The intracellular sensor NLRP3 mediates key innate and healing responses to influenza A virus via the regulation of caspase-1.

Virus-induced interlukin-1beta (IL-1beta) and IL-18 production in macrophages are mediated via caspase-1 pathway. Multiple microbial components, including viral RNA, are thought to trigger assembly of the cryopyrin inflammasome resulting in caspase-1 activation. Here, we demonstrated that Nlrp3(-/-) and Casp1(-/-) mice were more susceptible than wild-type mice after infection with a pathogenic influenza A virus. This enhanced morbidity correlated with decreased neutrophil and monocyte recruitment and reduced cytokine and chemokine production. Despite the effect on innate immunity, cryopyrin-deficiency was not associated with any obvious defect in virus control or on the later emergence of the adaptive response. Early epithelial necrosis was, however, more severe in the infected mutants, with extensive collagen deposition leading to later respiratory compromise. These findings reveal a function of the cryopyrin inflammasome in healing responses. Thus, cryopyrin and caspase-1 are central to both innate immunity and to moderating lung pathology in influenza pneumonia.

Association of RIG-I with innate immunity of ducks to influenza.

Ducks and wild waterfowl perpetuate all strains of influenza viruses in nature. In their natural host, influenza viruses typically cause asymptomatic infection and little pathology. Ducks are often resistant to influenza viruses capable of killing chickens. Here, we show that the influenza virus sensor, RIG-I, is present in ducks and plays a role in clearing an influenza infection. We show evidence suggesting that RIG-I may be absent in chickens, providing a plausible explanation for their increased susceptibility to influenza viruses compared with ducks. RIG-I detects RNA ligands derived from uncapped viral transcripts and initiates the IFN response. In this study, we show that the chicken embryonic fibroblast cell line, DF-1, cannot respond to a RIG-I ligand. However, transfection of duck RIG-I into DF-1 cells rescues the detection of ligand and induces IFN-beta promoter activity. Additionally, DF-1 cells expressing duck RIG-I have an augmented IFN response resulting in decreased influenza replication after challenge with either low or highly pathogenic avian influenza virus. Implicating RIG-I in the antiviral response to an infection in vivo, we found that RIG-I expression is induced 200 fold, early in an innate immune response in ducks challenged with the H5N1 virus A/Vietnam/1203/04. Finding this natural disease resistance gene in ducks opens the possibility of increasing influenza resistance through creation of a transgenic chicken.

Puzzling inefficiency of H5N1 influenza vaccines in Egyptian poultry.

In Egypt, efforts to control highly pathogenic H5N1 avian influenza virus in poultry and in humans have failed despite increased biosecurity, quarantine, and vaccination at poultry farms. The ongoing circulation of HP H5N1 avian influenza in Egypt has caused >100 human infections and remains an unresolved threat to veterinary and public health. Here, we describe that the failure of commercially available H5 poultry vaccines in Egypt may be caused in part by the passive transfer of maternal H5N1 antibodies to chicks, inhibiting their immune response to vaccination. We propose that the induction of a protective immune response to H5N1 is suppressed for an extended period in young chickens. This issue, among others, must be resolved and additional steps must be taken before the outbreaks in Egypt can be controlled.

Fatal outcome of pandemic H1N1 2009 influenza virus infection is associated with immunopathology and impaired lung repair, not enhanced viral burden, in pregnant mice.

