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1.  Autophagy inducing protein Beclin-1 in dendritic cells regulates CD4 T cell responses and disease severity during RSV infection 
Journal of immunology (Baltimore, Md. : 1950)  2013;191(5):10.4049/jimmunol.1300477.
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.
doi:10.4049/jimmunol.1300477
PMCID: PMC3811020  PMID: 23894198
2.  IL-25 induces type 2 cytokine production in a novel, steroid resistant IL-17RB+ myeloid population that exacerbates asthmatic pathology 
Nature Medicine  2012;18(5):751-758.
This study identifies the IL-25 receptor, IL-17RB, is an important mediator of both innate and adaptive pulmonary type 2 immune responses. Allergen exposure upregulated IL-25 and induced type 2 cytokine production in a novel granulocytic population, termed Type 2 Myeloid (T2M) cells. Il17rb−/− mice exhibited reduced lung pathology following chronic allergen exposure and decreased cytokine production in T2M cells and CD4+ T-lymphocytes. Airway instillation of IL-25 induced IL-4 and IL-13 production exclusively in T2M cells demonstrating their importance in generating T cell-independent inflammation. The adoptive transfer of T2M cells reconstituted IL-25-mediated responses in Il17rb−/− mice. High dose dexamethasone treatment did not reduce the IL-25-induced T2M pulmonary response. Finally, a similar IL-4/IL-13 producing granulocytic population was identified in peripheral blood of asthmatics. These data establish IL-25/IL-17RB as targets for innate and adaptive immune responses in chronic allergic airways disease, and identify T2M cells as a novel steroid-resistant cell population.
doi:10.1038/nm.2735
PMCID: PMC3378776  PMID: 22543263
3.  Autophagy-mediated DC activation is essential for innate cytokine production and APC function with Respiratory Syncytial Virus responses 
The regulation of innate immune responses during viral infection is a crucial step to promote anti-viralreactions. Recent studies have drawn attention to a strong relationship of pathogen associated molecular patterns (PAMP) recognition with autophagy for activation of APC function. Our initial observations indicated that autophagosomes formed in response to RSV infection of DC. To further investigate whether RSV-induced DC activation and innate cytokine production was associated with autophagy, we utilized several methods to block autophagosome formation. Using 3-MA,siRNA inhibition of LC3,or Beclin +/- mouse derived DC,studies establisheda relationship between RSV-induced autophagy and enhanced type I IFN, TNF, IL-6, and IL-12p40expression. Moreover, autophagosome formation induced by starvation also promoted innate cytokine expression in DC. The induction of starvation-induced autophagy in combination with RSV infection synergistically enhanced DC cytokine expressionthat was blocked by an autophagy inhibitor. The latter synergistic responses were differentially altered in DC from MyD88-/- and TRIF-/-mice supporting the concept of autophagy-mediated TLR signaling. In addition, blockade of autophagy in RSV-infected DC inhibited the maturation of DCs as assessed by MHC Class II and co-stimulatory molecule expression. Subsequently, we demonstrated that inhibition of autophagy in DCsused to stimulateprimary ovalbumin-induced and secondary RSV-infected responses significantly attenuatedcytokine production by CD4+ T cells. Thus, these studies have outlined that autophagy in DC afterRSV infection isa crucial mechanism for driving innate cytokine productionleading to alteredacquired immune responses.
doi:10.4049/jimmunol.1100524
PMCID: PMC3186849  PMID: 21911604
4.  Notch ligand Delta-like 4 regulates development and pathogenesis of allergic airway responses by modulating IL-2 production and Th2 immunity 
Activation of the canonical Notch pathways has been implicated in Th cell differentiation, but the role of specific Notch ligands in Th2 mediated allergic airway responses has not been completely elucidated. In this study, we show that delta-like 4 (Dll4) was up-regulated on dendritic cells in response to cockroach allergen. Blocking Dll4 in vivo during either the primary or secondary response enhanced allergen-induced pathogenic consequences including airway hyperresponsiveness (AHR) and mucus production via increased Th2 cytokines. In vitro assays demonstrated that Dll4 regulates IL-2 in T cells from established Th2 responses as well as during primary stimulation. Interestingly, Dll4 blockade during the primary, but not the secondary response, increased IL-2 levels in lung and lymph node of allergic mice. The in vivo neutralization of Dll4 was associated with increased expansion and decreased apoptosis during the primary allergen sensitization. Moreover, Dll4-mediated Notch activation of T cells during primary stimulation in vitro increased apoptosis during the contraction/resting phase of the response, which could be rescued by exogenous IL-2. Consistent with the role for Dll4-mediated IL-2 regulation in overall T cell function, the frequency of IL-4 producing cells were also significantly altered by Dll4 both in vivo and in vitro. These data demonstrate a regulatory role of Dll4 on both initial Th2 differentiation as well as on Th2 cytokine production in established allergic responses.
