Endosomal TLRs play an important role in innate immune response as well as in autoimmune processes. In the therapy of systemic lupus erythematosus, antimalarial drugs chloroquine, hydroxychloroquine, and quinacrine have been used for a long time. Their suppression of endosomal TLR activation has been attributed to the inhibition of endosomal acidification, which is a prerequisite for the activation of these receptors. We discovered that chloroquine inhibits only activation of endosomal TLRs by nucleic acids, whereas it augments activation of TLR8 by a small synthetic compound, R848. We detected direct binding of antimalarials to nucleic acids by spectroscopic experiments and determined their cellular colocalization. Further analysis revealed that other nucleic acidbinding compounds, such as propidium iodide, also inhibited activation of endosomal TLRs and colocalized with nucleic acids to endosomes. We found that imidazoquinolines, which are TLR7/8 agonists, inhibit TLR9 and TLR3 even in the absence of TLR7 or TLR8, and their mechanism of inhibition is similar to the antimalarials. In contrast to bafilomycin, none of the tested antimalarials and imidazoquinolines inhibited endosomal proteolysis or increased the endosomal pH, confirming that inhibition of pH acidification is not the underlying cause of inhibition. We conclude that the direct binding of inhibitors to nucleic acids mask their TLRbinding epitope and may explain the efficiency of those compounds in the treatment of autoimmune diseases.

Microbial as well as endogenous nucleic acids are recognized by a group of endosomal Toll-like receptors TLR3, TLR7, TLR8 and TLR9. Recent discoveries significantly improved our understanding of molecular mechanism of their activation and their physiological role. Those include recognition of dsRNA through two nucleic acid binding sites of TLR3 ectodomain, activation of TLR9 by phosphodiester backbone of ssDNA, independent of the nucleotide sequence and phosphorothioate modified bonds, and the role of proteolysis in activation of TLR9. In addition, proteins that chaperone nucleic acids, such as HMGB1 or LL-37, have been described to mediate TLR activation. There is growing evidence that supports involvement of endosomal TLRs in a number of autoimmune diseases, suggesting a therapeutic potential of immunomodulatory endosomal TLR ligands. So far, inhibitory nucleic acids against TLR7, TLR8 and TLR9 as well as small compounds targeting downstream signal transduction of single or several endosomal TLRs have been reported. TLR-targeting drugs have been included in clinical trials as vaccine adjuvants or as therapeutic agents for the treatment of diseases, ranging from cancer, infections, asthma and allergy to autoimmune diseases.

Cysteine cathepsins, an important group of lysosomal proteolytic enzymes [1,2], have been implicated in a number of steps in tumor progression, including processes of cell transformation and differentiation, motility, adhesion, invasion, angiogenesis and metastasis [3,4]. In particular, high activity of cathepsin B has been identified as an important tumor promoting factor capable of degrading proteins of the basement membrane and extracellular matrix (ECM) and enhancing progression of malignant disease. It has been demonstrated that, besides the extracellular cathepsin B, its intracellular fraction is involved in degrading the ECM, which is internalized by tumor cells and exposed to lysosomes [5,6].We and others have demonstrated that inhibitors that are able to enter cells, and thus inactivate lysosomal cathepsin B, effectively reduce ECM degradation and consequently cell invasiveness [6]. However, the uptake by aggressive tumor cells of cathepsin B inhibitors, either small molecules, protein inhibitors or neutralizing monoclonal antibodies, is a rather slow process with very limited final efficacy. A strategy to speed up the uptake and to target the inhibitors to the lysosomes would be most desirable. Cathepsin B, however, possesses several functions with respect to physiological processes of normal cells, such as intracellular protein catabolism, pro-hormone processing and regulation of cytotoxic immunity [7][8][9] Breast cancer cells exhibit excessive proteolysis, which is responsible for extensive extracellular matrix degradation, invasion and metastasis. Besides other proteases, lysosomal cysteine protease cathepsin B has been implicated in these processes and the impairment of its intracellular activity was suggested to reduce harmful proteolysis and hence diminish progression of breast tumors. Here, we present an effective system composed of poly(d,llactide-coglycolide) nanoparticles, a specific anti-cytokeratin monoclonal IgG and cystatin, a potent protease inhibitor, that can neutralize the excessive intracellular proteolytic activity as well as invasive potential of breast tumor cells. The delivery system distinguishes between breast and other cells due to the monoclonal antibody specifically recognizing cytokeratines on the membrane of breast tumor cells. Bound nanoparticles are rapidly internalized by means of endocytosis releasing the inhibitor cargo within the lysosomes. This enables intracellular cathepsin B proteolytic activity to be inhibited, reducing the invasive and metastatic potential of tumor cells without affecting proteolytic functions in normal cells and processes. This approach may be applied for treatment of breast and other tumors in which intracellular proteolytic activity is a part of the process of malignant progression.Abbreviations ECM, extracellular matrix; EDC, 1-ethyl-3-(3-dimethylaminopopyl)carbodiimide hydrocloride; FITC, fluorescein isothiocyanate; PLGA, poly(D,L-lactide-coglycolide); TAA, tumor-associated antigen.