OX 40 ligand newly expressed on bronchiolar progenitors mediates influenza infection and further exacerbates pneumonia
2016; Springer Nature; Volume: 8; Issue: 4 Linguagem: Inglês
10.15252/emmm.201506154
ISSN1757-4684
AutoresTaizou Hirano, Toshiaki Kikuchi, Naoki Tode, Arif Santoso, Mitsuhiro Yamada, Yoshiya Mitsuhashi, Riyo Komatsu, Takeshi Kawabe, Takeshi Tanimoto, Naoto Ishii, Yuetsu Tanaka, Hidekazu Nishimura, Toshihiro Nukiwa, Akira Watanabe, Masakazu Ichinose,
Tópico(s)Immune Response and Inflammation
ResumoReport14 March 2016Open Access Source DataTransparent process OX40 ligand newly expressed on bronchiolar progenitors mediates influenza infection and further exacerbates pneumonia Taizou Hirano Taizou Hirano Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Toshiaki Kikuchi Corresponding Author Toshiaki Kikuchi Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Naoki Tode Naoki Tode Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Arif Santoso Arif Santoso Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Mitsuhiro Yamada Mitsuhiro Yamada Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Yoshiya Mitsuhashi Yoshiya Mitsuhashi Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Riyo Komatsu Riyo Komatsu Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Takeshi Kawabe Takeshi Kawabe Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Takeshi Tanimoto Takeshi Tanimoto Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Japan Search for more papers by this author Naoto Ishii Naoto Ishii Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Yuetsu Tanaka Yuetsu Tanaka Department of Immunology, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan Search for more papers by this author Hidekazu Nishimura Hidekazu Nishimura Virus Research Center, Sendai Medical Center, National Hospital Organization, Sendai, Japan Search for more papers by this author Toshihiro Nukiwa Toshihiro Nukiwa Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Akira Watanabe Akira Watanabe Research Division for Development of Anti-Infective Agents, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan Search for more papers by this author Masakazu Ichinose Masakazu Ichinose Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Taizou Hirano Taizou Hirano Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Toshiaki Kikuchi Corresponding Author Toshiaki Kikuchi Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Naoki Tode Naoki Tode Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Arif Santoso Arif Santoso Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Mitsuhiro Yamada Mitsuhiro Yamada Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Yoshiya Mitsuhashi Yoshiya Mitsuhashi Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Riyo Komatsu Riyo Komatsu Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Takeshi Kawabe Takeshi Kawabe Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Takeshi Tanimoto Takeshi Tanimoto Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Japan Search for more papers by this author Naoto Ishii Naoto Ishii Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Yuetsu Tanaka Yuetsu Tanaka Department of Immunology, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan Search for more papers by this author Hidekazu Nishimura Hidekazu Nishimura Virus Research Center, Sendai Medical Center, National Hospital Organization, Sendai, Japan Search for more papers by this author Toshihiro Nukiwa Toshihiro Nukiwa Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Akira Watanabe Akira Watanabe Research Division for Development of Anti-Infective Agents, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan Search for more papers by this author Masakazu Ichinose Masakazu Ichinose Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Search for more papers by this author Author Information Taizou Hirano1, Toshiaki Kikuchi 1,7, Naoki Tode1, Arif Santoso1, Mitsuhiro Yamada1, Yoshiya Mitsuhashi1, Riyo Komatsu1, Takeshi Kawabe2, Takeshi Tanimoto3, Naoto Ishii2, Yuetsu Tanaka4, Hidekazu Nishimura5, Toshihiro Nukiwa1, Akira Watanabe6 and Masakazu Ichinose1 1Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan 2Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, Sendai, Japan 3Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Japan 4Department of Immunology, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan 5Virus Research Center, Sendai Medical Center, National Hospital Organization, Sendai, Japan 6Research Division for Development of Anti-Infective Agents, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan 7Present