OVA-induced Allergic Airway Inflammation Research Articles

Characteristics of alveolar macrophages from murine models of OVA-induced allergic airway inflammation and LPS-induced acute airway inflammation

ABSTRACTBackground: Macrophages include the classically activated pro-inflammatory M1 macrophages (M1s) and alternatively activated anti-inflammatory M2 macrophages (M2s). The M1s are activated by both interferon-γ and Toll-like receptor ligands, including lipopolysaccharide (LPS), and have potent pro-inflammatory activity. In contrast, Th2 cytokines activate the M2s, which are involved in the immune response to parasites, promotion of tissue remodeling, and immune regulatory functions. Although alveolar macrophages (AMs) play an essential role in the pulmonary immune system, little is known about their phenotypes. Methods: Quantitative reverse transcription polymerase chain reaction and flow cytometry were used to define the characteristics of alveolar macrophages derived from untreated naïve mice and from murine models of both ovalbumin (OVA)-induced allergic airway inflammation and LPS-induced acute airway inflammation. AMs were co-cultured with CD4+ T cells and were pulsed with tritiated thymidine to assess proliferative responses. Results: We characterized in detail murine AMs and found that these cells were not completely consistent with the current M1 versus M2-polarization model. OVA-induced allergic and LPS-induced acute airway inflammation promoted the polarization of AMs towards the current M2-skewed and M1-skewed phenotypes, respectively. Moreover, our data also show that CD11c+ CD11b+ AMs from the LPS-treated mice play a regulatory role in antigen-specific T-cell proliferation in vitro. Conclusions: These characteristics of AMs depend on the incoming pathogens they encounter and on the phase of inflammation and do not correspond to the current M1 versus M2-polarization model. These findings may facilitate an understanding of their contributions to the pulmonary immune system in airway inflammation.

Role of atypical protein kinase on Th17 differentiation and function (IRM15P.606)

Abstract We have reported that PKCλ/ι, a member of atypical protein kinase family, is critical for the control of cell polarity during T cell activation, Th2 cytokine production and allergic airway inflammation. However, the effect of PKCλ/ι on Th17 cells remains unknown. By the use of mice with conditional PKCλ/ι knockout in activated T cells, we investigated the potential roles of this protein kinase on Th17 differentiation, effector cytokine secretion and allergic airway inflammation. We found that loss of PKCλ/ι in activated CD4+ T cells impaired Th17-differentiation in vitro. The secretion of Th17 effector cytokines was down-regulated from PKCλ/ι-deficient T cells either under non-polarized or Th17-polarizd conditions. Furthermore, PKCλ/ι-deficiency inhibited OVA-induced allergic airway inflammation in vivo, where Th17 effector cytokines were also reduced along with Th2 cytokines. Further studies are ongoing for underlying mechanisms whereby PKCλ/ι controls Th17 cells. Our preliminary findings indicate that PKCλ/ι might be a novel regulator for Th17 cells.

TLR2-dependent amelioration of allergic airway inflammation by parasitic nematode type II MIF in mice.

In our previous studies, the recombinant type II macrophage migration inhibitory factor homologue (rAs-MIF) secreted from Anisakis simplex suppressed experimental inflammation mouse model through IL-10 production and CD4(+)CD25(+)Foxp3(+) T-cell recruitment. Also, TLR2 gene expression was significantly increased following rAs-MIF treatment. To know the relation between TLR2 and amelioration mechanisms of rAs-MIF, we induced allergic airway inflammation by ovalbumin and alum with or without rAs-MIF under TLR2 blocking systems [anti-TLR2-specific antibody (α-mTLR2 Ab) treatment and using TLR2 knockout mice]. As a result, the amelioration effects of rAs-MIF in allergic airway inflammation model (diminished inflammation and Th2 response in the lung, increased IL-10 secretion, CD4(+)CD25(+)Foxp3(+) T-cell recruitment) were diminished under two of the TLR2 blocking model. The expression of TLR2 on the surface of lung epithelial cell was significantly elevated by rAs-MIF treatment or Pam3CSK (TLR2-specific agonist) treatment, but they might have some competition effect on the elevation of TLR2 expression. In addition, the elevation of IL-10 gene expression by rAs-MIF treatment was significantly inhibited by α-mTLR2 Ab or Pam3CSK pretreatment. In conclusion, anti-inflammatory effects of the rAs-MIF on OVA-induced allergic airway inflammation might be closely related to TLR2.

