Innate immune response to adenoviral vectors is mediated by both Toll-like receptor-dependent and -independent pathways

Recombinant adenoviral vectors have been widely used for gene therapy applications and as vaccine vehicles for treating infectious diseases such as human immunodeficiency virus disease. The innate immune response to adenoviruses represents the most significant hurdle in clinical application of adenoviral vectors for gene therapy, but it is an attractive feature for vaccine development. How adenovirus activates innate immunity remains largely unknown. Here we showed that adenovirus elicited innate immune response through the induction of high levels of type I interferons (IFNs) by both plasmacytoid dendritic cells (pDCs) and non-pDCs such as conventional DCs and macrophages. The innate immune recognition of adenovirus by pDCs was mediated by Toll-like receptor 9 (TLR9) and was dependent on MyD88, whereas that by non-pDCs was TLR independent through cytosolic sensing of adenoviral DNA. Furthermore, type I IFNs were pivotal in innate and adaptive immune responses to adenovirus in vivo, and type I IFN blockade diminished immune responses, resulting in more stable transgene expression and reduction of inflammation. These findings indicate that adenovirus activates innate immunity by its DNA through TLR-dependent and -independent pathways in a cell type-specific fashion, and they highlight a critical role for type I IFNs in innate and adaptive immune responses to adenoviral vectors. Our results that suggest strategies to interfere with type I IFN pathway may improve the outcome of adenovirus-mediated gene therapy, whereas approaches to activate the type I IFN pathway may enhance vaccine potency.     

Microglia-mediated nitric oxide cytotoxicity of T cells following amyloid beta-peptide presentation to Th1 cells

Alzheimer's disease is marked by progressive accumulation of amyloid beta-peptide (Abeta) which appears to trigger neurotoxic and inflammatory cascades. Substantial activation of microglia as part of a local innate immune response is prominent at sites of Abeta plaques in the CNS. However, the role of activated microglia as Abeta APCs and the induction of adaptive immune responses has not been investigated. We have used primary microglial cultures to characterize Abeta-Ag presentation and interaction with Abeta-specific T cells. We found that IFN-gamma-treated microglia serve as efficient Abeta APCs of both Abeta1-40 and Abeta1-42, mediating CD86-dependent proliferation of Abeta-reactive T cells. When cultured with Th1 and Th2 subsets of Abeta-reactive T cells, Th1, but not Th2, cells, underwent apoptosis after stimulation, which was accompanied by increased levels of IFN-gamma, NO, and caspase-3. T cell apoptosis was prevented in the presence of an inducible NO synthase type 2 inhibitor. Microglia-mediated proliferation of Abeta-reactive Th2 cells was associated with expression of the Th2 cytokines IL-4 and IL-10, which counterbalanced the toxic levels of NO induced by Abeta. Our results demonstrate NO-dependent apoptosis of T cells by Abeta-stimulated microglia which may enhance CNS innate immune responses and neurotoxicity in Alzheimer's disease. Secretion of NO by stimulated microglia may underlie a more general pathway of T cell death in the CNS seen in neurodegenerative diseases. Furthermore, Th2 type T cell responses may have a beneficial effect on this process by down-regulation of NO and the proinflammatory environment.     

Modulation of innate and adaptive immune responses by tofacitinib (CP-690,550)

