Acute Influenza Virus Infection Research Articles

Uncompromised NK cell activation is essential for virus-specific CTL activity during acute influenza virus infection

Natural killer (NK) cells are indispensable components of both the innate and adaptive immune response. However, their precise roles in the cross-talk between innate and adaptive immunity during influenza virus infection remain controversial. By comparing NK cell dynamics and activity under a sub-lethal dose and high dose of influenza virus infection, we showed that influenza virus PR8 directly infected NK cells during natural infection, which was consistent with our previous findings obtained from an in vitro investigation of human NK cells. The impairments in cytotoxicity and IFN-γ production by spleen NK cells following high-dose infection were accompanied by decreased virus-specific killing mediated by cytotoxic T lymphocytes (CTLs). Importantly, the weakened CTL activity could be reversed by adoptive transfer of spleen NK cells harvested from low-dose-infected mice but not healthy donors. Taken together, our data provide direct evidence supporting the contribution of NK cells to antiviral T-cell responses. This study also indicates that a novel NK-targeted immune evasion strategy is used by influenza virus to shrink both innate and adaptive immune responses.

Respiratory Influenza Virus Infection Induces Memory-like Liver NK Cells in Mice.

Although NK cells are classified as innate immune cells, recent studies have demonstrated the transformation of NK cells into long-lived memory cells that contribute to secondary immune responses in certain mouse models. However, whether NK cells mount an Ag-specific memory response to acute influenza virus infection has not yet been examined. Here, we show that, consistent with previous studies, lung NK cells play an important role in controlling viral proliferation after primary influenza virus infection. However, although lung NK cells display a memory phenotype at the late stage of infection, these cells do not protect mice against secondary influenza virus infection. Interestingly, liver NK cells from influenza virus-infected mice possess a memory phenotype and protect mice against secondary influenza virus infection. Memory-like liver NK cells display a CD49a+DX5- phenotype, and the adoptive transfer of purified liver CD49a+DX5- NK cells into naive mice followed by viral infection results in protective immunity and decreased viral titer. Moreover, we demonstrate that primary inactivated influenza virus induces memory NK cells residing in the liver of Rag1-/- mice. Collectively, these data suggest that liver CD49a+DX5- NK cells remember encountered Ag from influenza virus after primary infection and are more protective upon subsequent infection.

Hierarchical effects of pro-inflammatory cytokines on the post-influenza susceptibility to pneumococcal coinfection.

In the course of influenza A virus (IAV) infections, a secondary bacterial infection frequently leads to serious respiratory conditions provoking high hospitalization and death tolls. Although abundant pro-inflammatory responses have been reported as key contributing factors for these severe dual infections, the relative contributions of cytokines remain largely unclear. In the current study, mathematical modelling based on murine experimental data dissects IFN-γ as a cytokine candidate responsible for impaired bacterial clearance, thereby promoting bacterial growth and systemic dissemination during acute IAV infection. We also found a time-dependent detrimental role of IL-6 in curtailing bacterial outgrowth which was not as distinct as for IFN-γ. Our numerical simulations suggested a detrimental effect of IFN-γ alone and in synergism with IL-6 but no conclusive pathogenic effect of IL-6 and TNF-α alone. This work provides a rationale to understand the potential impact of how to manipulate temporal immune components, facilitating the formulation of hypotheses about potential therapeutic strategies to treat coinfections.

The Effects of Acute Neutrophil Depletion on Resolution of Acute Influenza Infection, Establishment of Tissue Resident Memory (TRM), and Heterosubtypic Immunity.

After disease resolution, a small subset of influenza specific CD8+ T cells can remain in the airways of the lung as a tissue resident memory population (TRM). These cells are critical for protection from subsequent infections with heterosubtypic influenza viruses. Although it is well established that expression of the collagen IV binding integrin alpha 1 is necessary for the retention and maintenance of TRM cells, other requirements allowing them to localize to the airways and persist are less well understood. We recently demonstrated that inhibition of neutrophils or neutrophil derived chemokine CXCL12 during acute influenza virus infection reduces the effector T cell response and affects the ability of these cells to localize to the airways. We therefore sought to determine whether the defects that occur in the absence of neutrophils would persist throughout resolution of the disease and impact the development of the TRM population. Interestingly, the early alterations in the CD8+ T cell response recover by two weeks post-infection, and mice form a protective population of TRM cells. Overall, these observations show that acute neutrophil depletion results in a delay in the effector CD8+ T cell response, but does not adversely impact the development of TRM.

