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Innate Immune Responses to Influenza Virus Infection
T. Chambers, D. Horohov
Department of Veterinary Sciences
The equine-1 (H7N7) influenza virus possesses the most characteristic molecular feature of the high-pathogenicity avian influenza viruses, i.e. the furin-cleavable connecting peptide between the subunits of the viral surface, protein, hemagglutinin. And indeed equine H7N7 viruses are lethal in chickens and mice-but not in horses. The reason for the difference in pathogenicity of these viruses in the different species is unclear.
Influenza pathogenesis is the outcome of interactions between the host innate immune responses-the first line of defense-and virus-coded factors, the properties of which are not uniform among influenza virus strains. Also, new knowledge is accumulating about the host genes and proteins that are collectively responsible for host innate immune responses.
The purpose of this proposed research is to decipher the basis for the differential pathogenicity of equine-1 influenza virus in different species, and to elucidate further the interaction of influenza factors with some newly recognized players in the innate response.
2011 Project Description
In the final year of the project, both specific aims were pursued.
Specific Aim 1: We did a pilot study to establish a method for analyzing how the molecular structure of influenza viral hemagglutinin (HA) affected the immune responses to virus infection.
We inoculated horses with plasmid DNA constructs expressing HA of equine influenza virus (EIV), and compared lymphocyte proliferation responses both following inoculation and after a follow-up challenge with wild-type EIV. The advantage of this approach is that site-specific mutations in plasmid-expressed HA are easily made. At various times peripheral blood mononuclear cells were isolated and stimulated in vitro with media alone or homologous EIV (Ohio/03 strain), followed by a pulse with tritiated thymidine. Mitogenic stimulation with ConA was used as positive control. Cell cultures were harvested and incorporated thymidine was measured.
Following DNA inoculation, horses did respond with antibody induction to HA. However the differences in mean lymphocyte proliferation values with Treatment (DNA vs negative controls, n=4 each) were not statistically significant. The differences in mean values with Timepoint were also not statistically significant. As expected, by 14 days post-challenge the lymphocyte proliferation index increased in both DNA-inoculated and control groups without statistically significant differences between them.
This pilot study suggested that either the approach is valid but undermined by limitations of measurement, or that HA-DNA does not appreciably stimulate lymphocyte proliferation even though it does stimulate antibody production.
Specific Aim 2: Since cytokines play a critical role in monocyte differentiation into dendritic cells (DC), production of IL-10, IL-12 and TGF-b which are known to influence DC development was analyzed based on their intracellular cytokine mRNA synthesis in mock or influenza virus infected monocytes. Mock infected cells had higher IL-12 mRNA levels than live influenza infected cells, whereas virus infection enhanced IL-10 mRNA transcription by 3-fold over mock infected cells. Another cytokine, TGF-b which favors DC differentiation was significantly down-regulated in influenza virus infected monocytes (p <0.05).
These data indicate that IL-12, a pro-inflammatory cytokine produced by DCs, was down-regulated in influenza virus infected monocytes whereas IL-10, a reciprocal regulatory cytokine to IL-12, was up-regulated which would be favorable for arresting monocyte differentiation into DCs. Other inflammatory cytokines, however, were differently influenced by the viral infection. Influenza virus infection significantly up-regulated gene expression of IFN-a and TNF-a as compared to mock infected monocytes (p <0.05).
This work has been presented at numerous seminars, and portions have been published in Boliar and Chambers 2010, A new strategy of immune evasion by influenza A virus: inhibition of monocyte differentiation into dendritic cells, Vet. Immunol. Immunopathol. 136:201-210. The HA-DNA constructs will be described in a forthcoming publication on their antibody-inducing properties.
As cytokines are largely responsible for the mediation of adjuvant responses, better understanding of influenza-induced cytokine responses will aid in development of more effective vaccines.
We previously showed that equine monocyte-derived DC can be infected by both subtypes of EIV, that infection using H7N7 virus was shown to inhibit monocyte differentiation into DC, and that at least limited virus replication is necessary for complete inhibition of differentiation. The corollary effects on pro-inflammatory cytokine responses to virus infection are shown in our latest work.
Our findings on viral inhibition of monocyte maturation into DC identify a new strategy by influenza virus to block activation of immediate innate and subsequent adaptive immune responses and evade antiviral immunity; and once this viral function is localized in the viral genome/proteome, it too can become a target for future exploitation as anti-influenza therapy.
The pilot study reported here for 2011 was an attempt to develop a technical approach to accomplish this localization, but was unsuccessful and either a different target than lymphocyte proliferation must be measured using this approach, or a different approach will be required.
Adams, A.A., Sturgill, Tracy L., Breathnach, C.C., Chambers, T.M., Siger, L., Minke, J.M., and Horohov, D.W. (2011) Humoral and cell-mediated immune responses of old horses following influenza recombinant canarypox virus vaccination and challenge. Veterinary Immunology and Immunopathology 139:128-140.
Bryant, N.A., Rash, A.S., Woodward, A.L., Medcalf, E., Helwegen, M., Wohlfender, F., Cruz, F., Herrmann, C., Borchers, K., Tiwari, A., Chambers, T.M., Newton, J.R., Mumford, J.A., and Elton, D.M. (2011) Isolation and characterisation of equine influenza viruses (H3N8) from Europe and North America from 2008 to 2009. Veterinary Microbiology 147:19-27.
Lewis, N.S., Daly, J.M., Russell, C.A., Horton, D.L., Skepner, E., Bryant, N.A., Rash, A.S., Mumford, J.A., Wood, J.L.N., Chambers, T.M., Fouchier, R.A.M., Elton, M.D., and Smith, D.J. (2011). The antigenic and genetic evolution of equine influenza A (H3N8) virus from 1968-2007. Journal of Virology (in press).
Chambers, T.M. (2011) Updating equine influenza. Equine Disease Quarterly, v.20 no.4.