Respiratory infection with a bacterial pathogen attenuates CNS autoimmunity through IL-10 induction.

Infection with viral or bacterial pathogens has been linked with the development of multiple sclerosis (MS), while infection with helminth parasites has been associated protection against MS and other autoimmune diseases. Here we have used a murine model of MS, experimental autoimmune encephalomyelitis (EAE), to examine the effect of infection with the respiratory pathogen Bordetella pertussis infection on development of CNS inflammation. The data demonstrate that infection of mice with B. pertussis significantly attenuates the clinical course of EAE induced by active immunization or cell transfer. This was reflected in a significant reduction in VLA-4 and LFA-1 expression on T cells and infiltration of IL-17(+), IFN-γ(+) and IFN-γ(+)IL-17(+) CD4 T cells into the CNS. Infection with B. pertussis induced IL-10 production from dendritic cells in vitro and enhanced the frequency of IL-10-producing CD25(-)Foxp3(+/-) CD4(+) T cells in vivo. Furthermore, the suppressive effects of B. pertussis infection on EAE were lost in IL-10(-/-) mice. Our findings demonstrate that a bacterial infection of the respiratory tract can attenuate EAE by promoting production of the anti-inflammatory cytokine IL-10 that may suppress licensing of autoaggressive T cells in the lungs, thereby preventing their migration into the CNS.

A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases.

The NOD-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome is a component of the inflammatory process, and its aberrant activation is pathogenic in inherited disorders such as cryopyrin-associated periodic syndrome (CAPS) and complex diseases such as multiple sclerosis, type 2 diabetes, Alzheimer's disease and atherosclerosis. We describe the development of MCC950, a potent, selective, small-molecule inhibitor of NLRP3. MCC950 blocked canonical and noncanonical NLRP3 activation at nanomolar concentrations. MCC950 specifically inhibited activation of NLRP3 but not the AIM2, NLRC4 or NLRP1 inflammasomes. MCC950 reduced interleukin-1ß (IL-1ß) production in vivo and attenuated the severity of experimental autoimmune encephalomyelitis (EAE), a disease model of multiple sclerosis. Furthermore, MCC950 treatment rescued neonatal lethality in a mouse model of CAPS and was active in ex vivo samples from individuals with Muckle-Wells syndrome. MCC950 is thus a potential therapeutic for NLRP3-associated syndromes, including autoinflammatory and autoimmune diseases, and a tool for further study of the NLRP3 inflammasome in human health and disease.

Conjugated linoleic acid suppresses dendritic cell activation and subsequent Th17 responses.

PUFAs (polyunsaturated fatty acids) can modify immune responses, so they may have potential therapeutic effects in inflammatory disorders. We previously demonstrated that the cis-9, trans-11 isomer of the PUFA conjugated linoleic acid (CLA) can modulate dendritic cell (DC) cytokine production. Since DCs play a central role in initiating inflammation by directing T helper (Th) cell differentiation, here we examined the effects of CLA on DC maturation and migration and the subsequent generation of Th cell responses. We examined the effect of CLA in vitro on the function of lipopolysaccharide (LPS)-activated bone marrow-derived DCs and ex vivo using cells from mice with high levels of CLA in their diet. We report that CLA inhibits DC migration and modulates TLR-induced production of key cytokines involved in Th cell differentiation both in vitro and in vivo. These changes were accompanied by a significant decrease in expression of MHCII, CD80 and CD86 on the DC surface. Exposure of DCs to CLA suppressed their ability to promote differentiation of naïve T cells into Th1 and/or Th17 cells in vitro and following their adoptive transfer in vivo. Furthermore, in a murine model of endotoxic shock, treatment with CLA suppressed LPS-induced induction of circulating IFN-γ, IL-12p40 and IL-1ß. This is the first study to demonstrate that exposure of antigen-presenting cells to CLA can modulate the subsequent Th cell response, and the findings may explain some of the beneficial effects of c9, t11-CLA in inflammatory diseases mediated by Th1 and Th17 cells.

