Increased inducible nitric oxide synthase expression in organs is associated with a higher severity of H5N1 influenza virus infection

Abstract

Background: The mechanisms of disease severity caused by H5N1 influenza virus infection remain somewhat unclear. Studies have indicated that a high viral load and an associated hyper inflammatory immune response are influential during the onset of infection. This dysregulated inflammatory response with increased levels of free radicals, such as nitric oxide (NO), appears likely to contribute to disease severity. However, enzymes of the nitric oxide synthase (NOS) family such as the inducible form of NOS (iNOS) generate NO, which serves as a potent anti-viral molecule to combat infection in combination with acute phase proteins and cytokines. Nevertheless, excessive production of iNOS and subsequent high levels of NO during H5N1 infection may have negative effects, acting with other damaging oxidants to promote excessive inflammation or induce apoptosis.

Methodology/principal findings: There are dramatic differences in the severity of disease between chickens and ducks following H5N1 influenza infection. Chickens show a high level of mortality and associated pathology, whilst ducks show relatively minor symptoms. It is not clear how this varying pathogenicty comes about, although it has been suggested that an overactive inflammatory immune response to infection in the chicken, compared to the duck response, may be to blame for the disparity in observed pathology. In this study, we identify and investigate iNOS gene expression in ducks and chickens during H5N1 influenza infection. Infected chickens show a marked increase in iNOS expression in a wide range of organs. Contrastingly, infected duck tissues have lower levels of tissue related iNOS expression.

Conclusions/significance: The differences in iNOS expression levels observed between chickens and ducks during H5N1 avian influenza infection may be important in the inflammatory response that contributes to the pathology. Understanding the regulation of iNOS expression and its role during H5N1 influenza infection may provide insights for the development of new therapeutic strategies in the treatment of avian influenza infection.

Figure 1. H5N1 influenza infection results in increased serum NO levels in both chickens and ducks.

Serum collected from chickens and ducks infected with the H5N1 strain Muscovy/duck/Vietnam/453, as assayed for NO production during the peak of influenza infection (chickens at 24 hours, ducks at 24 and 72 hours post infection). Displayed values are the mean of 2 experiments with 4 birds in each group. A single asterisk indicates statistically significant differences of means with p<0.05, double asterisk indicates statistically significant differences of means with p<0.01.

Figure 2. Duck iNOS amino acid sequence alignment.

The open reading frame of the duck iNOS (DKINOS) sequence was analyzed through the Clustal W program and the predicted amino acid translation is shown in comparison to that of human (HUINOS) and chicken (CHINOS) iNOS. An asterisk (*) indicates identical amino acid residues while a colon (:) indicates a strongly conserved amino acid substitution and a dot (.) represents a weakly conserved amino acid substitution. Dashed sections (−) represent gaps introduced to optimize the alignment and numbers represent aa number. The nucleotide sequence was subsequently deposited to GeneBank (accession No. FJ966247).

Figure 3. Unrooted phylogenetic tree of the iNOS gene from a range of species.

An unrooted tree was constructed based on 1000 bootstrapped amino acid alignments of various iNOS members using the neighbour joining method. GeneBank accession numbers: chicken (Gallus gallus) NM_20496, cow (Bos taurus) DQ_676956, mouse (Mus musculus) NM_010927, dog (Canine) NM_001003186, rat (Rattus novegicus) NM_012611, human (Homo sapien) NM_000625, trout (Oncorhynchus mykiss) AJ_300555, pig (Sus suscrofa) NM_001143690.

Figure 4. Comparison of basal iNOS levels in chicken and duck tissues measured by QRT-PCR.

iNOS mRNA was measured by QRT-PCR in chicken and duck organs. GAPDH was used as a housekeeping gene to standardize results. Expression is shown as the basal fold change increase of iNOS as relative to muscle tissue. Experiments were performed in triplicate with the data representative of 3 independent experiments. An asterisk indicates statistically significant differences of means with p<0.05.

Figure 5. Activated chicken and duck splenocytes measured for iNOS expression levels by QRT-PCR.

Chicken and duck splenocytes were cultured for 20 hours with a range of concentrations of LPS (1, 10, and 100 µg/ml) or a single concentration (10 µg/ml) of poly(I:C) or recombinant chicken IFNγ protein. RNA was collected from the cells and quantified iNOS gene levels determined. Obtained iNOS mRNA values were normalized to GAPDH and expression levels are shown as mean fold expression change relative to un-stimulated controls. Experiments were performed in triplicate and data are representative of 3 independent experiments.

