An Expandable Mechanopharmaceutical Device (2): Drug Induced Granulomas Maximize the Cargo Sequestering Capacity of Macrophages in the Liver.

PURPOSE: Drug-induced liver injuries (DILI) comprise a significant proportion of adverse drug reactions leading to hospitalizations and death. One frequent DILI is granulomatous inflammation from exposure to harmful metabolites that activate inflammatory pathways of immune cells of the liver, which may act as a barrier to isolate the irritating stimulus and limit tissue damage. METHODS: Paralleling the accumulation of CFZ precipitates in the liver, granulomatous inflammation was studied to gain insight into its effect on liver structure and function. A structural analog that does not precipitate within macrophages was also studied using micro-analytical approaches. Depleting macrophages was used to inhibit granuloma formation and assess its effect on drug bioaccumulation and toxicity. RESULTS: Granuloma-associated macrophages showed a distinct phenotype, differentiating them from non-granuloma macrophages. Granulomas were induced by insoluble CFZ cargo, but not by the more soluble analog, pointing to precipitation being a factor driving granulomatous inflammation. Granuloma-associated macrophages showed increased activation of lysosomal master-regulator transcription factor EB (TFEB). Inhibiting granuloma formation increased hepatic necrosis and systemic toxicity in CFZ-treated animals. CONCLUSIONS: Granuloma-associated macrophages are a specialized cell population equipped to actively sequester and stabilize cytotoxic chemotherapeutic agents. Thus, drug-induced granulomas may function as drug sequestering "organoids" -an induced, specialized sub-compartment- to limit tissue damage.

An Expandable Mechanopharmaceutical Device (1): Measuring the Cargo Capacity of Macrophages in a Living Organism.

PURPOSE: Clofazimine (CFZ) is an FDA-approved, poorly soluble small molecule drug that precipitates as crystal-like drug inclusions (CLDIs) which accumulate in acidic cytoplasmic organelles of macrophages. In this study, we considered CLDIs as an expandable mechanopharmaceutical device, to study how macrophages respond to an increasingly massive load of endophagolysosomal cargo. METHODS: First, we experimentally tested how the accumulation of CFZ in CLDIs impacted different immune cell subpopulations of different organs. Second, to further investigate the mechanism of CLDI formation, we asked whether specific accumulation of CFZ hydrochloride crystals in lysosomes could be explained as a passive, thermodynamic equilibrium phenomenon. A cellular pharmacokinetic model was constructed, simulating CFZ accumulation driven by pH-dependent ion trapping of the protonated drug in the acidic lysosomes, followed by the precipitation of CFZ hydrochloride salt via a common ion effect caused by high chloride concentrations. RESULTS: While lower loads of CFZ were mostly accommodated in lung macrophages, increased CFZ loading was accompanied by organ-specific changes in macrophage numbers, size and intracellular membrane architecture, maximizing the cargo storage capabilities. With increasing loads, the total cargo mass and concentrations of CFZ in different organs diverged, while that of individual macrophages converged. The simulation results support the notion that the proton and chloride ion concentrations of macrophage lysosomes are sufficient to drive the massive, cell type-selective accumulation and growth of CFZ hydrochloride biocrystals. CONCLUSION: CLDIs effectively function as an expandable mechanopharmaceutical device, revealing the coordinated response of the macrophage population to an increasingly massive, whole-organism endophagolysosomal cargo load.

Detecting ordered small molecule drug aggregates in live macrophages: a multi-parameter microscope image data acquisition and analysis strategy.

Following prolonged administration, certain orally bioavailable but poorly soluble small molecule drugs are prone to precipitate out and form crystal-like drug inclusions (CLDIs) within the cells of living organisms. In this research, we present a quantitative multi-parameter imaging platform for measuring the fluorescence and polarization diattenuation signals of cells harboring intracellular CLDIs. To validate the imaging system, the FDA-approved drug clofazimine (CFZ) was used as a model compound. Our results demonstrated that a quantitative multi-parameter microscopy image analysis platform can be used to study drug sequestering macrophages, and to detect the formation of ordered molecular aggregates formed by poorly soluble small molecule drugs in animals.