Bioavailability and Allergoprotective Capacity of Milk-Associated Conjugated Linoleic Acid in a Murine Model of Allergic Airway Inflammation

Background: Cross-sectional epidemiological studies have demonstrated that farm milk from traditional farm settings possesses allergoprotective properties. Up to now, it has not been clarified which milk ingredient is responsible for protection against allergic diseases. As farm milk is rich in conjugated linoleic acids (CLA), it is hypothesized that this n-3 polyunsaturated fatty acid family contributes to the allergoprotective capacity of farm milk. We aim to prove this hypothesis in a murine model of allergic airway inflammation. Methods: To prove the bioavailability and allergoprotective capacity of milk-associated CLA in a standardized protocol, milk batches that differed significantly in terms of their CLA content were spray dried and incorporated into a basic diet by substituting the regular sunflower fat fraction. Initially, the milk CLA uptake from the diet was monitored via measurement of the CLA content in plasma and erythrocyte membranes obtained from supplemented mice. To determine whether a milk CLA-enriched diet possesses allergoprotective properties, female Balb/c mice were fed the milk CLA-enriched diet ahead of sensitization and a challenge with ovalbumin (OVA) and the parameters of airway inflammation and eisosanoid pattern were measured. Results: In animals, supplementation with a diet rich in milk CLA resulted in elevated CLA levels in plasma and erythrocyte membranes, indicating bioavailability of milk fatty acids. Though membrane-associated phospholipid patterns were affected by supplementation with milk CLA, this application neither reduced the hallmarks of allergic airway inflammation in sensitized and OVA-challenged mice nor modified the eiconsanoid pattern in the bronchoalveolar lavage fluid of these animals. Conclusion: Milk-associated CLA was not capable of preventing murine allergic airway inflammation in an animal model of OVA-induced allergic airway inflammation.

A CCL chemokine-derived peptide (CDIP-2) exerts anti-inflammatory activity via CCR1, CCR2 and CCR3 chemokine receptors: Implications as a potential therapeutic treatment of asthma

Allergic asthma is a chronic inflammatory disease characterized by the accumulation of eosinophils, Th2 cells and mononuclear cells in the airways, leading to changes in lung architecture and subsequently reduced respiratory function. We have previously demonstrated that CDIP-2, a chemokine derived peptide, reduced in vitro chemotaxis and decreased cellular infiltration in a murine model of allergic airway inflammation. However, the mechanisms involved in this process have not been identified yet. Now, we found that CDIP-2 reduces chemokine-mediated functions via interactions with CCR1, CCR2 and CCR3. Moreover, using bone marrow-derived eosinophils, we demonstrated that CDIP-2 modifies the calcium fluxes induced by CCL11 and down-modulated CCR3 expression. Finally, CDIP-2 treatment in a murine model of OVA-induced allergic airway inflammation reduced leukocyte recruitment and decreases production of cytokines. These data suggest that chemokine-derived peptides represent new therapeutic tools to generate more effective antiinflammatory drugs.

TRIF Deficiency does not Affect Severity of Ovalbumin-induced Airway Inflammation in Mice.

Allergic asthma is a chronic pulmonary inflammatory disease characterized by reversible airway obstruction, hyperresponsiveness and eosinophils infiltration. Toll-like receptors (TLRs) signaling are closely associated with asthma and have emerged as a novel therapeutic target in allergic disease. The functions of TLR3 and TLR4 in allergic airway inflammation have been studied; however, the precise role of TIR-domain-containing adapter-inducing interferon-β (TRIF), the adaptor molecule for both TLR3 and TLR4, is not yet fully understood. To investigate this, we developed a mouse model of OVA-induced allergic airway inflammation and compared the severity of allergic airway inflammation in WT and TRIF-/- mice. Histopathological assessment revealed that the severity of inflammation in airway inflammation in TRIF-deficient mice was comparable to that in WT mice. The total number of cells recovered from bronchoalveolar lavage fluid did not differ between WT and TRIF-deficient mice. Moreover, TRIF deficiency did not affect Th1 and Th2 cytokine production in lung tissue nor the level of serum OVA-specific IgE, IgG1 and IgG2c. These findings suggest that TRIF-mediated signaling may not be critical for the development of allergic airway inflammation.

