Entry Inhibition and Modulation of Pro-Inflammatory Immune Response Against Influenza A Virus by a Recombinant Truncated Surfactant Protein D

Abstract

Surfactant protein D (SP-D) is expressed in the mucosal secretion of the lung and contributes to the innate host defense against a variety of pathogens, including influenza A virus (IAV). SP-D can inhibit hemagglutination and infectivity of IAV, in addition to reducing neuraminidase (NA) activity via its carbohydrate recognition domain (CRD) binding to carbohydrate patterns (N-linked mannosylated) on NA and hemagglutinin (HA) of IAV. Here, we demonstrate that a recombinant fragment of human SP-D (rfhSP-D), containing homotrimeric neck and CRD regions, acts as an entry inhibitor of IAV and downregulates M1 expression considerably in A549 cells challenged with IAV of H1N1 and H3N2 subtypes at 2 h treatment. In addition, rfhSP-D downregulated mRNA levels of TNF-α, IFN-α, IFN-β, IL-6, and RANTES, particularly during the initial stage of IAV infection of A549 cell line. rfhSP-D also interfered with IAV infection of Madin Darby canine kidney (MDCK) cells through HA binding. Furthermore, rfhSP-D was found to reduce luciferase reporter activity in MDCK cells transduced with H1+N1 pseudotyped lentiviral particles, where 50% of reduction was observed with 10 µg/ml rfhSP-D, suggestive of a critical role of rfhSP-D as an entry inhibitor against IAV infectivity. Multiplex cytokine array revealed that rfhSP-D treatment of IAV challenged A549 cells led to a dramatic suppression of key pro-inflammatory cytokines and chemokines. In the case of pH1N1, TNF-α, IFN-α, IL-10, IL-12 (p40), VEGF, GM-CSF, and eotaxin were considerably suppressed by rfhSP-D treatment at 24 h. However, these suppressive effects on IL-10, VEGF, eotaxin and IL-12 (p40) were not so evident in the case of H3N2 subtype, with the exception of TNF-α, IFN-α, and GM-CSF. These data seem to suggest that the extent of immunomodulatory effect of SP-D on host cells can vary considerably in a IAV subtype-specific manner. Thus, rfhSP-D treatment can downregulate pro-inflammatory milieu encouraged by IAV that otherwise causes aberrant inflammatory cell recruitment leading to cell death and lung damage.

Influenza virus N-linked glycosylation and innate immunity

“…For example, engineered human SP-D enhanced IAV binding and clearance and murine survival in vivo [174]. A recombinant fragment of human SP-D, containing only neck and CRD regions, showed ability to act as virus entry inhibitor as well as ability to down-regulate excessive virus-induced pro-inflammatory responses [175]. Recombinant porcine SP-D (either as the intact protein or as a fragment containing the neck and CRD only) inhibited IAV and IBV infection in epithelial cells of human trachea [94] and A(H3N2) IAV in ex vivo cultures of respiratory tract tissue [176,177], and was more effective than recombinant human SP-D.…”

Complement-Independent Modulation of Influenza A Virus Infection by Factor H

“…pI.18-N2 [N2 from A/Texas/50/2012/(H3N2)] plasmid was a gift from Nigel Temperton (University of Kent). Anti-influenza antibodies used were obtained from BEI Resources, NIAID, NIH, USA, and used as previously described (38); these include polyclonal anti-influenza Virus H3 HA, A/Hong Kong/1/1968 and monoclonal anti-influenza virus H1 HA, A/California /04/2009.…”

“…Post infection, supernatant was subjected to ultra-centrifugation (25,000 × g) for 90 min at 4 • C. Purified viral particles were then resuspended in PBS, and purity of the virus was analyzed by SDS-PAGE and western blotting. Production of pseudotyped particles was carried out, as described earlier (38). Briefly, HEK293T cells were co-transfected with 20 µg of respective IAV pCDNAs, including pcDNA3.1-swineH1-flag (H1 from swine H1N1 A/California/04/09) (Invitrogen), pcDNA3.1-swine N1flag (N1 from swine H1N1 A/California/04/09) (Invitrogen), pcDNA-H3 [H3 from A/Denmark/70/03/(H3N2)], pI.18-N2 [N2 from A/Texas/50/2012/(H3N2)], pHIV-Luciferase backbone (Addgene), and psPAX2 (Addgene).…”

Severity of Coronavirus Disease 2019 (COVID-19): Does Surfactant Matter?

“…In line, SP-A deficient mice exhibited impaired clearance against viruses (e.g., RSV) and showed greater pulmonary infiltration with polymorphonuclear leukocytes (LeVine et al, 1999). Similarly, SP-A inhibited influenza A virus infection of lung epithelial cells (Al-Qahtani et al, 2019), while a recombinant fragment of human SP-D was sufficient to act as an entry inhibitor of influenza A virus in vitro (Al-Ahdal et al, 2018). SP-A protein levels are increased in the serum, plasma and sputum of smokers (Kida et al, 1997;Nomori et al, 1998;Mazur et al, 2011), providing another rationale for the proposed beneficial effects of smoking or nicotine consumption in preventing COVID-19 (Farsalinos et al, 2020a).…”