address: Department of Respiratory Medicine and Infectious Diseases, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan *Corresponding author. Tel: +81 25 368 9321; Fax: +81 25 368 9326; E-mail: [email protected] EMBO Mol Med (2016)8:422-436https://doi.org/10.15252/emmm.201506154 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Influenza virus epidemics potentially cause pneumonia, which is responsible for much of the mortality due to the excessive immune responses. The role of costimulatory OX40–OX40 ligand (OX40L) interactions has been explored in the non-infectious pathology of influenza pneumonia. Here, we describe a critical contribution of OX40L to infectious pathology, with OX40L deficiency, but not OX40 deficiency, resulting in decreased susceptibility to influenza viral infection. Upon infection, bronchiolar progenitors increase in number for repairing the influenza-damaged epithelia. The OX40L expression is induced on the progenitors for the antiviral immunity during the infectious process. However, these defense-like host responses lead to more extensive infection owing to the induced OX40L with α-2,6 sialic acid modification, which augments the interaction with the viral hemagglutinin. In fact, the specific antibody against the sialylated site of OX40L exhibited therapeutic potency in mitigating the OX40L-mediated susceptibility to influenza. Our data illustrate that the influenza-induced expression of OX40L on bronchiolar progenitors has pathogenic value to develop a novel therapeutic approach against influenza. Synopsis OX40 ligand (OX40L) is a costimulatory molecule known to be expressed preferentially on antigen-presenting cells for T-cell priming. Here, an unprecedented mechanism is found by which OX40L exacerbates influenza pneumonia on bronchiolar progenitors by functioning as the virus receptor. In lower respiratory infection with influenza virus, the number of bronchiolar progenitors is increased for repair of damaged epithelial cells. The OX40L expression is enhanced on the influenza-expanded bronchiolar progenitors to bolster the host immune response. The infection is paradoxically worsen by accelerated viral binding to the bronchiolar progenitors via the α-2,6 sialic acid of glycosylated OX40L protein. Introduction Influenza viruses are RNA viruses belonging to the Orthomyxoviridae family and are responsible for seasonal epidemics that yearly cause 3-5 million clinical infections and 250,000–500,000 deaths (Barik, 2012; Kuiken et al, 2012; van de Sandt et al, 2012; Hayden, 2013). Recognizing that excessive immune responses are associated with the life-threatening immunopathology of influenza, several studies have been conducted to better understand the contributions of costimulatory molecules in regulating the host immune response during influenza infection (Kim et al, 2011; Barik, 2012; Braciale et al, 2012; Damjanovic et al, 2012). OX40 (also known as CD134, TNFRSF4) is a 50-kDa type 1 transmembrane protein, which is predominantly expressed on activated T cells and has a costimulatory function promoting T-cell proliferation and survival (Watts, 2005; Cavanagh & Hussell, 2008; Croft, 2010; Goulding et al, 2011). In a mouse model of sublethal influenza infection, blockade of OX40 costimulation has been shown to reduce T-cell accumulation within the lung and to diminish the destruction of lung tissue, which is correlated with the prevention of weight loss (Kopf et al, 1999; Humphreys et al, 2003). The OX40's binding partner, OX40 ligand (OX40L, also known as gp34, CD252, TNFSF4), is a type II glycoprotein of 183 amino acids with 133 extracellular amino acids in the carboxyl terminus (Croft, 2010). The OX40L expression is constitutively low, but can be induced preferentially on professional antigen-presenting cells for T-cell priming via OX40 engagement after antigen recognition (Watts, 2005; Croft, 2010). Additional findings that non-hematopoietic cell types such as endothelial cells and smooth muscle cells also have the potential to express OX40L suggest that OX40–OX40L interactions can participate in several aspects of the physiological links between T cells and non-hematopoietic cells (Imura et al, 1996; Burgess et al, 2004; Croft et al, 2009). In the present study, because the impact of OX40 as well as OX40L on influenza viral infection has not yet been extensively investigated in lethal disease situations, we examined whether blocking OX40–OX40L interactions can actually reduce the mortality of influenza-infected mice. We found that deficiency of OX40L, but not that of OX40, markedly improved the