Toll‐like receptor 4 agonists adsorbed to aluminium hydroxide adjuvant attenuate ovalbumin‐specific allergic airway disease: role of MyD88 adaptor molecule and interleukin‐12/interferon‐γ axis

Background Epidemiological and experimental data suggest that bacterial lipopolysaccharides (LPS) can either protect from or exacerbate allergic asthma. Lipopolysaccharides trigger immune responses through toll‐like receptor 4 (TLR4) that in turn activates two major signalling pathways via either MyD88 or TRIF adaptor proteins. The LPS is a pro‐Type 1 T helper cells (Th1) adjuvant while aluminium hydroxide (alum) is a strong Type 2 T helper cells (Th2) adjuvant, but the effect of the mixing of both adjuvants on the development of lung allergy has not been investigated. Objective We determined whether natural (LPS) or synthetic (ER‐803022) TLR4 agonists adsorbed onto alum adjuvant affect allergen sensitization and development of airway allergic disease. To dissect LPS‐induced molecular pathways, we used TLR4‐, MyD88‐, TRIF‐, or IL‐12/IFN‐γ‐deficient mice. Methods Mice were sensitized with subcutaneous injections of ovalbumin (OVA) with or without TLR4 agonists co‐adsorbed onto alum and challenged with intranasally with OVA. The development of allergic lung disease was evaluated 24 h after last OVA challenge. Results Sensitization with OVA plus LPS co‐adsorbed onto alum impaired in dose‐dependent manner OVA‐induced Th2‐mediated allergic responses such as airway eosinophilia, type‐2 cytokines secretion, airway hyper‐reactivity, mucus hyper production and serum levels of IgE or IgG1 anaphylactic antibodies. Although the levels of IgG2a, Th1‐affiliated isotype increased, investigation into the lung‐specific effects revealed that LPS did not induce a Th1 pattern of inflammation. Lipopolysaccharides impaired the development of Th2 immunity, signaling via TLR4 and MyD88 molecules and via the IL‐12/IFN‐γ axis, but not through TRIF pathway. Moreover, the synthetic TLR4 agonists that proved to have a less systemic inflammatory response than LPS also protected against allergic asthma development. Conclusion Toll‐like receptor 4 agonists co‐adsorbed with allergen onto alum down‐modulate allergic lung disease and prevent the development of polarized T cell‐mediated airway inflammation.     

The CD20 homolog Ms4a8a integrates pro‐ and anti‐inflammatory signals in novel M2‐like macrophages and is expressed in parasite infection

Recently, we identified the CD20 homolog Ms4a8a as a novel molecule expressed by tumor‐associated macrophages that directly enhances tumor growth. Here, we analyzed Ms4a8a⁺ macrophages in M2‐associated infectious pathologies. In late‐stage Trypanosoma congolense and Taenia crassiceps infections, Ms4a8a expression was detected in hepatic and peritoneal macrophages respectively. Innate immunity in these infections is modulated by Toll‐like receptor (TLR) signaling and TLR2/4/7 agonists strongly induced Ms4a8a expression in bone marrow derived macrophages (BMDMs) treated with M2 mediators (glucocorticoids/IL‐4). LPS/dexamethasone/IL‐4‐induced Ms4a8a⁺ BMDMs were characterized by strong expression of mRNA of mannose receptor (Mmr), arginase 1, and CD163, and by decreased iNOS expression. Coinduction of Ms4a8a by M2 mediators and TLR agonists involved the classical TLR signaling cascade via activation of MyD88/TRIF and NF‐κB. Forced overexpression of Ms4a8a modulated the TLR4 response of RAW264.7 cells as shown by gene expression profiling. Upregulation of Hdc, Tcfec, and Sla was confirmed both in primary LPS/dexamethasone/IL‐4‐stimulated Ms4a8a⁺ BMDMs and in peritoneal macrophages from late‐stage Taenia crassiceps infection. In conclusion, we show that TLR signaling skews the typical alternative macrophage activation program to induce a special M2‐like macrophage subset in vitro that also occurs in immunomodulatory immune reactions in vivo, a process directly involving the CD20 homolog Ms4a8a.     

