Immunosuppressive components of Ascaris suum down-regulate expression of costimulatory molecules and function of antigen-presenting cells via an IL-10-mediated mechanism

High-molecular-weight components (PI) of Ascaris suum suppress both cell-mediated and humoral responses against ovalbumin (OVA) via an IL-4/IL-10-dependent mechanism. The aim of this work was to investigate the effect of PI on the ability of APC to activate T cells and the role of IL-10 in this process. Flow cytometry analyses of MHC class II, CD80, CD86 and CD40 molecules on LN cells from mice immunized with OVA or OVA+PI showed that PI inhibits expression of these molecules on unfractionated cells and on purified CD11c(+) cells. A low proliferative response was obtained when OVA-specific TCR-Tg T cells were incubated with CD11c(+) cells from OVA+PI-immunized mice pulsed with OVA, when compared to those incubated with cells from OVA-immunized mice. Similar results were obtained using as APC CD11c(+) cells from OVA-immunized mice pulsed with OVA+PI, which also expressed less of the four markers. The inhibitory effect of PI on both the expression of costimulatory molecules and the induction of T cell proliferation was abolished in IL-10-deficient mice. Our data indicate that the potent immunosuppressive effect of A. suum extract components on the host immune system is primarily related to their property of down-regulating the Ag-presenting ability of DC via an IL-10-mediated mechanism.     

Sphingosine-kinase 1 and 2 contribute to oral sensitization and effector phase in a mouse model of food allergy

Background

Sphingosine-1-phosphate (S1P) influences activation, migration and death of immune cells. Further, S1P was proposed to play a major role in the induction and promotion of allergic diseases. However, to date only limited information is available on the role of S1P in food allergy.

Objective

We aimed to investigate the role of sphingosine-kinase (SphK) 1 and 2, the enzymes responsible for endogenous S1P production, on the induction of food allergy.

Methods and results

Human epithelial colorectal CaCo2 cells stimulated in vitro with S1P revealed a decrease of transepithelial resistance and enhanced transport of FITC labeled OVA. We studied the effect of genetic deletion of the enzymes involved in S1P production on food allergy induction using a mouse model of food allergy based on intragastrically (i.g.) administered ovalbumin (OVA) with concomitant acid-suppression. Wild-type (WT), SphK1^−/− and SphK2^−/− mice immunized with OVA alone i.g. or intraperitoneally (i.p.) were used as negative or positive controls, respectively. SphK1- and SphK2-deficient mice fed with OVA under acid-suppression showed reduced induction of OVA specific IgE and IgG compared to WT mice, but had normal responses when immunized by the intraperitoneal route. Flow cytometric analysis of spleen cells revealed a significantly reduced proportion of CD4⁺ effector T-cells in both SphK deficient animals after oral sensitization. This was accompanied by a reduced accumulation of mast cells in the gastric mucosa in SphK-deficient animals compared to WT mice. Furthermore, mouse mast cell protease-1 (mMCP-1) levels, an IgE-mediated anaphylaxis marker, were reliably elevated in allergic WT animals.

Conclusion

Modulation of the S1P homeostasis by deletion of either SphK1 or SphK2 alters the sensitization and effector phase of food allergy.     

Toll‐like receptor 4 signalling through MyD88 is essential to control Salmonella enterica serovar Typhimurium infection,but not for the initiation of bacterial clearance

Toll-like receptor-4 (TLR4) is important in protection against lethal Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. Control of the early stages of sublethal S. Typhimurium infection in mice depends on TLR4-dependent activation of macrophages and natural killer (NK) cells to drive an inflammatory response. TLR4 signals through the adapter proteins Mal/MyD88 and TRIF-related adaptor molecule (TRAM)/TIR-domain-containing adaptor-inducing interferon-b (TRIF). In the mouse typhoid model we showed that TLR4 and MyD88, but not Mal or TRIF, are essential for the control of exponential S. Typhimurium growth. TRIF^−/− mice have a higher bacterial load in comparison with wild-type mice during a sublethal infection because TRIF is important for bacterial killing during the first day of systemic disease. Minimal pro-inflammatory responses were induced by S. Typhimurium infection of macrophages from TLR4^−/−, MyD88^−/− and TRIF^−/− mice in vitro. Pro-inflammatory responses from Mal^−/− macrophages were similar to those from wild-type cells. The pro-inflammatory responses of TRIF^−/− macrophages were partially restored by the addition of interferon-γ (IFN-γ), and TRIF^−/− mice produced markedly enhanced IFN-γ levels, in comparison to wild-type mice, probably explaining why bacterial growth can be controlled in these mice. TLR4^−/−, MyD88^−/−, TRIF^−/− and Mal^−/− mice all initiated clearance of S. Typhimurium, suggesting that TLR4 signalling is not important in driving bacterial clearance in comparison to its critical role in controlling early bacterial growth in mouse typhoid.     

