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Formation of reactive oxygen species in granulocytes is not inflammation.

Increasing evidence suggests that moisture and fungal growth in indoor environments contribute to exacerbation and possibly also development of pulmonary disorders. However, there are still considerable knowledge gaps, and studies investigating the causal relationship between moisture/mold exposure and human health effects are highly warranted. Frankel et al. (2014) at the National Research Centre for the Working Environment (Denmark) recently reported in Indoor Air that relative humidity affects aerosolization and inflammatory potential of fungal particles from dust-inoculated gypsum boards. The authors claim that high relative humidity (HRH) gives rise to fewer aerosolized particles but that these are more inflammatory than fungal particles from moistened gypsum boards incubated under low relative humidity (LRH). Although the study by Frankel et al. (2014) presents some interesting results, for instance that fungal particles from gypsum boards incubated at HRH contain higher levels of b-glucan, it is difficult from a toxicological point of view to see that the authors have sufficient support for their conclusions, especially on the particles’ ability to induce inflammation. In their study, moistened gypsum boards were incubated with fungi-containing dust samples for 27 days at HRH or for 21 days at HRH followed by 6 days at LRH (the latter samples referred to only as LRH). The authors observed no difference in distribution of fungal growth on the gypsum boards under the two regimes. However, LRH gave rise to more aerosolized particles and more fungi colony forming units (CFU), while the particles from HRH were smaller and contained more b-glucan. It would have been interesting also to know the b-glucan level on the gypsum boards (i.e. in the fungi prior to aerosolization) and to analyze the aerosolized particles by scanning electron microscopy (SEM), to determine whether the drying process merely affected how and which fungi particles were aerosolized or (as the authors discussed) whether it altered production of reactive compounds by the fungi. Including a negative control (moisture damaged gypsum boards in the absence of fungi) could also have verified that the higher particle level from LRH was not affected by gypsum board particles. That possibility should be considered by the authors in case of a follow-up study. More importantly, to assess inflammatory potential, Frankel et al. (2014) utilized what they claim to be “a cell-based assay that can measure the total inflammatory potential (TIP) of various microbial agents simultaneously”. This TIP assay was apparently developed to assess microbial contamination in medicines and is basically an assay for formation of reactive oxygen species (ROS) in granulocytes (more specifically in differentiated HL-60 cells, a human leukemia derived cell line). ROS formation in granulocytes has limited relevance to assess inflammatory effects and certainly does not reflect the “total inflammatory potential” of a particle. Ideally, to support conclusions about effects on total inflammatory potential, the particles should have been thoroughly tested in vivo. Effects should at least have been assessed on a large array of pro-inflammatory genes or proteins, and even if this had been done, the results would have had considerable limitations as long as the test was performed in a single cell line. Moreover, choosing granulocytes may not be the optimal model as resident macrophages, epithelia cells, and sensory neurons are the primary targets of inhaled noxious compounds. It also appears from the paper that the authors tested and compared effects of low concentrations of HRH particles with high concentrations of LRH particles. Although they observed higher TIP values (ROS levels) for the LRH particles, the relative TIP per number of particles was higher for gypsum boards incubated at HRH. The authors therefore suggest that HRH particles may be more reactive (more “inflammatory”). However, without any evaluation of concentration–effect relationship, it is difficult to conclude with certainty the relative reactivity. As concentration–effect curves tend to be S-shaped, comparing effects from a high concentration of one material with effects from a low concentration of another is problematic. There is a risk of obtaining a relatively lower effect of the high concentration due to saturation of the assay used or the attainment of maximum levels of the biological response. Alternatively, the relative effect of the low concentration could be