Influenza vaccine is able to prevent neuroinflammation triggered by H7N7 IAV infection

Abstract

Influenza A virus (IAV) subtypes are a major cause of illness and mortality worldwide and pose a threat to human health. Although IAV infection is considered a self-limiting respiratory syndrome, an expanded spectrum of cerebral manifestations has been reported following IAV infection. Neurotropic IAVs, such as the H7N7 subtype, are capable of invading the central nervous system (CNS) and replicating in brain cells, resulting in microglia-induced neuroinflammation. Microglial cells, the brain's resident immune cells, are instrumental in the inflammatory response to viral infection. While activation of microglia is important to initially contain the virus, excessive activation of these cells leads to neuronal damage. Previous studies have shown that acute and even long-term IAV-induced neuroinflammation leads to CNS damage. Therefore, the search for possible preventive or therapeutic strategies is of great importance. In this study, we investigated the potential effect of vaccination against acute neuroinflammation induced by H7N7 infection and subsequent neuronal damage in the hippocampus, a particularly vulnerable brain region, comparing young and aged mice. Immunosenescence is one of the striking pathophysiological changes during mammalian aging that leads to "inflammaging" and critically limits the protection by vaccines in the elderly. The results suggest that formalin-inactivated H7N7 vaccine has a preventive effect against the inflammatory responses in the periphery and also in the CNS after H7N7 infection. Cytokine and chemokine levels, increased microglial density, and cell volume after H7N7 infection were all attenuated by vaccination. Further structural analysis of microglial cells also revealed a change in branching complexity after H7N7 infection, most likely reflecting the neuroprotective effect of the vaccination. In addition, synapse loss was prevented in vaccinated mice. Remarkably, engulfment of post-synaptic compartments by microglia can be proposed as the underlying mechanism for spine loss triggered by H7N7 infection, which was partially modulated by vaccination. Although young mice showed better protection against neuroinflammation and the resulting deleterious neuronal effects upon vaccination, a beneficial role of the vaccine was also observed in the brains of older mice. Therefore, vaccination can be proposed as an important strategy to prevent neurological sequelae of H7N7 infection.

Keywords: dendritic spine; inactivated influenza vaccine; influenza infection; microglia; neuroinflammation.

FIGURE 1

Immunization of young and old C57BL/6JRj mice with formalin-inactivated H7N7 IAV vaccine against H7N7 IAV infection. (A) Schematic overview of the immunization schedule for young and old mice followed by infection with 10 FFU influenza A/Seal/Mass/1/80 rSC35M (H7N7) virus. During the peak of infection, brain and lungs were harvested from both young (8dpi) and old (10 dpi) mice for further analysis (Created with BioRender.com). In 2-month-old mice (B) after immune priming, (C) after first immune boost, and (D) after second immune boost, the body weight of the animals was comparable and, after an initial decrease, steadily increased slightly in the following days, although the initial decrease was more pronounced in the vaccinated animals (n = 28–32 animals per group). (E) While the body weight loss was relatively constant after PBS inoculation in both the Ctrl and VAC groups, the body weight of the mice infected with the H7N7 IAV began to decrease uniformly by day 6 post-infection. Between days 6 and 8 post-infection, the body weight of the vaccinated animals returned to the control level, while the Ctrl mice infected with H7N7 IAV lost even more weight (n = 10–14 animals per group). In 15-month-old mice, (F) body weight loss after immune priming was more pronounced in the vaccinated group. This was also the case (G) after the first immune boost and (H) after the second immune boost, but not as pronounced as with immune priming (n = 13–15 animals per group). (I) After H7N7 IAV infection, both Ctrl-PBS and VAC-PBS showed similar changes in body weight. However, both control and vaccinated groups infected with H7N7 IAV showed body weight loss from day 4, which was even significantly greater in the previously vaccinated mice (n = 6–8 animals per group). Data are presented as mean ± SEM and were analyzed with the repeated measures two-way ANOVA followed by Fisher’s LSD test; *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 2