Influenza A Virus Infection Predisposes Hosts to Secondary Infection with Different Streptococcus pneumoniae Serotypes with Similar Outcome but Serotype-Specific Manifestation.

Influenza A virus (IAV) and Streptococcus pneumoniae are major causes of respiratory tract infections, particularly during coinfection. The synergism between these two pathogens is characterized by a complex network of dysregulated immune responses, some of which last until recovery following IAV infection. Despite the high serotype diversity of S. pneumoniae and the serotype replacement observed since the introduction of conjugate vaccines, little is known about pneumococcal strain dependency in the enhanced susceptibility to severe secondary S. pneumoniae infection following IAV infection. Thus, we studied how preinfection with IAV alters host susceptibility to different S. pneumoniae strains with various degrees of invasiveness using a highly invasive serotype 4 strain, an invasive serotype 7F strain, and a carrier serotype 19F strain. A murine model of pneumococcal coinfection during the acute phase of IAV infection showed a significantly increased degree of pneumonia and mortality for all tested pneumococcal strains at otherwise sublethal doses. The incidence and kinetics of systemic dissemination, however, remained bacterial strain dependent. Furthermore, we observed strain-specific alterations in the pulmonary levels of alveolar macrophages, neutrophils, and inflammatory mediators ultimately affecting immunopathology. During the recovery phase following IAV infection, bacterial growth in the lungs and systemic dissemination were enhanced in a strain-dependent manner. Altogether, this study shows that acute IAV infection predisposes the host to lethal S. pneumoniae infection irrespective of the pneumococcal serotype, while the long-lasting synergism between IAV and S. pneumoniae is bacterial strain dependent. These results hold implications for developing tailored therapeutic treatment regimens for dual infections during future IAV outbreaks.

LAG-3 expressing antigen-specific CD4+ T cells attenuate lung inflammation during acute influenza virus infection

Abstract The host immune system responds to influenza virus infection for eradication of the virus but the immune response associated inflammation causes tissue injury and damage to the host as well. In our antigen-specific mouse model system for influenza virus infection, influenza hemagglutinin antigen-specific CD4+ T cells responded to acute infection with Th1 effector phenotype. A significant proportion of them were also with concurrent expression of LAG-3, CTLA-4, GITR or CD25. Adoptive transfer of purified LAG-3 expressing hemagglutinin antigen-specific CD4+ T cells controlled lung inflammation with acute influenza virus infection, as represented by the activation of co-transferred influenza hemagglutinin antigen-specific CD8+ T cells. Control of the inflammation by LAG-3 expressing antigen-specific Th1 CD4+ T cells was not necessarily associated with impaired capacity of virus eradication. These results reveal, besides effector function for virus eradication, subsets of CD4+ T cells activated by acute influenza virus infection also acquired regulatory function to attenuate inflammation for reducing the damage to the host. These subsets of CD4+ T cells may be employed as the practical handle for management of severe influenza disease.

ECM remodelling by ADAMTS5 regulates influenza-specific immunity following acute infection

Abstract Members of the ADAMTS (A disintegrin-like and metalloproteinase with thrombospondin-1 motifs) metalloproteinase family remodel the extracellular matrix (ECM) to facilitate immune cell migration and cell adhesion. In this current study, we infected Adamts5−/− mice with X31 influenza virus (104pfu/mouse)and measured weight loss, viraemia and T cell immunity following acute infection. Adamts5−/− mice showed greater weight loss and increased viraemia when compared to WT counterparts. Furthermore, we observed reduced NP366 and PA224 influenza specific CD8+ T cells in the spleen and lungs of Adamts5−/− mice with a corresponding accumulation of these T cell specificities in the mediastinal lymph node (MLN) 10 days post infection. Critically, these responses correlated with increased expression of a key ADAMTS5 substrate; versican, in the MLNs of Adamts5−/− mice. We therefore hypothesize that versican accumulation in the MLNs of Adamts5−/− mice results in T cell accumulation and that ADAMTS5 is a key enzyme involved in facilitating T cell migration from the MLN to the periphery following acute influenza virus infection.