doi:10.4049/jimmunol.1000175
PMCID: PMC2980697  PMID: 20944009
5.  Notch ligand Dll4 enhances T cell differentiation by promoting IL-17 production and RORγT expression 
The activation and differentiation of T cells are dependent upon numerous initiating events that are influenced by the immune environment, nature of the antigen, as well as the activation state of APCs. In the present studies we have investigated the role of a specific notch ligand, delta-like 4 (Dll4). In particular, our data have indicated that Dll4 is inducible by pathogen-associated signals through TLR activation on DC but not early response inflammatory cytokines, IL-1 and IL-18 that also activate cells via MyD88 adapter pathway. Our observations from in vitro cultures with ovalbumin specific TCR transgenic cells (DO11.10) confirmed earlier reports demonstrating that Dll4 inhibits Th2 cytokine production. Furthermore, Dll4 enhances the generation of IL-17 producing T cells in the presence of additional skewing cytokines, IL-6 and TGFβ. In the absence of notch signals IL17 production was significantly reduced even under specific skewing conditions. These studies further demonstrate that Dll4 upregulates RORγt expression in T cells and that both RORγt and IL17 gene promoters are direct transcriptional notch targets that augment the differentiation of Th17 cell populations. Thus, facilitation of efficient T cell differentiation may depend upon the activation of T cells via specific notch ligand stimulation.
doi:10.4049/jimmunol.0804322
PMCID: PMC2980695  PMID: 19494260
6.  Discovery of a potent nanoparticle P-selectin antagonist with anti-inflammatory effects in allergic airway disease 
The severity of allergic asthma is dependent, in part, on the intensity of peribronchial inflammation. P-selectin is known to play a role in the development of allergen-induced peribronchial inflammation and airway hyperreactivity. Selective inhibitors of P-selectin-mediated leukocyte endothelial-cell interactions may therefore attenuate the inflammatory processes associated with allergic airway disease. Novel P-selectin inhibitors were created using a polyvalent polymer nanoparticle capable of displaying multiple synthetic, low molecular weight ligands. By assembling a particle that presents an array of groups, which as monomers interact with only low affinity, we created a construct that binds extremely efficiently to P-selectin. The ligands acted as mimetics of the key binding elements responsible for the high-avidity adhesion of P-selectin to the physiologic ligand, PSGL-1. The inhibitors were initially evaluated using an in vitro shear assay system in which interactions between circulating cells and P-selectin-coated capillary tubes were measured. The nanoparticles were shown to preferentially bind to selectins expressed on activated endothelial cells. We subsequently demonstrated that nanoparticles displaying P-selectin blocking arrays were functionally active in vivo, significantly reducing allergen-induced airway hyperreactivity and peribronchial eosinophilic inflammation in a murine model of asthma.
doi:10.1096/fj.03-0166fje
PMCID: PMC2839900  PMID: 14563683
selectin inhibitor; asthma; eosinophil; lung
7.  Thymic stromal lymphopoietin is induced by respiratory syncytial virus–infected airway epithelial cells and promotes a type 2 response to infection 
Background
Respiratory viral infection, including respiratory syncytial virus (RSV) and rhinovirus, has been linked to respiratory disease in pediatric patients, including severe acute bronchiolitis and asthma exacerbation.
Objective
The study examined the role of the epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) in the response to RSV infection.
Methods
Infection of human airway epithelial cells was used to examine TSLP induction after RSV infection. Air–liquid interface cultures from healthy children and children with asthma were also tested for TSLP production after infection. Finally, a mouse model was used to directly test the role of TSLP signaling in the response to RSV infection.
Results
Infection of airway epithelial cells with RSV led to the production of TSLP via activation of an innate signaling pathway that involved retinoic acid induced gene I, interferon promoter-stimulating factor 1, and nuclear factor-κB. Consistent with this observation, airway epithelial cells from asthmatic children a produced significantly greater levels of TSLP after RSV infection than cells from healthy children. In mouse models, RSV-induced TSLP expression was found to be critical for the development of immunopathology.
Conclusion
These findings suggest that RSV can use an innate antiviral signaling pathway to drive a potentially nonproductive immune response and has important implications for the role of TSLP in viral immune responses in general.
doi:10.1016/j.jaci.2012.07.031
PMCID: PMC4284103  PMID: 22981788
TSLP; RSV; asthma; epithelium; TH2
9.  IL-17A inhibits airway reactivity induced by RSV infection during allergic airway inflammation 
Thorax  2013;68(8):717-723.
Background
Viral infections are the most frequent cause of asthma exacerbations and are linked to increased airway reactivity (AR) and inflammation. Mice infected with respiratory syncytial virus (RSV) during ovalbumin (OVA)-induced allergic airway inflammation (OVA/RSV) had increased AR compared to OVA or RSV mice alone. Further, IL-17A was only increased in OVA/RSV mice.
