The NODScid dyW mouse is immuno-deficient and lacks Laminin-α2 protein
To produce an immunodeficient mouse model of LAMA2-CMD, NODScid mice were bred with dyW+/- animals. Along with the test group NODScid dyW, we also generated wild type, NODScid and dyW control groups. Muscles from wild type, NODScid, dyW, and NODScid dyW were harvested at 6-weeks of age and immunofluorescence used to detect the Laminin-α2 chain. While strong signal for Laminin-α2 immunofluorescence was observed in wild-type muscle, little signal was detected in dyW and NODScid dyW muscle (Figure 1A). These results confirmed NODScid dyW lacked Laminin-α2 in skeletal muscle.
To determine if NODScid dyW lacked an adaptive immune system, we next isolated serum from 6-week old mice and performed an ELISA to detect serum immunoglobulin G (IgGs). Our results show that while wild type and dyW mice had high levels of IgG in serum, NODScid and the NODScid dyW serum had no detectable IgGs (Figure 1B). These results confirmed that NODScid dyW animals lack functional B-cells and are unable to produce immunoglobulin.
Next, we used fluorescence-activated-cell sorting (FACS) to quantify circulating levels of T and B cells in blood. Hematopoietic cells (CD45+) from sera of wild type and NODScid dyW were co-labeled with T-cell marker (CD3ε+) and B-cell marker (CD19+) (Figure 1C). Results showed that in wild type 31.6% of CD45+ cells were CD3ε+ and 38.4% were CD19+. In NODScid dyW, 0.88% were CD3ε+ and 1.08% were CD19+. These results show that NODScid dyW mice lack functional T- and B-cells and therefore lack an adaptive immune response.
Muscular dystrophy in NODScid dyW is comparable to the dyW mouse model of LAMA2-CMD
We next performed a survival study using wild type, NODScid, dyW and NODScid dyW experimental groups. We observed that neither the female nor the male NODScid dyW groups had a significant increase in lifespan compared to dyW (Figure 2A and 2B). Male and female NODScid showed reduced lifespan compared to wild-type mice, consistent with reports on radio-sensitivity induced lymphomas [14]. Symptoms of lymphomas were not observed in the NODScid dyW mouse. Weekly body mass showed no significant gender differences between NODScid dyW and dyW animals (Figure 2C and 2D). These data indicate growth and survival were similar between immune competent and immune deficient dyW animals.
To determine if loss of the immune system affected muscle strength in dyW mice, a forelimb grip-strength test was performed at 6 weeks of age and normalized to body weight as previously described [15]. As expected, 6-week old wild type and NODScid mice exhibited an average of 3-fold increase in muscle grip strength compared to dystrophic dyW and NODScid dyW groups. Interestingly, 6-week-old NODScid dyW males showed a 1.4-fold increase in grip strength compared to dyW males (Figure 3A; N = 6 and 7, respectively, p-value <0.0001). In contrast, dyW and NODScid dyW females did not show any differences (Figure 3B). These results suggest that the immune response in dystrophic muscle contributes to the lower grip strength observed in male dyW animals.
To determine if the immune response in dystrophic muscle contributed to fibrosis observed in LAMA2-CMD, we used Sirius red to stain sections from TA muscles (Supplemental Figure 1A) and quantified levels of hydroxyproline as previously described [16] (Supplemental Figure 1B, C) in quadriceps of all groups. Sirius Red staining indicated more fibrosis in TA muscle sections from dyW and NODScid dyW mice compared to wild type and NODScid muscle. This was confirmed and quantified using a hydroxyproline (HOP) assay. Wild-type and NODScid muscle had approximately 1.5-fold less HOP in their TA muscles compared to dystrophic dyW and NODScid dyW mice. There was no difference in HOP levels between males and females in the dyW and NODScid dyW experimental groups. These results indicate the immune response did not play a major role in the development of TA muscle fibrosis in 6-week-old dyW mice.
