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. Author manuscript; available in PMC: 2011 Jan 20.
Published in final edited form as: Virology. 2009 Nov 12;396(2):178–188. doi: 10.1016/j.virol.2009.10.016

Innate Immune Response to H3N2 and H1N1 Influenza Virus Infection in a Human Lung Organ Culture Model

Wenxin Wu †,*, J Leland Booth †, Elizabeth S Duggan †, Shuhua Wu †,#, Krupa B Patel †, K Mark Coggeshall §, Jordan P Metcalf †,‡
PMCID: PMC2789846  NIHMSID: NIHMS155720  PMID: 19913271

Abstract

We studied cytokine responses to influenza virus PR8 (H1N1) and Oklahoma/309/06 (OK/06, H3N2) in a novel human lung tissue model. Exposure of the model to influenza virus rapidly activated the mitogen activated protein kinase signaling (MAPK) pathways ERK, p38 and JNK. In addition, RNase protection assay demonstrated the induction of several cytokine and chemokine mRNAs by virus. This finding was reflected at the translational level as IL-6, MCP-1, MIP-1α/β, IL-8 and IP-10 proteins were induced as determined by ELISA. Immunohistochemistry for IP-10 and MIP-1α revealed that alveolar epithelial cells and macrophages were the source of these two cytokines. Taken together, both PR8 and OK/06 cause similar induction of cytokines in human lung, although OK/06 is less effective at inducing the chemokines MCP-1 and IL-8. This human organ culture model should thus provide a relevant platform to study the biological responses of human lung to influenza virus infection.

Keywords: influenza virus, lung, cytokine, chemokine, MAPK, IP-10, MIP-1α

Introduction

In the United States, influenza annually infects 5% to 20% of the population, hospitalizes 200,000, and kills 36,000. Thus, it is a leading cause of death (Izurieta et al., 2000; Thompson et al., 2004). The devastating Spanish influenza A virus infected about a third of the world’s population and killed 40 million people during the pandemic of 1918.

Following replication in the superficial cells of the respiratory tract, influenza virus causes a spectrum of acute clinical syndromes that can progress to a fatal outcome. The clinical syndromes include asymptomatic infection and primary viral or secondary bacterial pneumonia. Therefore, although the virus infects the entire respiratory tract, morbidity and mortality is associated with lower respiratory tract involvement (Hers, 1966; Hers et al., 1958; Hers and Mulder, 1961).

Human influenza virus belongs to the orthomyxoviridae family, which consists of four genera: influenza A, B, and C virus, and Thogovirus. Only influenza virus A and B are pathogenic among human beings. Influenza A viruses are further subdivided into subtypes based on the antigenicity of two transmembrane glycoproteins, hemagglutinin (HA) and neuraminidase (NA). There are 16 HA and 9 NA subtypes in influenza virus A. Viruses with HA types H1-H3 and NA types N1 and N2 are found in humans.

Epithelial cells are the primary site of viral replication for influenza, although monocytes/macrophages and other leukocytes can also be infected (Ronni et al., 1997). Influenza virus specific antigen has been found in type 1 and type 2 alveolar epithelial cells, as well as in alveolar macrophages. Viruses initiate infection by binding of the viral HA to sialic acid on the cell surface and enter the cells by receptor-mediated endocytosis. Once inside the cells, influenza virus shuts off host cell protein synthesis and replicates in a fast and efficient way. This process results in host cell apoptosis or death by cytolysis. However, the host cells respond in several ways to limit viral spreading. The most significant response is production of cytokines and chemokines by epithelial cells and leukocytes via activation of multiple transcriptional and posttranslational systems (Julkunen et al., 2000). Cytokines are extracellular signal proteins that stimulate adjacent and distant cells to activate host antiviral defense. Chemokines are low molecular weight chemoattractant cytokines which bind to their specific receptors in leukocytes, recruit inflammatory cells to the site of infection, and activate innate immune responses (Baggiolini, 1998). Cytokines are released from lung epithelial cell lines during influenza virus infection. These include Type 1 IFN (Ronni et al., 1997), IL-6 and the chemokines MCP-1, RANTES, and IL-8 (Adachi et al., 1997; Kawaguchi et al., 2000; Matsukura et al., 1996). In addition, IL-6, TNF-α, MIP-1α, MIP-1β and IL-8 have also been detected in the nasopharyngeal secretions of influenza A virus infected patients (Fritz et al., 1999; Kaiser et al., 2001). Monocytes also secrete chemokines during influenza exposure, including MIP-1α, MIP-1β, MIP-3α, MCP-1, MCP-3, RANTES, IP-10 and cytokines TNF-α, IL-1β, IL-6, IL-18 and IFN α/β (Bussfeld et al., 1998; Garn et al., 2002; Pirhonen et al., 2001; Sprenger et al., 1996).

We have developed a human lung organ culture model in order to study the local lung response to human pathogens (Booth et al., 2004; Chakrabarty et al., 2007). Precision-cut lung slices have frequently been used in toxicology studies and have advantages over the use of isolated, cultured epithelial cells for infectious disease studies. The structural integrity of lung tissue is maintained (Placke and Fisher, 1987) and this allows for cell-cell interaction in a more complex and native three-dimensional system. Detailed mechanistic studies of intracellular processes such as signal pathway activation can be examined in human tissue without risk to the host.

The primary strategy against viral epidemics and pandemics has been development of vaccines. Alternate strategies are likely to be necessary for strains for which development of a vaccine is problematic. One step in developing these strategies is to understand local, i.e. pulmonary, responses to these strains. As nearly all prior cytokine induction studies have been carried out with H1N1 (mostly A/ PR8/34) strains, very little is known about the pulmonary responses to influenza virus subtype H3N2. Even in the cases of H1N1 virus, few studies have been done in humans, and none have examined the pathophysiology of human lung infection. Therefore, this paper will investigate the innate immune response of the human lung to both a recently isolated, unadapted H3N2 strain, A/Oklahoma/309/06 (OK/06) and the commonly used, mouse adapted, H1N1 strain (PR8).

