Airborne lipid antigens mobilize resident intravascular NKT cells to induce allergic airway inflammation

Airborne exposure to microbial cell wall lipids such as lipopolysaccharide triggers innate immune responses that regulate susceptibility to allergic airway inflammation. α-Glycosylceramides represent another widespread class of microbial lipids that directly stimulate innate-like, IL-4- and IL-13-producing, CD1d-restricted NKT cells. In this study, we demonstrate that NKT cells constitutively accumulate and reside in the microvasculature of the mouse lung. After a single airborne exposure to lipid antigen, they promptly extravasate to orchestrate the formation of peribronchiolar and interstitial lymphohistiocytic granulomas containing numerous eosinophils. Concomitant airborne exposure to ovalbumin (OVA) induces the priming of OVA-specific Th2 cells and IgE antibodies by the same dendritic cell coexpressing CD1d and MHC class II. Although NKT cell activation remains confined to the lipid-exposed lung and draining lymph nodes, Th2 cells recirculate and seed the lung of a parabiotic partner, conferring susceptibility to OVA challenge months after the initial exposure, in a manner independent of NKT cells and CD1d. Thus, transient recruitment and activation of lung-resident intravascular NKT cells can trigger long-term susceptibility to allergic airway inflammation.     

Activation or anergy: NKT cells are stunned by alpha-galactosylceramide

Invariant natural killer T (iNKT) cells are T lymphocytes that behave similarly to cells of the innate immune system. The glycolipid alpha-galactosylceramide (alpha-GalCer) is a potent and specific activator of mouse and human iNKT cells and has been used in cancer clinical trials to drive NKT cell-mediated immune responses. However, little is known about the dynamics of the iNKT cell response to alpha-GalCer in vivo. In this issue of the JCI, Parekh and colleagues demonstrate that administration of alpha-GalCer causes iNKT cells to become unresponsive, for at least 1 month, in mice. This leads us to ask, should sequential administration of alpha-GalCer still be used to activate iNKT cells given the anergic state it has been shown here to induce? This intriguing article raises the issue of the avoidance of anergy induction in the design of treatment regimens that use alpha-GalCer as a specific activator of iNKT cells.     

Invariant NKT cells reduce the immunosuppressive activity of influenza A virus–induced myeloid-derived suppressor cells in mice and humans

Infection with influenza A virus (IAV) presents a substantial threat to public health worldwide, with young, elderly, and immunodeficient individuals being particularly susceptible. Inflammatory responses play an important role in the fatal outcome of IAV infection, but the mechanism remains unclear. We demonstrate here that the absence of invariant NKT (iNKT) cells in mice during IAV infection resulted in the expansion of myeloid-derived suppressor cells (MDSCs), which suppressed IAV-specific immune responses through the expression of both arginase and NOS, resulting in high IAV titer and increased mortality. Adoptive transfer of iNKT cells abolished the suppressive activity of MDSCs, restored IAV-specific immune responses, reduced IAV titer, and increased survival rate. The crosstalk between iNKT and MDSCs was CD1d- and CD40-dependent. Furthermore, IAV infection and exposure to TLR agonists relieved the suppressive activity of MDSCs. Finally, we extended these results to humans by demonstrating the presence of myeloid cells with suppressive activity in the PBLs of individuals infected with IAV and showed that their suppressive activity is substantially reduced by iNKT cell activation. These findings identify what we believe to be a novel immunomodulatory role of iNKT cells, which we suggest could be harnessed to abolish the immunosuppressive activity of MDSCs during IAV infection.     

T cells, but not B cells, are required for bowel inflammation in interleukin 2-deficient mice

Interleukin-2 (IL-2)-deficient (IL-2-/-) mice develop anemia and colonic inflammatory bowel disease. To elucidate the mechanism of this disease, we have bred IL-2-/- mice to two strains of immunodeficient mice, RAG-2-deficient (RAG-2-/-, lacking B and T cells) and JH- deficient mice (JH-/-, lacking B cells). IL-2-/-, RAG-2-/- double- mutant mice are disease free, while IL-2-/-, JH-/- double-mutant mice succumb to bowel disease at the same rate as IL-2-/- littermates. IL-2- /-, JH-/- mice do not, however, succumb to anemia. Thus, spontaneous intestinal inflammation in IL-2-/- mice requires mature T cells, not B cells, while anemia is dependent on B cells.     