Effects of vaccination on the release of cytokines and chemokines in the lung induced by H7N7 IAV infection. In the lungs of young mice, levels of (A) CCL2 and (B) IFNγ increased after H7N7 IAV infection, whereas vaccination prevented this increase and resulted in a significant decrease in the release of these mediators in infected mice. H7N7 IAV infection led to a significant increase in the levels of (C) IL1β and (D) IL-6 only in the unvaccinated mice. H7N7 IAV infection resulted in increased levels of (E) IL-10 and (F) TNFα in the lungs of young mice, regardless of whether they were vaccinated or not (n = 3–4 animals per group). In the lungs of older mice, H7N7 IAV infection resulted in increased release of (G) CCL2, (H) IFNγ, and (I) IL1β independent of vaccination. CCL2 levels were even higher in the lungs of infected mice that had been previously vaccinated. (J) IL-6 levels increased only in the lungs of old unvaccinated mice after H7N7 IAV infection. (K) IL-10 levels increased only in the lungs of vaccinated mice after infection. H7N7 IAV infection resulted in increased (L) TNFα levels in the lungs of aged mice, whether vaccinated or not (n = 3–4 animals per group). Data are presented as mean ± SEM and were analyzed with an ordinary two-way ANOVA followed by Fisher’s LSD test; *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 3

Effects of vaccination on the release of cytokines and chemokines in the brain induced by H7N7 IAV infection. In the brains of young mice, levels of (A) CCL2, (B) IFNγ, (C) IL1β, (D) IL-6, and (F) TNFα increased after H7N7 IAV infection, whereas vaccination prevented this increase and resulted in a lower release of these mediators in the brain of infected mice. (E) IL-10 was not increased in the brains of H7N7 IAV infected mice, regardless of whether they were vaccinated or not (n = 3–4 animals per group). In the brains of older mice, H7N7 IAV infection resulted in increased release of (G) CCL2, (H) IFNγ, and (I) IL1β independent of vaccination. CCL2 and IFNγ levels were even higher in the brains of infected mice that had been previously vaccinated. Levels of (J) IL-6 and (L) TNFα were higher in the brains of unvaccinated mice after H7N7 IAV infection than in the Ctrl-PBS group. (K) IL-10 levels increased only in the brains of unvaccinated mice after H7N7 IAV infection (n = 3 animals per group). Data are presented as mean ± SEM and were analyzed with an ordinary two-way ANOVA followed by Fisher’s LSD test; *p < 0.05, and **p < 0.01.

FIGURE 4

Effects of vaccination on hippocampal neuroinflammation induced by H7N7 IAV infection. In young mice (A) microglial density in the CA1 subregion of the hippocampus was significantly increased 8 days after H7N7 IAV infection in unvaccinated mice compared with the corresponding control. In vaccinated mice, this increase was not present after H7N7 IAV infection. Vaccination itself did not result in the significant increase in microglial density in CA1 (n = 4–6 animals per group). (B) Infection with the H7N7 IAV resulted in a significant increase in microglial density in the dentate gyrus subregion in unvaccinated mice compared with the control group. This increase was not seen after H7N7 IAV infection in previously vaccinated mice. Vaccination itself did not lead to the significant increase in microglial density in DG (n = 4–6 animals per group). (C) Representative examples of IBA-1 immunostaining in the dentate gyrus subregion at 8 dpi (20X); scale = 50 μm. In aged mice (D) 10 days after H7N7 IAV infection, microglia density in the CA1 subregion of the hippocampus was comparable between unvaccinated infected and PBS-inoculated mice. Microglia density was significantly increased in vaccinated mice receiving PBS compared with unvaccinated mice receiving PBS. However, H7N7 IAV infection did not further increase microglial cell density in vaccinated mice (n = 3–4 animals per group). (E) In the DG subregion, H7N7 IAV infection in unvaccinated mice resulted in increased microglia density compared with corresponding control mice. Microglial density was also significantly increased in this subregion of the hippocampus in vaccinated mice receiving PBS compared with unvaccinated mice receiving PBS. However, infection with H7N7 IAV did not further increase microglial cell density in vaccinated mice (n = 3–4 animals per group). (F) Representative examples of IBA-1 immunostaining in the dentate gyrus subregion at 10 dpi (20X); scale = 50 μm. Data are presented as mean ± SEM and were analyzed with an ordinary two-way ANOVA followed by Fisher’s LSD test; *p < 0.05, **p < 0.01, and **p < 0.01.