ADAMTS5: A critical enzyme for the maintenance of influenza-specific CD8+ T cell memory

Abstract Extracellular matrix (ECM) enzymes, such as the ADAMTS (A disintegrin-like and metalloproteinase with thrombospondin-1 motifs) family, play a significant role in ECM remodelling. ECM remodelling facilitates immune cell migration and adhesion. ADAMTS5, a member of the ADAMTS family, cleaves ECM proteoglycans, such as aggrecan and versican, and has a well-characterised role in osteoarthritis and embryogenesis. However, ADAMTS5 has an un-identified role throughout the immune response. Our previous studies indicated that ADAMTS5 expression is required to mediate T cell migration during acute influenza virus infection. In this current study, we analysed secondary acute responses (day 7) and primary memory (day 30) following influenza infection of Adamts5−/− mice. Here, we primed Adamts5−/− mice with PR8 influenza virus (1.5×107 pfu/mouse) and challenged 8 weeks later with X31 influenza virus (104 pfu/mouse). Following secondary infection, Adamts5−/− mice displayed greater weight loss and expressed fewer IFNγ+NP366-372+CD8+T cells. Critically, we observed a reduced number of influenza-specific CD8+ memory cells (CD62LhiCD44lo) in Adamts5−/− mice after primary infection, which may result in the lower number of IFNγ+NP366-372+CD8+T cells observed in secondary acute infection. We therefore hypothesize that the ADAMTS5 enzyme, through its ECM remodelling capabilities, is essential for the maintenance of memory and recall of functional immunity following secondary infection.

Indelible labeling and tracking of influenza virus infected antigen presenting cells

Abstract Influenza A virus is a seasonal pathogen with the potential to unpredictably cause catastrophic pandemics. The virus primarily replicates in epithelial cells of the upper and lower respiratory tract but can also infect a spectrum of other cell lineages, including cells of the immune system. Classically infected cells have been tracked through detection of virus products (RNA or protein) or through virus-derived reporters. However, these methods cannot define any potentially remaining infected cells after active replication has ceased. Using a novel virus expressing Cre recombinase to indelibly label infected cells in reporter mice, we unexpectedly found that epithelial cells were capable of surviving influenza virus infection long-term (Heaton and Langlois et al J. Ex. Med. 2014). We have subsequently discovered that infected CD45+ immune cells are also capable of surviving acute influenza virus infection in vivo. Importantly this virus-reporter system is able to distinguish between immune cells that are directly infected versus those that acquire virus protein/antigen exogenously. We find that both dendritic cells and macrophages, but not B cells, are directly infected within the lung and that the number of these cells increases during the acute infection phase. However, infected dendritic cells and macrophages do not persist in the lungs nor do they migrate systemically. Together these data demonstrate a new tool to track infected cells and defines the migration, turnover, and survival of infected antigen presenting cells.

Human nasal epithelial cells derived from multiple subjects exhibit differential responses to H3N2 influenza virus infection in vitro

Human nasal epithelial cells derived from multiple subjects exhibit differential responses to H3N2 influenza virus infection in vitro

CD8+ cytotoxic T lymphocytes in human influenza virus infection.

CD8+ cytotoxic T lymphocytes in human influenza virus infection.

