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Supplementary Figure from Chronic Exposure to Waterpipe Smoke Elicits Immunomodulatory and Carcinogenic Effects in the Lung

Effects of waterpipe smoking on lung pathobiology and carcinogenesis remain sparse despite the worldwide emergence of this tobacco vector. To address this gap, we investigated the effects of chronic waterpipe smoke (WPS) exposure on lung pathobiology, host immunity, and tumorigenesis using an experimental animal model that is prone to tobacco carcinogens and an exploratory observational analysis of human waterpipe smokers and nonsmokers. Mice exhibited elevated incidence of lung tumors following heavy WPS exposure (5 days/week for 20 weeks) compared to littermates with light WPS (once/week for 20 weeks) or control air. Lungs of mice exposed to heavy WPS showed augmented CD8+ and CD4+ T cell counts along with elevated protumor immune phenotypes including increased IL17A in T/B cells, PD-L1 on tumor and immune cells, and the proinflammatory cytokine IL1β in myeloid cells. RNA-sequencing (RNA-seq) analysis showed reduced antitumor immune gene signatures in animals exposed to heavy WPS relative to control air. We also performed RNA-seq analysis of airway epithelia from bronchial brushings of cancer-free waterpipe smokers and nonsmokers undergoing diagnostic bronchoscopy. Transcriptomes of normal airway cells in waterpipe smokers, relative to waterpipe nonsmokers, harbored gene programs that were associated with poor clinical outcomes in patients with lung adenocarcinoma, alluding to a WPS-associated molecular injury, like that established in response to cigarette smoking. Our findings support the notion that WPS exhibits carcinogenic effects and constitutes a possible risk factor for lung cancer as well as warrant future studies that can guide evidence-based policies for mitigating waterpipe smoking.Prevention Relevance:Potential carcinogenic effects of waterpipe smoking are very poorly understood despite its emergence as a socially acceptable form of smoking. Our work highlights carcinogenic effects of waterpipe smoking in the lung and, thus, accentuate the need for inclusion of individuals with exclusive waterpipe smoking in prevention and smoking cessation studies.

Effects of waterpipe smoking on lung pathobiology and carcinogenesis remain sparse despite the worldwide emergence of this tobacco vector. To address this gap, we investigated the effects of chronic waterpipe smoke (WPS) exposure on lung pathobiology, host immunity, and tumorigenesis using an experimental animal model that is prone to tobacco carcinogens and an exploratory observational analysis of human waterpipe smokers and nonsmokers. Mice exhibited elevated incidence of lung tumors following heavy WPS exposure (5 days/week for 20 weeks) compared to littermates with light WPS (once/week for 20 weeks) or control air. Lungs of mice exposed to heavy WPS showed augmented CD8+ and CD4+ T cell counts along with elevated protumor immune phenotypes including increased IL17A in T/B cells, PD-L1 on tumor and immune cells, and the proinflammatory cytokine IL1β in myeloid cells. RNA-sequencing (RNA-seq) analysis showed reduced antitumor immune gene signatures in animals exposed to heavy WPS relative to control air. We also performed RNA-seq analysis of airway epithelia from bronchial brushings of cancer-free waterpipe smokers and nonsmokers undergoing diagnostic bronchoscopy. Transcriptomes of normal airway cells in waterpipe smokers, relative to waterpipe nonsmokers, harbored gene programs that were associated with poor clinical outcomes in patients with lung adenocarcinoma, alluding to a WPS-associated molecular injury, like that established in response to cigarette smoking. Our findings support the notion that WPS exhibits carcinogenic effects and constitutes a possible risk factor for lung cancer as well as warrant future studies that can guide evidence-based policies for mitigating waterpipe smoking. Prevention Relevance: Potential carcinogenic effects of waterpipe smoking are very poorly understood despite its emergence as a socially acceptable form of smoking. Our work highlights carcinogenic effects of waterpipe smoking in the lung and, thus, accentuate the need for inclusion of individuals with exclusive waterpipe smoking in prevention and smoking cessation studies.

