Lipoteichoic acids from Lactobacillus strains elicit strong tumor necrosis factor alpha-inducing activities in macrophages through Toll-like receptor 2

Lactobacilli are nonpathogenic gram-positive inhabitants of microflora. At least some Lactobacillus strains have been postulated to have health beneficial effects, such as the stimulation of the immune system. Here we examined the stimulatory effects of lactobacilli on mouse immune cells. All six heat-killed Lactobacillus strains examined induced the secretion of tumor necrosis factor alpha (TNF-alpha) from mouse splenic mononuclear cells, albeit to various degrees. When fractionated subcellular fractions of Lactobacillus casei were tested for NF-kappaB activation and TNF-alpha production in RAW264.7, a mouse macrophage cell line, the activity was found to be as follows: protoplast > cell wall > polysaccharide-peptidoglycan complex. Both crude extracts and purified lipoteichoic acids (LTAs) from two Lactobacillus strains, L. casei and L. fermentum, significantly induced TNF-alpha secretion from RAW264.7 cells and splenocytes of C57BL/6, C3H/HeN, and C3H/HeJ mice but not from splenocytes of C57BL/6 TLR2(-/-) mice. Lactobacillus LTA induced activation of c-Jun N-terminal kinase activation in RAW264.7 cells. Furthermore, in HEK293T cells transected with a combination of CD14 and Toll-like receptor 2 (TLR2), NF-kappaB was activated in response to Lactobacillus LTA. Taken together, these data suggest that LTAs from lactobacilli elicit proinflammatory activities through TLR2.     

Toll-like receptor 9-dependent macrophage activation by Entamoeba histolytica DNA

Activation of the innate immune system by bacterial DNA and DNA of other invertebrates represents a pathogen recognition mechanism. In this study we investigated macrophage responses to DNA from the intestinal protozoan parasite Entamoeba histolytica. E. histolytica genomic DNA was purified from log-phase trophozoites and tested with the mouse macrophage cell line RAW 264.7. RAW cells treated with E. histolytica DNA demonstrated an increase in levels of tumor necrosis factor alpha (TNF-α) mRNA and protein production. TNF-α production was blocked by pretreatment with chloroquine or monensin. In fact, an NF-κB luciferase reporter assay in HEK cells transfected with human TLR9 demonstrated that E. histolytica DNA signaled through Toll-like receptor 9 (TLR9) in a manner similar to that seen with CpG-ODN. Immunofluorescence assays confirmed NF-κB activation in RAW cells, as seen by nuclear translocation of the p65 subunit. Western blot analysis demonstrated mitogen-activated protein kinase activation by E. histolytica DNA. E. histolytica DNA effects were abolished in MYD88^−/− mouse-derived macrophages. In the context of disease, immunization with E. histolytica DNA protected gerbils from an E. histolytica challenge infection. Taken together, these results demonstrate that E. histolytica DNA is recognized by TLR9 to activate macrophages and may provide an innate defense mechanism characterized by the induction of the inflammatory mediator TNF-α.     

Effects of propofol on lipopolysaccharide-induced expression and release of HMGB1 in macrophages

This study aimed to determine the effects of different concentrations of propofol (2,6-diisopropylphenol) on lipopolysaccharide (LPS)-induced expression and release of high-mobility group box 1 protein (HMGB1) in mouse macrophages. Mouse macrophage cell line RAW264.7 cells were randomly divided into 5 treatment groups. Expression levels of HMGB1 mRNA were detected using RT-PCR, and cell culture supernatant HMGB1 protein levels were detected using enzyme-linked immunosorbent assay (ELISA). Translocation of HMGB1 from the nucleus to the cytoplasm in macrophages was observed by Western blotting and activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus was detected using ELISA. HMGB1 mRNA expression levels increased significantly in the cell culture supernatant and in cells after 24 h of stimulating RAW264.7 cells with LPS (500 ng/mL). However, HMGB1 mRNA expression levels in the P2 and P3 groups, which received 500 ng/mL LPS with 25 or 50 μmol/mL propofol, respectively, were significantly lower than those in the group receiving LPS stimulation (P<0.05). After stimulation by LPS, HMGB1 protein levels were reduced significantly in the nucleus but were increased in the cytoplasm (P<0.05). Simultaneously, the activity of NF-κB was enhanced significantly (P<0.05). After propofol intervention, HMGB1 translocation from the nucleus to the cytoplasm and NF-κB activity were inhibited significantly (each P<0.05). Thus, propofol can inhibit the LPS-induced expression and release of HMGB1 by inhibiting HMGB1 translocation and NF-κB activity in RAW264.7 cells, suggesting propofol may be protective in patients with sepsis.     

