TLR1/TLR2 agonist induces tumor regression by reciprocal modulation of effector and regulatory T cells

Using TLR agonists in cancer treatment can have either beneficial or detrimental effects. Therefore, it is important to determine their effect on the tumor growth and understand the underlying mechanisms in animal tumor models. In this study, we report a general immunotherapeutic activity of a synthetic bacterial lipoprotein (BLP), a TLR1/TLR2 agonist, on established lung carcinoma, leukemia, and melanoma in mice. Systemic treatment of 3LL tumor-bearing mice with BLP, but not LPS, led to a dose-dependent tumor regression and a long-lasting protective response against tumor rechallenge. The BLP-mediated tumor remission was neither mediated by a direct tumoricidal activity nor by innate immune cells, because it lacked therapeutic effect in immunodeficient SCID mice. Instead, BLP treatment reduced the suppressive function of Foxp3(+) regulatory T cells (Tregs) and enhanced the cytotoxicity of tumor-specific CTL in vitro and in vivo. Furthermore, adoptive cotransfer of BLP-pretreated but not untreated CTL and Tregs from wild-type but not from TLR2(-/-) mice was sufficient to restore antitumor immunity in SCID mice by reciprocally modulating Treg and CTL function. These results demonstrate that the TLR1/TLR2 agonist BLP may have a general tumor therapeutic property involving reciprocal downregulation of Treg and upregulation of CTL function. This property may play an important role in the development of novel antitumor strategies.     

TLR2 agonist ameliorates murine experimental allergic conjunctivitis by inducing CD4 positive T-cell apoptosis rather than by affecting the Th1/Th2 balance

Innate immune responses that operate through Toll-like receptors (TLRs) are actively involved in the development of diseases predominantly mediated by adaptive immune responses. This is true also for allergic disease, as TLRs have been found to be involved in the development of allergic airway inflammation. We investigated whether stimulating TLR2 also abrogates murine allergic conjunctivitis by upregulating Th1 responses. We found that treating mice during the efferent phase with the TLR2 agonist Pam3CSK4 significantly suppressed eosinophil infiltration into the conjunctiva. However, Pam3CSK4 treatment inhibited both the Th1 and Th2 responses in the mice, and also suppressed eosinophil infiltration in IFN-gamma knockout mice. Flow cytometric analysis demonstrated that Pam3CSK4 treatment significantly elevated the numbers of annexin V-positive splenocytes, especially CD4 positive T cells. Thus, the stimulation of TLR2 during the efferent phase of murine allergic conjunctivitis suppresses eosinophil infiltration by inducing CD4 positive T-cell apoptosis rather than upregulating Th1 responses.     

Regulatory T cells more effectively suppress Th1-induced airway inflammation compared with Th2

Asthma is a syndrome with different inflammatory phenotypes. Animal models have shown that, after sensitization and allergen challenge, Th2 and Th1 cells contribute to the development of allergic airway disease. We have previously demonstrated that naturally occurring regulatory T cells (nTregs) can only marginally suppress Th2-induced airway inflammation and airway hyperresponsiveness. In this study, we investigated nTreg-mediated suppression of Th2-induced and Th1-induced acute allergic airway disease. We demonstrate in vivo that nTregs exert their suppressive potency via cAMP transfer on Th2- and Th1-induced airway disease. A comparison of both phenotypes revealed that, despite similar cAMP transfers, Th1-driven airway hyperresponsiveness and inflammation are more susceptible to nTreg-dependent suppression, suggesting that potential nTreg-based therapeutic strategies might be more effective in patients with predominantly neutrophilic airway inflammation based on deregulated Th1 response.     

Atorvastatin ameliorates experimental autoimmune neuritis by decreased Th1/Th17 cytokines and up-regulated T regulatory cells

Statins have anti-inflammatory and immune-regulating properties. To investigate the effects of atorvastatin on experimental autoimmune neuritis (EAN), an animal model of Guillain–Barré syndrome (GBS), atorvastatin was administered to Lewis rats immunized with bovine peripheral myelin in complete Freund’s adjuvant. We found that atorvastatin ameliorated the clinical symptoms of EAN, decreased the numbers of inflammatory cells as well as IFN-γ⁺ and IL-17⁺ cells in sciatic nerves, decreased the CD80 expression and increased the number of CD25⁺Foxp3⁺ cells in mononuclear cells (MNC), and decreased the levels of IFN-γ in MNC culture supernatants. These data provide strong evidence that atorvastatin can act as an inhibitor in EAN by inhibiting the immune response of Th1 and Th17, decreasing the expression of co-stimulatory molecule, and up-regulating the number of T regulatory cells. These data demonstrated that statins could be used as a therapeutic strategy in human GBS in future.     

