Suppressor of cytokine signaling 1 DNA administration inhibits inflammatory and pathogenic responses in autoimmune myocarditis

Myocarditis and subsequent dilated cardiomyopathy are major causes of heart failure in young adults. Myocarditis in humans is highly heterogeneous in etiology. Recent studies have indicated that a subgroup of myocarditis patients may benefit from immune-targeted therapies, because autoimmunity plays an important role in myocarditis as well as contributing to the progression to cardiomyopathy and heart failure. Suppressor of cytokine signaling (SOCS) 1 plays a key role in the negative regulation of both TLR- and cytokine receptor-mediated signaling, which is involved in innate immunity and subsequent adaptive immunity. In this study, we investigated the therapeutic effect of SOCS1 DNA administration on experimental autoimmune myocarditis (EAM) in mice. EAM was induced by s.c. immunization with cardiac-specific peptides derived from α myosin H chain in BALB/c mice. In contrast to control myocarditis mice, SOCS1 DNA-injected mice were protected from development of EAM and heart failure. SOCS1 DNA administration was effective for reducing the activation of autoreactive CD4(+) T cells by inhibition of the function of Ag-presenting dendritic cells. Our findings suggest that SOCS1 DNA administration has considerable therapeutic potential in individuals with autoimmune myocarditis and dilated cardiomyopathy.     

TLR ligands act directly upon T cells to restore proliferation in the absence of protein kinase C-theta signaling and promote autoimmune myocarditis

The serine/threonine kinase, protein kinase C-theta (PKC-theta), plays a central role in the activation and differentiation of Th2 cells while being redundant in CD4+ and CD8+ antiviral responses. Recent evidence indicates that PKC-theta may however be required for some T cell-driven autoimmune responses. We have investigated the role of PKC-theta in the induction of autoimmune myocarditis induced by either Coxsackie B3 virus infection or immunization with alpha-myosin/CFA (experimental autoimmune myocarditis (EAM)). PKC-theta-deficient mice did not develop EAM as shown by impaired inflammatory cell infiltration into the heart, reduced CD4+ T cell IL-17 production, and the absence of a myosin-specific Ab response. Comparatively, PKC-theta was not essential for both early and late-phase Coxsackie virus-induced myocarditis. We sought to find alternate pathways of immune stimulation that might reconcile the differential requirements for PKC-theta in these two disease models. We found systemic administration of the TLR ligand CpG restored EAM in PKC-theta-deficient mice. CpG could act directly upon TLR9-expressing T cells to restore proliferation and up-regulation of Bcl-x(L), but exogenous IL-6 and TGF-beta was required for Th17 cell differentiation. Taken together, these results indicate that TLR-mediated activation of T cells can directly overcome the requirement for PKC-theta signaling and, combined with the dendritic cell-derived cytokine milieu, can promote the development of autoimmunity.     

Cutting edge: TLR ligands are not sufficient to break cross-tolerance to self-antigens

Cross-presentation of peripheral self-Ags by dendritic cells (DC) can induce deletion of autoreactive CTL by a mechanism termed cross-tolerance. Activation of DC by microbial TLR ligands is thought to result in adaptive immunity. However, activation of tolerogenic DC may cause autoimmunity by stimulating instead of deleting autoreactive CTL. To investigate this scenario, we have monitored the response of autoreactive CTL in specific for the transgenic self Ag, OVA, expressed in pancreatic islets of RIP-mOVA mice injected with ligands of TLR2, 3, 4, and 9. This somewhat enhanced proliferation and cytokine production, and moderately reduced the CTL number able to induce autoimmunity. Nevertheless, physiological CTL numbers were deleted before disease ensued, unless specific CD4 T cell help was provided. In conclusion, DC activation by TLR ligands was insufficient to break peripheral cross-tolerance in the absence of specific CD4 T cell help, and triggered autoimmunity by stimulating the early effector phase of autoreactive CTL only when their precursor frequency was extremely high.     