Augmentation of Cellular Immunity and Reduction of Influenza Virus Titer in Aged Mice Fed Lactobacillus casei Strain Shirota

We investigated whether oral administration of Lactobacillus casei strain Shirota activates the cellular immune system and ameliorates influenza virus (IFV) titer in the nasal site in upper respiratory IFV infection by using aged mice. Natural killer activity of splenocytes and lung cells of aged mice fed an L. casei strain Shirota diet (L.casei strain Shirota group) was significantly (P < 0.01 and P < 0.05) increased compared to those fed a control diet (control group). The increases were 1.5- and 2.5-fold, respectively. In aged mice fed an XL.casei strain Shirota diet, potent induction of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α), which play a very important role in excluding IFV, was evident in nasal lymphocytes. IFN-γ and TNF-α production increased 12- and 3.5-fold, respectively. In this model of upper respiratory IFV infection, the titer of IFV in the nasal washings of aged mice fed an L.casei strain Shirota diet was significantly (P < 0.05) lower than that in aged mice fed a control diet (10^{1.6 ± 0.6} and 10^{2.2 ± 0.5}, respectively). These findings suggest that oral administration of L.casei strain Shirota activates not only systemic cellular immunity but also local cellular immunity and that it ameliorates IFV infection.     

Immunomodulatory effects of dead Lactobacillus on murine splenocytes and macrophages

To elucidate the immunomodulation effects of dead lactobacilli, whole cells and gastrointestinal enzymatic hydrolysates of supernatants and precipitates from Lactobacillus paracasei subsp. paracasei NTU 101 and L. plantarum NTU 102 on RAW264.7 macrophages and splenocytes were investigated. Increased NO, COX-2 expression, IL-10 and IL-12 were observed in high-dose precipitates and whole cells of both strains after 24-h stimulation. All of the hydrolysates and whole cells from both strains induced lower pro-inflammatory cytokines (IL-1β and IL-6) than LPS. The supernatants activated cell division to the S phase or promoted advance to the G2/M phase. Regardless of the Lactobacillus strains, higher levels of TNF-α, IL-6, IL-10 and IL-12 in splenocytes were induced by the precipitates. Supernatant of NTU 101 increased the amounts of IFN-γ than precipitate in splenocytes. It shows that hydrolysates of NTU 101 induce the proliferations of macrophage and splenocyte and the release of IL-10 and IL-12 cytokines to modulate the innate and adaptive immune systems and inflammatory response.     

Nitric Oxide-Mediated Inhibition of the Ability of Rickettsia prowazekii To Infect Mouse Fibroblasts and Mouse Macrophagelike Cells

