Background: Laminin-α2-related congenital muscular dystrophy (LAMA2-CMD) is a devastating genetic disease caused by mutations in the LAMA2 gene. These mutations result in progressive muscle wasting and inflammation leading to delayed milestones, and reduced lifespan in affected patients. There is currently no cure or treatment for LAMA2-CMD. Preclinical studies have demonstrated that mouse laminin-111 can serve as an effective protein replacement therapy in a mouse model of LAMA2-CMD. Methods: In this study, we generated a novel immunocompromised dy mouse model of LAMA2-CMD to study the role the immune system plays in muscle disease progression. We used this immune-deficient dy mouse model to test the therapeutic benefits of recombinant human laminin-111 and laminin-211 protein therapy on laminin-α2-deficient muscle disease progression. Results: We show that immunodeficient laminin-α2 null mice demonstrate subtle differences in muscle regeneration compared to immunocompetent animals during early disease stages but overall exhibit a comparable muscle disease progression. We found human laminin-111 and laminin-211 could serve as effective protein replacement strategies with mice showing improvements in muscle pathology and function. We observed that human laminin-111 and laminin-211 exhibit differences on satellite and myoblast cell populations and differentially affect muscle repair. Conclusions: This study describes the generation of a novel immunodeficient mouse model that allows investigation of the role the immune system plays in LAMA2-CMD. This model can be used to assess the therapeutic potential of heterologous therapies that would elicit an immune response. Using this model, we show that recombinant human laminin-111 can serve as effective protein replacement therapy for the treatment of LAMA2-CMD. Keywords: LAMA2-CMD, MDC1A, Laminin-111, Immune system * Correspondence: firstname.lastname@example.org Department of Pharmacology, University of Nevada, Reno School of Medicine, Reno, NV 89557, USA © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Barraza-Flores et al. Skeletal Muscle (2020) 10:18 Page 2 of 12 Introduction contributes to initial muscle disease in LAMA2-CMD, Laminin-α2-related congenital muscular dystrophy but that other non-immune-related mechanisms con- (LAMA2-CMD) also known as merosin-deficient congeni- tribute to long-term muscle disease progression. tal muscular dystrophy type 1A (MDC1A) is a severe gen- Laminin-111 is a form of laminin that is structurally etic disease with an incidence estimated at 1–9/100,000 and functionally similar to laminin-211 and 221 and people and representing 10–30% of all congenital dystro- has been shown to rescue mouse models of LAMA2- phies [1–3]. LAMA2-CMD patients present with neonatal CMD [10–13]. We next determined the efficacy of hypotonia, muscle wasting resulting in wheelchair confine- recombinant human laminin-111 (HsLAM-111) and ment, and requiring respiratory support at a young age. laminin-211 (HsLAM-211) protein therapies to prevent There is currently no effective cure or treatment for muscle disease progression using this immunodeficient LAMA2-CMD , and patients often die from respiratory dy mice model. Our results show that treatment with insufficiency as early as during their first decade of life . HsLAM-111 and HsLAM-211 improved muscle func- Although mutations can cause partial or reduced tion and pathology, but results show HsLAM-111 and expression of LAMA2, mutations that result in a HsLAM-211 had different efficacies on muscle regener- complete absence of the laminin-α2 protein chain cause ation. Together these studies indicate a role for the the most severe muscle disease and clinical outcomes in adaptive immune response in LAMA2-CMD and sup- patients. Laminin-α2 is a critical component of the lam- port the idea of laminin protein replacement therapies inin heterotrimer, which along with laminin-β1or β2 as a treatment option for LAMA2-CMD. and laminin-γ1 form the structural glycoproteins laminin-211 and laminin-221 in skeletal muscle. Results Laminin-211 and -221 polymerize with each other and The NODScid dy mouse is immuno-deficient and lacks interact with nidogen and collagen-IV to form the laminin-α2 protein muscle basal lamina. Laminin-211 and laminin-221 bind To produce an immunodeficient mouse model of to muscle cell surface through the α7β1 integrin and α- W+/− LAMA2-CMD, NODScid mice were bred with dy dystroglycan of the dystrophin glycoprotein complex via animals. Along with the test group NODScid dy ,we their globular C-terminal domains. This