Type I IFN signalling blockade does not enhance the frequency of IL-17A+ NKT and conventional CD4 T cells

The protumoral activity of γδT17 cells has recently emerged in a wide variety of solid malignancies, including breast cancer. These cells exert their detrimental functions by promoting tumor growth, angiogenesis, and subsequent metastasis development. However, the intratumoral factors that regulate the biology of γδT17cells within the tumor microenvironment are less well understood. Here, using two experimental models of breast cancer, we reinforced the concept that tumor-infiltrating γδT17 cells are endowed with protumoral functions, which promote tumor progression and metastasis development. More importantly, we demonstrated a critical role for type I IFN signaling in controlling the preferential accumulation in the tumor bed of a peculiar subset of γδT17 cells displaying a CD27− CD3bright phenotype (previously associated with the invariant Vγ6Vδ1+ TCR). Interestingly, this effect was indirect and partially relied on the IFNAR1-dependent control of IL7 secretion, a factor that triggers proliferation and activating functions of deleterious γδT17 cells. Our work therefore identifies a key role of the type I IFN/IL7 axis in the regulation of intratumoral γδT17-cell functions and in the development of primary breast tumor growth and metastasis.Significance: Tumor-derived IL7 can represent a therapeutic target to prevent accumulation of immune cells endowed with potent protumoral activities. Cancer Res; 78(1); 195–204. ©2017 AACR.

Background. This report aims to detail the use of phosphorescence oxygen analyzer for in vitro investigation of thymic response to pharmaceutical agents, in particular immunosuppressants and immunomodulators. Sirolimus (a highly specific inhibitor of the ‘molecular target of rapamycin’, mTOR) and ozanimod (an agonist of the sphingosine 1-phosphate receptor, a recently approved drug for treatment of multiple sclerosis and ulcerative colitis) are used for this purpose. Methods. Thymic fragments from mice were placed in vials containing phosphate-buffered saline, bovine albumin, and Pd(II) meso-tetra (sulfophenyl) tetrabenzoporphyrin. The vials were sealed from air, and the cellular oxygen consumption was monitored as function of time. Results. The decline of dissolved oxygen concentration with time (d[O2]/dt) was linear; thus, its rate (thymocyte respiration) was expressed as µM O2 min-1. Cyanide inhibited respiration, confirming the oxygen consumption was in cytochrome oxidase. In age-matched mice, the rate of thymocyte respiration (mean ± SD, in µM O2 min-1 mg-1) was 0.046 ± 0.011 (median = 0.043, range = 0.028 to 0.062, n = 10). In thymic fragments from littermates, this rate was inhibited in the presence of sirolimus (16% lower) or ozanimod (29% lower). Conclusions. Thymocyte respiration can serve as a surrogate biomarker for studying the mode-of-action and the cytotoxicity of immunotoxins and immunosuppressants.

BackgroundThis report aims to detail the use of phosphorescence oxygen analyzer for in vitro investigation of the thymic response to pharmaceutical agents, in particular immunosuppressants and immunomodulators. Sirolimus (a highly specific inhibitor of the ‘molecular target of rapamycin’, mTOR) and ozanimod (an agonist of the sphingosine 1-phosphate receptor, a recently approved drug for treatment of multiple sclerosis and ulcerative colitis) were investigated for this purpose. The detection method in this prescribed analytical instrument is based on the principle that oxygen quenches the phosphorescence of Pd(II) meso-tetra (sulfophenyl) tetrabenzoporphyrin.MethodsThymic fragments from mice were placed in vials containing 2.0 mL phosphate-buffered saline, 0.5% fat-free bovine albumin, and 3 µM Pd phosphor (with or without added glucose). The vials were then sealed from air and their dissolved oxygen concentration was monitored at 25oC as a function of time.ResultsThe kinetics of oxygen consumption was zero-order; thus, its rate (in µM O2 min− 1) was the negative of d[O2]/dt. The addition of cyanide inhibited respiration, confirming oxygen was consumed in cytochrome oxidase of the mitochondrial respiratory chain. In age-matched mice, the rate of thymocyte (cellular) respiration (mean ± SD, in µM O2 min− 1 mg− 1) was 0.046 ± 0.011 (median = 0.043, range = 0.028 to 0.062, n = 10). In thymic fragments from littermates, this rate was inhibited in the presence of sirolimus (16% lower) or ozanimod (29% lower).ConclusionsRespiration can serve as a surrogate biomarker for studying the mode-of-action and cytotoxicity of immunotoxins and immunosuppressants in thymic fragments collected from mice.