NKT cells promote antibody-induced joint inflammation by suppressing transforming growth factor beta1 production

Although NKT cells has been known to exert protective roles in the development of autoimmune diseases, the functional roles of NKT cells in the downstream events of antibody-induced joint inflammation remain unknown. Thus, we explored the functional roles of NKT cells in antibody-induced arthritis using the K/BxN serum transfer model. NKT cell-deficient mice were resistant to the development of arthritis, and wild-type mice administrated with alpha-galactosyl ceramide, a potent NKT cell activator, aggravated arthritis. In CD1d-/- mice, transforming growth factor (TGF)-beta1 was found to be elevated in joint tissues, and the blockade of TGF-beta1 using neutralizing monoclonal antibodies restored arthritis. The administration of recombinant TGF-beta1 into C57BL/6 mice reduced joint inflammation. Moreover, the adoptive transfer of NKT cells into CD1d-/- mice restored arthritis and reduced TGF-beta1 production. In vitro assay demonstrated that interleukin (IL)-4 and interferon (IFN)-gamma were involved in suppressing TGF-beta1 production in joint cells. The adoptive transfer of NKT cells from IL-4-/- or IFN-gamma-/- mice did not reverse arthritis and TGF-beta1 production in CD1d-/- mice. In conclusion, NKT cells producing IL-4 and IFN-gamma play a role in immune complex-induced joint inflammation by regulating TGF-beta1.     

Suppression of allergic airway inflammation by helminth-induced regulatory T cells

Allergic diseases mediated by T helper type (Th) 2 cell immune responses are rising dramatically in most developed countries. Exaggerated Th2 cell reactivity could result, for example, from diminished exposure to Th1 cell-inducing microbial infections. Epidemiological studies, however, indicate that Th2 cell-stimulating helminth parasites may also counteract allergies, possibly by generating regulatory T cells which suppress both Th1 and Th2 arms of immunity. We therefore tested the ability of the Th2 cell-inducing gastrointestinal nematode Heligmosomoides polygyrus to influence experimentally induced airway allergy to ovalbumin and the house dust mite allergen Der p 1. Inflammatory cell infiltrates in the lung were suppressed in infected mice compared with uninfected controls. Suppression was reversed in mice treated with antibodies to CD25. Most notably, suppression was transferable with mesenteric lymph node cells (MLNC) from infected animals to uninfected sensitized mice, demonstrating that the effector phase was targeted. MLNC from infected animals contained elevated numbers of CD4(+)CD25(+)Foxp3(+) T cells, higher TGF-beta expression, and produced strong interleukin (IL)-10 responses to parasite antigen. However, MLNC from IL-10-deficient animals transferred suppression to sensitized hosts, indicating that IL-10 is not the primary modulator of the allergic response. Suppression was associated with CD4(+) T cells from MLNC, with the CD4(+)CD25(+) marker defining the most active population. These data support the contention that helminth infections elicit a regulatory T cell population able to down-regulate allergen induced lung pathology in vivo.     

FcgammaRIII engagement provides activating signals to NKT cells in antibody-induced joint inflammation