FIGURE 5

Effects of vaccination on microglial morphological changes induced by H7N7 IAV infection. In young mice (A) microglial cell volume in the CA1 subregion of the hippocampus was increased 8 days after infection with the H7N7 IAV in both unvaccinated and vaccinated mice compared with the corresponding control groups receiving PBS (n = 4–6 animals per group). (B) Infection with the H7N7 IAV resulted in an increase in the volume of microglial cells in the dentate gyrus subregion in unvaccinated mice compared with the control group. This increase was seen to a lesser extent after infection with the H7N7 IAV in previously vaccinated mice. Vaccination itself did not significantly increase microglial cell volume in either subregion (n = 4–6 animals per group). (C) Representative 3D reconstruction of a microglial cell volume in the dentate gyrus subregion of unvaccinated and vaccinated older animals after infection compared with PBS inoculation; scale = 7 μm. In aged mice, at 10 days after H7N7 IAV infection, microglial cell volume increased in (D) CA1 and (E) DG subregions compared with unvaccinated PBS-inoculated mice. Microglial cell volume was significantly increased in the CA1 subregion of vaccinated mice infected with H7N7 IAV compared with vaccinated mice receiving PBS. Vaccination itself did not significantly alter microglial cell volume in either subregion (n = 3–4 animals per group). (F) Representative 3D reconstruction of soma (green), filaments (pink), and branch points (small green dots in filaments) in the dentate gyrus subregion of unvaccinated and vaccinated older animals after infection compared with PBS inoculation; scale = 7 μm. In young mice, (G) the number of branch points of microglial processes in the CA1 subregion increased after H7N7 IAV infection in previously vaccinated mice compared with vaccinated mice receiving PBS. (H) In the DG subregion, infection induced a higher number of branch points in microglial processes in previously vaccinated mice compared with unvaccinated, infected mice (n = 4–6 animals per group). In aged mice, (I) H7N7 IAV infection resulted in a substantial increase in branch points of microglial processes in the CA1 subregion in previously vaccinated mice compared with vaccinated mice receiving PBS. (J) In the DG subregion, infection resulted in a significantly increased number of branch points of microglial processes in the unvaccinated mice compared with the corresponding control. However, in the vaccinated mice, infection resulted in a decreased number of branch points of microglial processes compared with the unvaccinated infected mice (n = 3–4 animals per group). Vaccination itself did not significantly alter the number of branch points of microglial processes in either subregion of the hippocampus, in either the young or old cohorts. Data are presented as mean ± SEM and were analyzed with an ordinary two-way ANOVA followed by Fisher’s LSD test; *p < 0.05.

FIGURE 6

Effects of vaccination on hippocampal neuron morphology changes caused by H7N7 IAV infection. In young mice (A) the density of dendritic spines in the CA1 subregion of the hippocampus did not change significantly 8 days after infection with the H7N7 IAV in unvaccinated mice compared with the corresponding control group. This was also the case in vaccinated mice. Vaccination itself did not cause the significant changes in dendritic spine density in CA1. (B) Infection with the H7N7 IAV resulted in a significant reduction in dendritic spine density in the dentate gyrus subregion in unvaccinated mice compared with the control group. This significant reduction was not observed after H7N7 IAV infection in previously vaccinated mice. Vaccination itself did not induce the significant changes in dendritic spine density in DG (n = 3–4 animals per group). (C) Representative images of Golgi-Cox staining showing dendrites of granule cells in the dentate gyrus superior subregion of all experimental groups of young mice (63X); scale = 5 μm. In aged mice (D), the dendritic spine density in the CA1 subregion of the hippocampus did not change significantly 10 days after infection with the H7N7 IAV in unvaccinated mice compared to the corresponding control group. This was also the case in vaccinated mice. Vaccination itself did not cause the significant changes in dendritic spine density in CA1. (E) Infection with the H7N7 IAV resulted in a significant reduction in dendritic spine density in the superior subregion of the dentate gyrus in unvaccinated mice compared to controls. This significant reduction was not observed after H7N7 IAV challenge in previously vaccinated mice. Vaccination itself did not lead to significant changes in dendritic spine density in DG (n = 3 animals per group). (F) Representative Golgi-Cox staining images showing dendrites of granule cells in the superior dentate gyrus subregion of all experimental groups of old mice (63X); Scale = 5 µm. Data are presented as mean ± SEM and were analyzed with an ordinary two-way ANOVA followed by Fisher’s LSD test; *p < 0.05.