survival in spite of the influenza A viral burden. This unexpected dissimilarity between OX40 and OX40L was thought to result from the influenza virus-binding capacity of OX40L on bronchiolar progenitors, which increased the cell numbers upon influenza infection to mediate epithelial repair and induced the OX40L expression on their surfaces to stimulate immune responses. Results Severity of lethal influenza pneumonia depends on OX40L rather than OX40 Blocking OX40 engagement has been shown to prevent immune-mediated lung damage in a sublethal influenza infection model by eliminating the influenza-induced CD4+ and CD8+ T-cell infiltration within the lung (Kopf et al, 1999; Humphreys et al, 2003; Croft, 2010). To dissect the implications of OX40–OX40L interactions for severe infection, we used a lethal influenza A pneumonia model for OX40L-deficient (OX40L−/−) and OX40-deficient (OX40−/−) mice (Fig 1A–C). Consistent with previous reports of sublethal influenza infection, OX40−/− mice showed decreases in the total numbers of BAL cells and amounts of cell infiltrate to the lungs as compared with wild-type mice (P < 0.01, Fig 1B and C). However, the survival of OX40−/− mice was comparable to that of wild-type mice (P > 0.1, Fig 1A). Comparable levels between OX40−/− mice and wild-type mice were also observed for interleukin (IL)-4, IL-6, and interferon (IFN)-γ in BAL fluids (IL-4, P > 0.3, Appendix Fig S1A; IL-6, P > 0.8, Appendix Fig S1B; IFN-γ, P > 0.5, Appendix Fig S1C). Interestingly, OX40L−/− mice significantly survived the lethal influenza A/H1N1 pneumonia as compared with OX40−/− mice and wild-type mice, showing decreased total numbers of BAL cells, amounts of lung leukocyte infiltration, and cytokine levels in BAL fluids except for IL-4 (P < 0.001, Fig 1A; P < 0.005, Fig 1B and C; IL-4, P > 0.8, Appendix Fig S1A; IL-6, P < 0.0001, Appendix Fig S1B; IFN-γ, P < 0.05, Appendix Fig S1C). Similar results were achieved using influenza A/H3N2 virus and reducing the viral load of influenza A/H1N1 by half, from 5 to 2.5 times the minimal lethal dose (OX40L−/− vs. OX40−/−: P < 0.05, Appendix Fig S2A; P < 0.005, day 6, Appendix Fig S2B; P < 0.001, Appendix Fig S3A; P < 0.01, day 6, Appendix Fig S3B). These results suggest that severe influenza pneumonia can be attributed to a biological property of OX40L that is not associated with the OX40 triggering. Figure 1. Mice lacking OX40L, especially on non-bone marrow-derived cells, are more resistant to lethal influenza infection than those lacking OX40 A–C. Wild-type (WT), OX40L−/−, and OX40−/− mice were intratracheally infected with a lethal dose of influenza A/H1N1 virus (PR8 strain). Controls included wild-type mice treated with saline (mock). D–F. The study was similar to that in panels (A–C), but wild-type and OX40L−/− mice that were transplanted with wild-type or OX40L−/− bone marrow (BM) were used. Data information: The susceptibility was determined by the survival of the mice (n = 12, A), body weight change (n = 4, D), total and differential cell counts in bronchoalveolar lavage (BAL; n = 3, B; n = 4, E), and histopathology of lung sections stained with hematoxylin and eosin (H&E, C and F; scale bar, 200 μm). For panels (B, D and E), data are shown as the mean ± standard error. In panel (F), lung sections were also immunostained with antibody to matrix protein 2 of influenza virus (M2, green), and the nuclei were identified with DAPI (blue, inset; scale bar, 200 μm). Kaplan–Meier analysis and the log rank statistic (A): OX40−/− vs. WT, P = 0.1410; OX40L−/− vs. WT, P = 0.0001; OX40L−/− vs. OX40−/−, P = 0.0003. Tukey's honestly significant difference test (B and D): OX40−/− vs. WT, P = 0.0099 (B); OX40L−/− vs. WT, P = 0.0001 (B); OX40L−/− vs. OX40−/−, P = 0.0039 (B); WT donor, P = 0.0109 (D); OX40L−/− donor, P = 0.0178 (D). Source data are available online for this figure. Source Data for Figure 1 [emmm201506154-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint OX40L on non-hematopoietic cells contributes to the severity We next investigated which of the hematopoietic and non-hematopoietic cells serve pathogenic OX40L for the lethal influenza A pneumonia (Fig 1D–F). After bone marrow reconstitution with wild-type or OX40L−/− donor cells, wild-type and OX40L−/− recipient mice were challenged with a lethal dose of influenza A/H1N1 virus. Regardless of the donor cell type, wild-type recipient mice lost over 25% of their body weight at the start of the experiment, whereas OX40L−/− recipient mice did not (wild-type recipients vs. OX40L−/− recipients on day 7: wild-type donor, P < 