Foxp3 -Treg Cells Enhanced by Repeated Low-Dose Gamma-Irradiation Attenuate Ovalbumin-Induced Allergic Asthma in Mice

Gamma radiation is used for several therapeutic indications such as cancers and autoimmune diseases. Low-dose whole-body γ irradiation has been shown to activate immune responses in several ways, however, the effect and mechanism of irradiation on allergic asthma remains poorly understood. This study investigated whether or not irradiation exacerbates allergic asthma responses and its potential mechanism. C57BL/6 mice were sensitized and challenged with ovalbumin (OVA) to induce asthma. The mice received whole-body irradiation once daily for 3 consecutive days with a dose of 0.667 Gy using (137)Cs γ rays 24 h before every OVA challenge. Repeated low-dose irradiation reduced OVA-specific IgE levels, the number of inflammatory cells including mast cells, goblet cell hyperplasia, collagen deposition, airway hyperresponsiveness, expression of inflammatory cytokines, CCL2/CCR2, as well as nuclear factor kappa B (NF-κB) and activator protein-1 activities. All of these factors were increased in BAL cells and lung tissue of OVA-challenged mice. Irradiation increased the number of Treg cells, expression of interleukin (IL)-10, IL-2 and IL-35 in BAL cells and lung tissue. Irradiation also increased Treg cell-expressed Foxp3 and IL-10 by NF-κB and RUNX1 in OVA-challenged mice. Furthermore, while Treg cell-expressing OX40 and IL-10 were enhanced in lung tissue or act-bone marrow-derived mast cells (BMMCs) with Treg cells, but BMMCs-expressing OX40L and TGF-β were decreased. The data suggest that irradiation enhances Foxp3(+)- and IL-10-producing Treg cells, which reduce OVA-induced allergic airway inflammation and tissue remodeling through the down-regulation of migration by the CCL2/CCR2 axis and activation of mast cells via OX40/OX40L in lung tissue of OVA-challenged mice.

The Inhibitory Role of Hydrogen Sulfide in Airway Hyperresponsiveness and Inflammation in a Mouse Model of Asthma

Cystathionine γ-lyase (CSE) is one of the major enzymes producing hydrogen sulfide (H2S) in lungs, participating in the regulation of respiratory functions. The role of CSE-derived H2S in eosinophil-dominant inflammation in allergic diseases has been unclear. The objective of this study was to explore the protective role of H2S against allergen-induced airway hyperresponsiveness (AHR) and inflammation. CSE expression and H2S production rate were assessed in mouse lung tissues with ovalbumin (OVA)-induced acute asthma. AHR, airway inflammation, and Th2 response in wild-type (WT) mice were compared with those in CSE gene knockout (KO) mice. The effect of NaHS, an exogenous H2S donor, was also evaluated on these parameters. CSE expression was absent and H2S production rate was significantly lower in the lungs of CSE KO mice when compared with WT littermates. OVA challenge decreased lung CSE expression and H2S production in WT mice. CSE deficiency resulted in aggravated AHR, increased airway inflammation, and elevated levels of Th2 cytokines such as IL-5, IL-13, and eotaxin-1 in bronchoalveolar lavage fluid after OVA challenge. The aforementioned alterations were reversed by exogenous H2S treatment. More importantly, NaHS supplement rescued CSE KO mice from the aggravated pathological process of asthma. The CSE/H2S system plays a critical protective role in the development of asthma. A new therapeutic potential for asthma via targeting CSE/H2S metabolism is indicated.

Superoxide dismutase 3 controls adaptive immune responses and contributes to the inhibition of OVA-induced allergic airway inflammation

The superoxide dismutase 3 (SOD3) is an isoform of SOD. Extensive studies have been focused on role of SOD3 as an antioxidant. However, the role of SOD3 in the immune responses that contribute to the inhibition of allergic lung inflammation has not been investigated. Here, we report for the first time that SOD3 specifically inhibits dendritic cell maturation. Subsequently, SOD3 controls T cell activation and proliferation, and Th2 and Th17 cell differentiation. As a consequence, the administration of SOD3 into mice alleviated Th2 cell- mediated OVA-induced allergic asthma.