Respiratory infection promotes T cell infiltration and amyloid-ß deposition in APP/PS1 mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by deposits of amyloid-ß and neurofibrillary tangles. It has been suggested that inflammatory changes are associated with disease; however, it has not been established whether these are a consequence of ongoing neurodegeneration or whether inflammation itself contributes to disease pathogenesis. Recent studies suggest that exposure to infection can accelerate cognitive decline in AD patients, and pathogens have been detected in the AD brain. However, the influence of infection on neuroinflammation and pathology remains poorly understood. In this study, we examined the effect of a peripheral infection on AD pathology in APP/PS1 mice. We found that, 8 weeks after infection with the Gram negative respiratory pathogen Bordetella pertussis, there was significant infiltration of IFNγ- and IL-17-producing T cells and NKT cells in older APP/PS1 mice. This was accompanied by increased glial activation and amyloid-ß deposition. The data suggest that infection is a critical factor in the progression of AD, emphasising the importance of early diagnosis and treatment of infections in elderly individuals.

Blocking retinoic acid receptor-α enhances the efficacy of a dendritic cell vaccine against tumours by suppressing the induction of regulatory T cells.

The immune system has evolved regulatory mechanisms to control immune responses to self-antigens. Regulatory T (Treg) cells play a pivotal role in maintaining immune tolerance, but tumour growth is associated with local immunosuppression, which can subvert effector immune responses. Indeed, the induction and recruitment of Treg cells by tumours is a major barrier in the development of effective immunotherapeutics and vaccines against cancer. Retinoic acid (RA) has been shown to promote conversion of naïve T cells into Treg cells. This study addresses the hypothesis that blocking RA receptor alpha (RARα) may enhance the efficacy of a tumour vaccine by inhibiting the induction of Treg cells. We found that RA significantly enhanced TGF-ß-induced expression of Foxp3 on naïve and committed T cells in vitro and that this was blocked by an antagonist of RARα (RARi). In addition, RARi significantly suppressed TGF-ß and IL-10 and enhanced IL-12 production by dendritic cells (DC) in response to killed tumour cells or TLR agonists. Furthermore, RARi augmented the efficacy of an antigen-pulsed and TLR-activated DC vaccine, significantly attenuating growth of B16 tumours in vivo and enhancing survival of mice. This protective effect was associated with significant reduction in tumour-infiltrating FoxP3(+) and IL-10(+) Treg cells and a corresponding increase in tumour-infiltrating CD4(+) and CD8(+) T cells that secreted IFN-γ. Our findings demonstrate that RARα is an important target for the development of effective anti-tumour immunotherapeutics and for improving the efficacy of cancer vaccines.

Gene silencing of TGF-ß1 enhances antitumor immunity induced with a dendritic cell vaccine by reducing tumor-associated regulatory T cells.

Active immunotherapy and cancer vaccines that promote host antitumor immune responses promise to be effective and less toxic alternatives to current cytotoxic drugs for the treatment of cancer. However, the success of tumor immunotherapeutics and vaccines is dependent on identifying approaches for circumventing the immunosuppressive effects of regulatory T (Treg) cells induced by the growing tumor and by immunotherapeutic molecules, including Toll-like receptor (TLR) agonists. Here, we show that tumors secrete high concentrations of active TGF-ß1, a cytokine that can convert naive T cells into Foxp3+ Treg cells. Silencing TGF-ß1 mRNA using small interfering RNA (siRNA) in tumor cells inhibited active TGF-ß1 production in vitro and restrained their growth in vivo. Prophylactic but not therapeutic administration of TGF-ß1 siRNA reduced the growth of CT26 tumors in vivo. Furthermore, suppressing TGF-ß1 expression at the site of a tumor, using siRNA before, during and after therapeutic administration of a TLR-activated antigen-pulsed dendritic cell vaccine significantly reduced the growth of B16 melanoma in mice. The protective effect of co-administering TGF-ß1 siRNA with the DC vaccine was associated with suppression of CD25+ Foxp3+ and CD25+ IL-10+ T cells and enhancement of tumor infiltrating CD4 and CD8 T cells. Our findings suggest that transient suppression of TGF-ß1 may be a promising approach for enhancing the efficacy of tumor vaccines in humans.

A deep rough type structure in Bordetella bronchiseptica lipopolysaccharide modulates host immune responses.

The present authors have previously obtained the Bordetella bronchiseptica mutant BbLP39, which contains a deep-rough lipopolysaccharide (LPS) instead the wild type smooth LPS with O antigen. This mutant was found to be altered in the expression of some proteins and in its ability to colonize mouse lungs. Particularly, in BbLP39 the expression of pertactin is decreased. To differentiate the contribution of each bacterial component to the observed phenotype, here mice defective in the LPS sensing receptor TLR4 (TLR4-defective mice) were used. In contrast to wild-type mice, infection of TLR4-defective mice with BbLP39 resulted in lung infection, which persisted for more than 10 days post-challenge. Comparative analysis of the immune responses induced by purified mutant and wild type LPSs showed that the mutant LPS induced significantly higher degrees of expression of TNF-α and IL-10 mRNA than did the wild type. UV matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry analysis revealed that both LPSs had the same penta-acylated lipid A structure. However, the lipid A from BbLP39 contained pyrophosphate instead of phosphate at position 1. This structural difference, in addition to the lack of O-antigen in BbLP39, may explain the functional differences between BbLP39 and wild type strains.