Microbial dysbiosis has emerged as a modulator of oncogenesis and response to therapy, particularly in lung cancer. Here, we investigate the evolution of the gut and lung microbiomes following exposure to a tobacco carcinogen. We performed 16S rRNA-Seq of fecal and lung samples collected prior to and at several timepoints following (nicotine-specific nitrosamine ketone/NNK) exposure in Gprc5a−/− mice that were previously shown to exhibit accelerated lung adenocarcinoma (LUAD) development following NNK exposure. We found significant progressive changes in human-relevant gut and lung microbiome members (e.g., Odoribacter, Alistipes, Akkermansia, and Ruminococus) that are closely associated with the phenotypic development of LUAD and immunotherapeutic response in human lung cancer patients. These changes were associated with decreased short-chain fatty acids (propionic acid and butyric acid) following exposure to NNK. We next sought to study the impact of Lcn2 expression, a bacterial growth inhibitor, given our previous findings on its protective role in LUAD development. Indeed, we found that the loss of Lcn2 was associated with widespread gut and lung microbiome changes at all timepoints, distinct from those observed in our Gprc5a−/− mouse model, including a decrease in abundance and diversity. Our overall findings apprise novel cues implicating microbial phenotypes in the development of tobacco-associated LUAD.

Effects of waterpipe smoking on lung pathobiology and carcinogenesis remain sparse despite the worldwide emergence of this tobacco vector as a socially acceptable form of smoking, particularly among the youth. To address this gap, we investigated the effects of chronic waterpipe smoke (WPS) exposure on lung pathobiology, host immunity, and tumorigenesis using both an experimental animal model and an exploratory observational analysis of human waterpipe smokers and non-smokers. Mice exposed to increasing doses of WPS (once or five times per week for 20 weeks), through an exclusively devised exposure system for this study, were more prone to develop lung tumors compared to control-air exposed littermates. This effect was accompanied by various pro-tumor immune phenotypes, including increased IL-17A+ levels in T/B cells as well as elevated expression of the immune checkpoint PD-L1 and the pro-inflammatory cytokine IL-1β in myeloid cells. While flow cytometry analysis revealed increased CD4 and CD8 T cell infiltration in WPS-exposed mice, gene signatures of cytotoxic and expanded immune response were, conversely, decreased in animals exposed to heavy WPS relative to control air. Interestingly, mice heavily exposed to WPS exhibited increased expression of Tmprss4, Cd55, and Ace2, cell receptors and mediators of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry and, thus, COVID-19 pathogenesis. We also perform RNA-sequencing analysis of bronchial airway epithelial brushings of cancer-free waterpipe smokers and non-smokers undergoing diagnostic bronchoscopy. Transcriptomes of normal airway cells in waterpipe smokers, relative to waterpipe non-smokers, harbored gene programs that were associated with poor clinical outcomes in lung adenocarcinoma (LUAD) patients, alluding to a WPS-associated molecular injury, like that established in response to cigarette smoking. Overall, our analyses demonstrate immunomodulatory and carcinogenic effects of WPS on the murine and human lung. Our study also shows that WPS exposure leads to a field of injury that may be associated with increased risk for lung cancer. This study is unique for interrogating carcinogenic effects of chronic exposure to WPS with the longest, to our knowledge, follow-up time in vivo. Our findings accentuate the need for additional studies that can guide evidence-based policies to counteract shortfalls in public health control of waterpipe smoking. Citation Format: Zahraa Rahal, Maya Hassane, Nareg Karaoghlanian, Jiexin Zhang, Ansam Sinjab, J. Jack Lee, Maria Gabriela Raso, Junya Fujimoto, Hassan Chami, Alan Shihadeh, Humam Kadara. Chronic exposure to waterpipe smoke elicits immunomodulatory and carcinogenic effects in the lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 223.

Rationale: Early pathogenesis of lung adenocarcinoma (LUAD) remains largely unknown. We found that, relative to wild-type littermates, the innate immunomodulator Lcn2 (lipocalin-2) was increased in...

International audience; Interleukin-7 (IL-7) is a critical cytokine in B- and T-lymphocyte development and maturation. Recent evidence suggests that IL-7 is a preferential homeostatic and survival factor for RORγt+ innate T cells such as natural killer T (NKT) cells, γδT cells, and mucosal-associated invariant T (MAIT) cells in the periphery. Given the important contribution of these populations in antibacterial immunity at barrier sites, we questioned whether IL-7 could be instrumental in boosting the local host immune response against respiratory bacterial infection. By using a cytokine-monoclonal antibody approach, we illustrated a role for topical IL-7 delivery in increasing the pool of RORγt+ IL-17A-producing innate T cells. Prophylactic IL-7 treatment prior to Streptococcus pneumoniae infection led to better bacterial containment, a process associated with increased neutrophilia and that depended on γδT cells and IL-17A. Last, combined delivery of IL-7 and α-galactosylceramide (α-GalCer), a potent agonist for invariant NKT (iNKT) cells, conferred an almost total protection in terms of survival, an effect associated with enhanced IL-17 production by innate T cells and neutrophilia. Collectively, we provide a proof of concept that IL-7 enables fine-tuning of innate T- cell functions. This might pave the way for considering IL-7 as an innovative biotherapeutic against bacterial infection.

