Abstract

A protease–antiprotease imbalance is a feature of many chronic lung diseases, with concentrations and activity of several MMPs (matrix metalloproteinases) correlating with disease pathology. MMP-12 plays a prominent role in lung tissue remodeling owing to its capacity to degrade elastin and other extracellular matrix constituents but is also increasingly recognized for immunomodulatory functions central to the regulation of innate immunity. MMP-12 is elevated in patients with asthma and, in some instances, associated with more severe forms of disease (1, 2). Mouse models of allergic airway disease (AAD) have defined a central role for MMP-12 in driving airway remodeling and recruitment of innate immune cells (3, 4). However, the role of MMP-12 in regulating adaptive immunity in chronic lung diseases has been neglected. In a mouse model of house dust mite (HDM)-induced AAD (Figure 1A) (5), C57BL/6 wild-type (WT) mice showed a strong induction of airwayMMP-12, which was absent inMmp12 (KO) mice (Figure 1B). Although numbers of CD3 T cells were comparable betweenWT and KO animals (Figure 1C), lung B cells were significantly reduced in HDM-treated KO animals (Figure 1D). Numbers of B cells in the lung draining mediastinal lymph nodes (Figure 1E) and peripheral blood (Figure 1F) were not significantly different in allergen-exposedWT and KOmice, indicating the reduced B cell response in KO animals was lung specific. Chronic HDM exposure induces the formation of tertiary lymphoid structures containing ectopic germinal centers in the lungs of mice. Significantly fewer germinal center B cells were present in the lungs of HDM-exposed KOmice relative toWT control mice (Figure 1G), but T follicular helper cell numbers were comparable (Figure 1H). Histological analysis revealed a reduction in the number and size of pulmonary B cell aggregates (Figures 1I–1K), andMMP-12 concentrations correlated with total PAX5 area (r=0.5098; P=0.0013). Interrogation of precision-cut lung slices by confocal microscopy demonstrated the colocalization of germinal center B cells and CD4 T cells (Figure 1L) and validated that the number of B cell aggregates (Figure 1M) and the individual aggregate volume (Figure 1N) were reduced in HDM-exposed KOmice. In keeping with the diminished pulmonary B cell aggregates in HDM-KOmice, concentrations of airway total IgE (HDM-specific IgE below limits of detection) and HDM-specific IgA were also significantly reduced (Figures 1O and 1P). Systemic HDM-specific IgE was also reduced in HDM-treated KOmice (Figure 1Q), whereas IgG1 was comparable toWTs (Figure 1R). AirwayMMP-12 positively correlated with IgE (r=0.612; P, 0.0001) and IgA (r=0.482; P=0.002). Furthermore, mast cell protease-1, a surrogate for IgE-induced mast cell degranulation, was reduced in HDMtreated KOmice relative toWT control mice and showed a significant correlation with IgE (r=0.681; P, 0.0001). These data show that MMP-12 is important for functional B cell aggregate formation and local antibody responses during AAD. CXCL13 (chemokine (C-X-Cmotif) ligand 13) is a B cell chemokine demonstrated to drive aggregate formation in tertiary lymphoid structures in mouse models of AAD (6), and CXCL12 has been demonstrated to be important in distinct lung inflammatory models (7). MMP-12 can modulate the activity of CXC chemokines by proteolytic processing (8). Although lung CXCL13 (Figure 2A) and CXCL12 (Figure 2B) concentrations were comparable between HDM-treatedWT and KOmice, MMP-12 proteolytically cleaves CXCL13, but not CXCL12, in vitro (Figure 2C). Mass spectrometry analysis demonstrated that recombinant CXCL13 (9,812 Da) yields a product of 8,239 Da upon exposure to MMP-12. Deconvolution of these data suggested that MMP-12 cleaves CXCL13 between Ser-94 (P1) and Leu-95 (P1) at the chemokine’s C-terminal domain (Figure 2D). MMP-12 cleavage of CXCL13 was subsequently assessed in vivo in allergen-exposed mice byWestern blot. Antibodies to CXCL13 frequently target its C-terminus and were thus found to bindMMP-12–cleaved CXCL13 with reduced affinity resulting in lower-intensity bands (Figures 2E and 2F). CXCL13 concentrations present in BAL fluid (BALF) and lungs of HDM-treated mice were below the limit of detection byWestern blot (Figures 2E and 2F). When BALF ofWTHDM-exposed mice was incubated ex vivowith CXCL13, it cleaved the chemokine in an analogous manner toMMP12; however, no cleavage was observed with KO BALF (Figure 2E). CXCL13 immunoprecipitations of lung homogenate fromHDMtreatedWT and KOmice were performed to enrich the chemokine. Full-length CXCL13 was detectable in immunoprecipitations of MMP-12 KOmice, whereas the truncated form was detectable inWT immunoprecipitations (Figure 2F). Therefore, MMP-12 cleavage of CXCL13 occurs in vivo in HDM-exposed mice. The excised fragment at the C-terminus of CXCL13 contains a binding site for the glycosaminoglycan heparan sulfate of the extracellular matrix (ECM) (9). Accordingly, MMP-12–cleaved CXCL13 demonstrated a reduced capacity to bind to biotinylated heparan sulfate (Figure 2G). Previous work demonstrated that cathepsin B operates within lymph nodes to cleave CXCL13 at a comparable site to MMP-12 and that ensuing cleavage-dependent This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0. For commercial usage and reprints, please e-mail Diane Gern ([email protected]).