interaction an- also generated wild-type, NODScid, and dy control chors muscle cells to the basal lamina and regulates groups. Muscles from wild-type, NODScid, dy , and mechanotransduction and cell signaling [6, 7]. Loss of NODScid dy were harvested at 6 weeks of age and im- these laminin-211 and laminin-221 in LAMA2-CMD munofluorescence used to detect the laminin-α2 chain. disrupts these molecular interactions and results in While strong signal for laminin-α2 immunofluorescence reduced muscle strength, failed muscle regeneration, was observed in wild-type muscle, little signal was inflammation, and fibrosis [1, 4, 5]. W W detected in dy and NODScid dy muscle (Fig. 1a). Severe inflammation is a hallmark of LAMA2-CMD These results confirmed NODScid dy lacked laminin- and muscle biopsies from mouse and LAMA2-CMD α2 in skeletal muscle. patients exhibit immune cell infiltration especially dur- To determine if NODScid dy lacked an adaptive im- ing early stages of the disease. However, in contrast with mune system, we next isolated serum from 6-week-old other muscular dystrophies, inflammatory infiltrate is mice and performed an ELISA to detect serum immuno- decreased during later stages of LAMA2-CMD muscle globulin G (IgGs). Our results show that while wild-type disease progression and its role in LAMA2-CMD muscle and dy mice had high levels of IgG in serum, NODScid disease remains unclear [5, 8, 9]. and the NODScid dy serum had no detectable IgGs In this study, to determine the role the immune (Fig. 1b). These results confirmed that NODScid dy response plays in LAMA2-CMD muscle disease, we pro- animals lack functional B cells and are unable to pro- duced an immunodeficient Lama2-null mouse on the duce immunoglobulin. dy strain background. This novel mouse line, which we Next, we used fluorescence-activated-cell sorting designated NODScid dy , lacks laminin-α2 and func- (FACS) to quantify circulating levels of T and B cells in tional B and T-immune cells. This new model was com- the blood. Hematopoietic cells (CD45 ) from sera of pared to the immunocompetent dy mouse model to wild-type and NODScid dy were co-labeled with T cell assess the impact of the loss of the immune response + + marker (CD3ε ) and B cell marker (CD19 ) (Fig. 1c). plays in LAMA2-CMD muscle disease. The immunode- Results showed that in wild-type, 31.6% of CD45 cells ficient NODScid dy mouse model showed reduced + + W were CD3ε and 38.4% were CD19 . In NODScid dy , levels of basal regenerating myofibers compared to dy + + 0.88% were CD3ε and 1.08% were CD19 . These results animals. There were no differences between immunode- show that NODScid dy mice lack functional T and B ficient and immunocompetent mice in terms of disease cells and therefore lack an adaptive immune response. progression. These results indicate the immune response Barraza-Flores et al. Skeletal Muscle (2020) 10:18 Page 3 of 12 Fig. 1 The NODScid dy mouse model of LAMA2-CMD shows downregulated levels of laminin-α2 protein chain and is immunodeficient. a W W W Detection of laminin-α2 (red) in tibialis anterior (TA) sections of wild-type, dy internal control, dy , and NODScid dy . Scale bar 100 μm. b W W Relative levels of immunoglobulin in sera of wild-type, NOD Scid, dy , and NODScid dy (N =9; p value < 0.0001). c Fluorescence-activated cell + W + sorting (FACS) gate analysis of hematopoietic cells (CD45 ) from sera of wild-type and NODScid dy co-labeled with T cell marker (CD3ε ) and B cell marker (CD19 ) Muscular dystrophy in NODScid dy is comparable to the males (Fig. 3a; N = 6 and 7, respectively, p value < W W W dy mouse model of LAMA2-CMD 0.0001). In contrast, dy and NODScid dy females did We next performed a survival study using wild-type, not show any differences (Fig. 3b). These results suggest W W NODScid, dy , and NODScid dy experimental groups. that the immune response in dystrophic muscle contrib- We observed that neither the female nor the male utes to the lower grip strength observed in male dy NODScid dy groups had a significant increase in life- animals. span compared to dy (Fig. 2a, b). Male and female To determine if the immune response in dystrophic NODScid showed reduced lifespan compared to wild- muscle contributed to fibrosis observed in LAMA2- type mice, consistent with reports on radio-sensitivity- CMD, we used Sirius red to stain sections from TA induced lymphomas . Symptoms of lymphomas were muscles (Supplemental Figure 1A) and quantified levels not observed in the NODScid dy mouse. Weekly body of hydroxyproline as previously described  (Supple- mass showed no significant