Th17 deviation under influence of Th1 response attenuates the inflammation in late phase of influenza virus infection (VIR5P.1138)

Abstract Overwhelming inflammation with cytokine storm has been postulated as a major cause of severe influenza disease in human. In our influenza Ag- specific mouse experimental system, we discovered a Th17 deviation of CD4+ T cell response with resultant attenuation of inflammation in the late phase of influenza infection. Naïve hemagglutinin (HA)-specific CD4+ T cells respond to acute influenza virus infection with Th1 phenotype. In the presence of antigen specific Th1 CD4+ T cells, naïve CD4+ T cells adoptively transferred 4 days after the acute infection are activated with deviation to the Th17 phenotype. The second batch of naïve HA-specific CD4+ T cells is activated in an environment enriched with Th1 cytokines and active TGF-β. They proliferate, secrete IL-17 but not IFN-γ and express LAG-3 on the cell surface. They can suppress T cell activation in vitro and decrease lung inflammation in vivo. The 6.5 mice, with monotonous TCR repertoire responsive to influenza HA, reveal a Th17 deviated CD4+ T cell response and significant survival benefit in influenza virus infection. Consecutive waves of naïve CD4+ T cells from the thymus in 6.5 mice are probably activated as subsequent batches of naïve CD4+ T cells, with Th17 deviation which attenuates the inflammation in late phase of influenza virus infection.

Simultaneous infection with gammaherpes and influenza viruses enhances the host immune defense.

We have studied the impact of simultaneous infection of mice with murine gammaherpesvirus (MHV) and influenza Avirus (IAV) on the immune response and pathogenesis of both infections. After apersistent MHV-68 herpesviral infection had been established, the same mice were super-infected with IAV. Individual parameters of MHV infection (viral DNA detection in organs and blood) and numbers of leukocytes in lungs and spleens were determined. With regard to the assumed reactivation of MHV-68 (mainly in lungs, spleen, thymus and peritoneal exudate cells) we focused our attention on the detection of transcripts, typical either for lytic infection (ORF50) and/or for latency (ORF73). Herpesviral DNA was detected in above mentioned organs in several intervals during the acute phase of IAV co-infection, but the expression of monitored transcripts was lower, i.e. it has decreased. Though the reason for such limited expression during acute influenza superinfection remains unclear, it is unambiguous that lower MHV-68 expression was detected in lungs and peritoneal exudate cells (PECs) from 3rd to 10th day after co-infection with IAV. Furthermore, our study showed that the ongoing gammaherpesvirus latency in co-infected mice affected the number of cytotoxic T-lymphocytes and neutrophils during the acute IAV infection and lowered their deviations from that of non-infected mice. Therefore, we suppose that co-infection with herpes and influenza viruses could be mutually beneficial for the host by promoting its defense against both viruses.

Crossreactive Epstein-Barr virus (EBV)-influenza A virus (IAV) specific CD8 memory T cells during acute symptomatic IAV infection. (VIR2P.1022)

We previously showed that crossreactivity is common between IAV and EBV in HLA-A2+ patients during infectious mononucleosis. IAV-M1 specific CD8 T cells, along with expanded populations of IAV-M1/EBV-BRLF-1 and IAV-M1/EBV-BMLF-1 double-tetramer+ cells were detected directly ex-vivo in 5 HLA-A2+ patients. Altered IAV-M1, EBV-BRLF1 and -BMLF1 TCR repertoires were observed over the course of infection and in comparison to healthy donors. After culture, cells were sorted and analyzed by gene array in order to assess global changes in immune responses following different stimulations, either cognate or crossreactive, in different patient populations. M1- and BRLF1-specific cells had similar immune-response gene signatures, but the BMLF1-specific CD8 cells were more similar to the two-crossreactive populations. Crossreactive M1/BRLF1 cells from the BRLF1 line were different in their activation status than the BRLF1-specific cells, consistent with BRLF1 ligand stimulation of different gene activation profiles in these two populations. These results suggest that during symptomatic IAV infection there is an expansion of EBV/IAV crossreactive memory CD8 T cell responses. Ongoing studies are investigating whether EBV-IAV cross-reactive CD8+ T cells may contribute to immunopathology during acute IAV infection.

