Neutrophil Recruitment Is Regulated By Adamts-13 in a Murine Model of Invasive Aspergillosis

Introduction: During inflammation von Willebrand factor (VWF) multimers are secreted as an acute phase protein whereupon the size and the prothrombotic activity play an essential role. The size of VWF multimers is regulated by the specific proteolytic activity of ADAMTS-13 (a disintegrin and metalloprotease with ThromboSpondin type 1 repeats-13) which is diminished under several pathological conditions. Employing a murine model of invasive pulmonary aspergillosis (IPA) we aimed to determine the relevance of this regulatory pathway for innate inflammatory responses and polymorphonuclear neutrophil (PMN) recruitment which is crucial for fungal clearance and survival.

Methods: IPA was induced by intratracheal application of Aspergillus fumigatus (A. fumigatus) conidia in wildtype (129/Sv/Pas) or Adamts13 deficient (Adamts13^-/-) mice. 24 h after infection some mice were sacrificed for analysis of fungal load and histology. To assess fungal load as CFU lung homogenates were plated and cultured on Sabourough agar plates. Paraffin sections of the lungs were prepared and stained with mouse complement component C3d antibody for histological analysis. In addition, degranulation, oxidative burst activity and CD62L shedding were assessed in PMN of wildtype and Adamts13^-/- mice. Chemotactic properties of A. fumigatus-activated and control serum from wildtype and knock-out mice was evaluated by migration of purified human PMN, isolated by dextran sedimentation and Histopaque® centrifugation, in a transwell assay (Corning® HTS Transwell®-96 well permeable support; 3µm).

Results: Adamts13 deficiency in mice was accompanied by a worse outcome of IPA infections compared to wildtype controls. Adamts13^-/- mice displayed more severe signs of disease and a lethal course was observed in about 24% of animals. Compared to Adamts13^-/- mice all neutropenic mice developed lethal IPA after infection, but all wild-type mice survived the disease. Besides, examination of the lungs revealed a higher fungal burden along with increased signs of acute lung injury, complement deposition and levels of pro-inflammatory cytokines in Adamts13^-/- mice. Furthermore a more pronounced perivascular leukocyte infiltration was observed in histological sections indicating a dysregulated inflammatory response in Adamts13^-/- mice. Importantly, the activation of neutrophil effector functions in response to TLR agonist or in PMN-mediated fungal killing of conidia or hyphae revealed no general defect in vitro. Despite enhanced complement deposition in the lungs in Adamts13^-/- mice, PMN migration towards complement-activated serum revealed unaltered chemotactic properties comparing A. fumigatus-activated serum of wildtype and knock-out mice.

Conclusion: In acute inflammatory processes such as fungal pneumonia the proteolytic regulation of VWF by ADAMTS-13 is an important mechanism to control PMN recruitment and outcome of infections.