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• W2896762729 abstract "Article15 October 2018Open Access Source DataTransparent process Loss of T-bet confers survival advantage to influenza–bacterial superinfection Jun Zhi Er Jun Zhi Er NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore City, Singapore Search for more papers by this author Ricky Abdi Gunawan Koean Ricky Abdi Gunawan Koean Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore City, Singapore Search for more papers by this author Jeak Ling Ding Corresponding Author Jeak Ling Ding [email protected] orcid.org/0000-0003-4578-370X NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore City, Singapore Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore City, Singapore Search for more papers by this author Jun Zhi Er Jun Zhi Er NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore City, Singapore Search for more papers by this author Ricky Abdi Gunawan Koean Ricky Abdi Gunawan Koean Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore City, Singapore Search for more papers by this author Jeak Ling Ding Corresponding Author Jeak Ling Ding [email protected] orcid.org/0000-0003-4578-370X NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore City, Singapore Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore City, Singapore Search for more papers by this author Author Information Jun Zhi Er1, Ricky Abdi Gunawan Koean2 and Jeak Ling Ding *,1,2 1NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore City, Singapore 2Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore City, Singapore *Corresponding author. Tel: +65 6516 2776; Fax: +65 6779 248; E-mail: [email protected] The EMBO Journal (2019)38:e99176https://doi.org/10.15252/embj.201899176 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 The transcription factor, T-bet, regulates type 1 inflammatory responses against a range of infections. Here, we demonstrate a previously unaddressed role of T-bet, to influenza virus and bacterial superinfection. Interestingly, we found that T-bet deficiency did not adversely affect the efficacy of viral clearance or recovery compared to wild-type hosts. Instead, increased infiltration of neutrophils and production of Th17 cytokines (IL-17 and IL-22), in lungs of influenza virus-infected T-bet−/− mice, were correlated with survival advantage against subsequent infection by Streptococcus pneumoniae. Neutralization of IL-17, but not IL-22, in T-bet−/− mice increased pulmonary bacterial load, concomitant with decreased neutrophil infiltration and reduced survival of T-bet−/− mice. IL-17 production by CD8+, CD4+ and γδ T cell types was identified to contribute to this protection against bacterial superinfection. We further showed that neutrophil depletion in T-bet−/− lungs increased pulmonary bacterial burden. These results thus indicate that despite the loss of T-bet, immune defences required for influenza viral clearance are fully functional, which in turn enhances protective type 17 immune responses against lethal bacterial superinfections. Synopsis T-bet transcription factor regulates vital immune functions for pathogen resistance. Unexpectedly, T-bet deficiency retained immune defence against flu, and resulting type 17 immune responses were protective against lethal bacterial superinfection. Loss of transcription factor T-bet does not reduce host protection against influenza virus infection. Immune response in influenza infected T-bet−/− mice is characterized by enhanced type 17 response and neutrophilia. Loss of T-bet protects hosts against lethal post-influenza secondary pneumococcus infection. Blocking of IL-17 or neutrophil depletion abrogates survival advantage of T-bet−/− hosts against post-influenza bacterial infection. Introduction Influenza viruses are respiratory intracellular pathogens that cause significant morbidity and mortality in human populations. Although vaccines and antiviral drugs have been developed against influenza viruses, rapid evolution and host adaptation of the virus underlie its constant threat for pandemic formation and debilitating disease (Taubenberger & Kash, 2010), notably from recent H5N1 and H7N9 strains. In individuals infected by influenza viruses, the