Mechanism of endosomal TLR inhibition by antimalarial drugs and imidazoquinolines.

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 acid-binding 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 TLR-binding epitope and may explain the efficiency of those compounds in the treatment of autoimmune diseases.

Immunonanoparticles--an effective tool to impair harmful proteolysis in invasive breast tumor cells.

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,L-lactide-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.

Therapeutic applications of nucleic acids as ligands for Toll-like receptors.

Intracellular TLRs, represented by TLRs 3, 7, 8 and 9, are specialized for the recognition of different types of microbe-derived nucleic acids. However, endogenous nucleic acids can also activate these TLRs, triggering autoimmunity. Nucleic acid-sensing TLRs initiate innate immune responses upon infection, but these receptors also initiate the development of protective adaptive immune responses. Therefore, TLR stimulation represents an attractive strategy for the development of therapeutic and prophylactic agents targeting microbial infections, cancers and allergies. The current use of stimulatory nucleic acids targeting TLRs is reviewed for applications ranging from vaccine adjuvants to anticancer, antiviral and anti-allergic agents. In addition, inhibitory nucleic acid ligands being evaluated for their ability to ameliorate autoimmune disorders and viremias, such as systemic lupus erythematosus and HIV infection, respectively, are described.