In our current study, we demonstrated that human lung responds to influenza virus by induction of the MAPK pathways ERK, SAP/JNK, and p38. This is followed by an increase in the production of several cytokines and chemokines at both the transcriptional and translational levels. The importance of signal pathway activation in cytokine induction was determined by measurement of the induction by virus in the presence or absence of chemical inhibitors of the pathways. The sources of cytokines MIP-1α and IP-10 are both alveolar macrophages and epithelia, with a possible, smaller contribution by interstitial cells.

Results

Human lung slices in culture support influenza virus replication and infection

Lung slices were processed by frozen section for detection of Cy3 labeled influenza virus. There was no detectable red fluorescence in tissue exposed to virus diluent (Fig. 1A, left panel). Exposure of the tissue to Cy3-OK/06 for 18 hours resulted in detection of fluorescently labeled virus associated with multiple cells, which appear morphologically to be alveolar epithelial cells (Fig. 1A, right panel). Thus, the human lung slices support infection during exposure to influenza virus.

FIG. 1.

FIG. 1

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Exposure of human lung tissue to influenza virus results in infection and replication of alveolar epithelial cells. (A) Infection assessed by the use of fluorescently labeled influenza virus. Labeled virus was separated from the unconjugated dye by dialysis against multiple changes of PBS at 4°C. Tissue slices exposed to virus diluent were used as a negative control for virus detection. The left panel shows no detection of red fluorescence in tissue exposed to virus diluent. The right panel shows exposure of the tissue to Cy3-virus for 18 h resulted in detection of virus in multiple cells. (B) Replication of influenza virus PR8 (H1N1) in the human lung organ culture model. Lung slices exposed to PR8 were cultured for various times and total mRNA in the lung slice was extracted. RT-PCR was performed using Oligo dT as the primer for the first strand synthesis. Primers specific for NS1 were used to examine NS1 mRNA expression.

Influenza virus infection was measured in tissue slices by semi-quantitive RT-PCR. The results demonstrated significant production of viral nonstructural protein 1 (NS1) mRNA with a peak by 8 hours, which remained elevated, but gradually declined as infection continued (Fig. 1B). This demonstrated that the human lung slice model was permissive for human influenza virus replication. This data also demonstrated the intra-slice reproducibility of total cell density, as shown by the similar levels of GAPDH, determined by semiquantitative RT-PCR from different individual slices.

RNA expression of cytokines and chemokines by human lung slices exposed to influenza virus

We first examined the innate immune cytokine and chemokine response of human lung tissue to influenza virus using RNase protection assay (RPA). Lung slices were exposed to 6 × 106 PFU/ml or virus diluent (negative control) for 4, 8, 12, 16, 20 and 24 h. RPA for cytokines demonstrated a 2 to 10 fold mRNA induction of IL-6, IL-8, MIP-1α, IP-10, TNF-α, MCP-1, and IFNγ. Influenza virus caused a significant time-dependent induction of cytokines IP-10 and IFNγ in lung tissue. The peak induction by PR8 occurred at 12 h post infection for most of the cytokines while OK/06 peak induction was later, at 16 h post infection (Fig. 2). The chemokine IP-10, whose receptors are found on activated T lymphocytes and natural killer cells (Dufour et al., 2002; Loetscher et al., 1996), was the most highly induced cytokine in terms of mRNA fold increase. As to the patterns of cytokines induced, there was no discernable difference in induction for the two viral strains. Thus, RPA results suggest a broad cytokine immune response, and also a monocyte and neutrophil chemokine response to influenza virus exposure by human lung.

FIG.2.

FIG.2

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Cytokine and chemokine mRNA response of human lung to influenza virus infection. Human lung tissue slices (3 slices per data point) were exposed to 6 × 106 PFU/ml of influenza virus PR8 (A) and OK/06 (B). Equal volumes of virus diluent were used as a negative control. mRNA expression levels were determined using a custol cytokine template (see Materials and Methods). Fold increase was determined by normalization for levels of housekeeping genes present in each sample.

Induction of cytokines in human lung tissue infected with influenza virus

RPA data indicated influenza induction of mRNA levels of several cytokines and chemokines. To confirm that this increase in mRNA was reflected at the level of translation, we tested for the corresponding cytokine protein in supernatants of lung slices exposed to influenza virus for 6, 20, 30 and 40 h using ELISA. Human lung slices were mock treated with virus diluent, treated with 6 × 106 PFU/ml of influenza virus, or treated with PMA (100 ng/ml) as a positive control. Consistent with the RPA results, we saw an increase in the cytokines IL-6 and the chemokines IL-8, MCP-1, IP-10 and MIP-1α/β with virus exposure (Fig. 3). Specifically, there was a peak fold increase over background in PR8 induced cytokine and chemokine levels of IL-6, IP-10, IL-8, MCP-1, and MIP-1α/β of 16, 75, 2, 12 and 50, respectively. MIP-1α/β maximum levels were seen at 20 h, IL-8 at 30 h while the maximum increase in IL-6, IP-10 and MCP-1 were seen at 40 h. At all the times tested, MCP-1 was stimulated to a greater extent than that seen with the positive control, 100 ng/ml of PMA (Fig. 3C). The most stimulated cytokine was IP-10, 75 fold over mock, which is consistent with the RPA result showing IP-10 is the most highly induced cytokine at the level of transcription. PMA is not a strong inducer for IP-10, which suggests that the human lung innate response involving IP-10 is specific to virus (Fig. 3E). Although we saw a significant induction of IFN-γ mRNA in the RPA, IFN-γ protein levels were low (less than 100 pg/ml) in lung slice supernatants, and undetectable in lung tissue extracts (data not shown). However, as IP-10 (10 kDa interferon-gamma-induced protein) is secreted by several cell types in response to IFN-γ (Luster et al., 1985), induction of IP-10 suggests that IFN-γ induction may have occurred in our system despite the ELISA results. For the most part, PR8 and OK/06 caused similar induction of most of the cytokines tested except MCP-1 and IL-8. OK/06 more weakly induces MCP-1 than PR8, at two of the four times tested. For IL-8 induction, OK/06 also appears to induce IL-8 less readily than PR8, but this only reached statistical significance at 30 h after infection. These results show that the induction of cytokine and chemokine genes, as determined by RPA, is consistently reflected at the protein level. As such, both influenza virus strains induced a broad innate immune chemokine and cytokine response in human lung.