eventual recovery depends on an efficient immune response spanning the innate and adaptive immune systems (Braciale et al, 2012; Iwasaki & Pillai, 2014), suggesting the robustness of host defences against influenza viruses. Conversely, overactivation of immune responses causes many influenza related deaths, indicating the need for proper balance in immune regulation during infection (Taubenberger & Morens, 2008; Newton et al, 2016). Hence, understanding and targeting of host immune responses during influenza virus infections is an important facet in tackling influenza pneumonia. Flu infections are frequently complicated by secondary bacterial infections, significantly increasing the risk of severe pneumonia (Metersky et al, 2012; Chertow & Memoli, 2013; McCullers, 2014). In fact, bacterial complicated influenza infections accounted for nearly all the deaths in the 1918 flu pandemic (Morens et al, 2008) and up to 55% of deaths from the 2009 H1N1 pandemic (Rice et al, 2012; Centers for Disease Control and Prevention (CDC), 2009; Mauad et al, 2010). Recent studies have identified interactions between host, virus and bacteria as a main cause of heightened host susceptibility (Sun & Metzger, 2008; Shahangian et al, 2009; Ghoneim et al, 2013; Cao et al, 2014; Ellis et al, 2015). Based on these insights, the identification and manipulation of factors central to viral–bacterial lethality will formulate a viable treatment strategy against post-influenza bacterial superinfection. The transcription factor T-bet is a central regulator of type 1 immune responses. Its functions are hitherto known to be mediated through the expression of cytokines IFNγ and IL-12, chemokines CCL3 and CCL4 as well as chemokine receptors such as CXCR3. Furthermore, the expression of T-bet is involved in various cellular functions—the development of CD4+ Th1 cells (Szabo et al, 2002); suppression of Th2 and Th17 immunity (Hwang et al, 2005; Djuretic et al, 2007; Lazarevic et al, 2011); maturation and cytolytic activity of natural killer (NK) and CD8+ T cells (Sullivan et al, 2003; Townsend et al, 2004); formation of memory immune cells (Intlekofer et al, 2005, 2007; Joshi et al, 2007; Marshall et al, 2011; Wang et al, 2012); and regulation of IgG class switching (Peng et al, 2002; Liu et al, 2003). As a result, T-bet is purportedly required for immune defence against many classes of pathogens. Indeed, the absence of T-bet has rendered hosts more susceptible to primary infections by the parasite, Leishmania major (Szabo et al, 2002), and bacterial species such as Mycobacterium tuberculosis (Sullivan et al, 2005), Staphylococcus aureus (Hultgren et al, 2004), Salmonella typhimurium (Ravindran et al, 2005) and Francisiella tularensis (Melillo et al, 2014). However, the role of T-bet in viral immunity is less well defined. Earlier studies demonstrated its requirement for control of Vaccina virus and herpesvirus infections (Matsui et al, 2005; Svensson et al, 2005; Rubtsova et al, 2013), while a recent study showed that loss of T-bet did not affect eventual viral clearance from rhinovirus infection, but is instead involved in suppression of viral-mediated allergic airway response (Glanville et al, 2016). Hence, the regulatory functions and role of T-bet in viral infections and coinfections of pathogens remain to be addressed. In influenza virus infections, T-bet-mediated functions in CD8+ T, CD4+ T, Treg and B cells have been described (Mayer et al, 2008; Bedoya et al, 2013; Dolfi et al, 2013; Dutta et al, 2013; Hua et al, 2013; Naradikian et al, 2016). Nevertheless, the outcome from deficiency of T-bet on pathogenesis or survival on hosts infected with influenza virus and subsequent bacterial complications have, to our knowledge, not been investigated. Given the wide immunoregulatory effects of T-bet on type 1 immunity, we unexpectedly found that T-bet-deficient mice could effectively clear influenza viruses and recover from sub-lethal dose of infection. Furthermore, the loss of T-bet led to increased neutrophil and eosinophil infiltration in the lungs during influenza virus infection with concomitant production of Th2 and Th17 cytokines. This viral-induced inflammation was required for increased protection