Inhibitors of the JAK family of nonreceptor tyrosine kinases have demonstrated clinical efficacy in rheumatoid arthritis and other inflammatory disorders; however, the precise mechanisms by which JAK inhibition improves inflammatory immune responses remain unclear. In this study, we examined the mode of action of tofacitinib (CP-690,550) on JAK/STAT signaling pathways involved in adaptive and innate immune responses. To determine the extent of inhibition of specific JAK/STAT-dependent pathways, we analyzed cytokine stimulation of mouse and human T cells in vitro. We also investigated the consequences of CP-690,550 treatment on Th cell differentiation of naive murine CD4(+) T cells. CP-690,550 inhibited IL-4-dependent Th2 cell differentiation and interestingly also interfered with Th17 cell differentiation. Expression of IL-23 receptor and the Th17 cytokines IL-17A, IL-17F, and IL-22 were blocked when naive Th cells were stimulated with IL-6 and IL-23. In contrast, IL-17A production was enhanced when Th17 cells were differentiated in the presence of TGF-β. Moreover, CP-690,550 also prevented the activation of STAT1, induction of T-bet, and subsequent generation of Th1 cells. In a model of established arthritis, CP-690,550 rapidly improved disease by inhibiting the production of inflammatory mediators and suppressing STAT1-dependent genes in joint tissue. Furthermore, efficacy in this disease model correlated with the inhibition of both JAK1 and JAK3 signaling pathways. CP-690,550 also modulated innate responses to LPS in vivo through a mechanism likely involving the inhibition of STAT1 signaling. Thus, CP-690,550 may improve autoimmune diseases and prevent transplant rejection by suppressing the differentiation of pathogenic Th1 and Th17 cells as well as innate immune cell signaling.     

Splenic stromal microenvironment negatively regulates virus-activated plasmacytoid dendritic cells through TGF-beta

Plasmacytoid dendritic cells (pDCs) secrete large amounts of IFN-alpha upon exposure to virus, subsequently promoting and regulating innate and adaptive immune responses. However, little is known about the functional regulation of virus-activated pDCs after they exert functions in secondary lymph organs. Our previous studies show that splenic stromal microenvironment can down-regulate the T cell response by inducing generation of regulatory myeloid dendritic cells; therefore, we wondered whether the splenic stromal microenvironment can regulate the function of virus-activated pDCs. In this study, we provide evidences that the splenic stromal microenvironment can chemoattract vesicular stomatitis virus (VSV)-activated pDCs via stromal cell-derived factor 1 (SDF-1), inhibit the secretion of IFN-alpha, IL-12, TNF-alpha, and expression of I-Ab, CD86, CD80, and CD40 by VSV-activated pDCs, and subsequently inhibit VSV-infected pDCs to activate NK cell IFN-gamma production and cytotoxicity. Stroma-derived TGF-beta participates in the negative regulation of VSV-activated pDCs. Therefore, we demonstrate that splenic stromal microenvironment negatively regulates the virus-activated pDCs through TGF-beta, outlining an additional mechanistic explanation for maintenance of immune homeostasis.     

IL-4-dependent Th2 collateral priming to inhaled antigens independent of Toll-like receptor 4 and myeloid differentiation factor 88

Allergic asthma is an inflammatory lung disease thought to be initiated and directed by type 2 helper T cells responding to environmental Ags. The mechanisms by which allergens induce Th2-adaptive immune responses are not well understood, although it is now clear that innate immune signals are required to promote DC activation and Th2 sensitization to inhaled proteins. However, the effect of ongoing Th2 inflammation, as seen in chronic asthma, on naive lymphocyte activation has not been explored. It has been noted that patients with atopic disorders demonstrate an increased risk of developing sensitivities to new allergens. This suggests that signals from an adaptive immune response may facilitate sensitization to new Ags. We used a Th2-adoptive transfer murine model of asthma to identify a novel mechanism, termed "collateral priming," in which naive CD4(+) T cells are activated by adaptive rather than innate immune signals. Th2 priming to newly encountered Ags was dependent on the production of IL-4 by the transferred Th2 population but was independent of Toll-like receptor 4 signaling and the myeloid differentiation factor 88 Toll-like receptor signaling pathway. These results identify a novel mechanism of T cell priming in which an Ag-specific adaptive immune response initiates distinct Ag-specific T cell responses in the absence of classical innate immune system triggering signals.     