Pandemic A (H1N1) 2009 influenza virus (pH1N1) infection in pregnant women can be severe. The mechanisms that affect infection outcome in this population are not well understood. To address this, pregnant and nonpregnant BALB/c mice were inoculated with the wild-type pH1N1 strain A/California/04/09. To determine whether innate immune responses are associated with severe infection, we measured the innate cells trafficking into the lungs of pregnant versus nonpregnant animals. Increased infiltration of pulmonary neutrophils and macrophages strongly correlated with an elevated mortality in pregnant mice. In agreement with this, the product of nitric oxide (nitrite) and several cytokines associated with recruitment and/or function of these cells were increased in the lungs of pregnant animals. Surprisingly, increased mortality in pregnant mice was not associated with higher virus load because equivalent virus titers and immunohistochemical staining were observed in the nasal cavities or lungs of all mice. To determine whether exacerbated inflammatory responses and elevated cellularity resulted in lung injury, epithelial regeneration was measured. The lungs of pregnant mice exhibited reduced epithelial regeneration, suggesting impaired lung repair. Despite these immunologic alterations, pregnant animals demonstrated equivalent percentages of pulmonary influenza virus-specific CD8(+) T lymphocytes, although they displayed elevated levels of T-regulator lymphocytes (Tregs) in the lung. Also, pregnant mice mounted equal antibody titers in response to virus or immunization with a monovalent inactivated pH1N1 A/California/07/09 vaccine. Therefore, immunopathology likely caused by elevated cellular recruitment is an implicated mechanism of severe pH1N1 infection in pregnant mice.

Influenza A virus-induced early activation of ERK and PI3K mediates V-ATPase-dependent intracellular pH change required for fusion.

The vacuolar (H+)-ATPases (V-ATPases) facilitate the release of influenza A virus (IAV) genome into the cytoplasm by acidifying the endosomal interior. The regulation of V-ATPases by signalling pathways has been demonstrated in various model systems. However, little is known about signalling-regulated V-ATPase activation during IAV infection. Here we show that V-ATPase activity is elevated during infection of cell monolayers with IAV, as measured by intracellular pH change, via a mechanism mediated by extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K). Inhibition of IAV-induced early activation of these kinases reduced V-ATPase activity and the acidification of intracellular compartments in infected cells. IAV-activated ERK and PI3K appear to interact directly, and they colocalize with the E subunit of V-ATPase V1 domain. Further, siRNAs targeting the E2 subunit isoform significantly reduced virus titres. Interestingly, suppression of PI3K early activation, but not that of ERK or V-ATPase, negatively affected virus internalization, suggesting the involvement of the pathway in earlier, V-ATPase-independent infection-promoting events. Cell treatment with a V-ATPase-specific inhibitor impaired the nuclear localization of incoming viral ribonucleoproteins, inhibiting replication/transcription of viral RNAs. These findings highlight the importance of IAV-induced ERK and PI3K early activation as signalling mediators in V-ATPase-stimulated endosomal acidification required for fusion.

TNF/iNOS-producing dendritic cells are the necessary evil of lethal influenza virus infection.

Respiratory infection with highly pathogenic influenza A viruses is characterized by the exuberant production of cytokines and chemokines and the enhanced recruitment of innate inflammatory cells. Here, we show that challenging mice with virulent influenza A viruses, including currently circulating H5N1 strains, causes the increased selective accumulation of a particular dendritic cell subset, the tipDCs, in the pneumonic airways. These tipDCs are required for the further proliferation of influenza-specific CD8(+) T cells in the infected lung, because blocking their recruitment in CCR2(-/-) mice decreases the numbers of CD8(+) effectors and ultimately compromises virus clearance. However, diminution rather than total elimination of tipDC trafficking by treatment with the peroxisome proliferator-activated receptor-gamma agonist pioglitazone moderates the potentially lethal consequences of excessive tipDC recruitment without abrogating CD8(+) T cell expansion or compromising virus control. Targeting the tipDCs in this way thus offers possibilities for therapeutic intervention in the face of a catastrophic pandemic.

Changes in H5N1 influenza virus hemagglutinin receptor binding domain affect systemic spread.

