Inducible costimulator regulates Th2-mediated inflammation, but not Th2 differentiation, in a model of allergic airway disease

A novel costimulatory molecule expressed on activated T cells, inducible costimulator (ICOS), and its ligand, B7-related protein-1 (B7RP-1), were recently identified. ICOS costimulation leads to the induction of Th2 cytokines without augmentation of IL-2 production, suggesting a role for ICOS in Th2 cell differentiation and expansion. In the present study, a soluble form of murine ICOS, ICOS-Ig, was used to block ICOS/B7RP-1 interactions in a Th2 model of allergic airway disease. In this model, mice are sensitized with inactivated Schistosoma mansoni eggs and are subsequently challenged with soluble S. mansoni egg Ag directly in the airways. Treatment of C57BL/6 mice with ICOS-Ig during sensitization and challenge attenuated airway inflammation, as demonstrated by a decrease in cellular infiltration into the lung tissue and airways, as well as by a decrease in local IL-5 production. These inhibitory effects were not due to a lack of T cell priming nor to a defect in Th2 differentiation. In addition, blockade of ICOS/B7RP-1 interactions during ex vivo restimulation of lung Th2 effector cells prevented cytokine production. Thus, blockade of ICOS signaling can significantly reduce airway inflammation without affecting Th2 differentiation in this model of allergic airway disease.     

B7RP-1-ICOS interactions are required for optimal infection-induced expansion of CD4+ Th1 and Th2 responses

Although initial reports linked the costimulatory molecule ICOS preferentially with the development of Th2 cells, there is evidence that it is not required for protective type 2 immunity to helminths and that it contributes to Th1 and Th2 responses to other parasites. To address the role of ICOS in the development of infection-induced polarized Th cells, ICOS(-/-) mice were infected with Trichuris muris or Toxoplasma gondii. Wild-type mice challenged with T. muris developed Th2 responses and expelled these helminths by day 18 postinfection, whereas ICOS(-/-) mice failed to clear worms and produced reduced levels of type 2 cytokines. However, by day 35 postinfection, ICOS(-/-) mice were able to mount an effective Th2 response and worms were expelled. This delay in protective immunity was associated with a defect in infection-induced increases in the number of activated and proliferating CD4+ T cells. Similarly, following challenge with T. gondii ICOS was required for optimal proliferation by CD4+ T cells. However, the reduced number of activated CD4+ T cells and associated defect in the production of IFN-gamma did not result in increased susceptibility to T. gondii, but rather resulted in decreased CNS pathology during the chronic phase of this infection. Taken together, these data are consistent with a model in which ICOS is not involved in dictating polarity of the Th response but rather regulates the expansion of these subsets.     

The interaction between ICOS and B7RP-1 is not required for the development of experimental murine allergic conjunctivitis

It is still unclear whether the interaction between inducible costimulator (ICOS) and its ligand, B7 related protein (B7RP)-1, is important for the development of allergic diseases. We investigated whether blocking the ICOS/B7RP-1 interaction affects the development of murine experimental allergic conjunctivitis (EC). EC was induced in Balb/c mice either by active immunization of ragweed (RW) or by transferring RW-primed splenocytes, followed by challenge with RW-containing eye drops. The mice were treated with anti-B7RP-1 antibody (Ab) or normal rat immunoglobulin G (IgG) during either the induction or effector phase. Regardless of the induction method or when the animals were treated, eosinophil infiltration into the conjunctiva was not affected by the anti-B7RP-1 Ab treatment. Splenocyte responses were not largely affected by this treatment. However, serum Ig levels were significantly reduced. These data suggest that blocking the ICOS/B7RP-1 in allergic diseases may not always be therapeutic.     