c-di-GMP Enhances Protective Innate Immunity in a Murine Model of Pertussis

Innate immunity represents the first line of defense against invading pathogens in the respiratory tract. Innate immune cells such as monocytes, macrophages, dendritic cells, NK cells, and granulocytes contain specific pathogen-recognition molecules which induce the production of cytokines and subsequently activate the adaptive immune response. c-di-GMP is a ubiquitous second messenger that stimulates innate immunity and regulates biofilm formation, motility and virulence in a diverse range of bacterial species with potent immunomodulatory properties. In the present study, c-di-GMP was used to enhance the innate immune response against pertussis, a respiratory infection mainly caused by Bordetella pertussis. Intranasal treatment with c-di-GMP resulted in the induction of robust innate immune responses to infection with B. pertussis characterized by enhanced recruitment of neutrophils, macrophages, natural killer cells and dendritic cells. The immune responses were associated with an earlier and more vigorous expression of Th1-type cytokines, as well as an increase in the induction of nitric oxide in the lungs of treated animals, resulting in significant reduction of bacterial numbers in the lungs of infected mice. These results demonstrate that c-di-GMP is a potent innate immune stimulatory molecule that can be used to enhance protection against bacterial respiratory infections. In addition, our data suggest that priming of the innate immune system by c-di-GMP could further skew the immune response towards a Th1 type phenotype during subsequent infection. Thus, our data suggest that c-di-GMP might be useful as an adjuvant for the next generation of acellular pertussis vaccine to mount a more protective Th1 phenotype immune response, and also in other systems where a Th1 type immune response is required.     

Influence of Human Papillomavirus E7 Oncoprotein on Maturation and Function of Plasmacytoid Dendritic Cells In Vitro

The major difficulties of human papillomavirus(HPV) treatment are its persistence and recurrence. The HPV E7 oncoprotein-loaded dendritic cells have been evaluated as cellular vaccine in previous reports. Plasmacytoid dendritic cells(pDCs) play an essential role of connecting the innate immune response and adaptive immune response in the immune system. But they function in HPV E7 loading is unclear. To investigate whether loading of the HPV type 6b, 11, and 16 E7 proteins affects the activity of pDCs, human peripheral blood-separated pDCs and mouse bone marrow-derived pDCs were pulsed with the HPV E7 proteins. The expression levels of CD40, CD80, CD86, and MHC II were significantly upregulated in pDCs upon HPV 6b/11 E7 protein pulse. The secretion and gene expression of type I IFN and IL-6 were both upregulated by HPV 6b/11 E7 proteins, more significant than HPV 16 E7 protein. The expression of essential factors of TLR signaling pathway and JNK/p38 MAP kinase signaling pathway were all increased in HPV 6b/11 E7 proteins pulsed pDCs. Our results suggest that HPV E7 proteins could promote the differentiation and maturation of pDCs and activate the TLR and MAPK pathway to induce host innate immune response. It might be conducive to explore novel immunotherapy targeting HPV infection with HPV E7 loaded pDC.     

Cutting Edge: Plasmacytoid dendritic cells provide innate immune protection against mucosal viral infection in situ

Dendritic cells (DCs) are powerful APCs capable of activating naive lymphocytes. Of the DC subfamilies, plasmacytoid DCs (pDCs) are unique in that they secrete high levels of type I IFNs in response to viruses but their role in inducing adaptive immunity remains divisive. In this study, we examined the importance of pDCs and their ability to recognize a virus through TLR9 in immunity against genital HSV-2 infection. We show that a low number of pDCs survey the vaginal mucosa at steady state. Upon infection, pDCs are recruited to the vagina and produce large amounts of type I IFNs in a TLR9-dependent manner and suppress local viral replication. Although pDCs are critical in innate defense against genital herpes challenge, adaptive Th1 immunity developed normally in the absence of pDCs. Thus, by way of migrating directly into the peripheral mucosa, pDCs act strictly as innate antiviral effector cells against mucosal viral infection in situ.     