TLR2 signaling improves immunoregulation to prevent type 1 diabetes

Signaling through TLR2 promotes inflammation and modulates CD4⁺CD25⁺ Tregs. We assessed mechanistically how this molecule would alter immunoregulation in type 1 diabetes (T1D). We also asked whether TLR2 may be involved in our recent discovery that viral infection can protect from autoimmune diabetes by expanding and invigorating Tregs. Treatment of prediabetic mice with a synthetic TLR2 agonist diminished T1D and increased the number and function of CD4⁺CD25⁺ Tregs, also conferring DCs with tolerogenic properties. TLR2 ligation also promoted the expansion of Tregs upon culture with DCs and ameliorated their capacity to prevent the disease. Protection from T1D by lymphocytic choriomeningitis virus (LCMV) infection depended on TLR2. LCMV increased the frequency of CD4⁺CD25⁺ Tregs and their production of TGF‐β more significantly in WT than TLR2‐deficient mice. Furthermore, LCMV infection in vivo or LCMV‐infected DCs in vitro rendered, via TLR2, CD4⁺CD25⁺ Tregs capable of diminishing T1D. We identify novel mechanisms by which TLR2 promotes immunoregulation and controls autoimmune diabetes in naïve or infected hosts. This work should help understand T1D etiology and develop novel immune‐based therapeutic interventions.     

CD8α⁺ dendritic cells improve collagen-induced arthritis in CC chemokine receptor (CCR)-2 deficient mice

Objective

Dendritic cells (DCs) have long been recognized as potential therapeutic targets of rheumatoid arthritis (RA). Increasing evidence has showed that DCs are capable of suppressing autoimmunity by expanding FoxP3⁺ regulatory T cells (T_reg), which in turn exert immunosuppression by increasing TGFβ-1. In the SKG mice, activated DC prime autoreactive T cells causing autoantibody production and an inflammatory arthritic response. Recently, we reported that CC-chemokine receptor-2 deficient (Ccr2^−/−) mice had impaired DCs migration and reduced CD8α⁺ DCs in the C57Bl/6J mice strain and that these mice were more susceptible to collagen antibody-induced arthritis (CAIA), compared to wild type mice. To examine the mechanism by which DCs contribute to the increased susceptibility of arthritis in Ccr2^−/− mice, we tested the hypothesis that CD8α⁺ DCs are protective (tolerogenic) against autoimmune arthritis by examining the role of CD8α⁺ DCs in Ccr2^−/− and SKG mice.

Methods

To examine the mechanism by which DCs defects lead to the development of arthritis, we used two murine models of experimental arthritis: collagen-induced arthritis (CIA) in DBA1/J mice and zymosan-induced arthritis in SKG mice. DBA1/J mice received recombinant fms-like tyrosine kinase 3 ligand (Flt3L) injections to expand endogenous DCs populations or adoptive transfers of CD8α⁺ DCs.

Results

Flt3L-mediated expansion of endogenous CD8α⁺ DCs resulted in heightened susceptibility of CIA. In contrast, supplementation with exogenous CD8α⁺ DCs ameliorated arthritis in Ccr2^−/− mice and enhanced TGFβ1 production by T cells. Furthermore, SKG mice with genetic inactivation of CCR2 did not affect the numbers of DCs nor improve the arthritis phenotype.

Conclusion

CD8α⁺ DCs were tolerogenic to the development of arthritis. CD8α⁺ DCs deficiency heightened the sensitivity to arthritis in Ccr2^−/− mice. Ccr2 deficiency did not alter the arthritic phenotype in SKG mice suggesting the arthritis in Ccr2^−/− mice was T cell-independent.     

