Olfactory bulb and hypothalamic acute-phase responses to influenza virus: effects of immunization

Abstract

Background: Within hours of intranasal challenge, mouse-adapted H1N1 A/Puerto Rico/8/34 (PR8) influenza genomic RNA is found in the olfactory bulb (OB) and OB pro-inflammatory cytokines are up-regulated. Severing the olfactory tract delays the acute-phase response (APR) and the APR is attenuated by immunization.

Objectives: To determine if immunization affects OB localization of influenza or the molecular brain mechanisms regulating APR.

Methods: Male mice were immunized with PR8 influenza, then OB viral RNA, APR, and influenza-related cytokine responses were determined after homologous viral challenge.

Results: Immunization did not prevent influenza OB viral invasion within 24 h of viral challenge. However, it greatly attenuated OB viral RNA 6 days after viral challenge and the APR including hypothermia and body weight loss responses. Within the OB, 24 h after influenza challenge, prior immunization blocked virus-induced up-regulation of toll-like receptor 7 and interferon (IFN) γ mRNAs. At this time, hypothalamic (HT) growth hormone-releasing hormone receptor and tumor necrosis factor-α mRNAs were greatly enhanced in immunized but not in positive control mice. By 6 days after viral challenge, OB and HT mRNAs returned towards baseline values. In the lung, mRNA up-regulation was greater than that in the brain and maximized 6 days after challenge. Lung IFNγ mRNA decreased at 24 h but increased 6 days after challenge in the positive compared to negative controls. Immunization prevented the up-regulation of most of the flu-related mRNAs measured in lungs.

Conclusion: Collectively, these data suggest a role for OB and HT involvement in immunization protection against influenza infection.

Figure 1. Body weight and core body temperatures after PR8 viral challenge

Body weight was not altered in negative control mice challenged with HI 2.5 × 10⁴ TCID₅₀ PR8 (A). Positive controls had a rapid loss in body weight (days 2–7) and died within 7 days of viral challenge. Immunized mice had a similar body weight response post-challenge (2.5 × 10⁴ TCID₅₀ PR8) as the negative control mice. Negative control and immunized mice showed normal cyclical temperature variation throughout the day and viral challenge did not alter core body temperature in these groups (B). Positive control mice had rapid loss of body temperature after the viral challenge. n = 4–6 per group. (*) = significant difference comparing positive controls to negative controls. On the x-axis, the black bar indicates dark cycle and white indicates light cycle (12 hour light:12 hour dark). Significance was set at p < 0.05.

Figure 2. Plaque Assay for Detection of Antibodies Specific to PR8

Plaque numbers for each treatment group are presented as means ± SE (n = 10 per group). No plaques were formed on the MDCK monolayer when no virus was applied. PR8 induced plaque formation if only virus was applied (i.e. no plasma). Significantly fewer plaques were formed when plasma from immunized mice was applied to the MDCK monolayer compared to when plasma from the other treatment groups was used, indicating the presence of PR8-specific antibodies in the immunized mice. Similar numbers of plaques were found in negative and positive control groups suggesting that plasma from both groups lacked anti-PR8 antibodies. (*) = significant when compared to MDCK monolayers that had no virus applied. (#) = significance when compared to positive control mice. Significance was set at p < 0.05.

Figure 3. Virus-related cytokines in the olfactory bulb, hypothalamus, and lung 24 hours and 6 days post-viral challenge

Influenza virus induced small enhancements of most cytokine mRNAs in the olfactory bulb (OB) (A) of the positive control mice 24 hours post-viral challenge. Immunization had little effect on altering mRNA levels in the OB after viral challenge with the exception of IFNγ and TLR7 mRNAs. In the hypothalamus (HT) (B), the influenza-induced enhancement of TLR7 was attenuated with immunization, although Mx1, TNFα, and GHRHR mRNA levels were enhanced. In the lung (C), immunization inhibited PR8-induced elevations in most of the mRNAs measured; this effect was more pronounced 6 days post-viral challenge. (*) = significant difference compared to negative controls. (+) = significant difference compared to positive controls. Significance was set at p < 0.05.