The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response

Recognition of viral dsRNA by Toll-like receptor 3 (TLR3) leads to induction of interferons (IFNs) and proinflammatory cytokines, and innate antiviral response. Here we identified the RNA-binding protein Mex3B as a positive regulator of TLR3-mediated signaling by expression cloning screens. Cells from Mex3b^−/− mice exhibited reduced production of IFN-β in response to the dsRNA analog poly(I:C) but not infection with RNA viruses. Mex3b^−/− mice injected with poly(I:C) was more resistant to poly(I:C)-induced death. Mex3B was associated with TLR3 in the endosomes. It bound to dsRNA and increased the dsRNA-binding activity of TLR3. Mex3B also promoted the proteolytic processing of TLR3, which is critical for its activation. Mutants of Mex3B lacking its RNA-binding activity inhibited TLR3-mediated IFN-β induction. These findings suggest that Mex3B acts as a coreceptor of TLR3 in innate antiviral response.     

Intranasal administration of poly(I:C) and LPS in BALB/c mice induces airway hyperresponsiveness and inflammation via different pathways

Background

Bacterial and viral infections are known to promote airway hyperresponsiveness (AHR) in asthmatic patients. The mechanism behind this reaction is poorly understood, but pattern recognizing Toll-like receptors (TLRs) have recently been suggested to play a role.

Materials and Methods

To explore the relation between infection-induced airway inflammation and the development of AHR, poly(I:C) activating TLR3 and LPS triggering TLR4, were chosen to represent viral and bacterial induced interactions, respectively. Female BALB/c or MyD88-deficient C57BL/6 mice were treated intranasally with either poly(I:C), LPS or PBS (vehicle for the control group), once a day, during 4 consecutive days.

Results

When methacholine challenge was performed on day 5, BALB/c mice responded with an increase in airway resistance. The maximal resistance was higher in the poly(I:C) and LPS treated groups than among the controls, indicating development of AHR in response to repeated TLR activation. The proportion of lymphocytes in broncheoalveolar lavage fluid (BALF) increased after poly(I:C) treatment whereas LPS enhanced the amount of neutrophils. A similar cellular pattern was seen in lung tissue. Analysis of 21 inflammatory mediators in BALF revealed that the TLR response was receptor-specific. MyD88-deficient C57BL/6 mice responded to poly (I:C) with an influx of lymphocytes, whereas LPS caused no inflammation.

Conclusion

In vivo activation of TLR3 and TLR4 in BALB/c mice both caused AHR in conjunction with a local inflammatory reaction. The AHR appeared to be identical regardless of which TLR that was activated, whereas the inflammation exhibited a receptor specific profile in terms of both recruited cells and inflammatory mediators. The inflammatory response caused by LPS appeared to be dependent on MyD88 pathway. Altogether the presented data indicate that the development of AHR and the induction of local inflammation might be the result of two parallel events, rather than one leading to another.     

Suppression of TLR4-mediated inflammatory response by macrophage class A scavenger receptor (CD204)

The class A scavenger receptor (SR-A, CD204), one of the principal receptors expressed on macrophages, has been found to regulate inflammatory response and attenuate septic endotoxemia. However, the detailed mechanism of this process has not yet been well characterized. To clarify the regulative mechanisms of lipopolysaccharide (LPS)-induced macrophage activation by SR-A, we evaluated the activation of Toll-like receptor 4 (TLR4)-mediated signaling molecules in SR-A-deficient (SR-A^−/−) macrophages. In a septic shock model, the blood levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and interferon (IFN)-β were significantly increased in SR-A^−/− mice compared to wild-type mice, and elevated nuclear factor kappa B (NFκB) activation was detected in SR-A^−/− macrophages. SR-A deletion increased the production of pro-inflammatory cytokines, and the phosphorylation of mitogen-activated protein kinase (MAPK) and NFκB in vitro. SR-A deletion also promoted the nuclear translocation of NFκB and IFN regulatory factor (IRF)-3. In addition, a competitive binding assay with acetylated low-density lipoprotein, an SR-A-specific ligand, and anti-SR-A antibody induced significant activation of TLR4-mediated signaling molecules in wild-type macrophages but not in SR-A^−/− macrophages. These results suggest that SR-A suppresses the macrophage activation by inhibiting the binding of LPS to TLR4 in a competitive manner and it plays a pivotal role in the regulation of the LPS-induced inflammatory response.     

