The healthy lung maintains a steady state of immune readiness to rapidly respond to injury from invaders. Integrins are important for setting the parameters of this resting state, particularly the epithelial-restricted αVβ6 integrin, which is upregulated during injury. Once expressed, αVβ6 moderates acute lung injury (ALI) through as yet undefined molecular mechanisms. We show that the upregulation of β6 during influenza infection is involved in disease pathogenesis. β6-deficient mice (β6 KO) have increased survival during influenza infection likely due to the limited viral spread into the alveolar spaces leading to reduced ALI. Although the β6 KO have morphologically normal lungs, they harbor constitutively activated lung CD11b+ alveolar macrophages (AM) and elevated type I IFN signaling activity, which we traced to the loss of β6-activated transforming growth factor-β (TGF-β). Administration of exogenous TGF-β to β6 KO mice leads to reduced numbers of CD11b+ AMs, decreased type I IFN signaling activity and loss of the protective phenotype during influenza infection. Protection extended to other respiratory pathogens such as Sendai virus and bacterial pneumonia. Our studies demonstrate that the loss of one epithelial protein, αVβ6 integrin, can alter the lung microenvironment during both homeostasis and respiratory infection leading to reduced lung injury and improved survival.
At each breath, the lung is challenged by a large number and diversity of microbes and other foreign material such as pollen and dust. Many inhaled microbes cause lethal infections if not contained by the lung immune system, which has evolved to balance rapid and efficient microbial clearance with protection of the delicate lung structure from excessive damage. Lung damage caused by microbial pathogens is the cause of acute lung injury (ALI), which leads to increased edema, alveolar permeability, and impaired oxygen exchange. In severe cases, ALI can result in impaired gas exchange function and ultimately death (acute respiratory distress syndrome or ARDS) [1]. To mitigate lung damage after infection, inflammation resolves, returning to homeostasis that restores normal lung function [2,3]. The air-interface structure of the lung involves a complex immune cell population including resident interstitial macrophages and dendritic cells, and GM-CSF-dependent alveolar macrophages [4–7]. A key question in understanding pulmonary immunity concerns how the balance between effective immune surveillance and maintenance of lung anatomy and physiology is achieved through life. Integrins are heterodimers composed of α and β subunits that regulate a plethora of cellular functions including cell-matrix and cell-cell adhesion, cell activation, and the recognition and post-translational processing of molecules [8]. In the lung microenvironment, β6 (encoded by Itgb6) exclusively pairs with the αV subunit and expression of the αVβ6 heterodimer is limited to epithelial cells. In this context, αVβ6 plays a key role in controlling several steps in lung homeostasis. Mice lacking the β6 subunit (β6 KO) have highlighted the central role αVβ6 integrin plays in balancing the pulmonary environment during injury [9,10]. For example, in both bleomycin-induced and PAR1-mediated ventilator-associated lung injury and airway hyper-responsiveness models, β6 KO mice were protected from ALI [11–13]. β6 KO mice also had reduced IL-1β-mediated lung injury in a model of bronchopulmonary dysplasia due to decreased inflammation and cellular infiltrate to the lung [14]. Alveolar macrophages isolated from β6 KO are characterized as large and foamy and express high amounts of MMP12 [15,16]. Severe respiratory infections are often associated with ALI/ARDS [17–19], and the pathophysiology is similar for a variety of infectious pathogens. For example, patients with SARS and MERS-CoV were found to have increased infiltration of inflammatory macrophages, accumulation of debris in the lung, and diffuse alveolar damage [20,21]. Similarly, elderly patients suffering from RSV sustained widespread alveolar damage [22]. Bacterial pneumonia can result in increased formation of hyaline membranes [23]. Deposition of collagen and fibrin, while necessary for lung repair, can also build up and block gas exchange [24]. In the case of influenza, the major complication leading to ALI/ARDS is pulmonary edema and impaired fluid clearance due to dysregulation of transporters that clear fluid from the alveolus such as ENaC and Na+K+ATPase [23,25,26]. The virus itself can also directly kill epithelial cells [27]. ALI/ARDS has a mortality rate of 30–50%, therefore it is critical from a public health perspective to understand the mechanisms at play [28,29]. Given the role of the β6 integrin in modulating ALI, we hypothesized that its upregulation during respiratory infections such as influenza would be important for viral pathogenesis. To test this, β6 KO mice were infected with the 2009 pandemic H1N1 influenza virus. β6-deficient mice were protected from influenza virus-induced disease as well as a broad range of respiratory pathogens, in most cases independent of effects on overall microbial numbers. Mechanistically, we found that epithelial β6 controls the homeostatic lung interferon response. In the absence of β6, type I interferon signaling is constitutive, causing the host to have an advantage over the spread of the virus. This protective phenotype was reversed by exogenous TGF-β1 or elimination of the type I interferon receptor, suggesting that αVβ6 controls a communication system between lung epithelia and immune cells through a TGF-β-dependent mechanism. These studies have important implications as transient inhibition of αVβ6 may represent a potential therapy for the management of acute lung injury.