Lung adenocarcinomas (LUADs) with mutations in the K-ras oncogene display dismal prognosis. Proinflammatory and immunomodulatory events that drive development of K-ras mutant LUAD are poorly understood. Here, we develop a lung epithelial specific K-ras mutant/Stat3 conditional knockout (LR/Stat3Δ/Δ) mouse model. Epithelial Stat3 deletion results in intriguing sex-associated discrepancies; K-ras mutant tumors are decreased in female LR/Stat3Δ/Δ mice whereas tumor burdens are increased in males. RNA-sequencing and tumor microenvironment (TME) analysis demonstrate increased anti-tumor immune responses following Stat3 deletion in females and, conversely, elevated pro-tumor immune pathways in males. While IL-6 blockade in male LR/Stat3Δ/Δ mice reduces lung tumorigenesis, inhibition of estrogen receptor signaling in female mice augments K-ras mutant oncogenesis and reprograms lung TME toward a pro-tumor phenotype. Our data underscore a critical sex-specific role for epithelial Stat3 signaling in K-ras mutant LUAD, thus paving the way for developing personalized (e.g. sex-based) immunotherapeutic strategies for this fatal disease., Proinflammatory and immunomodulatory events that drive development of K-ras mutant lung adenocarcinoma (LUAD) are poorly understood. Here they develop a lung epithelial specific K-ras mutant/Stat3 conditional knockout mouse model and show a sex-specific role for epithelial Stat3 signaling in K-ras-mutant LUAD.

Lung adenocarcinomas (LUADs) with somatic mutations in the KRAS oncogene comprise the most common molecular subtype of lung cancer in smokers and present with overall dismal prognosis and resistance to most therapies. Our group recently demonstrated that tobacco carcinogen-exposed mice with knockout of the airway lineage G-protein coupled receptor, Gprc5a, develop LUADs with somatic mutations in Kras. Earlier work has suggested that cancer stem cells (CSCs) play crucial roles in clonal evolution of tumors and in therapy resistance. To date, our understanding of CSCs in LUADs with somatic Kras mutations remains lagging. Here we derived CSCs (as spheres in 3D cultures) with self-renewal properties from a murine Kras-mutant LUAD cell line we previously established from a tobacco carcinogen-exposed Gprc5a−/− mouse. Using syngeneic Gprc5a−/− models, we found that these CSCs, compared to their parental isoforms, exhibited increased tumorigenic potential in vivo, particularly in female animals. Using whole-transcriptome sequencing coupled with pathways analysis and confirmatory PCR, we identified gene features (n = 2,600) differentially expressed in the CSCs compared to parental cells and that were enriched with functional modules associated with an augmented malignant phenotype including stemness, tumor-promoting inflammation and anti-oxidant responses. Further, based on in silico predicted activation of GSK3β in CSCs, we found that tideglusib, an irreversible inhibitor of the kinase, exhibited marked anti-growth effects in the cultured CSCs. Our study underscores molecular cues in the pathogenesis of Kras-mutant LUAD and presents new models to study the evolution, and thus high-potential targets, of this aggressive malignancy.