Autoreactive preplasma cells break tolerance in the absence of regulation by dendritic cells and macrophages

The ability to induce Ab responses to pathogens while maintaining the quiescence of autoreactive cells is an important aspect of immune tolerance. During activation of TLR4, dendritic cells (DCs) and macrophages (MFs) repress autoantibody production through their secretion of IL-6 and soluble CD40L (sCD40L). These soluble mediators selectively repress B cells chronically exposed to Ag, but not naive cells, suggesting a means to maintain tolerance during TLR4 stimulation, yet allow immunity. In this study, we identify TNF-α as a third repressive factor, which together with IL-6 and CD40L account for nearly all the repression conferred by DCs and MFs. Similar to IL-6 and sCD40L, TNF-α did not alter B cell proliferation or survival. Instead, it reduced the number of Ab-secreting cells. To address whether the soluble mediators secreted by DCs and MFs functioned in vivo, we generated mice lacking IL-6, CD40L, and TNF-α. Compared to wild-type mice, these mice showed prolonged anti-nuclear Ab responses following TLR4 stimulation. Furthermore, adoptive transfer of autoreactive B cells into chimeric IL-6(-/-) × CD40L(-/-) × TNF-α(-/-) mice showed that preplasma cells secreted autoantibodies independent of germinal center formation or extrafollicular foci. These data indicate that in the absence of genetic predisposition to autoimmunity, loss of endogenous IL-6, CD40L, and TNF-α promotes autoantibody secretion during TLR4 stimulation.     

Deficiency in TLR2 but not in TLR4 impairs dendritic cells derived IL-10 responses to schistosome antigens

The purpose of this study was to observe the diverse functions of Toll-like receptors (TLRs) in responses to specific schistosome antigens. Bone marrow-derived dendritic cells (BMDCs) from TLR2-deficient (TLR2(-/-)) or TLR4-deficient (TLR4(-/-)) mice were activated with soluble schistosomule antigen (SSA) or soluble egg antigen (SEA). TLR2 mRNA expression was significantly increased in B6 BMDCs following SEA stimulation. TLR2-deficient BMDCs showed enhanced MHCII expression following SSA and SEA stimulation. TLR2-deficient but not TLR4-deficient BMDC failed to produce IL-12p70 and IL-10 in response to schistosome antigens. TLR2-deficient BMDCs induced a stronger CD4(+) T cell proliferative response. IL-4 and IL-10 expression was inhibited in CD4(+) T cells primed with TLR2-deficient BMDCs, while enhanced in TLR4-deficient BMDCs-primed CD4(+) T cells. These results suggest that TLR2 is essential for the establishment of the DC production of IL-12p70 and IL-10.     

Different toll-like receptor stimuli have a profound impact on cytokines required to break tolerance and induce autoimmunity

Although toll-like receptor (TLR) signals are critical for promoting antigen presenting cell maturation, it remains unclear how stimulation via different TLRs influence dendritic cell (DC) function and the subsequent adaptive response in vivo. Furthermore, the relationship between TLR-induced cytokine production by DCs and the consequences on the induction of a functional immune response is not clear. We have established a murine model to examine whether TLR3 or TLR4 mediated DC maturation has an impact on the cytokines required to break tolerance and induce T-cell-mediated autoimmunity. Our study demonstrates that IL-12 is not absolutely required for the induction of a CD8 T-cell-mediated tissue specific immune response, but rather the requirement for IL-12 is determined by the stimuli used to mature the DCs. Furthermore, we found that IFNα is a critical pathogenic component of the cytokine milieu that circumvents the requirement for IL-12 in the induction of autoimmunity. These studies illustrate how different TLR stimuli have an impact on DC function and the induction of immunity.     

