TLR signaling mediated by MyD88 is required for a protective innate immune response by neutrophils to Citrobacter rodentium

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium are classified as attaching and effacing pathogens based on their ability to adhere to intestinal epithelium via actin-filled membranous protrusions (pedestals). Infection of mice with C. rodentium causes breach of the colonic epithelial barrier, a vigorous Th1 inflammatory response, and colitis. Ultimately, an adaptive immune response leads to clearance of the bacteria. Whereas much is known about the adaptive response to C. rodentium, the role of the innate immune response remains unclear. In this study, we demonstrate for the first time that the TLR adaptor MyD88 is essential for survival and optimal immunity following infection. MyD88(-/-) mice suffer from bacteremia, gangrenous mucosal necrosis, severe colitis, and death following infection. Although an adaptive response occurs, MyD88-dependent signaling is necessary for efficient clearance of the pathogen. Based on reciprocal bone marrow transplants in conjunction with assessment of intestinal mucosal pathology, repair, and cytokine production, our findings suggest a model in which TLR signaling in hemopoietic and nonhemopoietic cells mediate three distinct processes: 1) induction of an epithelial repair response that maintains the protective barrier and limits access of bacteria to the lamina propria; 2) production of KC or other chemokines that attract neutrophils and thus facilitate killing of bacteria; and 3) efficient activation of an adaptive response that facilitates Ab-mediated clearance of the infection. Taken together, these experiments provide evidence for a protective role of innate immune signaling in infections caused by attaching and effacing pathogens.     

Prophylactic and therapeutic use of antibodies for protection against respiratory infection with Francisella tularensis

The role of Abs in protection against respiratory infection with the intracellular bacterium Francisella tularensis is not clear. To investigate the ability of Abs to clear bacteria from the lungs and prevent systemic spread, immune serum was passively administered i.p. to naive mice before intranasal F. tularensis live vaccine strain infection. It was found that immune serum treatment provided 100% protection against lethal challenge while normal serum or Ig-depleted immune serum provided no protection. Protective efficacy was correlated with increased clearance of bacteria from the lung and required expression of FcgammaR on phagocytes, including macrophages and neutrophils. However, complement was not required for protection. In vitro experiments demonstrated that macrophages were more readily infected by Ab-opsonized bacteria but became highly efficient in killing upon activation by IFN-gamma. Consistent with this finding, in vivo Ab-mediated protection was found to be dependent upon IFN-gamma. SCID mice were not protected by passive Ab transfer, suggesting that T cells but not NK cells serve as the primary source for IFN-gamma. These data suggest that a critical interaction of humoral and cellular immune responses is necessary to provide sterilizing immunity against F. tularensis. Of considerable interest was the finding that serum Abs were capable of conferring protection against lethal respiratory tularemia when given 24-48 h postexposure. Thus, this study provides the first evidence for the therapeutic use of Abs in Francisella-infected individuals.     

TLR2-mediated activation of neutrophils in response to German cockroach frass

It is becoming increasingly clear that innate immune mediators play a role in regulating adaptive immune responses in asthma pathogenesis. Cockroach exposure is a major risk factor for the development of asthma. In this study we asked whether German cockroach (GC) feces (frass) could initiate an innate immune response. Naive BALB/c mice were challenged with a single intratracheal inhalation of GC frass. Proinflammatory cytokines were significantly increased in the bronchoalveolar lavage fluid at 3 h and were maintained at higher than baseline levels for at least 24 h. Neutrophil migration into the airways was evident as early as 3 h but was maximal between 6 and 24 h postinhalation. The early increase in cytokine expression was independent of TLR2 or TLR4. Newly infiltrated airway neutrophils were responsible for maintaining high levels of cytokines in the airways. Using neutrophils as an early marker of the innate immune response, we show that show that neutrophils isolated from the airways following GC frass inhalation express TLR2 and release cytokines. GC frass directly affected neutrophil cytokine production via TLR2, but not TLR4, as evidenced by the use of TLR-neutralizing Abs and neutrophils from TLR-deficient mice. Activation of cytokine expression occurred via GC frass-induced NF-kappaB translocation and DNA binding. These data show that GC frass contains a TLR2 agonist and, to our knowledge, this is the first report of an allergen directly activating cells of the innate immune system via TLR2 and suggests an important link between innate and adaptive immunity.     