Modulation of TNFSF expression in lymphoid tissue inducer cells by dendritic cells activated with Toll-like receptor ligands

Toll-like receptors (TLRs), which recognize structurally conserved components among pathogens, are mainly expressed by antigen-presenting cells such as dendritic cells (DCs), B cells, and macrophages. Recognition through TLRs triggers innate immune responses and influences antigen-specific adaptive immune responses. Although studies on the expression and functions of TLRs in antigen-presenting cells have been extensively reported, studies in lymphoid tissue inducer (LTi) cells have been limited. In this study, we observed that LTi cells expressed TLR2 and TLR4 mRNA as well as TLR2 protein and upregulated OX40L, CD30L, and TRANCE expression after stimulation with the TLR2 ligand zymosan or TLR4 ligand LPS. The expression of tumor necrosis factor superfamily (TNFSF) members was significantly upregulated when cells were cocultured with DCs, suggesting that upregulated TNFSF expression may contribute to antigen-specific adaptive immune responses.     

Plasmacytoid dendritic cells in antiviral immunity and autoimmunity

Plasmacytoid dendritic cells (pDCs) represent a unique and crucial immune cell population capable of producing large amounts of type I interferons (IFNs) in response to viral infection. The function of pDCs as the professional type I IFN-producing cells is linked to their selective expression of Toll-like receptor 7 (TLR7) and TLR9, which sense viral nucleic acids within the endosomal compartments. Type I IFNs produced by pDCs not only directly inhibit viral replication but also play an essential role in linking the innate and adaptive immune system. The aberrant activation of pDCs by self nucleic acids through TLR signaling and the ongoing production of type I IFNs do occur in some autoimmune diseases. Therefore, pDC may serve as an attractive target for therapeutic manipulations of the immune system to treat viral infectious diseases and autoimmune diseases.     

Cutting edge: Peyer's patch plasmacytoid dendritic cells (pDCs) produce low levels of type I interferons: possible role for IL-10, TGFbeta, and prostaglandin E2 in conditioning a unique mucosal pDC phenotype

The organized lymphoid tissues of the intestine likely play an important role in the balance between tolerance harmless mucosal Ags and commensal bacteria and immunity to mucosal pathogens. We examined the phenotype and function of plasmacytoid dendritic cells (pDCs) from murine Peyer's patches (PPs). When stimulated with CpG-enriched oligodeoxynucleotides in vitro, PPs and spleen pDCs made equivalent levels of IL-12, yet PP pDCs were incapable of producing significant levels of type I IFNs. Three regulatory factors associated with mucosal tissues, PGE(2), IL-10, and TGFbeta, inhibited the ability of spleen pDCs to produce type I IFN in a dose-dependent fashion. These studies suggest that mucosal factors may regulate the production of type I IFN as well as IL-12 by pDCs. In the intestine, this may be beneficial in preventing harmful innate and adaptive immune responses to commensal microorganisms.     

Ligation of Notch receptors in human conventional and plasmacytoid dendritic cells differentially regulates cytokine and chemokine secretion and modulates Th cell polarization

Notch signaling is involved in multiple cellular processes. Recent data also support the prominent role of Notch signaling in the regulation of the immune response. In this study, we analyzed the expression and function of Notch receptors and ligands on both human blood conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs). The expression and modulation upon TLR activation of Notch molecules partially differed between cDCs and pDCs, but functional involvement of the Notch pathway in both cell types was clearly revealed by specific inhibition using DAPT. Beyond the induction of Notch target genes and modulation of maturation markers, Notch pathway was also involved in a differential secretion of some specific cytokines/chemokines by DC subsets. Whereas Notch ligation induced IL-10 and CCL19 secretion in cDCs, Notch inhibition resulted in a diminished production of these proteins. With regard to pDCs, Notch activation induced TNF-α whereas Notch inhibition significantly abrogated the secretion of CCL19, CXCL9, CXCL10, and TNF-α. Additionally, Notch modulation of DC subsets differentially affected Th polarization of allostimulated T cells. Our results suggest that the Notch pathway may function as an additional mechanism controlling human DC responses, with differential activity on cDCs and pDCs. This control mechanism may ultimately contribute to define the local milieu promoted by these cells under the particular conditions of the immune response.     