The role of the nitric oxide synthase (NOS) pathway in inhibiting the ability of Rickettsia prowazekii to initially infect (invade) mouse cytokine-treated, fibroblastic L929 cells and macrophagelike RAW264.7 cells and the ability of nitric oxide (NO) to damage isolated rickettsiae were investigated. Substantial amounts of nitrite (a degradation product of NO) were produced and the initial rickettsial infection was suppressed in cultures of L929 cells treated with crude lymphokine preparations (LK) or with gamma interferon (IFN-γ) plus tumor necrosis factor alpha (TNF-α) but not in L929 cell cultures treated with IFN-γ alone or TNF-α alone. The NOS inhibitors N^G-methyl-l-arginine and aminoguanidine both inhibited nitrite production and prevented the suppression of the initial rickettsial infection. Antibody-mediated neutralization of the IFN-γ in the LK also inhibited both nitrite production and suppression of the initial rickettsial infection. Cultures of RAW264.7 cells treated with IFN-γ plus lipopolysaccharide exhibited suppression of the initial rickettsial infection, and the suppression was relieved by aminoguanidine. Addition of oxyhemoglobin (a scavenger of extracellular NO) during the rickettsial infection alleviated the suppression of the initial rickettsial infection observed in appropriately treated L929 cells and RAW264.7 cells. In addition, the oxyhemoglobin restored the rickettsia-mediated, rapid killing of the treated RAW264.7 cells. Incubation of isolated rickettsiae with NO inhibited their ability to infect L929 and IFN-γ-treated RAW264.7 cells and to rapidly kill IFN-γ-treated RAW264.7 cells. In contrast, incubation of L929 cells with a solution that contained NO and/or degradation products of NO did not affect their ability to be infected by rickettsiae. The data are consistent with the hypothesis that NO released from appropriately stimulated potential host cells kills extracellular rickettsiae and thus prevents the rickettsiae from infecting the cells.Rickettsia prowazekii, the etiologic agent of epidemic typhus, Brill-Zinsser disease, and flying-squirrel-associated typhus in humans, is an obligate intracytoplasmic bacterium. In vivo R. prowazekii grows within the endothelial cells that line the small blood vessels (39); this organism can also grow within macrophages (9).The cytokines gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) alter the interactions between R. prowazekii and host cells such as fibroblasts, endothelial cells, and macrophages, and these cytokines may play roles not only in host defense but also in the pathogenesis of R. prowazekii infections (reviewed in references 26, 30, and 37). The effects of these cytokines on R. prowazekii infection in cultured cells include inhibition of rickettsial growth, killing of some of the rickettsiae, cytotoxic effects on the host cells, and (in some instances) inhibition of the initial infection of host cells by the rickettsiae (reviewed in reference 30).Studies delineating the role of these cytokines in R. prowazekii infection in vivo are not available. However, studies of Rickettsia conorii infections in mice have demonstrated that both IFN-γ and TNF-α are important host defenses against infections with this Rickettsia species (7). How these cytokines protect the host is not completely understood; however, experimental evidence suggests the importance of nitric oxide synthase (NOS)-dependent alterations in rickettsia-host cell interactions (8, 31).Many informative studies of how IFN-γ and/or TNF-α do (and do not) alter R. prowazekii-host cell interactions have employed fibroblasts and macrophagelike cell lines (reviewed in references 26, 30, and 37). Such studies allow for evaluation of the effects of cytokines on rickettsial infections and the mechanisms of these effects in experimental systems that are easily manipulated and not affected by many of the variables inherent in other, more complex systems. Moreover, some questions about the effects of cytokines on rickettsia-host cell interactions that can be answered through studies with cultured cells would be very difficult (or even impossible) to answer through studies with intact animals. Studies with cultured cells demonstrated that multiplication of R. prowazekii is inhibited in mouse L929 cells treated with IFN-γ and/or TNF-α and that the combination of IFN-γ and/or TNF-α treatment and R. prowazekii infection has a cytotoxic effect on the host cells (20, 28). In addition, such investigations determined that a lack of the amino acid tryptophan does not explain the ability of IFN-γ to limit the growth of R. prowazekii in either mouse L929 cells (in which tryptophan is not depleted in response to IFN-γ) or human fibroblasts (in which reconstitution of the depleted tryptophan pool in IFN-γ-treated cells does not alleviate the inhibition of rickettsial growth) (25). Furthermore, such studies revealed (i) that both NOS-independent and NOS-dependent mechanisms are involved in suppressing the growth of R. prowazekii in mouse L929 cells treated with IFN-γ plus TNF-α (28), (ii) that the inhibition of growth of R. prowazekii observed in L929 cells treated with either IFN-γ alone or TNF-α alone is largely NOS independent (28), (iii) that the mechanism responsible for IFN-γ-induced, NOS-independent inhibition of growth of R. prowazekii in mouse L929 cells is likely to exist also in IFN-γ-treated human fibroblasts (30), and (iv) that the cytotoxic effects on host cells brought about by the combination of cytokine treatment and R. prowazekii infection are not dependent on NOS (28, 29). These effects of cytokine treatment on R. prowazekii-host cell interactions are observed in host cells infected with either the virulent Breinl strain or the avirulent Madrid E strain of R. prowazekii.Under some conditions, the ability of R. prowazekii to initially infect (invade) host cells is suppressed after the latter are treated with cytokines (23, 24, 26). For example, if L929 cells are treated for 24 h with high concentrations of lymphokines (LK) (supernatant fluids collected from cultures of antigen- or mitogen-stimulated mouse spleen cells), the initial infection (invasion) of the host cells by R. prowazekii (Madrid E and Breinl strains) is reduced (24, 26). Curiously, IFN-γ alone does not cause suppression of the initial infection in L929 cells; however, neutralization of the IFN-γ in the LK abolishes the ability of the LK to cause suppression (24, 26). It has not been determined if TNF-α alone (or in combination with IFN-γ) causes suppression of the initial R. prowazekii infection in L929 cells.In RAW264.7 cell cultures pretreated with a low concentration of IFN-γ plus lipopolysaccharide (LPS), decreased R. prowazekii (Madrid E)-mediated, rapid killing of the RAW264.7 cells is associated with increased production of nitric oxide (NO), and addition of NOS inhibitors restores the R. prowazekii-mediated killing of the RAW264.7 cells in such cultures to a level comparable to that observed in RAW264.7 cultures pretreated with a low concentration of IFN-γ alone (29). Because the initial rickettsial infections were not monitored in these experiments, it is not known if the decreased R. prowazekii-mediated, rapid killing of the RAW264.7 cells observed in cultures treated with IFN-γ plus LPS is associated with lower initial rickettsial infection. However, macrophagelike RAW264.7 cell cultures pretreated with high concentrations of LK or IFN-γ exhibit markedly lower initial R. prowazekii (Breinl) infections and significantly less rickettsia-mediated, rapid killing of the RAW264.7 cells than comparably infected RAW264.7 cell cultures that have been pretreated with low concentrations of LK or IFN-γ (23). The possible role of the NOS pathway in mediating these suppressive effects of high concentrations of cytokines has not been evaluated.Infection (invasion) of a host cell by R. prowazekii requires metabolic activity on the part of both the host cell and the rickettsia (32, 33). The infection process involves both phagocytosis and a phospholipase A activity, which is presumed to allow the rickettsia to escape from the phagosome into the cytoplasm of the host cell (36). Since R. prowazekii is an obligate intracellular bacterium, preventing it from infecting a host cell prevents it from growing.Because cytokine-induced inhibition of the initial infection of potential host cells by R. prowazekii may represent an important antirickettsial host defense, we sought to obtain information about the mechanism of this inhibition by evaluating the possible role of the NOS pathway. This study reports the NOS dependence of cytokine-induced suppression of the initial R. prowazekii infection in L929 cells and RAW264.7 cells and implicates extracellularly released NO and/or NO products in causing the suppression. Furthermore, experiments with isolated rickettsiae exposed to NO in the absence of host cells permitted evaluation of the effects of NO on the rickettsiae themselves. The results provide evidence that isolated rickettsiae exposed to NO are killed, as judged by their reduced ability to infect L929 cells and their decreased capacity for infecting and rapidly killing IFN-γ-treated RAW264.7 cells.     