against secondary respiratory infection by Streptococcus pneumoniae, as neutralization of IL-17 or neutrophil depletion led to increased susceptibility to secondary bacterial infection. These findings shed new perspectives on T-bet under influenza virus infection and subsequent bacterial superinfections, with implications for modulation of T-bet as a strategy to improve survival outcomes from post-influenza bacterial pneumonia. Results T-bet−/− mice clear influenza virus infection with increased cellular infiltration into the lungs To determine whether global loss of T-bet had an impact on host susceptibility during influenza virus infection, we followed and compared the body weights of T-bet−/− and wild-type mice throughout the course of infection. Both wild-type and T-bet−/− mice survived sub-lethal doses of the H1N1 PR8 mouse-adapted influenza virus with no significant differences in percentage weight loss at all timepoints (Fig 1A). Similar to wild-type mice, infected T-bet−/− mice started to recover body weight after day 11 post-infection and regained its original body weight by day 25. Weight loss and survival under lethal dose of influenza infection were also not significantly different between wild-type and T-bet−/− hosts (Fig EV1A and B). Survival and recovery of both wild-type and T-bet−/− mice under sub-lethal infection were correlated with their ability to clear the virus by day 9 post-infection, as measured by the amount of PR8 nucleoprotein (NP) mRNA in the lungs (Fig 1B). In contrast to wild-type counterparts, histology of the T-bet−/− lungs revealed notably higher cellular infiltration into the alveolar spaces at days 5, 9 and 15 post-infection (Fig 1C), an observation which was further exemplified by increased numbers of CD45+ immune cells in the lung tissue and bronchoalveolar lavage (BAL; Fig 1D). Pulmonary injury was more severe at day 9 post-infection in T-bet−/− lungs. Nevertheless, the increased infiltration of immune cells and heightened inflammation did not cause increased cytotoxicity in T-bet−/− lungs as reflected by the level of lactate dehydrogenase (LDH) in the BAL (Fig 1E). In fact, T-bet−/− lungs displayed better recovery at day 15 post-infection, with increased repair of alveolar walls and bronchiole epithelium and reduced pulmonary injury (Figs 1C and EV1C). To further assess tissue repair in T-bet−/− lungs, podoplanin (PDPN), a glycoprotein expressed on alveolar type 1 epithelial cells and marker of lung injury and repair (Li et al, 2015; Zuo et al, 2015), was stained by Western blot (Figs 1F and EV1D) and immunohistochemistry (Fig EV1E). Days 15 and 25 post-infected T-bet−/− lungs displayed equal expression of podoplanin to wild-type lungs, confirming that increased inflammation at earlier timepoints did not affect subsequent tissue recovery and repair. Decreased leakiness of T-bet−/− lungs at day 25 post-infection, as assessed by the concentration of total protein and albumin in the BAL (Fig 1G and H), further supports intact tissue homeostasis in T-bet−/− mice. Together, these observations suggest that T-bet deficiency does not affect host ability to clear influenza virus infections, and increased inflammation in T-bet−/− lungs does not affect subsequent tissue repair and homeostasis. Figure 1. T-bet-deficient lungs exhibit increased cellular infiltration and reduced leakiness following influenza virus infectionMice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows. Percentage body weight loss. See also Fig EV1A and B. Relative amount of PR8 viral NP mRNA, by qPCR. Histology of lungs by H&E staining with pathology scores. Scale bars = 200 μm. See also Fig EV1C. Absolute number of live CD45+ cells in whole lungs and BAL, by flow cytometry. Level of LDH in BAL, by LDH cytotoxicity assay. Expression of PDPN in lungs, by Western blot. See also Fig EV1D and E. Total protein in BAL, by Bradford assay. Concentration of albumin in BAL, by ELISA. Data information: Data are representative of three independent experiments with 3–5 mice per group and represented as mean ± SEM. Statistical significance in (D), (G) and (H) was determined with unpaired Student's t-test. Source data are available online for this figure. Source Data for Figure 1 [embj201899176-sup-0004-SDataFig1.