TLR5 functions as an endocytic receptor to enhance flagellin-specific adaptive immunity

Innate immune activation via TLR induces dendritic cell maturation and secretion of inflammatory mediators, generating favorable conditions for naïve T‐cell activation. Here, we demonstrate a previously unknown function for TLR5, namely that it enhances MHC class‐II presentation of flagellin epitopes to CD4⁺ T cells and is required for induction of robust flagellin‐specific adaptive immune responses. Flagellin‐specific CD4⁺ T cells expanded poorly in TLR5‐deficient mice immunized with flagellin, a deficiency that persisted even when additional TLR agonists were provided. Flagellin‐specific IgG responses were similarly depressed in the absence of TLR5. In marked contrast, TLR5‐deficient mice developed robust flagellin‐specific T‐cell responses when immunized with processed flagellin peptide. Surprisingly, the adaptor molecule Myd88 was not required for robust CD4⁺ T‐cell responses to flagellin, indicating that TLR5 enhances flagellin‐specific CD4⁺ T‐cell responses in the absence of conventional TLR signaling. A requirement for TLR5 in generating flagellin‐specific CD4⁺ T‐cell activation was also observed when using an in vitro dendritic cell culture system. Together, these data uncover an Myd88‐independent function for dendritic cell TLR5 in enhancing the presentation of peptides to flagellin‐specific CD4⁺ T cells.     

Requirement for TLR2 in the development of albuminuria,inflammation and fibrosis in experimental diabetic nephropathy

Inflammation and fibrosis are essential elements of diabetic nephropathy (DN). We tested the hypothesis that these elements are dependent upon Toll-like receptor 2 (TLR2) signalling by examining WT and TLR2^-/- mice in an experimental model of DN. Diabetes was induced in WT and TLR2^-/- mice by i.p. injection of streptozotocin. Kidney injury was assessed at 6, 12 and 24 weeks after induction of diabetes. Gene expression of TLR2, its endogenous ligands and downstream cytokines, chemokines and fibrogenic molecules were upregulated in kidneys from WT mice with streptozotocin diabetes. TLR2^-/- mice were protected against the development of DN, exhibiting less albuminuria, inflammation, glomerular hypertrophy and hypercellularity, podocyte and tubular injury as compared to diabetic WT controls. Marked reductions in interstitial collagen deposition, myofibroblast activation (α-SMA) and expression of fibrogenic genes (TGF-β and fibronectin) were also evident in TLR2 deficient mice. Consistent with our in vivo results, high glucose directly promoted TLR2 activation in podocytes and tubular epithelial cells (TECs) in vitro, resulting in NF-κB activation, inflammation and TGF-β production. We conclude that TLR2 was required for the full development of inflammation, kidney damage and fibrosis in this model of DN. As TLR2 is known to be expressed by intrinsic kidney cells and as high concentration glucose stimulated podocytes and TECs in vitro to express TLR2 and TLR2 ligands, pro-inflammatory and pro-fibrotic cytokines in a TLR2 dependent manner in the present study, it appears likely that TLR2 signalling in intrinsic kidney cells contributes to the pathogenesis of diabetic nephropathy.     

Elevated CD14 (Cluster of Differentiation 14) and Toll‐Like Receptor (TLR) 4 Signaling Deteriorate Periapical Inflammation in TLR2 Deficient Mice

Apical periodontitis (periapical lesions) is an infection‐induced chronic inflammation in the jaw, ultimately resulting in the destruction of apical periodontal tissue. Toll‐like receptors (TLRs) are prominent in the initial recognition of pathogens. Our previous study showed that TLR4 signaling is proinflammatory in periapical lesions induced by a polymicrobial endodontic infection. In contrast, the functional role of TLR2 in regulation of periapical tissue destruction is still not fully understood. Using TLR2 deficient (KO), TLR2/TLR4 double deficient (dKO), and wild‐type (WT) mice, we demonstrate that TLR2 KO mice are highly responsive to polymicrobial infection‐induced periapical lesion caused by over activation of TLR4 signal transduction pathway that resulted in elevation of NF‐kB (nuclear factor kappa B) and proinflammatory cytokine production. The altered TLR4 signaling is caused by TLR2 deficiency‐dependent elevation of CD14 (cluster of differentiation 14), which is a co‐receptor of TLR4. Indeed, neutralization of CD14 strikingly suppresses TLR2 deficiency‐dependent inflammation and tissue destruction in vitro and in vivo. Our findings suggest that a network of TLR2, TLR4, and CD14 is a key factor in regulation of polymicrobial dentoalveolar infection and subsequent tissue destruction. Anat Rec, 299:1281–1292, 2016. © 2016 Wiley Periodicals, Inc.     