Relative to other tumors in smokers, KRAS-mutant lung adenocarcinomas (LUADs) display dismal prognosis warranting the need for early management of this disease. Limiting these advances is our gap in knowledge of events that drive KRAS-mutant LUAD oncogenesis. Host defense systems, such as those elicited by the gut microbiome, were recently shown to influence tumors external to the gastrointestinal tract (e.g. melanomas), thus highlighting the microbiome as an orchestrator of oncogenesis. Yet, gut microbiome changes in early stages of KRAS-mutant LUAD development are not known. We recently reported that mice with knockout of G-protein coupled receptor 5A (Gprc5a-/-), in contrast to wild type (WT) littermates, develop LUADs which are accelerated following exposure to tobacco carcinogen (nicotine-specific nitrosamine ketone/NNK). LUADs in the tobacco exposed Gprc5a-/- mouse model exhibit high somatic mutation burdens, driver Kras variants and other co-occurring drivers, features constituting a “perfect storm” for LUAD pathogenesis in smokers. Using this human-relevant model, we performed 16S-Seq of fecal samples collected prior to and at several time points post-NNK exposure during LUAD development. We found significant, some progressive, microbial changes during the pathogenesis of LUAD, including abolishment of phylum Verrucomicrobia, progressive increase in tumor-promoting genera Odoribacter spp., gradual decrease in Akkermansia spp., which was previously shown to be associated with response to PD-1 blockade, as well as reduced abundance of Ruminococcus which was previously reported to be suppressed during colon carcinogenesis. Additionally, and prior to tumor onset, lungs and immune cells of these Gprc5a-/- mice exhibited markedly elevated expression of lipocalin 2 (LCN2), an antimicrobial protein released from host cells during microbiome imbalance and inflammation. We further found that Gprc5a-/- mice with knockout of Lcn2 exhibited increased tumors compared to similarly exposed Gprc5a-/- with intact Lcn2. These effects were accompanied by widespread changes in the gut microbiome including increased abundance of tumor-promoting Alistipes spp. and, conversely, reduced abundance of Lactobacillus spp. Our data show that host defense mediated by LCN2 counteracts Kras-mutant LUAD development by restricting gut microbiome imbalance (i.e., maintaining gut microbiome homeostasis and diversity), suggesting a protective role during Kras-mutant LUAD development. Our overall findings inform on novel pathways implicating antimicrobial host defense mechanisms in the development of smoking-associated Kras-mutant LUAD. Efforts are underway to discern specific microbiome profiles that are likely causally related to smoking-associated Kras-mutant LUAD development as well as to develop and test preclinical microbiome intervention strategies for this malignancy. Citation Format: Warapen Treekitkarnmongkol, Casey Finnicum, Ansam Sinjab, Maya Hassane, Christel Davis, Gareth E. Davies, Kristi L. Hoffman, Junya Fujimoto, Florencia McAllister, Boris Sepesi, Tina Cascone, Robert R. Jenq, Joseph Petrosino, Erik Ehli, Seyed J. Moghaddam, Humam Kadara. Evolution of the gut microbiome during the pathogenesis of smoking-associated Kras-mutant lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3349.

The protumoral activity of γδT17 cells has recently emerged in a wide variety of solid malignancies, including breast cancer. These cells exert their detrimental functions by promoting tumor growth, angiogenesis, and subsequent metastasis development. However, the intratumoral factors that regulate the biology of γδT17cells within the tumor microenvironment are less well understood. Here, using two experimental models of breast cancer, we reinforced the concept that tumor-infiltrating γδT17 cells are endowed with protumoral functions, which promote tumor progression and metastasis development. More importantly, we demonstrated a critical role for type I IFN signaling in controlling the preferential accumulation in the tumor bed of a peculiar subset of γδT17 cells displaying a CD27− CD3bright phenotype (previously associated with the invariant Vγ6Vδ1+ TCR). Interestingly, this effect was indirect and partially relied on the IFNAR1-dependent control of IL7 secretion, a factor that triggers proliferation and activating functions of deleterious γδT17 cells. Our work therefore identifies a key role of the type I IFN/IL7 axis in the regulation of intratumoral γδT17-cell functions and in the development of primary breast tumor growth and metastasis. Significance: Tumor-derived IL7 can represent a therapeutic target to prevent accumulation of immune cells endowed with potent protumoral activities. Cancer Res; 78(1); 195–204. ©2017 AACR.

Traditionally regarded as simple foot soldiers of the innate immune response limited to the eradication of pathogens, neutrophils recently emerged as more complex cells endowed with a set of immunoregulatory functions. Using a model of invasive pneumococcal disease, we highlighted an unexpected key role for neutrophils as accessory cells in innate interleukin (IL)-17A production by lung resident Vγ6Vδ1(+) T cells via nucleotide-binding oligomerization domain receptor, pyrin-containing 3 (NLRP3) inflammasome-dependent IL-1β secretion. In vivo activation of the NLRP3 inflammasome in neutrophils required both host-derived and bacterial-derived signals. Elaborately, it relies on (i) alveolar macrophage-secreted TNF-α for priming and (ii) subsequent exposure to bacterial pneumolysin for activation. Interestingly, this mechanism can be translated to human neutrophils. Our work revealed the cellular and molecular dynamic events leading to γδT17 cell activation, and highlighted for the first time the existence of a fully functional NLRP3 inflammasome in lung neutrophils. This immune axis thus regulates the development of a protective host response to respiratory bacterial infections.Mucosal Immunology advance online publication, 4 January 2017; doi:10.1038/mi.2016.113.

