Resistance to a peritoneal lymphoma graft induced by heat-killed S or R Brucella abortus

Summary Mice were injected either with the rough (R) or smooth (S) strains of killed Brucella abortus, after which, at various times, they were given an i.p. injection of a strain-specific lymphoma (EL4). In parallel, samples of peritoneal exudate cells were taken from the Brucella-injected mice, and their in vitro cytostatic activity against EL4 tumour cells was investigated. Protection against the lymphoma graft lasted up to the 7th day with S and the 20th with R. In contrast, cytostatic activity was more intense and lasted longer with S than with R. The parallelism between in vivo resistance to the lymphoma graft and augmentation of macrophage cytostatic activity was satisfactory with R but not with S. The differential effects of S and R on EL4 lymphoma growth could not be simply explained on the basis of a difference in macrophage activation.     

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.     

Lipoproteins, not lipopolysaccharide, are the key mediators of the proinflammatory response elicited by heat-killed Brucella abortus

Inflammation is a hallmark of brucellosis. Although Brucella abortus, one of the disease's etiologic agents, possesses cytokine-stimulatory properties, the mechanism by which this bacterium triggers a proinflammatory response is not known. We examined the mechanism whereby heat-killed B. abortus (HKBA), as well as its LPS, induces production of inflammatory cytokines in monocytes/macrophages. Polymyxin B, a specific inhibitor of LPS activity, did not inhibit the production of TNF-alpha- and IL-6-induced HKBA in the human monocytic cell line THP-1. HKBA induced the production of these cytokines in peritoneal macrophages of both C3H/HeJ and C3H/HeN mice, whereas B. abortus LPS only stimulated cells from C3H/HeN mice. Anti-TLR2 Ab, but not anti-TLR4 Ab, blocked HKBA-mediated TNF-alpha and IL-6 production in THP-1 cells. Because bacterial lipoproteins, a TLR2 ligand, have potent inherent stimulatory properties, we investigated the capacity of two B. abortus lipoproteins, outer membrane protein 19 (Omp19) and Omp16, to elicit a proinflammatory response. Lipidated (L)-Omp16 and L-Omp19, but not their unlipidated forms, induced the secretion of TNF-alpha, IL-6, IL-10, and IL-12 in a time- and dose-dependent fashion. Preincubation of THP-1 cells with anti-TLR2 Ab blocked L-Omp19-mediated TNF-alpha and IL-6 production. Together, these results entail a mechanism whereby B. abortus can stimulate cells from the innate immune system and induce cytokine-mediated inflammation in brucellosis. We submit that LPS is not the cause of inflammation in brucellosis; rather, lipoproteins of this organism trigger the production of proinflammatory cytokines, and TLR2 is involved in this process.     

Lymph node hypertrophy following Leishmania major infection is dependent on TLR9

Control of the protozoan parasite Leishmania major is dependent on establishing a robust T cell response. An early event in the development of an effective T cell response is the expansion (or hypertrophy) of the lymph node draining the site of infection, although the mechanisms involved in this response are not completely understood. In this study, we show that lymph node hypertrophy following L. major infection in mice is associated with increased recruitment of lymphocytes to the lymph node from the blood, and that CD62L-deficient mice, which are unable to recruit cells to the lymph node, develop a chronic infection with L. major. Injection of L. major-activated dendritic cells promoted lymph node hypertrophy, and this correlated with an increase in the expression of CCR7 on dendritic cells, although the upregulation of CCR7 occurred on the bystander (uninfected) dendritic cells rather than those containing parasites. We found that increased CCR7 expression was TLR9-dependent, that TLR9(-/-) dendritic cells migrated less efficiently to the draining lymph node, and that TLR9(-/-) mice exhibited a deficit in lymph node expansion following L. major infection, as well as increased susceptibility. Taken together, to our knowledge, these results are the first to demonstrate that activation of dendritic cells via TLR9 is essential for the induction of lymph node hypertrophy in leishmaniasis.     