CCR8 is not essential for the development of inflammation in a mouse model of allergic airway disease

Chemokine receptors play an important role in the trafficking of various immune cell types to sites of inflammation. Several chemokine receptors are differentially expressed in Th1 and Th2 effector populations. Th2 cells selectively express CCR3, CCR4, and CCR8, which could direct their trafficking to sites of allergic inflammation. Additionally, increased expression of the CCR8 ligand, TCA-3, has been detected in affected lungs in a mouse model of asthma. In this study, CCR8-deficient mice were generated to address the biological role of CCR8 in a model of allergic airway disease. Using two different protocols of allergen challenge, we demonstrate that absence of CCR8 does not affect the development of pulmonary eosinophilia and Th2 cytokine responses. In addition, administration of anti-TCA-3-neutralizing Ab during allergen sensitization and rechallenge failed to inhibit airway allergic inflammation. These results suggest that CCR8 does not play an essential role in the pathogenesis of inflammation in this mouse model of allergic airway disease.     

Preproenkephalin is a Th2 cytokine but is not required for Th2 differentiation in vitro.

Preproenkephalin (PPNK) mRNA expression has been detected in many cells of the immune system, including monocytes and lymphocytes. In the present paper, the expression of PPNK mRNA in purified CD4+ Th1 and Th2 lymphocyte subpopulations is investigated and correlated with the presence of the opioid neuropeptides leu- and met-enkephalin. We found that PPNK mRNA and met-enkephalin were present at higher levels in the Th2 cultures compared with the Th1 cultures. Lymphocytes from PPNK-deficient mice were then used to look at the role of PPNK in Th2 lymphocyte differentiation. Lymphocytes from these mice could be driven into a Th2 phenotype, suggesting that cultures containing IL-4 do not require PPNK for Th2 differentiation.     

Granulocyte-macrophage colony-stimulating factor is required for bronchial eosinophilia in a murine model of allergic airway inflammation

GM-CSF plays an important role in inflammation by promoting the production, activation, and survival of granulocytes and macrophages. In this study, GM-CSF knockout (GM-CSF(-/-)) mice were used to investigate the role of GM-CSF in a model of allergic airway inflammation. In allergic GM-CSF(-/-) mice, eosinophil recruitment to the airways showed a striking pattern, with eosinophils present in perivascular areas, but almost completely absent in peribronchial areas, whereas in wild-type mice, eosinophil infiltration appeared in both areas. In the GM-CSF(-/-) mice, mucus production in the airways was also reduced, and eosinophil numbers were markedly reduced in the bronchoalveolar lavage (BAL)(3) fluid. IL-5 production was reduced in the lung tissue and BAL fluid of GM-CSF(-/-) mice, but IL-4 and IL-13 production, airway hyperresponsiveness, and serum IgE levels were not affected. The presence of eosinophils in perivascular but not peribronchial regions was suggestive of a cell migration defect in the airways of GM-CSF(-/-) mice. The CCR3 agonists CCL5 (RANTES) and CCL11 (eotaxin-1) were expressed at similar levels in GM-CSF(-/-) and wild-type mice. However, IFN-gamma mRNA and protein were increased in the lung tissue and BAL fluid in GM-CSF(-/-) mice, as were mRNA levels of the IFN-gamma-inducible chemokines CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-Tac). Interestingly, these IFN-gamma-inducible chemokines are natural antagonists of CCR3, suggesting that their overproduction in GM-CSF(-/-) mice contributes to the lack of airway eosinophils. These findings demonstrate distinctive abnormalities to a model of allergic asthma in the absence of GM-CSF.     

Osteopontin is not required for the development of Th1 responses and viral immunity

Osteopontin (OPN) has been defined as a key cytokine promoting the release of IL-12 and hence inducing the development of protective cell-mediated immunity to viruses and intracellular pathogens. To further characterize the role of OPN in antiviral immunity, OPN-deficient (OPN-/-) mice were analyzed after infection with influenza virus and vaccinia virus. Surprisingly, we found that viral clearance, lung inflammation, and recruitment of effector T cells to the lung were unaffected in OPN-/- mice after influenza infection. Furthermore, effector status of T cells was normal as demonstrated by normal IFN-gamma production and CTL lytic activity. Moreover, activation and Th1 differentiation of naive TCR transgenic CD4+ T cells by dendritic cells and cognate Ag was normal in the absence of OPN in vitro. Contrary to a previous report, we found that OPN-/- mice mounted a normal immune response to Listeria monocytogenes. In conclusion, OPN is dispensable for antiviral immune responses against influenza virus and vaccinia virus.     