Antigen delivery to plasmacytoid dendritic cells via BST2 induces protective T cell-mediated immunity

Plasmacytoid dendritic cells (PDCs) are capable of presenting Ags to T cells in a tolerogenic or immunogenic manner depending on the formulation of the Ag and the mode of stimulation. It has not been investigated whether effective adaptive immune responses useful for vaccination can be induced by Ab-mediated Ag targeting to PDCs in vivo. In this study, we show that Ag delivered to murine PDCs via bone marrow stromal cell Ag 2 (BST2)/CD317 in combination with TLR agonists as adjuvants is specifically presented by PDCs in vivo and elicits strong cellular and humoral immune responses. These include IFN-γ production by CD4(+) T cells and high Ab titers with a broad range of IgG isotypes. In addition, BST2-mediated Ag delivery in the presence of polyinosinic-polycytidylic acid as adjuvant induces cytotoxic T lymphocytes that are functional in vivo. A single immunization with Ag-fused anti-BST2 Ab together with polyinosinic-polycytidylic acid as adjuvant is sufficient to trigger protective immunity against subsequent viral infection and tumor growth. We conclude that despite the potential tolerogenic properties of PDCs, Ag targeting to PDCs in combination with TLR agonists as adjuvants is an effective vaccination strategy.     

Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus

The mammalian Toll-like receptor 4, TLR4, is an important component in the innate immune response to gram-negative bacterial infection. The role of TLR4 in antiviral immunity has been largely unexplored. In this study, the in vivo immune responses to respiratory syncytial virus (RSV) and influenza virus infection were examined in TLR4-deficient (C57BL/10ScNCr) and TLR4-expressing (C57BL/10Sn) mice. TLR4-deficient mice challenged with RSV, but not influenza virus, exhibited impaired natural killer (NK) cell and CD14(+) cell pulmonary trafficking, deficient NK cell function, impaired interleukin-12 expression, and impaired virus clearance compared to mice expressing TLR4. These findings suggest that Toll signaling pathways have an important role in innate immunity to RSV.     

A predominant role for antibody in acquired immunity to chlamydial genital tract reinfection

Acquired immunity to murine Chlamydia trachomatis genital tract reinfection has long been assumed to be solely dependent on cell-mediated immunity. However, in this study, we identify a previously unrecognized protective role for Ab. Immunity develops in Ab-deficient mice following the resolution of primary chlamydial genital infection. Subsequent depletion of CD4+ T cells, but not CD8+ T cells, in those immune Ab-deficient mice before secondary infectious challenge, resulted in an infection that did not resolve. Passive immunization with immune (convalescent) serum conferred a marked level of protective immunity to reinfection, which was characterized by a striking decrease in bacterial shedding, from >100,000 inclusion forming units to fewer than 10 inclusion forming units, and a shortened duration of infection. Furthermore, mAbs to the chlamydial major outer membrane protein and LPS conferred significant levels of immunity to reinfection and reduced chlamydial shedding by >100-fold. Anti-heat shock protein 60 mAb had no protective effect. In contrast to the marked protective efficacy of immune serum on reinfection, the course of primary infection was essentially unaltered by the passive transfer of immune serum. Our results convincingly demonstrate that Abs contribute importantly to immunity to chlamydial genital tract reinfection, and that Ab-mediated protection is highly dependent on CD4+ T cell-mediated adaptive changes that occur in the local genital tract tissues during primary infection. These results impact our understanding of immunity to chlamydial genital infection and may provide important insight into vaccine development.     