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. T-bet-deficient mice exhibit comparable disease outcomes to wild-type hosts, with increased infiltration of pulmonary granulocytes and lymphocytes, related to Fig 1 A, B. (A) Weight loss and (B) survival of mice infected with lethal influenza virus dose (500 pfu per mice). C–E. Mice were infected with sub-lethal dose of influenza virus. (C) Histopathology of H&E-stained lungs. Scale bars = 500 μm. Mean pathology scores from 3 to 5 H&E-stained mouse lungs, with detailed assessment of inflammation, pulmonary injury and tissue responses in the lungs shown. Scores for each sub-category range from 0 (least evident) to 5 (most evident). (D) Expression levels of PDPN in mouse lungs, assessed by densitometry of Western blots. (E) Immunohistochemistry staining of PDPN on mouse lungs. Arrow heads indicate tissue repair of damaged areas. To assess specificity of antibody staining, negative controls with no anti-goat HRP secondary antibody (1° antibody only) or no anti-PDPN antibody (2° antibody only) were processed simultaneously. Scale bars = 200 μm. Data information: Survival data from (A and B) are combined from two independent experiments, n = 10 mice per experimental group. Data from (C–E) are representative of three independent experiments with 3–5 mice per group. Data in (D) are represented as mean ± SEM. Source data are available online for this figure. Download figure Download PowerPoint Increased infiltration of granulocytes and B cells into lungs of T-bet−/− mice during influenza virus infection To date, studies on infection of T-bet−/− mice with various pathogens have mostly reported some form of susceptibility to infection, which is congruent with the central role of T-bet in regulating type 1 immune responses. Recently, it was reported that T-bet-deficient Th1 cells display an aberrant amplification of type I interferon (IFN) response (Iwata et al, 2017). To determine whether T-bet−/− mice were able to survive influenza infection due to increased antiviral responses, we quantified the expression of type I IFNs (IFNα and IFNβ) at the mRNA and protein levels in the lungs and BAL, respectively, at days 5 and 9 post-influenza infection (Fig EV2A and B). T-bet deficiency did not result in a notable change in expression of these cytokines. Furthermore, mRNA expression of a number of interferon response genes (ISGs) was equally expressed in wild-type and T-bet−/− lungs at day 5 post-influenza infection (Fig EV2A) where viral load is at its peak (Fig 1B). In fact, expression of Isg15, Ifit3, Oasl1 and Irf7 was reduced at day 9 post-influenza infection in T-bet−/− lungs relative to wild-type lungs. Hence, assessment of global antiviral responses did not reveal elevated type I IFN responses which would have conferred additional protective immunity against influenza in T-bet−/− mice. To further understand immune responses of T-bet deficiency in clearing influenza viruses and identify specific immune cells infiltrating T-bet−/− lungs, we performed flow cytometry of single cells from influenza virus-infected lungs. Strikingly, T-bet−/− lungs had increased numbers and frequencies of neutrophils and eosinophils on days 9 and 15 post-influenza infection (Fig 2A and B) while wild-type mice lungs displayed a decline in these cell types. These results imply dysregulated immune responses or compensatory mechanisms for combating influenza virus infection in T-bet−/− mice. Despite their increase in the earlier phase of infection, the numbers of neutrophils and eosinophils were reduced to basal levels by day 25 post-infection in both wild-type and T-bet−/− lungs. No difference in numbers of monocytes was detected. Click here to expand this figure. Figure EV2. Analysis of wild-type and T-bet−/− immune response in the lungs following influenza virus infection, related to Fig 2 A, B. Mice were infected with sub-lethal dose of influenza virus. (A) Relative expression of type I IFNs and antiviral mRNA in lungs of mice, as assessed by qPCR. (B) Protein concentrations of type I IFN in BAL, as assessed by ProcartaPlex assay. C. Representative flow cytometry gating strategy used for identification of immune cells in the