In this study a combination of imaging and material science techniques were utilized to comprehensively scrutinize lung tissue samples, and for the first time thoroughly characterize welding-related NP in situ in lung tissue from welders. Moreover, given the tissue localization and the effects of such NP on macrophages in vitro, the data here strongly suggests that these NP could be responsible at least in part for the pulmonary inflammation observed in welders.
To the best of our knowledge, only a very limited number of studies has been performed in welders, identifying the presence of nanoparticles [19–22]. However, although using the valuable combined (S)TEM/EDX analysis as developed by Abraham and colleagues [23–26], these studies present some major limitations, such as a small number of patient(s) [19, 21], or a very specific exposure (aluminum factory, not representative of the general occupational exposure of welders) . Our study in the other hand is the first to provide persuasive evidence not only of the presence of NP in situ in human lung tissue samples, but also the identification of their chemical composition. As expected given the occupational exposure of the patients, iron was the major element present in excess in the arc welders evaluated in our study. Indeed, if the exact chemical composition of welding fumes is dependent on the material being welded and the electrode, in manual arc welding fumes and in accordance with our findings, the elemental composition is predominantly iron present as metal oxide NP of the form (M, Fe)3O4, where M may be substituted for Mn and Cr essentially [9, 27–30]. These results led us to chemically synthesize 4 different NP, representative of those found in the welders lungs; 2 speciation of pure iron oxide (Fe2O3 and Fe3O4), a mixed Mn/Fe oxide (MnFe2O4), and an oxyhydroxide CrOOH NP. We chose to generate both Fe2O3 and Fe3O4 because, although our data didn’t provide any evidence of the exact speciation of these iron oxides, such species have been largely described in the literature regarding welding fume chemical composition [9, 27, 30]. Moreover, given the observed co-localization of Mn and Fe signals in our lung samples as well as data from the literature , we chose to generate MnFe2O4 NP as a mix metal oxide relevant to welding fume exposure. Finally, we chose to generate CrOOH oxyhydroid NP that present the same + III oxidation state and particle size in the same range as the other NP. Indeed, FeCr2O4 particles can be synthesized but not at the nanoscale, making them of no interest for our study.
We demonstrated that exposure of macrophages to the synthesized representative NP results in the production of a pro-inflammatory secretome, by itself able to further activate the migration of macrophages in vitro. Although we did not conduct experiments to identify the specific protein(s) responsible for the activation of macrophage migration, several candidates present in macrophage pro-inflammatory secretome can be proposed; CCL-2, CCL-3, CXCL-8, as they directly act as efficient monocyte, lymphocyte and neutrophil chemoattractant [32–34]. Moreover, it is important to underline that mediators such as TNF-α and IL-1ß, highly secreted by macrophages in response to NP (Figure 4), could also participate in the perpetuation of inflammation initiated by NP exposure since they have been shown to further induce the expression of chemotactic cytokines such as CCL-2, −3 and CXCL-2, thus sustaining the pulmonary increases of neutrophils, lymphocytes, and macrophages [32, 35, 36]. Similar events have been described in BAL fluid from animals exposed to welding fumes or other dusts [5, 13, 37–42]. These events could happen in welders, given that we were able to detect some of these cytokines in lung tissue sections of the patients analyzed in our study. Not all NP produced the same effects in vitro, although utilized at the same mass concentration; Fe3O4 was overall the less reactive NP, particularly as compared to Fe2O3, which underlines the importance of NP speciation in their individual effects. Such differences in NP effects could be related to different degrees of solubility. However, observation by TEM of 3 months old NP suspensions revealed that all nanoparticles appear similar to those observed in the initial suspension, except for MnFe2O4 nanoparticles that appear slightly amorphous at their surface (data not shown). This most probably indicates a very small or no solubility at all of the nanoparticle of interest, as we should have observed a diminution of the nanoparticle diameter in case of solubility. This is also in accordance with data from literature demonstrating that iron and chromium oxides are very poorly soluble in water [43–47]. Interestingly, we did not observe any synergistic effect between NP when used as a 1:1:1:1 mix, suggesting a common biological mechanism of action. An LPS contamination of our NP could have been responsible for the M1-like phenotype of THP-1 macrophages in vitro. However, the endotoxin content of the NP solutions was evaluated and we didn’t measure any detectable levels of endotoxin, thus ruling out endotoxin contamination (data not shown). Altogether, our data strongly suggests a role for the NP identified in lung tissue samples from welders in the development of their pulmonary inflammation. This could have great implications in other populations of workers occupationally exposed to NP.
As stated previously, the majority of the patients studied here were either current or former smokers which could have been troublesome during the interpretation of the data. Indeed, the presence of NP has been described in cigarette smoke . Therefore, the welding origin of the NP found in welders lungs could be questioned. However, only Al and Si NP were observed in control patients, irrespective of their smoking status, thus ruling out a potential misinterpretation of the findings related to cigarette smoke NP. Moreover, we did not observe any synergistic effect between cigarette smoke and metallic NP exposure in vitro. Although we observed only a limited effect of cigarette smoke exposure alone, this underlines the unique effect of welding-related NP.
NP-loaded macrophages were present not only in the alveolar lumen but also in the fibrous tissue of lung tissue sections from welders. In chronic repetitive injury situations leading to fibrosis development (such as welding fume exposure in an occupational context), it is known that inflammation invariably precedes fibrosis . Since we detected a pro-inflammatory secretome in macrophages exposed to NP, we hypothesized that this secretome could induce fibroblast differentiation into myofibroblasts. Indeed, myofibroblasts represent the major cellular effector of fibrogenic reactions, and fibroblasts have long been considered as the only cell type of origin for myofiboblasts . However, the data here did not indicate any myofibroblastic differentiation. These findings could be explained by the absence of TGF-ß production in NP-exposed macrophage supernatant, as TGF-ß is the main cytokine involved in fibroblasts differentiation . Moreover, one can’t rule out the influence of kinetic aspects of our experimental set-up, where macrophages were exposed for 48 hours, leading to a M1-like pro-inflammatory phenotype, without the possibility of developing a later M2 phenotype, where macrophages could act as the primary effectors of later stages of repair and/or later proliferative and remodeling phases. Beside fibroblasts, fibrocytes and epithelial cells have also been described as sources of myofibroblasts [50, 51]. Their response to supernatant from NP-exposed macrophages could have been interesting to evaluate given the presence of specific cytokines such as CCL-2 and CCL-3. Indeed, CCL-2 is a chemoattractant for fibrocytes , and CCL-3 has been involved in bleomycin-induced recruitment of bone marrow-derived macrophages and fibrocytes, and the subsequent development of pulmonary fibrosis . This should deserve further studies.