gender differences between mental Figure 1B, C) in quadriceps of all groups. Sirius W W NODScid dy and dy animals (Fig. 2c, d). These data Red staining indicated more fibrosis in TA muscle sec- W W indicate growth and survival were similar between im- tions from dy and NODScid dy mice compared to munocompetent and immunodeficient dy animals. wild-type and NODScid muscle. This was confirmed To determine if loss of the immune system affected and quantified using a hydroxyproline (HOP) assay. muscle strength in dy mice, a forelimb grip-strength Wild-type and NODScid muscle had approximately 1.5- test was performed at 6 weeks of age and normalized to fold less HOP in their TA muscles compared to dys- W W body weight as previously described . As expected, trophic dy and NODScid dy mice. There was no dif- 6-week-old wild-type and NODScid mice exhibited an ference in HOP levels between males and females in the W W average of 3-fold increase in muscle grip strength com- dy and NODScid dy experimental groups. These W W pared to dystrophic dy and NODScid dy groups. results indicate the immune response did not play a Interestingly, 6-week-old NODScid dy males showed a major role in the development of TA muscle fibrosis in W W 1.4-fold increase in grip strength compared to dy 6-week-old dy mice. Barraza-Flores et al. Skeletal Muscle (2020) 10:18 Page 4 of 12 Fig. 2 Survival and weight did not change in immunodeficient dy mice compared to immuno-competent mouse models of LAMA2-CMD. W W Survival study curves of male (a) and female (b) wild-type, NOD Scid, dy , and NODScid dy . c Weekly weights throughout the life span of male W W and female (d) wild-type, NOD Scid, dy , and NODScid dy To assess for the presence of other inflammatory cells, Immunodeficient dy mice exhibit decreased muscle we used immunofluorescence to detect the three major repair myeloidal cell infiltrates: eosinophils, macrophages Laminin-α2 deficiency leads to failed muscle regeneration (CD11B) (Supplemental Figure 2), and neutrophils and early apoptosis of regenerating myofibers [1, 18–20]. (LysC) (Supplemental Figure 3) associated with muscular To assess differences in levels of ongoing regeneration in dystrophy [8, 17]. Our results showed presence of innate the NODScid dy , we quantified embryonic myosin heavy W W immune infiltrates in NODScid dy and dy muscle, chain (eMHC), a marker for muscle regeneration, in TA suggesting genetic ablation of the adaptive response sections (Fig. 4a). When compared to dy , we found that through NODScid immune suppression did not affect both male and female NODScid dy groups showed a the innate immune infiltration in these animals. decrease in eMHC-positive fibers: from 8.77 ± 0.81% in Fig. 3 Grip strength is increased in immunodeficient male mice compared to immunocompetent LAMA2-CMD mouse models. Normalized force W W measurements of grip strength in wild-type, NOD Scid, dy , and NODScid dy a male (N = 4, 5, 7, 6, respectively; p value < 0.0001) and b female (N =5; p value < 0.05*, < 0.0001***) Barraza-Flores et al. Skeletal Muscle (2020) 10:18 Page 5 of 12 Fig. 4 Basal regeneration is decreased in immunodeficient compared to immunocompetent mouse models of LAMA2-CMD. a Detection of W W embryonic Myosin Heavy Chain (eMHC) in TA sections of dy and NODScid dy mice. Quantification of eMHC-positive fibers in male (N =7, 5, W W respectively; p value of 0.01; scale bar 100 μm) (b) and female (N = 5, 4, respectively; p value of 0.02). c dy and NODScid dy mice. Frequency histogram of minimum Feret’s diameters in TA muscle of male (N = 4, 5, respectively; p value < 0.0001) (d) and female (N =5; p value < 0.0001) W W (e)dy and NODScid dy mice W W dy and 5.86 ± 0.535% in NODScid dy males (N =7, 5, towards increased myofiber diameter, from a mean of W W W respectively, p value 0.01) and 6.09 ± 0.50% in dy and 30.11 μmindy to 34.81 μm in NODScid dy (N =4, 4.31 ± 0.40% in NODScid dy females (N = 5,4,respect- 5, respectively, p value < 0.0001). TA muscle from female ively, p value 0.02) (Fig. 4b, c). mice, however, did not show a change in myofiber size Feret minimal diameters were used to measure myofi- with a mean of 34.00 μmindy to 34.98 μm in NODS- ber size. TA muscle from male mice showed a shift cid dy (N =5, p value 0.114) (Fig. 4d, e). This data Barraza-Flores et al. Skeletal Muscle (2020) 10:18 Page 6 of 12 suggests that NODScid dy muscle exhibits a lower due to production availability of HsLAM-111 and level of basal muscle damage compared to dy . This HsLAM-211 (BioLamina, Sundyberg, Sweden) at 0.1 mg/ may indicate that suppression of