FIG.3.

FIG.3

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Influenza virus stimulates chemokine and cytokine release in human lung. For each data point, multiple lung slices were exposed to 6 × 106 PFU/ml of influenza virus PR8 and OK/06, and allowed to incubate at 37°C, 5% CO2 for the indicated periods Virus diluent was used as a negative control, and PMA (100 ng/ml) was used as a positive control. Chemokine and cytokine protein levels were determined by ELISA on lung slice supernatants. Data are expressed as the means ± SEM from three separate lung slice donor experiments. Statistical significance was determined by ANOVA. Means were compared to data from the negative control group. *P < 0.05; **P < 0.01; NS represents no significant difference.

Induction of MAPK signaling pathways in human lung slices exposed to influenza virus

To determine the role of signal pathway activation in cytokine and chemokine induction by influenza virus in human lung, we next studied the kinetics of virus-induced activation of the MAPK signaling cascades. We assessed ERK, SAPK/JNK, and p38 activation as exhibited by phosphorylation in human lung infected with virus (6 × 106 PFU/ml). Stimulation with PMA (100 ng/ml) for 1 h served as a positive control for phosphorylation, and mock-infected, unstimulated lysates were prepared at 1 and 8 h as a negative control. At various times after infection, lung lysates were prepared, and total and phosphorylated ERK, SAPK/JNK, and p38 levels were assessed by western blot analysis. Membranes were also probed for total ERK, p38 and JNK MAPK as a control for protein loading (Rodriguez-Viciana et al., 2006; Williams et al., 2001). Phosphorylation was assessed by determining the ratio of phosphorylated (Fig. 4 A, D and H) to total kinase (Fig. 4 B, E and I), and the resultant activities were graphed (Fig. 4 C, F and J). The ratio corrects for variations in the amount of sample protein loaded onto SDS-PAGE gels. These results revealed that ERK, SAPK/JNK, and p38 were all activated by either, or both, of the influenza virus strains in the human lung organ culture model.

FIG. 4.

FIG. 4

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Kinetics of ERK1/2, SAPK/JNK, and p38 phosphorylation by influenza virus in human lung slices. Human lung tissue slices were exposed to 6 × 106 PFU/ml of influenza virus PR8 and OK/06 for the times indicated. Lung lysates were prepared, and levels of total and phosphorylated ERK1/2 (A, B, and C), p38 (D, E, and F), and SAPK/JNK (H, I, and J) were assessed by western blot analysis. PMA (100 ng/ml) was used as positive controls, and virus diluent was used as a negative control. Western blots were probed with antibody specific for phosphorylated ERK1/2 (A), p38 (D), or SAPK/JNK (H) are shown. Identically prepared blots were also probed with pan-anti-kinase antibody against ERK1/2 (B), p38 (E), or SAPK/JNK (I). Activation as determined by the ratio of phosphorylated kinase/total kinase for ERK1/2 (C), p38 (F), and SAPK/JNK (J) is graphed as shown.

For PR8, p38 and JNK were activated, and were greatest at the last time point measured (8h). Activation of p38 and SAPK/JNK at that time was 2.7 and 1.9 fold, respectively, over mock-infected controls. PR8-induced ERK phosphorylation exhibited a constant, moderate increase over the negative control at all times tested, ranging from 1.1 to 1.5 fold. For OK/06, ERK phosphorylation also exhibited a constant, moderate increase over the negative control at most times tested, ranging from 1.2 to 1.7 fold. With this strain, in contrast to PR8, activation of p38 occurred soon after infection, and declined thereafter. Also, in contrast to PR8, no induction of JNK was evident during the experiment. These results indicate that exposure to influenza virus elicits a modest increase in ERK, SAPK/JNK, and p38 activation which precedes cytokine mRNA induction and depends on the influenza virus strain used.

Requirement for activation of multiple signaling pathways in cytokine induction by influenza virus

The findings described above demonstrated that the MAPK signaling pathways are activated when human lung slices are exposed to influenza virus. We next sought to determine whether the activation of these signaling pathways is essential for the induction of cytokines. Lung slices were preincubated for 4 h in medium with 50 μM of the ERK pathway inhibitor U0126, 0.5 μM of the p38 pathway inhibitor SB203580, or 0.5 μM of the SAPK/JNK pathway inhibitor SP600125. For human lung, these doses were sufficient to inhibit induction of their corresponding signaling pathways by PMA/LPS. A combination of the three inhibitors was also used. The inhibitors remained in the medium throughout the duration of the experiment, and slices that were not treated with inhibitors were exposed to an inhibitor solvent (DMSO) as an additional control. The lung slices were incubated for 20 h either with virus (6×106 PFU/ml) or with PMA (100 ng/ml) prior to collection of the supernatants for cytokine and chemokine determination.

The data revealed differences in the requirements for signaling pathway activation for the induction of specific cytokines by influenza virus (Fig. 5). With the exception of virus induction of IL-8 and MIP-1α/β, addition of any of the three inhibitors diminished cytokine and chemokine induction by influenza virus. In the case of MIP-1α/β, inhibition of the p38 pathway individually did not alter induction of this chemokine by virus. For all cytokines and chemokines tested, inhibition of all three pathways appeared to enhance blockade of virus-mediated cytokine and chemokine induction.

FIG.5.