IL-27 mediates the response to IFN-ß therapy in multiple sclerosis patients by inhibiting Th17 cells.

Interferon (IFN)-ß is a commonly used therapy for relapsing remitting multiple sclerosis (RRMS). However its protective mechanism is still unclear and the failure of many patients to respond has not been explained. We have found that IFN-ß suppressed IL-23 and IL-1ß production and increased IL-10 production by human dendritic cells (DC) activated with the TLR2 and dectin-1 agonist zymosan. Furthermore, IFN-ß impaired the ability of DC to promote IL-17 production by CD4(+) T cells, but did not affect IFN-γ production. IFN-ß induced IL-27 expression by DC, and neutralisation of IL-27 abrogated the suppressive effects of IFN-ß on zymosan-induced IL-1 and IL-23 production and the generation of Th17 cells in vitro. Complementary in vivo studies in a mouse model showed that treatment with IFN-ß enhanced expression of IL-27, and reduced IL-17 in the CNS and periphery and attenuated the clinical signs of experimental autoimmune encephalomyelitis (EAE). In addition, the significant suppressive effect of IFN-ß on the ability of DC to promote Th17 cells was lost in cells from IL-27 receptor deficient mice. Finally, we showed that PBMC from non-responder RRMS patients produced significantly less IL-27 in response to IFN-ß than patients who responded to IFN-ß therapy. Our findings suggest that IFN-ß mediates its therapeutic effects in MS at least in part via the induction of IL-27, and that IL-27 may represent an alternative therapy for MS patients that do not respond to IFN-ß.

The role of regulatory T cells in respiratory infections and allergy and asthma.

The role of distinct CD4(+) T-cell populations in regulating the nature and strength of immune responses is well documented and in the past has principally focused on the cross-regulation of T-helper type 1 (Th1) and Th2 cells, which secrete interferon-gamma and interleukin-4, respectively. However, the identification of T cells capable of suppressing responses mediated by Th1 and Th2 cells, termed regulatory T cells (Treg cells), has prompted a paradigm shift in our understanding of the regulation of immune responses to infection and environmental antigens. This article focuses on the role of Treg cells in the lungs following infection with respiratory pathogens and discusses the targeting of Treg cells in the development of new therapies for immune-mediated respiratory diseases, such as allergy and asthma.

TLR, NLR Agonists, and Other Immune Modulators as Infectious Disease Vaccine Adjuvants.

Vaccines based on attenuated or killed viruses and bacteria are highly effective in preventing infection with a range of pathogens, but can have safety issues. Therefore, a move is underway toward the development of subunit vaccines based on recombinant proteins or naked DNA. However, protein subunit vaccines are typically poorly immunogenic when administered alone and therefore require coadministration with adjuvants to boost the immune response. For many decades, very little progress was made in understanding the mechanism of action of adjuvants, but recently several significant breakthroughs have occurred in this area. The binding of pathogen-derived molecules to different immune sensors, including Toll-like receptors (TLR), nucleotide-binding oligomerization domain-like receptors (NLR), and retinoic acid-inducible gene (RIG)-1-like receptors (RLR), activates important innate immune pathways and provides not only an understanding of how current vaccines and adjuvants work, but also potential targets for novel adjuvant development.

Therapeutic vaccination with dendritic cells pulsed with tumor-derived Hsp70 and a COX-2 inhibitor induces protective immunity against B16 melanoma.

Prophylactic immunization of mice with autologous tumor-derived heat shock proteins (Hsp) generates effective anti-tumor immunity. However, this approach is ineffective when used therapeutically, partly due to the immunosuppressive effects of the growing tumor. Here we sought to overcome this problem by therapeutic vaccination with dendritic cells (DC) pulsed with Hsp70 and a COX-2 inhibitor. We found that Hsp70 induces IL-6 and IL-10 production and suppressed expression of CD40 on DC. Incubation of DC with tumor-conditioned medium attenuated Hsp70-induced expression of CD80 and induced expression of COX-2. Inhibition of COX-2 partially reversed the stimulatory effect of Hsp70 on DC IL-6 and IL-10 production and enhanced expression of CD80 and MHC classes I and II. Therapeutic administration of DC pulsed in vitro with Hsp70 in the presence of a COX-2 inhibitor significantly reduced progression of B16 tumors in mice and significantly enhanced survival. This was associated with a reduction in the frequency of IL-10-producing CD4(+) T cells and enhancement of IFN-gamma-producing CD8(+) T cells. Our findings provide a novel immunotherapeutic approach against cancer based on attenuation of COX-2-mediated immunosuppression using in vitro modulated DC.