TLR2 signaling depletes IRAK1 and inhibits induction of type I IFN by TLR7/9

Pathogens may signal through multiple TLRs with synergistic or antagonistic effects on the induction of cytokines, including type I IFN (IFN-I). IFN-I is typically induced by TLR9, but not TLR2. Moreover, we previously reported that TLR2 signaling by Mycobacterium tuberculosis or other TLR2 agonists inhibited TLR9 induction of IFN-I and IFN-I-dependent MHC-I Ag cross processing. The current studies revealed that lipopeptide-induced TLR2 signaling inhibited induction of first-wave IFN-α and IFN-β mRNA by TLR9, whereas induction of second-wave IFN-I mRNA was not inhibited. TLR2 also inhibited induction of IFN-I by TLR7, another MyD88-dependent IFN-I-inducing receptor, but did not inhibit IFN-I induction by TLR3 or TLR4 (both Toll/IL-1R domain-containing adapter-inducing IFN-β dependent, MyD88 independent). The inhibitory effect of TLR2 was not dependent on new protein synthesis or intercellular signaling. IL-1R-associated kinase 1 (IRAK1) was depleted rapidly (within 10 min) by TLR2 agonist, but not until later (e.g., 2 h) by TLR9 agonist. Because IRAK1 is required for TLR7/9-induced IFN-I production, we propose that TLR2 signaling induces rapid depletion of IRAK1, which impairs IFN-I induction by TLR7/9. This novel mechanism, whereby TLR2 inhibits IFN-I induction by TLR7/9, may shape immune responses to microbes that express ligands for both TLR2 and TLR7/TLR9, or responses to bacteria/virus coinfection.     

Effects of liver-derived dendritic cell progenitors on Th1- and Th2-like cytokine responses in vitro and in vivo

There is evidence that donor-derived dendritic cells (DC), particularly those at a precursor/immature stage, may play a role in the immune privilege of liver allografts. Underlying mechanisms are poorly understood. We have examined the influence of in vitro generated mouse liver-derived DC progenitors (DCp) on proliferative, cytotoxic, and Th1/Th2 cytokine responses induced in allogeneic T cells. Liver DCp, propagated in GM-CSF from C57B10 mice (H2b), induced only minimal proliferation, and weak cytotoxic responses in allogeneic (C3H; H2k) T cells compared with mature bone marrow (BM)-derived DC. Flow-cytometric analysis of intracellular cytokine staining revealed that mature BM DC, but not liver DCp, elicited CD4+ T cell production of IFN-gamma. Intracellular expression of IL-10 was very low in both BM DC- and liver DCp-stimulated CD4+ T cells. Only stimulation by liver DCp was associated with IL-10 secretion in primary MLR. Notably, these liver DCp cocultured with allogeneic T cells stained strongly for IL-10. Following local (s.c. ) injection in allogeneic recipients, both BM DC and liver DCp homed to T cell areas of draining lymph nodes and spleen, where they were readily detected by immunohistochemistry up to 2 wk postinjection. Liver DCp induced clusters of IL-10- and IL-4-secreting mononuclear cells, whereas Th2 cytokine-secreting cells were not detected in mice injected with mature BM DC. By contrast, comparatively high numbers of IFN-gamma+ cells were induced by BM DC. Modulation of Th2 cytokine production by donor-derived DCp may contribute to the comparative immune privilege of hepatic allografts.     

Interleukin-18 is a unique cytokine that stimulates both Th1 and Th2 responses depending on its cytokine milieu

IL-18 is a potent proinflammatory cytokine able to induce IFNgamma, GM-CSF, TNFalpha and IL-1 in immunocompetent cells, to activate killing by lymphocytes, and to up-regulate the expression of certain chemokine receptors. IL-18 is also essential to host defences against severe infections. In particular, the clearance of intracellular bacteria, fungi and protozoa requires the induction of host-derived IFNgamma, which evokes effector molecules such as nitric oxide. Also, IL-18 plays a part in the clearance of viruses, partly by the induction of cytotoxic T cells, and the expulsion of viruses is impaired in IL-18-deficient mice. IL-18 also enhances tumour rejection by its potent capacity to augment the cytotoxic activity of NK and T cells in vivo. In contrast, recent studies also demonstrate a convincing role for IL-18 in atopic responses, including atopic asthma. IL-18 induces naive T cells to develop into Th2 cells. Moreover, IL-18 also induces IL-13 and/or IL-4 production by NK cells, mast cells and basophils. Therefore, IL-18 should be seen as a unique cytokine that enhances innate immunity and both Th1- and Th2-driven immune responses.     

Phosphatidylethanolamine synthesis is required for optimal virulence of Brucella abortus

The Brucella cell envelope contains the zwitterionic phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Synthesis of PC occurs exclusively via the PC synthase pathway, implying that the pathogen depends on the choline synthesized by the host cell to form PC. Notably, PC is necessary to sustain a chronic infection process, which suggests that the membrane lipid content is relevant for Brucella virulence. In this study we investigated the first step of PE biosynthesis in B. abortus, which is catalyzed by phosphatidylserine synthase (PssA). Disruption of pssA abrogated the synthesis of PE without affecting the growth in rich complex medium. In minimal medium, however, the mutant required choline supplementation for growth, suggesting that at least PE or PC is necessary for Brucella viability. The absence of PE altered cell surface properties, but most importantly, it impaired several virulence traits of B. abortus, such as intracellular survival in both macrophages and HeLa cells, the maturation of the replicative Brucella-containing vacuole, and mouse colonization. These results suggest that membrane phospholipid composition is critical for the interaction of B. abortus with the host cell.     