β6 expression is induced upon mechanical or inflammatory injury and is an important mediator of ALI [10,12,14,30]. Thus, we hypothesized that upregulation of the β6 integrin during respiratory infections would be involved in viral pathogenesis. To test this, we measured β6 expression by quantitative RT-PCR at different times post-influenza infection. Consistent with lung injury models, β6 mRNA was significantly upregulated in whole lung homogenates by 3 days post-infection (dpi) and expression remained elevated through 5 dpi (p < 0. 0001) correlating with the appearance of ALI (Fig 1A). We then intranasally infected WT and Itgb6-deficient mice (β6 KO mice) with A/California/04/2009 (CA/09) H1N1 virus and monitored morbidity for 12 dpi. Compared to WT controls, β6 KO mice lost significantly less weight (4,6, and 8 dpi p < 0. 0001) and began to recover by 10 dpi, while WT mice lost weight and either succumbed to infection or were euthanized due to morbidity between 6–10 dpi (Fig 1B and 1C). The dose of CA/09 virus used (104 TCID50) was lethal to all WT mice, while 70% β6 KO mice survived the infection (p = 0. 0090). Mouse lethal dose 50 (MLD50) studies demonstrated the resistance of the β6 KO to influenza infection, with WT mice having an MLD50 of 102. 5 versus 105. 3 for the KO to the CA/09 virus. Protection was not limited to H1N1 infection; β6 KO mice were also protected from an emerging strain of avian influenza virus associated with severe and even fatal human respiratory disease [17], A/Anhui/1/2013 (H7N9) influenza virus (p = 0. 0326, Fig 1D). β6 KO mice were also significantly protected from a lethal challenge of Sendai virus (p = 0. 006, Fig 1E) and Streptococcus pneumonia (p = 0. 0082, Fig 1F). A major risk factor for influenza infection is secondary bacterial pneumonia [31]. As β6 KO mice were protected from individual influenza and S. pneumoniae challenges, we tested the β6 KO mice with a secondary bacterial challenge model. Mice were inoculated intranasally with a sublethal dose of A/Puerto Rico/8/34 H1N1 influenza virus, and 7 dpi administered a low dose of S. pneumoniae (D39X strain) [32]. This dual infection model is generally lethal in WT mice, although neither challenge alone causes death [33]. Survival of WT mice after secondary challenge was ~30%, while β6 KO mice were significantly protected (p = 0. 0226) with 70% survival (Fig 1G) highlighting the importance of β6 integrin in the pathogenesis of respiratory infections. To determine if the enhanced survival seen in the β6 KO mice was associated with decreased ALI, we examined histological sections for evidence of tissue injury, alterations of the alveolar capillary barrier, and inflammatory responses [1]. Although groups were histologically similar at 3 dpi, at 7 dpi inflammation and thickened septa involving extensive areas of alveolar parenchyma was seen in WT lungs. In marked contrast, evidence of inflammation and tissue damage in the β6 KO lungs was generally limited to terminal bronchioles and small numbers of adjacent alveoli (Fig 2A). Infected WT mice had increased TNF-α and IL-6 throughout the duration of infection, while the inflammatory cytokine response in β6 KO mice was less robust (Fig 2B). Evidence of alterations to the alveolar capillary barrier was found in infected WT mice beginning at 5 dpi as compared to KO mice (Fig 2C–2F) including heavier lungs as determined by wet/dry weight ratios (5 dpi p = 0. 0077,7 dpi p = 0. 0063) indicating increased edema (Fig 2C), accompanied by increased total protein (p < 0. 0001, Fig 2D) and albumin in the bronchoalveolar lavage fluid (BALF) (7 dpi p < 0. 0001, Fig 2E) and accumulation of Evans blue dye in the lungs of WT mice (p = 0. 0071, Fig 2F). Overall, β6 KO had decreased inflammation, acute lung injury, and improved survival during influenza infection. The most likely mechanism of protection in influenza infected β6 KO mice was decreased viral titers. However, tissue culture infectious dose 50 (TCID50) and quantitative real-time PCR showed no significant differences in viral titers, although RT-PCR suggested a trend towards lower titers in β6 KO mice (Fig 3A and 3B). We therefore spatially monitored viral spread in the lungs by immunohistochemistry for viral nucleoprotein (NP) and use of a reporter virus [34,35]. Viral spread into the alveolar spaces was reduced in β6 KO mice in comparison to WT mice, which had extensive NP staining of type II pneumocytes and macrophages in the alveolar spaces by 5 dpi. In contrast, NP staining in β6 KO mice was largely restricted to the terminal bronchiolar epithelium and adjacent alveoli (Fig 3C). This is even more evident using our NLuc CA/09 reporter virus, which clearly demonstrates decreased virus in β6 KO ([34,35], S1 Fig). Finally, WT lungs had a higher percentage of sites of active infection at 5 and 7 dpi as well as more viral antigen detected in the alveolar spaces (Fig 3D and 3E). Collectively, viral spread in KO lungs was limited compared to WT mice, with less extensive involvement of alveolar epithelial cells possibly explaining the improved lung function relative to controls. These