FIG.5

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Inhibition of influenza virus induction of cytokines and chemokines by signal pathway inhibitors. Human lung tissue slices were preincubated with 0.5 μM of SB203580, 0.5 μM of SP600125 and 50 μM of U0126, or all three together (mixture) or inhibitor solvent (Mock), and the concentrations of these reagents was maintained throughout. Slices were then exposed to virus diluent, 6 × 106 PFU/ml of influenza virus PR8 and OK/06, or PMA (100 ng/ml) for 20 h prior to the measurement of cytokines and chemokines by ELISA. The data are expressed as the means ± SEM of four experiments, and 3 tissue slices were used per experiment. Statistical significance was determined by ANOVA. Means were compared to data from the virus-infected control group without inhibitors. *P < 0.05; **P < 0.01.

OK/06 was more sensitive to the p38 inhibitor, SB203580, for IL-6 induction than PR8, but not for induction of MIP-1α/β, MCP-1, IP-10 and IL-8. PR8 induced MCP-1 and IP-10 were p38 dependent while MIP-1α/β induction was likely independent of activation of this pathway. The different sensitivities of the two strains to inhibitors were not likely due to inhibitor-caused viral replication defects since NS1 gene expression of both strains was not affected in inhibitor treated tissue (data not shown).

Cellular source of cytokine induction by influenza virus

To determine the lung cellular elements that participate in the lung innate immune cytokine response to influenza virus, we performed immunohistochemistry on virus-exposed lung slices. Lung slices were exposed to virus at 6 × 106 PFU/ml or virus buffers for 24 h in the presence of Brefeldin A to enhance detection of cytokines. The slices were then processed for immunohistochemistry for detection of influenza virus nucleoprotein (NP) and the cytokines MIP-1α and IP-10. Macrophages were also detected by using an anti-CD 68 polyclonal antibody (De Groot et al., 1997; Noorman et al., 1997). Tissues exposed to virus diluent were used to demonstrate basal cytokine and chemokine detection. An additional negative control was performed for MIP-1α and IP-10 detection by using the same staining protocol but with the MIP-1α or IP-10 primary absence of antibody omitted. There was minimal background immunofluorescence in the absence of MIP-1α and IP-10 primary antibodies (Figs, S1 and S2). MIP-1α and IP-10 detection were significantly enhanced by influenza virus infection (Fig. 6, 7, Panels C). Both MIP-1α and IP-10 were detected in both epithelial cells and CD 68+ alveolar macrophages (Fig. 6, 7, Panels F). There were also scattered interstitial cells that also stained positive for MIP-1α and IP-10. The results indicate that both lung epithelia and alveolar macrophages contribute to the innate immune response through induction of cytokines. Additional interstitial cells may also contribute to this response. There is no obvious cell specificity difference for cytokine expression between the two influenza strains.

FIG.6.

FIG.6

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Cellular source of MIP-1α induction by influenza virus in human lung. Lung slices were exposed to 6 × 106 PFU/ml of influenza virus PR8 and OK/06 or virus diluent for 24 h in the presence of BFA to enhance detection of cytokines. The slices were then processed for immunohistochemistry for detection of the chemokine MIP-1α using goat polyclonal antibodies, viral nucleoprotein (NP) using rabbit polyclonal antibody and macrophages using an anti-CD 68 polyclonal antibody. Panels E are brightfield images that demonstrate that lung architecture is preserved during the experiment. The rest of the panels are fluorescent images that demonstrate nuclei (Panels A, blue), NP (Panels B, red), MIP-1α (Panels C, green) and macrophages (Panels D, cyan). Panels F, are overlays of the fluorescent images and demonstrate that the primary cellular sources of the cytokines are alveolar macrophages (arrows) and epithelial cells. Some interstitial cells are also positive for MIP-1α. The bar represents 100 μm.

FIG.7.

FIG.7

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Cellular source of IP-10 induction by influenza virus. Lung slices were exposed to 6 × 106 PFU/ml of influenza virus PR8 and OK/06 or virus diluents for 24 h in the presence of BFA to enhance detection of cytokines. The slices were then processed for immunohistochemistry for detection of the chemokine IP-10 using goat polyclonal antibodies, viral NP using rabbit polyclonal antibody and macrophages using an anti-CD 68 polyclonal antibody. Panels E are brightfield images that demonstrate that lung architecture is preserved during the experiment. The rest of the panels are fluorescent images that demonstrate nuclei (Panels A, blue), NP (Panels B, red), IP-10 (Panels C, green) and macrophages (Panels D, cyan). Panels F, are overlays of the fluorescent images and demonstrate that the primary cellular sources of the cytokines are alveolar macrophages (arrows) and epithelial cells. Some interstitial cells are also positive for IP-10. The bar represents 100 μm.

Discussion

Host innate immunity is the first line of protection against infection by virus and is essential in local control of invading microbes. The innate immune system is composed of macrophages, neutrophils, natural killer cells and dendritic cells which play crucial roles in the initiation and subsequent direction the of adaptive immune response, as well as in promoting localized inflammation and producing cytokines that recruit additional leukocytes to the site of infection. However, unrestrained or excessive stimulation of the innate immune response can be harmful. The recent study from Kobasa et al. using reconstructed 1918 influenza virus in macaques revealed that the unprecedented lethality of the 1918 pandemic virus is linked to an aberrant innate immune response (Kobasa et al., 2007). Similar analysis of mice infected with the reconstructed 1918 influenza virus also demonstrated an increased and accelerated activation of host immune response genes associated with severe pulmonary pathology (Kash et al., 2006). With increased mobility of potentially infected subjects due to modern travel, concern has been raised about the introduction of highly pathogenic avian influenza viruses into humans and the possibility, if adaptation of the virus enables person-to-person spread, of a resultant worldwide pandemic. Therefore, a comprehensive understanding of the human innate immune response to influenza virus is important. Moreover, understanding the contribution of host immune responses to virulent influenza virus infections is an important starting point for the identification of prognostic indicators and the development of novel antiviral therapies.