Relative to other lung adenocarcinomas (LUADs), Kras mutant LUAD displays dismal prognosis warranting the need for new strategies for early treatment of this lung cancer subtype. Limiting these advances is our poor understanding of events that drive Kras mutant LUAD development. In efforts to fill this void, we recently found that mice with knockout of G-protein coupled receptor 5A (Gprc5a-/-) and after tobacco carcinogen exposure (nicotine-specific nitrosamine ketone/NNK) developed LUADs harboring driver somatic Kras mutations. To understand early events preceding the onset of these Kras mutant LUADs, we performed RNA-sequencing (RNA-Seq) of normal airways in Gprc5a-/- mice and wild type (WT) littermates prior to NNK exposure. We found markedly elevated expression of the antimicrobial lipocalin 2 (Lcn2) gene in normal airways of Gprc5a-/- mice. Also, analysis of publicly available human datasets revealed elevated expression of LCN2 in human LUADs relative to normal lung tissues particularly in KRAS mutant tumors. We then sought to probe the role of Lcn2 in the pathogenesis of Kras mutant LUAD. Mice with knockout of host Lcn2 (Gprc5a-/-/Lcn2-/-) showed increased lung tumor development compared to Gprc5a-/- littermates. RNA-Seq of whole lungs at baseline and at seven months post-NNK (n = 9-10 mice each group) pinpointed differential expression profiles that denoted decreased anti-tumor immunity in Gprc5a-/-/Lcn2-/- mice. Computational estimation of immune infiltrates demonstrated increased levels of neutrophils post-NNK in Gprc5a-/-/Lcn2-/- mice but not in Gprc5a-/-littermates. By flow cytometry analysis of lung tissues, we found elevated numbers of various lymphoid and myeloid immune cells in both groups following NNK exposure and increased neutrophil counts in Gprc5a-/-/Lcn2-/- mice relative to Gprc5a-/- littermates. Histopathological assessment also revealed increased numbers of pro-inflammatory lesions in mice lacking Lcn2. Gprc5a-/-/Lcn2-/- mice at three months post-NNK were also found to exhibit increased production of IL-17A by lung-resident CD4+ and CD8+ T cells relative to Gprc5a-/- littermates concomitant with decreased expression of the anti-tumor Th1/cytolytic immune markers Ifnγ and Gzmb. Preliminary analysis also showed elevated expression of the major immune checkpoint PD-L1 on alveolar macrophages in Gprc5a-/-/Lcn2-/- mice relative to Gprc5a-/- mice with WT Lcn2. Lastly, we preliminarily found, by bacterial 16S rRNA sequencing of temporally collected fecal samples, globally altered microbiome profiles in mice lacking Lcn2. Our findings underscore novel cues in lung oncogenesis and suggest that Lcn2 decreases anti-tumor immunity, perturbs the host microbiome and promotes development of Kras mutant LUAD thereby offering new venues for early treatment of this fatal malignancy. Citation Format: Maya Hassane, Warapen Treekitkarnmongkol, Tina L. McDowell, Smruthy Sivakumar, Wenhua Lang, Joshua K. Ochieng, Sayuri Nunomura-Nakamura, Casey Finnicum, Christel Davis, Gareth E. Davies, Junya Fukuoka, Tina Cascone, Florencia McAllister, Ignacio I. Wistuba, Erik Ehli, Seyed J. Moghaddam, Paul Scheet, Junya Fujimoto, Humam Kadara. Host lipocalin 2 protects against Kras mutant lung cancer development by maintaining an anti-tumor immune contexture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4952.

