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.
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.
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.
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.
Inflammation; infection; mucus; cytokines; lymphocytes
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.
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.
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.
selectin inhibitor; asthma; eosinophil; lung
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.
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.
asthma; BALB/c; childhood; gob-5; IL-13
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.
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.
T helper lymphocytes; immune regulation; hygiene hypothesis; soluble egg antigen; sialyl Lewisx glycans
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.
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.
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
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.
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.
Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia in young children worldwide, and no vaccine is currently available. Inactivated RSV vaccines tested in the 1960's led to vaccine-enhanced disease upon viral challenge, which has undermined RSV vaccine development. RSV infection is increasingly being recognized as an important pathogen in the elderly, as well as other individuals with compromised pulmonary immunity. A safe and effective inactivated RSV vaccine would be of tremendous therapeutic benefit to many of these populations.
In these preclinical studies, a mouse model was utilized to assess the efficacy of a novel, nanoemulsion-adjuvanted, inactivated mucosal RSV vaccine. Our results demonstrate that NE-RSV immunization induced durable, RSV-specific humoral responses, both systemically and in the lungs. Vaccinated mice exhibited increased protection against subsequent live viral challenge, which was associated with an enhanced Th1/Th17 response. In these studies, NE-RSV vaccinated mice displayed no evidence of Th2 mediated immunopotentiation, as has been previously described for other inactivated RSV vaccines.
These studies indicate that nanoemulsion-based inactivated RSV vaccination can augment viral-specific immunity, decrease mucus production and increase viral clearance, without evidence of Th2 immune mediated pathology.
Cellular recruitment during inflammatory/immune responses is tightly regulated. The ability to dampen inflammation is imperative for prevention of chronic immune responses, as in asthma. Here we investigated the ability of lipoxin A4 (LXA4) stable analogs to regulate airway responses in two allergen-driven models of inflammation. A 15-epi-LXA4 analog (ATLa) and a 3-oxa-15-epi-LXA4 analog (ZK-994) prevented excessive eosinophil and T lymphocyte accumulation and activation after mice were sensitized and aerosol-challenged with ovalbumin. At <0.5 mg/kg, these LXA4 analogs reduced leukocyte trafficking into the lung by >50% and to a greater extent than equivalent doses of the CysLT1 receptor antagonist montelukast. Distinct from montelukast, ATLa treatment led to marked reductions in cysteinyl leukotrienes, interleukin-4 (IL-4), and IL-10, and both ATLa and ZK-994 inhibited levels of IL-13. In cockroach allergen-induced airway responses, both intraperitoneal and oral administration of ZK-994 significantly reduced parameters of airway inflammation and hyper-responsiveness in a dose-dependent manner. ZK-994 also significantly changed the balance of Th1/Th2-specific cytokine levels. Thus, the ATLa/LXA4 analog actions are distinct from CysLT1 antagonism and potently block both allergic airway inflammation and hyper-reactivity. Moreover, these results demonstrate these analogs’ therapeutic potential as new agonists for the resolution of inflammation.
resolution; lipid mediators; leukocytes
Rationale: Accumulating evidence supports the hypothesis that the continuous host response to a persistent challenge can polarize the cytokine environment toward a Th2 cytokine phenotype, but the mechanisms responsible for this skewing are not clear.
Objectives: We investigated the role of Toll-like receptor 9 (TLR9) in a Th2-driven pulmonary granulomatous response initiated via the embolization of Schistosoma mansoni eggs to the lungs of mice.
Methods: Mice were intravenously injected with S. mansoni eggs. Histological and flow cytometric analysis, cytokine measurement, adoptive transfer of bone marrow (BM)-derived dendritic cells (DCs), and in vitro T-cell treatments with antigen-presenting cells were examined.
Measurements and Main Results: In comparison to wild-type mice, TLR9−/− mice showed increased pulmonary granuloma size, augmented collagen deposition, increased Th2 cytokine phenotype, and impaired accumulation of DCs. BM-derived DCs, but not macrophages, recovered from animals with developed Th2-type lung granulomas promoted the production of type 2 cytokines from CD4+ T cells. BM-derived DCs from TLR9−/− mice induced impaired Th1 cytokine and enhanced Th2 cytokine production by T cells, compared with DCs from WT mice. Macrophages from TLR9−/− mice expressed a significantly higher alternatively activated (M2) phenotype characterized by increased “found in inflammatory zone-1” (FIZZ1) and arginase-1 expression. The adoptive transfer of BM-derived DCs from syngeneic WT mice into TLR9−/− mice restored the granuloma phenotype seen in WT mice.
Conclusions: These studies suggest that TLR9 plays an important mechanistic role in the maintenance of the pulmonary granulomatous response.
granuloma; pulmonary fibrosis; innate immunity; dendritic cell; macrophage
Influenza virus is a common cause of respiratory infection and morbidity, which is often due to deleterious host immune responses directed against the pathogen. We investigated the role of IL-1 receptor-associated kinase-M (IRAK-M), an inhibitor of MyD88-dependent TLR signaling, in modulating the innate inflammatory response during influenza pneumonia using a murine model. The intranasal administration of influenza resulted in the upregulation of IRAK-M mRNA and protein levels in the lungs within 2 d after infectious challenge. Pulmonary influenza infection in mice deficient in IRAK-M (IRAK-M−/−) resulted in substantially increased mortality compared with similarly treated wild-type animals. Increased mortality in IRAK-M−/− mice was associated with enhanced early influx of neutrophils, high permeability edema, apoptosis of lung epithelial cells, markedly increased expression of inflammatory cytokines/chemokines, and release of neutrophil-derived enzymes, including myeloperoxidase and neutrophil elastase. Early viral clearance was not different in mutant mice, whereas viral titers in lungs and blood were significantly higher in IRAK-M−/− mice compared with wild-type animals. Increased lethality observed in IRAK-M−/− mice after influenza challenge was abrogated by Ab-mediated blockade of CXCR2. Collectively, our findings indicate that IRAK-M is critical to preventing deleterious neutrophil-dependent lung injury during influenza infection of the respiratory tract.
