Severe respiratory syncytial virus (RSV) infections are characterized by airway epithelial cell damage, mucus hypersecretion, and Th2 cytokine production. Less is known about the role of IL-17. We observed increased IL-6 and IL-17 levels in tracheal aspirate samples from severely ill infants with RSV infection. In a mouse model of RSV infection, time-dependent increases in pulmonary IL-6, IL-23, and IL-17 expression were observed. Neutralization of IL-17 during infection and observations from IL-17(-/-) knockout mice resulted in significant inhibition of mucus production during RSV infection. RSV-infected animals treated with anti-IL-17 had reduced inflammation and decreased viral load, compared with control antibody-treated mice. Blocking IL-17 during infection resulted in significantly increased RSV-specific CD8 T cells. Factors associated with CD8 cytotoxic T lymphocytes, T-bet, IFN-γ, eomesodermin, and granzyme B were significantly up-regulated after IL-17 blockade. Additionally, in vitro analyses suggest that IL-17 directly inhibits T-bet, eomesodermin, and IFN-γ in CD8 T cells. The role of IL-17 was also investigated in RSV-induced exacerbation of allergic airway responses, in which neutralization of IL-17 led to a significant decrease in the exacerbated disease, including reduced mucus production and Th2 cytokines, with decreased viral proteins. Taken together, our data demonstrate that IL-17 plays a pathogenic role during RSV infections.

With a 5-year survival rate of <5%, pancreatic cancer is one of the most rapidly fatal malignancies. Current protocols for the treatment of pancreas cancer are not as effective as we desire. In this study, we show that a novel Mucin-1 (MUC1)-based vaccine in combination with a cyclooxygenase-2 inhibitor (celecoxib), and low-dose chemotherapy (gemcitabine) was effective in preventing the progression of preneoplastic intraepithelial lesions to invasive pancreatic ductal adenocarcinomas. The study was conducted in an appropriate triple transgenic model of spontaneous pancreatic cancer induced by the KRASG12D mutation and that expresses human MUC1 as a self molecule. The combination treatment elicited robust antitumor cellular and humoral immune responses and was associated with increased apoptosis in the tumor. The mechanism for the increased immune response was attributed to the down-regulation of circulating prostaglandin E2 and indoleamine 2, 3,-dioxygenase enzymatic activity, as well as decreased levels of T regulatory and myeloid suppressor cells within the tumor microenvironment. The preclinical data provide the rationale to design clinical trials with a combination of MUC1-based vaccine, celecoxib, and gemcitabine for the treatment of pancreatic cancer.

Macrophages (MΦ) play an essential role in innate immune responses and can either display a pro-inflammatory, classically activated phenotype (M1) or undergo an alternative activation program (M2) promoting immune regulation. M-CSF is used to differentiate monocytes into MΦ and IFN-γ or IL-4+IL-13 to further polarize these cells towards M1 or M2, respectively. Recently, differentiation using only GM-CSF or M-CSF has been described to induce a M1- or M2-like phenotype, respectively. In this study, we combined both approaches by differentiating human MΦ in GM-CSF or M-CSF followed by polarization with either IFN-γ or IL-4+IL-13. We describe the phenotypic differences between CD14hi CD163hi CD206int FOLR2-expressing M-CSF MΦ and CD14lo CD163lo CD206hi GM-CSF MΦ but show that both macrophage populations reacted similarly to further polarization with IFN-γ or IL-4+IL-13 with up- and down-regulation of common M1 and M2 marker genes. We also show that high expression of the mannose receptor (CD206), a marker of alternative activation, is a distinct feature of GM-CSF MΦ. Changes of the chromatin structure carried out by chromatin modification enzymes (CME) have been shown to regulate myeloid differentiation. We analyzed the expression patterns of CME during MΦ polarization and show that M1 up-regulate the histone methyltransferase MLL and demethylase KDM6B, while resting and M2 MΦ were characterized by DNA methyltransferases and histone deacetylases. We demonstrate that MLL regulates CXCL10 expression and that this effect could be abrogated using a MLL-Menin inhibitor. Taken together we describe the distinct phenotypic differences of GM-CSF or M-CSF MΦ and demonstrate that MΦ polarization is regulated by specific epigenetic mechanisms. In addition, we describe a novel role for MLL as marker for classical activation. Our findings provide new insights into MΦ polarization that could be helpful to distinguish MΦ activation states.