Differential Regulation of Proinflammatory Cytokine Expression by Mitogen-Activated Protein Kinases in Macrophages in Response to Intestinal Parasite Infection

Blastocystis is a common enteric protistan parasite that can cause acute, as well as chronic, infection and is associated with irritable bowel syndrome (IBS). However, the pathogenic status of Blastocystis infection remains unclear. In this study, we found that Blastocystis antigens induced abundant expression of proinflammatory cytokines, including interleukin 1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α), in mouse intestinal explants, in mouse colitis colon, and in macrophages. Further investigation utilizing RAW264.7 murine macrophages showed that Blastocystis treatment in RAW264.7 macrophages induced the activation of ERK, JNK, and p38, the three major groups of mammalian mitogen-activated protein (MAP) kinases that play essential roles in the expression of proinflammatory cytokines. ERK inhibition in macrophages significantly suppressed both mRNA and protein expression of IL-6 and TNF-α and mRNA expression of IL-1β. On the other hand, JNK inhibition resulted in reductions in both c-Jun and ERK activation and significant suppression of all three proinflammatory cytokines at both the mRNA and protein levels. Inhibition of p38 suppressed only IL-6 protein expression with no effect on the expression of IL-1β and TNF-α. Furthermore, we found that serine proteases produced by Blastocystis play an important role in the induction of ERK activation and proinflammatory cytokine expression by macrophages. Our study thus demonstrated for the first time that Blastocystis could induce the expression of various proinflammatory cytokines via the activation of MAP kinases and that infection with Blastocystis may contribute to the pathogenesis of inflammatory intestinal diseases through the activation of inflammatory pathways in host immune cells, such as macrophages.     

Toll-like receptor 2 recognition of the microsporidia Encephalitozoon spp. induces nuclear translocation of NF-kappaB and subsequent inflammatory responses

Microsporidia are obligate intracellular parasites that are ubiquitous in nature and have been recognized as causing an important emerging disease among immunocompromised individuals. Limited knowledge exists about the immune response against these organisms, and virtually nothing is known about the receptors involved in host recognition. Toll-like receptors (TLR) are pattern recognition receptors that bind to specific molecules found on pathogens and signal a variety of inflammatory responses. In this study, we show that both Encephalitozoon cuniculi and Encephalitozoon intestinalis are preferentially recognized by TLR2 and not by TLR4 in primary human macrophages. This is the first demonstration of host receptor recognition of any microsporidian species. TLR2 ligation is known to activate NF-κB, resulting in inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8). We found that the infection of primary human macrophages leads to the nuclear translocation of NF-κB in as early as 1 h and the subsequent production of TNF-α and IL-8. To verify the direct role of TLR2 parasite recognition in the production of these cytokines, the receptor was knocked down in primary human macrophages using small interfering RNA. This knockdown resulted in decreases in both the nuclear translocation of NF-κB and the levels of TNF-α and IL-8 after challenge with spores. Taken together, these experiments directly link the initial inflammatory response induced by Encephalitozoon spp. to TLR2 stimulation in human macrophages.     

