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. 2015 Dec 8:5:17999.
doi: 10.1038/srep17999.

Early apoptosis of porcine alveolar macrophages limits avian influenza virus replication and pro-inflammatory dysregulation

Pengxiang Chang  1 , Suresh V Kuchipudi  2 , Kenneth H Mellits  3 , Sujith Sebastian  1 , Joe James  4 , Jinhua Liu  5 , Holly Shelton  4 , Kin-Chow Chang  1
Affiliations

Early apoptosis of porcine alveolar macrophages limits avian influenza virus replication and pro-inflammatory dysregulation

Pengxiang Chang et al. Sci Rep. .

Abstract

Pigs are evidently more resistant to avian than swine influenza A viruses, mediated in part through frontline epithelial cells and alveolar macrophages (AM). Although porcine AM (PAM) are crucial in influenza virus control, their mode of control is unclear. To gain insight into the possible role of PAM in the mediation of avian influenza virus resistance, we compared the host effects and replication of two avian (H2N3 and H6N1) and three mammalian (swine H1N1, human H1N1 and pandemic H1N1) influenza viruses in PAM. We found that PAM were readily susceptible to initial infection with all five avian and mammalian influenza viruses but only avian viruses caused early and extensive apoptosis (by 6 h of infection) resulting in reduced virus progeny and moderated pro-inflammation. Full length viral PB1-F2 present only in avian influenza viruses is a virulence factor that targets AM for mitochondrial-associated apoptotic cell death. With the use of reverse genetics on an avian H5N1 virus, we found that full length PB1-F2 contributed to increased apoptosis and pro-inflammation but not to reduced virus replication. Taken together, we propose that early apoptosis of PAM limits the spread of avian influenza viruses and that PB1-F2 could play a contributory role in the process.

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Figures

Figure 1
Figure 1. Avian and mammalian influenza viruses showed comparable early infectivity in PAM.
CD172a (mouse anti-pig CD172a at 1:200 dilution, AbD Serotec) positive PAM as revealed by FITC conjugated secondary antibody (green) (A) co-expressed avian SAαa2,3-Gal (MAAII lectin binding) linked and human SAαa2,6-Gal (SNA lectin binding) linked receptors (B). Blue = DAPI nuclear detection. (C) Immunodetection of viral NP in PAM infected with (1) LPAI H2N3, (2) LPAI H6N1, (3) swine H1N1, (4) USSR H1N1, (5) pandemic H1N1 2009 at 1.0 MOI for 5 h or (6) no virus control showed that all viruses assessed were comparably infective in PAM.
Figure 2
Figure 2. Avian but not mammalian influenza viruses caused severe damage to PAM.
Avian viruses LPAI H2N3 (A,A’) and LPAI H6N1 (B,B’), and mammalian viruses swine H1N1 (C,C’), USSR H1N1 (D,D’) and pandemic H1N1 2009 (E,E’) were used at 1.0 MOI (based on focus forming units [FFU] titration on MDCK cells) to infect PAM for 6 h with mock infection as control (F and F’). (A–F) are bright field images and (A’–F’) are corresponding viral NP immunostained images. By 6 h of infection, widespread damage of PAM (cytopathogenicity, cell fragmentation and blebbings) was evident in PAM infected with avian influenza viruses ((A,B), (A’,B’)). By contrast, PAM infected with mammalian influenza viruses remained visibly normal ((C–E) and (C’–E’)), even beyond 24 h of infection (data not shown).
Figure 3
Figure 3. Avian influenza virus infected PAM displayed rapid apoptosis and reduced progeny virus release.
PAM were infected with avian and mammalian viruses as indicated at MOI of 1.0 for 6 h to determine host responses in (A) caspase 3/7 activation, (B) relative presentation of annexin V and uptake of propidium iodide (PI), (C) messenger RNA expression of Mcl1, and (D) infectious virus release into culture supernatants. PAM infected with avian influenza viruses showed (A) much higher caspase3/7 activation, and (B) more detectable annexin V with little evidence of necrosis as indicated by lack of PI uptake. (C) Marked apoptosis detected in avian virus infected PAM coincided with the down-regulation of Mcl1 (expression normalized to 18S rRNA) by avian H2N3 and H6N1 viruses. (D) Less avian than mammalian progeny virus was released from 6 h infected PAM, as determined by virus titration on MDCK cells with culture supernatants. (E,F) PAM were infected with LPAI H2N3 or LPAI H6N1 virus at 1.0 MOI; after 1 h of initial virus incubation the cells were washed with PBS and incubated in fresh infection culture media with 20 μM Q-VD-OPh, a pan-caspase inhibitor, for a further 5 h. (E) Infected PAM in the presence Q-VD-OPh showed no caspase 3/7 activation and no apparent cytopathic or apoptotic damage, unlike infected DMSO treated PAM. (F) Q-VD-OPh inhibition of apoptosis in PAM increased viable progeny release of H2N3 and H6N1 viruses. Data shown are the means of triplicate wells. Error bar = standard error of mean. *P < 0.05, **P < 0.001 (ANOVA with the use of Prism [GraphPad]).
Figure 4
Figure 4. Avian influenza virus infected PAM appeared more pro-inflammatory than those infected with human and pig influenza viruses.
PAM were infected with influenza viruses as indicated at 1.0 MOI for 6 h. PAM infected with avian influenza viruses exhibited more TNF-α and IL-6 transcription than with mammalian influenza viruses (A,C). Conversely, IL10 expression in PAM was strongly down-regulated by avian but not mammalian influenza viruses (B). There was no clear difference in the induction of IP-10 (D) and IFN-β (E) in PAM between avian and mammalian influenza viruses. Mammalian influenza viruses, however, strongly up-regulated the expression of IFN-inducible genes Mx1 and OAS1 (F,G). All RNA expression results were normalised to 18S rRNA. Data shown are the means of triplicate wells. Error bar = standard error of mean. *P < 0.05, **P < 0.001 (ANOVA).
Figure 5
Figure 5. Contribution of viral PB1-F2 protein to avian influenza promotion of apoptosis and pro-inflammation in PAM.
Recombinant H5N1 (H5N1-w, H5N1-57 and H5N1-del) and mammalian influenza viruses, as indicated, were used to infect PAM at MOI of 1.0 for 6 h. (A) Both truncated (H5N1-57) and deleted (H5N1-del) PB1-F2 mutants showed significantly reduced activation of caspase 3/7 in PAM relative to the wild type virus (H5N1-w) but the levels were not down to the same basal levels as those from mammalian viruses. Fold changes were in relation to mock control. (B,B’) The two PB1-F2 mutants conferred reduced TNF-α induction of RNA (B) and protein (B’) in PAM compared with the wild type virus. (C) H5N1-del mutant also maintained elevated expression of IL-10 in PAM, unlike that of the wild type which suppressed 1L-10 expression. Hence PB1-F2 mutations in H5N1-w virus appeared to dampen apoptotic and pro-inflammatory responses. (D–F) The induction of IL-6, IP-10 and IFN-β by the three recombinant H5N1 viruses, however, followed a less clear-cut pattern. (E) Induction of IL-6 and IP-10 was weakest with the truncated PB1-F2 mutant (H5N1-57), and was similarly high between the deleted mutant (H5N1-del) and wild type virus. H5N1-w (wild type) virus induced the strongest IFN-β response (F) but the induction of OAS1 (G) and Mx1 (H) did not follow the same trend. (I) PB1-F2 mutant viruses in PAM produced less progeny virus than the parental wild type. Data shown are the means of triplicate wells. Error bar = standard error of mean. *P < 0.05, **P < 0.001 (ANOVA).
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