findings are consistent with studies suggesting that increased survival during influenza infection can be independent of changes in viral titers [36–40]. To understand the underlying mechanism (s) for the decreased viral spread within the lungs of β6 KO mice, we performed flow cytometry on BALF and lungs at different times post-infection. No significant differences were noted in numbers of TNF-α/iNOS-producing (tip) DCs, neutrophils, CD4+ T cells, CD8+ T cells, or influenza PB1-specific CD8+ T cells in either BALF (Fig 4A–4E) or whole lung (Fig 4F–4J). However, we noted substantial differences in the resident lung F480+ CD11c+ CD11b+ populations (macrophages and dendritic cells), namely that β6 KO mice lacked a conventional CD11c+ alveolar macrophage (AM) population (Fig 5A). AMs have characteristic flow cytometry properties as compared to dendritic cells (DCs) or incoming activated bone marrow-derived inflammatory monocytes including autofluoresence in the FITC channel, high constitutive expression of CD11c, F4/80 and SiglecF and low expression of CD11b [4,41,42]. By contrast, incoming activated bone marrow-derived inflammatory monocytes express CD11b, providing a means to distinguish between resident AM, inflammatory bone marrow-derived monocytes and DCs [4,43]. When we examined the lung macrophage/DC populations in uninfected β6 KO mice we found they had higher numbers of autofluorescent cells indicating AMs (Fig 5B) but instead of the normal CD11chi CD11b- phenotype, the vast majority of the β6 KO AM were MerTK+, CD64+, CD11c+, and uniformly CD11b+ (Fig 5C; See S2 and S3 Figs for gating strategies). These cells maintained expression of F4/80 and SiglecF, suggesting they are AMs with altered properties. They were also phenotypically distinct from those of WT mice, with a ‘foamy’ appearance typical of activated macrophages (Fig 5D). Importantly, the β6 KO CD11c+ CD11b+ AM were present at the earliest times tested (d3 after birth), continued to be present at all points of adulthood (S4A Fig), and were not affected by reducing neonatal exposure to airborne particulate matter by using HEPA-filtered cages or low dust cage bedding (S4B Fig). Even throughout the course of influenza infection very few CD11chi CD11b- cells were present in the lungs of β6 KO mice (Fig 5E). As the lungs from β6 KO were overtly normal, we concluded the CD11c+ CD11b+ cells were sufficient to perform the normal activities of lung homeostasis [12,14,16,30]. Most pulmonary AM originate from embryonic erythro-myeloid progenitors whose replacement by blood monocytes under steady state is extremely slow [42,44]. To test whether the β6 KO CD11c+ CD11b+ AM were predominantly derived from embryonic progenitors, we created mice where the blood monocyte pool was severely depleted by loss of the chemokine receptor CCR2, which is the predominant mediator for monocyte egress from the bone marrow. In the lungs of β6/CCR2 doubly-deficient mice, CD11c+ CD11b+ AM were present in identical amounts compared with those of β6 KO mice, arguing these cells are unlikely to be derived from peripheral blood monocytes (S4C Fig). Although other unknown pathways to recruit monocytes to the lung cannot be discounted, they are not as well characterized or understood as the CCR2 pathway. Taken together, we concluded loss of an epithelial integrin alters the phenotype of lung AMs. Next we tested whether the β6 KO CD11c+ CD11b+ AM were a consequence of exposure to an altered lung microenvironment caused by loss of β6, or were caused by a cell-intrinsic mechanism. Adoptive transfer of congenically marked WT AM into β6 KO animals led to the CD11c+ CD11b+ AM phenotype after 7 days while transfer of β6 KO AMs into WT animals results in a WT CD11chi CD11b- phenotype (Fig 6A–6C). Importantly, the effect of the β6 KO lung microenvironment was not unique to transferred AM. Adoptive transfer of peritoneal resident macrophages into β6 KO animals resulted in conversion into the CD11c+ CD11b+ AM phenotype (Fig 6D and 6E) highlighting that it is the altered lung microenvironment of the β6 KO that controls AM phenotype. Donor cell recovery was consistent for all experiments (S5 Fig). What could be different about the β6 KO lung microenvironment? β6 integrin is the primary way to endogenously activate transforming growth factor-β (TGF-β) in the lung, which is known to restrain AM activity [16,45]. Thus, we next asked if the altered macrophage phenotype in the β6 KO mice was TGF-β1 dependent. As expected, endogenous lung TGF-β1 activity was reduced in β6 KO mice as compared to WT (Fig 7A). To test the hypothesis that reduced TGF-β1 activity contributed to the altered lung microenvironment of β6 KO mice, we intranasally administered exogenous TGF-β1 to WT and β6 KO mice every other day for 3 weeks (Fig 7B) as described [46]. β6 KO mice treated with TGF-β1 had increased numbers of CD11c+CD11b- macrophages (Fig 7C, quantification Fig 7D), partially reversing the CD11c+CD11b+ phenotype, and were no longer protected from influenza infection (Fig 7E) suggesting that the loss of β6-mediated TGF-β1activation is important for both protection