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Alveolar macrophage (AM) responses are commonly induced in inhalation toxicology studies, typically being observed as an increase in number or a vacuolated 'foamy' morphology. Discriminating between adaptive AM responses and adverse events during nonclinical and clinical development is a major scientific challenge. When measuring and interpreting induced AM responses, an understanding of macrophage biology is essential; this includes 'sub-types' of AMs with different roles in health and disease and mechanisms of induction/resolution of AM responses to inhalation of pharmaceutical aerosols. In this context, emerging assay techniques, the utility of toxicokinetics and the requirement for new biomarkers are considered. Risk assessment for nonclinical toxicology findings and their translation to effects in humans is discussed from a scientific and regulatory perspective. At present, when apparently adaptive macrophage-only responses to inhaled investigational products are observed in nonclinical studies, this poses a challenge for risk assessment and an improved understanding of induced AM responses to inhaled pharmaceuticals is required.

BackgroundParticulate matter has been shown to stimulate the innate immune system and induce acute inflammation. Therefore, while nanotechnology has the potential to provide therapeutic formulations with improved efficacy, there are concerns such pharmaceutical preparations could induce unwanted inflammatory side effects. Accordingly, we aim to examine the utility of using the proteolytic activity signatures of cysteine proteases, caspase 1 and cathepsin S (CTSS), as biomarkers to assess particulate-induced inflammation.MethodsPrimary peritoneal macrophages and bone marrow-derived macrophages from C57BL/6 mice and ctss −/− mice were exposed to micro- and nanoparticulates and also the lysosomotropic agent, L-leucyl-L-leucine methyl ester (LLOME). ELISA and immunoblot analyses were used to measure the IL-1β response in cells, generated by lysosomal rupture. Affinity-binding probes (ABPs), which irreversibly bind to the active site thiol of cysteine proteases, were then used to detect active caspase 1 and CTSS following lysosomal rupture. Reporter substrates were also used to quantify the proteolytic activity of these enzymes, as measured by substrate turnover.ResultsWe demonstrate that exposure to silica, alum and polystyrene particulates induces IL-1β release from macrophages, through lysosomal destabilization. IL-1β secretion positively correlated with an increase in the proteolytic activity signatures of intracellular caspase 1 and extracellular CTSS, which were detected using ABPs and reporter substrates. Interestingly IL-1β release was significantly reduced in primary macrophages from ctss −/− mice.ConclusionsThis study supports the emerging significance of CTSS as a regulator of the innate immune response, highlighting its role in regulating IL-1β release. Crucially, the results demonstrate the utility of intracellular caspase 1 and extracellular CTSS proteolytic activities as surrogate biomarkers of lysosomal rupture and acute inflammation. In the future, activity-based detection of these enzymes may prove useful for the real-time assessment of particle-induced inflammation and toxicity assessment during the development of nanotherapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-016-0129-5) contains supplementary material, which is available to authorized users.

Acute and repeated inhalation exposures (for 28 days) to polymeric methylene diphenyl diisocyanate (PMDI) were performed in rats. Investigations were made at the end of exposures and after 3, 10 and 30 days of recovery following single acute exposures and after 30 days of recovery following 28 days of exposure. Acute exposures to 10, 30 or 100 mg m(-3) PMDI produced clinical signs in all animals that were consistent with exposure to irritant aerosols. An exposure concentration-related body weight loss and increase in lung weight were seen post-exposure, with complete recovery by day 8. The time course of changes in the lung over the initial days following exposure consisted of a pattern of initial toxicity, rapid and heavy influx of inflammatory cells and soluble markers of inflammation and cell damage, increased lung surfactant, a subsequent recovery and epithelial proliferative phase and, finally, a return to the normal status quo of the lung. During these stages there was evidence for perturbation of lung surfactant homeostasis, demonstrated by increased amounts of crystalline surfactant and increased number and size of lamellar bodies within type II alveolar cells. Repeated exposure over 28 days to the less toxic concentrations of 1, 4 or 10 mg m(-3) PMDI produced no clinical signs or body weight changes, but an increase in lung weight was seen in animals exposed to 10 mg m(-3), which resolved following the 30-day recovery period. Other effects seen were again consistent with exposure to irritant aerosols, but were less severe than those seen in the acute study. Analysis of bronchoalveolar lavage fluid revealed similar changes to those seen in the acute study. At both 10 and 4 mg m(-3) PMDI increased numbers of 'foamy' macrophages in lung lavage cell pellet correlated with the increased phospholipid content of the pellet. Changes in lung lavage parameters and electron microscopic evidence again suggested perturbations in surfactant homeostasis. Histologically, bronchiolitis and thickening of the central acinar regions was seen at 10 and 4 mg m(-3), reflecting changes in cell proliferation in the terminal bronchioles and centro-acinar regions. Almost all effects seen had recovered by day 30 post-exposure. Both acute and subacute studies demonstrate rapid recovery of effects in the lung following exposure to PMDI, with no progression of these effects even at concentrations higher than those shown to produce tumours in a chronic study. These findings add weight to the hypothesis that pulmonary tumours seen following chronic exposure to PMDI are most likely due to a combination of the chronic irritant effects of repeated exposure, coupled with the presence of insoluble polyureas formed by polymerization of PMDI (found in studies reported here and previous chronic studies), and therefore acute or short-term exposures to PMDI are likely to be of little concern for long-term pulmonary health.

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