is in clinical development for the treatment of acute heart failure . In the RELAX-AHF phase III clinical trial, patients treated with relaxin-2 for 48 hours experienced improved heart failure symptoms and a 37% reduction in mortality, leading to a breakthrough therapy designation by the Food and Drugs Administration (FDA). Finally, Insl3 has been shown to have cardiac regenerative properties in a zebrafish model . Thus the insulin-like/relaxin family members represent a potentially interesting source of factors for the treatment of heart failure and muscle weakness, two processes that are inter-related in the pathological condition that is referred to as cardiac cachexia.
In contrast to the well-described anabolic functions of the IGF isoforms [34, 35], Insl6 overexpression neither stimulates myogenesis in normal muscle nor induces C2C12 cell hypertrophy in culture . Insl6 promotes muscle regeneration only after injury, and this effect is associated with an increase in satellite cell activation and the diminution of inflammatory marker expression . Consistent with these observations, the Insl6-deficient mice generated by Burnicka-Turek and colleagues  have morphologically and functionally normal muscle at baseline. However, after muscle stress or damage, significantly impaired regeneration and deteriorated pathology were observed in the Insl6-deficient animals.
Many of the immunopathogenic processes behind myositis remain poorly understood. However, it is generally accepted that cytokines and chemokines are essential regulators of leukocyte activation and migration [3, 11, 36]. In animal EAM models, it has been reported that IL-6 or IL-1 blockade diminishes the severity of myositis . Regulatory T cells (Tregs) have been identified as a pivotal cell population in the control of autoimmunity . Treg-deficiency causes over-proliferation of lymphocytes and multi-organ autoimmunity in both humans and mice. Treg abundance is significantly reduced in skin lesions and peripheral blood of patients with dermatomyositis . In Treg-depleted mice, a more severe phenotype was observed in the experimental autoimmune myositis model, whereas the injection of Tregs at the time of immunization significantly improved the disease .
To date, evidence for the utility of Insl6 as a potential therapy for inflammatory myositis has been limited to experimental settings using genetic models and plasmid-driven overexpression in vivo. Future studies will involve the development of a well-characterized, biologically-active recombinant form of Insl6 to assess the pharmacological activity of this protein and further elucidate its mechanism of action.
Using mouse genetic models of Insl6-deficiency and overexpression, this study has identified a role for Insl6 in experimental autoimmune myositis. Insl6 improves skeletal muscle function and reduces inflammation in this murine model. In vitro studies revealed that Insl6 inhibits T cell proliferation and activation. Alterations in Insl6 expression in samples taken from both human and murine myositis-affected muscle highlight the potential for translational impact. Further investigation into the anti-inflammatory and muscle regenerative pathways mediated by Insl6 is warranted.
analysis of variance
C protein-induced myositis
complete Freund’s adjuvant
Dulbecco’s modified Eagle medium
experimental autoimmune myositis
enzyme-linked immunosorbent assay
fetal bovine serum
Food and Drug Administration
hematoxylin and eosin
insulin-like growth factor
multiplicity of infection
polymerase chain reaction
phorbol 12-myristate 13-acetate
radioimmunoprecipitation assay buffer
human recombinant relaxin-2 protein
Tris-buffered saline with Tween
tumor necrosis factor
regulatory T cells
This work was supported by National Institutes of Health grants AG34972, HL81587 and HL68758 to KW.
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