CMRF-35-like molecule 3 preferentially promotes TLR9-triggered proinflammatory cytokine production in macrophages by enhancing TNF receptor-associated factor 6 ubiquitination

TLRs are critical innate immune sensors in the induction of proinflammatory cytokines to eliminate invading pathogens. However, the mechanisms for the full activation of TLR-triggered innate immune response need to be fully understood. The murine CMRF-35-like molecule (CLM)-3 is a representative of CLM family belonging to the Ig superfamily. Considering that CLM-3 is selectively expressed in macrophages and the roles of CLM members in innate immune response remain unclear, in this study we investigated the role of CLM-3 in the regulation of TLR-triggered innate response. We found that CLM-3 was an endosome/lysosome-localized molecule, and was downregulated in macrophages by stimulation with TLR9 ligand, but not TLR4 and TLR3 ligands. Interestingly, CLM-3 selectively promoted production of TNF-α and IL-6 in macrophages triggered by TLR9, but not TLR4 or TLR3. CLM-3 enhanced activation of MAPKs and NF-κB pathways in TLR9-triggered macrophages. Furthermore, CLM-3-transgenic mice were generated, and CLM-3 expression was confirmed by mAb against CLM-3 that we prepared. Accordingly, the macrophages derived from CLM-3-transgenic mice were more sensitive to TLR9 ligand stimulation, with more pronounced production of TNF-α, IL-6, and increased activation of MAPKs and NF-κB pathways. Moreover, ubiquitination of TNFR-associated factor 6, a crucial signaling transducer of TLR-triggered MAPKs and NF-κB activation, was found to be significantly promoted by CLM-3 in macrophages. Collectively, the endosome/lysosome-localized CLM-3 can promote full activation of TLR9-triggered innate responses by enhancing TNFR-associated factor 6 ubiquitination and subsequently activating MAPKs and NF-κB.     

TLR2 synergizes with both TLR4 and TLR9 for induction of the MyD88-dependent splenic cytokine and chemokine response to Streptococcus pneumoniae

We previously demonstrated that induction of splenic cytokine and chemokine secretion in response to Streptococcus pneumoniae (Pn) is MyD88-, but not critically TLR2-dependent, suggesting a role for additional TLRs. In this study, we investigated the role of TLR2, TLR4, and/or TLR9 in mediating this response. We show that a single deficiency in TLR2, TLR4, or TLR9 has only modest, selective effects on cytokine and chemokine secretion, whereas substantial defects were observed in TLR2(-/-)xTLR9(-/-) and TLR2(-/-)xTLR4(-/-) mice, though not as severe as in MyD88(-/-) mice. Chloroquine, which inhibits the function of intracellular TLRs, including TLR9, completely abrogated detectable cytokine and chemokine release in spleen cells from TLR2(-/-)xTLR4(-/-) mice, similar to what is observed for mice deficient in MyD88. These data demonstrate significant synergy between TLR2 and both TLR4 and TLR9 for induction of the MyD88-dependent splenic cytokine and chemokine response to Pn.     

MyD88 regulation of Fusarium keratitis is dependent on TLR4 and IL-1R1 but not TLR2

The fungal pathogens Fusarium solani and Fusarium oxysporum cause severe corneal disease in the United States and worldwide and were the causative organisms in a recent outbreak of contact lens-associated keratitis. To characterize innate immunity in Fusarium keratitis, we developed a murine model in which conidia are injected into the corneal stroma. Immunocompetent C57BL/6 mice rapidly developed severe corneal opacification associated with neutrophil infiltration and clearance of Fusarium hyphae. In contrast, neutrophil infiltration was delayed in MyD88-/- mice, resulting in uncontrolled growth of Fusarium hyphae in the corneal stroma and anterior chamber, and eventually resulting in corneal perforation. Corneal opacification scores in TLR2-/-, TLR4-/-, and TLR2/4-/- mice were similar to those of C57BL/6 mice; however, TLR4-/- and TLR2/4-/- mice had impaired antifungal responses. The phenotype of infected IL-1R1-/- mice was similar to that of MyD88-/- mice, with uncontrolled fungal growth resulting in corneal perforation. IL-1R1-/- mice also produced significantly less CXCL1/KC in the corneal stroma compared with C57BL/6 mice consistent with delayed neutrophil recruitment to the corneal stroma. Together, these findings indicate that IL-1R1 and MyD88 regulate CXC chemokine production and neutrophil recruitment to the cornea, and that TLR4 has an important role in controlling growth and replication of these pathogenic fungi.     