TLR4 Ligands Augment Antigen-Specific CD8+ T Lymphocyte Responses Elicited by a Viral Vaccine Vector

Toll-like receptor (TLR) ligands are critical activators of innate immunity and are being developed as vaccine adjuvants. However, their utility in conjunction with viral vector-based vaccines remains unclear. In this study, we evaluated the impact of a variety of TLR ligands on antigen-specific CD8⁺ T lymphocyte responses elicited by a recombinant adenovirus serotype 26 (rAd26) vector expressing simian immunodeficiency virus Gag in mice. The TLR3 ligand poly(I:C) suppressed Gag-specific cellular immune responses, whereas the TLR4 ligands lipopolysaccharide and monophosphoryl lipid A substantially augmented the magnitude and functionality of these responses by a MyD88- and TRIF-dependent mechanism. These data demonstrate that TLR ligands can modulate the immunogenicity of viral vaccine vectors both positively and negatively. Moreover, these findings suggest the potential utility of TLR4 ligands as adjuvants for rAd vector-based vaccines.Toll-like receptors (TLRs) are critical sensors of infection with a fundamental role in the activation of innate immune responses and the subsequent modulation of pathogen-specific adaptive immunity (2). TLR ligands have therefore emerged as potential vaccine adjuvants, particularly in the context of peptide, protein, and DNA vaccines (17). In particular, TLR agonists are widely reported to modulate antibody and T helper lymphocyte responses, and in some cases CD8⁺ T lymphocyte responses, elicited by protein-based vaccines (5, 19, 33, 41). However, far less is known about the impact of TLR ligands on the immunogenicity of viral vector-based vaccines.Compared with DNA vaccines, viral vectors are typically more immunogenic, presumably as a result of the activation of innate immunity via multiple TLRs or other pattern recognition receptors (29). Viral vectors elicit robust T lymphocyte responses and thus are attractive vaccine candidates for pathogens such as human immunodeficiency virus type 1 (HIV-1) and malaria (10). Whether the addition of exogenous TLR agonists might further enhance the immunogenicity of viral vectors, however, remains unclear. The few studies that have explored the utility of TLR adjuvants with viral vectors have typically shown no or mild enhancement of antibody and T lymphocyte responses (7, 26). We therefore sought to determine systematically whether TLR ligands can modulate cellular immune responses elicited by a recombinant adenovirus serotype 26 (rAd26) vector in mice.C57BL/6 mice (n = 7 to 8/group) were immunized with a single injection of 3 × 10⁸ viral particles (vp) rAd26-Gag alone or combined with various TLR ligands (1). Vectors were mixed with soluble TLR agonists 1 h prior to intramuscular (i.m.) injection into both quadriceps muscles. Cellular immune responses were assessed by D^b/AL11 tetramer binding assays (3, 6), gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assays (6), and multiparameter intracellular cytokine staining (ICS) assays (14). As shown in Fig. ​Fig.11 A, immunization with rAd26-Gag plus either 20 μg Pam3CSK (TLR1/2 ligand) (25), 20 μg Pam2CSK (TLR2/6 ligand) (9, 20), 10 μg flagellin (TLR5 ligand) (5, 8), 100 μg CLO97 (TLR7 ligand) (41), or 40 μg CpG (TLR9 ligand) (40) (all obtained from InvivoGen, San Diego, CA) elicited AL11-specific tetramer-positive responses (3, 6) that were similar to those detected in the unadjuvanted groups.Open in a separate windowFIG. 1.Antigen-specific CD8⁺ T cell responses elicited by rAd26-Gag are modulated by soluble TLR ligands. (A) C57BL/6 mice (n = 7 to 8 mice/group) were immunized once with 3 × 10⁸ vp rAd26-Gag alone or 3 × 10⁸ vp rAd26-Gag combined with the following TLR ligands: 20 μg synthetic triacylated lipoprotein (Pam3CSK; TLR1/2 ligand), 20 μg synthetic diacylated lipoprotein (Pam2CSK; TLR 2/6 ligand), 100 μg poly(I:C) (TLR3 ligand), 10 μg LPS (TLR4 ligand), 10 μg flagellin (TLR5 ligand), 100 μg CLO97 (TLR7 ligand), or 40 μg unmethylated CpG-oligodeoxynucleotides (CpG; TLR9 ligand). Gag-specific cellular immune responses were assayed by D^b/AL11 tetramer binding assays at multiple time points following injection. (B) At week 4 following immunization, functional immune responses from mice immunized with rAd26 vaccine alone or with 10 μg LPS or 100 μg poly(I:C) were assessed by IFN-γ ELISPOT assays in response to pooled Gag peptides, the CD8⁺ T lymphocyte epitopes AL11 and KV9, and the CD4⁺ T lymphocyte epitope DD13. (C) Assessment of the dose response of LPS (10 μg, 2 μg, 0.4 μg) and poly(I:C) (100 μg, 20 μg, 4 μg) with rAd26-Gag (n = 4 mice/group) by D^b/AL11 tetramer binding assays. (D) Mice were immunized once