Rapid CD4+ T‐cell responses to bacterial flagellin require dendritic cell expression of Syk and CARD9

Toll‐like receptors (TLRs) can recognize microbial patterns and utilize adaptor molecules, such as‐MyD88 or (TRIF TIR‐domain‐containing adapter‐inducing interferon‐β), to initiate downstream signaling that ultimately affects the initiation of adaptive immunity. In addition to this inflammatory role, TLR5 expression on dendritic cells can favor antigen presentation of flagellin peptides and thus increase the sensitivity of flagellin‐specific T‐cell responses in vitro and in vivo. Here, we examined the role of alternative signaling pathways that might regulate flagellin antigen presentation in addition to MyD88. These studies suggest a requirement for spleen tyrosine kinase, a noncanonical TLR‐signaling adaptor molecule, and its downstream molecule CARD9 in regulating the sensitivity of flagellin‐specific CD4⁺ T‐cell responses in vitro and in vivo. Thus, a previously unappreciated signaling pathway plays an important role in regulating the dominance of flagellin‐specific T‐cell responses.     

SARM inhibits both TRIF‐ and MyD88‐mediated AP‐1 activation

SARM (sterile α‐ and armadillo‐motif‐containing protein), the fifth identified TIR (Toll–interleukin 1 receptor (IL‐1R)) domain‐containing adaptors in humans, downregulates NF‐κB and IRF3 (interferon‐regulatory factor 3)‐mediated TLR3 and TLR4 signaling. SARM was characterized as a negative regulator of the TRIF (TIR‐domain‐containing adaptor protein inducing IFN‐β)‐dependent pathway via its interaction with TRIF. However, the precise mechanism of action of SARM remains unclear. Here, we demonstrate that SARM inhibits MAPK activation in human embryonic kidney 293 cells, and U937 cells. Both the TRIF‐ and MyD88‐mediated, as well as basal MAPK activity, were repressed, indicating that SARM‐mediated inhibition may not be exclusively directed at TRIF or MyD88, but that SARM may also directly inhibit MAPK phosphorylation. The MAPK inhibition effect was verified by RNAi, which increased the basal level of AP‐1. Furthermore, LPS challenge upregulated SARM at both the mRNA and protein levels. Finally, we provide evidence to show that truncated SARM changes its subcellular localization, suggesting the importance of the N‐terminal and sterile alpha motif domains in the autoregulation of SARM activity.     

Identification of a TLR4- and TRIF-dependent activation program of dendritic cells

Dendritic cell activation by Toll-like receptors (TLR) is crucial for the generation of protective immune responses. In addition to the common myeloid differentiation factor 88 (MyD88)-dependent signaling pathway, TLR4 engages the adaptor protein Toll/IL-1 receptor (TIR)-domain-containing adaptor inducing IFN-beta (TRIF), leading to interferon regulatory factor 3 (IRF-3) activation and type I interferon production. Using microarray expression profiling we now identify TRIF as a major regulator of the TLR4-triggered activation program of dendritic cells. We show that the expression of 47% of the genes that are responsive to TLR4 stimulation in wild-type dendritic cells is significantly altered in cells carrying a loss-of-function mutation of TRIF. Specifically, expression of IL-12, IL-18, and IL-23 was impaired in the absence of functional TRIF, suggesting that TLR4-promoted Th1 responses are TRIF-dependent. Furthermore, we provide evidence that TRIF regulates TLR4-mediated gene expression both by type I IFN-dependent and -independent mechanisms. Whereas dendritic cell production of CXCL10 and CCL12 was dependent on both TRIF and the type I interferon receptor, expression of IL-6 required TRIF but not type I interferon activity. Functional TRIF was also required for the normal induction of numerous genes considered important for host defense against diverse pathogens.Together, these data therefore identify TRIF as a crucial regulator of TLR4-dependent dendritic cell responses.     