Signaling through toll-like receptor 7/8 induces the differentiation of human bone marrow CD34+ progenitor cells along the myeloid lineage

Toll-like receptors (TLRs) play a key role in pathogen recognition and regulation of the innate and adaptive immune responses. Although TLR expression and signaling have been investigated in blood cells, it is currently unknown whether their bone marrow ancestors express TLRs and respond to their ligands. Here we found that TLRs (e.g. TLR4, TLR7 and TLR8) were expressed by freshly isolated human bone marrow (BM) hematopoietic CD34+ progenitor cells. Incubation of these primitive cells with TLR ligands such as immunostimulatory small interfering RNAs and R848, a specific ligand for TLR7/8, induced cytokine production (e.g. IL1-beta, IL6, IL8, TNF-alpha, GM-CSF). Moreover, TLR7/8 signaling induced the differentiation of BM CD34+ progenitors into cells with the morphology of macrophages and monocytic dendritic precursors characterized by the expression of CD13, CD14 and/or CD11c markers. By contrast, R848 ligand did not induce the expression of glycophorin A, an early marker for erythropoiesis. Collectively, the data indicate for the first time that human BM CD34+ progenitor cells constitutively express functional TLR7/TLR8, whose ligation can induce leukopoiesis without the addition of any exogenous cytokines. Thus, TLR signaling may regulate BM cell development in humans.     

Combined TLR/CD40 stimulation mediates potent cellular immunity by regulating dendritic cell expression of CD70 in vivo

We previously showed that immunization with a combination of TLR and CD40 agonists (combined TLR/CD40 agonist immunization) resulted in an expansion of Ag-specific CD8 T cells exponentially greater than the expansion observed to immunization with either agonist alone. We now show that the mechanism behind this expansion of T cells is the regulated expression of CD70 on dendritic cells. In contrast to previous results in vitro, the expression of CD70 on dendritic cells in vivo requires combined TLR/CD40 stimulation and is not significantly induced by stimulation of either pathway alone. Moreover, the exponential expansion of CD8(+) T cells following combined TLR/CD40 agonist immunization is CD70 dependent. Thus, the transition from innate stimuli (TLRs) to adaptive immunity is controlled by the regulated expression of CD70.     

Sex-specific signaling through Toll-Like Receptors 2 and 4 contributes to survival outcome of Coxsackievirus B3 infection in C57Bl/6 mice

Background

Coxsackievirus B3 (CVB3) induces myocarditis, an inflammatory heart disease, which affects men more than women. Toll-like receptor (TLR) signaling has been shown to determine the severity of CVB3-induced myocarditis. No direct role for signaling through TLR2 had been shown in myocarditis although published studies show that cardiac myosin is an endogenous TLR2 ligand and stimulates pro-inflammatory cytokine expression by dendritic cells in vitro. The goal of this study is to determine which TLRs show differential expression in CVB3 infected mice corresponding to male susceptibility and female resistance in this disease.

Methods

Male and female C57Bl/6 mice were infected with 10² PFU CVB3 and killed on day 3 or 6 post infection. Hearts were evaluated for virus titer, myocardial inflammation, and TLR mRNA expression by PCR array and microarray analysis. Splenic lymphocytes only were evaluated by flow cytometry for the number of TLR+/CD3+, TLR+/CD4+, TLR+F4/80+ and TLR+/CD11c+ subpopulations and the mean fluorescence intensity to assess upregulation of TLR expression on these cells. Mice were additionally treated with PAM3CSK4 (TLR2 agonist) or ultrapure LPS (TLR4 agonist) on the same day as CVB3 infection or 3 days post infection to confirm their role in myocarditis susceptibility.

Results

Despite equivalent viral titers, male C57Bl/6 mice develop more severe myocarditis than females by day 6 after infection. Microarray analysis shows a differential expression of TLR2 at day 3 with female mice having higher levels of TLR2 gene expression compared to males. Disease severity correlates to greater TLR4 protein expression on splenic lymphocytes in male mice 3 days after infection while resistance in females correlates to preferential TLR2 expression, especially in spleen lymphocytes. Treating male mice with PAM reduced mortality from 55% in control CVB3 infected animals to 10%. Treating female mice with LPS increased mortality from 0% in control infected animals to 60%.