0.05; OX40L−/− donor, P < 0.05; Fig 1D). Similar results were achieved in the differential cell counts in BAL and assessment of lung histology with the viral burden (Fig 1E and F), showing that a deficiency of OX40L on non-bone marrow-derived (i.e., non-hematopoietic) cells results in less viral production and less lung inflammation compared to that on bone marrow-derived (i.e., hematopoietic) cells. Increased number of OX40L-positive lung cells including bronchiolar progenitors To characterize non-hematopoietic cells expressing OX40L, which has a key role in regulating the responses, we undertook a flow cytometry approach to analyze OX40L-positive cells in lungs exposed to a lethal load of influenza A virus (Fig 2A–C). The flow cytometry data revealed that the frequency and the number of OX40L-positive (OX40Lpos) lung cells increased in influenza-infected mice (more than sixfold, P < 0.0001, Fig 2A). The OX40Lpos lung cells were fractionated using the endothelial marker CD31 and the pan-hematopoietic marker CD45, and one quarter of them were negative for both markers (Fig 2B). When the early hematopoietic/pan-endothelial marker CD34 was further included among the markers for the lineage-negative selection (Linneg, CD31negCD45negCD34neg), nearly 80% of OX40LposLinneg lung cells could be fractionated for subpopulations of bronchiolar progenitors featured as low Sca-1 and low autofluorescence (Sca-1lowAFlow, Fig 2C). These results implicate bronchiolar progenitors in the increased OX40L expression on non-hematopoietic lung cells exposed to influenza A virus. Figure 2. Both the number and the OX40L expression level of bronchiolar progenitors were increased by influenza infection. Wild-type mice were intratracheally infected with a lethal dose of influenza A/H1N1 virus (Flu) or saline (Mock), and 7 days later, their lung cells and sections were evaluated A. Cell counts of OX40L-positive cells in whole lung cells. B. Status of CD31 and CD45 expression in lung OX40L-positive cells. C. Status of Sca-1 expression and autofluorescence in lung OX40L-positive Lin-negative (i.e., OX40LposCD31negCD45negCD34neg) cells. D, E. (D) Proportion of bronchiolar progenitors (LinnegSca-1lowAFlow, red box) and club cells (LinnegSca-1lowAFhigh, blue box), and (E) their cell counts. F, G. (F) Lung sections stained with antibodies to CCSP (red) and SPC (green), and (G) quantitative analysis of these imaging data. Scale bar, 50 μm. H. Cell counts of OX40L-positive cells in bronchiolar progenitors and club cells. I. OX40L gene expression in bronchiolar progenitors and club cells. By quantitative RT–PCR, the gene expression levels were analyzed relative to the bronchiolar progenitors of mock-infected mice. ND, not determined for scarcity of club cells after influenza infection. Data information: Data are presented as the mean ± standard error of n = 4 (A and E) or n = 3 (I) per group. Student's unpaired two-tailed t-test (A and I): P = 0.0001 (A); progenitors, P = 0.0102 (I). Tukey's honestly significant difference test (E): progenitors, P = 0.0001; club cells, P = 0.0097. Source data are available online for this figure. Source Data for Figure 2 [emmm201506154-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Increased number of bronchiolar progenitors We then sought to determine whether the size of the bronchiolar progenitor population is increased in influenza-infected lungs (Fig 2D and E). Lung infection with influenza A/H1N1 virus increased the frequency of bronchiolar progenitors exhibiting a Sca-1lowAFlow phenotype within the lung Linneg fraction, but decreased that of club cells, formerly known as Clara cells, exhibiting a Sca-1lowAFhigh phenotype within the same fraction (Fig 2D). Concomitantly, influenza A/H1N1 infection caused a significant increase in the number of LinnegSca-1lowAFlow bronchiolar progenitors, and a significant decrease in that of LinnegSca-1lowAFhigh club cells (progenitors, P < 0.001; club cells, P < 0.01; Fig 2E). Similar results were also achieved using influenza A/H3N2 virus (progenitors, P < 0.001; club cells, P < 0.05; Fig EV1A). Click here to expand this figure. Figure EV1. Influenza A/H3N2 virus infection, which is enhanced by human OX40L expression in vitro, increases both the number and the OX40L expression level of bronchiolar progenitors in vivo A, B. Wild-type mice were intratracheally infected with a lethal dose of influenza A/H3N2 virus (Flu) or saline (Mock), and 7 days later, their lung cells were evaluated for cell counts of bronchiolar progenitors and club cells (A), and OX40L-positive cells in bronchiolar progenitors and