CARMA1 is critical for the development of allergic airway inflammation in a murine model of asthma

CARMA1 has been shown to be important for Ag-stimulated activation of NF-kappaB in lymphocytes in vitro and thus could be a novel therapeutic target in inflammatory diseases such as asthma. In the present study, we demonstrate that mice with deletion in the CARMA1 gene (CARMA1(-/-)) do not develop inflammation in a murine model of asthma. Compared with wild-type controls, CARMA1(-/-) mice did not develop airway eosinophilia, had no significant T cell recruitment into the airways, and had no evidence for T cell activation in the lung or draining lymph nodes. In addition, the CARMA1(-/-) mice had significantly decreased levels of IL-4, IL-5, and IL-13, did not produce IgE, and did not develop airway hyperresponsiveness or mucus cell hypertrophy. However, adoptive transfer of wild-type Th2 cells into CARMA1(-/-) mice restored eosinophilic airway inflammation, cytokine production, airway hyperresponsiveness, and mucus production. This is the first demonstration of an in vivo role for CARMA1 in a disease process. Furthermore, the data clearly show that CARMA1 is essential for the development of allergic airway inflammation through its role in T lymphocytes, and may provide a novel means to inhibit NF-kappaB for therapy in asthma.     

Th1 and Th2 cells are required for both eosinophil- and neutrophil-associated airway inflammatory responses in mice

Most current animal models focus on eosinophil-mediated asthma, despite compelling evidence that a neutrophil-mediated disease occurs in some asthma patients. Using intranasal challenge of mice sensitized either orally or nasally with whole peanut protein extract in the presence of cholera toxin, we developed mouse models of eosinophil- and neutrophil-mediated asthma, respectively. In this study, mice deficient in Th1 (IL-12 and IFN-gamma) or Th2 (IL-4 and IL-13) pathways were used to characterize the role played by Th1 and Th2 cytokines during the initial priming phase in the two models. Antigen-specific Ab responses were controlled primarily by Th2 cytokines in mice sensitized by the oral route, whereas Th1 cytokines appeared to play a predominant role in mice sensitized by the nasal route. Furthermore, the absence of key Th1 or Th2 cytokines during the initial phase of priming reduced lung reactivity in both mouse models of airway inflammation.     

CpG oligodeoxynucleotides can reverse Th2-associated allergic airway responses and alter the B7.1/B7.2 expression in a murine model of asthma

CpG oligodeoxynucleotides (CpG-ODN) administered during Ag sensitization or before Ag challenge can inhibit allergic pulmonary inflammation and airway hyperreactivity in murine models of asthma. In this study, we investigated whether CpG-ODN can reverse an ongoing allergic pulmonary reaction in a mouse model of asthma. AKR mice were sensitized with conalbumin followed by two intratracheal challenges at weekly intervals. CpG-ODN was administered 24 h after the first Ag challenge. CpG-ODN administration reduced Ag-specific IgE levels, bronchoalveolar lavage fluid eosinophils, mucus production, and airway hyperreactivity. We found that postchallenge CpG-ODN treatment significantly increased IFN-gamma concentrations and decreased IL-13, IL-4, and IL-5 concentrations in bronchoalveolar lavage fluids and spleen cell culture supernatants. Postchallenge CpG-ODN treatment also increased B7.1 mRNA expression and decreased B7.2 mRNA expression in lung tissues. These results suggest that CpG-ODN may have potential for treatment of allergic asthma by suppressing Th2 responses during IgE-dependent allergic airway reactions. The down-regulation of Th2 responses by CPG-ODN may be associated with regulation of the costimulatory factors B7.1 and B7.2.     