Conclusion

CVB3 infection causes an up-regulation of TLR2 in female and of TLR4 in male mice and this differential expression between the sexes contributes to disease resistance of females and susceptibility of males. While previous reports demonstrated a pathogenic role for TLR4 this is the first report that TLR2 is preferentially up-regulated in CVB3 infected female mice or that signaling through this TLR directly causes myocarditis resistance.

     

CD4 T cells play major effector role and CD8 T cells initiating role in spontaneous autoimmune myocarditis of HLA-DQ8 transgenic IAb knockout nonobese diabetic mice

In humans, spontaneous autoimmune attack against cardiomyocytes often leads to idiopathic dilated cardiomyopathy (IDCM) and life-threatening heart failure. HLA-DQ8 transgenic IAb knockout NOD mice (NOD.DQ8/Ab(0); DQA1*0301, DQB1*0302) develop spontaneous anticardiomyocyte autoimmunity with pathology very similar to human IDCM, but why the heart is targeted is unknown. In the present study, we first investigated whether NOD/Ab(0) mice transgenic for a different DQ allele, DQ6, (DQA1*0102, DQB1*0602) would also develop myocarditis. NOD.DQ6/Ab(0) animals showed no cardiac pathology, implying that DQ8 is specifically required for the myocarditis phenotype. To further characterize the cellular immune mechanisms, we established crosses of our NOD.DQ8/Ab(0) animals with Rag1 knockout (Rag1(0)), Ig H chain knockout (IgH(0)), and beta(2)-microglobulin knockout (beta(2)m(0)) lines. Adoptive transfer of purified CD4 T cells from NOD.DQ8/Ab(0) mice with complete heart block (an indication of advanced myocarditis) into younger NOD.DQ8/Ab(0) Rag1(0) animals induced cardiac pathology in all recipients, whereas adoptive transfer of purified CD8 T cells or B lymphocytes had no effect. Despite the absence of B lymphocytes, NOD.DQ8/Ab(0)IgH(0) animals still developed complete heart block, whereas NOD.DQ8/Ab(0)beta(2)m(0) mice (which lack CD8 T cells) failed to develop any cardiac pathology. CD8 T cells (and possibly NK cells) seem to be necessary to initiate disease, whereas once initiated, CD4 T cells alone can orchestrate the cardiac pathology, likely through their capacity to recruit and activate macrophages. Understanding the cellular immune mechanisms causing spontaneous myocarditis/IDCM in this relevant animal model will facilitate the development and testing of new therapies for this devastating disease.     

The endoplasmic reticulum-resident heat shock protein Gp96 activates dendritic cells via the Toll-like receptor 2/4 pathway

The heat shock protein Gp96 has been shown to induce specific immune responses. On one hand, this phenomenon is based on the specific interaction with CD91 that mediates endocytosis and results in major histocompatibility complex class I-restricted representation of the Gp96-associated peptides. On the other hand, Gp96 induces activation of professional antigen-presenting cells, resulting in the production of pro-inflammatory cytokines and up-regulation of costimulatory molecules by unknown mechanisms. In this study, we have analyzed the consequences of Gp96 interaction with cells expressing different Toll-like receptors (TLRs) and with bone marrow-derived dendritic cells from mice lacking functional TLR2 and/or TLR4 molecules. We find that the Gp96-TLR2/4 interaction results in activation of nuclear factor kappaB-driven reporter genes and mitogen- and stress-activated protein kinases and induces IkappaBalpha degradation. Bone marrow-derived dendritic cells of C3H/HeJ and more pronounced C3H/HeJ/TLR2(-/-) mice fail to respond to Gp96. Interestingly, activation of bone marrow-derived dendritic cells depends on endocytosis of Gp96 molecules. Our results provide, for the first time, the molecular basis for understanding the Gp96-mediated activation of antigen-presenting cells by describing the simultaneous stimulation of the innate and adaptive immune system. This feature explains the remarkable ability of Gp96 to induce specific immune responses against tumors and pathogens.     