Campylobacter-induced interleukin-8 secretion in polarized human intestinal epithelial cells requires Campylobacter-secreted cytolethal distending toxin- and Toll-like receptor-mediated activation of NF-kappaB

Campylobacter jejuni and Campylobacter coli colonize and infect the intestinal epithelium and cause acute inflammatory diarrhea. The intestinal epithelium serves as a physical barrier to, and a sensor of, bacterial infection by secreting proinflammatory cytokines. This study examined the mechanisms for Campylobacter-induced secretion of the proinflammatory chemokine interleukin-8 (IL-8) by using polarized T84 human colonic epithelial cells as a model. C. jejuni increased the secretion of both IL-8 and tumor necrosis factor alpha (TNF-α) in polarized epithelial cells. However, the increase in IL-8 secretion was independent of Campylobacter-stimulated TNF-α secretion. Polarized T84 cells secreted IL-8 predominantly to the basolateral medium independently of the inoculation direction. While there was a significant correlation between the levels of IL-8 secretion and Campylobacter invasion, all 11 strains tested increased IL-8 secretion by polarized T84 cells despite their differences in adherence, invasion, and transcytosis efficiencies. Cell-free supernatants of Campylobacter-T84-cell culture increased IL-8 secretion to levels similar to those induced by live bacterial inoculation. The ability of the supernatant to induce IL-8 secretion was reduced by flagellum and cytolethal distending toxin (CDT) gene mutants, treatment of the supernatant with protease K or heat, or treatment of T84 cells with the Toll-like receptor (TLR) inhibitor MyD88 inhibitory peptide or chloroquine. NF-κB inhibitors or cdtB mutation plus MyD88 inhibitor, but not flaA cdtB double mutations, abolished the ability of the supernatant to induce IL-8 secretion. Taken together, our results demonstrate that Campylobacter-induced IL-8 secretion requires functional flagella and CDT and depends on the activation of NF-κB through TLR signaling and CDT in human intestinal epithelial cells.     

Regulatory and Effector Helper T-Cell Profile after Nerve Xenografting in the Toll-Like Receptor-Deficient Mice

Introduction: The balance between regulatory T cells (Tregs) and effector T help cells (Th cells) is critical for the control of adaptive immune response during nerve transplantation. However, whether the homeostasis of immune regulation between Tregs and Th cells requires toll-like receptor (TLR) signaling is unclear. The aim of this study is to profile the distribution of spleen Tregs and Th cells in a mouse model of nerve xenografting in the TLR2 and NF-κB gene knockout mice.Methods: The sciatic nerve was taken from a SD rat or an allogeneic mouse and transplanted to a right back leg of recipient C57BL/6, TLR2^-/-, or NF-κB^-/- mice by subcutaneous transplantation. After 7 days, the T lymphocytes were then isolated from spleen, stained with phenotyping kits, and analyzed by flow cytometry.Results: The results showed that Tregs were decreased after nerve xenografting in the recipient C57BL/6 mouse. In addition, nerve xenografting also increased the Th1 and Th17 but not the Th2 cell populations. In contrast, amelioration of the Tregs elimination was found in TLR2^-/- and NF-κB^-/- mice after transplantation of the nerve xenograft. Moreover, the mice lacking TLR2 or NF-κB showed attenuation of the increase in Th1 and Th17 cells after nerve xenografting.Conclusions: TLR signaling is involved in T cell population regulation during tissue transplantation. Knock-out of TLR2 and NF-κB prevented Tregs elimination and inhibited Th1- and Th17-driven immune response after nerve xenografting. This study highlighted the potential of inhibiting TLR signaling to modulate T cell-mediated immune regulation to facilitate tolerance to nerve transplantation.     

Antigen presenting cells may be able to distinguish between normal and radiated Schistosoma japonicum cercaria: an in vitro observation

Objective

To observe the discrepancies of responses induced by Schistosoma japonicum (S. japonicum) normal cercaria antigen (NCA) and ultraviolet (UV) -radiation-attenuated cercaria antigen (UVACA) in an in vitro system.

Methods

S. japonicum cercariae were collected and UVACA and NCA were prepared. Mouse macrophage model cells (RAW 264.7) were treated with medium, NCA (40 µg/mL) or UVACA (40 µg/mL) in the presence or absence of recombinant mouse interferon gamma (rmIFN-γ; 4 ng/mL) for 48 h. Cell surface staining and flow cytometry were used to assess the major histocompatibility complex (MHC)γ; 4 ng/mL) for 48 h. Cell surface staining and flow cytometry were used to assess the major histocompatibility complex (MHC) II expression, and data were expressed as mean fluorescence intensities (MFI). Interleukin (IL) -10, IL-6 and prostaglandin E2 (PGE₂) in cell culture supernatant were evaluated by commercial enzyme-linked immunosorbent assays.