Interferon regulatory factor-1 in flagellin-induced reprogramming: potential protective role of CXCL10 in cornea innate defense against Pseudomonas aeruginosa infection.

PURPOSE: We previously showed that pre-exposure of the cornea to Toll-like receptor (TLR)5 ligand flagellin induces strong protective innate defense against microbial pathogens and hypothesized that flagellin modulates gene expression at the transcriptional levels. Thus, we sought to determine the role of one transcription factor, interferon regulatory factor (IRF1), and its target gene CXCL10 therein. METHODS: Superarray was used to identify transcription factors differentially expressed in Pseudomonas aeruginosa-challenged human corneal epithelial cells (CECs) with or without flagellin pretreatment. The expression of CXCL10, IRF1, LI-8(CXCL2), and IFNγ was determined by PCR, immunohistochemistry, Western/dot blotting, and/or ELISA. IRF1 knockout mice, CXCL10 and IFNγ neutralization, and NK cell depletion were used to define in vivo regulation and function of CXCL10. The severity of P. aeruginosa was assessed using clinical scoring, slit-lamp microscopy, bacterial counting, polymorphonuclear leukocytes (PMN) infiltration, and macrophage inflammatory protein 2/Chemokine (C-X-C motif) ligand 2 (MIP-2/CXCL2) expression. RESULTS: Flagellin pretreatment drastically affected P. aeruginosa-induced IRF1 expression in human CECs. However, flagellin pretreatment augmented the P. aeruginosa-induced expression of Irf1 and its target gene Cxcl10 in B6 mouse corneas. Irf1 deficiency reduced infection-triggered CXCL10 expression, increased keratitis severity, and attenuated flagellin-elicited protection compared to values in wild-type (WT) controls. CXCL10 neutralization in the cornea of WT mice displayed pathogenesis similar to that of IRF1⁻/⁻ mice. IFNγ receptor neutralization and NK cell depletion prevented flagellin-augmented IRF1 and CXCL10 expression and increased the susceptibility to P. aeruginosa infection in mouse corneas. CONCLUSIONS: IRF1 plays a role in the corneal innate immune response by regulating CXCL10 expression. IFNγ-producing NK cells augment the epithelial expression of IRF1 and CXCL10 and thus contribute to the innate defense of the cornea against P. aeruginosa infection.

Dendritic cell-epithelium interplay is a determinant factor for corneal epithelial wound repair.

The functions of intraepithelial dendritic cells (DCs) are critical for mucosal innate and adaptive immunity, but little is known about the role of tissue-specific DCs in epithelial homeostasis and tissue repair. By using the epithelial debridement wound model and CD11c-diphtheria toxin receptor mice that express a CD11c promoter-driven diphtheria toxin receptor, we showed that DCs migrate along with the epithelial sheet to cover the wound and that local depletion of DCs resulted in a significant delay in epithelial wound closure. In response to wounding, migratory epithelia produce CXCL10, thymic stromal lymphopoietin, and IL-1ß and its antagonist soluble IL-1 receptor antagonist (sIL-1Ra); depletion of corneal DCs reversed their elevated expressions to a different extent, suggesting a DC-mediated positive feedback loop in epithelial gene expression. Furthermore, both CXCL10 and thymic stromal lymphopoietin were localized in migratory epithelia, suggesting that epithelial cells play a key role in DC infiltration and activation in injured corneas. On the other hand, DC depletion resulted in suppressed epithelial AKT activation, increased cell apoptosis, and decreased polymorphonuclear leukocyte infiltration in the healing cornea. These results indicate that DCs and epithelium form a functional entity at mucosal surfaces for maintaining corneal homeostasis and for tissue repair.