adaptive immunity in ml compared to EHS laminin-111 (Thermo Fisher, laminin-α2-deficient skeletal muscle results in decreased Waltham MA) at 1 mg/ml. At 6 weeks of age, mice were muscle damage that results in muscle hypertrophy in humanely euthanized, and TA, gastrocnemius, quadriceps, male animals. and triceps were harvested. TA muscle sections were tested for presence of Human laminin-111 and laminin-211 protein therapy laminin protein using immunofluorescence. Two anti- increase muscle repair in NODScid dy bodies directed against the carboxyl-terminal and rod Previous research has shown that treatment with domains of human laminin-α1 chain were used for natural Englebreth-Holm-Swam (EHS) murine immunofluorescence using muscle from vehicle and sarcoma-derived laminin-111 enhances muscle regen- HsLAM-111 treated groups. Supplemental Fig. 4A shows eration and prevents myopathy of mouse models of positive immunofluorescence for both domains in LAMA2-CMD and Duchenne muscular dystrophy HsLAM-111 compared to PBS-treated groups. Addition- (DMD) [10, 11, 16, 21–23]. To test whether human ally, we used western analysis to show these antibodies laminin-111 has the same effect, we treated NODS- are specific for the human isoform of laminin-111 cid dy mice with HsLAM-111. We also used (Supplemental Fig. 4B). We were unable to detect HsLAM-211 to investigate if it could completely sub- HsLAM-211 as antibodies against laminin-α2 were not stitute for the loss of laminin-α2 in LAMA2-CMD. specific for the human isoform and the dy mouse Female NODScid dy mice were treated from 2 to 6 model expresses a low level of truncated laminin-α2 weeks of age with weekly retro-orbital injections of 1 mg/ protein. kg HsLAM-111, HsLAM-211, or vehicle (Fig. 5a). This TA cryosections were subjected to immunofluores- dose was 10-fold lower than previous studies [13, 16, 22] cence for eMHC-positive fibers. We found that Fig. 5 Treatment with human recombinant laminin-111 (HsLAM-111) and human recombinant laminin-211 (HsLAM-211) increased muscle regeneration in a mouse model of LAMA2-CMD. a NODScid dy mice were treated with weekly retro-orbital injections of 1 mg/kg HsLAM-111, HsLAM-211, or PBS from 2 weeks to 6 weeks of age. b Quantification of eMHC-positive fibers in PBS-, HsLAM-111-, and HsLAM-211-treated NODScid dy (N = 7, 5, 6, respectively; p value < 0.05*, 0.0049**). c Frequency histogram of minimum Feret’s diameters in TA muscle of PBS-, HsLAM-111-, and HsLAM-211-treated NODScid dy (N = 7, 5, 7, respectively; p value < 0.0001) Barraza-Flores et al. Skeletal Muscle (2020) 10:18 Page 7 of 12 treatment with both HsLAM-111 and HsLAM-211 re- showed no differences between vehicle and treatment sulted in a ~ 1.7-fold increase in levels of eMHC-positive groups. fibers in laminin-treated muscle compared to vehicle Together, these data suggest that treatment with treatment (Fig. 5b). Where PBS-treated mice showed HsLAM-111 and HsLAM-211 increased muscle regener- 3.03 ± 0.48% eMHC-positive fibers, HsLAM-111 treat- ation and reduced fiber diameter variability. ment resulted in a 5.192 ± 19% (N = 7, 5, respectively, p value < 0.05) and HsLAM-211 treatment in a 5.37 ± Human laminin-111 and laminin-211 differentially 0.23% (N =7,6; p value of 0.004). affected myogenic cells in laminin-α2-deficient muscle We next measured myofiber size using Feret’s To test the effect of HsLAM-111 and HsLAM-211 on minimal diameters and observed a change in the mean muscle repair, we next quantified satellite and myogenic from 23.68 μm in PBS-treated animals, 18.09 μmfor cells in TA muscle. For satellite cells, we conducted im- HsLAM-111 treatment, and 20.76 μminHsLAM-211 munofluorescence for the paired-box transcription factor treatment (N = 7, 5, 7, respectively; p value < 0.0001). 7 (Pax7) and counted the number of Pax7-positive cells We observed a decrease in the standard deviations of located adjacent to the myofiber under the basal lamina myofiber size in laminin-treated muscles (SD) from (Fig. 6a). Treatment with HsLAM-111 resulted in a sig- 8.35 μminPBS to 7.6 μm HsLAM-111 and 7.17 μmin nificant decrease in the number of satellite cells from HsLAM-211, indicating laminin treatment promoted a 2.06 ± 0.14 cells per frame in PBS to 1.38 ± 0.19 Pax7- reduction in myofiber size variability (Fig. 5c). Cen- positive cells (N = 8, 6, respectively, p value < 0.05). In trally located nuclei (CLNs) fibers were also quantified contrast, we observed a significant increase in satellite as a measure of ongoing repair (data not shown) but cells in muscle with HsLAM-211 treatment to 2.70 ± Fig. 6 