i.m. with 3 × 10⁸ vp rAd26-Gag alone, rAd26-Gag with 2 μg LPS, or rAd26-Gag with 20 μg poly(I:C) (n = 4 to 8 mice/group), and Gag-specific CD8⁺ T cell responses in splenocytes were assessed 4 weeks after vaccination by intracellular cytokine assays for IFN-γ, TNF-α, IL-2, and CD107. Responses to pooled Gag peptides are presented for each individual combination of functions and collated as the number of functions elaborated as a percent of total CD8⁺ T lymphocytes (insert; bar graph) and as the fraction of Gag-specific CD8⁺ T lymphocytes (insert; pie charts). Mean responses with standard errors are shown (*, P < 0.001; **, P < 0.05; two-tailed t test).The TLR3 ligand poly(I:C) (InvivoGen, San Diego, CA), however, markedly suppressed responses to the rAd26-Gag vaccine (Fig. ​(Fig.1A).1A). This finding contrasts with prior reports demonstrating its adjuvanticity for protein antigen vaccines (22, 34, 37). By day 28, mice that received the vaccine plus 100 μg poly(I:C) developed Gag-specific CD8⁺ T lymphocyte responses that were significantly lower (1.7%) than those of mice that received the vaccine alone (5.4%; P < 0.001; two-tailed t test). Similarly, IFN-γ ELISPOT responses in mice that received poly(I:C) were lower than those observed in the unadjuvanted group (Fig. ​(Fig.1B)1B) (6). In a dose response study (Fig. ​(Fig.1C),1C), 100-μg, 20-μg, and 4-μg doses of poly(I:C) all resulted in diminished tetramer-positive responses.In contrast, the TLR4 ligand lipopolysaccharide (LPS) (Ultrapure LPS from Escherichia coli 0111:B4; InvivoGen, San Diego, CA) substantially enhanced Gag-specific CD8⁺ T lymphocyte responses elicited by the rAd26-Gag vaccine (Fig. ​(Fig.1A).1A). At day 28, tetramer-positive responses in mice that received the vaccine plus 10 μg LPS (9.6%) were significantly higher than those in the unadjuvanted group (5.4%; P = 0.04). Moreover, IFN-γ ELISPOT responses (6, 21) to pooled Gag peptides, the CD8⁺ T lymphocyte epitopes AL11 and KV9, and the CD4⁺ T lymphocyte epitope DD13 were greater in mice that received the vaccine with LPS than in mice that received the vaccine alone at week 4 after immunization (P = 0.02) (Fig. ​(Fig.1B).1B). To further quantify this effect, mice were immunized once i.m. (n = 4 mice/group) with rAd26-Gag with various doses of LPS (10 μg, 2 μg, 0.4 μg). Tetramer-positive responses were enhanced by 10 μg and 2 μg LPS but not by 0.4 μg LPS (Fig. ​(Fig.1C),1C), indicating that this LPS effect was dose dependent. No overt clinical toxicities were observed by using these doses of LPS in mice.We next evaluated the functionality of CD8⁺ T lymphocyte responses by multiparameter ICS assays that assessed IFN-γ, tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), and the cytotoxic degranulation marker CD107 expression at week 4 following immunization with rAd26-Gag alone, rAd26-Gag with 2 μg LPS, or rAd26-Gag with 20 μg poly(I:C) (n = 4 to 8 mice/group) (15). As shown in Fig. ​Fig.1D,1D, the addition of LPS significantly enhanced not only the overall magnitude of Gag-specific CD8⁺ T lymphocyte responses (P = 0.04) but also the fraction of Gag-specific CD8⁺ T lymphocytes that expressed two or more effector functions (P = 0.04). In particular, the LPS-adjuvanted group induced higher levels of single-function CD107⁺, 2-function TNF-α⁺ CD107⁺, as well as 3-function IFN-γ⁺ TNF-α⁺ CD107⁺ CD8⁺ T lymphocytes than mice that received rAd26-Gag alone. These data show that LPS enhanced both the magnitude and functionality of antigen-specific cellular responses elicited by rAd26-Gag. In contrast, the addition of poly(I:C) diminished both the overall magnitude of Gag-specific responses and the fraction of these responses that were multifunctional.We further characterized the opposing effects of poly(I:C) and LPS by administering the rAd26-Gag vaccine with both poly(I:C) and LPS. C57BL/6 mice (n = 4 mice/group) were immunized with a single injection of rAd26-Gag alone or with 10 μg LPS, 60 μg poly(I:C), or both TLR ligands. As shown in Fig. ​Fig.22 A, administration of both TLR ligands resulted in reduced Gag-specific responses, suggesting that the suppressive effect of poly(I:C) was dominant over the enhancing effect of LPS. To determine the durability of the effects of poly(I:C) and LPS, C57BL/6 mice were primed with rAd26-Gag alone or with 2 μg LPS or 20 μg poly(I:C) (n = 4 mice/group) and were boosted on day 35 with a single i.m. injection of the heterologous vector rAd5HVR48(1-7) also expressing simian immunodeficiency virus (SIV) Gag (32). As shown in Fig. ​Fig.2B,2B, the mice that received poly(I:C) with the priming immunization responded to the boosting immunization with Gag-specific responses that were comparable to those observed in the mice that received rAd26-Gag alone. In contrast, mice that received LPS with the priming immunization exhibited sustained enhanced Gag-specific tetramer and ELISPOT responses, demonstrating the proliferative potential of antigen-specific CD8⁺ T lymphocytes elicited by the LPS-adjuvanted rAd26-Gag vaccine.Open in a separate windowFIG. 