TLR2 agonist ameliorates established allergic airway inflammation by promoting Th1 response and not via regulatory T cells

TLRs are primary sensors of both innate and adaptive immune systems, where they play a pivotal role in the response directed against structurally conserved components of pathogens. Synthetic bacterial lipopeptide Pam3CSK4 is a TLR2 agonist capable of modulating Th1 and Th2 responses. This study examines the therapeutic effect of Pam3CSK4 in established airway inflammation in a murine model of asthma. In mice previously sensitized and challenged with OVA, Pam3CSK4 given i.p. markedly reduced the total inflammatory cell infiltrate and eosinophilia in bronchoalveolar lavage fluid. Pam3CSK4 therapy was associated with a reduction in OVA-induced IL-4 and IL-5 secretion from thoracic lymph node culture, airways inflammation, bronchial hyperresponsiveness, and serum levels of IgE. Pam3CSK4 therapy was also associated with an increase in OVA-induced IFN-gamma, IL-12, and IL-10 production. However, the anti-inflammatory effect of Pam3CSK4 was independent of IL-10 or TGF-beta, but was critically dependent on IL-12, the production of which by dendritic cells was enhanced by Pam3CSK4 in vitro. Our results provide direct evidence that Pam3CSK4 could represent a novel therapeutic agent in allergic airways disease.     

Synchronization of dendritic cell activation and antigen exposure is required for the induction of Th1/Th17 responses

The dendritic cell (DC) targeting/activation patterns required to elicit Th1/Th17 responses remain undefined. One postulated requirement was that of a physical linkage between Ags and immunomodulators. Accordingly, the separate same-site administration of Ag85B-ESAT-6 (hybrid-1 protein; H1), a mycobacterial fusion Ag, and the CAF01 liposome-based adjuvant induced similar Ab and weak Th2 responses as those of coformulated H1/CAF01 but failed to elicit Th1/Th17 responses. Yet, this separate same-site injection generated the same type and number of activated Ag(+)/adjuvant(+) DCs in the draining lymph nodes (LN) as that of protective H1/CAF01 immunization. Thus, targeting/activating the same DC population by Ag and adjuvant is not sufficient to elicit Th1/Th17 responses. To identify the determinants of Th1/Th17 adjuvanticity, in vivo tracking experiments using fluorescently labeled Ag and adjuvant identified that a separate same-site administration elicits an additional early Ag(+)/adjuvant(-) DC population with a nonactivated phenotype, resulting from the earlier targeting of LN DCs by H1 than by CAF01 molecules. This asynchronous targeting pattern was mimicked by the injection of free H1 prior to or with, but not after, H1/CAF01 or H1/CpG/ aluminum hydroxide immunization. The injection of soluble OVA similarly prevented the induction of Th1 responses by OVA/CAF01. Using adoptively transferred OT-2 cells, we show that the Ag targeting of LN DCs prior to their activation generates nonactivated Ag-pulsed DCs that recruit Ag-specific T cells, trigger their initial proliferation, but interfere with Th1 induction in a dose-dependent manner. Thus, the synchronization of DC targeting and activation is a critical determinant for Th1/Th17 adjuvanticity.     

A PAF receptor antagonist inhibits acute airway inflammation and late-phase responses but not chronic airway inflammation and hyperresponsiveness in a primate model of asthma

We have examined the effects of a PAF receptor antagonist, WEB 2170, on several indices of acute and chronic airway inflammation and associated changes in lung function in a primate model of allergic asthma. A single oral administration WEB 2170 provided dose related inhibition of the release of leukotriene C(4) (LTC(4)) and prostaglandin D(2) (PGD(2)) recovered and quantified in bronchoalveolar lavage (BAL) fluid obtained during the acute phase response to inhaled antigen. In addition, oral WEB 2170 treatment in dual responder primates blocked the acute influx of neutrophils into the airways as well as the associated late-phase airway obstruction occurring 6 h after antigen inhalation. In contrast, a multiple dosing regime with WEB 2170 (once a day for 7 consecutive days) failed to reduce the chronic airway inflammation (eosinophilic) and associated airway hyperresponsiveness to inhaled methacholine that is characteristic of dual responder monkeys. Thus, we conclude that the generation of PAF following antigen inhalation contributes to the development of lipid mediators, acute airway inflammation and associated late-phase airway obstruction in dual responder primates; however, PAF does not play a significant role in the maintenance of chronic airway inflammation and associated airway hyperresponsiveness in this primate model.     