NKT cells promote antibody-induced arthritis, but the mechanism by which NKT cells are activated in this model remains unclear. It has been proposed that Fcgamma receptor (FcgammaR) contributes to NKT cell activation in antibody-induced arthritis. To address this issue, we explored the functions of FcgammaR on NKT cells in antibody-induced arthritis. RT-PCR and flow cytometric analysis demonstrated that NKT cells constitutively express surface FcgammaRIII but not FcgammaRI, -II, or -IV. FcgammaRIII engagement by aggregated IgG on NKT cells enhanced CD25 and CD69 expression, whereas FcgammaR(-/-) mouse NKT cells did not enhance activation. FcgammaRIII engagement on NKT cells enhanced the production of IL-4, IL-10, IL-13, and IFN-gamma, whereas FcgammaR-deficient NKT cells did not alter the production of these cytokines after aggregated IgG treatment. However, FcgammaR-deficient NKT cells were functionally intact in terms of TCR-induced activation. Moreover, adoptive transfer of FcgammaR-deficient NKT cells could not restore inflammation or TGF-beta production in the joint tissues of CD1d(-/-) mice, whereas adoptive transfer of wild-type NKT cells induced arthritis and reduced TGF-beta production in joint tissues. We conclude that FcgammaRIII engagement by IgG in joint tissues provides activating signals to NKT cells in antibody-induced arthritis.     

Mast cells are required for experimental oral allergen-induced diarrhea

Gastrointestinal allergic disorders represent a diverse spectrum of inflammatory diseases that are occurring with increasing incidence and severity. An essential question concerning these disorders is to determine the specific cells and mediators responsible for specific clinical manifestations. With this in mind, we developed a murine model of oral allergen-induced intestinal inflammation accompanied by strong Th2-associated humoral and cellular responses and focused on the immunopathogenesis of allergic diarrhea. Exposure of OVA/alum-sensitized mice to repeated doses of intragastric OVA induced genetically restricted, dose-dependent, acute diarrhea associated with increased intestinal permeability, eosinophilia, and mastocytosis. Mice developed limited systemic manifestations of anaphylaxis, even though they developed marked intestinal mucosal mast cell degranulation. Notably, experiments involving mast cell depletion (with anti-c-kit mAb), anti-IgE treatment, and Fc epsilon RI-deficient mice indicated a critical effector role for mast cells in mediating allergic diarrhea. Furthermore, allergic diarrhea was dependent upon synergistic signaling induced by serotonin and platelet-activating factor (PAF), but not histamine. These results demonstrate that oral allergen-induced diarrhea associated with experimental Th2 intestinal inflammation is largely mast cell, IgE, serotonin, and PAF dependent.     

Interleukin 12 p40 production by barrier epithelial cells during airway inflammation

Human airway epithelial cells appear specially programmed for expression of immune response genes implicated in immunity and inflammation. To better determine how this epithelial system operates in vivo, we analyzed its behavior in mouse models that allow for in vitro versus in vivo comparison and genetic modification. Initial comparisons indicated that tumor necrosis factor alpha induction of epithelial intercellular adhesion molecule 1 required sequential induction of interleukin (IL)-12 (p70) and interferon gamma, and unexpectedly localized IL-12 production to airway epithelial cells. Epithelial IL-12 was also inducible during paramyxoviral bronchitis, but in this case, initial IL-12 p70 expression was followed by 75-fold greater expression of IL-12 p40 (as monomer and homodimer). Induction of IL-12 p40 was even further increased in IL-12 p35-deficient mice, and in this case, was associated with increased mortality and epithelial macrophage accumulation. The results placed epithelial cell overgeneration of IL-12 p40 as a key intermediate for virus-inducible inflammation and a candidate for epithelial immune response genes that are abnormally programmed in inflammatory disease. This possibility was further supported when we observed IL-12 p40 overexpression selectively in airway epithelial cells in subjects with asthma and concomitant increases in airway levels of IL-12 p40 (as homodimer) and airway macrophages. Taken together, these results suggest a novel role for epithelial-derived IL-12 p40 in modifying the level of airway inflammation during mucosal defense and disease.     