TLR9 contributes to antiviral immunity during gammaherpesvirus infection

The human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and EBV cause important infections. As pathogenetic studies of the human infections are restricted, murine gammaherpesvirus 68 serves as a model to study gammaherpesvirus pathogenesis. TLRs are a conserved family of receptors detecting microbial molecular patterns. Among the TLRs, TLR9 recognizes unmethylated CpG DNA motifs present in bacterial and viral DNA. The aim of this study was to assess the role of TLR9 in gammaherpesvirus pathogenesis. Upon stimulation with murine gammaherpesvirus 68, Flt3L-cultured bone marrow cells (dendritic cells) from TLR9-/- mice secreted reduced levels of IL-12, IFN-alpha, and IL-6, when compared with dendritic cells from wild-type mice. Intranasal infection of TLR9-/- and wild-type mice did not reveal any differences during lytic and latent infection. In contrast, when infected i.p., TLR9-/- mice showed markedly higher viral loads both during lytic and latent infection. Thus, we show for the first time that TLR9 is involved in gammaherpesvirus pathogenesis and contributes to organ-specific immunity.     

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

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

Down-regulation of Toll-like receptor expression in monocyte-derived Langerhans cell-like cells: implications of low-responsiveness to bacterial components in the epidermal Langerhans cells

In the skin, there are unique dendritic cells called Langerhans cells, however, it remains unclear why this particular type of dendritic cell resides in the epidermis. Langerhans cell-like dendritic cells (LCs) can be generated from CD14(+) monocytes in the presence of GM-CSF, IL-4, and TGF-beta1. We compared LCs with monocyte-derived dendritic cells (DCs) generated from CD14(+) monocytes in the presence of GM-CSF and IL-4 and examined the effect of exposure to two distinct bacterial stimuli via Toll-like receptors (TLRs), such as peptidoglycan (PGN) and lipopolysaccharide (LPS) on LCs and DCs. Although stimulation with both ligands induced a marked up-regulation of CD83 expression on DCs, PGN but not LPS elicited up-regulation of expression CD83 on LCs. Consistent with these results, TLR2 and TLR4 were expressed on DCs, whereas only TLR2 was weakly detected on LCs. These findings suggest the actual feature of epidermal Langerhans cells with low-responsiveness to skin commensals.     

Tonsil-derived mesenchymal stromal cells produce CXCR2-binding chemokines and acquire follicular dendritic cell-like phenotypes under TLR3 stimulation

We previously isolated mesenchymal stromal cells from human tonsils (T-MSCs) and showed the potential of these cells to differentiate into the mesodermal lineage and acquire a follicular dendritic cell (FDC) phenotype under cytokine stimulation. Because these T-MSCs were originally isolated from inflamed tonsillar tissues, we were curious about their activation status in response to innate immune stimuli, such as Toll-like receptors (TLRs). Therefore, we analyzed the expression profile of TLRs in T-MSCs and stimulated the T-MSCs with TLR agonists. TLR3 stimuli induced C–C chemokine receptor type 6 expression in T-MSCs after 24 h. Furthermore, results from cytokine arrays showed increases in epithelial neutrophil-activating peptide-78/C-X-C motif chemokine (CXCL) 5, granulocyte chemotactic protein-2/CXCL6, growth-related oncogene-α/CXCL1, interleukin-8/CXCL8, and interferon gamma-induced protein-10/CXCL10. CD54 expression was also increased after TLR3 stimulation. However, co-culturing T-MSCs with human B cells did not induce B-cell proliferation. This suggests that TLR3 stimulates the differentiation of T-MSCs into FDC-like cells and induces chemokine secretion, possibly by recruiting C–X–C chemokine receptor 2-expressing immune cells. In addition, T-MSCs also appeared to exert immunomodulatory effects by inhibiting B-cell proliferation, possibly by down-regulating CD18.     