Lack of SIGIRR/TIR8 aggravates hydrocarbon oil‐induced lupus nephritis

Multiple genetic factors contribute to the clinical variability of spontaneous systemic lupus erythematosus (SLE) but their role in drug‐induced SLE remain largely unknown. Hydrocarbon oil‐induced SLE depends on mesothelial cell apoptosis and Toll‐like receptor (TLR)‐7‐mediated induction of type I interferons. Hence, we hypothesized that TIR8/SIGIRR, an endogenous TLR inhibitor, prevents oil‐induced SLE. Sigirr‐deficient dendritic cells expressed higher TLR7 mRNA levels and TLR7 activation resulted in increased IL‐12 production in vitro. In vivo, lack of SIGIRR increased surface CD40 expression on spleen CD11c⁺ dendritic cells and MX‐1, TNF, IL‐12, BAFF and BCL‐2 mRNA expression 6 months after pristane injection. Spleen cell counts of CD4^?/CD8^? ‘autoreactive’ T cells and B220⁺ B cells were also increased in Sigirr^?/? mice. Serum autoantibody analysis revealed that Sigirr deficiency specifically enhanced the production of rheumatoid factor (from 4 months of age) and anti‐snRNP IgG (from 5 months of age), while anti‐Smith IgG or anti‐dsDNA IgG were independent of the Sigirr genotype. This effect was sufficient to significantly aggravate lupus nephritis in Sigirr‐deficient mice. Structure model prediction identified the BB loop of SIGIRR's intracellular TIR domain to interact with TLR7 and MyD88. BB loop deletion was sufficient to completely abrogate SIGIRR's inhibitory effect on TLR7 signalling. Thus, TIR8/SIGIRR protects from hydrocarbon oil‐induced lupus by suppressing the TLR7‐mediated activation of dendritic cells, via its intracellular BB loop. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

MyD88 and TRIF synergistic interaction is required for TH1‐cell polarization with a synthetic TLR4 agonist adjuvant

Glucopyranosyl lipid adjuvant‐stable emulsion (GLA‐SE) is a synthetic adjuvant TLR4 agonist that promotes potent poly‐functional T_H1 responses. Different TLR4 agonists may preferentially signal via MyD88 or TIR‐domain‐containing adapter inducing IFN‐beta (TRIF) to exert adjuvant effects; however, the contribution of MyD88 and TRIF signaling to the induction of polyclonal T_H1 responses by TLR4 agonist adjuvants has not been studied in vivo. To determine whether GLA‐SE preferentially signals through MyD88 or TRIF, we evaluated the immune response against a candidate tuberculosis (TB) vaccine Ag following immunization of mice lacking either signaling adapter compared with that of wild‐type mice. We find that both MyD88 and TRIF are necessary for GLA‐SE to induce a poly‐functional T_H1 immune response characterized by CD4⁺ T cells producing IFN‐γ, TNF, and IL‐2, as well as IgG2c class switching, when paired with the TB vaccine Ag ID93. Accordingly, the protective efficacy of ID93/GLA‐SE immunization against aerosolized Mycobacterium tuberculosis was lost when either signaling molecule was ablated. We demonstrate that MyD88 and TRIF must be expressed in the same cell for the in vivo T_H1‐skewing adjuvant activity, indicating that these two signaling pathways cooperate on an intracellular level. Thus engagement of both the MyD88 and TRIF signaling pathways are essential for the effective adjuvant activity of this TLR4 agonist.     