Toll-like receptor 4 dependence of innate and adaptive immunity to Salmonella: importance of the Kupffer cell network

Mammalian cells recognize LPS from Gram-negative bacteria via the Toll-like receptor 4 (TLR4) complex. During experimental Salmonella infection, C3H/HeJ mice carrying a dominant-negative mutation in TLR4 exhibited delayed chemokine production, impaired NO generation, and attenuated cellular immune responses. However, dramatically enhanced bacterial growth within the Kupffer cell network before the recruitment of inflammatory cells appeared to be primarily responsible for the early demise of Salmonella-infected TLR4-deficient mice. LPS-TLR4 signaling plays an essential role in the generation of both innate and adaptive immune responses throughout the course of infection with Gram-negative bacteria. Alternative pattern-recognition receptors cannot completely compensate for the loss of TLR4, and compensation occurs at the expense of an increased microbial burden.     

Resident Vdelta1+ gammadelta T cells control early infiltration of neutrophils after Escherichia coli infection via IL-17 production

Neutrophils infiltrate the site of infection and play critical roles in host defense, especially against extracellular bacteria. In the present study, we found a rapid and transient production of IL-17 after i.p. infection with Escherichia coli, preceding the influx of neutrophils. Neutralization of IL-17 resulted in a reduced infiltration of neutrophils and an impaired bacterial clearance. Ex vivo intracellular cytokine flow cytometric analysis revealed that gammadelta T cell population was the major source of IL-17. Mice depleted of gammadelta T cells by mAb treatment or mice genetically lacking Vdelta1 showed diminished IL-17 production and reduced neutrophil infiltration after E. coli infection, indicating an importance of Vdelta1(+) gammadelta T cells as the source of IL-17. It was further revealed that gammadelta T cells in the peritoneal cavity of naive mice produced IL-17 in response to IL-23, which was induced rapidly after E. coli infection in a TLR4 signaling-dependent manner. Thus, although gammadelta T cells are generally regarded as a part of early induced immune responses, which bridge innate and adaptive immune responses, our study demonstrated a novel role of gammadelta T cells as a first line of host defense controlling neutrophil-mediated innate immune responses.     

Ly6G+ neutrophils are dispensable for defense against systemic Listeria monocytogenes infection

Listeria monocytogenes is a facultative intracellular bacterium that causes systemic infections in immunocompromised hosts. Early recruitment of myeloid cells, including inflammatory monocytes and neutrophils, to sites of L. monocytogenes infection is essential for the control of infection and host survival. Because previous experimental studies used depleting or blocking Abs that affected both inflammatory monocytes and neutrophils, the relative contributions of these cell populations to defense against L. monocytogenes infection remain incompletely defined. In this article, we used highly selective depletion strategies to either deplete inflammatory monocytes or neutrophils from L. monocytogenes-infected mice and demonstrate that neutrophils are dispensable for early and late control of infection. In contrast, inflammatory monocytes are essential for bacterial clearance during the innate and adaptive phases of the immune response to L. monocytogenes infection.     

Bacterial clearance and survival are dependent on CXC chemokine receptor-2 ligands in a murine model of pulmonary Nocardia asteroides infection

Survival from murine pulmonary nocardiosis is highly dependent on CXC chemokine receptor-2 (CXCR2) ligand-mediated neutrophil chemotaxis and subsequent clearance of the infectious agent Nocardia asteroides. Intratracheal inoculation of N. asteroides rapidly up-regulated the CXC chemokines macrophage inflammatory protein-2 (MIP-2) and KC within 24 h, with levels remaining elevated through day 3 before returning to near baseline levels by day 7. Coinciding with elevated MIP-2 and KC were the rapid recruitment of neutrophils and clearance of the organism. Anti-Ly-6G Ab-mediated neutrophil depletion before bacterial challenge resulted in strikingly increased mortality to N. asteroides infection. The relative contribution of MIP-2 in neutrophil recruitment was examined by anti-MIP-2 Ab treatment before nocardial infection. MIP-2 neutralization had no detrimental effects on survival, neutrophil recruitment, or bacterial clearance, suggesting the usage of additional or alternative CXCR2-binding ligands. The importance of the CXC family of chemokines was determined by the administration of an anti-CXCR2 Ab capable of blocking ligand binding in vivo. Anti-CXCR2 treatment greatly increased mortality by preventing neutrophil migration into the lung. Paralleling this impaired neutrophil recruitment was a 100-fold increase in lung bacterial burden. Combined, these observations indicate a critical role for neutrophils and CXC chemokines during nocardial pneumonia. These data directly link CXCR2 ligands and neutrophil recruitment and lend further support to the concept of CXC chemokine redundancy. For infections highly dependent on neutrophils, such as nocardial pneumonia, this is of critical importance.     