Rac1 and superoxide are required for the expression of cell adhesion molecules induced by tumor necrosis factor-alpha in endothelial cells

Oxidative signals play an important role in the regulation of endothelial cell adhesion molecule expression. Small GTP-binding protein Rac1 is activated by various proinflammatory substances and regulates superoxide generation in endothelial cells. In the present study, we demonstrate that adenoviral-mediated expression of dominant negative N17Rac1 (Ad.N17Rac1) suppresses tumor necrosis factor-alpha (TNF-alpha)-induced vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin gene expression in a dose-dependent manner. Ad.N17Rac1 did not inhibit TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) binding activity or inhibitor of NF-kappaB-alpha degradation. In contrast, Ad.N17Rac1 inhibited TNF-alpha-induced NF-kappaB-driven HIV(kappaB)(4)-CAT and p288VCAM-Luc promoter activity, suggesting that N17Rac1 inhibits TNF-alpha-induced VCAM-1, E-selectin, and ICAM-1 through suppressing NF-kappaB-mediated transactivation. In addition, expression of superoxide dismutase by adenovirus suppressed TNF-alpha-induced VCAM-1, E-selectin, and ICAM-1 mRNA accumulation. However, adenoviral-mediated expression of catalase only partially inhibited TNF-alpha-induced E-selectin gene expression and had no effect on VCAM-1 and ICAM-1 gene expression. These data suggest that Rac1 and superoxide play crucial roles in the regulation of expression of cell adhesion molecules in endothelial cells.     

Helper T cells are required for the polyclonal stimulation of cytotoxic T cells by concanavalin A

Concanavalin A stimulation of T-cell cytotoxicity has been shown to be absolutely dependent on helper T-cell collaboration. Thymocytes stimulated with ConA do not differentiate to yield cytotoxic effector cells. However, thymocytes cocultured with irradiated spleen cells as helpers and ConA yield high levels of cytotoxicity. The helper cell bears theta antigens on its surface, is not an adherent cell, and does not require any adherent cell functions in our culture conditions. The ConA-dependent helper cells appear to be polyclonal in specificity. Thus, polyclonal stimulation of cytotoxicity by ConA requires T helper-T precursor collaboration in analogy to antigen-specific T helper-T precursor interactions. Unlike the antigen-specific interacitons, the ConA-driven cytotoxicity does not appear to require linked associative recognition for induction of cytotoxicity.     

MMPs are required for recruitment of antigen-nonspecific mononuclear cells into the liver by CTLs

We recently showed that antigen-nonspecific inflammatory cells are recruited into the liver when hepatitis B virus (HBV)-specific CTLs are injected into HBV transgenic mice, and that this process amplifies the severity of liver disease. We also showed that the severity of CTL-induced liver disease is ameliorated by the depletion of Gr-1(+) cells (Gr-1 is an antigen highly expressed by neutrophils), which, secondarily, abolishes the intrahepatic recruitment of all antigen-nonspecific Gr-1(-) mononuclear cells (NK and NKT cells, T and B lymphocytes, monocytes, macrophages, dendritic cells) despite the strong induction of chemokine gene expression. Those results suggested that in addition to chemokine expression, CTL-induced functions are necessary for mononuclear cell recruitment to occur. We now report that MMPs known to be produced by Gr-1(+) cells are rapidly induced in the livers of CTL-injected mice. The inhibition of MMP activity reduced the intrahepatic recruitment of antigen-nonspecific mononuclear cells and much of the attending liver disease without affecting the migration or antiviral potential of antigen-specific CTLs. The notion that the inhibition of MMP activity is associated with maintenance of antiviral effects but diminished tissue damage may be significant for the development of immunotherapeutic approaches for the treatment of chronic HBV infection.     

Lymphotoxin is required for maintaining physiological levels of serum IgE that minimizes Th1-mediated airway inflammation

Although elevated levels of IgE in asthmatic patients are strongly associated with lung infiltration by activated T helper (Th) 2 cells, the physiological role of immunoglobulin E (IgE) in the airway remains largely undefined. Lymphotoxin-deficient alpha (LTalpha-/-) mice exhibit increased airway inflammation, paradoxically accompanied by diminished levels of IgE and reduced airway hyperresponsiveness in response to both environmental and induced antigen challenge. The severe lung inflammation in LTalpha-/- mice is Th1 in nature and can be alleviated by IgE reconstitution. Conversely, depletion of IgE in wild-type mice recapitulates the lung pathologies of LTalpha-/- mice. Therefore, this work has revealed that lymphotoxin is essential for IgE production, and a physiological role of IgE in the airway may consist of maintaining the balance of Th1 and Th2 responses to prevent aberrant inflammation.     

