Research Accomplishment Reports 2010

Innate Immune Responses to Influenza Virus Infection

T. Chambers, D. Horohov
Department of Veterinary Sciences

 

Non-Technical Summary

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.

2010 Project Description

Progress on both specific aims was made in the fourth year of the project.

Specific Aim 1: Following up on our finding H7N7 equine influenza (EIV), but not H3N8 EIV, produced intense inflammatory cytokine responses in infected Balb/c mice but not in horses, we hypothesized that these intense responses are part of the pathologic mechanism responsible for the high mortality in mice. This is in line with current thinking about the pathologic mechanism of the 1918 Spanish Flu virus that killed 20+ million humans. Therefore we tested whether hyperbaric oxygen treatment (HBOT), which sometimes causes reductions in LPS-induced pro-inflammatory cytokine expression in Balb/c mice, would reduce the high mortality caused by equine H7N7 virus infection in mice. A virus dose of about 1 LD50/mouse was used to infect 80 mice, after which the experimental groups (2x20) received daily 1-hr HBOT treatments.

Virus titrations in mouse lungs showed that compared to untreated controls, initial virus replication was reduced in HBOT-treated mice, but by Day +5 this had reversed. Virus-induced lung neutrophilia was not significantly affected by HBOT. Cytokines in bronchio-alveolar lavages were measured by cytometric bead assay. Early on (Day +1), some pro-inflammatory cytokines (IL-, IL-10, TNF-alpha) were heightened by HBOT treatment, although at later times they were not significantly different from controls. Importantly, overall mortality was increased in the treatment group (12/20), compared to the untreated controls (6/20).

Specific Aim 2: Evasion of cellular innate immune responses at the level of dendritic cells (DC). Here we wished to explore whether inhibition is dependent upon virus subtype, and whether the influenza NS protein, which possesses a variety of host-inhibitory functions, is also responsible for inhibition of monocyte differentiation. An H3N8 equine influenza virus (KY/02) and its NS1-truncated variant (KY/02 NS1-73) were utilized in order to investigate these questions. Similar to the experiments with NY/73 virus, expression of DC markers such as CD11c and CD172a was measured.

The mock infected cells, as expected, enhanced their expression of CD11c significantly (p <0.05) with respect to monocytes. But its expression on both wild type and NS1-truncated virus infected monocytes was either comparable to or less than monocytes and significantly lower than mock infected cells (p<0.05). Similarly, fewer wild type virus (KY/02) infected monocytes expressed CD172a on their surface than mock infected cells. The expression of this myeloid marker was even lower in NS1-truncated virus infected monocytes. Experiments are in progress to evaluate the cytokine expression profiles associated with DC differentiation-arrest. This work was disseminated at 1st International Symposium on Neglected Influenza Viruses, Amelia Island FL, February 5, 2010; American Society for Microbiology annual meeting, San Diego, CA, May 25, 2010; 3rd International Symposium on Emerging Infectious Diseases, Ulaanbaatar, Mongolia. July 29-30, 2010; and Association of Veterinary Microbiologists, 35th annual meeting, Lexington KY, August 6, 2010.

2010 Impact

Specific Aim 1: Contrary to expectation, HBOT treatment exacerbated H7N7 virus mortality instead of reducing it. This may be associated with exacerbation of some pro-inflammatory cytokines early in infection. This does not refute our underlying hypothesis on the pathologic mechanism responsible for H7N7 viral lethality for mice, but shows that if indeed a pro-inflammatory cytokine cascade is responsible, HBOT treatment did not reduce it.

Specific Aim 2: Both H3N8 wild type and NS1-defective influenza viruses were able to hinder monocytes from attaining distinctive DC phenotypes. This shows that, as expected, the DC-inhibition effect is not specific to equine H7N7 virus, and more importantly that it is not mediated by the viral NS1 protein. 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 when this viral function is localized in the viral genome/proteome, it may become a target for future exploitation as anti-influenza therapy.

2010 Publications

Horohov, D.W., Adams, A.A., and Chambers, T.M. (2010). Immunosenescence of the Equine Immune System. J. Comp. Path. 142:S78-S84.

Boliar, S. and Chambers, T.M.(2010). A new strategy of immune evasion by influenza A virus: inhibition of monocyte differentiation into dendritic cells. Vet. Immunol. Immunopathol. 136:201-210.

Lin, C.B., Holland, R.E., Donofrio, J.C., McCoy, M.H., and Chambers, T.M.(2010). Infectivity of equine influenza virus in bovine turbinate cells and cattle. Influenza and Other Respiratory Viruses 4:357-361.

Lu, Z., Dubovi, E.J., Zylich, N.C., Crawford, P.C., Sells, S., Go, Y.Y., Loynachan, A.T., Timoney, P.J., Chambers, T.M., and Balasuriya, U.B.R. (2010). Diagnostic application of the H3N8 specific equine influenza real-time RT-PCR assays for the detection of canine influenza virus in clinical specimens. J. Vet. Diag. Invest. 22:942-945.