Effects of bee venom-PLA2 on Regulatory T cells in asthma mice model (173.37)

Abstract CD4+CD25+Foxp3+ regulatory T cell (Treg) plays an important role in immune tolerance which can reduce inflammation and allergic reactions through its inhibitory functions. Bee venom (BV) has been widely used in the treatment of chronic inflammatory diseases such as Rheumatoid Arthritis. In this study, we hypothesized that phospholipase A2 (PLA2), major constituent of BV, could increase Treg expression and treat OVA-induced allergic asthma in mice. In vitro study, the percentage of Treg increased significantly by PLA2 treatment in CD4+T cell compared to saline treatment. In OVA induced allergic asthma model, PLA2-treatment significantly suppressed eosinophil and lymphocyte infiltration in BALF. In addition, PLA2 inhibited the production of IL-4, IL-13, INF-γ, IgE and OVA-specific IgE, along with suppressing airway hyper-responsiveness. Furthermore, CD4+CD25+Foxp3+ population was significantly increased by PLA2 treatment. Interestingly, these anti-asthmatic effects by PLA2 were totally abolished by Treg depletion via anti-CD25 antibody injection, suggesting pharmaceutical action of PLA2 was medicated by Treg. Our data suggest that PLA2 inhibits OVA-induced allergic airway inflammation by modulating Treg function.

Silymarin Reduces OVA-induced Allergic Airway Inflammation

RationaleInexpensive, readily available drugs are needed for prevention and treatment of asthma. Oxidative stress, or an imbalance of reactive oxygen species (ROS) and antioxidants, occurs in asthma with infiltration of inflammatory cells, such as eosinophils, into the airways resulting in inflammation and tissue damage. Silymarin, a flavonoid complex from Silybum marianum (milk thistle plant), reduces ROS by increasing antioxidant enzymes, such as catalase, in human hepatotoxicity and cancer. However, the effect of silymarin on allergic airway inflammation and asthma is unknown. We hypothesized silymarin decreases ovalbumin (OVA)-induced airway inflammation in BALB/c mice by increasing catalase levels.MethodsBALB/c female mice were sensitized to OVA conjugated with aluminum hydroxide on day -14. Two weeks later, mice were challenged with 1% OVA aerosol daily on days 0 to 3. One hour prior to each OVA challenge mice were orally given silymarin (50-200mg/kg body weight) or vehicle (0.4% methylcellulose). On day 4, blood, bronchoalveolar lavage (BAL) fluid and lungs were harvested. Serum IgE levels and IL-13, a Th2 cytokine upregulated with allergic airway inflammation, were determined by ELISA. Infiltration of inflammatory cells was examined in BAL fluid. Catalase mRNA expression was examined by real-time PCR.ResultsSilymarin significantly decreased lymphocytes and eosinophils in the BAL fluid (n=11-12, p<0.05) and decreased BAL fluid levels of IL-13. IgE serum levels were not altered with silymarin (p=0.013). Silymarin increased mRNA expression levels of catalase (n=11-12, p<0.05).ConclusionsSilymarin attenuated OVA-induced allergic airway inflammation, but had no effect on IgE levels. Therefore, silymarin may be a potential therapeutic for asthma. RationaleInexpensive, readily available drugs are needed for prevention and treatment of asthma. Oxidative stress, or an imbalance of reactive oxygen species (ROS) and antioxidants, occurs in asthma with infiltration of inflammatory cells, such as eosinophils, into the airways resulting in inflammation and tissue damage. Silymarin, a flavonoid complex from Silybum marianum (milk thistle plant), reduces ROS by increasing antioxidant enzymes, such as catalase, in human hepatotoxicity and cancer. However, the effect of silymarin on allergic airway inflammation and asthma is unknown. We hypothesized silymarin decreases ovalbumin (OVA)-induced airway inflammation in BALB/c mice by increasing catalase levels. Inexpensive, readily available drugs are needed for prevention and treatment of asthma. Oxidative stress, or an imbalance of reactive oxygen species (ROS) and antioxidants, occurs in asthma with infiltration of inflammatory cells, such as eosinophils, into the airways resulting in inflammation and tissue damage. Silymarin, a flavonoid complex from Silybum marianum (milk thistle plant), reduces ROS by increasing antioxidant enzymes, such as catalase, in human hepatotoxicity and cancer. However, the effect of silymarin on allergic airway inflammation and asthma is unknown. We hypothesized silymarin decreases ovalbumin (OVA)-induced airway inflammation in BALB/c mice by increasing catalase levels. MethodsBALB/c female mice were sensitized to OVA conjugated with aluminum hydroxide on day -14. Two weeks later, mice were challenged with 1% OVA aerosol daily on days 0 to 3. One hour prior to each OVA challenge mice were orally given silymarin (50-200mg/kg body weight) or vehicle (0.4% methylcellulose). On day 4, blood, bronchoalveolar lavage (BAL) fluid and lungs were harvested. Serum IgE levels and IL-13, a Th2 cytokine upregulated with allergic airway inflammation, were determined by ELISA. Infiltration of inflammatory cells was examined in BAL fluid. Catalase mRNA expression was examined by real-time PCR. BALB/c female mice were sensitized to OVA conjugated with aluminum hydroxide on day -14. Two weeks later, mice were challenged with 1% OVA aerosol daily on days 0 to 3. One hour prior to each OVA challenge mice were orally given silymarin (50-200mg/kg body weight) or vehicle (0.4% methylcellulose). On day 4, blood, bronchoalveolar lavage (BAL) fluid and lungs were harvested. Serum IgE levels and IL-13, a Th2 cytokine upregulated with allergic airway inflammation, were determined by ELISA. Infiltration of inflammatory cells was examined in BAL fluid. Catalase mRNA expression was examined by real-time PCR. ResultsSilymarin significantly decreased lymphocytes and eosinophils in the BAL fluid (n=11-12, p<0.05) and decreased BAL fluid levels of IL-13. IgE serum levels were not altered with silymarin (p=0.013). Silymarin increased mRNA expression levels of catalase (n=11-12, p<0.05). Silymarin significantly decreased lymphocytes and eosinophils in the BAL fluid (n=11-12, p<0.05) and decreased BAL fluid levels of IL-13. IgE serum levels were not altered with silymarin (p=0.013). Silymarin increased mRNA expression levels of catalase (n=11-12, p<0.05). ConclusionsSilymarin attenuated OVA-induced allergic airway inflammation, but had no effect on IgE levels. Therefore, silymarin may be a potential therapeutic for asthma. Silymarin attenuated OVA-induced allergic airway inflammation, but had no effect on IgE levels. Therefore, silymarin may be a potential therapeutic for asthma.

