Gene Therapy for Occupational Lung Disease: Steering Macrophages in the Right Direction?

Pulmonary fibrosis (PF) has continued to claim the lives and lungs of those diagnosed. The immune and cellular mechanisms underpinning disease progression have proven complicated and difficult to elucidate. One patient spoke of her diagnosis, saying, “we didn’t know how to cope in the early stages. There were lots of scans and biopsies, but frustratingly more questions than answers” (1). In many ways, scientists studying PF echo the sentiments of patients. Recent work in this issue of the Journal from Zhe Zhang and colleagues (pp. 213–227) points to FcγRIIB (Fc γ receptor IIb) as a possible therapeutic target and suggests that gene therapy to prevent pulmonary fibrosis may be a promising avenue for future research (2).

Zhang and colleagues used a preclinical model of silicosis by instilling a silica suspension intratracheally to reliably model the pathology of a fibrosing lung condition. Silicosis is a clinically well-described interstitial lung disease stemming from the inhalation of aerosolized silica. It is often associated with workplace hazards. On spirometry, silicosis typically presents as a mixed restrictive/obstructive lung disease. On radiographs, characteristic egg-shell calcification of silica-containing nodules may be seen. Current therapies depend on presentation and consist of removing the subject from further exposure, avoiding ongoing insults, smoking cessation, and nonspecific treatments such as bronchodilators or glucocorticoids (like prednisolone) (3–5). Because of limited clinical success, several experimental therapies have been attempted. Whole lung lavage was recently promoted with some demonstrable benefit (6, 7). Furthermore, inhibition of IL-1 and TNF, as well as the use of antioxidants and inhibitors of apoptosis, have been proposed and attempted (8, 9). However, mortality from progressive fibrosis remains high without a lung transplant, which remains the best option for patients despite its limited availability and associated risks (10).

Clinical silicosis studies, as well as preclinical models of the disease, have strongly implicated macrophages in disease pathogenesis, given the robust recruitment and apoptosis of macrophages in the disease (11). The subtypes of macrophages involved, and the immune signaling that results in PF and subsequent loss of lung function, are subjects of ongoing research (12). Recent reports suggest that a population of profibrotic, tissue-resident macrophages within the interstitial compartment contribute substantially to the proliferation of fibroblasts in the lung (13, 14). Research has demonstrated the existence of a signaling axis among immune-regulatory cells, interstitial macrophages, and fibroblasts. Finally, macrophage-derived exomes have been linked to the attenuation of fibrosis and improved clinical outcomes, suggesting a possible antifibrotic role for some macrophage populations (15). However, the markers of each macrophage subset, and their role in the immune response to infection and vaccination, as well as in autoimmunity and response to irritants (bleomycin, silica, etc.), demand further research.

As the diverse roles of interstitial and alveolar lung macrophages become more evident, work to modulate their activity for therapeutic means is propitious. The current work by Zhang and colleagues suggests that FcγRIIB is a candidate for the modulation of inflammation, fibrosis, and the decrease in lung function after silica exposure (2). They show after silica exposure that there is consistent upregulation of Fcgr2b mRNA transcripts, as well as the protein itself, in human silicosis samples as well as in their mouse model of induced silicosis. Zhang and colleagues further show colocalization of FcγRIIB and CD68, a macrophage marker, and colocalization of FcγRIIB and Siglec F, an alveolar macrophage (AM) marker, by immunohistochemistry, thus establishing target cells for FcγRIIB action. Using single-cell RNA sequencing, they show altered transcriptional regulation in a further subset of AMs. This establishes a meaningful link between silica exposure, the expression of Fcgr2b, and FcγRIIB localization in a subset of AMs. FcγRIIB is somewhat unique among Fc receptors. Most Fc receptors express an immunoreceptor tyrosine-based activation motif in their cytoplasmic tail, but FcγRIIB encodes an immunoreceptor tyrosine-based inhibitory motif that can inhibit macrophage activation that has been shown to have a role in disease susceptibility and tolerance (16).

Subsequent gene deletion experiments confirmed the antiinflammatory role they hypothesized for FcγRIIB. The authors probed the function of FcγRIIB by examining how its absence, globally induced using an Fcgr2b^−/− mouse, impacted markers of inflammation, fibrosis, and lung function after either intratracheal silica installation or vehicle control. They show that wild-type mice exposed to silica develop significant deficits in lung function, increased markers of inflammation, and more histologically observable fibrosis and inflammation than those treated with the vehicle alone. Furthermore, Fcgr2b^−/− mice exposed to silica fared significantly worse with higher degrees of inflammatory markers, and they exhibited a greater degree of lung inflammation and fibrosis histologically. To better understand the cell-intrinsic effects of FcγRIIB, they replicated the above findings in conditional knockout mice lacking Fcgr2b in cells of myeloid lineage by employing Lyz2-Cre and loxP sites flanking exons 3–5 of Fcgr2b.

Next, the authors build an understanding of the genetic and cellular basis of FcγRIIB’s negative regulation of fibrosis after silica exposure. By employing FACS and quantitative PCR, they were able to compare the expression of pro- and antifibrotic markers in tissue-resident and monocyte-derived AMs. This work indicated that tissue-resident AMs were responsible for increased inflammation after silica exposure, whereas monocyte-derived AMs had a dual role acting to both attenuate and perpetuate inflammation. Using single-cell RNA sequencing, they identified many genes that showed evidence of regulation by FcγRIIB at multiple time points. They narrowed this list down by examining only genes that appeared to be regulated at both 3 and 6 weeks and that had been previously identified in the literature for their involvement in PF. Using this methodology, they determined that Spp1 and Ctss transcription was significantly inhibited by FcγRIIB in AMs.

Having established how FcγRIIB in monocyte-derived AMs may be exerting their antifibrotic and antiinflammatory effects after silica exposure, the authors went on to examine the possibility of targeting FcγRIIB as an antifibrotic therapy. They examined how overexpression of the receptor was protective in two ways. First, they generated a transgenic mouse line that overexpresses FcγRIIB in macrophages by crossing a Lyz2-Cre line with an Fcgr2b transgenic line, and these mice were resistant to the previously demonstrated adverse effects of silica exposure. They next explored whether gene therapy-based overexpression could be achieved by using a recombinant adenoviral vector encoding Fcgr2b. They assessed the efficiency of the transduction using FACS and controlled for the effects of the virus itself and its installation by comparing it to an empty viral vector. After the challenge with silica, mice that received AdFcgr2b developed less fibrosis and inflammation by histological analysis, less compromised lung function (including lessened signs of pulmonary hypertension), and decreased concentrations of inflammatory cytokines in BAL fluid. These studies identify gene therapy as a potential therapy or prophylaxis for silicosis and suggest FcγRIIB as a potential target.

This work opens the door to gene therapy and macrophage modulation in the treatment of silicosis and, from a less translational perspective, underscores the diversity of lung interstitial macrophages. It is exciting to consider the use of gene therapy in occupational lung diseases, and it is tempting to connect the findings here to other fibrosing lung conditions. Ultimately, a single silica exposure is not silicosis as it exists clinically. The model is not of a progressive fibrosing condition in which intervention to halt the progression of fibrosis (without evidence of reversal and restoration of lung function) would be most helpful. The appeal of relevance to idiopathic pulmonary fibrosis has not been fully demonstrated. However, it is only fair to acknowledge that these limitations are illustrative of the difficulty of studying a relatively rare and difficult-to-model set of conditions and should be seen as future directions.