2.Dominant suppressive effect of poly(I:C) over LPS with the rAd26-Gag vaccine. (A) Mice were immunized once i.m. with 3 × 10⁸ vp rAd26-Gag alone or with 20 μg poly(I:C), 2 μg LPS, or both poly(I:C) and LPS (n = 4 mice/group). Gag-specific CD8⁺ T lymphocyte responses were assessed by D^b/AL11 tetramer binding assays and IFN-γ ELISPOT assays 4 weeks after immunization. (B) Mice were primed once with 3 × 10⁸ vp rAd26-Gag alone or with 2 μg LPS or 20 μg poly(I:C) and then boosted (↓) with 3 × 10⁸ vp rAd5HVR48(1-7) at week 5. Gag-specific cellular immune responses were assessed by D^b/AL11 tetramer binding assays and by IFN-γ ELISPOT responses at week 4 postboost. Mean responses with standard errors are shown.We next investigated whether the mechanism underlying the immunomodulatory effects of LPS and poly(I:C) involved the expected TLR signaling pathways. Although LPS and poly(I:C) are chiefly considered TLR ligands, poly(I:C) can also signal through the intracellular sensor MDA-5 (14), and both LPS and poly(I:C) may activate inflammasomes through Nalp3 (12, 28). To explore whether the effects of LPS and poly(I:C) involved TLR signaling, we utilized C57BL/6 mice lacking TRIF (Jackson Laboratory, Bar Harbor, ME), which is utilized by TLR3, or C57BL/6 mice lacking MyD88 (provided by S. Akira and B. Pulendran), which is utilized by the majority of TLRs. In particular, TLR4 signals through both TRIF and MyD88. Wild-type, MyD88^−/−, and TRIF^−/− mice (n = 4 mice/group) were immunized with rAd26-Gag vaccine alone or with 2 μg LPS or 20 μg poly(I:C). As shown in Fig. ​Fig.3,3, the adjuvant activity of LPS was abrogated in both MyD88^−/− and TRIF^−/− mice (Fig. 3A and B), suggesting that the adjuvanticity of the TLR4 ligand LPS was dependent on both MyD88 and TRIF, as expected. In contrast, the suppressive effect of poly(I:C) was observed in MyD88^−/− mice but not in TRIF^−/− mice (Fig. 3A and B), indicating that the suppressive effect of the TLR3 ligand poly(I:C) was dependent on TRIF, rather than MDA-5 or nonspecific effects (14, 39). These data confirm that the immunomodulatory effects of LPS and poly(I:C) were dependent on the expected TLR signaling pathways.Open in a separate windowFIG. 3.The immunomodulatory effects of poly(I:C) and LPS are TLR dependent. MyD88−/− and TRIF−/− mice (n = 4 mice/group) were immunized once i.m. with 3 × 10⁸ vp rAd26-Gag alone or with 2 μg LPS or 20 μg poly(I:C). (A) D^b/AL11 tetramer binding assays were performed at multiple time points following injection, and (B) IFN-γ ELISPOT responses were assessed 4 weeks after immunization. Mean responses with standard errors are shown.LPS is not a likely adjuvant for clinical development as a result of its toxicities, and alternative TLR4 ligands have been developed for potential clinical use. In particular, monophosphoryl lipid A (MPLA) is an LPS derivative that retains the immunologically active lipid A portion of the parent molecule (23, 27). The reduced toxicity of MPLA is attributed to the preferential recruitment of TRIF upon TLR4 activation, resulting in decreased induction of inflammatory cytokines (18). To determine if MPLA can similarly adjuvant cellular immune responses elicited by rAd26-Gag, C57BL/6 mice were immunized with 3 × 10⁷, 3 × 10⁸, or 3 × 10⁹ vp rAd26-Gag alone or with 5 μg MPLA (derived from Salmonella enterica serovar Minnesota R595 LPS; InvivoGen, San Diego, CA) (n = 4 mice/group). This optimal dose of MPLA was selected by dose response studies (data not shown). As shown in Fig. ​Fig.44 A, Gag-specific IFN-γ ELISPOT responses to the lowest dose of vector were essentially undetectable in the unadjuvanted group, consistent with prior observations (1). In contrast, clear responses were observed in the mice that received 3 × 10⁷ vp rAd26-Gag with MPLA (P < 0.01; two-tailed t test). Mice that received the 3 × 10⁸ vp and 3 × 10⁹ vp doses of rAd26-Gag with MPLA also exhibited higher Gag-specific cellular immune responses than the unadjuvanted groups (P < 0.01). Functionality of these Gag-specific CD8⁺ T lymphocyte responses, as measured by multiparameter ICS assays assessing IFN-γ, TNF-α, IL-2, and CD107 expression, was also greater in mice that received rAd26-Gag with MPLA compared with rAd26-Gag (P < 0.05 for the lowest dose group) (Fig. ​(Fig.4B).4B). Thus, the TLR4 ligand MPLA also augmented antigen-specific CD8⁺ T lymphocyte responses elicited by rAd26-Gag.Open in a separate windowFIG. 