IL-33 is a crucial amplifier of innate rather than acquired immunity

IL-33, a member of the IL-1-related cytokines, is considered to be a proallergic cytokine that is especially involved in Th2-type immune responses. Moreover, like IL-1α, IL-33 has been suggested to act as an "alarmin" that amplifies immune responses during tissue injury. In contrast to IL-1, however, the precise roles of IL-33 in those settings are poorly understood. Using IL-1- and IL-33-deficient mice, we found that IL-1, but not IL-33, played a substantial role in induction of T cell-mediated type IV hypersensitivity such as contact and delayed-type hypersensitivity and autoimmune diseases such as experimental autoimmune encephalomyelitis. Most notably, however, IL-33 was important for innate-type mucosal immunity in the lungs and gut. That is, IL-33 was essential for manifestation of T cell-independent protease allergen-induced airway inflammation as well as OVA-induced allergic topical airway inflammation, without affecting acquisition of antigen-specific memory T cells. IL-33 was significantly involved in the development of dextran-induced colitis accompanied by T cell-independent epithelial cell damage, but not in streptozocin-induced diabetes or Con A-induced hepatitis characterized by T cell-mediated apoptotic tissue destruction. In addition, IL-33-deficient mice showed a substantially diminished LPS-induced systemic inflammatory response. These observations indicate that IL-33 is a crucial amplifier of mucosal and systemic innate, rather than acquired, immune responses.