T regulatory cells 1 inhibit a Th2-specific response in vivo

We recently described a new population of CD4(+) regulatory T cells (Tr1) that inhibits proliferative responses of bystander T cells and prevents colitis induction in vivo through the secretion of IL-10. IL-10, which had been primarily described as a Th2-specific cytokine inhibiting Th1 responses, has displayed in several models a more general immune suppression on both types of effector T cell responses. Using an immediate hypersensitivity model in which BALB/c mice immunized with OVA (alum) normally generate Th2-dominated responses, we examined the ability of OVA-specific Tr1 T cell clones to inhibit OVA-specific cytokines and Ab responses. In contrast to Th2 or Th1 T cell clones, transfer of Tr1 T cell clones coincident with OVA immunization inhibited Ag-specific serum IgE responses, whereas IgG1 and IgG2a synthesis were not affected. This specific inhibition was mediated in part through IL-10 secretion as anti-IL-10 receptor Abs treatment reverted the inhibitory effect of Tr1 T cell clones. Although specifically targeted to IgE responses, Tr1 clones' inhibitory effects were more profound as they affected Ag-specific Th2 cell priming both in term of proliferative responses and cytokine secretion. These results suggest that regulatory T cells may play a fundamental role in maintaining the balance of the immune system to prevent allergic disorders.     

Inhibition of Th1- and Th2-mediated airway inflammation by the sphingosine 1-phosphate receptor agonist FTY720

The sphingosine 1-phosphate receptor agonist FTY720 is a novel immunomodulator that sequesters lymphocytes in secondary lymphoid organs and thereby prevents their migration to sites of inflammation. However, there is currently no information available on whether this drug affects Th1 or Th2 cell-mediated lung-inflammatory responses. The effect of FTY720 was therefore investigated in a murine airway inflammation model using OVA-specific, in vitro differentiated, and adoptively transferred Th1 and Th2 cells. Both Th1 and Th2 cells express a similar pattern of FTY720-targeted sphingosine 1-phosphate receptors. The OVA-induced Th1-mediated airway inflammation characterized by increased numbers of lymphocytes and neutrophils in bronchoalveolar lavage fluid was significantly inhibited by oral FTY720 treatment. Similarly, FTY720 suppressed the Th2 cell-induced bronchoalveolar lavage fluid eosinophilia and the infiltration of T lymphocytes and eosinophils into the bronchial tissue. Moreover, the Ag-induced bronchial hyperresponsiveness to inhaled metacholine was almost completely blocked. The inhibitory effect of FTY720 on airway inflammation, induction of bronchial hyperresponsiveness, and goblet cell hyperplasia could be confirmed in an actively Ag-sensitized murine asthma model, clearly indicating that Th2 cell-driven allergic diseases such as asthma could benefit from such treatment.     

Heme oxygenase-1-mediated CD4+CD25high regulatory T cells suppress allergic airway inflammation

Heme oxygenase-1 (HO-1) has anti-inflammatory effects in asthma. CD4+CD25(high) regulatory T cells (Treg) are a potent immunoregulator that suppresses the immune response. We studied the effects of HO-1-mediated CD4+CD25(high) Treg on suppression of allergic airway inflammation by comparing mice treated with hemin, OVA, Sn-protoporphyrin (SnPP), and hemin plus SnPP. Airway responsiveness, airway eosinophil infiltration, the level of OVA-specific IgE, and the numbers of cells in general and eosinophils in particular in bronchial alveolar lavage fluid were lower in the hemin group than in the OVA, SnPP, and hemin plus SnPP groups. The expressions of HO-1 mRNA and protein in the lung were increased by repeated administrations of hemin and SnPP. However, the activity of HO-1 was highest in hemin mice. The percentage and suppressive function of CD4+CD25(high) Treg and the expression of Foxp3 mRNA were obviously enhanced after treatment with hemin. This increase was diminished by the administration of SnPP. The concentration of serum IL-10 was higher in the hemin group than in the other groups, whereas the level of serum TGF-beta did not significantly differ across groups. Furthermore, the ratio of IFN-gamma/IL-4 mRNA in the lung was higher in hemin-treated mice than in OVA and SnPP mice. The suppressive capacity of CD4+CD25(high) Treg was not enhanced in the IL-10-deficient mice treated with hemin. In conclusion, our experiments in the animal model demonstrated that HO-1 has anti-inflammatory effects, probably via enhancement of the secretion of IL-10 and promotion of the percentage of CD4+CD25(high) Treg.     