4.The TLR4 ligand MPLA augments the immunogenicity of rAd26-Gag. C57BL/6 mice (n = 4 mice/group) were immunized once i.m. with 3 × 10⁷, 3 × 10⁸, or 3 × 10⁹ vp rAd26-Gag with or without 5 μg MPLA. Gag-specific cellular immune responses were assessed 4 weeks after immunization by IFN-γ ELISPOT responses (*, P < 0.01 for responses to pooled Gag peptides; two-tailed t test) (A) and by ICS for IFN-γ, TNF-α, IL-2, and CD107 (B). Responses to pooled Gag peptides in mice immunized with 3 × 10⁷ vp rAd26-Gag with or without 5 μg MPLA are presented for each individual combination of functions and collated as the number of functions as a fraction of the total Gag-specific CD8⁺ T lymphocyte response (insert; pie charts) (**, P < 0.05). (C) Cytokine levels were measured in sera of mice 8 h after immunization with 3 × 10⁸ vp rAd26-Gag alone or 3 × 10⁸ vp rAd26-Gag with 5 μg MPLA or 2 μg LPS (n = 4 mice/group). Mean responses with standard errors are shown.To explore differences in acute inflammatory responses following MPLA and LPS administration, serum levels of IL-1α, IL-6, granulocyte colony-stimulating factor (G-CSF), and IP-10 were assessed 8 h after vaccination in duplicate using multiplexed fluorescent bead-based immunoassays (Millipore, Billerica, MA) and analyzed on the Luminex 100 IS (Luminex, Austin, TX). As shown in Fig. ​Fig.4C,4C, mice that received MPLA had lower levels of the MyD88-associated acute proinflammatory cytokines IL-1α and IL-6 than mice that received LPS, as expected. Levels of IP-10 and G-CSF, which are associated with TRIF activation (18), were comparable (Fig. ​(Fig.4B).4B). These data confirm that MPLA resulted in lower levels of systemic inflammatory cytokine secretion than LPS.Optimization of the immunogenicity of viral vectors is an important research priority. However, there have been few reports addressing the potential use of adjuvants together with viral vectors. Combining alum with rAd35 elicited improved antibody responses to a malaria antigen (24), and the addition of TLR9 agonists (CpGs) resulted in paradoxically diminished immune responses elicited by a rAd5 vector but improved protection against a cancer antigen (13). Most recently, Appledorn et al. reported enhanced antigen-specific T lymphocyte responses with the coadministration of a rAd vector engineered to express a novel TLR5 agonist (4). Our study extends these findings and represents the first systematic investigation of the capacity of a panel of soluble TLR ligands to modulate rAd-elicited CD8⁺ T lymphocyte responses.The TLR agonists that modulated vaccine-elicited immune responses in this study included poly(I:C), LPS, and MPLA. These ligands have all been reported to augment CD8⁺ T lymphocyte responses elicited by peptide or protein vaccines (11, 22, 31, 33, 42), presumably through enhanced cross-presentation (34, 35). TLR signaling has been shown to be important for virus-elicited CD8⁺ T lymphocyte responses (38), often through activation of multiple TLRs or other pattern recognition receptors (30). The activation of TLR4 by LPS or MPLA with a viral vector most likely provides an additive or synergistic signal, probably resulting in enhanced APC maturation in the appropriate cytokine milieu. Moreover, immunization of the viral vector and LPS at different sites abrogated the observed adjuvanticity (data not shown), indicating that TLR4 adjuvanticity involves a local mechanism of action. However, the mechanism by which a TLR3 agonist suppresses immunogenicity of a viral vector remains unclear. It is possible that the high levels of type I interferon elicited by poly(I:C) (data not shown) may limit expression from the rAd26 vector. Alternatively, poly(I:C) has been reported to elicit IL-10 secretion, and this suppressive cytokine may limit CD8⁺ T cell proliferation (22, 36). The unexpected suppressive activity of poly(I:C) illustrates the inherent complexity of viral vectors compared to protein-based vaccines (16, 37).Our data demonstrate that antigen-specific CD8⁺ T lymphocyte responses elicited by a rAd26-Gag vaccine vector can be both positively and negatively modulated by soluble TLR ligands, and the mechanism underlying these observations involves the expected TRIF and MyD88 signaling pathways. In particular, the TLR4 ligands LPS and MPLA substantially augmented the magnitude and functionality of antigen-specific cellular immune responses elicited by this vaccine vector. These findings suggest that TLR ligands, particularly MPLA, deserve further exploration as potential adjuvants to improve the immunogenicity and protective efficacy of viral vaccine vectors.     

