Here we have shown that a loss of immune tolerance to a lung self antigen results in spontaneous interstitial lung disease. We used a relatively unbiased approach to identify the lung protein vomeromodulin as a key autoantigen in spontaneous ILD that occurs in several strains of Aireo/o mice. We demonstrated that immune responses directed against VM are sufficient to induce lung autoimmunity and found that VM is expressed within the thymus in an Aire-dependent manner. This places VM among similar organ specific autoantigens previously identified in the Aire-deficient model and illustrates the importance of central tolerance in the prevention of ILD. Our results show how an immune response against a lung antigen can cause ILD both in the setting of multi-organ autoimmunity and as an isolated organ-specific disease. We extended these findings to an APS1 patient with lung disease that histologically mirrors ILD in the mouse and demonstrated autoreactivity to a human antigen that is similar to VM, providing evidence for a potential diagnostic biomarker and therapeutic target in this subset of patients.
To date it has been difficult to conclusively show that autoimmune mechanisms play an important role in ILD pathogenesis. Several studies report the presence of activated lymphocytes in the lung or lavage fluid of ILD patients, but the underlying cause for their presence is unknown (7
). Given the lung's extensive vascular network and exposed mucosal surface, there are numerous factors that may drive immune cells to the lung in ILD. Hypotheses for this range from infectious causes, vascular injury or a hypersensitivity reaction to inhaled antigens (5
). We have now demonstrated that an autoimmune response to a lung self-antigen can provide a sufficient trigger for interstitial lung disease. Characterization of the cells infiltrating the lung showed that many are TH
1 polarized CD4+
T cells (), consistent with a human study that identified a similar phenotype in patients with idiopathic ILD (7
The lymphocyte accumulation in lung resulted in a bronchiolitis and cellular interstitial pneumonia in Aireo/o
mice that was similar to the lung disease in the APS1 patient and in APS1 patients described in the literature (12
). Importantly, these histologic patterns are also frequently seen in other human autoimmune syndromes (3
). With our animal model we can define the natural history of ILD, a task that has been difficult to accomplish in patients. For instance, the airway-centered infiltrates appear to precede interstitial pneumonia in Aireo/o
mice, suggesting that a similar temporal pattern may occur in ILD patients since these features are sometimes seen simultaneously in human disease (4
Aire-mediated control of peripheral tissue antigen expression in the thymus is a fundamental mechanism by which immune tolerance to self-antigens is established. We showed that the thymic expression of our lung autoantigen is controlled by Aire. In Aireo/o
mice, the VM transcript in the thymus was nearly absent (), which likely facilitates the escape of VM-specific T cells from negative selection and their subsequent release into the periphery (). In support of a T cell response to VM in lung disease, we showed that the adoptive transfer of activated VM-specific cells was capable of inducing lung infiltrates. Previous work confirms that Aire-mediated control of tissue-specific self-antigens is critical for the prevention of organ specific autoimmune diseases. We reported in a prior study that loss of the Aire-regulated eye autoantigen, interphotoreceptor retinoid-binding protein (IRBP), in the thymus is sufficient to induce a T cell-mediated autoimmune uveitis (17
). Another study identified the Aire-dependent protein seminal vesicle secretory protein 2 (SVS2) as a primary target of a T cell-mediated autoimmune prostatitis (18
). Despite the different expression patterns of these tissue-specific antigens, a notable feature linking VM, IRBP and SVS2 is that they are all secreted into the extracellular space. We speculate that this commonality is due to their increased accessibility as autoantigens in the periphery, although Aire does not appear to preferentially regulate the transcription of secreted proteins (29
). It should also be noted that the immunogenicity of secreted antigens could potentially be offset by dendritic cells that pick up the protein and traffic to the thymus (31
). Nevertheless, the expression and presentation of tissue antigens to developing thymocytes is an important component of the immune tolerance mechanism mediated by Aire, and this pathway is likely important in the control of autoimmune diseases other than APS1. For example, in Type 1 Diabetes and myasthenia gravis the thymic abundance of the target autoantigens (insulin and the α-subunit of the muscle acetylcholine receptor, respectively) correlates with susceptibility to disease (32
). This same principle may govern the abundance of thymic lung antigens and determine the propensity of APS1 patients and other patients with autoimmune disease to develop ILD.
There are no reports implicating vomeromodulin in autoimmunity and much remains unknown about the protein and its function. Vomeromodulin was discovered in the lateral nasal glands of the rat, where it is secreted into the mucus overlying the respiratory epithelium of the nasal passages, including the vomeronasal organ, and primarily localized to the olfactory epithelium (21
). Given this distribution the authors hypothesized the protein might be involved in pheromone transport to the vomeronasal organ. Similar to VM, the PLUNC proteins are BPI homologs that are secreted into the mucus covering the respiratory epithelium of larger airways. They are thought to have antimicrobial properties and be important for innate immune responses (34
). Recombinant BPI peptides may exhibit anti-fungal properties (34
), and one might speculate that autoimmune responses against PLUNCs may induce a specific immunodeficiency for fungal infections. Indeed, APS1 patients frequently develop candidiasis.
Our work follows a paper that provides evidence for lung-specific autoimmunity in a subset of APS1 patients with antibodies to KCNRG, an antigen that is also expressed in the bronchial epithelium. Nearly all of the patients with autoantibodies to KCNRG had pulmonary disease, defined broadly as respiratory symptoms. Three children underwent lung biopsies, which demonstrated a lymphocytic infiltrate surrounding small and large airways that was similar to the early disease we saw in Aireo/o
). The function of the KCNRG protein is not currently understood; structurally it has little resemblance to VM or LPLUNC1. By immunofluorescence the distribution of KCNRG in the lung mirrors that of both LPLUNC1 and VM. We did not detect autoreactivity to a protein that migrated at the weight of mouse KCNRG on immunoblotting with Aireo/o
serum. We also did not isolate KCNRG from our immunoaffinity purification, and KCNRG does not appear to be a major autoantigen in the mouse system. Nevertheless, it is clear that bronchiolar epithelium is a primary target for autoimmune reactions, and it will be interesting to determine whether APS1 patients with KCNRG reactivity also have autoantibodies to the LPLUNC1 antigen.
Finally, an important outcome of our work is the development of an inducible autoimmune lung disease model through immunization to the self-antigen VM. This system could be utilized similarly to the EAE model of multiple sclerosis. The EAE system has been invaluable in demonstrating the many effector pathways in autoimmune central nervous system disease such as the recently highlighted IL-17 secreting T cells (36
). Our ability to cause lung disease through adjuvant immunization suggests that Aire-mediated tolerance to the VM protein is not complete. The low numbers of VM-specific T cells we identified in wild-type mice () indicates that other tolerance mechanisms may be preventing these cells from becoming activated and causing disease. This may serve as a clue to a process by which even subtle defects in central tolerance can lead to the development of ILD, possibly as a cumulative result of multiple factors including, for instance, a failure of peripheral tolerance mechanisms or a lung insult caused by inhalational injury. Further investigation will be needed to explore the potential role of autoimmune responses to lung antigens in interstitial lung diseases. Taken together, our results here provide a framework by which these diseases may arise and the likely role of central tolerance in preventing them.