lungs. Back-gating of myeloid cells in the FSC-SSC plot is shown in the top right-hand corner. D. Increased CD11c expression in eosinophils from infected lungs compared to uninfected lungs. Identification of eosinophils was further confirmed with CD64−CD24hi phenotype. Data information: Data are representative of three (A) and two (B) independent experiments with four mice per group and are represented as mean ± SEM. Statistical significance in (A) and (B) was determined with unpaired Student's t-test. n.s., not significant. Download figure Download PowerPoint Figure 2. Increased infiltration of neutrophils, eosinophils and B cells in T-bet-deficient lungs infected with influenza virus Absolute cell counts from lungs of sub-lethally influenza virus-infected mice following analysis by flow cytometry. After gating out alveolar macrophages by forward and side scatter, immune cells are defined as follows: neutrophil (Ly6G+CD11b+SiglecF−Ly6Cint), eosinophil (SiglecF+CD11c−/+F4/80−CD64−), monocyte (Ly6G−CD11b+MHCII−SiglecF−), natural killer cell (NK, B220−/intNK1.1+), B cell (B220hiNK1.1−), CD8+ T cell (TCRβ+CD8+), CD4+ T cell (TCRβ+CD4+). See also Fig EV2C and D. Flow cytometry plots of neutrophils and eosinophils in lungs at days 9 and 15 post-sub-lethal influenza virus infection. Numbers in plots indicate frequency of gated immune cells in each throughput. Data information: Data are representative of three independent experiments with 3–4 mice per group and represented as mean ± SEM. Statistical significance in (A) was determined with unpaired Student's t-test. n.s., not significant. Download figure Download PowerPoint We observed a reduction of natural killer (NK) cells in T-bet−/− lungs compared to wild-type lungs at days 0, 9 and 25 post-influenza virus infection (Fig 2A), consistent with the role of T-bet in NK homeostasis and maturation (Townsend et al, 2004). Nevertheless, pulmonary NK cells in T-bet−/− mice at day 5 post-infection were comparable to wild-type lungs, suggesting intact recruitment of NKs during the early phase of infection. The numbers of CD4+ T and CD8+ T cells between wild-type and T-bet−/− lungs were largely similar, although a consistent trend of their reduced numbers in T-bet−/− lungs (with P > 0.05) across independent experiments was observed at day 9 post-infection (Fig 2A). We observed a 10-fold increase of B cells in T-bet−/− lungs relative to wild-type lungs specifically at day 15 post-influenza infection, suggesting a role for T-bet in regulating proliferation or migration of B cells. Collectively, the results demonstrate that absence of T-bet does not abrogate innate or adaptive lymphocyte responses in the lungs. Overall, analysis of immune responses revealed increased granulocytes and B cells in influenza-infected T-bet−/− lungs, while concomitant intact type I IFN responses and recruitment of cytotoxic and helper lymphocytes likely contributed to the effective clearance of influenza virus. T-bet deficiency confers survival advantage to post-influenza bacterial superinfection Excessive pulmonary infiltration of neutrophils and eosinophils has been observed in influenza virus-induced pneumonia and deaths (Tumpey et al, 2005; Taubenberger & Morens, 2008; Jeon et al, 2010; Larrañaga et al, 2016), which was taken to indicate lung impairment. However, our results thus far did not suggest increased susceptibility or damage to lung tissues despite heightened infiltration of neutrophils and eosinophils in T-bet−/− lungs. We then asked how this increase in granulocytes in influenza-infected T-bet−/− hosts might respond under bacterial superinfection, a frequent complication following influenza infections. To investigate responses of T-bet−/− mice to post-influenza bacterial superinfection compared to wild-type hosts, mice were infected with a sub-lethal dose of PR8 influenza virus, followed by administration of a sub-lethal dose of S. pneumoniae serotype 19F, at day 7 post-influenza infection (Fig 3A). Remarkably, T-bet−/− mice displayed significantly enhanced survival following secondary bacterial infection, compared to 85% lethality of wild-type mice (Fig 3B). Furthermore, bacterial load in T-bet−/− lungs was lower than wild-type lungs (Fig 3C). These