Protease‐activated receptor 2 signalling promotes dendritic cell antigen transport and T‐cell activation in vivo

Deficiency of protease-activated receptor-2 (PAR2) modulates inflammation in several models of inflammatory and autoimmune disease, although the underlying mechanism(s) are not understood. PAR2 is expressed on endothelial and immune cells, and is implicated in dendritic cell (DC) differentiation. We investigated in vivo the impact of PAR2 activation on DCs and T cells in PAR2 wild-type (WT) and knockout (KO) mice using a specific PAR2 agonist peptide (AP2). PAR2 activation significantly increased the frequency of mature CD11c^high DCs in draining lymph nodes 24 hr after AP2 administration. Furthermore, these DCs exhibited increased expression of major histocompatibility complex (MHC) class II and CD86. A significant increase in activated (CD44⁺ CD62⁻) CD4⁺ and CD8⁺ T-cell frequencies was also observed in draining lymph nodes 48 hr after AP2 injection. No detectable change in DC or T-cell activation profiles was observed in the spleen. The influence of PAR2 signalling on antigen transport to draining lymph nodes was assessed in the context of delayed-type hypersensitivity. PAR2 WT mice that were sensitized by skin-painting with fluorescein isothiocyanate (FITC) to induce delayed-type hypersensitivity possessed elevated proportion of FITC⁺ DCs in draining lymph nodes 24 hr after FITC painting when compared with PAR2 KO mice (0·95% versus 0·47% of total lymph node cells). Collectively, these results demonstrate that PAR2 signalling promotes DC trafficking to the lymph nodes and subsequent T-cell activation, and thus provides an explanation for the pro-inflammatory effect of PAR2 in animal models of inflammation.     

Subcutaneous immunization with Streptococcus pneumoniae GAPDH confers effective protection in mice via TLR2 and TLR4

The surface localized Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Streptococcus pneumoniae is best known as housekeeping protein. Currently, GAPDH has been recognized as moonlighting protein and virulent factor. Therefore, we investigate whether GAPDH can act as a suitable vaccine candidate protein to prevent pneumococcal infection. In this study, mice received subcutaneous vaccination with recombinant GAPDH followed by challenge with D39 and 19F showing higher survival rate and lower bacterial loads in nasal washes and lung homogenates than control. Meanwhile, high titers of rGAPDH specific antibody and elevated titers of IgG subtype indicated that rGAPDH could elicit immune response in mice. Then, we investigated the mechanism that immunization with rGAPDH conferred protection against Streptococcus pneumoniae in host. In vitro experiments, rGAPDH induced phenotypic and functional maturation of BMDCs, because the high expression of CD40, CD86 and MHC II and the production of IL-12p70, IL-6 and TNF-α were observed after treatment with rGAPDH. However, the costimulatory molecules and cytokines declined significantly in TLR2^−/− and TLR4^−/− mice, indicating rGAPDH can be a potential ligand for both TLR2 and TLR4. Subsequent investigations suggested that rGAPDH could also activate the phosphorylation of MAPKs, PI3K-Akt and NF-κB. Meantime, upregulation of mir-146a and downregulation of mir-27a in BMDCs were observed. Taken together, our findings confirm that rGAPDH, a housekeeping protein, is also qualified as a vaccine candidate protein and rGAPDH activates BMDCs in a TLR2 and TLR4 dependent manner.     

Pneumococcal DnaJ modulates dendritic cell-mediated Th1 and Th17 immune responses through Toll-like receptor 4 signaling pathway

Pneumococcal DnaJ was recently shown to be a potential protein vaccine antigen that induces strong Th1 and Th17 immune response against streptococcus pneumoniae infection in mice. However, how DnaJ mediates T cell immune response against S. pneumoniae infection has not been addressed. Here, we investigate whether DnaJ contributes to the development of T cell immunity through the activation of bone marrow-derived dendritic cells (BMDCs). We found that endotoxin-free recombinant DnaJ (rDnaJ) induced activation and maturation of BMDCs via recognition of Toll-like receptor 4 (TLR4) and activation of MAPKs, NF-κB and PI3K-Akt pathways. rDnaJ-treated BMDCs effectively stimulated naïve CD4⁺ T cells to secrete IFN-γ and IL-17A. Splenocytes from mice that were adoptively transferred with rDnaJ-pulsed BMDCs secreted higher levels of IFN-γ and IL-17A compared with those that received PBS-activated BMDCs. Splenocytes from TLR4^−/− mice immunized with rDnaJ produced lower levels of IFN-γ and IL-17A compared with those from wild type mice. Our findings indicate that DnaJ can induce Th1 and Th17 immune responses against S. pneumoniae through activation of BMDCs in a TLR4-dependent manner.     