and the ‘activated’ macrophage phenotype. β6 KO mice had an altered lung environment resulting in decreased viral spread. Type I IFNs are amongst the most potent cytokines limiting viral spread. Thus, we tested the hypothesis that β6 KO mice had increased type I IFN than WT. We used phosphorylated STAT1 levels (pSTAT1) as a marker for type I IFN signaling. Increased pSTAT1 along with increased type I IFN gene expression and unchanged expression of type II IFN-dependent targets such as IDO1 are indicative of a type I IFN dominated environment. Further, STAT1 is a robust marker of type I IFN signaling due to its weak activation by other cytokines such as IL-6 [47]. Immunoblot analysis of whole lung homogenates showed the β6 KO mice had robust type I IFN signaling at baseline that increased 3–5 dpi (Fig 8A). We sought to determine the source of type I IFNs and how epithelial-expressed β6 regulated the IFN response. We noted that type I IFN mRNAs were not substantially altered in uninfected β6 KO mice (S6A–S6D Figs). Furthermore, ELISA measurements for type I IFNs did not show substantial differences between WT and β6 KO mice within whole lung homogenates. Therefore, we generated β6 KO mice crossed to an IFN-β-YFP reporter strain. In these mice, YFP expression in the whole lung was predominantly found in CD45+ cells and increased in the absence of β6 (Fig 8B); although immunofluorescent microscopy of frozen lung sections from WT and β6 KO YFP reporter mice showed increased YFP+ cells scattered throughout the lung; including in the epithelia (Fig 8C and 8D). Further delineation of the exact cell types in this system expressing YFP is precluded by the strong autofluorescence in the FITC channel in lung macrophages [4]. Thus, we examined isolated AMs and primary epithelial cells grown at the air-liquid interface from β6 KO and WT mice for increased type I IFN signaling. Sorting autofluorescent CD11c+CD11b- AM from uninfected WT mice and comparing them by microarray to autofluorescent CD11c+CD11b+ ‘AM’ from β6 KO demonstrated that β6 KO AM showed a striking enrichment of gene ontogeny and pathway terms linked to immune responses (S7 Fig), particularly genes associated with type I IFN responses (Fig 8E, Table 1). There was no difference in IDO1 expression, a cardinal IFN-γ regulated gene [48] (Table 1). These data suggest lung macrophages in the absence of β6 had increased IFN signaling. To test this further, we measured phosphorylation of IRF3 and STAT1, hallmarks of the type I IFN response, in the alveolar macrophage populations from WT and β6 KO mice. CD11c+ CD11b+ macrophages from uninfected β6 KO mice had increased phospho-IRF3 and phospho-STAT1 relative to WT CD11c+CD11b- AM (Fig 8F). To determine if type I IFN signaling was also increased in epithelial cells, primary bronchotracheal epithelial cells (mTEC) from WT and β6 KO mice were isolated, differentiated and grown at the air-liquid interface prior to infection with CA/09 virus (MOI 0. 1). Phosphorylated and total levels of STAT1 and IRF3 were quantitated at 24 hours post-infection by immunoblot (Fig 8H). β6 KO mTEC exhibited significant increases in total STAT1 and IRF3 levels as compared to WT mTEC after infection as well as at baseline, although differences in phosphorylated STAT1 and IRF3 were minimal (Fig 8H and S6E Fig). Based on these data, we concluded that regulation of type I IFNs was dysregulated in the lungs of β6 KO mice at baseline, and that hematopoietic cells as well as epithelial cells may be involved. Finally, to determine if this is regulated by TGF-β1, lung homogenates from mice administered exogenous TGF-β1 were monitored for total and phosphorylated STAT1. Treatment decreased phospho-STAT1 in whole lung homogenate (Fig 8I). Macrophages isolated from TGF-β1-treated β6 KO mice also displayed significantly decreased mRNA encoding levels of the IFN associated transcripts Irf7, Ifit1, and Oas1g that were comparable to levels in WT macrophages (Fig 8G) highlighting that the loss of β6 activated TGF-β results in an altered lung macrophage population and increased type I IFN activation. We question whether this was the reason for the enhanced protection from influenza infection. To determine if the increased type I IFN signaling in β6 KO mice was responsible for the enhanced protection, we crossed β6 KO with Ifnar-/- mice (β6/IFNAR double KO), which lack the type I IFN receptor and all type I IFN signaling, then challenged these mice and their cognate controls created from the same heterozygote crosses with 104 TCID50 of CA/09 virus. Mice were monitored for morbidity and mortality for 12 days. β6/IFNAR double KO mice did not survive infection (p = 0. 0039 compared to β6 KO, Fig 9A) and exhibited similar mortality to WT mice. Further, imaging WT, β6 KO, IFNAR KO, and β6/IFNAR double KO mice infected with CA/09-NLuc reporter virus at 3 and 7 dpi showed that the ββ6 KO mice had reduced viral spread in the lungs as compared to WT controls (S1A and S1B Fig). In contrast, viral spread in β6/IFNAR double KO mice was comparable to WT and IFNAR KO mice, highlighting the importance of type I IFN signaling in limiting viral spread during infection. However, the ‘activated’ CD11c+CD11b+ macrophage phenotype of the β6 KO crosses remained the same as β6/IFNAR double KO (Fig 9B) suggesting the protective anti-viral phenotype of the β6 KO is type I IFNAR-dependent, but another pathway regulates the phenotype of the resident macrophages in the β6 KO independent of type I IFNs.