Results

NCA significantly suppressed IFN-γ-induced MHC II expression on RAW 264.7 cells. In the presence of IFN-γ, NCA significantly promoted IL-6, IL-10 and PGE₂ secretion from RAW 264.7 cells. In the presence of IFN-γ, UVACA significantly promoted IL-10 but not IL-6 and PGE₂ secretion from RAW 264.7 cells and showed no effect on IFN-γ-induced MHC II expression. Compared with UVACA, NCA significantly suppressed IFN-γ-induced MHC II expression and significantly promoted IL-6, PGE₂ and IL-10 secretion from RAW 264.7 cells.

Conclusion

RAW 264.7 cells respond differently to NCA and UVACA. NCA can significantly suppress IFN-γ-induced MHC II expression and significantly promote IL-6, IL-10 and PGE₂ secretion from RAW 264.7 cells compared with UVACA.     

Macrophage-elicited osteoclastogenesis in response to bacterial stimulation requires Toll-like receptor 2-dependent tumor necrosis factor-alpha production

The receptor activator of NF-κB ligand (RANKL) and the proinflammatory cytokines are believed to play important roles in osteoclastogenesis. We recently reported that the innate immune recognition receptor, Toll-like receptor 2 (TLR2), is crucial for inflammatory bone loss in response to infection by Porphyromonas gingivalis, the primary organism associated with chronic inflammatory periodontal disease. However, the contribution of macrophage-expressed TLRs to osteoclastogenesis has not been defined. In this study, we defined a requirement for TLR2 in tumor necrosis factor-alpha (TNF-α)-elicited osteoclastogenesis in response to exposure to P. gingivalis. Culture supernatant (CS) fluids from P. gingivalis-stimulated macrophages induced bone marrow macrophage-derived osteoclastogenesis. This activity was dependent on TNF-α and occurred independently of RANKL, interleukin-1β (IL-1β), and IL-6. CS fluids from P. gingivalis-stimulated TLR2^−/− macrophages failed to express TNF-α, and these fluids induced significantly less osteoclast formation compared with that of the wild-type or the TLR4^−/− macrophages. In addition, P. gingivalis exposure induced up-regulation of TLR2 expression on the cell surface of macrophages, which was demonstrated to functionally react to reexposure to P. gingivalis, as measured by a further increase in TNF-α production. These results demonstrate that macrophage-dependent TLR2 signaling is crucial for TNF-α-dependent/RANKL-independent osteoclastogenesis in response to P. gingivalis infection. Furthermore, the ability of P. gingivalis to induce the cell surface expression of TLR2 may contribute to the chronic inflammatory state induced by this pathogen.     

Lactobacilli and Streptococci Induce Interleukin-12 (IL-12), IL-18, and Gamma Interferon Production in Human Peripheral Blood Mononuclear Cells

Human peripheral blood mononuclear cells (PBMC) were stimulated with three nonpathogenic Lactobacillus strains and with one pathogenic Streptococcus pyogenes strain, and cytokine gene expression and protein production were analyzed. All bacteria strongly induced interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha mRNA expression and protein production. S. pyogenes was the most potent inducer of secretion of IL-12 and gamma interferon (IFN-γ), and two of three Lactobacillus strains induced IL-12 and IFN-γ production. All strains induced IL-18 protein production. IL-10 and IL-4 production was induced weakly and not at all, respectively. Our data show that nonpathogenic lactobacilli and pathogenic streptococci can induce Th1 type cytokines IL-12, IL-18, and IFN-γ in human PBMC.     

Mycobacterium bovis bacillus Calmette-Guerin induces CCL5 secretion via the Toll-like receptor 2-NF-kappaB and -Jun N-terminal kinase signaling pathways