The HA of influenza virus is a receptor-binding and fusion protein that is required to initiate infection. The HA receptor-binding domain determines the species of sialyl receptors recognized by influenza viruses. Here, we demonstrate that changes in the HA receptor-binding domain alter the ability of the H5N1 virus to spread systemically in mice. The A/Vietnam/1203/04 (VN1203) and A/Hong Kong/213/03 (HK213) viruses are consistently lethal to domestic chickens but differ in their pathogenicity to mammals. Insertion of the VN1203 HA and neuraminidase (NA) genes into recombinant HK213 virus expanded its tissue tropism and increased its lethality in mice; conversely, insertion of HK213 HA and NA genes into recombinant VN1203 virus decreased its systemic spread and lethality. The VN1203 and HK213 HAs differ by 10 aa, and HK213 HA has shown greater binding affinity for synthetic alpha2,6-linked sialyl receptor. Introduction of an S227N change and removal of N-linked glycosylation at residue 158 increased the alpha2,6-binding affinity of VN1203 HA. Recombinant VN1203 virus carrying the S227N change alone or with the residue-158 glycosylation site removed showed reduced lethality and systemic spread in mice but not in domestic chickens. Wild-type VN1203 virus exhibited the greatest efficiency in systemic spread after intramuscular inoculation and in infection of mouse bone marrow-derived dendritic cells and conventional pulmonary dendritic cells. These results show that VN1203 HA glycoprotein confers pathogenicity by facilitating systemic spread in mice; they also suggest that a minor change in receptor binding domain may modulate the virulence of H5N1 viruses.

Three amino acid changes in PB1-F2 of highly pathogenic H5N1 avian influenza virus affect pathogenicity in mallard ducks.

Despite reports that the PB1-F2 protein contributes to influenza virus pathogenicity in the mouse model, little is known about its significance in avian hosts. In our previous study, the A/Vietnam/1203/04 (H5N1) wild-type virus (wtVN1203) was more lethal to mallard ducks than a reverse genetics (rg)-derived VN1203. In search of potential viral factors responsible for this discrepancy, we found that synonymous mutations (SMs) had been inadvertently introduced into three genes of the rgVN1203 (rgVN1203/SM-3). Of 11 SMs in the PB1 gene, three resided in the PB1-F2 open reading frame, caused amino acid (aa) substitutions in the PB1-F2 protein, and reduced virus lethality in mallard ducks. The wtVN1203 and recombinant viruses with repairs to these three aa's (rgVN1203/R-PB1-F2) or with repairs to all 11 SMs (rgVN1203/R-PB1) were significantly more pathogenic than rgVN1203/SM-3. In cultured cells, repairing three mutations in PB1-F2 increased viral polymerase activity and expression levels of viral RNA.

Duck innate immune responses to high and low pathogenicity H5 avian influenza viruses.

Ducks are the reservoir host of influenza A viruses, and are permissive for replication of most strains, yet can elicit robust innate immune responses to highly pathogenic strains. Tissue tropism and viral amino acid differences affect virulence, but we have limited knowledge about how viral differences influence the host innate immune response. Here we compare the innate immune response in Pekin ducks to a recombinant highly-pathogenic avian influenza (HPAI) H5N1 virus and a naturally arising attenuated variant of this strain that differs at one amino acid in polymerase A (T515A), as well as ducks infected with two different H5 strains of low pathogenic avian influenza (LPAI). Using qPCR we examined the relative abundance of transcripts for RIG-I and interferon-beta (IFNß), and downstream interferon stimulated genes (ISGs). The polymerase PA (T515A) mutation did not significantly affect replication in vivo but greatly attenuated host interferon responses. ISG induction was robust for both H5N1 strains, but was three times lower for the PA mutant strain. Low pathogenic viruses elicited detectable induction of RIG-I, IFNß and ISGs in lung and intestine tissues that correlated with the recovery of viruses from tracheal or cloacal swabs. Several genes in the MAVS signaling pathway were also upregulated by H5N1, which contributed to further amplification of the signal. We also examined hematoxylin-eosin stained tissue sections and observe evidence of lung pathology and splenocyte depletion with both H5N1 viruses at 3 dpi, and recovery by 6 dpi. However, for both H5N1 strains we observed inflammation around neurons in brain, with increased cytokine expression in some individuals. Our findings reveal HPAI H5N1 viruses induced stronger innate immune responses to the infection, while LPAI viruses elicit a milder response.

Balb/Cj male mice do not feminize after infection with larval Taenia crassiceps.

