Transforming Growth Factor-β: Activation by Neuraminidase and Role in Highly Pathogenic H5N1 Influenza Pathogenesis

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Abstract
Transforming growth factor-beta (TGF-β), a multifunctional cytokine regulating several immunologic processes, is expressed by virtually all cells as a biologically inactive molecule termed latent TGF-β (LTGF-β). We have previously shown that TGF-β activity increases during influenza virus infection in mice and suggested that the neuraminidase (NA) protein mediates this activation. In the current study, we determined the mechanism of activation of LTGF-β by NA from the influenza virus A/Gray Teal/Australia/2/1979 by mobility shift and enzyme inhibition assays. We also investigated whether exogenous TGF-β administered via a replication-deficient adenovirus vector provides protection from H5N1 influenza pathogenesis and whether depletion of TGF-β during virus infection increases morbidity in mice. We found that both the influenza and bacterial NA activate LTGF-β by removing sialic acid motifs from LTGF-β, each NA being specific for the sialic acid linkages cleaved. Further, NA likely activates LTGF-β primarily via its enzymatic activity, but proteases might also play a role in this process. Several influenza A virus subtypes (H1N1, H1N2, H3N2, H5N9, H6N1, and H7N3) except the highly pathogenic H5N1 strains activated LTGF-β in vitro and in vivo. Addition of exogenous TGF-β to H5N1 influenza virus–infected mice delayed mortality and reduced viral titers whereas neutralization of TGF-β during H5N1 and pandemic 2009 H1N1 infection increased morbidity. Together, these data show that microbe-associated NAs can directly activate LTGF-β and that TGF-β plays a pivotal role protecting the host from influenza pathogenesis. Transforming growth factor-beta (TGF-β) is a multifunctional protein that serves as a global regulator of immunity by controlling the initiation and resolution of inflammatory responses. A pathogen that can regulate TGF-β activation could promote an immune-privileged state for itself within its host. Indeed, multiple parasitic, bacterial, and fungal pathogens successfully evade immune responses by regulating TGF-β. We demonstrate that the neuraminidase proteins from influenza A viruses and Clostridium perfringens convert biologically inactive TGF-β to its active form. Importantly, modulation of TGF-β activity during influenza infection affects viral titers and disease outcome in mice, suggesting that TGF-β plays an important role in influenza pathogenesis, particularly in protecting the host during infection. These studies suggest that neuraminidases from diverse microbes may be able to directly regulate TGF-β, which may in turn play an important role in disease.