To assess for the presence of other inflammatory cells, we used immunofluorescence to detect the three major myeloidal cell infiltrates: Eosinophils, macrophages (CD11B) (Supplemental Figure 2) and neutrophils (LysC) (Supplemental Figure 3) associated with muscular dystrophy [8,17]. Our results showed presence of innate immune infiltrates in NODScid dyW and dyW muscle, suggesting genetic ablation of the adaptive response through NODScid immune suppression did not affect greatly the innate immune infiltration in these animals.
Immune deficient dyW mice exhibit decreased muscle repair
Laminin-α2 deficiency leads to failed muscle regeneration and early apoptosis of regenerating myofibers [1,18–20]. To assess differences in levels of ongoing regeneration in the NODScid dyW, we quantified embryonic Myosin Heavy Chain (eMHC), a marker for muscle regeneration, in TA sections (Figure 4A). When compared to dyW, we found that both male and female NODScid dyW groups showed a decrease in eMHC positive fibers: from 8.77±0.81% in dyW and 5.86±0.535% in NODScid dyW males (N = 7, 5 respectively, p-value 0.01) and 6.09±0.50% in dyW and 4.31±0.40% in NODScid dyW females (N = 5, 4 respectively, p-value 0.02) (Figure 4B and 4C).
Feret minimal diameters were used to measure myofiber size. Male myofibers showed a shift towards increased in myofiber diameter, from a mean of 30.11µm in dyW to 34.81µm in NODScid dyW (N = 4, 5 respectively, p-value <0.0001). Female myofibers, however, did not show a shift with a mean of 34.00µm in dyW to 34.98µm in NODScid dyW (N = 5, p-value 0.114) (Figure 4D and 4E). This data suggests that NODScid dyW muscle exhibits a lower level of basal muscle damage compared to dyW. This may indicate that suppression of adaptive immunity in Laminin-α2 deficient skeletal muscle results in decreased muscle damage that results in muscle hypertrophy in male animals.
Human Laminin–111 and Laminin–211 protein therapy increase muscle repair in NODScid dyW
Previous research has shown that treatment with natural Englebreth-Holm-Swam (EHS) murine sarcoma derived Laminin–111 enhances muscle regeneration and prevents myopathy of mouse models of LAMA2-CMD and Duchenne Muscular Dystrophy (DMD) [10,11,16,21–23]. To test whether human Laminin–111 has the same effect, we treated NODScid dyW mice with HsLAM–111. We also used HsLAM–211 to investigate if it could completely substitute for the loss of Laminin-α2 in LAMA2-CMD.
Female NODScid dyW mice were treated from 2 to 6 weeks of age with weekly retro-orbital injections of 1 mg/kg HsLAM–111, HsLAM–211 or vehicle (Figure 5A). This dose was 10-fold lower than previous studies [13,16,22] due to production availability of HsLAM–111 and HsLAM–211 (BioLamina, Sundeberg, Sweden) at 0.1 mg/ml compared to EHS Laminin–111 (Thermo Fisher, Waltham MA) at 1 mg/ml. At 6 weeks of age, mice were humanely euthanized, and TA, gastrocnemius, quadriceps and triceps were harvested.
TA muscle sections were tested for presence of laminin protein using immunofluorescence. Two antibodies directed against the carboxyl-terminal and rod domains of human Laminin-β1 chain were used for immunofluorescence using muscle from vehicle and HsLAM–111 treated groups. Supplemental Fig.4A shows positive immunofluorescence for both domains in HsLAM–111 compared to PBS treated groups. Additionally, we used western analysis to show these antibodies are specific for the human isoform of Laminin–111 (Supplemental Fig. 4B). We were unable to detect HsLAM–211 as antibodies against laminin–2 were not specific for the human isoform and the dyW mouse model expresses a low level of truncated laminin–2 protein.
TA cryosections were subjected to immunofluorescence for eMHC-positive fibers. We found that treatment with both HsLAM–111 and HsLAM–211 resulted in a ~1.7-fold increase in levels of eMHC-positive fibers in laminin treated muscle compared to vehicle treatment (Figure 5B). Where PBS treated mice showed 3.03± 0.48% eMHC-positive fibers, HsLAM–111 treatment resulted in a 5.192± 19% (N = 7, 5 respectively, p-value <0.05) and HsLAM–211 treatment in a 5.37±0.23% (N = 7, 6; p-value of 0.004).