Balb/cJ mice fail to mount an immune response capable of clearing infection with larval Taenia crassiceps. Additionally, male Balb/cJ mice display a lag in larval growth of approximately 3 wk as compared to growth in female mice. It has been reported that male Balb/ cAnN mice generate a protective immune response early in infection, and become permissive to larval growth after they feminize (200-fold increase in serum estradiol and 90% decrease in serum testosterone). To determine if a different strain of Balb/c mice (Balb/cJ) also feminize, serum was collected from infected male mice for 16 wk and levels of 17-beta-estradiol and testosterone were measured via ELISA. In addition, the mounting responses of 12- and 16-wk infected male mice, as well as uninfected control mice, were determined after isolation with a female mouse. The results of these experiments show that male Balb/cJ mice do not feminize during infection with larval T. crassiceps. There was no significant change in serum levels of either 17-beta-estradiol or testosterone during the course of infection (> 16 wk). Moreover, there was no significant decrease in the number of times infected male mice mounted the female mouse as compared to uninfected controls. These results suggest that there may be variances between the substrains of Balb/c mice that lead to the phenotypic differences reported for male Balb/cJ and Balb/cAnN mice.

Uptake and secretion of host proteins by Taenia crassiceps metacestodes.

Although the presence of intact host proteins in the cyst fluid of cyclophyllidean metacestodes has been well documented, the underlying reason for protein uptake is poorly understood. To investigate this discrepancy, both the cyst fluid (CF) and excreted/secreted (E/S) proteins were collected in vitro from Taenia crassiceps metacestodes 16 wk postinfection in Balb/cJ female mice. The CF and E/S were subsequently immunoblotted using rabbit anti-mouse whole serum antibodies as a probe. The results show that whole host proteins were not only internalized by metacestodes but also secreted as well. The predominant secreted host protein was 66 kDa and was confirmed to be mouse serum albumin. The amount of secreted albumin decreased daily, whereas the concentration of albumin in the cyst fluid remained consistent. This suggests that the secretion of albumin is a coordinated function rather than a random event. It is probable that albumin cycling may be an evolved mechanism providing multiple benefits for the larvae, including osmoregulation and protection from innate immune responses.

Duck TRIM27-L enhances MAVS signaling and is absent in chickens and turkeys.

Wild waterfowl, including mallard ducks, are the natural reservoir of avian influenza A virus and they are resistant to strains that would cause fatal infection in chickens. Here we investigate potential involvement of TRIM proteins in the differential response of ducks and chickens to influenza. We examine a cluster of TRIM genes located on a single scaffold in the duck genome, which is a conserved synteny group with a TRIM cluster located in the extended MHC region in chickens and turkeys. We note a TRIM27-like gene is present in ducks, and absent in chickens and turkeys. Orthologous genes are predicted in many birds and reptiles, suggesting the gene has been lost in chickens and turkeys. Using quantitative real-time PCR (qPCR) we show that TRIM27-L, and the related TRIM27.1, are upregulated 5- and 9-fold at 1 day post-infection with highly pathogenic A/Vietnam/1203/2004. To assess whether TRIM27.1 or TRIM27-L are involved in modulation of antiviral gene expression, we overexpressed them in DF1 chicken cells, and neither show any direct effect on innate immune gene expression. However, when co-transfected with duck RIG-I-N (d2CARD) to constitutively activate the MAVS pathway, TRIM27.1 weakly decreases, while TRIM27-L strongly activates innate immune signaling leading to increased transcription of antiviral genes MX1 and IFN-ß. Furthermore, when both are co-expressed, the activation of the MAVS signaling pathway by TRIM27-L over-rides the inhibition by TRIM27.1. Thus, ducks have an activating TRIM27-L to augment MAVS signaling following RIG-I detection, while chickens lack both TRIM27-L and RIG-I itself.

Macrophage biology and their activation by protozoan-derived glycosylphosphatidylinositol anchors and hemozoin.