Fc receptor-mediated phagocytosis makes a significant contribution to clearance of influenza virus infections

Fc receptors for IgG expressed on macrophages and NK cells are important mediators of opsonophagocytosis and Ab-dependent cell-mediated cytotoxicity. Phagocyte-mediated opsonophagocytosis is pivotal for protection against bacteria, but its importance in recovery from infection with intracellular pathogens is unclear. We have now investigated the role of opsonophagocytosis in protection against lethal influenza virus infection by using FcR gamma(-/-) mice. Absence of the FcR gamma-chain did not affect the expression of IFN-gamma and IL-10 in the lungs and spleens after intranasal immunization with an influenza subunit vaccine. Titers of serum and respiratory Abs of the IgM, IgG1, IgG2a, and IgA isotypes in FcR gamma(-/-) mice were similar to levels seen in FcR gamma(+/+) mice. Nevertheless, FcR gamma(-/-) mice were highly susceptible to influenza infection, even in the presence of anti-influenza Abs from immune FcR gamma(+/+) mice. NK cells were not necessary for the observed Ab-mediated viral clearance, but macrophages were found to be capable of actively ingesting opsonized virus particles. We conclude that Fc receptor-mediated phagocytosis plays a pivotal role in clearance of respiratory virus infections.     

Induction of a novel mechanism of accelerated bacterial clearance by lipopolysaccharide in CD14-deficient and Toll-like receptor 4-deficient mice

Despite the lack of a proinflammatory response to LPS, CD14-deficient mice clear Gram-negative bacteria (Escherichia coli 0111) at least 10 times more efficiently than normal mice. In this study, we show that this is due to an early and intense recruitment of neutrophils following the injection of Gram-negative bacteria or LPS in CD14-deficient mice; in contrast, neutrophil infiltration is delayed by 24 h in normal mice. Similar results of early LPS-induced PMN infiltration and enhanced clearance of E. coli were seen in Toll-like receptor (TLR) 4-deficient mice. Furthermore, the lipid A moiety of LPS induced early neutrophil infiltration not only in CD14-deficient and TLR-4-deficient mice, but also in normal mice. In conclusion, the lipid A component of LPS stimulates a unique and critical pathway of innate immune responses that is independent of CD14 and TLR4 and results in early neutrophil infiltration and enhanced bacterial clearance.     

The major surface protein of Wolbachia endosymbionts in filarial nematodes elicits immune responses through TLR2 and TLR4

More than 150 million humans in tropical countries are infected by filarial nematodes which harbor intracellular bacterial endosymbionts of the genus Wolbachia (Rickettsiales). These bacteria have been implicated in adverse effects of drug treatment in filariasis. The present study provides evidence that purified major Wolbachia surface protein (rWSP) acts as an inducer of the innate immune system through TLR2 and TLR4: 1) recombinant, stringently purified rWSP elicited the release of TNF-alpha, IL-12, and IL-8 from cultured blood cells of both Onchocerca volvulus-infected and uninfected people; 2) the inflammatory response to rWSP challenge was TLR2- and TLR4-dependent as demonstrated with TLR-transfected fibroblastoid cells, as well as macrophages and dendritic cells from functional TLR-deficient mice; 3) blood cells of onchocerciasis patients exposed to rWSP also generated down-regulating mediators IL-10 and PGE(2) after 6 days of culture; 4) furthermore, rWSP-reactive IgG1 Abs were present in sera of O. volvulus-infected people but not in those of uninfected Europeans. The lack of rWSP-reactive IgE and IgG4 in serum indicated a bias toward a Th1-type adaptive immune response. Abs against rWSP stained endobacteria in living and degenerating adult O. volvulus filariae, tissue microfilariae and host tissue macrophages that apparently had engulfed microfilariae. Thus, filarial helminths, through products of their endobacteria such as WSP, acquire characteristics of a typical microbial pathogen inducing immune responses via TLR2 and TLR4.     