Influenza A virus infection can predispose to potentially devastating secondary bacterial infections. Invariant natural killer T (iNKT) cells are unconventional, lipid-reactive T lymphocytes that exert potent immunostimulatory functions. Using a mouse model of postinfluenza invasive secondary pneumococcal infection, we sought to establish whether α-galactosylceramide (α-GalCer [a potent iNKT cell agonist that is currently in clinical development]) could limit bacterial superinfection. Our results highlighted the presence of a critical time window during which α-GalCer treatment can trigger iNKT cell activation and influence resistance to postinfluenza secondary pneumococcal infection. Intranasal treatment with α-GalCer during the acute phase (on day 7) of influenza virus H3N2 and H1N1 infection failed to activate (gamma interferon [IFN-γ] and interleukin-17A [IL-17A]) iNKT cells; this effect was associated with a strongly reduced number of conventional CD103+ dendritic cells in the respiratory tract. In contrast, α-GalCer treatment during the early phase (on day 4) or during the resolution phase (day 14) of influenza was associated with lower pneumococcal outgrowth and dissemination. Less intense viral-bacterial pneumonia and a lower morbidity rate were observed in superinfected mice treated with both α-GalCer (day 14) and the corticosteroid dexamethasone. Our results open the way to alternative (nonantiviral/nonantibiotic) iNKT-cell-based approaches for limiting postinfluenza secondary bacterial infections., IMPORTANCE Despite the application of vaccination programs and antiviral drugs, influenza A virus (IAV) infection is responsible for widespread morbidity and mortality (500,000 deaths/year). Influenza infections can also result in sporadic pandemics that can be devastating: the 1918 pandemic led to the death of 50 million people. Severe bacterial infections are commonly associated with influenza and are significant contributors to the excess morbidity and mortality of influenza. Today’s treatments of secondary bacterial (pneumococcal) infections are still not effective enough, and antibiotic resistance is a major issue. Hence, there is an urgent need for novel therapies. In the present study, we set out to evaluate the efficacy of α-galactosylceramide (α-GalCer)—a potent agonist of invariant NKT cells that is currently in clinical development—in a mouse model of postinfluenza, highly invasive pneumococcal pneumonia. Our data indicate that treatment with α-GalCer reduces susceptibility to superinfections and, when combined with the corticosteroid dexamethasone, reduces viral-bacterial pneumonia.

Interleukin-17A is a pro-inflammatory cytokine that plays an important role at mucosal sites in a wide range of immune responses including infection, allergy and auto-immunity. γΔ T cells are recognized as IL-17 producers, but based on the level of CD3 expression we have recently defined the remarkable ability of a pulmonary CD3 bright Vγ6/VΔ1 + T cell subset with an effector memory phenotype to rapidly produce IL-17A. During pulmonary pneumococcal infection, we show that CD3 bright Vγ6/VΔ1 + γΔ T cells are the major source of IL-17A during the early course of infection in a mechanism involving IL-23 and IL-1β. These two cytokines are preferentially produced by conventional dendritic cells and neutrophils. Moreover their rapid response to infection might be explained by their particular localization in the lung tissue. Unlike, other innate-like T cells that preferentially reside in the lung tissue as marginated cells, CD3 bright γΔ T cells are almost exclusively represented in the interstitial compartment. Finally, we are currently investigating the role of IL-17-producing CD3 bright γΔ T cells during acute pneumococcal infection. In the future, this might lead to the design of new prophylactic/therapeutic strategies employing tailored γΔ T cell-based immunotherapy.