Acute symptomatic influenza A virus (IAV) infection in humans leads to expansion of highly diverse CD8 T cell repertoires crossreactive with persistent Epstein Barr virus (EBV) (HUM8P.344)

The competence of T cell responses predominantly depends on how efficient T cell receptors (TCRs) are at recognizing antigenic epitopes. We show here that during acute severely symptomatic IAV infection there was an expansion of IAV-M1/EBV-BRLF1 and IAV-M1/EBV-BMLF1 double-tetramer+ cells directly ex-vivo in 5 HLA-A2+ patients. We questioned whether this expansion specific to these two different crossreactive responses would lead to alterations in the TCR repertoire of the IAV-M1-58, EBV-BRLF1-109 and BMLF1-280 from before, during and following acute IAV infection. Using staining with Vb MAb we found that T cell responses generated to these epitopes became surprisingly more polyclonal, with the sharing of Vb between M1, BMLF1 and BRLF1 populations which is not seen in healthy donors and which decreased 2 months later consistent with crossreactive expansion. Furthermore, by using single-cell analysis of TCRα and TCRβ repertoire of tetramer sorted IAV-M1 cells we showed dramatic changes in specific clonotype usage and in JA and JB family usage during acute IAV infection compared to before infection. In summary, these changes in TCR repertoire during acute symptomatic IAV infection suggest that during severe infection there is a preferential expansion of highly diverse crossreactive responses between IAV and the persistent virus, EBV, which leads to permanent changes in TCR repertoires to both of these two viruses.

Long-term survival of influenza virus infected club cells drives immunopathology.

Respiratory infection of influenza A virus (IAV) is frequently characterized by extensive immunopathology and proinflammatory signaling that can persist after virus clearance. In this report, we identify cells that become infected, but survive, acute influenza virus infection. We demonstrate that these cells, known as club cells, elicit a robust transcriptional response to virus infection, show increased interferon stimulation, and induce high levels of proinflammatory cytokines after successful viral clearance. Specific depletion of these surviving cells leads to a reduction in lung tissue damage associated with IAV infection. We propose a model in which infected, surviving club cells establish a proinflammatory environment aimed at controlling virus levels, but at the same time contribute to lung pathology.

Attenuation of immune-mediated influenza pneumonia by targeting the inducible co-stimulator (ICOS) molecule on T cells.

Inducible Co-stimulator (ICOS) plays a critical role in mediating T cell differentiation and function and is considered a key player in balancing T effector and T regulatory (Treg) cell responses. Here we show that activation of the ICOS signalling pathway during acute influenza A virus (IAV) infection by application of an agonistic ICOS antibody reduced the frequency of CD8+ T cells in the respiratory tract of IAV infected animals and delayed pathogen elimination. In line with this, immune-mediated influenza pneumonia was significantly ameliorated in mice that received ICOS agonist as indicated by significantly reduced alveolar infiltrations and bronchointerstitial pneumonia, while at the same time virus-related pathology remained unaffected. Importantly, ICOS agonist treatment resulted in expansion of CD4+Foxp3+ Tregs in IAV infected mice, which was associated with elevated levels of the immunosuppressive cytokine IL-10 in the alveolar space. Together, our findings suggest a prominent role of ICOS signaling during acute IAV infection by increasing the Treg/CD8+ T cell ratio with beneficial outcome on immune-mediated pneumonia and underline the suitability of ICOS as potential therapeutic target for immune intervention in those infectious conditions characterized by strong immunopathology rather than virus-mediated cytopathic effects.

A prospective study of Romanian agriculture workers for zoonotic influenza infections.