The response to respiratory syncytial virus (RSV), negative strand ssRNA virus, depends upon the ability to recognize specific pathogen-associated targets. In the current study, the role of TLR7 that recognizes ssRNA was examined. Using TLR7−/− mice, we found that the response to RSV infection in the lung was more pathogenic as assessed by significant increases in inflammation and mucus production. Although there appeared to be no effect of TLR7 deficiency on type I IFN, the pathology was associated with an alteration in T cell responses with increases in mucogenic cytokines IL-4, IL-13, and IL-17. Examination of dendritic cells from TLR7−/− animals indicated a preferential activation of IL-23 (a Th17-promoting cytokine) and a decrease in IL-12 production. Neutralization of IL-17 in the TLR7−/− mice resulted in a significant decrease in the mucogenic response in the lungs of the RSV-infected mice. Thus, without TLR7-mediated responses, an altered immune environment ensued with a significant effect on airway epithelial cell remodeling and goblet cell hyper/metaplasia, leading to increased mucus production.

Recent work has demonstrated the importance of macroautophagy in dendritic cell (DC) maturation and innate cytokine production upon viral infection through delivery of cytoplasmic viral components to intracellular toll-like receptors. To study the functional consequences of impaired autophagosome formation during a Respiratory Syncytial Virus (RSV) infection, mice harboring significant autophagy defects due to Beclin-1 haploinsufficiency (Beclin-1+/−) were used. Upon RSV infection in vivo, lungs of Beclin-1+/− mice showed increased Th2 cytokine production, mucus secretion, and lung infiltration of eosinophils and inflammatory DCs. While isolated airway epithelial cells from Beclin-1+/− mice demonstrated little change compared to wildtype, Beclin-1+/− pulmonary and bone marrow-derived DCs (BMDCs) showed decreased expression of MHC-II and innate cytokine production upon RSV infection. Further examination indicated that Beclin-1+/− DC stimulated less IFNγ and IL-17 production by co-cultured CD4+ T cells and increased Th2 cytokine production in comparison to wild-type controls. Finally, adoptive transfer of RSV-infected Beclin-1+/− DCs into the airways of wild-type mice produced severe lung pathology and increased Th2 cytokine production upon subsequent RSV challenge compared to wild-type DC transfer controls. These results indicate a critical role of autophagy in dendritic cells during pulmonary viral infection, facilitating appropriate antiviral adaptive immune responses.

The cytosolic RNA helicases melanoma differentiation-associated gene 5 (MdA5) and retinoic acid-inducible gene-I (RIG-I) and their adaptor IFNβ promotor stimulator (IPS-1) have been implicated in the recognition of viral RNA and the production of type I interferon (IFN). Complementing the endosomal Toll-like receptors (TLR), Mda5 and RIG-I provide alternative mechanisms for viral detection in cells with reduced phagocytosis or autophagy. The infection route of Respiratory Syncytial Virus (RSV) - via fusion of virus particles with the cell membrane - points to IPS-1 signaling as the pathway of choice for downstream antiviral responses. In the present study, viral clearance and inflammation resolution were indeed strongly affected by the absence of an initial IPS-1-mediated IFNβ response. Despite the blunted inflammatory response in IPS-1 deficient alveolar epithelial cells, pulmonary macrophages and CD11b+ dendritic cells (DC), lungs of RSV-infected IPS-1 knockout (KO) mice showed augmented recruitment of inflammatory neutrophils, monocytes and DC. Interestingly, pulmonary CD103+ DC could functionally compensate for IPS-1 deficiency with the up-regulation of certain inflammatory cytokines and chemokines, possibly via TLR3 and TLR7 signaling. The increased inflammation and reduced viral clearance in IPS-1 KO mice was accompanied by increased T-cell activation and IFNγ production. Experiments with bone marrow chimeras indicated that RSV-induced lung pathology was most severe when IPS-1 expression was lacking in both immune and non-immune cell populations. Similarly, viral clearance was rescued upon restored IPS-1 signaling in either the non-immune or the immune compartment. These data support a non-redundant function for IPS-1 in controlling RSV-induced inflammation and viral replication.