Our current study examines the interaction of influenza virus with human lung tissue. Influenza virus induces a strong pro-inflammatory cytokine and a monocyte and lymphocyte chemokine response in human lung tissue, as shown by our RPA and ELISA results (Fig. 2 and 3). Specifically, this response includes induction of the cytokines IL-6 and IFN-γ, the monocyte chemokines MIP-1α/β and MCP-1, the neutrophil chemokine IL-8, and the lymphocyte chemokine IP-10. Immunohistochemistry data clearly shows that MIP-1α and IP-10 are induced not only in macrophages, as is traditionally thought, but also in alveolar epithelial cells. This discovery suggested epithelial cells may play more important roles in contributing to the pathogenesis of airway inflammation caused by influenza virus infection. Prior to the data presented in this work, only IL-6, IL-8, MCP-1, Eotaxin and RANTES have been shown to be induced by influenza in isolated human epithelial cells, or cell lines in culture (Adachi et al., 1997; Choi and Jacoby, 1992; Kawaguchi et al., 2000; Matsukura et al., 1996). Macrophages and dendritic cells have been the traditional focal points for study of the initial innate immune response to influenza virus infection, and are, of course, important in the response to the pathogen. However, the lung epithelium is the first site of exposure for virus, and contains a large, 80 m2 surface area for viral contact. Thus, due to the sheer number of the cells involved, and our demonstration that these cells respond to the virus with elaboration of cytokine, and chemokines, the contribution of these cells to the innate immune response is likely significant.

Our human lung organ culture model, unlike artificial models using differentiated cell lines, reproduces the normal lung architecture as it maintains the three dimensional structure present in native tissue. Also, current cell culture models lack the diversity of cell types found in normal lung. Thus, complex interactions of the different cell types in the native lung are not accurately modeled in cultured cell lines, but are closely replicated and reproduced structurally in our model. It is likely that the diverse cytokine and chemokine responses to influenza presented herein more accurately describe the actual lung innate immune response to the viruses.

One limitation of our study is that some of the lung tissue used was from patients who were undergoing resection for lung cancer. Thus, they are older, frequently suffer from COPD, and usually have previous cigarette smoke exposure. These patient-specific factors have variable effects on cytokine responses to stimuli. However, the overall responses, though not the magnitude or exact timing of the cytokine responses, are frequently comparable (Gaschler et al., 2008; Hackett et al., 2008). We have found that in the general group of patients from whom we have obtained tissues there are consistent cytokine responses to stimuli not only from slice to slice, but also from donor to donor. Thus, we believe that although the magnitude of the responses might be different, the overall characteristics of the responses are likely similar to those that occur in patients affected with influenza virus. Furthermore, we included in the inhibitor experiment both tissues from surgeries and from National Disease Research Interchange (NDRI). The overall cytokine responses of the lung from resection are comparable to a healthy human lung (Fig. 5 vs. Fig. 3 at 20 h after infection).

The initial phase of influenza virus infection is characterized by neutrophilic infiltration followed by a mixed monocytic/neutrophilic infiltration from the peripheral blood across the endo-/epithelial barrier into the alveolar air space (Toms et al., 1977). Later, the airways are filled with exudates containing monocytes and lymphocytes (Haesebrouck et al., 1985; Van Reeth, 2000). Induction of the neutrophil chemotaxin IL-8 is consistent with the finding of neutrophil infiltration in the initial phase. The induction of monocyte chemotaxins MIP-1α/β and MCP-1 is consistent with monocyte infiltration that occurs later in the course of infection. Finally, the robust expression of the lymphocyte chemotaxin IP-10 is likely important in recruiting lymphocytes that are prominent in the airway at latter stages of disease, and participate in the adaptive immune response to influenza. It is important to recognize that strict correlation of the autopsy findings with our present study is limited by the fact that autopsy studies represent findings of the terminal stages of influenza infection, while this current study examines virus-host interactions throughout the course of the illness.

The elucidation of intracellular signaling pathways that are activated by influenza A virus infection is important for the understanding of both viral replication strategies and host defense mechanisms. Here, we show that productive infection of lung slices results in a moderate activation of all the subgroup of MAPK signaling pathways, p38, ERK and JNK. Inhibition experiments showed that individual pathways are important in induction of specific cytokines and chemokines. However, as addition of all three inhibitors together provided additional suppression of the cytokine and chemokine responses, it is likely that multiple pathways play a role in cytokine and chemokine induction by influenza virus. Our data extends studies that show that p38 MAP kinase and JNK regulates RANTES production by bronchial epithelial cells induced by influenza virus A. Also p38 MAP kinase has been identified as important for hyperinduction of TNF-α expression in human macrophages due to the avian H5N1 influenza virus (Kujime et al., 2000; Lee et al., 2005). Ludwig et al. have also demonstrated that influenza virus-induced activation of JNK and AP-1 appears to be part of the innate antiviral response of the MDCK and U937 cells (Ludwig et al., 2001). This is the first report that all the three MAPK signaling pathways are involved in cytokine induction by influenza virus in cultured human lung tissue.