Nontoxic Shiga toxin derivatives from Escherichia coli possess adjuvant activity for the augmentation of antigen-specific immune responses via dendritic cell activation

Shiga toxin (Stx) derivatives, such as the Stx1 B subunit (StxB1), which mediates toxin binding to the membrane, and mutant Stx1 (mStx1), which is a nontoxic doubly mutated Stx1 harboring amino acid substitutions in the A subunit, possess adjuvant activity via the activation of dendritic cells (DCs). Our results showed that StxB1 and mStx1, but not native Stx1 (nStx1), resulted in enhanced expression of CD86, CD40, and major histocompatibility complex (MHC) class II molecules and, to some extent, also enhanced the expression of CD80 on bone marrow-derived DCs. StxB1-treated DCs exhibited an increase in tumor necrosis factor alpha and interleukin-12 (IL-12) production, a stimulation of DO11.10 T-cell proliferation, and the production of both Th1 and Th2 cytokines, including gamma interferon (IFN-gamma), IL-4, IL-5, IL-6, and IL-10. When mice were given StxB1 subcutaneously, the levels of CD80, CD86, and CD40, as well as MHC class II expression by splenic DCs, were enhanced. The subcutaneous immunization of mice with ovalbumin (OVA) plus mStx1 or StxB1 induced high titers of OVA-specific immunoglobulin M (IgM), IgG1, and IgG2a in serum. OVA-specific CD4+ T cells isolated from mice immunized with OVA plus mStx1 or StxB1 produced IFN-gamma, IL-4, IL-5, IL-6, and IL-10, indicating that mStx1 and StxB1 elicit both Th1- and Th2-type responses. Importantly, mice immunized subcutaneously with tetanus toxoid plus mStx1 or StxB1 were protected from a lethal challenge with tetanus toxin. These results suggest that nontoxic Stx derivatives, including both StxB1 and mStx1, could be effective adjuvants for the induction of mixed Th-type CD4+ T-cell-mediated antigen-specific antibody responses via the activation of DCs.     

In vivo anti- and pro-tumour activities of the TLR2 ligand FSL-1

TLR ligands as Th1 inducers have been investigated as potential anti-tumour agents. However, few attempts have been made to investigate the anti-tumour activity of TLR ligands as Th2 inducers. This study, therefore, was carried out to determine whether the TLR2 ligand FSL-1 as a Th2 inducers affects the growth of a QRsP tumour, a fibrosarcoma derived from the C57BL/6 (TLR2^+/+) mouse in vivo. Tumour volumes in TLR2^+/+ mice immunized with both FSL-1 and tumour-associated antigens were significantly smaller than those in control mice. Immunization with both FSL-1 and tumour-associated antigens increased the survival rate of TLR2^+/+ mice. However, surprisingly, immunization with FSL-1 alone significantly enhanced the growth of tumour. Both anti- and pro-tumour activities of FSL-1 were not observed in TLR2^−/− mice. Immunization of both FSL-1 and tumour-associated antigens induced tumour-associated antigen-specific cytolytic T cells, antibody-dependent cell-mediated cytotoxicity of natural killer cells by production of the tumour-specific antibodies, tumour lysis by complement activation and reduction of the number of regulatory T cells in the draining lymph node. Immunization with FSL-1 alone increased the number of regulatory T cells in the draining lymph node, and in vivo administration of anti-CD25 antibody into mice abrogated the pro-tumour activity of FSL-1, suggesting that regulatory T cells are involved in the pro-tumour activity.This study demonstrated that FSL-1 exhibited TLR2-mediated anti- and pro-tumour activities when immunized with and without tumour-associated antigens, respectively.     