Despite recent advances in medical technology and a global effort to improve public health and hygiene, parasitic infections remain a major health and economic burden worldwide. The World Health Organization estimates that about 1/3 of the world's population is currently infected with a soil-transmitted helminth, and millions more suffer from diseases caused by protozoan parasites including Plasmodium, Trypanosoma, and Leishmania species. Due to the selective pressure applied by parasitic and other infections, animals have evolved an intricate immune system; however, the current worldwide prevalence of parasitic infections clearly indicates that these pathogens have adapted equally well. Thus, developing a better understanding of the host-parasite relationship, particularly by focusing on the host immune response and the mechanisms by which parasites evade this response, is a critical first step in mitigating the detrimental effects of parasitic diseases. Macrophages are critical contributors during the host response to protozoan parasites, and the success or failure of these cells often tips the balance in favor of the host or parasite. Herein, we briefly discuss macrophage biology and provide an update on our current understanding of how these cells recognize glycosylphosphatidylinositol anchors from protozoan parasites as well as malarial hemozoin.

Identification of avian RIG-I responsive genes during influenza infection.

Ducks can survive infection with highly pathogenic avian influenza viruses that are lethal to chickens. We showed that the influenza detector, RIG-I can initiate antiviral responses in ducks, but this gene is absent in chickens. We can reconstitute this pathway by transfecting chicken DF-1 embryonic fibroblast cells with duck RIG-I, which augments their antiviral response to influenza and decreases viral titer. However, the genes downstream of duck RIG-I that mediate this antiviral response to influenza are not known. Using microarrays, we compared the transcriptional profile of chicken embryonic fibroblasts transfected with duck RIG-I or empty vector, and infected with low or highly pathogenic avian influenza viruses. Transfected duck RIG-I expressed in chicken cells was associated with the marked induction of many antiviral innate immune genes upon infection with both viruses. We used real-time PCR to confirm upregulation of a subset of these antiviral genes including MX1, PKR, IFIT5, OASL, IFNB, and downregulation of the influenza matrix gene. These results provide some insight into the genes induced by duck RIG-I upon influenza infection, and provide evidence that duck RIG-I can function to elicit an interferon-driven, antiviral response against influenza in chicken embryonic fibroblasts.

The duck genome and transcriptome provide insight into an avian influenza virus reservoir species.

The duck (Anas platyrhynchos) is one of the principal natural hosts of influenza A viruses. We present the duck genome sequence and perform deep transcriptome analyses to investigate immune-related genes. Our data indicate that the duck possesses a contractive immune gene repertoire, as in chicken and zebra finch, and this repertoire has been shaped through lineage-specific duplications. We identify genes that are responsive to influenza A viruses using the lung transcriptomes of control ducks and ones that were infected with either a highly pathogenic (A/duck/Hubei/49/05) or a weakly pathogenic (A/goose/Hubei/65/05) H5N1 virus. Further, we show how the duck's defense mechanisms against influenza infection have been optimized through the diversification of its ß-defensin and butyrophilin-like repertoires. These analyses, in combination with the genomic and transcriptomic data, provide a resource for characterizing the interaction between host and influenza viruses.

Avian influenza rapidly induces antiviral genes in duck lung and intestine.

Ducks are the natural reservoir of influenza A and survive infection by most strains. To characterize the duck immune response to influenza, we sought to identify innate immune genes expressed early in an infection. We used suppressive subtractive hybridization (SSH) to construct 3 libraries enriched in differentially expressed genes from lung RNA of a duck infected with highly pathogenic avian influenza virus A/Vietnam/1203/04 (H5N1), or lung and intestine RNA of a duck infected with low pathogenic avian influenza A/mallard/BC/500/05 (H5N2) compared to a mock-infected duck. Sequencing of 1687 clones identified a transcription profile enriched in genes involved in antiviral defense and other cellular processes. Major histocompatibility complex class I (MHC I), interferon induced protein with tricopeptide repeats 5 (IFIT5), and 2'-5' oligoadenylate synthetase-like gene (OASL) were increased more than 1000-fold in relative transcript abundance in duck lung at 1dpi with highly pathogenic VN1203. These genes were induced much less in lung or intestine following infection with low pathogenic BC500. The expression of these genes following infection suggests that ducks initiate an immediate and robust response to a potentially lethal influenza strain, and a minimal response to a low pathogenic strain.