Objective
To determine if IL-17A increases AR and inflammation in the OVA/RSV model.
Methods
Wild-type BALB/c and IL-17A KO mice underwent mock, RSV, OVA, or OVA/RSV protocols. Lungs, bronchoalveolar lavage (BAL) fluid, and/or mediastinal lymph nodes (MLNs) were harvested post infection. Cytokine expression was determined by flow cytometry and ELISA in the lungs or BAL fluid. MLNs were restimulated with either OVA (323–229) peptide or RSV M2 (127–135) peptide and IL-17A protein expression was analyzed. AR was determined by methacholine challenge.
Results
RSV increased IL-17A protein expression by OVA-specific T cells 6 days post infection. OVA/RSV mice had decreased IFN-α and IFN-β protein expression compared to RSV mice. OVA/RSV mice had increased IL-23 mRNA expression in lung homogenates compared to mock, OVA, or RSV mice. Unexpectedly, IL-17A KO OVA/RSV mice had increased AR compared to WT OVA/RSV mice. Further, IL-17A KO OVA/RSV mice had increased eosinophils, lymphocytes, and IL-13 protein expression in BAL fluid compared to WT OVA/RSV mice.
Conclusions
IL-17A negatively regulated AR and airway inflammation in OVA/RSV mice. This finding is important because IL-17A has been identified as a potential therapeutic target in asthma, and inhibiting IL-17A in the setting of virally induced asthma exacerbations may have adverse consequences.
doi:10.1136/thoraxjnl-2012-202404
PMCID: PMC3916091  PMID: 23422214
IL-17A; airway reactivity; CD4+ T cells; allergic inflammation; RSV
10.  Anti-CCL2 treatment inhibits Theiler's murine encephalomyelitis virus-induced demyelinating disease 
Journal of neurovirology  2006;12(4):251-261.
Theiler's murine encephalomyelitis virus induces a demyelinating disease (TMEV-IDD) of the central nervous system (CNS) in susceptible mouse strains with accompanying histopathology characterized by mononuclear cell infiltrates. In susceptible strains of mice such as SJL, virus establishes a persistent infection in macrophages, induces a CNS infiltration by macrophages, T cells, and B cells, which results in chronic-progressive paralysis. In the present report the authors have investigated the functional role of CCL2 (monocyte chemotactic protein-1) in the induction and progression of demyelinating disease. Treatment of infected mice at day 0, 14, or 28 with anti-CCL2 resulted in a significant decrease in the clinical disease progression. Further analysis of anti-CCL2–treated mice revealed decreased CNS inflammation and mononuclear cell infiltration with an accompanying change in inflammatory cytokine responses. There was an overall decrease in the absolute numbers of CNS-infiltrating CD4+ T cells, macrophages, and B cells. Finally, anti-CCL2 treatment resulted in decreased viral load in the CNS. These data directly demonstrate a role for CCL2 in the pathogenesis of TMEV-IDD.
doi:10.1080/13550280600873819
PMCID: PMC4040265  PMID: 16966216
cell trafficking; chemokines; demyelinating disease; multiple sclerosis; neuroimmunology; Theiler's virus
11.  IPS-1 Signaling Has a Non-Redundant Role in Mediating Antiviral Responses and the Clearance of Respiratory Syncytial Virus 
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.
doi:10.4049/jimmunol.1201763
PMCID: PMC3888965  PMID: 23136205
12.  STAT3 mediated IL-17 production by post-septic T cells exacerbates viral immunopathology of the lung 
Shock (Augusta, Ga.)  2012;38(5):515-523.
Survivors of severe sepsis exhibit increased morbidity and mortality in response to secondary infections. Although bacterial secondary infections have been widely studied, there remains a paucity of data concerning viral infections post-sepsis. In an experimental mouse model of severe sepsis (cecal ligation and puncture, CLP) followed by respiratory syncytial virus (RSV) infection, exacerbated immunopathology was observed in the lungs of CLP mice compared to RSV-infected sham surgery mice. This virus-associated immunopathology was evidenced by increased mucus production in the lungs of RSV-infected CLP mice and correlated with increased IL-17 production in the lungs. RSV infected CLP mice exhibited increased levels of Th2 cytokines and reduced IFNγ in the lungs and lymph nodes compared to RSV-infected sham mice. In addition, CD4 T cells from CLP mice produced increased IL-17 in vitro irrespective of the presence of exogenous cytokines or blocking antibodies. This increased IL-17 production correlated wth increased STAT3 transcription factor binding to the IL-17 promoter in CD4 T cells from CLP mice. Further, in vivo neutralization of IL-17 prior to RSV infection led to a significant reduction in virus induced mucus production and Th2 cytokines. Taken together, these data provide evidence that post septic CD4+T cells are primed toward IL-17 production via increased STAT3-mediated gene transcription, which may contribute to the immunopathology of a secondary viral infection.
doi:10.1097/SHK.0b013e31826f862c
PMCID: PMC3475732  PMID: 23042197
Inflammation; infection; mucus; cytokines; lymphocytes
13.  Toll Like Receptor 3 Plays a Critical Role in the Progression and Severity of Acetaminophen-Induced Hepatotoxicity 
PLoS ONE  2013;8(6):e65899.