B and CD4+ T‐cell expression of TLR2 is critical for optimal induction of a T‐cell‐dependent humoral immune response to intact Streptococcus pneumoniae

TLR2^?/? mice immunized with Streptococcus pneumoniae (Pn) elicit normal IgM, but defective CD4⁺ T‐cell‐dependent type 1 IgG isotype production, associated with a largely intact innate immune response. We studied the T‐cell‐dependent phosphorylcholine (PC)‐specific IgG3 versus the T‐cell‐independent IgM response to Pn to determine whether TLR2 signals directly via the adaptive immune system. Pn‐activated TLR2^?/? BMDC have only a modest defect in cytokine secretion, undergo normal maturation, and when transferred into naïve WT mice elicit a normal IgM and IgG3 anti‐PC response, relative to WT BMDC. Pn synergizes with BCR and TCR signaling for DNA synthesis in purified WT B and CD4⁺T cells, respectively, but is defective in cells lacking TLR2. Pn primes TLR2^?/? mice for a normal CD4⁺ T‐cell IFN‐γ recall response. Notably, TLR2^?/? B cells transferred into RAG‐2^?/? mice with WT CD4⁺T cells, or TLR2^?/? CD4⁺T cells transferred into athymic nude mice, each elicit a defective IgG3, in contrast to normal IgM, anti‐PC response relative to WT cells. These data are the first to demonstrate a major role for B‐cell and CD4⁺ T‐cell expression of TLR2 for eliciting an anti‐bacterial humoral immune response.     

Ingested soluble CD14 contributes to the functional pool of circulating sCD14 in mice

Soluble CD14 (sCD14) is a pattern recognition receptor and Toll-like co-receptor observed in human milk (5–26 μg/mL) and other bodily fluids such as blood (3 μg/mL). The most well defined role of sCD14 is to recognize lipopolysaccharide of Gram-negative bacteria and signal an immune response through Toll-like receptor 4 (TLR4). Previous research has shown ingested sCD14 to transfer from the gastrointestinal tract and into the blood stream in neonatal rats. The contribution of human milk sCD14 to circulating levels in the infant and the functionality of the protein, however, remained unknown. Using CD14^−/− mouse pups fostered to wild type (WT) mothers expressing sCD14 in their milk, we show herein that ingestion of sCD14 resulted in blood sCD14 levels up 0.16 ± 0.09 μg/mL. This represents almost one-third (26.7%) of the circulating sCD14 observed in WT pups fostered to WT mothers (0.60 ± 0.14 μg/mL). We also demonstrate that ingested-sCD14 transferred to the blood remains functional in its ability to recognize lipopolysaccharide as demonstrated by a significant increase in immune response (IL-6 and TNF-α) in CD14^−/− pups fostered to WT mothers in comparison to control animals (P = 0.002 and P = 0.007, respectively). Using human intestinal cells (Caco-2), we also observed a significant decrease in sCD14 transcytosis when TLR4 was knocked down (P < 0.001), suggesting sCD14 transfer involves TLR4. The bioavailability of human milk sCD14 established in this report confirms the importance of human milk proteins for the infant and demonstrates the need to improve infant formulas which are lacking in immune proteins such as sCD14.     

TLR2-deficiency of cKit+ bone marrow cells is associated with augmented potency to stimulate angiogenic processes

Objective: Toll-like receptor 2 (TLR2)-deficiency is associated with the preservation of vascular function and TLR2-deficient (TLR2^-/-) mice exhibit increased neovascularization following induction of hindlimb ischemia. Hematopoietic stem cells play an important role in ischemia-induced angiogenesis and we now investigated whether the effects observed in TLR2^-/- mice may be attributed to TLR2 deficiency on bone marrow-derived stem cells. Approach and Results: cKit-positive (cKit⁺) bone marrow cells (BMC) were isolated from wild type (WT) and TLR2^-/- mice employing MACS-bead technology. Co-incubation of TLR2^-/-cKit⁺ BMC with mature endothelial cells (ECs) resulted in increased tube formation of ECs on matrigel, augmented sprouting in a 3D-collagen matrix and increased migratory capacity compared to co-incubation with WT cKit⁺ BMC. In an in vivo matrigel plug assay, TLR2^-/-cKit⁺ BMC exhibited enhanced formation of capillary-like networks. In a murine model of hindlimb ischemia, administration of TLR2^-/- cKit⁺ BMC to WT mice augmented capillary density and reperfusion of ischemic M. gastrocnemius muscle tissue to the level of TLR2^-/- mice. Western Blot analysis revealed comparable expression of CXCR4 on TLR2^-/-cKit⁺ BMC but increased activation of the PI3K downstream signaling molecule protein kinase B (PKB/AKT) compared to WT cKit⁺ cells. Conclusions: The absence of TLR2 on cKit⁺ BMC is associated with augmented potency to support angiogenic processes in vitro and in vivo. Functional inhibition of TLR2 may therefore provide a novel tool to enhance stem cell function for the treatment of vascular diseases.     