We propose a model where during pulmonary homeostasis, β6 integrin, possibly by activation of endogenous TGF-β1, suppresses CD11b expression on alveolar macrophages and type I IFN signaling within the lung microenvironment. However, in the absence of β6 integrin, CD11b expression is increased as is type I IFN signaling inducing a ‘primed’ antiviral state in the absence of infection (Fig 10). Through the use of TGF-β1 ‘rescue’ experiments and β6 KO mice lacking type I IFN signaling, we were able to demonstrate the resistance of β6 KO mice to influenza depended on the loss of TGF-β1 and this elevated type I IFN signaling, while the phenotypic changes in lung macrophages were independent of the type I IFN pathway at least at the level of type I IFN signaling as shown in the β6/IFNAR double KO mice. Overall, our data argue β6 expression leads to changes in the lung microenvironment that are mediated immediately after birth and include alterations in the AM population, and repressed type I IFN signaling in the lung most likely through a major contribution from TGF-β1. This pathway might lead to selective regulation of pathogen colonization or spread, depending on the pathogen’s sensitivity to the pre-existing interferon milieu. Two caveats to the model proposed in Fig 10 include (i) the fact that β6 may regulate factors other than TGF-β1 that cause the downstream effects, and (ii) that we cannot yet definitively distinguish whether β6 negatively regulates type I IFN signaling, type I IFN production or both pathways. In our hands, assays quantitating type I IFN amounts by ELISA or bead-based assays are variable, lacking sensitivity or reproducibility. Furthermore, the potential increase in type I receptor expression on β6 KO macrophages (Table 1) could cause increased uptake of type I IFNs and not accurately reflect the soluble amounts present in the lung at a given time in homeostasis. Thus, the identification of the point at which β6 (with or without TGF-β1) negatively regulates IFN signaling will require new tools and approaches investigating regulation in both macrophages and epithelial cells. Mice lacking the β6 integrin are protected from disease caused by influenza infection. Compared to WT controls, β6 KO mice develop less severe ALI, as characterized by edema, inflammatory cytokine expression, and vascular permeability of the lung epithelium. β6 KO mice also have altered baseline homeostasis possessing high baseline CD11b+ CD11c+ macrophages as opposed to CD11b- CD11c+ alveolar macrophages. A reasonable interpretation of these data is that β6 KO mice are ‘primed’ for a more efficient response to respiratory pathogens, due to constitutively increased type I IFN signaling at baseline. Importantly, protection extended beyond pandemic H1N1 influenza virus strain to include other influenza virus strains, Sendai virus, and S. pneumoniae infection. However, protection did not extend to all infections tested. We challenged β6 KO mice with Francisella tularensis Schu4 strain using 25 live organisms via the intranasal route. Schu4-infected β6 KO mice had no differences in survival compared to controls consistent with experiments performed in the Ifnar-/- mice [49] (CB, personal communication) (S8A Fig). Although β6 KO mice were significantly protected from disease caused by H7N9 virus (Fig 1D) they were not protected from the highly pathogenic avian influenza (HPAI) A/Hong Kong/483/1997 H5N1 infection (HK/483, S8B Fig). HPAI H5N1 viruses are unique in their ability to rapidly spread beyond the respiratory tract and cause systemic, including neurological, disease [18] and productively replicate in alveolar macrophages [50]. It is possible that HPAI H5N1 viruses are more resistant to type I IFN, that mice succumb due to direct viral cytopathic effect, or complications of systemic infection. Further studies are needed to understand why β6 KO mice are not protected against HPAI H5N1 infection. Taken together, these data suggest β6-mediated modulation of IFN responses affects responses to multiple respiratory pathogens whereas protective responses depend on IFN signaling. The airway epithelium is a complex barrier comprised of multiple cell types that acts as an interface between the external environment and the internal lung milieu. It serves three main functions in a healthy lung; providing a tight mechanical barrier that rapidly repairs upon insult, mediating innate immune activity to limit foreign antigen invasion, and initiating an inflammatory response through production of cytokines and chemokines [51]. A variety of experimental models and human studies demonstrate the host response to respiratory infection involves initiation, resolution, and restoration phases, all of which must