Human rhinovirus (RV) infection is responsible for the majority of virus-induced asthma exacerbations. Using a mouse model of human RV infection, we sought to determine the requirement of CXCR2, the receptor for ELR-positive CXC chemokines, for RV-induced airway neutrophilia and hyperresponsiveness. Wild-type and CXCR2 −/− mice were inoculated intranasally with RV1B or sham HeLa cell supernatant. Following RV1B infection, CXCR2 −/− mice showed reduced airway and lung neutrophils and cholinergic responsiveness compared to wild-type mice. Similar results were obtained in mice treated with neutralizing antibody to Ly6G, a neutrophil-depleting antibody. Lungs from RV-infected, CXCR2 −/− mice showed significantly reduced production of tumor necrosis factor (TNF)-α, MIP-2/CXCL2 and KC/CXCL1, and lower expression of MUC5B, compared to RV-treated wild-type mice. The requirement of TNF-α for RV1B-induced airways responses was tested using TNF receptor (TNFR)-1 −/− mice. TNFR1 −/− animals displayed reduced airways responsiveness to RV1B, even when exogenous MIP-2 was added to the airways. We conclude that CXCR2 is required for RV-induced neutrophilic airway inflammation, and that neutrophil TNF-α release is required for airways hyperresponsiveness.
Chemokines are important mediators of the immune response to pathogens, but can also promote chronic inflammatory states. CCR6 is a chemokine receptor found on dendritic cells and T cells, suggesting its role in both innate and adaptive immunity. We investigated the role of CCR6 in a pulmonary viral infection caused by respiratory syncytial virus (RSV), a ubiquitous virus that can cause severe pulmonary complications. We found that CCR6−/− mice had reduced pathophysiology and lower numbers of activated T cells in the lungs post-RSV challenge. Analysis of RSV-specific cytokine responses showed that CCR6−/− mice skewed preferentially toward a Th1 effector response. Correspondingly, viral clearance was rapid and early in CCR6−/− mice, suggesting an efficient immune response. Early timepoint analysis revealed that CCR6−/− mice had significantly fewer conventional DCs (cDCs) recruited into the lung, but the same number of plasmacytoid DCs. A pathogenic phenotype could be reconstituted in CCR6−/− mice by supplying cDCs into the airway, indicating that mere number of cDCs dictates the adverse response. Our data suggest that CCR6 deficiency provides an environment whereby the balance of innate immune cells mediates the efficient antiviral response to RSV.
dendritic cells; viral; chemokines; inflammation; lung
Chemokines are a superfamily of chemotactic cytokines that play an important role in leukocyte trafficking and have been implicated as functional mediators of immunopathology in experimental autoimmune encephalomyelitis (EAE). In the present study, we investigated the role of the CCL20 receptor, CCR6, in chronic EAE. After immunization with myelin oligodendrocyte glycoprotein 35–55 in CFA, CCR6-/- mice developed a significantly more severe chronic EAE as compared to wild type immunized animals. CCR6 expression was not required by T cells to induce EAE. Measurement of peripheral T cell responses showed differences in IFN-γ and IL-17 responses between CCR6-/- and wild type mice. At the time when CCR6-/- mice showed significantly more severe chronic EAE there was a significant decrease in PD-L1-expressing mDC in the spleens and no differences in Foxp3 Treg. Furthermore, add back of mDC with increased PD-L1 expression to CCR6-/- mice reduced the severe chronic EAE disease phase to that of wild type controls. The results suggest a role for CCR6-expressing PD-L1+ mDC in regulating EAE progression.
multiple sclerosis; chemokine; CCL20; CCR6; EAE; central nervous system
Studies have shown that Notch is essential for the maintenance of a T cell Th2 phenotype in vivo. It has also been shown that Notch ligands have diverse functions during T cell activation. We chose to investigate the role of Notch ligands during the Th2 response.
We studied the relationship of two Notch ligands, delta-like 4 and jagged-1, to T cell proliferation in C57 Bl/6 mice. Our findings indicate that jagged-1 does not affect the rate of T cell proliferation in any subset examined. However, delta-like 4 causes an increase in the expansion of Th2 memory cells and a decrease in effector cell proliferation. Our in vivo studies indicate that the Notch system is dynamically regulated, and that blocking one Notch ligand increases the effective concentration of other Notch ligands, thus altering the response. Examination of genes related to the Notch pathway revealed that the Notch receptors were increased in memory T cells. Expression of BMI1, a gene involved in T cell proliferation, was also higher in memory T cells. Further experiments demonstrated that Notch directly regulates the expression of the BMI1 gene in T cells and may govern T cell proliferation through this pathway.
From these experiments we can make several novel conclusions about the role of Notch ligands in T cell biology. The first is that delta-like 4 suppresses effector cell proliferation and enhances Th2 memory cell proliferation. The second is that blocking one Notch ligand in vivo effectively increases the concentration of other Notch ligands, which can then alter the response.