Toll-like receptor (TLR) activation has been implicated in acetaminophen (APAP)-induced hepatotoxicity. Herein, we hypothesize that TLR3 activation significantly contributed to APAP-induced liver injury. In fasted wildtype (WT) mice, APAP caused significant cellular necrosis, edema, and inflammation in the liver, and the de novo expression and activation of TLR3 was found to be necessary for APAP-induced liver failure. Specifically, liver tissues from similarly fasted TLR3-deficient (tlr3−/−) mice exhibited significantly less histological and biochemical evidence of injury after APAP challenge. Similar protective effects were observed in WT mice in which TLR3 was targeted through immunoneutralization at 3 h post-APAP challenge. Among three important death ligands (i.e. TNFα, TRAIL, and FASL) known to promote hepatocyte death after APAP challenge, TNFα was the only ligand that was significantly reduced in APAP-challenged tlr3−/− mice compared with APAP-challenged WT controls. In vivo studies demonstrated that TLR3 activation contributed to TNFα production in the liver presumably via F4/80+ and CD11c+ immune cells. In vitro studies indicated that there was cooperation between TNFα and TLR3 in the activation of JNK signaling in isolated and cultured liver epithelial cells (i.e. nMuLi). Moreover, TLR3 activation enhanced the expression of phosphorylated JNK in APAP injured livers. Thus, the current study demonstrates that TLR3 activation contributes to APAP-induced hepatotoxicity.
doi:10.1371/journal.pone.0065899
PMCID: PMC3676358  PMID: 23762449
14.  Vaccine-Elicited CD8+ T Cells Protect against Respiratory Syncytial Virus Strain A2-Line19F-Induced Pathogenesis in BALB/c Mice 
Journal of Virology  2012;86(23):13016-13024.
CD8+ T cells may contribute to vaccines for respiratory syncytial virus (RSV). Compared to CD8+ T cells responding to RSV infection, vaccine-elicited anti-RSV CD8+ T cells are less well defined. We used a peptide vaccine to test the hypothesis that vaccine-elicited RSV-specific CD8+ T cells are protective against RSV pathogenesis. BALB/c mice were treated with a mixture (previously termed TriVax) of an M282-90 peptide representing an immunodominant CD8 epitope, the Toll-like receptor (TLR) agonist poly(I·C), and a costimulatory anti-CD40 antibody. TriVax vaccination induced potent effector anti-RSV CD8+ cytotoxic T lymphocytes (CTL). Mice were challenged with RSV strain A2-line19F, a model of RSV pathogenesis leading to airway mucin expression. Mice were protected against RSV infection and against RSV-induced airway mucin expression and cellular lung inflammation when challenged 6 days after vaccination. Compared to A2-line19F infection alone, TriVax vaccination followed by challenge resulted in effector CD8+ T cells with greater cytokine expression and the more rapid appearance of RSV-specific CD8+ T cells in the lung. When challenged 42 days after TriVax vaccination, memory CD8+ T cells were elicited with RSV-specific tetramer responses equivalent to TriVax-induced effector CD8+ T cells. These memory CD8+ T cells had lower cytokine expression than effector CD8+ T cells, and protection against A2-line19F was partial during the memory phase. We found that vaccine-elicited effector anti-RSV CD8+ T cells protected mice against RSV infection and pathogenesis, and waning protection correlated with reduced CD8+ T cell cytokine expression.
doi:10.1128/JVI.01770-12
PMCID: PMC3497630  PMID: 23015695
15.  Neonatal rhinovirus infection induces mucous metaplasia and airways hyperresponsiveness 
Recent studies link early rhinovirus (RV) infections to later asthma development. We hypothesized that neonatal RV infection leads to an IL-13-driven asthma-like phenotype in mice. BALB/c mice were inoculated with RV1B or sham on day 7 of life. Viral RNA persisted in the neonatal lung up to 7 days after infection. Within this time frame, IFNs-α, -β and -γ peaked 1 day after infection, whereas IFN-λ levels persisted. Next, we examined mice on day 35 of life, 28 days after initial infection. Compared to sham-treated controls, virus-inoculated mice demonstrated airways hyperresponsiveness. Lungs from RV-infected mice showed increases in several immune cell populations, as well as the percentages of CD4-positive T cells expressing IFN-γ and of NKp46/CD335+, TCR-β+ cells expressing IL-13. Periodic acid-Schiff and immunohistochemical staining revealed mucous cell metaplasia and muc5AC expression in RV1B- but not sham-inoculated lungs. Mucous metaplasia was accompanied by induction of gob-5, MUC5AC, MUC5B and IL-13 mRNA. By comparison, adult mice infected with RV1B showed no change in IL-13 expression, mucus production or airways responsiveness 28 days after infection. Intraperitoneal administration of anti-IL13 neutralizing antibody attenuated RV-induced mucous metaplasia and methacholine responses, and IL-4R null mice failed to show RV-induced mucous metaplasia. Finally, neonatal RV increased the inflammatory response to subsequent allergic sensitization and challenge. We conclude that neonatal RV1B infection leads to persistent airways inflammation, mucous metaplasia and hyperresponsiveness which are mediated, at least in part, by IL-13.