Monophosphoryl lipid A induced innate immune responses via TLR4 to enhance clearance of nontypeable Haemophilus influenzae and Moraxella catarrhalis from the nasopharynx in mice

Acute otitis media (AOM) is one of the most common infectious diseases in children. Nontypeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis, Gram-negative bacteria, are considered major pathogens of AOM and respiratory tract infections. In this study, we used monophosphoryl lipid A (MPL) as a Toll-like receptor (TLR4) agonist to induce innate immune responses before challenge with NTHi and M.?catarrhalis to enhance bacterial clearance from the nasopharynx. Mice were intranasally administered 40, 10, or 1?μg of MPL and challenged with NTHi and M.?catarrhalis 12 and 24?h later. At 6 and 12?h after the bacterial challenge, the mice were killed and nasal washes were collected. The numbers of NTHi, M.?catarrhalis, and inflammatory cells were quantitated. Inoculation of MPL produced a significant reduction in the number of bacteria recovered from the nasopharynx at 6 and/or 12?h after the bacterial challenge, when compared with control mice. The effect was dose dependent. MPL inoculation also induced the early accumulation of neutrophils in the nasopharynx after exposure to bacteria. MPL is effective for eliciting clearance of both NTHi and M.?catarrhalis from the nasopharynx. These results indicate the possibility of a new strategy against Gram-negative bacterial infection that involves the stimulation of the innate immune system by TLR4 agonists such as MPL.     

The specific innate immune receptor CEACAM3 triggers neutrophil bactericidal activities via a Syk kinase-dependent pathway

The human-restricted pathogens Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae and Moraxella catarrhalis colonize host tissues via carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs). One such receptor, CEACAM3, acts in a host-protective manner by orchestrating the capture and engulfment of invasive bacteria by human neutrophils. Herein, we show that bacterial binding to CEACAM3 causes recruitment of the cytoplasmic tyrosine kinase Syk, resulting in the phosphorylation of both CEACAM3 and Syk. This interaction is specific for the immunoreceptor tyrosine-based activation motif (ITAM) in the CEACAM3 cytoplasmic domain. While dispensable for the phagocytic uptake of single bacteria by CEACAM3, Syk is necessary for internalization when cargo size increases or when the density of CEACAM-binding ligand on the cargo surface is below a critical threshold. Moreover, Syk engagement is required for an effective bacterial killing response, including the neutrophil oxidative burst and degranulation functions in response to N. gonorrhoeae. These data reveal CEACAM3 as a specific innate immune receptor that mediates the opsonin-independent clearance of CEACAM-binding bacteria via Syk, a molecular trigger for functional immunoreceptor responses of both the adaptive (TCR, BCR, FcR) and innate (Dectin-1, CEACAM3) immune systems.     