The MyD88 protein 88 pathway is differently involved in immune responses induced by distinct substrains of Leishmania major

Host resistance to Leishmania major is highly dependent on the development of a Th1 immune response. The TLR adaptator myeloid differentiation protein 88 (MyD88) has been implicated in the Th1 immune response associated with the resistant phenotype observed in C57BL/6 mice after infection with L. major. To investigate whether the MyD88 pathway is differentially used by distinct substrains of parasites, MyD88^−/− C57BL/6 mice were infected with two substrains of L. major, namely L. major LV39 and L. major IR75. MyD88^−/− mice were susceptible to both substrains of L. major, although with different kinetics of infection. The mechanisms involved during the immune response associated with susceptibility of MyD88^−/− mice to L. major is however, parasite substrain‐dependent. Susceptibility of MyD88^−/− mice infected with L. major IR75 is a consequence of Th2 immune‐deviation, whereas susceptibility of MyD88^−/− mice to infection with L. major LV39 resulted from an impaired Th1 response. Depletion of regulatory T cells (Treg) partially restored IFN‐γ secretion and the Th1 immune response in MyD88^−/− mice infected with L. major LV39, demonstrating a role of Treg activity in the development of an impaired Th1 response in these mice.     

Toll-like receptor and tumour necrosis factor dependent endotoxin-induced acute lung injury

Recent studies on endotoxin/lipopolysaccharide (LPS)-induced acute inflammatory response in the lung are reviewed. The acute airway inflammatory response to inhaled endotoxin is mediated through Toll-like receptor 4 (TLR4) and CD14 signalling as mice deficient for TLR4 or CD14 are unresponsive to endotoxin. Acute bronchoconstriction, tumour necrosis factor (TNF), interleukin (IL)-12 and keratinocyte-derived chemokine (KC) production, protein leak and neutrophil recruitment in the lung are abrogated in mice deficient for the adaptor molecules myeloid differentiation factor 88 (MyD88) and Toll/Interleukin-1 receptor (TIR)-domain-containing adaptor protein (TIRAP), but independent of TIR-domain-containing adaptor-inducing interferon-beta (TRIF). In particular, LPS-induced TNF is required for bronchoconstriction, but dispensable for inflammatory cell recruitment. Lipopolysaccharide induces activation of the p38 mitogen-activated protein kinase (MAPK). Inhibition of pulmonary MAPK activity abrogates LPS-induced TNF production, bronchoconstriction, neutrophil recruitment into the lungs and broncho-alveolar space. In conclusion, TLR4-mediated, bronchoconstriction and acute inflammatory lung pathology to inhaled endotoxin are dependent on TLR4/CD14/MD2 expression using the adapter proteins TIRAP and MyD88, while TRIF, IL-1R1 or IL-18R signalling pathways are dispensable. Further downstream in this axis of signalling, TNF blockade reduces only acute bronchoconstriction, while MAPK inhibition abrogates completely endotoxin-induced inflammation.     

Chitin induces upregulation of B7‐H1 on macrophages and inhibits T‐cell proliferation

Chitin is a highly abundant glycopolymer, which serves as structural component in fungi, arthropods and crustaceans but is not synthesized by vertebrates. However, vertebrates express chitinases and chitinase‐like proteins, some of which are induced by infection with helminths suggesting that chitinous structures may be targets of the immune system. The chitin‐induced modulations of the innate and adaptive immune responses are not well understood. Here, we demonstrate that intranasal administration of OVA and chitin resulted in diminished T‐cell expansion and Th2 polarization as compared with OVA administration alone. Chitin did not promote nor attenuate Th2 polarization in vitro. Chitin‐exposed macrophages inhibited proliferation of CD4⁺ T cells in a cell–cell contact‐dependent manner. Chitin induced upregulation of the inhibitory ligand B7‐H1 (PD‐L1) on macrophages independently of MyD88, TRIF, TLR2, TLR3, TLR4 and Stat6. Inhibition of T‐cell proliferation was largely dependent on B7‐H1, as the effect was not observed in cocultures with cells from B7‐H1‐deficient mice.     

