Autophagy, Inflammation and Innate Immunity in Inflammatory Myopathies

<div><p>Autophagy has a large range of physiological functions and its dysregulation contributes to several human disorders, including autoinflammatory/autoimmune diseases such as inflammatory myopathies (IIMs). In order to better understand the pathogenetic mechanisms of these muscular disorders, we sought to define the role of autophagic processes and their relation with the innate immune system in the three main subtypes of IIM, specifically sporadic inclusion body myositis (sIBM), polymyositis (PM), dermatomyositis (DM) and juvenile dermatomyositis (JDM). We found that although the mRNA transcript levels of the autophagy-related genes BECN1, ATG5 and FBXO32 were similar in IIM and controls, autophagy activation in all IIM subgroups was suggested by immunoblotting results and confirmed by immunofluorescence. TLR4 and TLR3, two potent inducers of autophagy, were highly increased in IIM, with TLR4 transcripts significantly more expressed in PM and DM than in JDM, sIBM and controls, and TLR3 transcripts highly up-regulated in all IIM subgroups compared to controls. Co-localization between autophagic marker, LC3, and TLR4 and TLR3 was observed not only in sIBM but also in PM, DM and JDM muscle tissues. Furthermore, a highly association with the autophagic processes was observed in all IIM subgroups also for some TLR4 ligands, endogenous and bacterial HSP60, other than the high-mobility group box 1 (HMGB1). These findings indicate that autophagic processes are active not only in sIBM but also in PM, DM and JDM, probably in response to an exogenous or endogenous ‘danger signal’. However, autophagic activation and regulation, and also interaction with the innate immune system, differ in each type of IIM. Better understanding of these differences may lead to new therapies for the different IIM types.</p></div

Representative micrographs showing immunolocalization of alarmins (A) HMGB1 and (B) HMGB2 in IIM and controls.

<p>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.</p

Co-localization of HLA-DR (MHC class II cell surface receptor) with LC3 in IIM and controls.

<p>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.</p

Endogenous and bacterial HSP60 in IIM and control muscles.

<p>Endogenous HSP60 is highly expressed in all IIM (<b>A–C</b>), 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 (<b>D</b>). Bacterial HSP60 was occasionally observed associated with muscle infiltrates in all IIM muscles (<b>E–G</b>), particularly PM (<b>E</b>) and sIBM (<b>F</b>), but not controls (<b>H</b>). Original magnification x40; bar: 20 µm.</p

Interaction among HSP60, LC3 and TLR4 in IIM and control muscles.

<p>(<b>A</b>) LC3-positive autophagosomes (red) are present in infiltrating cells (white arrow), within muscle fibers (asterisks), and blood vessels (blue arrows) (<b>A</b>, top panel). Vesicles positive for endogenous HSP60 (green) and LC3 (red) are present mainly in association with infiltrates and capillaries (<b>A</b>, top panel). Cells positive for bacterial HSP60 (green) in IIM muscles are also immunopositive for LC3 (red) (<b>A</b>, top panel). (<b>B</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).</p