Pathological versus protective functions of IL-22 in airway inflammation are regulated by IL-17A

IL-22 has both proinflammatory and tissue-protective properties depending on the context in which it is expressed. However, the factors that influence the functional outcomes of IL-22 expression remain poorly defined. We demonstrate that after administration of a high dose of bleomycin that induces acute tissue damage and airway inflammation and is lethal to wild-type (WT) mice, Th17 cell–derived IL-22 and IL-17A are expressed in the lung. Bleomycin-induced disease was ameliorated in Il22^−/− mice or after anti–IL-22 monoclonal antibody (mAb) treatment of WT mice, indicating a proinflammatory/pathological role for IL-22 in airway inflammation. However, despite increased bleomycin-induced IL-22 production, Il17a^−/− mice were protected from airway inflammation, suggesting that IL-17A may regulate the expression and/or proinflammatory properties of IL-22. Consistent with this, IL-17A inhibited IL-22 production by Th17 cells, and exogenous administration of IL-22 could only promote airway inflammation in vivo by acting in synergy with IL-17A. Anti–IL-22 mAb was delivered to Il17a^−/− mice and was found to exacerbate bleomycin-induced airway inflammation, indicating that IL-22 is tissue protective in the absence of IL-17A. Finally, in an in vitro culture system, IL-22 administration protected airway epithelial cells from bleomycin-induced apoptosis, and this protection was reversed after coadministration of IL-17A. These data identify that IL-17A can regulate the expression, proinflammatory properties, and tissue-protective functions of IL-22, and indicate that the presence or absence of IL-17A governs the proinflammatory versus tissue-protective properties of IL-22 in a model of airway damage and inflammation.IL-22 is a member of the IL-10 cytokine family and plays critical roles in inflammation, immune surveillance, and tissue homeostasis at mucosal sites (Ouyang et al., 2008; Colonna, 2009). IL-22 is produced by CD4⁺ Th17 cells, NK cells, CD11c⁺ myeloid cells, and lymphoid tissue inducer–like cells (Liang et al., 2006; Zheng et al., 2008; Cella et al., 2009; Takatori et al., 2009). The IL-22 receptor is composed of the IL-22R and IL-10R2 subunits, and receptor ligation results in phosphorylation of STAT1, STAT3, and STAT5 and activation of the p38 mitogen-activated protein kinase pathway (Kotenko et al., 2001; Lejeune et al., 2002). The IL-22 receptor is found on cells of nonhematopoietic origin in the skin, kidney, liver, lung, and gut, allowing for IL-22–mediated regulation of local epithelial, endothelial, and stromal cell responses after infection or exposure to inflammatory stimuli (Wolk et al., 2004; Ouyang et al., 2008). Despite significant insights into IL-22–IL-22R interactions, reports on the in vivo functions of this pathway have been conflicting (Zenewicz and Flavell, 2008). For example, after infection with Gram-negative bacteria, IL-22 can enhance maintenance of the epithelial barrier and act in synergy with the Th17 cell–coexpressed cytokine IL-17A to promote host protective immunity against infection (Liang et al., 2006; Aujla et al., 2008; Zheng et al., 2008). In addition to antimicrobial properties, several studies have reported tissue-protective properties of IL-22 in mouse models of inflammatory bowel disease and hepatitis (Pan et al., 2004; Radaeva et al., 2004; Zenewicz et al., 2007, 2008; Sugimoto et al., 2008; Pickert et al., 2009). In contrast, other studies have demonstrated that IL-22 has proinflammatory/pathological properties after Toxoplasma gondii infection and in mouse models of psoriasis and arthritis (Zheng et al., 2007; Ma et al., 2008; Geboes et al., 2009; Muñoz et al., 2009).Although IL-22 is known to induce expression of antimicrobial peptides after Klebsiella pneumoniae infection in the lung (Aujla et al., 2008), the influence of the IL-22 pathway on the development, progression, and resolution of airway inflammation has not yet been examined. Using a model of high-dose bleomycin–induced acute tissue damage and airway inflammation (Snider et al., 1978; Nagai et al., 1992; Huaux et al., 2003; Matute-Bello et al., 2008), we demonstrate that a CD4⁺ Th17 cell response ensues after treatment of WT mice, characterized by the production of IL-22 and IL-17A in the lung. Administration of anti–IL-22 neutralizing mAb in WT mice or use of Il22^−/− mice revealed a reduction in bleomycin-induced disease, indicative of a proinflammatory/pathological role for IL-22 in airway inflammation. As IL-17A and IL-22 are coexpressed and have been shown to act cooperatively (Liang et al., 2006; Aujla et al., 2008), we investigated the influence of IL-17A on IL-22 expression and function in the lung by using Il17a^−/− mice. Il17a^−/− mice exhibited enhanced levels of bleomycin-induced IL-22 expression because of a loss of IL-17A–mediated suppression of IL-22 production in Th17 cells. Despite increased IL-22 expression, Il17a^−/− mice were protected from bleomycin-induced airway inflammation, indicating that IL-22 acts in synergy with IL-17A to promote airway inflammation. Consistent with this, exogenous IL-22 could only promote airway inflammation when coadministered with IL-17A. Treatment of Il17a^−/− mice with anti–IL-22 mAb exacerbated bleomycin-induced inflammation, supporting a tissue-protective role for IL-22 in the absence of IL-17A. Furthermore, IL-22 protected airway epithelial cells against bleomycin-induced apoptosis, and this property was reversed with the coadministration of IL-17A. Collectively, these data are the first to demonstrate a pathological role for IL-22 in a model of airway inflammation and identify that IL-17A can govern the proinflammatory/pathological versus tissue-protective properties of IL-22 in the lung.     