Bordetella pertussis expresses a functional type III secretion system that subverts protective innate and adaptive immune responses.

Certain bacteria use a type III secretion system (TTSS) to deliver effector proteins that interfere with cell function into host cells. While transcription of genes encoding TTSS components has been demonstrated, studies to date have failed to identify TTSS effector proteins in Bordetella pertussis. Here we present the first evidence of a functionally active TTSS in B. pertussis. Three known TTSS effectors, Bsp22, BopN, and BopD, were identified as TTSS substrates in B. pertussis 12743. We found expression of Bsp22 in a significant proportion of clinical isolates but not in common laboratory-adapted strains of B. pertussis. We generated a TTSS mutant of B. pertussis 12743 and showed that it induced significantly lower respiratory tract colonization in mice than the wild-type bacteria. Respiratory infection of mice with the mutant bacteria induced significantly greater innate proinflammatory cytokine production in the lungs soon after challenge, and this correlated with significantly higher antigen-specific interleukin-17, gamma interferon, and immunoglobulin G responses later in infection. Our findings suggest that the TTSS subverts innate and adaptive immune responses during infection of the lungs and may be a functionally important virulence factor for B. pertussis infection of humans.

TLR4 mediates vaccine-induced protective cellular immunity to Bordetella pertussis: role of IL-17-producing T cells.

Whole cell pertussis vaccines (Pw) induce Th1 responses and protect against Bordetella pertussis infection, whereas pertussis acellular vaccines (Pa) induce Ab and Th2-biased responses and also protect against severe disease. In this study, we show that Pw failed to generate protective immunity in TLR4-defective C3H/HeJ mice. In contrast, protection induced with Pa was compromised, but not completely abrogated, in C3H/HeJ mice. Immunization with Pw, but not Pa, induced a population of IL-17-producing T cells (Th-17), as well as Th1 cells. Ag-specific IL-17 and IFN-gamma production was significantly lower in Pw-immunized TLR4-defective mice. Furthermore, treatment with neutralizing anti-IL-17 Ab immediately before and after B. pertussis challenge significantly reduced the protective efficacy of Pw. Stimulation of dendritic cells (DC) with Pw promoted IL-23, IL-12, IL-1beta, and TNF-alpha production, which was impaired in DC from TLR4-defective mice. B. pertussis LPS, which is present in high concentrations in Pw, induced IL-23 production by DC, which enhanced IL-17 secretion by T cells, but the induction of Th-17 cells was also dependent on IL-1. In addition, we identified a new effector function for IL-17, activating macrophage killing of B. pertussis, and this bactericidal activity was less efficient in macrophages from TLR4-defective mice. These data provide the first definitive evidence of a role for TLRs in protective immunity induced by a human vaccine. Our findings also demonstrate that activation of innate immune cells through TLR4 helps to direct the induction of Th1 and Th-17 cells, which mediate protective cellular immunity to B. pertussis.

Regulatory cells and the control of respiratory infection.

The role of distinct CD4+ T-cell populations in regulating the nature and strength of immune responses is well documented, and has in the past principally focused on the mutual antagonism between Th1 and Th2 cells, which secrete interferon (IFN)-gamma and interleukin (IL)-4, respectively. However, the recent identification of T cells that secrete high levels of IL-10 and/or transforming growth factor-b, but not IFN-g or IL-4, called regulatory T (Tr) cells has prompted a paradigm shift in our understanding of the regulation of immune responses following infection. In this review, we focus on the role of antigen-specific Tr cells in the lungs following infection with various respiratory pathogens and discuss the targeting of Tr in the development of new therapies for immune-mediated diseases, such as allergy.

Effects of cholera toxin on innate and adaptive immunity and its application as an immunomodulatory agent.