In response to Mycobacterium bovis bacillus Calmette-Guérin (BCG), CC chemokines are secreted from host cells to attract components of the innate and adaptive immune systems to the site of infection. Toll-like receptor 2 (TLR2) has been shown to recognize M. bovis BCG and to initiate signaling pathways that result in enhanced secretion of CC chemokines. Despite the essential requirement of TLR2 in M. bovis BCG infection, the mechanisms by which it induces secretion of CC chemokines are not well defined. In this study, we report that stimulation of HEK293 cells expressing human TLR2 with M. bovis BCG resulted in increased CCL2 and CCL5 secretion, as determined by an enzyme-linked immunosorbent assay. M. bovis BCG infection resulted in the activation of c-Jun N-terminal kinase (JNK), and the inhibition of JNK activity had a significant effect on M. bovis BCG-dependent CCL5 secretion in TLR2-expressing cells but no effect on M. bovis BCG-dependent CCL2 secretion from infected HEK293 cells expressing human TLR2. The M. bovis BCG-induced CCL5 release was attenuated by sulfasalazine (a well-described inhibitor of NF-κB activity), BAY 11-7082 (an IκB phosphorylation inhibitor), and ALLN (a well-described inhibitor of NF-κB activation that prevents degradation of IκB and eventually results in a lack of translocated NF-κB in the nucleus). In addition, stimulation of TLR2-expressing cells with M. bovis BCG resulted in translocation of NF-κB subunits from the cytoplasmic to the nuclear fraction, and stimulation of cells with M. bovis BCG activated IκB kinase αβ. These findings indicate that M. bovis BCG induces CCL5 production through mechanisms that include a TLR2-dependent component that requires JNK and NF-κB activities.     

Enhanced innate immune responsiveness to pulmonary Cryptococcus neoformans infection is associated with resistance to progressive infection

Genetically regulated mechanisms of host defense against Cryptococcus neoformans infection are not well understood. In this study, pulmonary infection with the moderately virulent C. neoformans strain 24067 was used to compare the host resistance phenotype of C57BL/6J with that of inbred mouse strain SJL/J. At 7 days or later after infection, C57BL/6J mice exhibited a significantly greater fungal burden in the lungs than SJL/J mice. Characterization of the pulmonary innate immune response at 3 h after cryptococcal infection revealed that resistant SJL/J mice exhibited significantly higher neutrophilia, with elevated levels of inflammatory cytokine tumor necrosis factor alpha (TNF-α) and keratinocyte-derived chemokine (KC)/CXCL1 in the airways, as well as increased whole-lung mRNA expression of chemokines KC/CXCL1, MIP-1α/CCL3, MIP-1β/CCL4, MIP-2/CXCL2, and MCP-1/CCL2 and cytokines interleukin 1β (IL-1β) and IL-1Ra. At 7 and 14 days after infection, SJL/J mice maintained significantly higher levels of TNF-α and KC/CXCL1 in the airways and exhibited a Th1 response characterized by elevated levels of lung gamma interferon (IFN-γ) and IL-12/IL-23p40, while C57BL/6J mice exhibited Th2 immunity as defined by eosinophilia and IL-4 production. Alveolar and resident peritoneal macrophages from SJL/J mice also secreted significantly greater amounts of TNF-α and KC/CXCL1 following in vitro stimulation with C. neoformans. Intracellular signaling analysis demonstrated that TNF-α and KC/CXCL1 production was regulated by NF-κB and phosphatidylinositol 3 kinase in both strains; however, SJL/J macrophages exhibited heightened and prolonged activation in response to C. neoformans infection compared to that of C57BL/6J. Taken together, these data demonstrate that an enhanced innate immune response against pulmonary C. neoformans infection in SJL/J mice is associated with natural resistance to progressive infection.     

Diverse Toll-Like Receptors Mediate Cytokine Production by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in Macrophages

Toll-like receptors (TLRs) orchestrate a repertoire of immune responses in macrophages against various pathogens. Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans are two important periodontal pathogens. In the present study, we investigated TLR signaling regulating cytokine production of macrophages in response to F. nucleatum and A. actinomycetemcomitans. TLR2 and TLR4 are redundant in the production of cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]) in F. nucleatum- and A. actinomycetemcomitans-infected macrophages. The production of cytokines by macrophages in response to F. nucleatum and A. actinomycetemcomitans infection was impaired in MyD88-deficient macrophages. Moreover, cytokine concentrations were lower in MyD88-deficient macrophages than in TLR2/TLR4 (TLR2/4) double-deficient cells. An endosomal TLR inhibitor, chloroquine, reduced cytokine production in TLR2/4-deficient macrophages in response to F. nucleatum and A. actinomycetemcomitans, and DNA from F. nucleatum or A. actinomycetemcomitans induced IL-6 production in bone marrow-derived macrophages (BMDMs), which was abolished by chloroquine. Western blot analysis revealed that TLR2/4 and MyD88 were required for optimal activation of NF-κB and mitogen-activated protein kinases (MAPKs) in macrophages in response to F. nucleatum and A. actinomycetemcomitans, with different kinetics. An inhibitor assay showed that NF-κB and all MAPKs (p38, extracellular signal-regulated kinase [ERK], and Jun N-terminal protein kinase [JNK]) mediate F. nucleatum-induced production of cytokines in macrophages, whereas NF-κB and p38, but not ERK and JNK, are involved in A. actinomycetemcomitans-mediated cytokine production. These findings suggest that multiple TLRs may participate in the cytokine production of macrophages against periodontal bacteria.     