be tightly regulated to prevent disease [52]. However, little is known about how interactions between epithelial cells, which are often the primary targets of respiratory pathogens, and alveolar macrophages, which play a critical role in controlling infections, and the regulation of these responses. Our data identify a pathway by which alveolar epithelial cells normally suppress the anti-microbial activity of alveolar macrophages through a pathway that may involve local activation of TGF-β and subsequent suppression of IFN signaling. In terms of homeostasis, little is known about how type I IFNs control the status of tissue microenvironments. Most work on type I IFN signaling has been associated with anti-viral responses and the regulation of inflammation over the first 18–24 months of life [16]. The implication of our findings is that the lung can tolerate a homeostatic increase in IFN signaling, which provides an advantage against viral spread, but likely comes with ‘costs’ that are not yet clear. We note a recent study has shown homeostatic IFN-β and IFNAR signaling in the brain has an essential role in suppressing neurodegeneration [53]. Therefore, a more comprehensive understanding of the hitherto unknown tissue-specific homeostatic roles of type I IFN signaling is warranted. During infection, although it would seem that upregulation of β6 would only have a negative impact on the host by limiting the anti-viral response, it could also be important for balancing the inflammatory response by suppressing activated macrophages through a TGF-β-dependent process [2,5, 15,45], initiating wound repair, or by recruiting macrophages to the site of infection. Attempts to address the role of TGF-β in influenza pathogenesis by systemic inhibition resulted in lethal infection [54]. Thus, future studies will focus on inhibiting the β6 integrin at different times post-infection to understand its precise role in viral infection and its potential as a therapeutic target. Why is the lung macrophage population different in the β6 KO mice? The αVβ6 integrin is expressed at low amounts in healthy adult tissue but is rapidly upregulated during development and in response to injury and inflammation [30]. Once expressed, β6 activates latent TGF-β1 by binding to the RGD motif in the latency-associated peptide (LAP) [10,55]. Upon activation, TGF-β1 regulates a variety of genes associated with the immune response and pulmonary fibrosis and is thought to negatively restrict inflammation in macrophages through an unknown mechanism [45]. However, systemic inhibition of all TGF-β isoforms by a broadly neutralizing antibody led to lethal influenza infection [54] suggesting that the role of TGF-β during infection may be complex and depend on both the cellular milieu and TGF-β isoform. Recent work has shown that the pulmonary microenvironment dictates the behavior of alveolar macrophages to a greater extent than the origin of the macrophages [42]. Based on our studies we propose that in the lung microenvironment, upregulation of the β6 integrin leads to localized activation of TGF-β1 that negatively regulates alveolar macrophages leading to decreased type I IFN activity in the microenvironment through an as yet undefined mechanism that will require further molecular investigation. An important extension of this model is that influenza viruses themselves activate latent TGF-β during infection [56] and global inhibition of TGF-β during infection is lethal, suggesting an important role in protection from influenza [54]. Therefore, it will be important to be able to experimentally segregate the host- versus virus-specific TGF-β processing events in mice expressing or lacking β6 integrin. Overall, loss of the β6 integrin resulted in protection from a variety of respiratory infections including influenza and Sendai viruses, bacterial pneumonia, and viral-bacterial co-infections. β6 KO mice had significantly reduced extent and severity of lung injury and inflammation, reduced collagen deposition despite no difference in viral titer, consistent with studies using β6 integrin function blocking antibodies [57], with virus infection being more restricted to airways instead of infiltrating the deep-lung alveolar spaces. β6 function blocking antibodies have been successfully used to improve outcome in idiopathic pulmonary fibrosis and currently recruiting for phase II human clinical trials [58,59]. While the β6 integrin represents an attractive therapeutic target to combat pulmonary disease, it does play a role in lung homeostasis. While transient inhibition of β6 in the lung results in resistance to infection, complete lack of β6 function for long periods of time could be detrimental. Over long periods of time, β6 KO mice over produce macrophage-derived matrix metalloprotease MMP12, leading to emphysema and destruction of pulmonary tissue [16]. However, for short-term protection against the adverse consequences of potentially lethal pulmonary viral infections, our work identifies the αVβ6 integrin as a potentially attractive therapeutic target for transient intervention in lung viral infection.