club cells (B). C. Human OX40L-transfected MDCK cells were infected in vitro with influenza A/H3N2 virus. The levels of influenza virus NP gene expression were analyzed by semiquantitative and quantitative RT–PCR 24 h after the infection. The intensity was quantified relative to pNull-transfected cells. Endogenous canine GAPDH mRNA expression was used as a control. D. The study was similar to that in panel C, but cells were pretreated with W66 anti-human OX40L antibody for 24 h before the infection. The levels of influenza virus NP gene expression were analyzed relative to those in control antibody-pretreated cells. Data information: Data are presented as the mean ± standard error of n = 4 (A) or n = 3 (C, D) per group. Download figure Download PowerPoint Because a CCSP and SPC dual-expressing phenotype has also been proposed as a characteristic of bronchiolar progenitors (Teisanu et al, 2009; Lau et al, 2012; Ardhanareeswaran & Mirotsou, 2013), we analyzed the CCSP/SPC dual expression by immunofluorescent staining of lung tissues from mice subjected to influenza A pulmonary infection (Fig 2F and G). The results showed that influenza A/H1N1 infection promoted the shedding of CCSP-immunoreactive club cells in terminal bronchioles and, instead, the appearance of CCSP/SPC dual-positive cells in such distal airways (Fig 2F). Correspondingly, the quantitative determination of the fluorescent signal intensity on the stained lung sections revealed that influenza A/H1N1 infection led to a twofold increase in the frequency of CCSP/SPC dual-positive cells as compared to mock infection (Fig 2G). Taken together, these data suggest that club cells in bronchioles are readily abolished by influenza A infection, whereas bronchiolar progenitors enlarge the population likely to repair the damaged epithelia. Influenza-induced OX40L expression on bronchiolar progenitors To further understand the expression dynamics of OX40L on bronchiolar progenitors, we analyzed the OX40L expression by flow cytometry and quantitative RT–PCR studies (Fig 2H and I). As determined by flow cytometry, the frequency of bronchiolar progenitors expressing OX40L on the cell surface was increased up to 41% following influenza A/H1N1 infection, whereas that of club cells was increased only up to 7% (Fig 2H). Similar results were observed following influenza A/H3N2 infection (Fig EV1B). The quantitative RT–PCR analysis using Gapdh as the internal control gene revealed the mRNA expression encoding OX40L to be significantly elevated in bronchiolar progenitors upon influenza A/H1N1 infection of the mouse airways (P < 0.05, Fig 2I). Similar results were achieved by using other internal control genes in the quantitative RT–PCR analysis (beta-2-microglobulin, P < 0.05; hypoxanthine phosphoribosyl-transferase 1, P < 0.01; Appendix Fig S4). These findings indicate that the pulmonary OX40L abundance associated with non-hematopoietic cells is augmented in influenza-infected mice as a result of both the enlarged population of bronchiolar progenitors and their increased expression of OX40L. OX40L-mediated susceptibility of bronchiolar progenitors to influenza A virus To examine the functional implications of bronchiolar progenitors and their OX40L, we sorted bronchiolar progenitors and club cells, or OX40Lpos and OX40Lneg lung cells, after influenza A/H1N1 lung infection (Fig 3A–E). The expression of the mRNA encoding influenza nucleoprotein (NP) was assayed for the susceptibility to influenza viral infection by semiquantitative and quantitative RT–PCR. In wild-type mice, bronchiolar progenitors displayed a significantly higher level of the viral nucleoprotein expression than club cells (P < 0.05, Fig 3A). The nucleoprotein expression level was significantly attenuated in OX40L-deficient bronchiolar progenitors as compared with wild-type ones (P < 0.05, Fig 3B). When analyzing the differences between the OX40Lpos and OX40Lneg lung cells of influenza-infected wild-type mice, we found an approximately sixfold increase in the nucleoprotein expression in OX40Lpos lung cells, which was consistent with the immunofluorescence studies showing matrix protein 2 (M2) of influenza virus only in OX40Lpos lung cells (P < 0.01, Fig 3C and D). The compatibility between OX40L and influenza A/H1N1 virus was confirmed by OX40L/M2 dual immunostaining, in which OX40L and influenza matrix protein 2 tended to merge with each other, especially in bronchiolar progenitors (high and low magnification, Fig 3E and F). In aggregate, these data show that bronchiolar progenitors are more susceptible to influenza A virus, at