Atypical protein kinase C (aPKC) isoforms ζ and λ/ι synergically control Th2 development and function (134.13)

Abstract The protein kinase C (PKC) family consists of three subfamilies: classical, novel, and atypical PKCs. The atypical PKC (aPKC) consists of two isoforms, PKCζ and PKCλ/ι. We have recently presented in vitro, ex vivo, and in vivo genetic evidence that PKCζ is necessary for optimal activation of the IL-4 signaling cascade upstream of Jak1. Since whole-body genetic deletion of PKCλ/ι is lethal, we have used a conditional gene knockout system by loxP-Cre recombination to generated conditional PKCλ/ι KO mice to selectively delete PKCλ/ι in activated T cells. With this system, we found that PKCλ/ι was necessary for Th2 cytokine production. Upon anti-CD3/CD28 stimulation, IL-4 and IL-13 production from CD4+ T cells was significantly reduced in conditional KO mice as compared to their WT littermates. The activation of the transcription factors NFAT and NF-κB was impaired in PKCλ/ι-deficient activated T cells. Furthermore, deficiency of PKCλ/ι inhibited OVA-induced allergic airway inflammation in vivo. Recently, we have introduced the activated T cell-specific conditional PKCλ/ι deficiency into a PKCζ-/- background to generate mice with PKCζ and PKCλ/ι double knockout (DKO) in activated T cells. From these T cells, the secretion of Th2 cytokines, IL-4, IL-5 and IL-13, was more dramatically reduced and the proliferation was more significantly impaired compared to T cells with single PKCζ or PKCλ/ι deficiency. PKCλ/ι, like PKCζ, emerges as a new critical regulator of Th2 cell activation.

Strain-dependent activation of NF-κB in the airway epithelium and its role in allergic airway inflammation

NF-kappaB activation in the airway epithelium has been established as a critical pathway in ovalbumin (Ova)-induced airway inflammation in BALB/c mice (Poynter ME, Cloots R, van Woerkom T, Butnor KJ, Vacek P, Taatjes DJ, Irvin CG, Janssen-Heininger YM. J Immunol 173: 7003-7009, 2004). BALB/c mice are susceptible to the development of allergic airway disease, whereas other strains of mice, such as C57BL/6, are considered more resistant. The goal of the present study was to determine the proximal signals required for NF-kappaB activation in the airway epithelium in allergic airway disease and to unravel whether these signals are strain-dependent. Our previous studies, conducted in the BALB/c mouse background, demonstrated that transgenic mice expressing a dominant-negative version of IkappaBalpha in the airway epithelium (CC10-IkappaBalpha(SR)) were protected from Ova-induced inflammation. In contrast to these earlier observations, we demonstrate here that CC10-IkappaBalpha(SR) transgenic mice on the C57BL/6 background were not protected from Ova-induced allergic airway inflammation. Consistent with this finding, Ova-induced nuclear localization of the RelA subunit of NF-kappaB was not observed in C57BL/6 mice, in contrast to the marked nuclear presence of RelA in BALB/c mice. Evaluation of cytokine profiles in bronchoalveolar lavage demonstrated elevated expression of TNF-alpha in BALB/c mice compared with C57BL/6 mice after an acute challenge with Ova. Finally, neutralization of TNF-alpha by a blocking antibody prevented nuclear localization of RelA in BALB/c mice after Ova challenge. These data suggest that the mechanism of response of the airway epithelium of immunized C57BL/6 mice to antigen challenge is fundamentally different from that of immunized BALB/c mice and highlight the potential importance of TNF-alpha in regulating epithelial NF-kappaB activation in allergic airway disease.

A20 Attenuates Allergic Airway Inflammation in Mice

TNF receptor 1 can activate signaling pathways leading to the activation of NF-kappaB. A20, an NF-kappaB-inducible protein, negatively regulates these signaling pathways and acts as an anti-inflammatory mediator. Therefore, A20 is viewed as a potential therapeutic target for inflammatory disease. In this study, we examined the effect of A20 on an OVA-induced allergic airway inflammation model in mice. We used an adenovirus containing A20 cDNA (Ad-A20) that was delivered intratracheally before OVA challenge. Single administration of Ad-A20 reduced airway inflammatory cell recruitment and peribronchiolar inflammation and suppressed the production of various cytokines in bronchoalveolar fluid. In addition, Ad-A20 suppressed mucus production and prevented the development of airway hyperresponsiveness. The protective effect of Ad-A20 was mediated by the inhibition of the NF-kappaB signaling pathway. Taken together, our results suggest that the development of an immunoregulatory strategy based on A20 may have therapeutic potential for the treatment of allergic asthma.