BackgroundIn this prospective study we sought to examine seroepidemiological evidence for acute zoonotic influenza virus infection among Romanian agricultural workers.MethodsSera were drawn upon enrollment (2009) and again at 12 and 24 months from 312 adult agriculture workers and 51 age-group matched controls. Participants were contacted monthly for 24 months and queried regarding episodes of acute influenza-like illnesses (ILI). Cohort members meeting ILI criteria permitted respiratory swab collections as well as acute and convalescent serum collection. Serologic assays were performed against 9 avian, 3 swine, and 3 human influenza viruses.ResultsDuring the two-year follow-up, a total of 23 ILI events were reported. Two subjects' specimens were identified as influenza A by rRT-PCR. During the follow-up period, three individuals experienced elevated microneutralization antibody titers ≥1∶80 against three (one each) avian influenza viruses: A/Teal/Hong Kong/w312/97(H6N1), A/Hong Kong/1073/1999(H9N2), or A/Duck/Alberta/60/1976(H12N5). However, none of these participants met the criteria for poultry exposure. A number of subjects demonstrated four-fold increases over time in hemagglutination inhibition (HI) assay titers for at least one of the three swine influenza viruses (SIVs); however, it seems likely that two of these three responses were due to cross-reacting antibody against human influenza. Only elevated antibody titers against A/Swine/Flanders/1/1998(H3N2) lacked evidence for such confounding. In examining risk factors for elevated antibody against this SIV with multiple logistic regression, swine exposure (adjusted OR = 1.8, 95% CI 1.1–2.8) and tobacco use (adjusted OR = 1.8; 95% CI 1.1–2.9) were important predictors.ConclusionsWhile Romania has recently experienced multiple incursions of highly pathogenic avian influenza among domestic poultry, this cohort of Romanian agriculture workers had sparse evidence of avian influenza virus infections. In contrast, there was evidence, especially among the swine exposed participants, of infections with human and one swine H3N2 influenza virus.

Acute symptomatic influenza A virus (IAV) infection in humans leads to expansion of highly diverse CD8 T cell repertoires crossreactive with persistent Epstein Barr virus (EBV) (HUM8P.344)

Abstract The competence of T cell responses predominantly depends on how efficient T cell receptors (TCRs) are at recognizing antigenic epitopes. We show here that during acute severely symptomatic IAV infection there was an expansion of IAV-M1/EBV-BRLF1 and IAV-M1/EBV-BMLF1 double-tetramer+ cells directly ex-vivo in 5 HLA-A2+ patients. We questioned whether this expansion specific to these two different crossreactive responses would lead to alterations in the TCR repertoire of the IAV-M1-58, EBV-BRLF1-109 and BMLF1-280 from before, during and following acute IAV infection. Using staining with Vb MAb we found that T cell responses generated to these epitopes became surprisingly more polyclonal, with the sharing of Vb between M1, BMLF1 and BRLF1 populations which is not seen in healthy donors and which decreased 2 months later consistent with crossreactive expansion. Furthermore, by using single-cell analysis of TCRα and TCRβ repertoire of tetramer sorted IAV-M1 cells we showed dramatic changes in specific clonotype usage and in JA and JB family usage during acute IAV infection compared to before infection. In summary, these changes in TCR repertoire during acute symptomatic IAV infection suggest that during severe infection there is a preferential expansion of highly diverse crossreactive responses between IAV and the persistent virus, EBV, which leads to permanent changes in TCR repertoires to both of these two viruses.

Crossreactive Epstein-Barr virus (EBV)-influenza A virus (IAV) specific CD8 memory T cells during acute symptomatic IAV infection. (VIR2P.1022)

Abstract We previously showed that crossreactivity is common between IAV and EBV in HLA-A2+ patients during infectious mononucleosis. IAV-M1 specific CD8 T cells, along with expanded populations of IAV-M1/EBV-BRLF-1 and IAV-M1/EBV-BMLF-1 double-tetramer+ cells were detected directly ex-vivo in 5 HLA-A2+ patients. Altered IAV-M1, EBV-BRLF1 and -BMLF1 TCR repertoires were observed over the course of infection and in comparison to healthy donors. After culture, cells were sorted and analyzed by gene array in order to assess global changes in immune responses following different stimulations, either cognate or crossreactive, in different patient populations. M1- and BRLF1-specific cells had similar immune-response gene signatures, but the BMLF1-specific CD8 cells were more similar to the two-crossreactive populations. Crossreactive M1/BRLF1 cells from the BRLF1 line were different in their activation status than the BRLF1-specific cells, consistent with BRLF1 ligand stimulation of different gene activation profiles in these two populations. These results suggest that during symptomatic IAV infection there is an expansion of EBV/IAV crossreactive memory CD8 T cell responses. Ongoing studies are investigating whether EBV-IAV cross-reactive CD8+ T cells may contribute to immunopathology during acute IAV infection.