doi:10.4049/jimmunol.1101391
PMCID: PMC3294163  PMID: 22331068
asthma; BALB/c; childhood; gob-5; IL-13
16.  TSLP Promotes Induction of Th2 Differentiation but Is Not Necessary during Established Allergen-Induced Pulmonary Disease 
PLoS ONE  2013;8(2):e56433.
Thymic stromal lymphopoietin (TSLP) has been implicated in the development of allergic inflammation by promoting Th2-type responses and has become a potential therapeutic target. Using in vitro T cell differentiation cultures we were able to validate that TSLP played a more critical role in the early development of Th2 immune responses with less significant enhancement of already developed Th2 responses. Adoptive transfer of naive DO11.10 ovalbumin-specific T cells followed by airway exposure to ovalbumin showed an early impairment of Th2 immune response in TSLP−/− mice compared to wild type mice during the development of a Th2 response. In contrast, transfer of already differentiated Th2 cells into TSLP−/− mice did not change lung pathology or Th2 cytokine production upon ovalbumin challenge compared to transfer into wild type mice. An allergen-induced Th2 airway model demonstrated that there was only a difference in gob5 expression (a mucus-associated gene) between wild type and TSLP−/− mice. Furthermore, when allergic animals with established disease were treated with a neutralizing anti-TSLP antibody there was no change in airway hyperreponsiveness (AHR) or Th2 cytokine production compared to the control antibody treated animals, whereas a change in gob5 gene expression was also observed similar to the TSLP−/− mouse studies. In contrast, when animals were treated with anti-TSLP during the initial stages of allergen sensitization there was a significant change in Th2 cytokines during the final allergen challenge. Collectively, these studies suggest that in mice TSLP has an important role during the early development of Th2 immune responses, whereas its role at later stages of allergic disease may not be as critical for maintaining the Th2-driven allergic disease.
doi:10.1371/journal.pone.0056433
PMCID: PMC3577905  PMID: 23437132
17.  IL-13 Regulates Th17 Secretion of IL-17A in an IL-10-Dependent Manner 
IL-13 is a central mediator of airway hyperreactivity and mucus expression, both hallmarks of asthma. IL-13 is found in the sputum of patients with asthma; therefore, IL-13 is an attractive drug target for treating asthma. We have previously shown that IL-13 inhibits Th17 cell production of IL-17A and IL-21 in vitro. Th17 cells are associated with autoimmune diseases, host immune responses, and severe asthma. In this study, we extend our in vitro findings and determine that IL-13 increases IL-10 production from Th17 polarized cells, and that IL-13-induced IL-10 production negatively regulates secretion of IL-17A and IL-21. To determine if IL-13 negatively regulates lung IL-17A expression via an IL-10 dependent mechanism in vivo, we used a model of respiratory syncytial virus (RSV) strain A2 infection in STAT1 KO mice which increases IL-17A and IL-13 lung expression, cytokines not produced during RSV infection in WT mice. To test the hypothesis that IL-13 negatively regulates lung IL-17A expression, we created STAT1/IL-13 double KO (DKO) mice. We found that RSV-infected STAT1/IL-13 DKO mice had significantly greater lung IL-17A expression compared to STAT1 KO mice, and increased IL-17A expression was abrogated by anti-IL-10 antibody treatment. RSV-infected STAT1/IL-13 DKO mice also had increased neutrophil infiltration RSV-infected STAT1 KO mice. Neutralizing IL-10 increased infiltration of inflammatory cells into the lungs of STAT1 KO mice but not STAT1/IL-13 DKO mice. These findings are vital to understanding potential side effects of therapeutics targeting IL-13. Inhibiting IL-13 may decrease IL-10 production and increase IL-17A production, thus potentiating IL-17A-associated diseases.
