Inistration of rAAV6:CMV-hPLAP results in increased expression of pro-inflammatory macrophages markers, and pro-inflammatory signaling pathway activation. (a) TA muscles were injected with the indicated doses of rAAV6:CMV-hPLAP or rAAV6:CMVMCS control. At 14 days post-injection, RNA was extracted from muscle tissue and EMR1 and ITGAX expression was analyzed. *, p,0.05 vs. control. (b) EMR expression, as well as other markers of inflammation, IL-1b, IL-6 and TNFa were analyzed over 14, 21 and 28 days after administration of 16109 genomes of rAAV6:CMV-hPLAP. *, p,0.05 vs. control. (c) The upregulation of IL-6 expression also correlates with increased phosphorylation of Stat3. JNK and IKK-b phosphorylation were also assessed by Western blot *, p,0.05 vs. control. (d) MyoD and miR-206 gene expression, as well as MEF-2 protein levels, were analyzed from tissue harvested 14 or 28 days after vector administration. *, p,0.05 vs. control. doi:10.1371/journal.pone.0051627.gReporter Genes Can Promote Inflammation in MuscleReporter Genes Can Promote Inflammation in MuscleFigure 3. 15481974 Substitution of rAAV6:CMV-hPLAP with a muscle-specific CK6 promoter does not ameliorate the effects of vectormediated hPLAP expression on muscle damage and inflammation (a) Designs of expression cassettes packaged into rAAV6:CMVhPLAP and rAAV6:CK6-hPLAP vectors (b) Vectors were injected into the TA muscles of mice, and their effects examined 14 or 28 days afterwards, using the same methods as in Figure 1. Damage and cellular infiltration was not evident in muscles examined 
14 days after injection with rAAV6:CK6-hPLAP, but was notable by 28 days post-injection. (c) EMR, IL-6 and IL-1b expression were assessed at 14 and 28 days after administration of rAAV6:CMV-MCS, rAAV6:CMV-hPLAP and rAAV6:CK6-hPLAP vectors. *, p,0.05 vs. control (d) Protein was extracted from muscles and phosphorylation levels of inflammatory mediators Stat3, JNK and IKK-b were determined by Western blot analysis. *, p,0.05 vs. control (e) MyoD and miR-206 expression was examined in muscles collected 14 or 28 days after administration of rAAV6:CMV-MCS, rAAV6:CMV-hPLAP and rAAV6:MedChemExpress LED-209 CK6hPLAP vectors. *, p,0.05 vs. control. doi:10.1371/journal.pone.0051627.gPBS for 90 minutes and rinsed in room temperature PBS. Excess liquid was removed from the sections, and NBT/BCIP substrate solution (Sigma) was applied to each section for 30 minutes at room temperature in the dark. Slides were rinsed three times in PBS and cover-slipped with PermountTM mounting media (Fisher Scientific).Results rAAV6:CMV-hPLAP Administration Induces Inflammation in Murine 
Skeletal Muscles in a Dose- and Timedependent MannerhPLAP has been used as a reporter gene in Biotin NHS web previous studies to determine transduction efficiency, and also as an experimental control when investigating the effects of manipulating genes of interest using vector-mediated gene delivery. When transduced into the TA muscles of mice, expression of protein from this transgene induces dose-dependent increases in inflammation and muscle damage which correlate with increases in hPLAP activity 14 days after rAAV6:hPLAP administration (Fig. 1a). Whilst no inflammatory response was observed from the lowest viral genome dose employed here, the local administration of 16109 vector genomes (or higher doses) was associated with marked evidence of cellular infiltration and tissue damage. This effect was also timedependent, as inflammation and tissue damage that was evident 14 days after.Inistration of rAAV6:CMV-hPLAP results in increased expression of pro-inflammatory macrophages markers, and pro-inflammatory signaling pathway activation. (a) TA muscles were injected with the indicated doses of rAAV6:CMV-hPLAP or rAAV6:CMVMCS control. At 14 days post-injection, RNA was extracted from muscle tissue and EMR1 and ITGAX expression was analyzed. *, p,0.05 vs. control. (b) EMR expression, as well as other markers of inflammation, IL-1b, IL-6 and TNFa were analyzed over 14, 21 and 28 days after administration of 16109 genomes of rAAV6:CMV-hPLAP. *, p,0.05 vs. control. (c) The upregulation of IL-6 expression also correlates with increased phosphorylation of Stat3. JNK and IKK-b phosphorylation were also assessed by Western blot *, p,0.05 vs. control. (d) MyoD and miR-206 gene expression, as well as MEF-2 protein levels, were analyzed from tissue harvested 14 or 28 days after vector administration. *, p,0.05 vs. control. doi:10.1371/journal.pone.0051627.gReporter Genes Can Promote Inflammation in MuscleReporter Genes Can Promote Inflammation in MuscleFigure 3. 15481974 Substitution of rAAV6:CMV-hPLAP with a muscle-specific CK6 promoter does not ameliorate the effects of vectormediated hPLAP expression on muscle damage and inflammation (a) Designs of expression cassettes packaged into rAAV6:CMVhPLAP and rAAV6:CK6-hPLAP vectors (b) Vectors were injected into the TA muscles of mice, and their effects examined 14 or 28 days afterwards, using the same methods as in Figure 1. Damage and cellular infiltration was not evident in muscles examined 14 days after injection with rAAV6:CK6-hPLAP, but was notable by 28 days post-injection. (c) EMR, IL-6 and IL-1b expression were assessed at 14 and 28 days after administration of rAAV6:CMV-MCS, rAAV6:CMV-hPLAP and rAAV6:CK6-hPLAP vectors. *, p,0.05 vs. control (d) Protein was extracted from muscles and phosphorylation levels of inflammatory mediators Stat3, JNK and IKK-b were determined by Western blot analysis. *, p,0.05 vs. control (e) MyoD and miR-206 expression was examined in muscles collected 14 or 28 days after administration of rAAV6:CMV-MCS, rAAV6:CMV-hPLAP and rAAV6:CK6hPLAP vectors. *, p,0.05 vs. control. doi:10.1371/journal.pone.0051627.gPBS for 90 minutes and rinsed in room temperature PBS. Excess liquid was removed from the sections, and NBT/BCIP substrate solution (Sigma) was applied to each section for 30 minutes at room temperature in the dark. Slides were rinsed three times in PBS and cover-slipped with PermountTM mounting media (Fisher Scientific).Results rAAV6:CMV-hPLAP Administration Induces Inflammation in Murine Skeletal Muscles in a Dose- and Timedependent MannerhPLAP has been used as a reporter gene in previous studies to determine transduction efficiency, and also as an experimental control when investigating the effects of manipulating genes of interest using vector-mediated gene delivery. When transduced into the TA muscles of mice, expression of protein from this transgene induces dose-dependent increases in inflammation and muscle damage which correlate with increases in hPLAP activity 14 days after rAAV6:hPLAP administration (Fig. 1a). Whilst no inflammatory response was observed from the lowest viral genome dose employed here, the local administration of 16109 vector genomes (or higher doses) was associated with marked evidence of cellular infiltration and tissue damage. This effect was also timedependent, as inflammation and tissue damage that was evident 14 days after.