This paper is from the lab of Dr. Paul C. Roberts, Department of immunology and microbiology, Wayne state university school of medicine, Michigan, USA.
Influenza A virus generally causes mild respiratory illness. Mortality in most cases is due to secondary bacterial super-infections. It has been observed that 44 to 57% of patients hospitalized with influenza virus, test positive for bacterial pneumonia. Streptococcus pneumoniae is one of the leading causes of community acquired pneumonia and also one of the leading causative agents in patients suffering from bacterial and viral co-infections.
Previous studies have shown that infection of mice with S. pneumoniae, who were previously infected with influenza virus showed increased colonization of the bacterium in the respiratory epithelium and decrease neutrophils recruitment. It has been proposed that influenza infection causes unmasking of Pneumococcal receptors by neuraminidase. The other idea is that influenza causes a depression in neutrophils function. However, majority of these previous studies were based on use of mouse adapted influenza A/PR8/34 virus for prior infection. This strain of mice is highly lethal to the mice and it does not mimic the typical mild course of disease progression observed in humans. Thus, in order to monitor the bacterial super-infection in a natural course of influenza infection in humans, the authors used the filamentous influenza A/Udorn/72 virus strain and S. pneumoniae serotype 3. This strain of influenza virus is highly filamentous and results in mild infection. Influenza A virus in general produces both spherical and filamentous virions. It has been shown that primary human isolates of influenza virus are predominantly filamentous. The S. pneumoniae serotype 3 was chosen as it is the most common serotype colonizing adult throats and is also associated with greater risks of death.
The authors found that filamentous influenza virus infection predisposes mice to fatal septicemia following super-infection with S. pneumoniae serotype 3. The authors also observed that high levels of granulocyte colony-stimulating factors (G-CSF) play a major role in synergistic exacerbation of disease.
The results of the experiments are described below:
1. Filamentous influenza A/Udorn/72 and S. pneumoniae serotype 3 both produced a mild, non-lethal infection in BALB/c mice when administered individually
a. Mice infected with A/Udorn/72 did not show overt clinical symptoms associated with influenza.
b. Minimum transient weight loss was observed.
c. Virus replication peaked at day three, followed by gradual clearance by day 4 – 6 and no virus detected by day 7.
d. At no time virus was isolated from brain, kidney or spleen.
e. Similarly, infection with S. pneumoniae serotype 3 resulted in a mild, self limiting infection.
f. Transient bacteremia was observed in30% on mice.
g. Systemic bacterial load declined rapidly and by day 7 bacteria had completely cleared from the body.
2. Type 3 pneumococcus administered during active influenza A/Udron/52 virus infection resulted in fatal septicemia: When mice infected with influenza A/Udorn/72 were super-infected with S. pneumoniae type 3, it was observed that
a. Super-infection at day 3 (peak of viral load) or day 5 (decline of viral load) caused bacteremia in 100% of mice and they died within three days.
b. Significantly higher bacterial loads in all organs including nasal tract and blood were observed in all mice.
c. There was delay in viral clearance from lungs; the viral loads were still detectable in two of the seven mice at day 8 of infection
3. The increase susceptibility to fatal septicemia is maintained well after virus clearance: Mice who were infected with influenza were allowed to recover and at day 14 they were inoculated with S. pneumoniae. This time point was chosen as there were no detectable virus beyond day 7, thus by day 14, mice had completely recovered from influenza.
a. Super-infection at day 14 resulted in 90% fatality rate.
b. Super-infected mice showed symptoms that were identical to those displayed by mice, which were super-infected with S. pneumoniae in presence of an active influenza infection.
c. Super-infected mice (at day 14) also showed higher bacterial loads in comparison to mice infected with pneumococcus alone.