MyD88-dependent activation of dendritic cells and CD4(+) T lymphocytes mediates symptoms, but is not required for the immunological control of parasites during rodent malaria

We investigated the role of different TLRs and MyD88 in host resistance to infection and malaria pathogenesis. TLR2(-/-), TLR4(-/-), TLR6(-/-), TLR9(-/-) or CD14(-/-) mice showed no change in phenotypes (parasitemia, body weight and temperature) when infected with Plasmodium chabaudi chabaudi (AS). MyD88(-/-) mice displayed comparable ability to wild type animals in controlling and clearing parasitemia. Importantly, MyD88(-/-) mice exhibited impaired production of TNF-alpha and IFN-gamma as well as attenuated symptoms, as indicated by changes in body weight and temperature during parasitemia. Consistently, CD11b(+) monocytes and CD11c(+) dendritic cells from infected MyD88(-/-) mice were shown impaired for production of pro-inflammatory cytokines, and in initiating CD4(+) T cell responses. Importantly, the inhibition of T cell activation with anti-CD134L, mostly inhibited IFN-gamma, partially inhibited TNF-alpha production, and protected the animals from malaria symptoms. Our findings suggest that MyD88 and possibly its associated TLRs expressed by dendritic cells play an important role in pro-inflammatory responses, T cell activation, and pathogenesis of malaria, but are not critical for the immunological control of the erythrocytic stage of P. chabaudi.     

TLR9 is required for the gut-associated lymphoid tissue response following oral infection of Toxoplasma gondii

TLRs expressed by a variety of cells, including epithelial cells, B cells, and dendritic cells, are important initiators of the immune response following stimulation with various microbial products. Several of the TLRs require the adaptor protein, MyD88, which is an important mediator for the immune response following Toxoplasma gondii infection. Previously, TLR9-mediated innate immune responses were predominantly associated with ligation of unmethylated bacterial CpG DNA. In this study, we show that TLR9 is required for the Th1-type inflammatory response that ensues following oral infection with T. gondii. After oral infection with T. gondii, susceptible wild-type (WT; C57BL/6) but not TLR9(-/-) (B6 background) mice develop a Th1-dependent acute lethal ileitis; TLR9(-/-) mice have higher parasite burdens than control WT mice, consistent with depressed IFN-gamma-dependent parasite killing. A reduction in the total T cell and IFN-gamma-producing T cell frequencies was observed in the lamina propria of the TLR9(-/-) parasite-infected mice. TLR9 and type I IFN production was observed by cells from infected intestines in WT mice. TLR9 expression by dendritic cell populations is essential for their expansion in the mesenteric lymph nodes of infected mice. Infection of chimeric mice deleted of TLR9 in either the hemopoietic or nonhemopoietic compartments demonstrated that TLR9 expression by cells from both compartments is important for efficient T cell responses to oral infection. These observations demonstrate that TLR9 mediates the innate response to oral parasite infection and is involved in the development of an effective Th1-type immune response.     

Pertussis toxin is superior to TLR ligands in enhancing pathogenic autoimmunity, targeted at a neo-self antigen, by triggering robust expansion of Th1 cells and their cytokine production

Microbial products are assumed to play a major role in triggering pathogenic autoimmunity. Recently accumulated data have shown that these products stimulate the immune system by interacting with TLRs, expressed on APCs. To examine the capacity of various TLR ligands to trigger pathogenic autoimmunity, we used a system in which naive CD4 cells, specific against hen egg lysozyme (HEL), are injected into recipient mice expressing HEL in their eyes. Only when stimulated, the naive cells acquire pathogenic capacity and induce ocular inflammation. Seven TLR ligands were tested in this system: lipoteichoic acid/peptidoglycan, zymosan, poly (I:C), LPS, pertussis toxin (PTX), flagellin, and CpG oligodeoxynucleotide. Treatment of recipient mice with HEL alone stimulated proliferation of the transferred cells, but no disease, whereas ocular inflammation did develop in recipient mice coinjected with HEL and any one of the seven TLR ligands. Inflammation induced by PTX surpassed by its severity those induced by all other tested TLR ligands and was accompanied by a dramatic increase in number of the transferred cells that acquired features of effector Th1 lymphocytes. Ocular inflammation and number of transferred cells in recipients injected with PTX and HEL were substantially reduced by treatment with Abs against IFN-gamma or IL-12, thus indicating the role of these cytokines in the PTX effect. Overall, our observations demonstrate that various TLR ligands are capable of triggering pathogenic autoimmunity and that PTX surpasses other microbial products in this activity, by stimulating excessive proliferation and polarization toward Th1 of naive T cells.     