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide: players in innate and adaptive immunity.

Recent reports identified and described neural pathways, both hard-wiring and soluble mediators, that control and adjust the peripheral immune response. Immune organs are innervated by fibers rich in neurotransmitters and neuropeptides released in inflammatory conditions. Here we focus on the immunomodulatory role of two peptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP). VIP/PACAP are present and released from both innervation and immune cells, particularly Th2 cells, and immune cells express receptors for VIP/PACAP. VIP/PACAP have a general anti-inflammatory effect, both in innate and adaptive immunity. In innate immunity, VIP/PACAP inhibit the production of pro-inflammatory cytokines and chemokines from macrophages, microglia and dendritic cells. In addition, VIP/PACAP reduce the expression of costimulatory molecules (particularly CD80 and CD86) on the antigen-presenting cells, and therefore reduce stimulation of antigen-specific CD4+ T cells. In terms of adaptive immunity, VIP/PACAP promote Th2-type responses, and reduce the pro-inflammatory Th1-type responses. Several of the molecular mechanisms involved in the inhibition of cytokine and chemokine expression, and in the preferential development and/or survival of Th2 effects, are discussed.     

MyD88-Dependent Signaling Contributes to Host Defense against Ehrlichial Infection

The ehrlichiae are small Gram-negative obligate intracellular bacteria in the family Anaplasmataceae. Ehrlichial infection in an accidental host may result in fatal diseases such as human monocytotropic ehrlichiosis, an emerging, tick-borne disease. Although the role of adaptive immune responses in the protection against ehrlichiosis has been well studied, the mechanism by which the innate immune system is activated is not fully understood. Using Ehrlichia muris as a model organism, we show here that MyD88-dependent signaling pathways play a pivotal role in the host defense against ehrlichial infection. Upon E. muris infection, MyD88-deficient mice had significantly impaired clearance of E. muris, as well as decreased inflammation, characterized by reduced splenomegaly and recruitment of macrophages and neutrophils. Furthermore, MyD88-deficient mice produced markedly lower levels of IL-12, which correlated well with an impaired Th1 immune response. In vitro, dendritic cells, but not macrophages, efficiently produced IL-12 upon E. muris infection through a MyD88-dependent mechanism. Therefore, MyD88-dependent signaling is required for controlling ehrlichial infection by playing an essential role in the immediate activation of the innate immune system and inflammatory cytokine production, as well as in the activation of the adaptive immune system at a later stage by providing for optimal Th1 immune responses.     

VIP balances innate and adaptive immune responses induced by specific stimulation of TLR2 and TLR4

Crohn's disease (CD) is a chronic intestinal inflammatory pathology, which develops as a result of innate immune signals, such as the activation of Toll-like receptors (TLRs), and adaptive immune signals, including Th1 cytokine release. We have recently demonstrated in TNBS-induced colitis, a murine model of CD, that VIP plays a homeostatic role, by reducing TNBS-induced TLR2 and TLR4 expression to control levels. The purpose of this paper is to elucidate for the first time, the physiological relevance of VIP specific control of innate and adaptive immune responses through TLR2 and TLR4 ligands. In addition, we investigated the effect of VIP on regulatory activity of T regulatory (Treg) cells in the TNBS-colitis model. First, we found that VIP downregulated the inflammatory response elicited in mesenteric lymph node cell cultures by treatment with the TLR2 ligand Pam3Cys, or the TLR4 ligand lipopolysaccharide (LPS), reducing the production of the chemokine CXCL1. Also, treatment with VIP impaired the induction of Th1 responses by decreasing p70 interleukin (IL)-12 and interferon gamma (IFN-γ) levels after TLR2/TLR4 stimulation in culture. Besides, VIP treatment restored in vivo the numbers of TLR2 and TLR4 positive CD4⁺CD25⁺ T lymphocytes, augmented by TNBS administration, and increased the expression of molecules involved in regulatory T cell function, such as Foxp3 and TGF-β. In conclusion, the ability of VIP to down-regulate uncontrolled inflammation by targeting TLR-mediated responses and regulatory T cell activity could be used as a new alternative therapy for intestinal inflammatory/autoimmune disorders.     