UvrD2 is essential in Mycobacterium tuberculosis, but its helicase activity is not required

UvrD is an SF1 family helicase involved in DNA repair that is widely conserved in bacteria. Mycobacterium tuberculosis has two annotated UvrD homologues; here we investigate the role of UvrD2. The uvrD2 gene at its native locus could be knocked out only in the presence of a second copy of the gene, demonstrating that uvrD2 is essential. Analysis of the putative protein domain structure of UvrD2 shows a distinctive domain architecture, with an extended C terminus containing an HRDC domain normally found in SF2 family helicases and a linking domain carrying a tetracysteine motif. Truncated constructs lacking the C-terminal domains of UvrD2 were able to compensate for the loss of the chromosomal copy, showing that these C-terminal domains are not essential. Although UvrD2 is a functional helicase, a mutant form of the protein lacking helicase activity was able to permit deletion of uvrD2 at its native locus. However, a mutant protein unable to hydrolyze ATP or translocate along DNA was not able to compensate for lack of the wild-type protein. Therefore, we concluded that the essential role played by UvrD2 is unlikely to involve its DNA unwinding activity and is more likely to involve DNA translocation and, possibly, protein displacement.     

Th type 1-stimulating activity of lung macrophages inhibits Th2-mediated allergic airway inflammation by an IFN-gamma-dependent mechanism

In the mucosal immune system, resident dendritic cells are specialized for priming Th2-polarized immunity, whereas the Ag-presenting activity of macrophages has been linked with the development of Th1 phenotype. As an immune switch toward Th1 can protect against Th2-mediated allergic response, this study investigated the capacity of lung macrophages to stimulate Th1 responses during the secondary exposure to inhaled allergen, thereby suppressing Th2-mediated allergic airway inflammation in a murine model of allergic asthma. Following airway macrophage depletion in OVA-sensitized mice, lung T cells defaulted to a phenotype that produced less Th1 (IFN-gamma) and more Th2 (IL-4 and IL-5) cytokines, leading to more severe airway hyperreactivity and inflammation after intranasal Ag challenge. After OVA pulsing and adoptive transfer, lung macrophages selectively promoted a Th1 response in Ag-sensitized recipients and did not induce pulmonary eosinophilia. By contrast, OVA pulsing and adoptive transfer of a lung cell preparation, consisting of dendritic cells, B cells, and macrophages, promoted a Th2 response with an associated inflammatory response that was suppressed when macrophages were present and pretreated with IFN-gamma, but exacerbated when macrophages were depleted before IFN-gamma treatment. In addition, Th1-promoting activity of lung macrophages was not related to the autocrine production of IL-12p40. These results suggest that the Th1-promoting APC activity may be an inherent property of the lung macrophage population, and may play an important role, upon stimulation by IFN-gamma, in antagonizing an ongoing Th2 immunity and Th2-dependent allergic responses.     

The C2 domain of phosphatidylserine decarboxylase 2 is not required for catalysis but is essential for in vivo function

Phosphatidylserine decarboxylase 2 (Psd2p) is currently being used to study lipid trafficking processes in intact and permeabilized yeast cells. The Psd2p contains a C2 homology domain and a putative Golgi retention/localization (GR) domain. C2 domains play important functions in membrane binding and docking reactions involving phospholipids and proteins. We constructed a C2 domain deletion variant (C2Delta) and a GR deletion variant (GRDelta) of Psd2p and examined their effects on in vivo function and catalysis. Immunoblotting confirmed that the predicted immature and mature forms of Psd2(C2Delta)p, Psd2(GRDelta)p, and wild type Psd2p were produced in vivo and that the proteins localized normally. Enzymology revealed that the Psd2(C2Delta)p and Psd2(GRDelta)p were catalytically active and could readily be expressed at levels 10-fold higher than endogenous Psd2p. Both Psd2p and Psd2(GRDelta)p expression complemented the growth defect of psd1Deltapsd2Delta strains and resulted in normal aminoglycerophospholipid metabolism. In contrast, the Psd2(C2Delta)p failed to complement psd1Deltapsd2Delta strains, and [(3)H]serine labeling revealed a severe defect in the formation of PtdEtn in both intact and permeabilized cells, indicative of disruption of lipid trafficking. These findings identify an essential, non-catalytic function of the C2 domain of Psd2p and raise the possibility that it plays a direct role in membrane docking and/or PtdSer transport to the enzyme.