Polysaccharide Agaricus blazei Murill stimulates myeloid derived suppressor cell differentiation from M2 to M1 type,which mediates inhibition of tumour immune‐evasion via the Toll‐like receptor 2 pathway

Gr‐1⁺ CD11b⁺ myeloid‐derived suppressor cells (MDSCs) accumulate in tumor‐bearing animals and play a critical negative role during tumor immunotherapy. Strategies for inhibition of MDSCs are expected to improve cancer immunotherapy. Polysaccharide Agaricus blazei Murill (pAbM) has been found to have anti‐cancer activity, but the underlying mechanism of this is poorly understood. Here, pAbM directly activated the purified MDSCs through inducing the expression of interleukin‐6 (IL‐6), IL‐12, tumour necrosis factor and inducible nitric oxide synthase (iNOS), CD86, MHC II, and pSTAT1 of it, and only affected natural killer and T cells in the presence of Gr‐1⁺ CD11b⁺ monocytic MDSCs. On further analysis, we demonstrated that pAbM could selectively block the Toll‐like receptor 2 (TLR2) signal of Gr‐1⁺ CD11b⁺ MDSCs and increased their M1‐type macrophage characteristics, such as producing IL‐12, lowering expression of Arginase 1 and increasing expression of iNOS. Extensive study showed that Gr‐1⁺ CD11b⁺ MDSCs by pAbM treatment had less ability to convert the CD4⁺ CD25^? cells into CD4⁺ CD25⁺ phenotype. Moreover, result from selective depletion of specific cell populations in xenograft mice model suggested that the anti‐tumour effect of pAbM was dependent on Gr‐1⁺ CD11b⁺ monocytes, nether CD8⁺ T cells nor CD4⁺ T cells. In addition to, pAbM did not inhibit tumour growth in TLR2^–/– mice. All together, these results suggested that pAbM, a natural product commonly used for cancer treatment, was a specific TLR2 agonist and had potent anti‐tumour effects through the opposite of the suppressive function of Gr‐1⁺ CD11b⁺ MDSCs.     

Children with atopic histories exhibit impaired lipopolysaccharide‐induced Toll‐like receptor‐4 signalling in peripheral monocytes

Background The hygiene hypothesis states that early exposure to bacterial products such as lipopolysaccharide (LPS) may be protective against the development of allergic diseases. Whether atopic disease affects the ability of immune cells to respond to LPS is unclear. Our laboratory has demonstrated previously that children express high levels of Toll‐like receptor (TLR)‐4 on CD4⁺ cells in nasal mucosa. Objective To determine if children with a history of allergic disease have impaired responses to LPS on circulating CD4⁺ leucocytes. Methods Peripheral blood mononuclear cells from children (aged 2–18) and adults with or without a history of atopic conditions were cultured with/without IL‐4 or LPS for up to 24 h. Expression of surface TLR‐4, CD14, CD4, CD3, as well as of intracellular phosphorylated ^p42/p44ERK and p38 mitogen‐activated protein kinase (MAPK) were assessed by flow cytometry. Results A history of atopy in children was associated with impaired LPS‐induced TLR‐4‐dependent phosphorylation of ^p42/44ERK and p38 MAPK by CD4⁺ monocytes. Decreased LPS signalling was reproduced by pre‐incubation of control cells with recombinant IL‐4. LPS stimulation also decreased TLR‐4 expression on monocytes from children without atopic histories but not from atopic subjects. CD4⁺ T lymphocytes showed limited LPS responsiveness, regardless of atopic status. In contrast with non‐atopic children, TLR‐4 expression on monocytes of children with atopic histories decreased as a function of age. Conclusions This study provides evidence for defective LPS recognition on circulating CD4⁺ leucocytes of subjects with atopic histories compared with those from non‐atopic children. CD4⁺TLR4⁺ monocytes from children with atopic histories failed to phosphorylate MAPKs. Our results suggest that a history of atopic disease is associated with impaired TLR‐4‐mediated innate immune function compared with non‐atopic children. Cite this as: D. Préfontaine, A.‐A. Banville‐Langelier, P.‐O. Fiset, J. Guay, J. An, M. Mazer, Q. Hamid and B. D. Mazer, Clinical & Experimental Allergy, 2010 (40) 1648–1657.     

