Abstract
The soluble C-type lectin surfactant protein (SP)-A mediates lung immune responses partially via its direct effects on alveolar macrophages (AM), the main resident leukocytes exposed to antigens. SP-A modulates the AM threshold of lipopolysaccharide (LPS) activity towards an anti-inflammatory phenotype both in vitro and in vivo through various mechanisms. LPS responses are tightly regulated via distinct pathways including subcellular TLR4 localization and thus ligand sensing. The cytosolic scaffold and signaling protein β-arrestin 2 acts as negative regulator of LPS-induced TLR4 activation. Here we show that SP-A neither increases TLR4 abundancy nor co-localizes with TLR4 in primary AM. SP-A significantly reduces the LPS-induced co-localization of TLR4 with the early endosome antigen (EEA) 1 by promoting the co-localization of TLR4 with the post-Golgi compartment marker Vti1b in freshly isolated AM from rats and wild-type (WT) mice, but not in β-arrestin 2−/− AM. Compared to WT mice pulmonary LPS-induced TNF-α release in β-arrestin 2−/− mice is accelerated and enhanced and exogenous SP-A fails to inhibit both lung LPS-induced TNF-α release and TLR4/EEA1 positioning. SP-A, but not LPS, enhances β-arrestin 2 protein expression in a time-dependent manner in primary rat AM. The constitutive expression of β-arrestin 2 in AM from SP-A−/− mice is significantly reduced compared to SP-A+/+ mice and is rescued by SP-A. Prolonged endosome retention of LPS-induced TLR4 in AM from SP-A−/− mice is restored by exogenous SP-A, and is antagonized by β-arrestin 2 blocking peptides. LPS induces β-arrestin 2/TLR4 association in primary AM which is further enhanced by SP-A. The data demonstrate that SP-A modulates LPS-induced TLR4 trafficking and signaling in vitro and in vivo engaging β-arrestin 2.
Highlights
The soluble human pulmonary C-type lectin, surfactant protein (SP)-A, is the most abundant surfactant-associated protein in the lung with important functions in lung homeostasis in vivo [1,2,3]
The cellular responsiveness to LPS is in part regulated by the cell surface membrane levels of TLR4 that are determined by the amount of TLR4 trafficking from the Golgi to the plasma membrane and the amount of TLR4 internalized into endosomes [5]
Animals and Ethics Statement Primary alveolar macrophages (AM) were obtained from pathogen-free male Sprague-Dawley rats, from C57BL/6J WT mice (Charles River Laboratories), from SP-A2/2 mice generated as described before [27], from b-arrestin22/2 mice kindly provided by Dr Robert Lefkowitz (Duke University) [28] and the corresponding WT C57BL/6 mice (Charles River Laboratories) as described previously [29]
Summary
The soluble human pulmonary C-type lectin, surfactant protein (SP)-A, is the most abundant surfactant-associated protein in the lung with important functions in lung homeostasis in vivo [1,2,3]. The cellular responsiveness to LPS is in part regulated by the cell surface membrane levels of TLR4 that are determined by the amount of TLR4 trafficking from the Golgi to the plasma membrane and the amount of TLR4 internalized into endosomes [5]. TLR4 translocates to the cell surface upon LPS exposure and the LPS/ TLR4/MD2 complex is internalized into early endosomes and delivered to recycling endosomes and Golgi for recycling or to lysosomes for degradation [6,7,8,9]. In a sequential manner TLR4 utilises specific adaptor proteins at the plasma membrane and in early endosomes activating the NF-kB pathway and the type 1 interferon pathway, respectively [5]. The role of tissue-specific microenvironmental factors in regulating intracellular TLR4 localization remains poorly defined
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