We chose to include an H3N2 influenza virus recently isolated from the community in our study. The emergence of H3N2 influenza virus strains as a major seasonal pathogen is a major public health concern. This is because vaccination and antiviral therapy are the mainstays of planning against yearly influenza outbreaks, and H3N2 vaccine production is problematic. Most human H3N2 influenza viruses isolated after 1992 have lost the ability to agglutinate chicken red blood cells and only bind to human or guinea pig erythrocytes (Gulati et al., 2005; Nobusawa et al., 2000). These viruses grow well in MDCK cells, but they are difficult to adapt to egg culture, which has caused vaccine shortages. H3N2 viruses account for over 90% of the strains that are adamantane resistant, which has created additional public health issues (MMWR Journal Article, 2006). Understanding the pathogenesis of H3N2 infection in order to develop novel therapies is particularly important. Our use of both the H1N1 and H3N2 strains will ensure that the results can be correlated with responses studied using adapted strains in other models, and with the responses that occur when the general public is exposed to non-adapted influenza. In isolated human type II alveolar epithelial cells (ATII), both H1N1 and H3N2 viruses induced release of proinflammatory cytokines such as IL-6, IL-8, RANTES, MCP-1, and MIP-1β. Although both viruses have similar ability to infect and replicate in ATIIs, the wild-type strain PR8 is a stronger inducer of chemokines and cytokines than A/Phil/82 (a H3N2 reassortant PR/8 virus, Wang et al., 2009). Furthermore, since the ferret is considered the most reproducible model similar to human in terms of pathological changes and cytokine responses to influenza (Smith and Sweet, 1988), analysis of the local ferret immune response to human influenza isolates of the H1N1 and H3N2 subtypes showed that cytokine responses are subtype-independent (Svitek et al., 2008). In A549 cells (a human type II alveolar epithelial adenocarcinoma cell line), influenza A H1N1-induced cytokine mRNA expression was detectable at 24 h after infection, while H3N2 infection induced mRNA expression after 12 h of infection. Although influenza A H1N1 virus induced somewhat lower chemokine mRNA expression compared to H3N2 or influenza B viruses, the chemokine protein induction was nearly equal for all the influenza viruses tested (Veckman et al., 2006). In this study, despite the large difference in these strains in terms of growth in egg culture systems and adamantane resistance, we found that there were only subtle differences in terms of the innate immune cytokine response to H1N1 and H3N2 in human lung.

One research group studied the induction of the MAPK signal cascade by two seasonal human influenza A viruses, A/HK/218449/06 (H3N2) and A/HK/218847/06 (H1N1). Infection with H3N2 virus resulted in substantially increased activation of ERK signaling compared to that caused by H1N1 (Marjuki et al., 2007). Our data agree with earlier results in primary macrophages that only modest ERK and p38 signaling was induced during H1N1 infection (Lee et al., 2005). Furthermore, modest JNK activation was also observed in our model by H1N1.

Taken together, our results indicate that the human lung responds to infection by the influenza virus by producing a robust cytokine and chemokine response which recruits neutrophils, monocytes and lymphocytes to participate in the innate immune response. We also demonstrate that the production of the pro-inflammatory cytokines and chemokines are causally related to the activation of the signaling pathways ERK, JNK, and p38. This is the first description of the initial human lung innate immune response to influenza virus in a human organ culture model.

Materials and Methods

Preparation of influenza virus stock

The viruses used in this study include A/Oklahoma/309/06, a Wisconsin/05 like H3N2 isolate, and A/PR/34, H1N1 virus. A/Oklahoma/309/06 was isolated in the University of Oklahoma Health Sciences Center clinical microbiology lab in 2006. The viruses have been passaged in Madin-Darby canine kidney (MDCK) cells. MDCK cells were cultured in supplemented Dulbecco’s modified Eagle’s medium (DMEM). Viruses were grown in MDCK cells in DMEM/F12 with ITS+ (BD Biosciences, Franklin Lakes NJ) and trypsin (Liu and Air, 1993), harvested at 72 h postinfection and titered by plaque assay in MDCK cells. There was no detectable endotoxin in the final viral preparations used in the experiments as determined by limulus amebocyte lysate assay (Cambrex, Walkersville, MD). The lower limit of detection of this assay is 0.1 EU/ml or approximately 20 pg/ml LPS.

Lung explant culture

Human lung tissue was obtained from patients undergoing lung resection for cancer in accordance with protocols approved by the Institutional Review Boards of the University of Oklahoma, Veterans Administration Hospital, Baptist-Integris Hospital, St. Anthony’s Hospital, and Mercy Health Center, all of Oklahoma City, OK. Only tissue that did not contain tumor was used for experiments. For experiments on MAPK inhibition, lung tissue from NDRI was also used for comparison with tissue from surgeries. The tumor free lung tissue was transported on ice in sterile Phosphate buffered saline (PBS) containing 200 μg of gentamicin/ml, 100 U of penicillin/ml, 100 μg of streptomycin/ml, and 2.5 μg of amphotericin B/ml (PBS+antibiotics), and processed immediately. The subsegmental bronchi of lung tissue with intact outer pleura were isolated and cannulated with a flexible Teflon catheter, and the lung segments were gently inflated with 37°C lung slice medium (LSM) containing 1.5% low-melting-low-gelling agarose (BioWhittaker, Rockland, ME). LSM consisted of minimal essential medium (Sigma Chemical Co., St. Louis, MO) supplemented with 1.0 μg of bovine insulin/ml, 0.1 μg of hydrocortisone/ml, 0.1 μg of retinyl acetate/ml, 200 μg of gentamicin/ml, 100 U of penicillin/ml, 100 μg of streptomycin/ml, and 1.25 μg of amphotericin B/ml. After the agarose inflated lung was solidified, 1 cm diameter tissue cores of non-emphysematous tissue were prepared and sliced into 500 μm thick sections using a Krumdieck Tissue Slicer (Alabama Research and Development, Munford, AL). During slicing, the cores were submerged in chilled PBS + antibiotics. Each slice was placed in 0.5 ml of LSM in a single well of a 24-well plate, then placed in a humidified incubator at 37°C in 5% CO2. The LSM was replaced prior to subjecting the slices to the experimental treatments.

Infection of human lung slices with influenza virus

After overnight incubation of the lung slices, the culture medium was replaced with fresh LSM. For each data point, three lung slices were each exposed to 6 × 106 PFU/ml of influenza virus PR8 and OK/06, and allowed to incubate at 37°C, 5% CO2 for the indicated periods. Virus diluent was used as a negative control, and PMA (100 ng/ml) was used as a positive control. Following stimulation for various times, media supernatants were harvested and stored at −20°C prior to ELISA.

OK/06 H3N2 virus was reacted with Cy3 dye in 0.1 M sodium carbonate buffer (pH 9.3) for 30 minutes at room temperature with occasional mixing according to the manufacturer’s protocol for labeling proteins. The labeled virus was then dialyzed in PBS containing 0.6 mM CaCl2 and 0.5mM MgCl2 (CaMg-PBS) for 72 h with three buffer changes to remove unlabeled dye. The tissue slices exposed to virus-free diluent were used as a negative control for influenza virus detection.