4. Neutrophils dominate the lung inflammatory cell infiltrate in influenza virus- predisposed mice: Inflammatory cell infiltrates obtained by broncheo-alveolar lavage (BAL) were examined in different groups of mice. These groups were singly infected (with either Influenza or pneumococcus), super-infected (at different time points) and control (treated with PBS).
a. BAL fluid from singly infected and control mice largely consisted of alveolar macrophages (AM).
b. BALF from super-infected mice (at day 5) consisted mainly of neutrophils
c. Singly infected mice, however, showed lymphocyte infiltrations; 16% in pneumococcus and 34% in influenza.
d. Similar to mice that were super-infected at day 5, super-infection at day 14 also resulted in significant influx of neutrophils into the lungs.
e. On day 17 post-infection, BALF from singly infected and control mice consisted mostly of AMs, while BAL from super-infected mice at this time point (i.e., day 3 of post super-infection) consisted predominantly of neutrophils (65%).
f. At day 17 post-infection in mice singly infected with influenza, 13% cells were lymphocytes.
5. Histo-pathological analysis confirmed neutrophils accumulation in tissues:
a. Micro-scopical examination of the hemoatoxyin-eosin stained lung sections showed edema in four out of seven and neutrophilic infiltrates in seven out of the seven tissue samples.
b. Lung sections of singly infected mice were same as that of control mice with no pathological changes.
6. Cytokine and chemokine gene expression levels were selectively up-regulated in super-infected mice:
a. In contrast to singly infected and control mice, the levels of pro-inflammatory cytokines (IL-1beta, IL-6, TNF-alpha) were significantly up-regulated in mice which were super-infected at day 5 post influenza.
b. Similarly, gene expression of anti-inflammatory cytokine IL-10 was up-regulated in mice which were super-infected at day 5 post influenza.
c. The chemo-attractants, MIP-1beta, IP-10, MIP-2 and KC were also up-regulated mice which were super-infected at day 5 post influenza.
d. In the brains of super-infected (day 5) mice, the mRNA levels of IL-1beta, MIP-2 and KC were up-regulated.
e. In the spleen of super-infected (day 5) mice, the mRNA levels of IL-6, Il-10 and KC were up-regulated.
f. The mRNA levels of G-CSF were significantly up-regulated in the spleen, lungs and brains of super-infected (day 5) mice.
g. In the lungs of super-infected animals which were super-infected at day 14 post influenza, mRNA levels of IL-6, Il-10 and MIP-2 were up-regulated. However, IL-1beta, TNF-alpha and KC were not significantly up-regulated.
h. The spleens and brains of mice who were super-infected at day 5 post influenza, also showed elevated levels of expression of MIP-2, KC and G-CSF, although the levels of their expression was reduced a factor of 3.
7. Cytokine and chemokine levels were synergistically up-regulated in super-infected mice: Sera and BALF from mice were analyzed for the presence of cytokine and chemokines at day 5 and day 14 post infection.
a. Increase in production of IL-10, IL-6, TNF-alpha and KC was observed in both BAL and sera at both day 5 and day 14.
b. IL-1 beta was found to be elevated in BAL but not in sera of super-infected animals.
c. G-CSF levels were elevated in both sera and BAL from mice super-infected at day 5 and 14.
Conclusions:
When mice are singly infected with either filamentous influenza A or with S. pneumoniae serotype 3, they are able to control the infection.
An active influenza A infection is synergistically exacerbated by super-infecting S. pneumoniae leading to fatal disease.
A prior influenza infection leads to the generation of a “predisposed state” and that active influenza infection is not essential for generation of this state. This predisposed state makes the mice susceptible for subsequent super-infection even after clearance of influenza.
Influenza virus induced lymphocyte filtration may be responsible for the generation of predisposed state leading to exacerbated responses following super-infection.
This predisposed state induced by a prior influenza infection can be maintained for at least 7 to 8 days following viral clearance and results in up-regulated secretion of the anti-inflammatory cytokine IL-10 together with several pro-inflammatory cytokines and especially G-CSF.
No comments:
Post a Comment