survival advantages in T-bet−/− mice were correlated to increased infiltration of neutrophils and eosinophils in the lungs at days 1 and 3 of secondary bacterial infection (Fig 3D), suggesting that they could be involved in immune defence. Compared to wild-type lungs, the number of NK cells transiently increased in T-bet−/− lungs 1 day after secondary bacterial infection, supporting enhanced trafficking or proliferation in response to bacterial challenge, while CD8+ T cells were reduced at day 3 post-infection. Similar to the viral infection only controls, B cell numbers were increased in T-bet−/− lungs following secondary bacterial infection, while CD4+ T cells are comparable to wild-type lungs. Enhanced protection and immune cell responses against secondary bacterial infection under T-bet deficiency were reproduced with a more virulent S. pneumoniae serotype (S3; Fig EV3B–D). Figure 3. Improved resistance of T-bet-deficient mice against post-influenza bacterial superinfection A. Model of post-influenza bacterial infection illustrating infection with sub-lethal dose of influenza virus (25 pfu) followed by sub-lethal dose of secondary bacterial challenge (Streptococcus pneumoniae serotype 19F, 2 × 105 cfu) 7 days later. Where appropriate, mice lungs were analysed at days 0, 1 and 3 post-secondary bacterial infection, corresponding to days 7, 8 and 10 post-influenza virus infection, respectively. B–G. Mice were superinfected as described in (A). (B) Survival of mice following bacterial superinfection. See also Fig EV3B. (C) Pneumococcal cfu in BAL of mice. See also Fig EV3C. (D) Absolute immune cell counts in lungs, assessed by flow cytometry. See also Fig EV3D. (E) Pneumococcal cfu in BAL of mice 12 h after infection with 2 × 105 cfu of S. pneumoniae serotype 19F alone (−Flu) or following influenza virus infection (+Flu). See also Fig EV3C. (F) Pneumococcal cfu in BAL 36 h after infection of naïve mice with 2 × 105 cfu of S. pneumoniae serotype 19F alone. (G) Absolute neutrophil and eosinophil cell count in lungs infected as described in (E), assessed by flow cytometry. Data information: Data from (B) are combined from two independent experiments, n = 13 mice per experimental group. Data from (C–G) are representative of at least two independent experiments with 3–5 (C, D and G) or 5–7 (E and F) mice per experimental group. All data are represented as mean ± SEM. Statistical significance was determined by log-rank test in (B), unpaired Student's t-test in (D) and (G) and Mann–Whitney U-test in (C) and (E). n.s., not significant. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Improved immune protection in T-bet-deficient mice following influenza virus infection and bacterial superinfection with Streptococcus pneumoniae serotype S3, related to Figs 3, 4B and 5B A. Wild-type mice were infected with indicated doses of S. pneumoniae serotype 19F or S3 bacteria alone and assessed for survival. B–F. Wild-type and T-bet−/− mice were infected with sub-lethal dose of influenza virus followed by sub-lethal dose of secondary bacterial challenge (S. pneumoniae serotype S3, 50 cfu) 7 days later. (B) Survival of mice following post-influenza bacterial infection. (C) Pneumococcal cfu in lung homogenates of mice infected with 50 cfu of S. pneumoniae serotype S3 alone or following influenza virus infection. (D) Absolute immune cell counts in lungs, as assessed by flow cytometry. (E) Concentrations of IL-17 and IL-22 in lung homogenates of mice, assessed by ELISA. (F) T-bet−/− mice were administered αIL-17 or IgG isotype control antibody every other day from day −2 post-secondary bacterial infection and monitored for survival. Data information: Data in (A–C) and (F) are combined from two independent experiments with 6–16 mice per group. Data in (D) and (E) are representative of two independent experiments with four mice per group. All data are represented as mean ± SEM. Statistical significance was determined by log-rank test in (B) and (F), Mann–Whitney U-test in (C) and unpaired Student's t-test in (D) and (E). Download figure Download PowerPoint To determine whether mortality in wild-type mice was due to viral-mediated suppression of antibacterial defences through T-bet, naïve or