Patency of Litomosoides sigmodontis infection depends on Toll‐like receptor 4 whereas Toll‐like receptor 2 signalling influences filarial‐specific CD4+ T‐cell responses

BALB/c mice develop a patent state [release of microfilariae (Mf), the transmission life‐stage, into the periphery] when exposed to the rodent filariae Litomosoides sigmodontis. Interestingly, only a portion of the infected mice become patent, which reflects the situation in human individuals infected with Wuchereria bancrofti. Since those individuals had differing filarial‐specific profiles, this study compared differences in immune responses between Mf⁺ and Mf^– infected BALB/c mice. We demonstrate that cultures of total spleen or mediastinal lymph node cells from Mf⁺ mice produce significantly more interleukin‐5 (IL‐5) to filarial antigens but equal levels of IL‐10 when compared with Mf^– mice. However, isolated CD4⁺ T cells from Mf⁺ mice produced significantly higher amounts of all measured cytokines, including IL‐10, when compared with CD4⁺ T‐cell responses from Mf^– mice. Since adaptive immune responses are influenced by triggering the innate immune system we further studied the immune profiles and parasitology in infected Toll‐like receptor‐2‐deficient (TLR2^?/?) and TLR4^?/? BALB/c mice. Ninety‐three per cent of L. sigmodontis‐exposed TLR4^?/? BALB/c mice became patent (Mf⁺) although worm numbers remained comparable to those in Mf⁺ wild‐type controls. Lack of TLR2 had no influence on patency outcome or worm burden but infected Mf⁺ mice had significantly lower numbers of Foxp3⁺ regulatory T cells and dampened peripheral immune responses. Interestingly, in vitro culturing of CD4⁺ T cells from infected wild‐type mice with granulocyte–macrophage colony‐stimulating factor‐derived TLR2^?/? dendritic cells resulted in an overall diminished cytokine profile to filarial antigens. Hence, triggering TLR4 or TLR2 during chronic filarial infection has a significant impact on patency and efficient CD4⁺ T‐cell responses, respectively.     

Radioresistant cells expressing TLR5 control the respiratory epithelium's innate immune responses to flagellin

Bacterial products (such as endotoxins and flagellin) trigger innate immune responses through TLRs. Flagellin‐induced signalling involves TLR5 and MyD88 and, according to some reports, TLR4. Whereas epithelial and dendritic cells are stimulated by flagellin in vitro, the cell contribution to the in vivo response is still unclear. Here, we studied the respective roles of radioresistant and radiosensitive cells in flagellin‐induced airway inflammation in mice. We found that i.n. delivery of flagellin elicits a transient change in respiratory function and an acute, pro‐inflammatory response in the lungs, characterized by TLR5‐ and MyD88‐dependent chemokine secretion and neutrophil recruitment. In contrast, TLR4, CD14 and TRIF were not essential for flagellin‐mediated responses, indicating that TLR4 does not cooperate with TLR5 in the lungs. Respiratory function, chemokine secretion and airway infiltration by neutrophils were dependent on radioresistant, TLR5‐expressing cells. Furthermore, lung haematopoietic cells also responded to flagellin by activating TNF‐α production. We suggest that the radioresistant lung epithelial cells are essential for initiating early, TLR5‐dependent signalling in response to flagellin and thus triggering the lung's innate immune responses.     