RNA preparation and RNase protection assay (RPA)

Lung slices were harvested by homogenization in TRIzol Reagent (Invitrogen, Carlsbad CA), and the total RNA was isolated according to the manufacturer’s protocol using glycogen (20 mg/ml) as the carrier. Triplicate slices yielded 8–10 μg total RNA, 6 μg of which were used for a single RPA reaction.

Relative gene expression was determined with the RiboQuant Multi-Probe RNase Protection Assay system (BD Biosciences). A custom cytokine set was used containing probes for TNF-α, IL-12/p35, IP-10, IFN-γ, MIP-1α, MCP-1, IL-8 and IL-6. The template set contained probes for ribosomal protein (L32) and GAPDH to use for normalization of RNA loading. Labeled riboprobe was made with the In Vitro Transcription Kit (BD Biosciences) and (α-32P) UTP. The RPA Kit (BD Biosciences) was used for hybridization of the probe with the target RNA in the samples, and for digestion of unpaired transcripts. Additional controls included a sample containing yeast total RNA, a sample with the custom template control RNA, and one with unprotected probe. The resulting mRNA duplexes were separated on a standard 50 cm long, 0.4 mm thick polyacrylamide gel. The gel was dried and imaged using a phosphorimager (Molecular Dynamics, Sunnyvale, CA). The image was analyzed with ImageQuant 5.0 software (Molecular Dynamics) using the volume quantitation method with histogram peak background subtraction. The identity of each protected band in a sample lane was determined from the position of the bands in the unprotected probe lane. Fold increase for each RNA species over control samples prepared at the same time points was determined after correction for loading using the L32 and GAPDH standards.

Signal pathway inhibition and cytokine and chemokine protein determination by ELISA

After overnight incubation of the lung slices, the culture medium was replaced with fresh LSM. Lung slices were exposed to 6 × 106 PFU/ml of influenza virus PR8 and OK/06 in triplicate wells of a 24-well plate and allowed to incubate at 37°C for 24 h. Virus diluent was used as a negative control, and PMA (100 ng/ml) was used as a positive control.

To determine the effect of inhibition of the ERK, p38, and JNK signaling pathways on cytokine induction, the specific inhibitors U0126, SB203580 and SP600125, (Calbiochem, San Diego CA) were used, respectively. Lung slices were pre-incubated with 0.5 μM of SB203580, 0.5 μM of SP600125 and 50 μM of U0126 for 4 h at 37°C. These optimized doses were determined using dose range experiments in lung slices stimulated with PMA/LPS. The medium was replaced with LSM, and the lung slices were exposed to 6 × 106 PFU/ml of influenza virus PR8 and OK/06 for 20 h at 37°C. The final concentration of the inhibitors was maintained throughout the experiment. PMA (100 ng/ml) was used as a positive control, and mock-infected slices were exposed to the same concentration of the inhibitor solvent, dimethyl sulfoxide (DMSO), as present when all three inhibitors were used. After incubation, the supernatants were collected, centrifuged at 10,000 × g for 2 minutes, transferred to a new tube and stored at −20°C.

Cytokine ELISA’s were performed using anti-cytokine monoclonal primary antibodies and biotinylated anti-cytokine polyclonal secondary antibodies (R&D Systems, Minneapolis MN). As the MIP-1α monoclonal antibody crossreacts with MIP-1β, the results were expressed as MIP-1α/β levels. Plates were developed using the TMB reagent (BD Biosciences).

Signaling pathway kinase assay

Human lung slices were maintained overnight at 37°C, 5% CO2 in 0.5 ml LSM and the medium was replaced prior to stimulation. Four to eight slices per condition were used for MAPK family assays. Slices were stimulated with either 6 × 106 PFU/ml of influenza virus or PMA. Mock-infected, negative control slices were exposed to an equivalent volume of virus-free diluent. After incubation at 37°C, 5% CO2 for the indicated times, the slices were harvested and homogenized in 100 μl cold RIPA lysis buffer. Clarified lung slice homogenates containing 20 to 30 μg protein were heat denatured in SDS-PAGE sample buffer. The samples were separated on a 10% SDS-PAGE gel and then electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes. To detect activated, phosphorylated ERK, p38 or SAPK/JNK, the membranes were blocked overnight in 5% powdered milk in Tris-buffered saline (TBS) and then immunoblotted with specific affinity-purified, rabbit polyclonal antibodies (Cell Signaling Technology, Beverly, MA). Identically prepared membranes were probed with either rabbit polyclonal anti-ERK, anti-p38 or anti-SAPK/JNK antibodies that recognized both phosphorylated and non-phosphorylated forms of the signaling proteins (Cell Signaling Technology). The membranes were developed with horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell Signaling Technology) and chemiluminescent reagents (Pierce Biotechnology, Rockford, IL). The developed membranes were exposed to x-ray film, and digital scans of the film were quantified using ImageQuant software (BD/Molecular Dynamics, Bedford, MA).

Cytokine Immunohistochemistry on Lung Tissue Explants

To examine which cell types in the lung tissue produced IP-10 and MIP-1α after influenza infection, lung slices were exposed to 6 × 106 PFU/ml of influenza virus or virus diluent in fresh LSM and incubated at 37° C for 24 h. Brefeldin A (L C Laboratories, Wofford MA) was added at a concentration of 5 μg/ml to block protein export in order to enhance cytokine detection. Following the incubation, the lung slices were fixed with 4% paraformaldehyde in PBS at room temperature for 30 minutes and were then imbedded in paraffin. Sections (3–5 μm) were mounted on glass slides and immuno-probed overnight at 4°C with a goat anti-human polyclonal antibody for IP-10 (R&D Systems) or MIP-1α (Abcam, Cambridge, MA), a polyclonal rabbit anti-NP serum (Zhang and Air, 1994) and an anti-CD 68 monoclonal antibody (Dakocytomation, Carpinteria CA). After washing, the sections were probed with a donkey anti-goat secondary antibody conjugated to Alexa Fluor 350, a donkey anti-rabbit secondary antibody conjugated to Alexa Fluor 546 and a donkey anti-mouse secondary antibody conjugated to Alexa Fluor 647, and the cell nuclei were stained with SYTOX green (all from Molecular Probes). Transmitted light and fluorescent microscopy images were obtained using a Leica SP2 MP Confocal microscope.