All animal work was approved by St Jude Children’s Research Hospital Institutional Animal Care and Use committee (protocol #513). Fransicella challenges were conducted under protocols approved by the NIAID Rocky Mountain Laboratories Animal Care and Use Committee. St Jude is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC-I) and has an approved Animal Welfare Assurance Statement with the Office of Laboratory Animal Welfare (A3077-01). These guidelines were established by the Institute of Laboratory Animal Resources and approved by the Governing Board of the U. S. National Research Council. C57BL/6, Ifnb1-YFP, CCR2 KO and CD45. 1 mice were obtained from Jackson Laboratories (Bar Harbor, ME). Itgb6-/- mice, generated as described [9] and backcrossed 10 generations onto the C57BL/6 background, were obtained from Dean Sheppard (UCSF) then bred at St Jude Children’s Research Hospital. Littermate controls from heterozygous crosses were used as controls. IFNAR KO mice were obtained from Dr. Laura Knoll (University of Wisconsin). Knockouts were confirmed by PCR using primer sets (CCR2, IFNAR) reported on the Jackson Laboratories website, or as previously described (β6) [16]. A/California/04/2009 (CA/09) H1N1, A/Puerto Rico/8/34 H1N1, A/Anhui/1/2013 H7N9 and A/Hong Kong/483/1991 H5N1 influenza viruses were propagated in 10-day old specific-pathogen-free embroynated chicken eggs and viral titers determined by tissue culture infectious dose 50 (TCID50) on Madin Darby canine kidney cells (MDCK; ATCC CCL-34, Manassas, VA) cells as described [50] and quantitated [60]. Enders strain Sendai virus was propagated as described [61]. D39X S. pneumoniae was grown in C + Y medium to log phase (O. D. 0. 4), pelleted, and diluted to the appropriate concentration in phosphate buffered saline pH 7. 4 and stored on ice until infection. MDCK cells were cultured in modified Eagle’s medium (MEM) (Corning, Manassas, VA) supplemented with 2 mM glutamine (Gibco, Grand Island, NY) and 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) at 37°C, 5% CO2. Deeply anesthetized mice were perfused with 10% neutral buffered formalin, tissues collected at the indicated time post-infection, and embedded in paraffin. H&E and immunohistochemical slides were blinded and scored by PV. Influenza NP staining was performed with anti-NP antibodies (05G, US Biological, Massachusetts, MA) and five separate lung sections quantitated using Aperio ePathology software (Buffalo Grove, IL). RNA from whole lung homogenates was isolated using a Trizol (Invitrogen) per manufacturer’s protocol. cDNA was synthesized using the SuperScript VILO cDNA synthesis kit (Invitrogen) and the following parameters: 25°C for 10 min, 42°C for 1 hour, and 85°C for 5 min. PCR was run using 2 μl of cDNA to detect Itgb6, Irf7, Ifit1, and Oas1g mRNA using the Quantitect primer/probe assay system (Qiagen) per manufacturer’s protocol. Samples were normalized to GAPDH mRNA as the internal control. Whole lungs were collected at different times post-infection and homogenized in RIPA buffer (50 mM TrisHCl pH 7. 5,150 mM NaCl, 0. 1% SDS, 0. 5% sodium deoxycholate, 1% TritonX-100) containing protease and phosphatase inhibitors (ThermoScientific, Rockford, IL). After centrifugation at 15000 rpm at 4°C, protein concentration was determined using the Bradford assay (Pierce, Rockford, IL) and equivalent amounts loaded onto 10% Tris-glycine SDS-PAGE gels (Lonza, Allendale, NJ). After transfer to nitrocellulose, membranes were probed for integrin β6 (0. 236 μg/ml, ch2A1 antibody, a kind gift from Biogen Idec, Cambridge, MA), anti-STAT1 (E-23, Santa Cruz, Dallas, TX, sc-346) (0. 1 μg/ml), anti-pSTAT1 (D4A7, Cell Signaling, Danvers, MA, 7649S) (1: 1000 dilution), anti-IRF3 (D83B9, Cell Signaling, 4947S) (1: 1000 dilution), or anti-pIRF3 (4D4G, Cell Signaling, 4302S) (1: 1000 dilution) and detected using HRP conjugated secondary goat anti-rabbit or goat anti-mouse antibodies (0. 1–0. 2 μg/ml, Jackson) and visualized with ECL. Anti-β-actin (AC-15, Sigma, St Louis, MO, A5441) (1 μg/ml) or anti-GAPDH (mAbcam 9484, Abcam, Cambridge, MA, ab9484) (1 μg/ml) served as loading controls. BALF or whole lung homogenate was collected from PBS control and influenza inoculated mice at 2,5, 6, or 7 dpi. Sorted populations of macrophages were administered intratracheally to WT or β6 KO mice. For the AM adoptive transfer, autofluorescent FITC+ F4/80+ lung cells were sorted from donor mice. 200,000 cells were transferred into each recipient mouse in 100 μl PBS. For transfer of naive PDM, the mouse peritoneal cavity was washed/flushed with 10 ml of PBS, and the F4/80+ population sorted. 