Rhinovirus Attenuates Non-typeable Hemophilus influenzae-stimulated IL-8 Responses via TLR2-dependent Degradation of IRAK-1

Bacterial infections following rhinovirus (RV), a common cold virus, are well documented, but pathogenic mechanisms are poorly understood. We developed animal and cell culture models to examine the effects of RV on subsequent infection with non-typeable Hemophilus influenzae (NTHi). We focused on NTHI-induced neutrophil chemoattractants expression that is essential for bacterial clearance. Mice infected with RV1B were superinfected with NTHi and lung bacterial density, chemokines and neutrophil counts determined. Human bronchial epithelial cells (BEAS-2B) or mouse alveolar macrophages (MH-S) were infected with RV and challenged with NHTi, TLR2 or TLR5 agonists. Chemokine levels were measured by ELISA and expression of IRAK-1, a component of MyD88-dependent TLR signaling, assessed by immunoblotting. While sham-infected mice cleared all NTHi from the lungs, RV-infected mice showed bacteria up to 72 h post-infection. However, animals in RV/NTHi cleared bacteria by day 7. Delayed bacterial clearance in RV/NTHi animals was associated with suppressed chemokine levels and neutrophil recruitment. RV-infected BEAS-2B and MH-S cells showed attenuated chemokine production after challenge with either NTHi or TLR agonists. Attenuated chemokine responses were associated with IRAK-1 protein degradation. Inhibition of RV-induced IRAK-1 degradation restored NTHi-stimulated IL-8 expression. Knockdown of TLR2, but not other MyD88-dependent TLRs, also restored IRAK-1, suggesting that TLR2 is required for RV-induced IRAK-1 degradation.In conclusion, we demonstrate for the first time that RV infection delays bacterial clearance in vivo and suppresses NTHi-stimulated chemokine responses via degradation of IRAK-1. Based on these observations, we speculate that modulation of TLR-dependent innate immune responses by RV may predispose the host to secondary bacterial infection, particularly in patients with underlying chronic respiratory disorders.     

Toll-like receptor 4 (TLR4) is required for protective immunity to larval Strongyloides stercoralis in mice

TLR4 is important for immunity to various unicellular organisms and has been implicated in the immune responses to helminth parasites. The immune response against helminths is generally Th2-mediated and studies have shown that TLR4 is required for the development of a Th2 response against allergens and helminth antigens in mice. C3H/HeJ mice, which have a point mutation in the Tlr4 gene, were used in this study to determine the role of TLR4 in protective immunity to the nematode Strongyloides stercoralis. It was demonstrated that TLR4 was not required for killing larval S. stercoralis during the innate immune response, but was required for killing the parasites during the adaptive immune response. No differences were seen in the IL-5 and IFN-gamma responses, antibody responses or cell recruitment between wild type and C3H/HeJ mice after immunization. Protective immunity was restored in immunized C3H/HeJ mice by the addition of wild type peritoneal exudate cells in the environment of the larvae. It was therefore concluded that the inability of TLR4-mutant mice to kill larval S. stercoralis during the adaptive immune response is due to a defect in the effector cells recruited to the microenvironment of the larvae.     

TLR2, but not TLR4, is required for effective host defence against Chlamydia respiratory tract infection in early life

Chlamydia pneumoniae commonly causes respiratory tract infections in children, and epidemiological investigations strongly link infection to the pathogenesis of asthma. The immune system in early life is immature and may not respond appropriately to pathogens. Toll-like receptor (TLR)2 and 4 are regarded as the primary pattern recognition receptors that sense bacteria, however their contribution to innate and adaptive immunity in early life remains poorly defined. We investigated the role of TLR2 and 4 in the induction of immune responses to Chlamydia muridarum respiratory infection, in neonatal wild-type (Wt) or TLR2-deficient ((-/-)), 4(-/-) or 2/4(-/-) BALB/c mice. Wt mice had moderate disease and infection. TLR2(-/-) mice had more severe disease and more intense and prolonged infection compared to other groups. TLR4(-/-) mice were asymptomatic. TLR2/4(-/-) mice had severe early disease and persistent infection, which resolved thereafter consistent with the absence of symptoms in TLR4(-/-) mice. Wt mice mounted robust innate and adaptive responses with an influx of natural killer (NK) cells, neutrophils, myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells, and activated CD4(+) and CD8(+) T-cells into the lungs. Wt mice also had effective production of interferon (IFN)γ in the lymph nodes and lung, and proliferation of lymph node T-cells. TLR2(-/-) mice had more intense and persistent innate (particularly neutrophil) and adaptive cell responses and IL-17 expression in the lung, however IFNγ responses and T-cell proliferation were reduced. TLR2/4(-/-) mice had reduced innate and adaptive responses. Most importantly, neutrophil phagocytosis was impaired in the absence of TLR2. Thus, TLR2 expression, particularly on neutrophils, is required for effective control of Chlamydia respiratory infection in early life. Loss of control of infection leads to enhanced but ineffective TLR4-mediated inflammatory responses that prolong disease symptoms. This indicates that TLR2 agonists may be beneficial in the treatment of early life Chlamydia infections and associated diseases.     