Treatment with HsLAM-111 and HsLAM-211 differentially affects satellite cells and myoblasts in skeletal muscle of LAMA2-CMD. a Quantification of Pax-7-positive satellite cells per frame in TA muscle sections of PBS-, HsLAM-111-, and HsLAM-211-treated NODScid dy (N =8, 6, 6, respectively; p value < 0.05* < 0.0001***). Scale bar 100 μm. b Detection of Pax7-positive cells adjacent to fibers in TA sections of NODScid dy mice. c Quantification of myogenin-positive myoblasts per frame in TA muscle sections of PBS-, HsLAM-111-, and HsLAM-211-treated W W NODScid dy (N =7; p value of 0.019). d Detection of myogenin-positive cells located interstitially in TA sections of NODScid dy mice Barraza-Flores et al. Skeletal Muscle (2020) 10:18 Page 8 of 12 0.17 satellite cells compared to HsLAM-111 or PBS this biologic, NODScid dy mice were treated with treatments (N =6, p value < 0.0001 and p < 0.05, HsLAM-111, HsLAM-211, or PBS for several weeks. respectively) (Fig. 6b). Mice were then subjected to a computer-controlled ac- To determine if recombinant human laminin treat- tivity assay as previously described  (Fig. 7a). Results ment affected myogenic differentiation, we counted showed a significant increase in distance traveled from a myogenin-positive cells (Fig. 6c). Our results showed no mean of 3394 ± 479 cm in PBS-treated mice to 5386 ± significant decrease of myogenin-positive cells from 8.1 281.7 cm in HsLAM-111 treatment (Fig. 7b) (N =7,7, ± 0.79 cells per area in PBS-treated animals to 7.56 ± respectively; p value < 0.05), but no increase in HsLAM- 0.89 in HsLAM-111-treated mice. However, we did see a 211-treated mice with 3211 ± 724.9 cm. Resting time significant decrease to 4.78 ± 0.95 cells per frame in showed no significant decrease from a mean of 236.8 ± HsLAM-211-treated mice (N = 6, 7, respectively, p value 54.4 s in PBS to 109.8 ± 12.38 s in HsLAM-111 and of 0.01) (Fig. 6d). 238.6 ± 41.66 in HsLAM-211 treatment groups (Fig. 7c). Together, these results may suggest that laminin-111 Finally, HsLAM-111-treated mice showed a significant and laminin-211 isoforms have an effect on muscle re- increase in vertical breaks from 11.2 ± 9.5 in PBS to pair in laminin-α2 null muscle, while differentially pro- 50 ± 11.3 in HsLAM-111-treated animals, indicative moting myogenic cell differentiation. of increased use of hindlimbs during the assay (Fig. 7d) (N =7,7; p value 0.0005). Grip strength was also Human laminin-111 treatment improves the activity of performed with no significant differences between LAMA2-CMD mice treatment groups (Supplemental Fig. 5)(N =6). Previous studies have shown that treatment with EHS- These data indicate treatment with recombinant hu- derived mouse laminin-111 improves muscle function in man laminin-111 improves mobility of laminin-α2- the dy mouse model. To test the human isoform of deficient mice. Fig. 7 Treatment with HsLAM-111 improved mouse activity in LAMA2-CMD mouse model. a PBS-, HsLAM-111-, and HsLAM-211-treated NODScid dy mice pictures and trajectory detected using computer-controlled activity assay. Quantification of distance traveled b, resting time c, and vertical breaks d in PBS-, HsLAM-111-, and HsLAM-211-treated NODScid dy (N = 7, 7, 6, respectively; p value < 0.05*, < 0.005**) Barraza-Flores et al. Skeletal Muscle (2020) 10:18 Page 9 of 12 Discussion regeneration and larger myofibers found in the NODS- Mouse laminin-111 has previously shown to be an cid dy mouse indicate of a role of an active immune effective protein-based therapy in the dy mouse model response in promoting muscle regeneration in LAMA2- of LAMA2-CMD [10, 13]. Although mouse and human CMD. laminin-111 have a high degree of homology, there are Together, these results suggest the NODScid dy is a ~ 30% differences in the amino acid sequence in addition viable immunocompromised model for LAMA2-CMD, to differences in glycosylation patterns. For this reason, that while presenting reduced levels of basal muscle re- treatment using the recombinant HsLAM-111 is likely generation, disease progression is comparable to the to generate an immune response in mice that could established dy model and similar to LAMA2-CMD complicate interpretation of the efficacy of the human patients. This immunodeficient mouse model of biologic. To investigate if recombinant human laminin- LAMA2-CMD might be beneficial to investigate the role 111 could prevent disease progression, we generated an the immune system plays in LAMA2-CMD, or investi- immunodeficient dy mouse model of laminin-α2- gate the efficacy of human cell-based or human biologics related congenital