TLR2 mediates immunity to experimental cysticercosis

Information concerning TLR-mediated antigen recognition and regulation of immune responses during helminth infections is scarce. TLR2 is a key molecule required for innate immunity and is involved in the recognition of a wide range of viruses, bacteria, fungi and parasites. Here, we evaluated the role of TLR2 in a Taenia crassiceps cysticercosis model. We compared the course of T. crassiceps infection in C57BL/6 TLR2 knockout mice (TLR2^-/-) with that in wild type C57BL/6 (TLR2^+/+) mice. In addition, we assessed serum antibody and cytokine profiles, splenic cellular responses and cytokine profiles and the recruitment of alternatively activated macrophages (AAMφs) to the site of the infection. Unlike wild type mice, TLR2^-/- mice failed to produce significant levels of inflammatory cytokines in either the serum or the spleen during the first two weeks of Taenia infection. TLR2^-/- mice developed a Th2-dominant immune response, whereas TLR2^+/+ mice developed a Th1-dominant immune response after Taenia infection. The insufficient production of inflammatory cytokines at early time points and the lack of Th1-dominant adaptive immunity in TLR2^-/- mice were associated with significantly elevated parasite burdens; in contrast, TLR2^+/+ mice were resistant to infection. Furthermore, increased recruitment of AAMφs expressing PD-L1, PD-L2, OX40L and mannose receptor was observed in TLR2^-/- mice. Collectively, these findings indicate that TLR2-dependent signaling pathways are involved in the recognition of T. crassiceps and in the subsequent activation of the innate immune system and production of inflammatory cytokines, which appear to be essential to limit infection during experimental cysticercosis.     

Genomic island 2 of Brucella melitensis is a major virulence determinant: functional analyses of genomic islands

Brucella genomic islands (GIs) share similarities in their genomic organization to pathogenicity islands from other bacteria and are likely acquired by lateral gene transfer. Here, we report the identification of a GI that is important for the pathogenicity of Brucella melitensis. The deletion of GI-1, GI-5, or GI-6 did not affect bacterial growth in macrophages as well as their virulence in interferon regulatory factor 1-deficient (IRF-1^−/−) mice, suggesting that these islands do not contribute to Brucella virulence. However, the deletion of GI-2 resulted in the attenuation of bacterial growth in macrophages and virulence in IRF-1^−/− mice. The GI-2 mutant also displayed a rough lipopolysaccharide (LPS) phenotype indicated by acriflavin agglutination, suggesting that in vitro and in vivo attenuation is a result of LPS alteration. Further, systematic analysis of the entire GI-2 revealed two open reading frames (ORFs), BMEI0997 and I0998, that encode hypothetical sugar transferases and contribute to LPS alteration, as the deletion of either of these ORFs resulted in a rough phenotype similar to that of the GI-2 mutant. Complementation analyses indicated that in addition to I0997 and I0998, I0999 is required to restore the smooth LPS in the GI-2 mutant as well as its full in vitro and in vivo virulence. The I0999 sequence analysis suggested that it might function as a transporter to help facilitate the transport or linking of the O antigen to the LPS. Our study also indicated that the rough LPS resulting from the GI-2 deletion may affect pathogen-associated molecular pattern recognition by Toll-like receptors.     

Humanized TLR4/MD-2 Mice Reveal LPS Recognition Differentially Impacts Susceptibility to Yersinia pestis and Salmonella enterica

Although lipopolysaccharide (LPS) stimulation through the Toll-like receptor (TLR)-4/MD-2 receptor complex activates host defense against Gram-negative bacterial pathogens, how species-specific differences in LPS recognition impact host defense remains undefined. Herein, we establish how temperature dependent shifts in the lipid A of Yersinia pestis LPS that differentially impact recognition by mouse versus human TLR4/MD-2 dictate infection susceptibility. When grown at 37°C, Y. pestis LPS is hypo-acylated and less stimulatory to human compared with murine TLR4/MD-2. By contrast, when grown at reduced temperatures, Y. pestis LPS is more acylated, and stimulates cells equally via human and mouse TLR4/MD-2. To investigate how these temperature dependent shifts in LPS impact infection susceptibility, transgenic mice expressing human rather than mouse TLR4/MD-2 were generated. We found the increased susceptibility to Y. pestis for “humanized” TLR4/MD-2 mice directly paralleled blunted inflammatory cytokine production in response to stimulation with purified LPS. By contrast, for other Gram-negative pathogens with highly acylated lipid A including Salmonella enterica or Escherichia coli, infection susceptibility and the response after stimulation with LPS were indistinguishable between mice expressing human or mouse TLR4/MD-2. Thus, Y. pestis exploits temperature-dependent shifts in LPS acylation to selectively evade recognition by human TLR4/MD-2 uncovered with “humanized” TLR4/MD-2 transgenic mice.     