The last century has witnessed incredible progresses in the control of infectious diseases. However, respiratory pathogens continue to threaten humans and cause serious public health and economic problems. Not only emerging pathogens but also viruses that have been known for a while continue to cause widespread issues (Summary analysis of 2014 survey of National Influenza Centres in the WHO Global Influenza Surveillance and Response System, 2014). Despite the relative efficacy of the inactivated vaccine against influenza A viruses (IAV) has been shown for many years, this virus is still responsible for seasonal widespread morbidity and mortality. These outbreaks are usually associated with mild symptoms but can also result in 3 to 5 million cases of severe illnesses, especially amongst elderly and young children. Influenza virus still accounts for 250,000 to 500,000 deaths each year during epidemic episodes (Summary analysis of 2014 survey of National Influenza Centres in the WHO Global Influenza Surveillance and Response System, 2014). This is due to the continuous antigenic drift that leads to vaccine mismatch and therefore negatively impacts on its effectiveness (Krammer and Palese, 2015). Moreover, more sporadically, pandemics due to highly virulent strains or transversal infections of animal strains to humans represent another important threat (Palese, 2004). In this context, it is of the utmost importance to urgently develop universal influenza vaccines. Current vaccines are yearly updated on the basis of the circulating strains but unfortunately mainly generate neutralizing antibodies (Abs) directed against epitopes contained in highly variable regions (Gerdil, 2003). Nevertheless pre-clinical studies have demonstrated promising data through the generation of neutralizing Abs against conserved regions such as the helix-A of the surface glycoprotein hemagglutinin (HA) (Eggink et al., 2014) but these observations have not yet been translated into humans. Amongst additional functions triggered by Abs, Ab-dependent cellular cytotoxicity (ADCC) mediates antiviral activities through the binding of cell surface Ags by IgG subtypes on target cells. This ultimately leads to the lysis of the target cells by secreted lytic enzymes from effector cells. This secretion follows the recognition of Fc region of surface bound Abs by FcγRIIIα (CD16)-expressing cytotoxic effector cells from both innate and adaptive immunity such as Natural Killer (NK) cells or CD8+ T cells. ADCC directed against IAV has been recognized for a long time(Jegaskanda et al., 2014). Since this class of Abs can target much more conserved proteins, understanding ADCC mechanisms during IAV infection might help in the development of universal influenza vaccines. For instance, a vaccine trial in HIV patients has been demonstrated protective in part through ADCC-mediating Abs(Madhavi et al., 2012). In this issue, Vanderven and colleagues evidence in human samples from healthy influenza-exposed adults or symptomatic donors the existence of IAV-specific Abs capable of ADCC activity through NK cells (Vanderven et al., 2016). Interestingly these opsonizing Abs are directed against the highly conserved internal viral proteins nucleoprotein (NP) and matrix 1(M1) (~ 90% of identity in amino acid sequences between strains) from two different strains of IAV (H1N1/2009pdm and H3N2/2005). An increase for such cross-protective Abs was found in sera of naturally IAV-exposed humans suggesting that this class of Abs can potentially mediate heterologous response to various IAV strains during active infection. However, authors failed to detect rise in NK cell-mediated ADCC activity in volunteers experimentally exposed to IAV (H3N2/2005). The reasons for such a discrepancy are currently unknown. It is possible that the route/dose of infection as well as the strain used matters in the development of NK cell activating Abs. Other clinical studies investigating the contribution of these parameters will be informative to answer this question. The presence and functionality of NP- and M1-specific NK cell-mediated ADCC in animal models including non-human primates has already been demonstrated (Jegaskanda et al., 2014). From a mechanistic point of view, the relevance of ADCC mediated against internal proteins remains puzzling. Given that NP and M1 are only minimally exposed at the cell surface during IAV life cycle, it will be important to determine the precise molecular/cellular mechanisms underlying NK cell-mediated ADCC. Although these Abs could mediate direct protection through opsonization of infected/dying cells, another explanation might involve indirect mechanisms (e.g. cytokine production or immune complex formation) that could lead to enhanced immune responses. The origin of this class of Abs is currently unknown. Do they arise from specific B cell subsets? As suggested in this study, it is tempting to answer in the affirmative. Actually while most of the donors (healthy influenza-exposed and symptomatic) presented detectable levels of persistent NP- and M1-specific ADCC-mediating Abs, authors failed to concomitantly detect classical neutralizing Abs. Therefore, it will be important to understand the mechanisms and B cell populations involved in the generation of cross-reactive Abs. For a long time, immunologists have focused their attention on the sole role of neutralizing Abs for vaccination efficacy. It is now clear that other Ab-dependent mechanisms can play relevant biological functions. The existence and persistence of Abs capable of NK cell-mediated ADCC against conserved viral proteins in patients with IAV infection is an important step in our comprehension of the immune responses against these viruses. This observation and their recognized relevance in experimental models of IAV infection should encourage a better investigation of ADCC-mediating Abs as a putative predictive parameter in vaccine efficacy and could further support the development of a “Universal influenza vaccine”.

International audience; Interleukin-17A (IL-17A) is a pro-inflammatory cytokine that has an important role at mucosal sites in a wide range of immune responses including infection, allergy and auto-immunity. γδ T cells are recognized as IL-17 producers, but based on the level of CD3 expression, we now define the remarkable ability of a CD3(bright) γδ T-cell subset with an effector memory phenotype to rapidly produce IL-17A, but not interferon-γ. CD3(bright) γδ T cells uniformly express the canonical germline encoded Vγ6/Vδ1(+) T-cell receptor. They are widely distributed with a preferential representation in the lungs and skin are negatively impacted in the absence of retinoic acid receptor-related orphan receptor gammat expression or endogenous flora. This population responded rapidly to various stimuli in a mechanism involving IL-23 and NOD-like receptor family, pyrin domain containing 3 (NLRP3)-inflammasome-dependent IL-1β. Finally, we demonstrated that IL-17-producing CD3(bright) γδ T cells responded promptly and strongly to pneumococcal infection and during skin inflammation. Here, we propose a new way to specifically analyze IL-17-producing Vγ6/Vδ1(+) T cells based on the level of CD3 signals. Using this gating strategy, our data reinforce the crucial role of this γδ T-cell subset in respiratory and skin disorders.