Figure 1.
Boxplots of relative gene expression values for the autophagy-related genes and TLRs investigated.
Dark horizontal lines represent means, with the box representing the 25th and 75th percentiles, the whiskers the 5th and 95th percentiles, and the dots the outliers. mRNA levels of the autophagy-related genes BECN1, ATG5, and FBXO32 do not differ significantly between groups (IIM and control). By contrast, TLR4 and TLR3 transcripts are upregulated in all IIM subgroups compared to controls; for TLR4, highest expression levels occur in PM and DM muscle (**P<0.01 and ***P<0.001, respectively). PM, polymyositis; sIBM, sporadic inclusion body myositis; DM, dermatomyositis; JDM, juvenile dermatomyositis; CTRL, controls.
Figure 2.
Representative micrographs illustrating localization of TLR4 (A) and TLR3 (B) in IIM and control muscles.
(A) TLR4 is expressed in association with infiltrating cells in endomysial and perimysial spaces, particularly in PM and sIBM (yellow arrows), on capillaries and large blood vessels, mainly in DM and JDM (white arrows), and with the sarcolemma and sarcoplasm of some muscle fibers. TLR4 positivity is also present on some myofibers in the areas of perifascicular atrophy in DM, mainly in JDM (asterisks). (B) TLR3 is present in association with muscle infiltrating cells (yellow arrows), some muscle fibers, particularly atrophic myofibers (asterisks), and vascular endothelial cells (white arrows) in IIM muscle. Weak positivity is also present in control muscle. LC3+ autophagosomes are associated with TLR4+ myofibers (blue arrows) (A), and with TLR4+ and TLR3+ inflammatory infiltrates (orange arrows) (B). Original magnification x40; bar: 20 µm.
Figure 3.
Representative micrographs showing immunolocalization of alarmins (A) HMGB1 and (B) HMGB2 in IIM and controls.
Both HMGB1 and HMGB2 are highly expressed in all IIM samples, mainly in association with immune infiltrates (white arrows), blood vessels (blue arrows), nuclei of myofibers, and occasionally with muscle fiber cytoplasm (white asterisks). Both HMGB1 and 2 co-localize with LC3 in association with muscle infiltrating cells and myofibers (white asterisks). In control muscle LC3 and HMGB1 and HMGB2 staining is weak or absent. Original magnification x40; bar: 20 µm.
Figure 4.
Endogenous and bacterial HSP60 in IIM and control muscles.
Endogenous HSP60 is highly expressed in all IIM (A–C), mainly in association with inflammatory infiltrates (white arrows), vascular endothelial cells (red asterisk), myofibers surrounded or invaded by immune infiltrates (yellow arrow). In control muscles only occasional capillaries were positive for endogenous HSP60 (D). Bacterial HSP60 was occasionally observed associated with muscle infiltrates in all IIM muscles (E–G), particularly PM (E) and sIBM (F), but not controls (H). Original magnification x40; bar: 20 µm.
Figure 5.
Interaction among HSP60, LC3 and TLR4 in IIM and control muscles.
(A) LC3-positive autophagosomes (red) are present in infiltrating cells (white arrow), within muscle fibers (asterisks), and blood vessels (blue arrows) (A, top panel). Vesicles positive for endogenous HSP60 (green) and LC3 (red) are present mainly in association with infiltrates and capillaries (A, top panel). Cells positive for bacterial HSP60 (green) in IIM muscles are also immunopositive for LC3 (red) (A, top panel). (B) Endogenous HSP60 (green) co-expresses with TLR4 (red) on some muscle fibers (asterisks), blood vessels (blue arrows) and in extracellular matrix (yellow arrows). Original magnification x40; bar: 20 µm. Original magnification of the insets x120; bars: 5 µm (PM) and 10 µm (sIBM).
Figure 6.
Co-localization of HLA-DR (MHC class II cell surface receptor) with LC3 in IIM and controls.
HLA-DR positivity is present on the sarcolemma of some muscle fibers (blue arrows), particularly those close to inflammatory infiltrates in PM and sIBM, and in association with vascular endothelial cells, particularly in DM and JDM (asterisks). HLA-DR-positive myofibers contain LC3-positive vesicles (white arrows) or are close to LC3-positive infiltrating cells (blue star). In control muscles, both positivity for HLA-DR and for LC3 is weak or absent. Original magnification x40; bar: 20 µm.