Respiratory syncytial virus (RSV) infection can result in severe disease partially due to its ability to interfere with the initiation of Th1 responses targeting the production of type I interferons (IFN) and promoting a Th2 immune environment. Epigenetic modulation of gene transcription has been shown to be important in regulating inflammatory pathways. RSV-infected bone marrow-derived DCs (BMDCs) upregulated expression of Kdm5b/Jarid1b H3K4 demethylase. Kdm5b-specific siRNA inhibition in BMDC led to a 10-fold increase in IFN-β as well as increases in IL-6 and TNF-α compared to control-transfected cells. The generation of Kdm5b fl/fl-CD11c-Cre+ mice recapitulated the latter results during in vitro DC activation showing innate cytokine modulation. In vivo, infection of Kdm5b fl/fl-CD11c-Cre+ mice with RSV resulted in higher production of IFN-γ and reduced IL-4 and IL-5 compared to littermate controls, with significantly decreased inflammation, IL-13, and mucus production in the lungs. Sensitization with RSV-infected DCs into the airways of naïve mice led to an exacerbated response when mice were challenged with live RSV infection. When Kdm5b was blocked in DCs with siRNA or DCs from Kdm5b fl/fl-CD11c-CRE mice were used, the exacerbated response was abrogated. Importantly, human monocyte-derived DCs treated with a chemical inhibitor for KDM5B resulted in increased innate cytokine levels as well as elicited decreased Th2 cytokines when co-cultured with RSV reactivated CD4+ T cells. These results suggest that KDM5B acts to repress type I IFN and other innate cytokines to promote an altered immune response following RSV infection that contributes to development of chronic disease.

IL-27 is a heterodimeric cytokine composed of the subunits p28 and Epstein-Barr virus induced gene (EBI)-3 and is known for its effects on T-cell function and differentiation. IL-27 signals through the widely expressed IL-27 receptor (IL-27R), composed of the ligand-specific IL-27Rα chain and gp130. Engagement of the IL-27R activates STAT1 signaling, induces the expression of the type 1 helper T-cell (Th1) cytokine, interferon γ, and suppresses the differentiation of Th2 and Th17 cells. This study investigates the role of IL-27 signaling in respiratory syncytial virus (RSV) infection using IL-27Rα-deficient mice (IL-27rKO). Analysis of lungs from RSV-infected IL-27rKO mice showed exacerbation of mucus secretion compared with wild type, as well as enhanced expression of Muc5ac and Gob5 mRNA, markers of goblet cell metaplasia/hyperplasia. When compared with wild-type mice, RSV-challenged IL-27rKO mice had enhanced expression of Th17-associated cytokine IL-17a and an imbalance between Th1 and Th2 cytokine levels. Neutralization of IL-17 in RSV-infected IL-27rKO mice resulted in a significant decrease in the pulmonary mucus response and inhibition of the Th2-associated cytokines. Interestingly, IL-17 blockage led to an increase in the expression of IL-27 subunits p28 and EBI-3 in the lungs and lymph nodes of RSV-infected mice. Thus, IL-27 functions as a regulatory cytokine during RSV pathogenesis by suppressing the development of Th17 cells, but it also appears to be regulated by IL-17 induced by the virus.