least in part due to their OX40L expression, than club cells. Figure 3. Bronchiolar progenitors are susceptible to influenza infection due to their OX40L expression. Wild-type and OX40L−/− mice were intratracheally infected with a lethal dose of influenza A/H1N1 virus, and their lung cells and sections were evaluated 3 days later except in panels (F–H) A–C. By semiquantitative and quantitative RT–PCR, the levels of influenza virus nucleoprotein (NP) gene expression were analyzed in (A) wild-type bronchiolar progenitors relative to club cells, (B) wild-type bronchiolar progenitors relative to OX40L−/− ones, and (C) wild-type OX40-positive cells relative to OX40-negative cells. Mouse glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA expression was used as a control. D. Cytospins prepared from sorted OX40L-positive and OX40L-negative lung cells of infected wild-type mice were immunostained with antibody to M2 protein of influenza virus, and the nuclei were identified with DAPI. Scale bar, 50 μm. E. The study was similar to that in panel (D), but sorted bronchiolar progenitors and club cells from influenza-infected wild-type mice were stained with antibody to OX40L (red) as well as M2 protein (green). Scale bar, 10 μm. F. This study was similar to that in panel (E), but bronchiolar progenitors and club cells were sorted at 7 days after the infection. Scale bar, 50 μm. G. Representative Western blot of influenza virus M2 protein in lungs of wild-type and OX40L−/− mice on the indicated days after influenza infection. β-Actin was used as a loading control. H. Lung sections from influenza-infected wild-type and OX40L−/− mice were immunostained with antibody to M2 protein of influenza virus, and the nuclei were identified with DAPI. Scale bar, 100 μm. Data information: For panels (A–C), data are presented as the mean ± standard error of n = 3 per group. Student's unpaired two-tailed t-test (A–C): P = 0.0179 (A); P = 0.0173 (B); P = 0.0082 (C). Source data are available online for this figure. Source Data for Figure 3 [emmm201506154-sup-0005-SDataFig3.pdf] Download figure Download PowerPoint Less productive replication of influenza virus in lung tissues of OX40L-deficient mice Given that almost complete protection against otherwise lethal influenza pneumonia is achieved in mice lacking OX40L, which is considered as a susceptibility factor to influenza A virus, we tested whether this characteristic of OX40L-deficient mice is associated with the viral replication in respiratory tissues (Fig 3G and H). To address this, we performed immunoblotting of whole-cell extracts from respiratory specimens of influenza-infected mice. The results revealed that influenza A/H1N1 virus-derived matrix protein 2 in wild-type mice markedly increased for 3 days post-infection and then decreased gradually, whereas that in OX40L-deficient mice similarly but modestly increased for 3 days and then decreased rapidly below detectable limits (Fig 3G). Consistently, at 7 days after the infection, viral matrix protein 2 was abundantly observed in bronchiolar and alveolar epithelial cells of wild-type mice in contrast to lung sections of OX40L-deficient mice where the viral protein was hardly observed (Fig 3H). The reduced viral replication in OX40L-deficient mice was also confirmed by lower numbers of influenza viral plaques in the BAL fluids (P < 0.5, Appendix Fig S5). These results strengthened our view that influenza-induced OX40L expression on bronchiolar progenitors promotes their susceptibility to influenza A virus, leading to viral growth in the infected lung. Basal status of the influenza virus receptor on bronchiolar progenitors Human-adapted influenza A virus has been shown to initiate the infection via their hemagglutinin, which binds to α-2,6 sialic acid, sialic acid linked to galactose by α-2,6 linkage (Wilks et al, 2012). We next investigated whether the sialic acid modification status is relevant to the cellular susceptibility by fluorescent staining with α-2,6 sialic acid-specific lectin of Sambucus nigra. At the basal level, bronchiolar progenitors had higher levels of α-2,6 sialic acid on their cell surfaces as compared to club cells, suggesting a mechanistic link between the sialic acid modification and the cellular susceptibility to influenza A/H1N1 virus (Fig 4A). However, the abundance of α-2,6 sialic acid in bronchiolar progenitors was not simply a result of the sialylation-catalyzing activity, as analysis of our microarray data, whose details will be available in the ArrayExpress datab
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