We next measured myofiber size using Feret’s minimal diameters, and observed a change in the mean from 23.68µm in PBS treated animals, 18.09µm for HsLAM–111 treatment and 20.76µm in HsLAM–211 treatment (N = 7, 5, 7 respectively; p-value < 0.0001). We observed a decrease in the standard deviations of myofiber size in laminin treated muscles (SD) from 8.35µm in PBS to 7.6µm HsLAM–111 and 7.17µm in HsLAM–211, indicating laminin treatment promoted a reduction in myofiber size variability (Figure 5C). Centrally located nuclei (CLNs) fibers were also quantified as a measure of ongoing repair (data not shown), but showed no differences between vehicle and treatment groups.
Together these data suggest that treatment with HsLAM–111 and HsLAM–211 increased muscle regeneration and reduced fiber diameter variability.
Human Laminin–111 and Laminin–211 differentially affected myogenic cells in Laminin-α2 deficient muscle
To test the effect of HsLAM–111 and HsLAM–211 on muscle repair, we next quantified satellite and myogenic cells in TA muscle. For satellite cells we conducted immunofluorescence for the Paired-box transcription factor 7 (Pax7) and counted the number of Pax7 positive cells located adjacent to the myofiber under the basal lamina (Figure 6A). Treatment with HsLAM–111 resulted in a significant decrease in the numbers of satellite cells from 2.06±0.14 cells per frame in PBS to 1.38±0.19 Pax7 positive cells (N = 8, 6 respectively, p-value <0.05). In contrast, we observed a significant increase in satellite cells in muscle with HsLAM–211 treatment to 2.70±0.17 satellite cells compared to HsLAM–111 or PBS treatments (N = 6, p-value <0.0001 and p<0.05 respectively) (Figure 6B).
To determine if recombinant human Laminin treatment affected myogenic differentiation, we counted myogenin positive cells (Figure 6C). Our results showed no significant decrease of myogenin-positive cells from 8.1±0.79 cells per area in PBS treated animals to 7.56±0.89 in HsLAM–111 treated mice. However, we did see a significant decrease to 4.78±0.95 cells per frame in HsLAM–211 treated mice (N = 6, 7 respectively, p-value of 0.01) (Figure 6D).
Together these results may suggest that Laminin–111 and Laminin–211 isoforms have an effect on muscle repair in Laminin-α2 null muscle, while differentially promoting myogenic cell differentiation.
Human Laminin–111 treatment improves the activity of LAMA2-CMD mice
Previous studies have shown that treatment with EHS derived mouse laminin–111 improves muscle function in the dyW mouse model. To test the human isoform of this biologic, NODScid dyW mice were treated with HsLAM–111, HsLAM–211 or PBS for several weeks. Mice were then subjected to a computer controlled activity assay as previously described [10] (Figure 7A). Results showed a significant increase in distance traveled from a mean of 3394±479cm in PBS treated mice to 5386±281.7cm in HsLAM–111 treatment (Figure 7B) (N = 7, 7 respectively; p-value <0.05), but no increase in HsLAM–211 treated mice with 3211±724.9cm. Resting time showed no significant decrease from a mean of 236.8±54.4 seconds in PBS to 109.8±12.38 seconds in HsLAM–111 and 238.6±41.66 in HsLAM–211 treatment groups (Figure 7C). Finally, HsLAM–111 treated mice showed a significant increase in vertical breaks from 11.2±9.5 in PBS to 50±11.3 in HsLAM–111 treated animals, indicative of increased use of hindlimbs during the assay (Figure 7D) (N = 7, 7; p-value 0.0005). Grip strength was also performed with no significant differences between treatment groups (Supplemental Fig, 5) (N = 6). These data indicate treatment with recombinant human laminin–111 improves mobility of Laminin-α2 deficient mice.