FIGURE 7

Synaptic engulfment by microglia induced by H7N7 IAV infection and its possible modulation by vaccination. (A) Representative reconstruction of a microglial cell, a lysosomal compartment, and Homer-1 positive postsynaptic terminals from the dentate gyrus hippocampal subregion of aged mice. The entire microglial cell is shown on the left image (Scale = 10 µm), while an enlarged section is shown on the right side. IBA-1 surface was remodeled in IMARIS, as well as LAMP-1 volume inside the IBA-1 and Homer-1 puncta, which were localized inside the LAMP-1 volume inside the IBA-1 surface (Scale = 2 µm). In young mice (B), the LAMP-1 labeled lysosome volume in the microglial cells in the CA1 subregion of the hippocampus was significantly increased 8 days after infection with the H7N7 IAV in unvaccinated mice compared to the corresponding control group. This increase was less pronounced in vaccinated mice. In the CA1 subregion, vaccination itself did not cause significant changes in lysosome volume within the microglial cells of either PBS-inoculated group. (C) No significant changes were observed in the dentate gyrus of young mice after H7N7-IAV challenge, either between unvaccinated mice or between vaccinated mice. Vaccination per se did not also cause significant differences in this subregion (n = 4–6 animals per group). In aged mice, (D) in the CA1 subregion, infection resulted in increased amounts of lysosome volume regardless of whether the mice had been previously vaccinated, although the difference was not statistically significant. (E) In the DG subregion, the volume of lysosomes in microglial cells increased in unvaccinated infected mice, which was not very evident in vaccinated mice. The vaccination itself did not lead to any significant changes (n = 3 animals per group). (F,G) In both hippocampal subregions of young mice, H7N7 IAV infection resulted in increased Homer 1-positive puncta engulfment by microglial cells in unvaccinated mice compared to matched control mice. However, this trend of increase was less pronounced in vaccinated infected mice than in vaccinated mice receiving PBS. Vaccination per se did not lead to any significant trends (n = 4–6 animals per group). In older mice, (H) Homer-1 puncta engulfment by microglial cells in the CA1 subregion did not change in all experimental groups, (I) but in the DG subregion, the number of Homer-1 puncta engulfment by microglial cells showed stronger increasing trends that were absent in vaccinated infected mice. Even in aged mice, vaccination per se did not lead to any significant trends in either subregion. Data are presented as mean ± SEM and were analyzed with an ordinary two-way ANOVA followed by Fisher’s LSD test; *p < 0.05.

FIGURE 8

Effects of vaccination on hippocampal GFAP expression levels induced by H7N7 IAV infection. In young mice (A) GFAP expression in the CA1 subregion of the hippocampus was significantly decreased 8 days after H7N7 IAV infection in unvaccinated mice compared with the corresponding control. This decrease was not present in vaccinated mice after H7N7 IAV infection. Vaccination itself did not significantly decrease GFAP expression levels in CA1 (n = 3 animals per group). (B) Infection with H7N7 IAV resulted in a significant decrease in GFAP expression levels in the dentate gyrus subregion in unvaccinated mice compared with the control group. This decrease was not observed after infection with the H7N7 IAV in previously vaccinated mice. Vaccination itself did not induce the significant changes in DG (n = 3 animals per group). (C) Representative examples of GFAP immunostaining in the dentate gyrus subregion at 8 dpi (20X); scale = 50 μm. In aged mice (D), 10 days after H7N7 IAV infection, GFAP expression in the CA1 subregion of the hippocampus was comparable between unvaccinated infected and PBS-vaccinated mice. GFAP expression was lower in vaccinated mice receiving PBS than in unvaccinated mice receiving PBS. However, infection with H7N7 IAV did not further decrease GFAP expression in vaccinated mice (n = 3 animals per group). (E) In the DG subregion, H7N7 IAV infection in unvaccinated mice resulted in slightly decreased GFAP expression compared to corresponding control mice. GFAP expression was also reduced in this subregion of the hippocampus in vaccinated mice receiving PBS compared with unvaccinated mice receiving PBS. However, infection with H7N7 IAV did not further reduce GFAP expression in vaccinated mice (n = 3 animals per group). (F) Representative examples of GFAP immunostaining in the dentate gyrus subregion at 10 dpi (20X); scale = 50 μm. Data are presented as mean ± SEM and were analyzed with an ordinary two-way ANOVA followed by Fisher’s LSD test; *p < 0.05, **p < 0.01, and **p < 0.01.