500,000 cells were transferred into each recipient mouse in 100 μl PBS. After 7 days, mice were sacrificed and AM populations assessed. Donors and recipients were distinguished by CD45. 1 and CD45. 2 markers. BALF or whole lung homogenate was collected from PBS control and influenza inoculated mice at the indicated time post-infection. TNF-α and IL-6 protein expression was determined using the Milliplex Mouse 25-plex Cytokine Detection System mouse cytokine kit (Millipore, Billerica, MA) on a Luminex100 109 reader (Luminex Corp. , Austin, TX) according to the manufacturer’s protocol. Cytokine concentration was calculated using a calibration curve obtained in each experiment using the respective recombinant proteins. Active TGF-β1 protein levels were quantified by ELISA assay (BioLegend) on whole lung homogenates according to manufacturer’s protocol. Mice were deeply anesthetized with isoflurane and intranasally administered 1 μg of recombinant TGF-β1 (PeproTech, Rocky Hill, NJ) or PBS as previously described [46]. After 18 h, mice were lightly anesthetized and intranasally inoculated with 104 TCID50 CA/09 virus. At 2 dpi, mice were given an additional dose of rTGF-β1 or PBS and monitored for morbidity as described. For homeostasis studies, 1 μg of TGF-β1 was administered every 48 hours for 3 weeks. RNA from whole lung homogenates was isolated using a Trizol (Invitrogen) per manufacturer’s protocol. cDNA was synthesized using the SuperScript VILO cDNA synthesis kit (Invitrogen) and the following parameters: 25°C for 10 min, 42°C for 1 hour, and 85°C for 5 min. PCR was run using 2 μl of diluted cDNA (1: 5 in dH2O) to detect IFNa2/IFNa11 or IFNb mRNA using the Taqman primer/probe assay system (Life Technologies) per manufacturer’s protocol. Samples were normalized to GAPDH mRNA expression as the internal control. IFN-α (PBL Assay Science, Piscataway Township, NJ) and IFN-β (BioLegend, San Diego, CA) protein levels were quantified by ELISA assay on whole lung homogenates according to manufacturer’s protocol. Statistical analysis was performed with GraphPad Prism (San Diego, CA) as described in the Fig legends. Statistical significance was defined as p < 0. 05. Microarray data has been deposited in the GEO database with accession number GSE68802.
The lung undergoes daily assault by microbes and other inhaled particulates and must maintain the balance between clearance of harmful microorganisms while protecting the delicate lung structure to avoid acute lung injury. Not surprisingly, this is a complex process requiring communication between the lung epithelial cells (first site of attack by invaders) and the cells of the intrinsic immune response. We demonstrate the αVβ6 integrin is an important player in this interface. Loss of αVβ6 during influenza infection, and other respiratory infections, leads to bolstered protection against severe lung disease and improved survival in mice. Even in the absence of infection, β6 KO animals have a distinct anti-microbial lung microenvironment as evidenced by increased type I IFN activity and activated alveolar macrophages that appear “poised to defend”. These studies explore how the epithelial-specific αVβ6 integrin regulates the lung microenvironment to alter alveolar macrophage activity through a TGF-β-dependent mechanism, leading to changes in both the homeostatic lung and responses to respiratory infections as a way of balancing microbial clearance with protection of the lung from excessive damage.
blood cells
medicine and health sciences
immune cells
respiratory infections
pathology and laboratory medicine
influenza
pathogens
immunology
microbiology
orthomyxoviruses
pulmonology
viruses
integrins
rna viruses
cellular structures and organelles
infectious diseases
white blood cells
animal cells
cell adhesion
alveolar macrophages
extracellular matrix
proteins
medical microbiology
microbial pathogens
biochemistry
signal transduction
cell biology
influenza viruses
viral pathogens
interferons
tgf-beta signaling cascade
biology and life sciences
cellular types
viral diseases
macrophages
cell signaling
organisms
signaling cascades
An Epithelial Integrin Regulates the Amplitude of Protective Lung Interferon Responses against Multiple Respiratory Pathogens