Clofazimine Biocrystal Accumulation in Macrophages Upregulates Interleukin 1 Receptor Antagonist Production To Induce a Systemic Anti-Inflammatory State

Abstract

Clofazimine (CFZ) is a poorly soluble antibiotic and anti-inflammatory drug indicated for the treatment of leprosy. In spite of its therapeutic value, CFZ therapy is accompanied by the formation of drug biocrystals that accumulate within resident tissue macrophages, without obvious toxicological manifestations. Therefore, to specifically elucidate the off-target consequences of drug bioaccumulation in macrophages, we compared the level of inflammasome activation in CFZ-accumulating organs (spleen, liver and lung) in mice after 2 and 8 weeks of CFZ treatment when the drug exists in soluble and insoluble (biocrystalline) forms, respectively. Surprisingly, the results showed a drastic reduction in caspase 1 and interleukin-1␤ (IL-1␤) cleavage in the livers of mice treated with CFZ for 8 weeks (8-week-CFZ-treated mice) compared to 2-week-CFZ-treated and control mice, which was accompanied by a 3-fold increase in hepatic IL-1 receptor antagonist (IL-1RA) production and a 21-fold increase in serum IL-1RA levels. In the lung and spleen, IL-1␤ cleavage and tumor necrosis factor alpha expression were unaffected by soluble or biocrystal CFZ forms. Functionally, there was a drastic reduction of carrageenan-and lipopolysaccharide-induced inflammation in the footpads and lungs, respectively, of 8-week-CFZ-treated mice. This immunomodulatory activity of CFZ biocrystal accumulation was attributable to the upregulation of IL-1RA, since CFZ accumulation had minimal effect in IL-1RA knockout mice or 2-week-CFZ-treated mice. In conclusion, CFZ accumulation and biocrystal formation in resident tissue macrophages profoundly altered the host's immune system and prompted an IL-1RA-dependent, systemic anti-inflammatory response.

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

“…Following eight weeks of sustained therapy with CFZ, the alveolar macrophages are loaded with large amounts of intracellular crystals of the drug, consistent with previous reports from our laboratory [29]. As a result of the accumulation of intracellular drug complexes, intracellular dichroism increased significantly (0.042 ± 0.013, p<0.01, TwoTailed Student's T-Test), revealing that CLDIs are highly organized, self-assembling supramolecular entities, and that the dichroism of these crystals can be detected within a cell.…”

“…Based off of previous work, it has been shown that CLDIs tend to be found within tissue macrophages, such as Kupffer cells of the liver, red pulp macrophages of the spleen, and alveolar macrophages within the lung [18,28,29]. Due to the extensive accumulation within alveolar macrophages, these cells were chosen to test this multi-parameter imaging and analysis system in live cells.…”

Macrophage-Mediated Clofazimine Sequestration Is Accompanied by a Shift in Host Energy Metabolism

“…2A, H&E), since CFZ exists in the soluble form. 26 However, between 2 and 4 weeks, CFZ crystalizes in the liver 22,26 and by 4 weeks, small granulomas (Mean±S.D. : 6596±2609 μm 2 ) are apparent (Fig.…”

“…2A and C). By 8 weeks, liver CFZ concentration reached its peak 22,26 , which was accompanied by a 2.4-fold increase in total cell number (per field) (Fig. 2C).…”

“…22,26 These CLDI-containing granulomas can modulate the immune response of mice by boosting the production of hepatic interleukin-1 receptor antagonist (IL-1RA), while suppressing inflammasome activation to provide protection from lethal injury. 22,26 The immunomodulatory properties of CLDIs coincide with the onset of macrophage granuloma formation, and therefore, it is reasonable to expect a metabolic consequence considering the relationship between metabolism and immunomodulation. 27–29 Moreover, the identification of host metabolic changes could enhance the mechanistic understanding of the macrophage-mediated drug sequestration response.…”