Cholera toxin (CT) is a potent vaccine adjuvant when administered via parenteral, mucosal, or transcutaneous routes. It also inhibits innate inflammatory responses induced by pathogen-derived molecules, such as lipopolysaccharide (LPS). We demonstrated previously that CT promotes the induction of regulatory type 1 T cells (Tr1) as well as T helper type 2 cells (Th2). T cells from mice immunized with antigen in the presence of CT produced high levels of interleukin (IL)-10 and IL-5 and low levels of IL-4 and interferon-gamma (IFN-gamma). Here, we demonstrate that immunization with antigen in the presence of CT induced a population of antigen-specific CD4(+) T cells that produced IL-10 in the absence of IL-4, in addition to cells that coexpressed IL-4 and IL-10 or produced IL-4 only. CT-generated Tr1 cells inhibited antigen-specific proliferation as well as IFN-gamma production by Th1 cells, and this suppression was cell contact-independent. It is interesting that coincubation with Th1 cells significantly enhanced IL-10 production by the Tr1 cells. As IL-10 can promote the differentiation of Tr1 cells, we investigated cytokine production by dendritic cells (DC) following exposure to CT. Previous data showed that CT can modulate the expression of costimulatory molecules and inhibit the production of chemokines and cytokines, including IL-12 and tumor necrosis factor alpha and enhance IL-10 production. Here, we show that CT synergizes with LPS to induce IL-6 and IL-1beta in addition to IL-10 production by immature DC. Therefore, CT may promote the induction of Th2 and Tr1 cells in part via selective modulation of DC cytokine production and costimulatory molecule expression.

Toll-like receptor 4-mediated innate IL-10 activates antigen-specific regulatory T cells and confers resistance to Bordetella pertussis by inhibiting inflammatory pathology.

Signaling through Toll-like receptors (TLR) activates dendritic cell (DC) maturation and IL-12 production, which directs the induction of Th1 cells. We found that the production of IL-10, in addition to inflammatory cytokines and chemokines, was significantly reduced in DCs from TLR4-defective C3H/HeJ mice in response to Bordetella pertussis. TLR4 was also required for B. pertussis LPS-induced maturation of DCs, but other B. pertussis components stimulated DC maturation independently of TLR4. The course of B. pertussis infection was more severe in C3H/HeJ than in C3H/HeN mice. Surprisingly, Ab- and Ag-specific IFN-gamma responses were enhanced at the peak of infection, whereas Ag-specific IL-10-producing T cells were significantly reduced in C3H/HeJ mice. This was associated with enhanced inflammatory cytokine production, cellular infiltration, and severe pathological changes in the lungs of TLR4-defective mice. Our findings suggest that TLR-4 signaling activates innate IL-10 production in response to B. pertussis, which both directly, and by promoting the induction of IL-10-secreting type 1 regulatory T cells, may inhibit Th1 responses and limit inflammatory pathology in the lungs during infection with B. pertussis.

Cholera toxin promotes the induction of regulatory T cells specific for bystander antigens by modulating dendritic cell activation.

It has previously been reported that cholera toxin (CT) is a potent mucosal adjuvant that enhances Th2 or mixed Th1/Th2 type responses to coadministered foreign Ag. Here we demonstrate that CT also promotes the generation of regulatory T (Tr) cells against bystander Ag. Parenteral immunization of mice with Ag in the presence of CT induced T cells that secreted high levels of IL-4 and IL-10 and lower levels of IL-5 and IFN-gamma. Ag-specific CD4(+) T cell lines and clones generated from these mice had cytokine profiles characteristic of Th2 or type 1 Tr cells, and these T cells suppressed IFN-gamma production by Th1 cells. Furthermore, adoptive transfer of bone marrow-derived dendritic cells (DC) incubated with Ag and CT induced T cells that secreted IL-4 and IL-10 and low concentrations of IL-5. It has previously been shown that IL-10 promotes the differentiation or expansion of type 1 Tr cells. Here we found that CT synergized with low doses of LPS to induce IL-10 production by immature DC. CT also enhanced the expression of CD80, CD86, and OX40 (CD134) on DC and induced the secretion of the chemokine, macrophage inflammatory protein-2 (MIP-2), but inhibited LPS-driven induction of CD40 and ICAM-I expression and production of the inflammatory cytokines/chemokines IL-12, TNF-alpha, MIP-1alpha, MIP-1beta, and monocyte chemoattractant protein-1. Our findings suggest that CT induces maturation of DC, but, by inducing IL-10, inhibiting IL-12, and selectively affecting surface marker expression, suppresses the generation of Th1 cells and promotes the induction of T cells with regulatory activity.