Peptidoglycan recognition protein Pglyrp2 protects mice from psoriasis-like skin inflammation by promoting regulatory T cells and limiting Th17 responses

Skin protects the body from the environment and is an important component of the innate and adaptive immune systems. Psoriasis is a frequent inflammatory skin disease of unknown cause determined by multigenic predisposition, environmental factors, and aberrant immune response. Peptidoglycan recognition proteins (Pglyrps) are expressed in the skin, and we report in this article that they modulate sensitivity in an experimentally induced mouse model of psoriasis. We demonstrate that Pglyrp2(-/-) mice (but not Pglyrp3(-/-) and Pglyrp4(-/-) mice) are more sensitive to the development of 12-O-tetradecanoylphorbol 13-acetate-induced psoriasis-like inflammation, whereas Pglyrp1(-/-) mice are less sensitive. The mechanism underlying this increased sensitivity of Pglyrp2(-/-) mice to 12-O-tetradecanoylphorbol 13-acetate-induced psoriasis-like inflammation is reduced recruitment of regulatory T cells to the skin and enhanced production and activation of Th17 cells in the skin in Pglyrp2(-/-) mice, which results in more severe inflammation and keratinocyte proliferation. Thus, in wild type mice, Pglyrp2 limits overactivation of Th17 cells by promoting accumulation of regulatory T cells at the site of inflammation, which protects the skin from the exaggerated inflammatory response.     

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.     

Differential regulatory function of resting and preactivated allergen-specific CD4+ CD25+ regulatory T cells in Th2-type airway inflammation

Although CD4(+)CD25(+) regulatory T (Treg) cells are known to suppress Th1 cell-mediated immune responses, their effect on Th2-type immune responses remains unclear. In this study we examined the role of Treg cells in Th2-type airway inflammation in mice. Depletion and reconstitution experiments demonstrated that the Treg cells of naive mice effectively suppressed the initiation and development of Th2-driven airway inflammation. Despite effective suppression of Th2-type airway inflammation in naive mice, adoptively transferred, allergen-specific Treg cells were unable to suppress airway inflammation in allergen-presensitized mice. Preactivated allergen-specific Treg cells, however, could suppress airway inflammation even in allergen-presensitized mice by accumulating in the lung, where they reduced the accumulation and proliferation of Th2 cells. Upon activation, allergen-specific Treg cells up-regulated CCR4, exhibited enhanced chemotactic responses to CCR4 ligands, and suppressed the proliferation of and cytokine production by polarized Th2 cells. Collectively, these results demonstrated that Treg cells are capable of suppressing Th2-driven airway inflammation even in allergen-presensitized mice in a manner dependent on their efficient migration into the inflammatory site and their regulation of Th2 cell activation and proliferation.     

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.     

Suppression of murine allergic airway disease by IL-2:anti-IL-2 monoclonal antibody-induced regulatory T cells

Regulatory T cells (Treg) play a decisive role in many diseases including asthma and allergen-induced lung inflammation. However, little progress has been made developing new therapeutic strategies for pulmonary disorders. In the current study we demonstrate that cytokine:antibody complexes of IL-2 and anti-IL-2 mAb reduce the severity of allergen-induced inflammation in the lung by expanding Tregs in vivo. Unlike rIL-2 or anti-IL-2 mAb treatment alone, IL-2:anti-IL-2 complexes dampened airway inflammation and eosinophilia while suppressing IL-5 and eotaxin-1 production. Mucus production, airway hyperresponsiveness to methacholine, and parenchymal tissue inflammation were also dramatically reduced following IL-2:anti-IL-2 treatment. The suppression in allergic airway disease was associated with a marked expansion of Tregs (IL-10(+)CD4(+)CD25(+) and Foxp3(+)CD4(+)CD25(+)) in the tissues, with a corresponding decrease in effector T cell responses. The ability of IL-2:anti-IL-2 complexes to suppress airway inflammation was dependent on Treg-derived IL-10, as IL-10(+/+), but not IL-10(-/-) Tregs, were capable of mediating the suppression. Furthermore, a therapeutic protocol using a model of established airway allergy highlighted the ability of IL-2:anti-IL-2 complexes to expand Tregs and prevent successive airway inflammation and airway hyperresponsiveness. This study suggests that endogenous Treg therapy may be a useful tool to combat the rising incidence of allergic airway disease.     