influenza-infected mice were infected with equal doses of bacteria. Wild-type mice infected with prior influenza virus suffered heavier bacterial burden compared to infection with bacteria alone, indicating lethality in viral–bacterial superinfection (Figs 3E and EV3C). While T-bet−/− mice appeared to control bacteria more efficiently than wild-type mice at 12 h post-bacteria-only infection (Fig 3E), both mice strains were able to clear bacteria by 36 h post-infection (Fig 3F), indicating suppression of antibacterial defences by T-bet during influenza and bacterial superinfections. Moreover, the numbers of neutrophils and eosinophils were significantly lower in the lungs of both bacteria-only infected wild-type and T-bet−/− mice, compared to secondary infected T-bet−/− lungs (Fig 3G). Together, these data demonstrate that enhanced inflammatory responses during primary influenza infection in T-bet-deficient lungs contribute to subsequent antibacterial defence and improved survival. Increase in Th2 and Th17 cytokines in T-bet−/− lungs are correlated to enhanced infiltration of neutrophils and eosinophils To further understand the underlying immune mechanisms in T-bet−/− mice during the inflammatory phase of influenza virus infection, the BAL of days 5 and 9 post-infection was screened for cytokines and chemokines using a protein bead array. Out of 24 analytes examined, significant differences in concentrations of 10 target proteins between wild-type and T-bet−/− lungs were observed (Fig 4A). Strikingly, Th2 cytokines, IL-9 and IL-13, as well as Th17 cytokines, IL-17 and IL-22, were significantly elevated in T-bet−/− lungs (Figs 4A and EV4A), consistent with the role of T-bet in repression of these immune responses (Hwang et al, 2005; Villarino et al, 2010; Lazarevic et al, 2011). Furthermore, IL-13 and IL-17 are known to drive pulmonary recruitment of eosinophils and neutrophils, respectively (Pope et al, 2001; Ye et al, 2001), which may explain the increased cellular infiltration of these cell types into T-bet-deficient lungs following infection. Secretion of pro-inflammatory cytokines, IL-6 and IL-18, is increased in T-bet-deficient lungs (Fig 4A), suggesting possible compensatory inflammatory mechanisms perhaps in view of reduced antiviral cytokine IL-28. Decrease in concentrations of T-bet target protein, CCL3, was observed in T-bet−/− lungs. IFNγ-induced T cell chemoattractants like CXCL9 (MIG) and CXCL10 (IP-10) were also reduced, which may provide an explanation for lowered numbers of pulmonary T cells in T-bet−/− lungs (Figs 2A and 3D). Interestingly, no difference was observed in protein levels of the T-bet target—IFNγ, as further confirmed by ELISA (Fig EV4B). Nevertheless, reduced expression of Ifng and Th1 cytokine Tnf mRNA was observed in T-bet−/− lungs compared to wild-type (Fig EV4C), implying post-transcriptional regulation of Th1 effector cytokines. Overall, these mechanisms suggest a shift towards Th2 and Th17 immune responses in the absence of T-bet during influenza virus infection, leading to increased infiltration of granulocytes. Figure 4. Increased IL-17 and IL-22 production in T-bet−/− lungs infected with influenza virus or superinfected with bacteria A. Concentrations of cytokines or chemokines in the BAL of days 5 and 9 post-sub-lethal influenza virus infe" @default.
• W2896762729 created "2018-10-26" @default.
• W2896762729 creator A5007620364 @default.
• W2896762729 creator A5016756488 @default.
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• W2896762729 date "2018-10-15" @default.
• W2896762729 modified "2023-10-18" @default.
• W2896762729 title "Loss of T‐bet confers survival advantage to influenza–bacterial superinfection" @default.
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• W2896762729 cites W2184323843 @default.
• W2896762729 doi "https://doi.org/10.15252/embj.201899176" @default.
• W2896762729 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6315292" @default.
• W2896762729 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/30322895" @default.
• W2896762729 hasPublicationYear "2018" @default.
• W2896762729 type Work @default.
• W2896762729 sameAs 2896762729 @default.

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