Role of the PKCλ/ι in Th2 establishment and airway hyperresponsiveness (90.11)

Abstract The differentiation of T cells along different lineages is central to the control of immunity. In order to investigate the role of PKCλ/ι, a member of atypical protein kinase C, in T cell function, we used a conditional gene knockout system by loxP-Cre recombination to selectively delete PKCλ/ι in activated T cells. With this system, we found that PKCλ/ι was necessary for Th2 cytokine production. Upon anti-CD3 plus anti-CD28 stimulation, IL-4 and IL-13 production from CD4+ T cells was significantly reduced in conditional KO mice as compared to their WT littermates. However, PKCλ/ι was not involved in Th1 cytokine secretion. T cell proliferation was impaired in conditional PKCλ/ι KO mice. Furthermore, deficiency of PKCλ/ι inhibited OVA-induced allergic airway inflammation in vivo. Our data demonstrated that the activation of the transcription factors NFAT and NF-¿B was impaired in PKCλ/ι-deficient activated T cells. In addition, we presented genetic knockout evidence in ex vivo experiments with primary T cells that PKCλ/ι was critical for the control of cell polarity during T cell activation. Therefore, PKCλ/ι emerges as a critical regulator of Th2 cell activation.

Toll-like Receptor 2 Down-regulation in Established Mouse Allergic Lungs Contributes to Decreased Mycoplasma Clearance

Respiratory Mycoplasma pneumoniae (Mp) infection is involved in asthma pathobiology, but whether the established allergic airway inflammation compromises lung innate immunity and subsequently predisposes patients with asthma to Mp infection remains unknown. To test whether the established allergic airway inflammation compromises host innate immunity (e.g., Toll-like receptor 2 [TLR2]) to hinder the elimination of Mp from the lungs. We used mouse models of ovalbumin (OVA)-induced allergic airway inflammation with an ensuing Mp infection, and cultures of mouse primary lung dendritic cells (DCs) and bone marrow-derived DCs. Lung Mp clearance in allergic mice and TLR2 and IL-6 levels in lung cells, including DCs as well as cultured primary lung DCs and bone marrow-derived DCs, were assessed. The established OVA-induced allergic airway inflammation, or the prominent Th2 cytokines IL-4 and IL-13, inhibited TLR2 expression and IL-6 production in lung cells, including lung DCs, and eventually led to impaired host defense against Mp. Studies in IL-6 knockout mice indicated that IL-6 directly promoted Mp clearance from the lungs. IL-4- and IL-13-induced suppression of TLR2 was mediated by inhibiting nuclear factor-kappaB activation through signal transducer and activator of transcription 6 (STAT6) signaling pathway. The established OVA-induced allergic airway inflammation impairs TLR2 expression and host defense cytokine (e.g., IL-6) production, and subsequently delays lung bacterial clearance. This could offer novel therapeutic strategies to reinstate TLR2 activation by using TLR2 ligands and/or blocking IL-4 and IL-13 to ameliorate persisting respiratory bacterial infections in allergic lungs.

Mast cell–derived TNF contributes to airway hyperreactivity, inflammation, and T H2 cytokine production in an asthma model in mice