Cationic amino acid transporter 2 enhances innate immunity during Helicobacter pylori infection

Once acquired, Helicobacter pylori infection is lifelong due to an inadequate innate and adaptive immune response. Our previous studies indicate that interactions among the various pathways of arginine metabolism in the host are critical determinants of outcomes following infection. Cationic amino acid transporter 2 (CAT2) is essential for transport of L-arginine (L-Arg) into monocytic immune cells during H. pylori infection. Once within the cell, this amino acid is utilized by opposing pathways that lead to elaboration of either bactericidal nitric oxide (NO) produced from inducible NO synthase (iNOS), or hydrogen peroxide, which causes macrophage apoptosis, via arginase and the polyamine pathway. Because of its central role in controlling L-Arg availability in macrophages, we investigated the importance of CAT2 in vivo during H. pylori infection. CAT2(-/-) mice infected for 4 months exhibited decreased gastritis and increased levels of colonization compared to wild type mice. We observed suppression of gastric macrophage levels, macrophage expression of iNOS, dendritic cell activation, and expression of granulocyte-colony stimulating factor in CAT2(-/-) mice suggesting that CAT2 is involved in enhancing the innate immune response. In addition, cytokine expression in CAT2(-/-) mice was altered from an antimicrobial Th1 response to a Th2 response, indicating that the transporter has downstream effects on adaptive immunity as well. These findings demonstrate that CAT2 is an important regulator of the immune response during H. pylori infection.     

Distinctive TLR7 signaling, type I IFN production, and attenuated innate and adaptive immune responses to yellow fever virus in a primate reservoir host

Why cross-species transmissions of zoonotic viral infections to humans are frequently associated with severe disease when viruses responsible for many zoonotic diseases appear to cause only benign infections in their reservoir hosts is unclear. Sooty mangabeys (SMs), a reservoir host for SIV, do not develop disease following SIV infection, unlike nonnatural HIV-infected human or SIV-infected rhesus macaque (RM) hosts. SIV infections of SMs are characterized by an absence of chronic immune activation, in association with significantly reduced IFN-α production by plasmacytoid dendritic cells (pDCs) following exposure to SIV or other defined TLR7 or TLR9 ligands. In this study, we demonstrate that SM pDCs produce significantly less IFN-α following ex vivo exposure to the live attenuated yellow fever virus 17D strain vaccine, a virus that we show is also recognized by TLR7, than do RM or human pDCs. Furthermore, in contrast to RMs, SMs mount limited activation of innate immune responses and adaptive T cell proliferative responses, along with only transient antiviral Ab responses, following infection with yellow fever vaccine 17D strain. However, SMs do raise significant and durable cellular and humoral immune responses comparable to those seen in RMs when infected with modified vaccinia Ankara, a virus whose immunogenicity does not require TLR7/9 recognition. Hence, differences in the pattern of TLR7 signaling and type I IFN production by pDCs between primate species play an important role in determining their ability to mount and maintain innate and adaptive immune responses to specific viruses, and they may also contribute to determining whether disease follows infection.     

Regulation of TLR7/9 signaling in plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs), also known as type I interferon (IFN)-producing cells, are specialized immune cells characterized by their extraordinary capabilities of mounting rapid and massive type I IFN response to nucleic acids derived from virus, bacteria or dead cells. PDCs selectively express endosomal Toll-like receptor (TLR) 7 and TLR9, which sense viral RNA and DNA respectively. Following type I IFN and cytokine responses, pDCs differentiate into antigen presenting cells and acquire the ability to regulate T cell-mediated adaptive immunity. The functions of pDCs have been implicated not only in antiviral innate immunity but also in immune tolerance, inflammation and tumor microenvironments. In this review, we will focus on TLR7/9 signaling and their regulation by pDC-specific receptors.