muscular dystrophy (LAMA2-CMD). for the treatment of LAMA2-CMD. We demonstrate that this mouse model has disrupted Using this novel mouse model of LAMA2-CMD, we expression of laminin-α2 protein and an ablated adaptive next tested the efficacy of recombinant human laminin- immune system. Histological and physiological 111 and laminin-211 to act as protein substitution ther- characterization of this new mouse model shows severe apies and prevent muscle disease progression for muscle disease progression comparable to the estab- LAMA2-CMD. Previous research has shown treatment lished dy model of LAMA2-CMD. with EHS murine laminin-111 can promote muscle The NOD Scid mouse has been shown to present regeneration and prevent muscular disease in mouse multiple defects in innate and adaptive immunity and models of muscular dystrophy. To our knowledge, this is has been used extensively in xeno engraftment studies the first study to investigate the therapeutic potential of [24, 25]. Reduced natural killer (NK) cell activity, de- recombinant human laminin-111 and laminin-211 in a crease in functionally mature macrophages, and ablation muscular dystrophy disease model. of functional lymphoid T and B cells are the main Our results showed that HsLAM-111 and HsLAM-211 immuno-deficiencies reported in NODScid mice [14, 26]. increased the regenerative capacity of muscle but that The generation of a laminin-α2-deficient mouse that is the mechanism of action of these laminin isoforms on also NODScid allows for a study of the role of the innate satellite cells and myoblast wAS different. Laminin-111 and adaptive immunity plays in LAMA2-CMD disease promoted muscle repair at the expense of satellite cells, progression. while laminin-211 preserved satellite cell populations in A case study of several laminin-α2-deficient patients re- skeletal muscle. This could indicate that HsLAM-111 ported high levels of T and B cell infiltration in skeletal treatment induces satellite cell activation, which may ex- muscle at an early age but decreased inflammatory infil- plain the increase in eMHC-positive regenerative fibers tration at later stages of the disease . This suggests that observed in this treatment group. However, continuous in contrast with other muscular dystrophies, the immune long-term treatment with HsLAM-111 could deplete sat- system in LAMA2-CMD may not play a major role in ellite cells. On the other hand, treatment with HsLAM- later stages of disease progression . The immune re- 211 increased satellite cells and decreased myogenin- sponse may, however, be important during neonatal stages positive cells suggesting this laminin isoform could sup- and may exacerbate muscle disease progression. Our stud- port the satellite cell niche in a way that preserves the ies demonstrate there were no changes in the survival or satellite cell population. It is also possible that the local weight of immunocompromised and immunocompetent concentrations of laminin isoforms are critical for prolif- dy animals. Lack of improved grip strength and hyper- eration vs differentiation of myoblasts. Finally, we show trophy in females as opposed to males is consistent with that treatment with HsLAM-111 improved the activity reports of more severe myopathy in females vs males in of laminin-α2 null mice. other LAMA2-CMD mouse models . Another possible This short-term study of HsLAM-111 and HsLAM- explanation may be sex-dependent differences in immune 211 in skeletal muscle provided preliminary data for the infiltration or timing between males and female during treatment of LAMA2-CMD. A long-term treatment disease stages. The observed differences between male and using these biologicals is necessary to assess improve- female myopathy and inflammation in LAMA2-CMD re- ments in survival, immune infiltration, and fibrosis. Add- main to be further explored. itionally, it is important to explore the long-term effect Loss of immune function did result in reduced muscle on satellite cell depletion and renewal, as well as the po- regeneration in NODScid dy mice as indicated with tential for the combination of HsLAM-111 and HsLAM- eMHC expression. The reduced levels of muscle 211 therapies. Barraza-Flores et al. Skeletal Muscle (2020) 10:18 Page 10 of 12 To our knowledge, this is the first report using a Laboratories, Burlingame, CA, 1:00) for 10 min. Slides laminin-α2-deficient mouse model to investigate the role were prepared using Vectashield mounting media with that the immune system plays in LAMA2-CMD. In DAPI stain (Vector Laboratories, Burlingame, CA). Im- addition, this is the first