Activation profile of Toll‐like receptors of peripheral blood lymphocytes in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease associated with aberrant activation of T and B lymphocytes for the production of inflammatory cytokines and autoreactive antibodies. Animal studies of SLE have indicated that Toll‐like receptors (TLR) are important in the pathogenesis of murine lupus. In the present clinical study, differential protein expressions of TLR‐1–9 of monocytes and different lymphocyte subsets from patients with SLE and normal control subjects were determined by flow cytometry. Results showed that the expression of intracellular TLRs (TLR‐3, ‐8, ‐9) and extracellular TLRs (TLR‐1, ‐2, ‐4, ‐5, ‐6) were elevated in monocytes, CD4⁺ T lymphocytes, CD8⁺ T lymphocytes and B lymphocytes of SLE patients compared to control subjects (all P < 0·001). Moreover, cell surface expression of TLR‐4 on CD4⁺ T lymphocytes and CD8⁺ T lymphocytes, and TLR‐6 on B lymphocytes, were correlated positively with SLE disease activity index (SLEDAI) (TLR‐4 on CD4⁺ T lymphocytes and CD8⁺ T lymphocytes: r = 0·536, P = 0·04; r = 0·713, P = 0·003; TLR‐6 in B lymphocytes: r = 0·572, P = 0·026). In concordance with the above results, there is an observable increased relative induction (%) of inflammatory cytokine interleukin (IL)‐1β, IL‐6, IL‐10 and IL‐12, chemokines CCL2, CXCL8, CCL5 and CXCL10 from peripheral blood mononuclear cells (PBMC) upon differential stimulation by PolyIC (TLR‐3 ligand), lipopolysaccharide (TLR‐4 ligand), peptidoglycan (TLR‐2 ligand), flagellin (TLR‐5 ligand), R837 (TLR‐7 ligand) and CpG DNA (TLR‐9 ligand) in SLE patients compared to controls. These results suggest that the innate immune response for extracellular pathogens and self‐originated DNA plays immunopathological roles via TLR activation in SLE.     

A genital tract peptide epitope vaccine targeting TLR-2 efficiently induces local and systemic CD8+ T cells and protects against herpes simplex virus type 2 challenge

The next generation of needle-free mucosal vaccines is being rationally designed according to rules that govern the way in which the epitopes are recognized by and stimulate the genital mucosal immune system. We hypothesized that synthetic peptide epitopes extended with an agonist of Toll-like receptor 2 (TLR-2), that are abundantly expressed by dendritic and epithelial cells of the vaginal mucosa, would lead to induction of protective immunity against genital herpes. To test this hypothesis, we intravaginally (IVAG) immunized wild-type B6, TLR-2 (TLR2^−/−) or myeloid differentiation factor 88 deficient (MyD88^−/−) mice with a herpes simplex virus type 2 (HSV-2) CD8⁺ T-cell peptide epitope extended by a palmitic acid moiety (a TLR-2 agonist). IVAG delivery of the lipopeptide generated HSV-2-specific memory CD8⁺ cytotoxic T cells both locally in the genital tract draining lymph nodes and systemically in the spleen. Moreover, lipopeptide-immunized TLR2^−/− and MyD88^−/− mice developed significantly less HSV-specific CD8⁺ T-cell response, earlier death, faster disease progression, and higher vaginal HSV-2 titers compared to lipopeptide-immunized wild-type B6 mice. IVAG immunization with self-adjuvanting lipid-tailed peptides appears to be a novel mucosal vaccine approach, which has attractive practical and immunological features.     

Identification of two subpopulations of purified human blood B cells, CD27- CD23+ and CD27high CD80+, that strongly express cell surface Toll-like receptor 9 and secrete high levels of interleukin-6

B‐cell expression of certain Toll‐like receptors (TLRs) is important in linking innate and adaptive immune responses in normal and pathological conditions. The expression of TLR9 plays a role in the recognition of conserved pathogen motifs in a manner that is dependent on B‐cell localization, deduced from B‐cell phenotype. The nature of TLR9 function is unclear. A first step in unravelling the function of this pattern recognition receptor is to discover the precise nature of the cell types that express TLR9. This study used three‐colour flow cytometry to characterize the B lymphocytes from human peripheral blood mononuclear cells (PBMCs) that express TLR9 on the surface. We sorted TLR9‐positive B and non‐B cells from the PBMC population and detected TLR9 expression on naïve and memory B cells. Moreover, we identified two discrete subpopulations of B cells: CD19⁺ CD27^? CD23⁺ cells and CD19⁺ CD27^high CD80⁺ cells. These subpopulations expressed high levels of membrane TLR9 and exhibited a strong in vitro response to binding a relevant CpG motif by secreting high levels of interleukin‐6 (compared to controls). Our finding that this pattern recognition receptor is expressed on a variety of cell subsets adds to the current understanding of the functional complexity of B‐cell membrane TLR9.