Mast cells, IgE, and TNF, which have been implicated in human atopic asthma, contribute significantly to the allergic airway inflammation induced by ovalbumin (OVA) challenge in mice sensitized with OVA without alum. However, it is not clear to what extent mast cells represent a significant source of TNF in this mouse model. We investigated the importance of mast cell-derived TNF in a mast cell-dependent model of OVA-induced airway hyperreactivity (AHR) and allergic airway inflammation. Features of this model of airway inflammation were analyzed in C57BL/6J-wild-type mice, mast cell-deficient C57BL/6J-Kit(W-sh)(/W-sh) mice, and C57BL/6J Kit(W-sh/W-sh) mice that had been systemically engrafted with bone marrow-derived cultured mast cells from C57BL/6J-wild-type or C57BL/6J-TNF(-/-) mice. Ovalbumin-induced AHR and airway inflammation were significantly reduced in mast cell-deficient Kit(W-sh/W-sh) mice versus wild-type mice. By contrast, Kit(W-sh/W-sh) mice that had been engrafted with wild-type but not with TNF(-/-) bone marrow-derived cultured mast cells exhibited responses very similar to those observed in wild-type mice. Mast cells and mast cell-derived TNF were not required for induction of OVA-specific memory T cells in the sensitization phase, but significantly enhanced lymphocyte recruitment and T(H)2 cytokine production in the challenge phase. Mast cell-derived TNF contributes significantly to the pathogenesis of mast cell-dependent and IgE-dependent, OVA-induced allergic inflammation and AHR in mice, perhaps in part by enhancing lymphocyte recruitment and T(H)2 cytokine production. Our findings in mice support the hypothesis that mast cell-derived TNF can promote allergic inflammation and AHR in asthma.

Cyclooxygenase-1 Overexpression Decreases Basal Airway Responsiveness but Not Allergic Inflammation

Pharmacological inhibition or genetic disruption of cyclooxygenase (COX)-1 or COX-2 exacerbates the inflammatory and functional responses of the lung to environmentally relevant stimuli. To further examine the contribution of COX-derived eicosanoids to basal lung function and to allergic lung inflammation, transgenic (Tr) mice were generated in which overexpression of human COX-1 was targeted to airway epithelium. Although no differences in basal respiratory or lung mechanical parameters were observed, COX-1 Tr mice had increased bronchoalveolar lavage fluid PGE(2) content compared with wild-type littermates (23.0 +/- 3.6 vs 8.4 +/- 1.4 pg/ml; p < 0.05) and exhibited decreased airway responsiveness to inhaled methacholine. In an OVA-induced allergic airway inflammation model, comparable up-regulation of COX-2 protein was observed in the lungs of allergic wild-type and COX-1 Tr mice. Furthermore, no genotype differences were observed in allergic mice in total cell number, eosinophil content (70 vs 76% of total cells, respectively), and inflammatory cytokine content of bronchoalveolar lavage fluid, or in airway responsiveness to inhaled methacholine (p > 0.05). To eliminate the presumed confounding effects of COX-2 up-regulation, COX-1 Tr mice were bred into a COX-2 null background. In these mice, the presence of the COX-1 transgene did not alter allergen-induced inflammation but significantly attenuated allergen-induced airway hyperresponsiveness, coincident with reduced airway leukotriene levels. Collectively, these data indicate that COX-1 overexpression attenuates airway responsiveness under basal conditions but does not influence allergic airway inflammation.

Allergic airway inflammation is exacerbated during acute influenza infection and correlates with increased allergen presentation and recruitment of allergen-specific T-helper type 2 cells.

Respiratory viral infections are a leading cause of the hospitalization of asthmatics, however, the cellular immunological interactions which underlie these two diseases remain elusive. We sought to characterize the effect influenza viral infection has on allergic airway inflammation and to identify the cellular pathways involved. We have used an ovalbumin (OVA) model of allergic airway inflammation, which involves sensitization of animals with OVA adsorbed in alum adjuvant followed by an intranasal challenge with OVA in phosphate-buffered saline. To study T cell recruitment into the lung, we adoptively transferred in vitro activated T cell receptor-transgenic T cells, which were subsequently identified by fluorescence-activated cell sorting (FACS) analysis. In addition, to study in vivo dendritic cell (DC) migration, we administered fluorescently labelled dextran and identified DCs that had phagocytosed it by FACS analysis. We found that different stages of influenza infection had contrasting effects upon the outcome of OVA-induced allergic airway inflammation. The allergic response against OVA was exacerbated during the acute stage of influenza infection; however, mice were protected against the development of airway eosinophilia at late time-points following infection. We investigated the mechanisms responsible for the virus-induced exacerbation and found that the response was partially independent of IL-4 and that there was increased delivery of inhaled allergens to the draining lymph node during the acute stage of the infection. In addition, virus-induced inflammation in the lung and draining lymph node resulted in the non-specific recruitment of circulating allergen-specific effector/memory cells. In addition to virus-mediated damage to the lung and airways, influenza viral infection can also enhance unrelated local allergic responses.