doi:10.4049/jimmunol.1102216
PMCID: PMC3262924  PMID: 22210911
18.  Chronic schistosome infection leads to modulation of granuloma formation and systemic immune suppression 
Schistosome worms have been infecting humans for millennia, but it is only in the last half century that we have begun to understand the complexities of this inter-relationship. As our sophistication about the inner workings of every aspect of the immune system has increased, it has also become obvious that schistosome infections have broad ranging effects on nearly all of the innate and adaptive immune response mechanisms. Selective pressures on both the worms and their hosts, has no doubt led to co-evolution of protective mechanisms, particularly those that favor granuloma formation around schistosome eggs and immune suppression during chronic infection. The immune modulatory effects that chronic schistosome infection and egg deposition elicit have been intensely studied, not only because of their major implications to public health issues, but also due to the emerging evidence that schistosome infection may protect humans from severe allergies and autoimmunity. Mouse models of schistosome infection have been extremely valuable for studying immune modulation and regulation, and in the discovery of novel aspects of immunity. A progression of immune reactions occurs during granuloma formation ranging from innate inflammation, to activation of each branch of adaptive immune response, and culminating in systemic immune suppression and granuloma fibrosis. Although molecular factors from schistosome eggs have been identified as mediators of immune modulation and suppressive functions of T and B cells, much work is still needed to define the mechanisms of the immune alteration and determine whether therapies for asthma or autoimmunity could be developed from these pathways.
doi:10.3389/fimmu.2013.00039
PMCID: PMC3576626  PMID: 23429492
T helper lymphocytes; immune regulation; hygiene hypothesis; soluble egg antigen; sialyl Lewisx glycans
19.  Delta-like Ligand 4 Regulates CNS T Cell Accumulation during Experimental Autoimmune Encephalomyelitis1 
Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell-mediated inflammatory demyelinating disease of the central nervous system (CNS) that serves as a model for multiple sclerosis (MS). Notch receptor signaling in T lymphocytes has been shown to regulate thymic selection and peripheral differentiation. In the present study we hypothesized that Notch ligand–receptor interaction affects EAE development by regulating encephalitogenic T cell trafficking. We demonstrate that CNS-infiltrating myeloid DC, macrophages, and resident microglia expressed Delta-like ligand 4 (DLL4) following EAE induction. Treatment of mice with a DLL4-specific blocking antibody significantly inhibited the development of clinical disease induced by active priming. Furthermore, the treatment resulted in decreased CNS accumulation of mononuclear cells in the CNS. Anti-DLL4 treatment did not significantly alter development of effector cytokine expression by antigen-specific T cells. In contrast, anti-DLL4 treatment reduced T cell mRNA and functional cell surface expression of the chemokine receptors CCR2 and CCR6. Adoptive transfer of antigen-specific T cells to mice treated with anti-DLL4 resulted in decreased clinical severity and diminished antigen-specific CD4+ T cell accumulation in the CNS. These results suggest a role for DLL4 regulation of EAE pathogenesis through modulation of T cell chemokine receptor expression and migration to the CNS.
doi:10.4049/jimmunol.1100160
PMCID: PMC3159801  PMID: 21788444
20.  Amelioration of the Cardiovascular Effects of Cocaine in Rhesus Monkeys by a Long-Acting Mutant Form of Cocaine Esterase 
Neuropsychopharmacology  2011;36(5):1047-1059.
A long-acting mutant form of a naturally occurring bacterial cocaine esterase (T172R/G173Q CocE; double mutant CocE (DM CocE)) has previously been shown to antagonize the reinforcing, convulsant, and lethal effects of cocaine in rodents. However, the effectiveness and therapeutic characteristics of DM CocE in nonhuman primates, in a more clinically relevant context, are unknown. The current studies were aimed at (1) characterizing the cardiovascular effects of cocaine in freely moving rhesus monkeys, (2) evaluating the capacity of DM CocE to ameliorate these cocaine-induced cardiovascular effects when administered 10 min after cocaine, and (3) assessing the immunological responses of monkeys to DM CocE following repeated administration. Intravenous administration of cocaine produced dose-dependent increases in mean arterial pressure (MAP) and heart rate (HR) that persisted throughout the 2-h observation period following a dose of 3.2 mg/kg cocaine. Cocaine failed to produce reliable changes in electrocardiograph (ECG) parameters, body temperature, and locomotor activity. DM CocE produced a rapid and dose-dependent amelioration of the cardiovascular effects, with saline-like MAP measures restored within 5–10 min, and saline-like HR measures restored within 20–40 min of DM CocE administration. Although administration of DM CocE produced increases in anti-CocE antibodies, they did not appear to have a neutralizing effect on the capacity of DM CocE to reverse the cardiovascular effects of cocaine. In conclusion, these findings in monkeys provide strong evidence to suggest that highly efficient cocaine esterases, such as DM CocE, can provide a potential therapeutic option for treatment of acute cocaine intoxication in humans.
