Intrapulmonary Autoantibodies to HSP72 Are Associated with Improved Outcomes in IPF

Abstract

Rationale: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic interstitial lung disease, with high mortality. Currently, the aetiology and the pathology of IPF are poorly understood, with both innate and adaptive responses previously being implicated in the disease pathogenesis. Heat shock proteins (Hsp) and antibodies to Hsp in patients with IPF have been suggested as therapeutic targets and prognostic biomarkers, respectively. We aimed to study the relationship between the expression of Hsp72 and anti-Hsp72 antibodies in the BAL fluid and serum Aw disease progression in patients with IPF.

Methods: A novel indirect ELISA to measure anti-Hsp72 IgG was developed and together with commercially available ELISAs used to detect Hsp72 IgG, Hsp72 IgGAM, and Hsp72 antigen, in the serum and BALf of a cohort of IPF (n = 107) and other interstitial lung disease (ILD) patients (n = 66). Immunohistochemistry was used to detect Hsp72 in lung tissue. The cytokine expression from monocyte-derived macrophages was measured by ELISA.

Results: Anti-Hsp72 IgG was detectable in the serum and BALf of IPF (n = 107) and other ILDs (n = 66). Total immunoglobulin concentrations in the BALf showed an excessive adaptive response in IPF compared to other ILDs and healthy controls (p = 0.026). Immunohistochemistry detection of C4d and Hsp72 showed that these antibodies may be targeting high expressing Hsp72 type II alveolar epithelial cells. However, detection of anti-Hsp72 antibodies in the BALf revealed that increasing concentrations were associated with improved patient survival (adjusted HR 0.62, 95% CI 0.45-0.85; p = 0.003). In vitro experiments demonstrate that anti-Hsp72 complexes stimulate macrophages to secrete CXCL8 and CCL18.

Conclusion: Our results indicate that intrapulmonary anti-Hsp72 antibodies are associated with improved outcomes in IPF. These may represent natural autoantibodies, and anti-Hsp72 IgM and IgA may provide a beneficial role in disease pathogenesis, though the mechanism of action for this has yet to be determined.

Figure 1

Total IgG and IgA concentrations in the serum and BALf were elevated in IPF. Total IgG was found to be significantly elevated in the serum of IPF patients compared to other ILD patients and healthy controls ((a) p = <0.05). IPF and other ILD patients had elevated IgA compared to healthy controls ((b) p = 0.016). BALf concentrations of IgG were elevated in IPF compared to other ILDs and healthy controls ((c) p = <0.001 and p = <0.0001, respectively; other ILDs to healthy controls not significant by Kruskal-Wallis). BALf concentrations of IgA were elevated in IPF compared to healthy controls ((d) p = 0.026; other ILDs to healthy controls not significant by Kruskal-Wallis). IPF progressors had elevated serum IgG compared to nonprogressors (p = 0.043). No differences were seen between IPF subgroups in serum IgA or BALf IgG or IgA. Statistical analysis between patient cohorts and healthy controls was performed using the Kruskal-Wallis test with Dunn's posttest. Comparison between IPF progressors and nonprogressors was analysed with the Mann-Whitney U test.

Figure 2

Elevated expression of Hsp72 in IPF patients occurred in lung epithelia and coincided with C4d deposition. IPF patient serum and BALf was measured for Hsp72 using a commercial ELISA (IPF n = 82, other ILDs n = 38, healthy controls n = 22). Serum concentrations of Hsp72 were elevated in ILD patients compared to healthy controls ((a) p = 0.002). Hsp72 concentrations in the BALf were normalised to total albumin content, and no difference was seen between ILD and healthy controls ((b) p = 0.059). Immunohistochemistry detection of Hsp72 and C4d was performed on 8 IPF biopsies with UIP ((c) unstained control, (d) Hsp72 detection, and (e) C4d detection). Detection of Hsp72 and C4d was seen in hyperplastic alveolar epithelia (denoted by blue arrows) near areas of fibrotic foci (FF). Statistics was performed using a Kruskal-Wallis test with Dunn's posttest.

Figure 3

Anti-Hsp72 IgG and IgGAM concentrations are elevated in the BALf of IPF patient nonprogressors compared to progressors. Anti-Hsp72 IgG was measured in the serum and BALf using the in-house optimised anti-Hsp72 IgG ELISA and commercial anti-Hsp72 IgGAM ELISA (IPF n = 82, other ILDs n = 46, healthy controls n = 17). BALf concentrations of anti-Hsp72 IgG and IgGAM are standardised to BALf total IgG. Between ILD subgroups and healthy controls, no significant difference was seen in the serum anti-Hsp72 IgG ((a) p = 0.72) or anti-Hsp72 IgGAM ((b) p = 0.24). No significant difference was seen between IPF progressor and nonprogressor patient serum anti-Hsp72 IgG ((a) p = 0.83) or serum anti-Hsp72 IgGAM ((b) p = 0.95) concentrations. Elevated concentrations of BALf anti-Hsp72 IgG ((c) p = 0.014) and anti-Hsp72 IgGAM ((d) p = 0.005) were seen in IPF nonprogressors compared to progressors. Statistical analysis was performed using Kruskal-Wallis with Dunn's posttest and Mann-Whitney U tests.

Figure 4

IPF patients with raised concentrations of BALf anti-Hsp72 IgGAM have longer survival rates. Total BALf IgG, IgA and anti-Hsp72 IgG IgGAM were split between high (red lines) and low (black lines) expression rates by the median value. A Kaplan-Meier survival curve from the date of the BAL sample was made to either death or the census date. No association with survival was seen in total IgG ((a) HR 0.82, 95% CI 0.45-1.49, p = 0.51) or anti-Hsp72 IgG ((c) HR 1.38, 95% CI 0.50-1.74, p = 0.33). Total IgA ((b) HR 2.09, 95% CI 1.01-4.32, p = 0.048) and anti-Hsp72 IgGAM ((d) HR 0.44, 95% CI 0.20-0.92, p = 0.018) associated with improved patient survival. Total IgA and anti-Hsp72 IgGAM were reanalysed using a Cox proportional hazard model adjusting for age, sex, smoking, and percentage predicted VC and T_LCO. After adjustment, total IgA showed no association with patient survival (adjusted HR 0.85, p = 0.77), whilst elevated anti-Hsp72 IgGAM did show a significant association with survival (adjusted HR 0.62, 95% CI 0.45-0.85, p = 0.003).

Figure 5

Hsp72-IgG complexes induce CXCL8 secretion from in vitro MDM culture. MDM cell culture supernatant was analysed for CXCL8 (a) and CCL18 (b) by ELISA after treatment with 1 μg/ml IgG isolated from BALf of IPF patients ± 100 ng/ml Hsp72 (IPF patient n = 6, repeated twice) for 24 hours. 1 μg/ml of BALf IgG did not lead to any overt fold change in CXCL8 secretion in comparison to untreated cells, but preincubation of IgG with Hsp72 led to increased CXCL8 secretion ((a), p = 0.031). A 4-fold increase in MDM CCL18 secretion was seen in response to BALf IgG, with Hsp72 preincubation having no effect ((b), p = 0.84). Statistical tests to compare BALf IgG with preincubated BALf IgG used a Wilcoxon test; no statistical comparison was made with untreated cells.