Figure 6. NO and iNOS levels increase in a similar fashion following LPS stimulation of chicken and duck splenocytes.

Chicken and duck splenocytes were cultured over a time-course of 6–48 hours with a concentration of LPS (10 µg/mL). (A) NO levels, measured by a colorimetric assay, were increased in a similar fashion in chicken and duck cell culture supernatants. (B) iNOS gene expression levels, measured by QRT-PCR, were increased in chicken and duck splenocytes. Levels of iNOS gene expression were determined in conjunction with the house keeping gene GAPDH and expression levels are shown as mean fold expression change relative to un-stimulated splenocytes. Experiments were performed in triplicate and data are representative of 3 independent experiments. An asterisk indicates statistically significant differences of means with p<0.05.

Figure 7. H5N1 influenza infection increases iNOS expression at higher levels in chickens compared to ducks.

QRT-PCR was carried out on various tissues from chickens and ducks infected with H5N1 and compared to uninfected controls. Chicken samples were taken at 24 hours post infection, duck samples were taken at 72 hours post infection. Data represents the mean fold expression of either duck or chicken iNOS relative to each uninfected tissue type with GAPDH used as the housekeeping gene. Displayed values are the mean of 2 experiments with 4 birds in each group. Single asterisk indicates statistically significant differences of means with p<0.05, double asterisk indicates statistically significant differences of means with p<0.01.

Figure 8. H5N1 infection appears more widespread in chicken tissues in comparison to duck tissues.

IHC for H5N1 antigen, left-hand panel shows staining in chicken tissues, right-hand panel shows staining in duck tissues (A) Chicken lung 24 hours post infection (h.p.i), with IHC stain showing H5N1 viral antigen as red/brown colour. (B) Duck lung 72 h.p.i., H5N1 antigen was less prevalent in duck lung tissue than in chicken. H5N1 antigen was detected in single cells scattered within the lung parenchyma and in the hyaline cartilage. (C) Chicken caecal tonsil 24 h.p.i., with H5N1 in caecal lymphoid follicles and submucosa. (D) Duck caecal tonsil 72 h.p.i., with similar H5N1 antigen staining. (E) Chicken liver tissue 24 h.p.i., with severe H5N1 antigen staining. (F) Duck liver 72 h.p.i., showed no signs of viral antigen. (G) Chicken heart tissue 24 h.p.i. showed H5N1 staining in the myocardium and typically near blood vessels. (H) Duck heart tissue 72 h.p.i., with IHC H5N1 antigen staining the myocardium. All scale bars  = 50 µm. (I) The graph shows viral replication efficiency between chickens and ducks across a range of organs following H5N1 Vt453 infection. In chickens 24 h.p.i, lung, caecal tonsil, liver and heart tissue showed between 5.5 and 7 log₁₀ TCID₅₀. Similarly, ducks had 7 log₁₀ TCID₅₀ of virus in heart tissue, but moderately less virus in other tissues, between 2.5 and 4 log₁₀ TCID₅₀.

Figure 9. During H5N1 Influenza infection iNOS appears more highly expressed in chicken tissues when compared to duck tissues.

IHC for iNOS antigen staining, with left-hand panel showing chicken tissues and right-hand panels showing duck tissues. (A) Chicken lung 24 h.p.i., IHC stain (red/brown) for iNOS mainly in the submucosa. (B) Duck lung 72 h.p.i., IHC iNOS staining in the submucosa. (C) Chicken caecal tonsil 24 h.p.i., with iNOS in lymphoid follicles, the submucosa and lymphoid aggregates. (D) Duck caecal tonsil 72 h.p.i., IHC staining of iNOS mainly in the submucosa. (E) Chicken liver tissue 24 h.p.i., iNOS present in the sinusoidal endothelium. (F) Duck liver 72 h.p.i., little or no iNOS detected. (G) Chicken heart 24 h.p.i., iNOS in the myocardium and proximity to blood vessels (presumably endothelial cells). (H) Duck heart 72 h.p.i., with low levels of IHC staining for iNOS. All scale bars  = 50 µm.