doi:10.1038/npp.2010.242
PMCID: PMC3076304  PMID: 21289605
rhesus monkey; cardiovascular; cocaine; acute cocaine toxicity; cocaine esterase; DM CocE; drug discovery/development; animal models; psychostimulants; addiction & substance abuse; rhesus monkey; cocaine; cardiovascular; acute toxicity; cocaine esterase
21.  Rhinovirus infection of allergen-sensitized and -challenged mice induces eotaxin release from functionally polarized macrophages 
Human rhinovirus is responsible for the majority of virus-induced asthma exacerbations. To determine the immunologic mechanisms underlying rhinovirus-induced asthma exacerbations, we combined mouse models of allergic airways disease and human rhinovirus infection. We inoculated ovalbumin-sensitized and challenged BALB/c mice with rhinovirus serotype 1B, a minor group strain capable of infecting mouse cells. Compared to sham-infected, ovalbumin-treated mice, virus-infected mice showed increased lung infiltration with neutrophils, eosinophils and macrophages, airway cholinergic hyperresponsiveness, and increased lung expression of cytokines including eotaxin-1/CCL11, IL-4, IL-13 and IFN-γ. Administration of anti-eotaxin-1 attenuated rhinovirus-induced airway eosinophilia and responsiveness. Immunohistochemistry showed eotaxin-1 in the lung macrophages of virus-infected, ovalbumin-treated mice, and confocal fluorescence microscopy revealed co-localization of rhinovirus, eotaxin-1 and IL-4 in CD68-positive cells. RV inoculation of lung macrophages from ovalbumin-treated, but not PBS-treated, mice induced expression of eotaxin-1, IL-4, and IL-13 ex vivo. Macrophages from ovalbumin-treated mice showed increased expression of arginase-1, Ym-1, Mgl-2 and IL-10, indicating a shift in macrophage activation status. Depletion of macrophages from ovalbumin-sensitized and -challenged mice reduced eosinophilic inflammation and airway hyperreactivity following RV infection. We conclude that augmented airway eosinophilic inflammation and hyperresponsiveness in RV-infected mice with allergic airways disease is directed in part by eotaxin-1. Airway macrophages from mice with allergic airways disease demonstrate a change in activation state characterized in part by altered eotaxin and IL-4 production in response to RV infection. These data provide a new paradigm to explain RV-induced asthma exacerbations.
doi:10.4049/jimmunol.1000286
PMCID: PMC3208235  PMID: 20644177
22.  The Critical Role of Notch Ligand Delta-like 1 in the Pathogenesis of Influenza A Virus (H1N1) Infection 
PLoS Pathogens  2011;7(11):e1002341.
Influenza A viral infections have been identified as the etiologic agents for historic pandemics, and contribute to the annual mortality associated with acute viral pneumonia. While both innate and acquired immunity are important in combating influenza virus infection, the mechanism connecting these arms of the immune system remains unknown. Recent data have indicated that the Notch system is an important bridge between antigen-presenting cells (APCs) and T cell communication circuits and plays a central role in driving the immune system to overcome disease. In the present study, we examine the role of Notch signaling during influenza H1N1 virus infection, focusing on APCs. We demonstrate here that macrophages, but not dendritic cells (DCs), increased Notch ligand Delta-like 1 (Dll1) expression following influenza virus challenge. Dll1 expression on macrophages was dependent on retinoic acid-inducible gene-I (RIG-I) induced type-I IFN pathway, and not on the TLR3-TRIF pathway. We also found that IFNα-Receptor knockout mice failed to induce Dll1 expression on lung macrophages and had enhanced mortality during influenza virus infection. Our results further showed that specific neutralization of Dll1 during influenza virus challenge induced higher mortality, impaired viral clearance, and decreased levels of IFN-γ. In addition, we blocked Notch signaling by using γ-secretase inhibitor (GSI), a Notch signaling inhibitor. Intranasal administration of GSI during influenza infection also led to higher mortality, and higher virus load with excessive inflammation and an impaired production of IFN-γ in lungs. Moreover, Dll1 expression on macrophages specifically regulates IFN-γ levels from CD4+and CD8+T cells, which are important for anti-viral immunity. Together, the results of this study show that Dll1 positively influences the development of anti-viral immunity, and may provide mechanistic approaches for modifying and controlling the immune response against influenza H1N1 virus infection.
Author Summary
Influenza viruses cause annual epidemics and occasional pandemics that have claimed the lives of millions. Both innate and acquired immunity are essential for protection against influenza virus, and Notch and Notch ligands provide a key bridge between innate and acquired immunity. However, the role of Notch system during influenza virus infection is unknown. Here, we show that Notch ligand Delta-like 1 (Dll1) expression was up-regulated in influenza virus H1N1 challenged macrophages, and was dependent on both retinoic-acid–inducible protein I (RIG-I) and IFNα receptor (IFNαR)-mediated pathways. IFNαR-deficient mice challenged with influenza virus in vivo also display a profoundly impaired Dll1 expression with increased mortality and abrogated IFN-γ production. Treatment of WT mice during influenza infection, with either neutralizing antibodies specific for Dll1 or a γ-secretase inhibitor (GSI), which blocks Notch signaling, resulted in increased mortality, impaired viral clearance, and lower IFN-γ production. In addition, Dll1 specifically regulated IFN-γ production from both CD4+and CD8+T cells in vitro. Together, these results suggest that Notch signaling through macrophage-dependent Dll1 is critical in providing an anti-viral response during influenza infection by linking innate and acquired immunity.
doi:10.1371/journal.ppat.1002341
PMCID: PMC3207886  PMID: 22072963
23.  Predictors of job satisfaction among Academic Faculty: Do instructional and clinical faculty differ? 
Medical education  2010;44(10):985-995.