Toll-like receptor (TLR) expression on CD4 and CD8 T-cells in patients chronically infected with hepatitis C virus

Toll-like receptor (TLR) expression on T-cells and the signalling pathways that lead to the production of cytokines may limit antigen-specific T-cell responses. Here, expression of TLR and retinoic acid inducible gene I (RIG-I) on T-cells were evaluated in patients chronically infected with hepatitis C virus (HCV), before and during pegylated interferon-α and ribavirin therapy. Expression of TLR2,3,4,7,9 and retinoic acid inducible gene (RIG)-I on different CD4⁺ and CD8⁺ T-cell sub-populations (naïve: CD45RA⁺CD57⁻; central memory: T_CM CD45RA⁻CD57⁻; effector memory: T_EM CD45RA⁻CD57⁺ and terminally differentiated effector memory: T_EMRA CD45RA⁺CD57⁺) were measured by flow cytometry. TLR7, TLR9 and RIG-I expression on CD4⁺ T-cells and RIG-I expression on CD8⁺ T-cells was higher in patients than healthy controls. Therapy increased expression of TLR2, TLR4 and TLR9 and this was observed for all T-cell sub-populations. Evaluation of TLR expression at baseline did not identify patients able to achieve sustained virological response following therapy.     

The Induction of Colitis and Ileitis in Mice Is Associated with Marked Increases in Intestinal Concentrations of Stimulants of TLRs 2, 4, and 5

Background

Inflammatory bowel diseases (IBDs) appear to be modulated by the interaction of pathogen-associated molecular patterns (PAMPs) derived from intestinal bacteria with their respective innate immune receptors, including Toll-like receptors (TLRs). We aimed to establish if intestinal concentrations of proinflammatory bacterial ligands of TLR2, TLR4, or TLR5 may be altered in murine IBD models, and to characterize which of the major bacterial groups may contribute to each signal.

Methodology/Principal Findings

PAMPs specific for TLR2 (lipopeptide equivalents), TLR4 (lipopolysaccharide equivalents), and TLR5 (flagellin equivalents) in human and murine fecal and intestinal samples were quantified using HEK-293 cells transfected with respective TLRs and calibrated with defined standard PAMPs. The induction of colitis in mice by dextran-sodium-sulphate treatment significantly increased colonic lipopeptide (fourfold) and LPS equivalent (550-fold) concentrations, while flagellin equivalent concentrations remained similar. The induction of ileitis by oral infection with Toxoplasma gondii dramatically increased ileal concentrations of lipopeptide (370-fold), LPS (3,300-fold), and flagellin equivalents (38-fold), all P<0.01. Analysis of representative strains of the major bacterial groups of the human intestine revealed that enterobacterial species are likely to be more significant contributors of soluble TLR2 and TLR4 stimulants to the intestinal milieu than Bacteroides species or Gram-positive Firmicutes.

Conclusions/Significance

We conclude that the induction of colitis or ileitis in mice is associated with significant disease-specific alterations to the PAMP profile of the gut microbiota.     

Inhibition of the phosphoinositide 3-kinase pathway decreases innate resistance to lipopolysaccharide toxicity in TLR4 deficient mice

Background

Upon lipopolysaccharide (LPS) stimulation, activation of both the Toll-like receptor 4 (TLR4) and phosphoinositide 3-kinase (PI3K) pathways serves to balance proinflammatory and anti-inflammatory responses. Although the antagonist to TLR4 represents an emerging promising target for the treatment of sepsis; however, the role of the PI3K pathway under TLR4-null conditions is not well understood. This goal of this study was to investigate the effect of inhibition of PI3K on innate resistance to LPS toxicity in a murine model.

Results

The overall survival of the cohorts receiving intraperitoneal injections of 100, 500, or 1000 μg LPS from Escherichia coli serotype 026:B6 after 7 d was 100%, 10%, and 10%, respectively. In contrast, no mortality was noted after 500-μg LPS injection in Tlr4^-/- mice. When the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 was injected (1 mg/25 g body weight) 1 h prior to the administration of LPS, the overall survival of the Tlr4^-/- mice was 30%. In the Tlr4^-/- mice, the LPS injection induced no NF-κB activation but an increased Akt phosphorylation in the lung and liver, when compared to that of the C57BL/6 mice. Injection of 500 μg LPS led to a significant induction in O₂^- detected by electron paramagnetic resonance (EPR) spin trapping spectroscopy in the lung and liver at 3 and 6 h in C57BL/6 but not Tlr4^-/- mice. Addition of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 only significantly increased the O₂^- level in the lung and liver of the Tlr4^-/- mice but not in the C57BL/6 mice following 500-μg LPS injection. In addition, the serum IL-1β and IL-2 levels were more elevated in C57BL/6 mice than in Tlr4^-/- mice. Notably, IL-1β and IL-2 were significantly increased in Tlr4^-/- mice but not in the C57BL/6 mice when the PI3K pathway was inhibited by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 prior to LPS injection.

Conclusions

In this study, we demonstrate that innate resistance to LPS toxicity in Tlr4^-/- mice is impaired by inhibition of the PI3K pathway, with a corresponding increase in mortality and production of tissue O₂^- and inflammatory cytokines.     

Surfactant Protein-A Modulates LPS-Induced TLR4 Localization and Signaling via β-Arrestin 2

The soluble C-type lectin surfactant protein (SP)-A mediates lung immune responses partially via its direct effects on alveolar macrophages (AM), the main resident leukocytes exposed to antigens. SP-A modulates the AM threshold of lipopolysaccharide (LPS) activity towards an anti-inflammatory phenotype both in vitro and in vivo through various mechanisms. LPS responses are tightly regulated via distinct pathways including subcellular TLR4 localization and thus ligand sensing. The cytosolic scaffold and signaling protein β-arrestin 2 acts as negative regulator of LPS-induced TLR4 activation. Here we show that SP-A neither increases TLR4 abundancy nor co-localizes with TLR4 in primary AM. SP-A significantly reduces the LPS-induced co-localization of TLR4 with the early endosome antigen (EEA) 1 by promoting the co-localization of TLR4 with the post-Golgi compartment marker Vti1b in freshly isolated AM from rats and wild-type (WT) mice, but not in β-arrestin 2^−/− AM. Compared to WT mice pulmonary LPS-induced TNF-α release in β-arrestin 2^−/− mice is accelerated and enhanced and exogenous SP-A fails to inhibit both lung LPS-induced TNF-α release and TLR4/EEA1 positioning. SP-A, but not LPS, enhances β-arrestin 2 protein expression in a time-dependent manner in primary rat AM. The constitutive expression of β-arrestin 2 in AM from SP-A^−/− mice is significantly reduced compared to SP-A^+/+ mice and is rescued by SP-A. Prolonged endosome retention of LPS-induced TLR4 in AM from SP-A^−/− mice is restored by exogenous SP-A, and is antagonized by β-arrestin 2 blocking peptides. LPS induces β-arrestin 2/TLR4 association in primary AM which is further enhanced by SP-A. The data demonstrate that SP-A modulates LPS-induced TLR4 trafficking and signaling in vitro and in vivo engaging β-arrestin 2.     

Salmonella enterica serovar Typhimurium ΔmsbB Triggers Exacerbated Inflammation in Nod2 Deficient Mice

The intracellular pathogen Salmonella enterica serovar Typhimurium causes intestinal inflammation characterized by edema, neutrophil influx and increased pro-inflammatory cytokine expression. A major bacterial factor inducing pro-inflammatory host responses is lipopolysaccharide (LPS). S. Typhimurium ΔmsbB possesses a modified lipid A, has reduced virulence in mice, and is being considered as a potential anti-cancer vaccine strain. The lack of a late myristoyl transferase, encoded by MsbB leads to attenuated TLR4 stimulation. However, whether other host receptor pathways are also altered remains unclear. Nod1 and Nod2 are cytosolic pattern recognition receptors recognizing bacterial peptidoglycan. They play important roles in the host''s immune response to enteric pathogens and in immune homeostasis. Here, we investigated how deletion of msbB affects Salmonella''s interaction with Nod1 and Nod2. S. Typhimurium Δ msbB-induced inflammation was significantly exacerbated in Nod2 ^−/− mice compared to C57Bl/6 mice. In addition, S. Typhimurium ΔmsbB maintained robust intestinal colonization in Nod2 ^−/− mice from day 2 to day 7 p.i., whereas colonization levels significantly decreased in C57Bl/6 mice during this time. Similarly, infection of Nod1 ^−/− and Nod1/Nod2 double-knockout mice revealed that both Nod1 and Nod2 play a protective role in S. Typhimurium ΔmsbB-induced colitis. To elucidate why S. Typhimurium ΔmsbB, but not wild-type S. Typhimurium, induced an exacerbated inflammatory response in Nod2 ^−/− mice, we used HEK293 cells which were transiently transfected with pathogen recognition receptors. Stimulation of TLR2-transfected cells with S. Typhimurium ΔmsbB resulted in increased IL-8 production compared to wild-type S. Typhimurium. Our results indicate that S. Typhimurium ΔmsbB triggers exacerbated colitis in the absence of Nod1 and/or Nod2, which is likely due to increased TLR2 stimulation. How bacteria with “genetically detoxified” LPS stimulate various innate responses has important implications for the development of safe and effective bacterial vaccines and adjuvants.     

TLR2 and its co-receptors determine responses of macrophages and dendritic cells to lipoproteins of Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) signals through Toll-like receptor 2 (TLR2) to regulate antigen presenting cells (APCs). Mtb lipoproteins, including LpqH, LprA, LprG and PhoS1, are TLR2 agonists, but their co-receptor requirements are unknown. We studied Mtb lipoprotein-induced responses in TLR2^−/−, TLR1^−/−, TLR6^−/−, CD14^−/− and CD36^−/− macrophages. Responses to LprA, LprG, LpqH and PhoS1 were completely dependent on TLR2. LprG, LpqH, and PhoS1 were dependent on TLR1, but LprA did not require TLR1. None of the lipoproteins required TLR6, although a redundant contribution by TLR6 cannot be excluded. CD14 contributed to detection of LprA, LprG and LpqH, whereas CD36 contributed only to detection of LprA. Studies of lung APC subsets revealed lower TLR2 expression by CD11b^high/CD11c^low lung macrophages than CD11b^low/CD11c^high alveolar macrophages, which correlated with hyporesponsiveness of lung macrophages to LpqH. Thus, lung APC subsets differ in TLR expression, which may determine differences in responses to Mtb.