The synthetic analogue of mycoplasmal lipoprotein FSL-1 induces dendritic cell maturation through Toll-like receptor 2

Granulocyte-macrophage colony-stimulating factor-differentiated bone marrow-derived dendritic cells were stimulated with the synthetic lipopeptide S-(2,3-bispalmitoyloxypropyl)-CGDPKHSPKSF (FSL-1) or the Escherichia coli lipopolysaccharide. FSL-1 induced the production of TNF-alpha and IL-12 by C57BL/6-derived bone marrow-derived dendritic cells but not by bone marrow-derived dendritic cells from Toll-like receptor 2-deficient (TLR2(-/-)) mice. Lipopolysaccharide induced the production of TNF-alpha and IL-12 by bone marrow-derived dendritic cells derived from either type of mice. FSL-1 did not induce production of IL-10 by bone marrow-derived dendritic cells from either type of mice, whereas lipopolysaccharide induced small amounts of IL-10 by bone marrow-derived dendritic cells from both types of mice. The upregulation by FSL-1 of the expression of CD80, CD86 and the MHC class II molecule IA(b) was dose- and time-dependent on the surfaces of C57BL/6-derived bone marrow-derived dendritic cells but not on the surface of TLR2(-/-)-derived bone marrow-derived dendritic cells. Lipopolysaccharide upregulated the expression of these molecules on the surfaces of bone marrow-derived dendritic cells from both types of mice. The expression of CD11c on the surfaces of C57BL/6-derived bone marrow-derived dendritic cells was upregulated by stimulation with both FSL-1 and lipopolysaccharide up to 12 h; thereafter, the expression was downregulated. The results suggest that FSL-1 can accelerate maturation of bone marrow-derived dendritic cells and this FSL-1 activity is mediated by TLR2.     

Stimulation by toll-like receptors inhibits osteoclast differentiation

Osteoclasts, the cells capable of resorbing bone, are derived from hemopoietic precursor cells of monocyte-macrophage lineage. The same precursor cells can also give rise to macrophages and dendritic cells, which are essential for proper immune responses to various pathogens. Immune responses to microbial pathogens are often triggered because various microbial components induce the maturation and activation of immunoregulatory cells such as macrophages or dendritic cells by stimulating Toll-like receptors (TLRs). Since osteoclasts arise from the same precursors as macrophages, we tested whether TLRs play any role during osteoclast differentiation. We showed here that osteoclast precursors prepared from mouse bone marrow cells expressed all known murine TLRs (TLR1-TLR9). Moreover, various TLR ligands (e.g., peptidoglycan, poly(I:C) dsRNA, LPS, and CpG motif of unmethylated DNA, which act as ligands for TLR2, 3, 4, and 9, respectively) induced NF-kappa B activation and up-regulated TNF-alpha production in osteoclast precursor cells. Unexpectedly, however, TLR stimulation of osteoclast precursors by these microbial products strongly inhibited their differentiation into multinucleated, mature osteoclasts induced by TNF-related activation-induced cytokine. Rather, TLR stimulation maintained the phagocytic activity of osteoclast precursors in the presence of osteoclastogenic stimuli M-CSF and TNF-related activation-induced cytokine. Taken together, these results suggest that TLR stimulation of osteoclast precursors inhibits their differentiation into noninflammatory mature osteoclasts during microbial infection. This process favors immune responses and may be critical to prevent pathogenic effects of microbial invasion on bone.