Objectives
To identify and compare predictors of job satisfaction between the instructional and clinical faculty tracks.
Method
A 61-item faculty job satisfaction survey was distributed to 1,898 academic faculty at the University of Michigan Medical School. The anonymous survey was web-based. Questions covered topics on departmental organization, research, clinical and teaching support, compensation, mentorship, and promotion. Levels of satisfaction were contrasted between the two tracks, and predictors of job satisfaction were identified using linear regression models.
Results
The response rates for the instructional and clinical tracks were 43.1% and 41.3%, respectively. Clinical faculty reported being less satisfied with how they are mentored, and fewer reported understanding the process for promotion. There was no significant difference in overall job satisfaction between faculty tracks. Surprisingly, clinical faculty with mentors were significantly less satisfied with how they were being mentored, with career advancement and overall job satisfaction, compared to instructional faculty mentees. Additionally, senior-level clinical faculty were significantly less satisfied with their opportunities to mentor junior faculty compared to senior-level instructional faculty. Significant predictors of job satisfaction for both tracks included areas of autonomy, meeting career expectations, work-life balance, and departmental leadership. Unique to the clinical track, compensation and career advancement variables also emerged as significant predictors.
Conclusion
Greater effort must be placed in the continued attention to faculty well-being both at the institutional level and at the level of departmental leadership. Success in enhancing job satisfaction is more likely if directed by locally designed assessments involving department chairs, specifically in fostering more effective mentoring relationships focused on making available career advancement activities such as research activities. Our findings show this strategy to significantly impact the job satisfaction and retention of clinical track faculty members.
doi:10.1111/j.1365-2923.2010.03766.x
PMCID: PMC2950106  PMID: 20880368
24.  Amelioration of the cardiovascular effects of cocaine in rhesus monkeys by a long-acting mutant form of cocaine esterase 
A long-acting mutant form of a naturally occurring bacterial cocaine esterase (T172R/G173Q CocE; DM CocE) has previously been shown to antagonize the reinforcing, convulsant, and lethal effects of cocaine in rodents. However, the effectiveness and therapeutic characteristics of DM CocE in nonhuman primates, in a more clinically relevant context, are unknown. The current studies were aimed at 1) characterizing the cardiovascular effects of cocaine in freely moving rhesus monkeys, 2) evaluating the capacity of DM CocE to ameliorate these cocaine-induced cardiovascular effects when administered 10 minutes after cocaine, and 3) assessing the monkeys’ immunologic responses to DM CocE following repeated administration. Intravenous administration of cocaine produced dose-dependent increases in mean arterial pressure (MAP) and heart rate (HR) that persisted throughout the two-hour observation period following a dose of 3.2 mg/kg cocaine. Cocaine failed to produce reliable changes in ECG parameters, body temperature, and locomotor activity. DM CocE produced a rapid and dose-dependent amelioration of the cardiovascular effects, with saline-like MAP measures restored within 5 to 10 minutes, and saline-like HR measures restored within 20 to 40 minutes of DM CocE administration. Although administration of DM CocE produced increases in anti-CocE antibodies, they did not appear to have a neutralizing effect on the capacity of DM CocE to reverse the cardiovascular effects of cocaine. In conclusion, these findings in monkeys provide strong evidence to suggest that highly efficient cocaine esterases, such as DM CocE, can provide a potential therapeutic option for treatment of acute cocaine intoxication in humans.
doi:10.1038/npp.2010.242
PMCID: PMC3076304  PMID: 21289605
Rhesus Monkey; Cardiovascular; Cocaine; Acute Cocaine Toxicity; Cocaine Esterase; DM CocE
25.  Respiratory Virus-induced TLR7 activation controls IL-17 associated Increase in mucus via IL-23 regulation 
The response to respiratory syncytial virus (RSV), negative strand ssRNA virus, depends upon the ability to recognize specific pathogen associated targets. In the present 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 hyper-secretion. While 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 DC from TLR7−/− animals indicated a preferential activation of IL-23 (a Th17 associated 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 mucus overproduction.
doi:10.4049/jimmunol.1000733
PMCID: PMC3006454  PMID: 20624950

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