Expression of duck CCL19 and CCL21 and CCR7 receptor in lymphoid and influenza-infected tissues.

Ducks are the natural host and reservoir of influenza viruses. We are interested in their immune responses to these viruses, to understand host-pathogen interactions and to develop effective agricultural vaccines. We identified duck homologues of the chemokines CCL19 and CCL21 and cloned their cognate receptor, CCR7. Conservation of key features, and expression in lymphoid tissues suggests that these chemokines are the direct orthologues of their mammalian counterparts. Mammalian CCL19 and CCL21 are responsible for the homing of dendritic cells and naïve lymphocytes to secondary lymphoid tissues. The contribution of local tertiary lymphoid tissues may be important during influenza infection in ducks. Consistent with leukocyte recruitment, CCL19 and CCL21 transcripts are abundant in lung tissues at 1 day post-infection with highly pathogenic avian influenza A/Vietnam/1203/04 (H5N1) (VN1203). In contrast, expression in lung or intestine tissues infected with low pathogenic A/mallard/BC/500/05 (H5N2) (BC500) is not significant. Recruitment and aggregation of leukocytes is visible in the vicinity of major airways 3 days after infection with VN1203. Chemokine gene expression may serve as a useful marker to evaluate duck immune responses to natural infections and vaccine strains.

Peroxisome proliferator-activated receptor and AMP-activated protein kinase agonists protect against lethal influenza virus challenge in mice.

BACKGROUND: A novel influenza A (H1N1) virus was isolated from humans in North America and has developed into the first pandemic of the 21st century. Reports of a global shortage of antiviral drugs, the evolution of drug-resistant influenza virus variants, and a 6-month delay in vaccine availability underline the need to develop new therapeutics that may be widely distributed during future pandemics. METHODS: In an effort to discover alternatives to the conventional therapeutic strategies available, we screened several classes of immunomodulatory agents possessing the potential to mitigate the effects of influenza virus-induced immunopathology. RESULTS: Here, we provide preliminary evidence that two classes of drugs, peroxisome proliferator-activated receptor-gamma agonists and AMP-activated protein kinase agonists, provide protection in mice infected with highly pathogenic and pandemic strains of influenza virus. CONCLUSIONS: The extensive production in the developed world, combined with the significant degree of protection described here, establishes these drugs as a potential therapeutic option that may be broadly implemented to combat serious disease caused by future influenza epidemics or pandemics.

Drugs in development for influenza.

The emergence and global spread of the 2009 pandemic H1N1 influenza virus reminds us that we are limited in the strategies available to control influenza infection. Vaccines are the best option for the prophylaxis and control of a pandemic; however, the lag time between virus identification and vaccine distribution exceeds 6 months and concerns regarding vaccine safety are a growing issue leading to vaccination refusal. In the short-term, antiviral therapy is vital to control the spread of influenza. However, we are currently limited to four licensed anti-influenza drugs: the neuraminidase inhibitors oseltamivir and zanamivir, and the M2 ion-channel inhibitors amantadine and rimantadine. The value of neuraminidase inhibitors was clearly established during the initial phases of the 2009 pandemic when vaccines were not available, i.e. stockpiles of antivirals are valuable. Unfortunately, as drug-resistant variants continue to emerge naturally and through selective pressure applied by use of antiviral drugs, the efficacy of these drugs declines. Because we cannot predict the strain of influenza virus that will cause the next epidemic or pandemic, it is important that we develop novel anti-influenza drugs with broad reactivity against all strains and subtypes, and consider moving to multiple drug therapy in the future. In this article we review the experimental data on investigational antiviral agents undergoing clinical trials (parenteral zanamivir and peramivir, long-acting neuraminidase inhibitors and the polymerase inhibitor favipiravir [T-705]) and experimental antiviral agents that target either the virus (the haemagglutinin inhibitor cyanovirin-N and thiazolides) or the host (fusion protein inhibitors [DAS181], cyclo-oxygenase-2 inhibitors and peroxisome proliferator-activated receptor agonists).