TLR2 engagement on CD4+ T cells enhances effector functions and protective responses to Mycobacterium tuberculosis

We have previously demonstrated that mycobacterial lipoproteins engage TLR2 on human CD4⁺ T cells and upregulate TCR‐triggered IFN‐γ secretion and cell proliferation in vitro. Here we examined the role of CD4⁺ T‐cell‐expressed TLR2 in Mycobacterium tuberculosis (MTB) Ag‐specific T‐cell priming and in protection against MTB infection in vivo. Like their human counterparts, mouse CD4⁺ T cells express TLR2 and respond to TLR2 costimulation in vitro. This Th1‐like response was observed in the context of both polyclonal and Ag‐specific TCR stimulation. To evaluate the role of T‐cell TLR2 in priming of CD4⁺ T cells in vivo, naive MTB Ag85B‐specific TCR transgenic CD4⁺ T cells (P25 TCR‐Tg) were adoptively transferred into Tlr2^?/? recipient C57BL/6 mice that were then immunized with Ag85B and with or without TLR2 ligand Pam₃Cys‐SKKKK. TLR2 engagement during priming resulted in increased numbers of IFN‐γ‐secreting P25 TCR‐Tg T cells 1 week after immunization. P25 TCR‐Tg T cells stimulated in vitro via TCR and TLR2 conferred more protection than T cells stimulated via TCR alone when adoptively transferred before MTB infection. Our findings indicate that TLR2 engagement on CD4⁺ T cells increases MTB Ag‐specific responses and may contribute to protection against MTB infection.     

Enhanced IL-10 production by CD4+ T cells primed in IL-15Rα-deficient mice

In this study, we investigated the functional outcomes of CD4(+) T cells primed in the absence of IL-15 transpresentation. Compared with their WT counterparts primed in WT mice, IL-15Rα KO CD4(+) T cells primed in KO mice were found to exclusively overproduce IL-10 upon in vitro restimulation(.) The comparable expression of IL-4 and Foxp3 in CD4(+) T cells primed in the WT and IL-15Rα KO mice indicated that this was neither due to T(H) 2- nor Treg cell-differentiation. IL-10 overproduction was also observed when OVA-specific TCR transgenic CD4(+) T (OT-II) cells were primed in KO mice, excluding an intrinsic deficiency of KO CD4(+) T cells. To investigate the WT and KO microenvironment, DCs from both WT and IL-15Rα KO mice were compared. DCs from both backgrounds were indistinguishable in their steady-state survival and in their expression of MHC class II and costimulatory molecules CD80, CD86, and CD40. However, IL-15Rα KO DCs primed OT-II cells in vitro to produce higher levels of IL-10 upon their restimulation. Additionally, IL-15Rα KO DCs produced significantly more IL-10 upon activation, and IL-10 neutralization during DC-mediated in vitro priming abolished IL-10 overproduction by CD4(+) T cells. Thus, IL-15Rα KO DCs provide an IL-10-enriched environment that preferentially primes CD4(+) T cells for more IL-10 production, highlighting a regulatory role for IL-15 transpresentation in CD4(+) T-cell priming.     

Primary CD8+ T-cell response to soluble ovalbumin is improved by chloroquine treatment in vivo

The efficiency of cross-presentation of exogenous antigens by dendritic cells (DCs) would seem to be related to the level of antigen escape from massive degradation mediated by lysosomal proteases in an acidic environment. Here, we demonstrate that a short course of treatment with chloroquine in mice during primary immunization with soluble antigens improved the cross-priming of naïve CD8⁺ T lymphocytes in vivo. More specifically, priming of chloroquine-treated mice with soluble ovalbumin (OVA), OVA associated with alum, or OVA pulsed on DCs was more effective in inducing OVA-specific CD8⁺ T lymphocytes than was priming of untreated mice. We conclude that chloroquine treatment improves the cross-presentation capacity of DCs and thus the size of effector and memory CD8⁺ T cells during vaccination.