report using an immunodefi- munostained sections were permeabilized using 0.2% cient preclinical model to explore the short-term efficacy Triton in PBS for 30 min before blocking, then primary of recombinant human laminin-111 and laminin-211 on antibodies anti-eMHC (Developmental Studies Hybri- LAMA2-CMD disease progression. Limitations of this droma Bank (DSHB), Iowa City, IA, BF-45, 1:40), CD11B approach include producing enough purified HsLAM- (Biolegend, San Diego, CA, 1:800) LysC (Biolegend, San 111 to treat LAMA2-CMD patients, potential immune Diego, CA, 1:800), Pax7 (DSHB, Iowa City, IA, Pax7, 1: responses to glycosylation differences between recom- 20) and Myogenin (DSHB, Iowa City, IA, F5D, 1:20) binant and native human laminin isoforms, and the ef- were incubated overnight. Rabbit polyclonal antibodies fects of long-term systemic treatment with HsLAM-111 against human laminin-α1C-terminal and rod domains protein. (Prothelia, Inc., Milford, MA) were used at a concentra- tion of 1:20 without permeabilization. All primary anti- Materials and methods bodies were followed by secondary antibody incubation Generation of immunodeficient NODScid dy mice for 1 h using FITC-anti-mouse or FITC-anti-rabbit anti- All animal studies were performed under an approved bodies (Jackson Laboratories, Sacramento, CA, 1:200), animal protocol (#00404) reviewed by the Institutional except for pre-labeled FITC-CD11b and FITC-LysC, and Animal Care and Use Committee (IACUC) at the Uni- lastly incubated for 10 min WGA stain (WGA-647 1: versity of Nevada, Reno. To generate immunodeficient 100). Slides were imaged using the Olympus Fluoview W W (+/−) dy mice, female dy mice were mated to male FV 1000 laser confocal microscope and analyzed using SCID NOD.CB17-Prkdc /J from Jackson Laboratories. Off- Image J-win32 software. Whole muscle cross-sections spring were genotyped for LacZ gene inserted in dy were imaged and used for quantification of fiber diame- and the NOD (non-obese diabetic) gene using PCR. The ters and eMHC. We obtained 5 to 10 images at × 40 Scid gene was genotyped using qPCR using primers rec- magnification to quantify Pax7- and myogenin-positive (+/−) (+/−) ommended by Jackson Laboratories. NOD Scid cells. To measure fibrosis TA slides were stained with W (+/−) dy were mated to generate breeding pairs Sirius Red (Sigma-Aldrich, St. Louis, MI) as previously (−/−) (−/−) W (+/−) (+/+) (+/+) W NOD Scid dy and NOD Scid dy described (Van Ry, 2014). Images were captured using (+/−) . Matings of these mice generated wild-type, NODS- Axiovision 4.8 software. W W cid, dy , and NODScid dy experimental groups. Immunoblotting Survival study At total of 1 mg of HsLam-111, HsLam-211, and EHS W W Wild-type, NODScid, dy , and NODScid dy were aged laminin-111 (Thermo Scientific, Waltham, MA) proteins until they reached morbidity or death. Morbidity was de- were separated in a NuPAGE 4 to 12% Bis-Tris gel scribed as loss of 10% weight from week to week or se- (Thermo Scientific, Waltham, MA) and transferred onto vere kyphosis combined with hind-limb myopathy as a Nitrocellulose membrane. Laminin-111 C-terminal defined within the approved IACUC protocol. and rod-terminal domains were detected using home- made antibodies at a 1:100 concentration incubated Generation and administration of recombinant laminins overnight followed by 1-h incubation with secondary HsLAM-111 and HsLAM-211 were purchased from Bio- antibody Alexa Fluor680 conjugated anti-rabbit (Invitro- Lamina (Sundyberg, Sweden) where they are produced gen, Carlsbad, CA). LI-COR imaging system was used to recombinantly at a stock concentration of 0.1 mg/ml. detect and image protein bands. HsLAM-111 and HsLAM-211 dialyzed overnight against PBS to remove preservatives. Mice were anesthetized using isoflurane and administered each treatment group IgG detection (HsLAM-111, HsLAM-211 or PBS) weekly via retro- An enzyme-linked immunoabsorbent assay (ELISA) was orbital injection. used to measure the relative levels of immunoglobulin (IgG) in mouse sera. Mouse sera were used to coat an Immunofluorescence Immulon 1B (Thermo Scientific, Waltham, MA) plate in Muscles were harvested and embedded in 2:3 ratio of triplicates overnight at 4C. After three 0.1%SDS washes, optimum cutting temperature (Fisher Scientific, Wal- samples were incubated with mouse anti-IgG antibody tham, MA) and 30% sucrose medium prior to flash- at 1:200 dilution in 1% BSA overnight at 4C. After three freezing. Tissues were cryosectioned at 10-μm thick and more washes, samples were incubated with secondary stained with wheat germ agglutinin (WGA) (Vector antibody FITC-anti-mouse (Jax labs, Bar Harbor, ME). Barraza-Flores et al. Skeletal Muscle (2020) 10:18 Page 11 of 12 After the final wash, plate was read using Victor photo- FITC: Fluorescein isothiocyanate; HOP: Hydroxyproline; HsLAM-111: Laminin- 111; HsLAM-211: Laminin-211; IACUC: Institutional Animal Care and Use spectrometer at 500 nm. Committee; LAMA2-CMD: Laminin-α2-related congenital muscular dystrophy; IgG: Immunoglobulin G; MDC1A: Merosin-deficient congenital muscular dystrophy type 1A; NOD: Non-obese diabetic; Pax7: Paired-box transcription Fluorescence-activated cell sorting (FACS) factor 7; PBS: Phosphate-buffered saline; PCR: Polymerase chain reaction; Blood samples were drawn from 6-week-old mice into qPCR: Quantitative polymerase chain reaction; p value: Probability value; EDTA K3 coated tubes. One million cells were diluted Scid: Severe combined immuno-deficiency; TA: Tibialis anterior; T cells: Thymus lymphocyte cells; WGA: Wheat germ agglutinin with pre-labeled primary antibodies CD45 (1:800), CD19 (1:800), and CD3ε (1:100) from BD Biosciences (San Acknowledgements Jose, CA) and incubated for 30 min in the dark at room The authors thank Dr. Bradley Hodges, Prothelia Inc., Milford, MA, for the temperature. Cells were lysed using FACS lysis solution rabbit anti-human laminin-α1 polyclonal antibodies used in this study. protocol (BD Biosciences Cat.no 349202) and washed in Authors’ contributions 0.5% fetal bovine serum in PBS. Cells were quantified P.B-F. and D.J.B: conceptualization and study design. P.B-F.: methodology, using the BD LSR II SORP cell analyzer and data was validation, and formal analysis. P.B-F., H.J.H, C.R.B, T.G.A., and T.T.G: investigation. D.J.B.: resources. P.B-F.: writing original draft. P.B-F. and D.J.B.: analyzed using FlowJo software. writing-review and editing. P.B-F: visualization and supervision. P.B-F. and D.J.B.: project administration. D.J.B.: funding acquisition. The authors read and approved the final manuscript. Statistics GraphPad Prism software was used for statistical calcula- Funding tions. Student’s t test was used to compare means be- This study was supported by NIH/NIAMS R01AR064338-01A1 to DJB. PBF and tween two groups and one-way ANOVA was used to CB were supported by Mick Hitchcock Scholarships. compare means between three or more groups. All Availability of data and materials ANOVA calculations were followed by Bonferroni post- Most of the data generated and analyzed during this study are included in test. Means of experimental groups were considered sta- the manuscript or supplemental data. Any data not included will be made available from the corresponding author upon request. tistically significant when p < 0.05. Ethics approval Supplementary information All vertebrate animal studies described in this manuscript were performed Supplementary information accompanies this paper at https://doi.org/10. under an animal protocol (#00404) approved by the University of Nevada, 1186/s13395-020-00235-4. Reno IACUC. Competing interests Additional file 1: Supplemental Figure 1. Fibrosis is not significantly The University of Nevada, Reno, has a patent on the therapeutic use of changed between immuno deficient and immuno competent LAMA2- laminin-111 and its derivatives. This patent has been licensed to Prothelia CMD muscle. (A) Detection of sirius red stain in TA sections of wild type, Inc., and the University of Nevada, Reno, has a small equity share in this NOD Scid, dyW and NODScid dyW mice. Quantification of hydroxyproline company. in quadriceps of male (N = 4, 6, 8, 5 respectively; p value < 0.05*, 0.004**) (B) and female male (N = 5, 4, 5, 6 respectively; p value < 0.05*, 0.0014**) Received: 14 May 2020 Accepted: 17 May 2020 (C) wild type, NOD Scid, dyW and NODScid dyW mice. Supplemental Fig- ure 2. Macrophages and eosinophils are not severely changed in immuno deficient compared to immuno competent LAMA2-CMD muscle. (A) De- References tection of CD11B positive cells in TA sections of NODScid dyW, dyW and 1. Mohassel P, Reghan Foley A, Bönnemann CG. Extracellular matrix-driven wild type. Scale bar 100 μm. Supplemental Figure 3. Neutrophils are not congenital muscular dystrophies [Internet]. Matrix Biol. 2018 [cited 2019 Apr severely changed in immuno deficient compared to immuno competent 25]. p. 188–204. Available from. https://doi.org/10.1016/j.matbio.2018.06.005. LAMA2-CMD muscle. (A) Detection of LysC positive cells in TA sections of 2. 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Skeletal Muscle – Springer Journals
Published: Jun 4, 2020