Mutated glutathione-S-transferase reduced airway inflammation by limiting oxidative stress and Th2 response

Oxidative stress is an important factor in the pathogenesis of asthma. Furthermore, antioxidants like GST are reduced in asthma patients. In the present study, the therapeutic effects of exogenous GST and mGST were evaluated in a mice model. GST mutated at residues 21/27 has reduced IgE binding with similar enzyme activity as that of GST. To evaluate the therapeutic effects of GST, BALB/c mice were immunized and challenged with ovalbumin. Mice were given GST, mGST, and alpha-lipoic acid by inhalation and sacrificed on Day 31 to evaluate inflammation and oxidative stress. Mice treated with mGST showed significantly reduced total cell count (P<0.01) and eosinophils (P<0.01) in BALF compared to GST- or PBS-treated groups. The lung inflammation score was lowest for the mGST-treated group along with reduced IL-4 (P<0.01) and OVA-specific IgE than other groups. Oxidative stress as per the lipid peroxidation level in BALF of mGST-treated mice was reduced significantly in comparison to PBS- or GST-treated mice. In conclusion, inhalation of mGST reduced airway inflammation in mice. Mutated GST with reduced allergenicity has better therapeutic potential and can be explored as an adjunct therapy in asthma.     

The TLR7 agonist R848 alleviates allergic inflammation by targeting invariant NKT cells to produce IFN-gamma

It has been documented that TLR7 stimulation triggers not only antiviral responses, but also alleviates experimental asthma. Considering the implication of invariant NKT (iNKT) cells in both situations, we postulated that they might contribute to the anti-inflammatory effect of TLR7 ligands. We show in this study that spleen cells activated by the TLR7 agonist resiquimod (R848) attenuate allergic inflammation upon adoptive transfer when they are recovered from wild-type, but not from iNKT cell-deficient Jα18(-/-) mice, which proves the specific involvement of this regulatory population. Furthermore, we provide evidence that IFN-γ is critical for the protective effect, which is lost when transferred iNKT cells are sorted from IFN-γ-deficient mice. In support of a direct activation of iNKT cells through TLR7 signaling in vivo, we observed a prompt increase of serum IFN-γ levels, associated with upregulation of CD69 expression on iNKT cells. Moreover, we demonstrate that iNKT cells effectively express TLR7 and respond to R848 in vitro by producing high levels of IFN-γ in the presence of IL-12, consistent with the conclusion that their contribution to the alleviation of allergic inflammation upon treatment with TLR7 ligands is mediated through IFN-γ.     

4-1BB triggering ameliorates experimental autoimmune encephalomyelitis by modulating the balance between Th17 and regulatory T cells

Agonistic anti-4-1BB Ab is known to ameliorate experimental autoimmune encephalomyelitis. 4-1BB triggering typically leads to the expansion of CD8(+) T cells, which produce abundant IFN-γ, and this in turn results in IDO-dependent suppression of autoimmune responses. However, because neutralization of IFN-γ or depletion of CD8(+) T cell only partially abrogates the effect of 4-1BB triggering, we sought to identify an additional mechanism of 4-1BB-triggered suppression of autoimmune responses using IFN-γ- or IFN-γR-deficient mice. 4-1BB triggering inhibited the generation of Th17 cells that is responsible for experimental autoimmune encephalomyelitis induction and progression, and increased Foxp3(+)CD4(+) regulatory T (Treg) cells, particularly among CD4(+) T cells. This was not due to a direct effect of 4-1BB signaling on CD4(+) T cell differentiation: 4-1BB signaling not only reduced Th17 cells and increased Treg cells in wild-type mice, which could be due to IFN-γ production by the CD8(+) T cells, but also did so in IFN-γ-deficient mice, in that case by downregulating IL-6 production. These results show that although secondary suppressive mechanisms evoked by 4-1BB triggering are usually masked by the strong effects of IFN-γ, 4-1BB signaling seems to modulate autoimmune responses by a number of mechanisms, and modulation of the Th17 versus Treg cell balance is one of those mechanisms.     

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.     

High-dose but not low-dose mainstream cigarette smoke suppresses allergic airway inflammation by inhibiting T cell function

Epidemiological studies have identified childhood exposure to environmental tobacco smoke as a significant risk factor for the onset and exacerbation of asthma, but studies of smoking in adults are less conclusive, and mainstream cigarette smoke (MCS) has been reported to both enhance and attenuate allergic airway inflammation in animal models. We sensitized mice to ovalbumin (OVA) and exposed them to MCS in a well-characterized exposure system. Exposure to MCS (600 mg/m(3) total suspended particulates, TSP) for 1 h/day suppresses the allergic airway response, with reductions in eosinophilia, tissue inflammation, goblet cell metaplasia, IL-4 and IL-5 in bronchoalveolar lavage (BAL) fluid, and OVA-specific antibodies. Suppression is associated with a loss of antigen-specific proliferation and cytokine production by T cells. However, exposure to a lower dose of MCS (77 mg/m(3) TSP) had no effect on the number of BAL eosinophils or OVA-specific antibodies. This is the first report to demonstrate, using identical smoking methodologies, that MCS inhibits immune responses in a dose-dependent manner and may explain the observation that, although smoking provokes a systemic inflammatory response, it also inhibits T cell-mediated responses involved in a number of diseases.