Membrane vesicles are immunogenic facsimiles of Salmonella typhimurium that potently activate dendritic cells, prime B and T cell responses, and stimulate protective immunity in vivo

Gram-negative bacteria produce membrane vesicles (MVs) from their outer membrane during growth, although the mechanism for MV production and the advantage that MVs provide for bacterial survival in vivo remain unknown. MVs function as an alternate secretion pathway for Gram-negative bacteria; therefore, MV production in vivo may be one method by which bacteria interact with eukaryotic cells. However, the interactions between MVs and cells of the innate and adaptive immune systems have not been studied extensively. In this study, we demonstrate that MVs from Salmonella typhimurium potently stimulated professional APCs in vitro. Similar to levels induced by bacterial cells, MV-stimulated macrophages and dendritic cells displayed increased surface expression of MHC-II and CD86 and enhanced production of the proinflammatory mediators NO, TNF-alpha, and IL-12. MV-mediated dendritic cell stimulation occurred by TLR4-dependent and -independent signals, indicating the stimulatory properties of Salmonella MVs, which contain LPS, do not strictly rely on signaling through TLR4. In addition to their strong proinflammatory properties, MVs contained Ags recognized by Salmonella-specific B cells and CD4(+) T cells; MV-vaccinated mice generated Salmonella-specific Ig and CD4(+) T cell responses in vivo and were significantly protected from infectious challenge with live Salmonella. Our findings demonstrate that MVs possess important inflammatory properties as well as B and T cell Ags known to influence the development of Salmonella-specific immunity to infection in vivo. Our findings also reveal MVs are a functional nonviable complex vaccine for Salmonella by their ability to prime protective B and T cell responses in vivo.     

Adenovirus vector-induced innate inflammatory mediators, MAPK signaling, as well as adaptive immune responses are dependent upon both TLR2 and TLR9 in vivo

Adenovirus (Ad) vectors are promising candidates for both gene transfer and vaccine applications. In this study, we investigated the role of TLR2 in innate and adaptive immune responses to Ad and/or the transgene it expresses following systemic injection. We found that Ad directly activates ERK1/2 in vivo, but that initiation of ERK1/2 activation is primarily a MyD88/TLR2-independent, but Kupffer cell-dependent, event. The complexity of Ad-induced innate immune responses was confirmed when we also found that both TLR2 and MyD88 functions are required for the sustained activation of ERK1/2. Although we found that the initial activation of NF-kappaB by Ads is dependent upon MyD88, but independent of TLR2 in (non-Kupffer cells) the liver, TLR2 significantly influenced the Ad-induced late phase NF-kappaB activation. These very rapid responses were positively correlated with subsequent innate immune responses to the Ad vector, as our results confirmed that the induction of several cytokines and chemokines, and the expression of innate immune response genes following Ad injection were TLR2 dependent in vivo. The requirement of TLR2 in Ad-induced innate responses also correlated with significantly altered adaptive immune responses. For example, our results demonstrate that the generation of Ad-neutralizing Abs, and anti-transgene-specific Abs elicited subsequent to Ad vector treatments, are both dependent upon TLR2 functionality. Finally, we found that several Ad-induced innate immune responses are dependent on both TLR2 and TLR9. Therefore, this study confirms that several (but not all) Ad-induced innate and adaptive immune responses are TLR dependent.