CD4+ T cells are required for development of a murine retrovirus- induced immunodeficiency syndrome (MAIDS)

Mice depleted in vivo of CD4+ Th cells by treatment with mAb GK1.5 were found to be resistant to the lymphoproliferative/immunodeficiency disease (MAIDS) induced in intact mice by infection with the mixture of LP-BM5 murine leukemia viruses. Depleted mice did not develop lymphadenopathy or splenomegaly, had normal serum IgM levels, normal CTL responses to alloantigens, and were able to generate PFC responses to Th-independent antigens even though frequencies of virus-producing spleen cells were comparable in depleted and intact mice. Depletion of CD4+ Th cells after infection resulted in a reversal of many abnormalities exhibited by infected controls; spleen weights, serum IgM levels, and allogeneic CTL responses of treated mice were comparable to those of uninfected controls. These results demonstrate that dysfunction of CD4+ Th cells is central to the induction and progression of both T and B cell abnormalities in MAIDS.     

Role of inflammatory cells in the development of airway inflammation

Chronic obstructive pulmonary disease(COPD) is a chronic airway disorder characterized by obstructive airflow limitation which is not completely reversible with treatment. Inflammatory changes in the peripheral airways, especially those with the diameter less than 2 mm(so-called small airway disease) have been speculated to be initial steps of COPD. However, it remains unclear which types of the cells play an essential role in the pathogenesis of these lesions. Studies with bronchoalveolar lavage demonstrated an increase in neutrophil numbers and the neutrophil chemoattractant interleukin-8. Tobacco smoke-induced IL-8 expression in the airway epithelial cells and macrophages results in neutrophil accumulation and activation. Immunohistochemical analysis recently demonstrated that T lymphocytes, especially CD8(+) cells are increased in the pathologic lesions. However, their role in the pathogenesis of inflammatory changes remains unelucidated. Further studies are necessary for the new development of treatment for this progressive lung disease.