Statistical analysis

Where applicable, the data have been expressed as the means ± standard error of the mean (SEM). Statistical significance was determined by one-way ANOVA with Student-Newman-Keuls post hoc correction for multiple comparisons. Significance was considered as P <0.05.

Supplementary Material

01

FIG.S1. Immunohistochemistry of MIP-1α induction by influenza virus. Lung slices were exposed to 6 × 106 PFU/ml of influenza virus PR8 and OK/06 or virus diluent for 24 h in the presence of BFA to enhance detection of cytokines. The slices were then processed for immunohistochemistry for detection of the chemokine MIP-1α using goat polyclonal antibodies as described in Materials and Methods. Panels A, D, G and J are brightfield images that demonstrate that lung architecture is preserved during the experiment. Panels B, E, H and K are fluorescent images that demonstrate nuclei by DAPI staining (blue) and MIP-1α (E and H) detection by Alexa-Fluor 546 (red) secondary antibody. Panels C, F, I and L are overlays of the brightfield and fluorescent images and demonstrate that the primary cellular sources of the cytokines are alveolar macrophages and epithelial cells. Some interstitial cells are also positive for MIP-1α. Panels J, K, and L are brightfield, fluorescent, and overlayed images of influenza virus-exposed tissue stained with secondary antibody only, and confirm that omission of the primary antibody results in a loss of signal. The bar represents 100 μm.

02

FIG.S2. Immunohistochemistry of IP-10 induction by influenza virus. Lung slices were exposed to 6 × 106 PFU/ml of influenza virus PR8 and OK/06 or virus diluent for 24 h in the presence of BFA to enhance detection of cytokines. The slices were then processed for immunohistochemistry for detection of the chemokine IP-10 using goat polyclonal antibodies as described in Materials and Methods. Panels A, D, G and J are brightfield images that demonstrate that lung architecture is preserved during the experiment. Panels B, E, H and K are fluorescent images that demonstrate nuclei by DAPI staining (blue) and IP-10 (E and H) detection by Alexa-Fluor 546 (red) secondary antibody. Panels C, F, I and L are overlays of the brightfield and fluorescent images and demonstrate that the primary cellular sources of the cytokines are alveolar epithelial cells and macrophages. Panels J, K, and L are brightfield, fluorescent, and overlayed images of influenza virus-exposed tissue stained with secondary antibody only, and confirm that omission of the primary antibody results in a loss of signal. The bar represents 100 μm.

Acknowledgments

We thank Dr. Gillian Air for providing us the influenza virus strains and the polyclonal rabbit anti-NP serum. The research described in this work was partially supported by a Clinical Innovator Award of Flight Attendant Medical Research Institute (to W. W.), and by the National Institute of Allergy and Infectious Diseases, project 1U19 AI62629 (to J.P.M. and K.M.C.).

We acknowledge the kind assistance of the Departments of Pathology of the Veterans Administration Hospital, the University of Oklahoma Medical Center, Baptist-Integris Hospital, St. Anthony’s Hospital, and Mercy Health Center, all of Oklahoma City, OK. We also acknowledge the assistance and expertise of Julie Maier of the Oklahoma Medical Research Foundation Imaging Analysis core facility.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01

FIG.S1. Immunohistochemistry of MIP-1α induction by influenza virus. Lung slices were exposed to 6 × 106 PFU/ml of influenza virus PR8 and OK/06 or virus diluent for 24 h in the presence of BFA to enhance detection of cytokines. The slices were then processed for immunohistochemistry for detection of the chemokine MIP-1α using goat polyclonal antibodies as described in Materials and Methods. Panels A, D, G and J are brightfield images that demonstrate that lung architecture is preserved during the experiment. Panels B, E, H and K are fluorescent images that demonstrate nuclei by DAPI staining (blue) and MIP-1α (E and H) detection by Alexa-Fluor 546 (red) secondary antibody. Panels C, F, I and L are overlays of the brightfield and fluorescent images and demonstrate that the primary cellular sources of the cytokines are alveolar macrophages and epithelial cells. Some interstitial cells are also positive for MIP-1α. Panels J, K, and L are brightfield, fluorescent, and overlayed images of influenza virus-exposed tissue stained with secondary antibody only, and confirm that omission of the primary antibody results in a loss of signal. The bar represents 100 μm.

NIHMS155720-supplement-01.tif (1.9MB, tif)
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FIG.S2. Immunohistochemistry of IP-10 induction by influenza virus. Lung slices were exposed to 6 × 106 PFU/ml of influenza virus PR8 and OK/06 or virus diluent for 24 h in the presence of BFA to enhance detection of cytokines. The slices were then processed for immunohistochemistry for detection of the chemokine IP-10 using goat polyclonal antibodies as described in Materials and Methods. Panels A, D, G and J are brightfield images that demonstrate that lung architecture is preserved during the experiment. Panels B, E, H and K are fluorescent images that demonstrate nuclei by DAPI staining (blue) and IP-10 (E and H) detection by Alexa-Fluor 546 (red) secondary antibody. Panels C, F, I and L are overlays of the brightfield and fluorescent images and demonstrate that the primary cellular sources of the cytokines are alveolar epithelial cells and macrophages. Panels J, K, and L are brightfield, fluorescent, and overlayed images of influenza virus-exposed tissue stained with secondary antibody only, and confirm that omission of the primary antibody results in a loss of signal. The bar represents 100 μm.

NIHMS155720-supplement-02.tif (1.8MB, tif)

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