Lipoteichoic acid is important in innate immune responses to gram-positive bacteria

To define the role of lipoteichoic acid (LTA) in innate immunity to gram-positive bacteria, we investigated the production of tumor necrosis factor alpha (TNF-α) by macrophages stimulated with gram-positive bacterial culture supernatants (GPCSs) after their LTA was removed or inactivated. GPCSs were obtained from three gram-positive species (pneumococci, staphylococci, and group B streptococci) during the exponential growth phase (designated early GPCSs) or at the senescent stage (designated late GPCSs). LTA was removed using an anti-LTA antibody or was inactivated by alkaline hydrolysis or platelet-activating factor acetylhydrolase (PAF-AH) treatment. Both early and late GPCSs from the three gram-positive bacteria stimulated macrophages to produce TNF-α primarily via Toll-like receptor 2 (TLR2), although late pneumococcal supernatant could stimulate macrophages via TLR4 as well. Following LTA inactivation by both methods, early GPCS lost about 85 to 100% of its activity and late GPCS lost about 50 to 90%. Both early and late culture supernatants from Escherichia coli could be inactivated by alkali hydrolysis but not by PAF-AH. In addition, removal of LTA from an early staphylococcal culture supernatant with a monoclonal antibody reduced about 70 to 85% of its potency. Reconstitution of inactivated early GPCS with a highly purified LTA restored its inflammatory activity, but the restored GPCS had higher activity than the pure LTA alone. These findings indicate that LTA is the primary TLR2 ligand in the early phase of gram-positive bacterial infection and remains a major ligand in the late phase when another TLR2 and TLR4 ligand(s) appears. In addition, our findings suggest that another gram-positive bacterial factor(s) synergizes with LTA in inducing inflammatory responses.     

Evasion of innate immune responses: evidence for mannose binding lectin inhibition of tumor necrosis factor alpha production by macrophages in response to Blastomyces dermatitidis

Serum factors, including mannose binding lectins (MBL), influence innate responses to microbes. Little is known about the effects of serum factors or MBL on the interaction of Blastomyces dermatitidis, a pulmonary fungal pathogen, with macrophages or on tumor necrosis factor alpha (TNF-α) production. Since macrophage production of TNF-α is an important innate immune response, we examined a mouse peritoneal macrophage (PM) cell line (RAW) and resident PM from CD-1 mice to study TNF-α production by PM stimulated with heat-killed (HK) or live B. dermatitidis yeast cells. Mouse serum and heat-inactivated mouse serum inhibited TNF-α production 94% when macrophages were stimulated by B. dermatitidis, whereas mouse immunoglobulin G (IgG) did not have this effect. HK B. dermatitidis incubated with serum and then washed also failed to stimulate significant TNF-α production by PM. By the sandwich immunofluorescent antibody (IFA) method with anti-mouse MBL (MBL-A or -C), we showed that serum MBL bound to B. dermatitidis. When serum was absorbed with HK B. dermatitidis or live B. dermatitidis, absorbed serum failed to significantly inhibit TNF-α production by RAW cells plus B. dermatitidis, and immunoblotting showed that absorbed serum was depleted of MBL-C. If serum was absorbed with live B. dermatitidis, unbound serum was eluted, and bound serum factor(s) (BS) was released with guanidine buffer, BS inhibited TNF-α production by PM plus B. dermatitidis in a concentration-dependent manner. BS contained MBL-C, which bound B. dermatitidis, as shown by IFA assay. 1,3-β-Glucan stimulated TNF-α production by PM, and this was inhibited by mouse serum. Treatment of B. dermatitidis with anti-1,3-β-glucan antibody inhibited TNF-α production by PM. With anti-1,3-β-glucan antibody, we showed by IFA assay that B. dermatitidis contained 1,3-β-glucan. In an IFA study with B. dermatitidis, serum with an anti-mouse IgG conjugate did not result in fluorescence, yet serum blocked IFA staining of B. dermatitidis by anti-1,3-β-glucan IgG antibody. This indicated that non-IgG serum factors binding to B. dermatitidis prevented access to 1,3-β-glucan by anti-1,3-β-glucan antibody. These results suggest that the mechanism of inhibition of the innate proinflammatory immune response of PM to B. dermatitidis is mediated by serum MBL binding to B. dermatitidis at 1,3-β-glucan sites or sterically masking 1,3-β-glucan sites, thus preventing 1,3-β-glucan stimulation of PM for TNF-α production.