Brucella control of dendritic cell maturation is dependent on the TIR-containing protein Btp1

Brucella is an intracellular pathogen able to persist for long periods of time within the host and establish a chronic disease. We show that soon after Brucella inoculation in intestinal loops, dendritic cells from ileal Peyer's patches become infected and constitute a cell target for this pathogen. In vitro, we found that Brucella replicates within dendritic cells and hinders their functional activation. In addition, we identified a new Brucella protein Btp1, which down-modulates maturation of infected dendritic cells by interfering with the TLR2 signaling pathway. These results show that intracellular Brucella is able to control dendritic cell function, which may have important consequences in the development of chronic brucellosis.     

Roquinimex-mediated protection effect on the development of chronic graft-versus-host disease in mice is associated with induction of Th1 cytokine production and inhibition of proinflammatory cytokine production

Roquinimex is an immunomodulator that can effectively inhibit the development of several autoimmune diseases in animal models, but the mechanism is still unknown. In this study, we investigated the effect of roquinimex on chronic graft-versus-host disease (GVHD) in mice, a well-established model for human systemic lupus erythematosus (SLE). Oral administration of roquinimex significantly suppressed the development of proteinuria and ameliorated nephritis symptoms in chronic GVHD mice. In addition, renal histopathology and immunohistochemistry studies revealed reduced glomerulonephritis and decreased IgG deposition in chronic GVHD mice treated with roquinimex. Chronic GVHD is characterized by a predominance of Th2 cytokines, and proinflammatory cytokines that also play an important role in the pathology of tissue damage. Therefore, we focused on the effect of roquinimex on cytokine production. Chronic GVHD mouse splenocytes exhibited severely reduced interferon (IFN)-gamma production in response to Concanavalin (Con A) stimulation and an overt Th2 skewness. Roquinimex treatment, however, induced IFN-gamma production and restored the Th1/Th2 cytokine balance, although only a minimal effect of roquinimex on interleukin (IL)-4 secretion was observed. The production of the proinflammatory cytokines TNF-alpha and IL-1 beta by peritoneal macrophages from lipopolysaccharide (LPS)-treated GVHD mice was significantly inhibited by roquinimex treatment. These data suggested that the beneficial effect of roquinimex on lupus might, at least in part, result from a restoration of Th1/Th2 cytokine balance and inhibition of inflammatory cytokine production.     

Immunomodulatory effects of inactivated parapoxvirus ovis (ORF virus) on human peripheral immune cells: induction of cytokine secretion in monocytes and Th1-like cells

Inactivated parapoxvirus ovis (Orf virus; PPVO) recently displayed strong immunostimulating and modulating capacities in several animal models for acute and chronic virus infections through the induction of gamma interferon (IFN-gamma) as a key mediator of antiviral activity. The data presented in this work demonstrate that inactivated PPVO has strong effects on cytokine secretion by human immune cells, including the upregulation of inflammatory and Th1-related cytokines (IFN-gamma, tumor necrosis factor alpha [TNF-alpha], interleukin 6 [IL-6], IL-8, IL-12, and IL-18) as well as anti-inflammatory and Th2-related cytokines (IL-4, IL-10, and IL-1 receptor antagonist [IL-1ra]). Studies on the mechanism of action revealed virus particles to be the effective components of the preparation. The virus particles activate monocytes or other antigen-presenting cells (APC), e.g., plasmacytoid dendritic cells, through signaling over CD14 and a Toll-like receptor and the intracellular presence of certain PPVO-specific components. The activation of monocytes or APC is followed by the release of early proinflammatory cytokines (TNF-alpha, IL-6, and IL-8) as well as the Th1-related cytokines IL-12 and IL-18. Both IL-18 and IL-12 are involved in PPVO-mediated IFN-gamma release by T cells and/or NK cells. The proinflammatory response is accompanied by the induction of anti-inflammatory and Th2-related cytokines (IL-4, IL-10, and IL-1ra), which exert a limiting efffect on the inflammatory response induced by PPVO. We conclude that the induction of a natural immune response with physiologically significant amounts of different cytokines and with antiviral potential might provide advantages over existing antiviral immunotherapies.     

Th1 and Th2 cytokine immunomodulation by gangliosides in experimental autoimmune encephalomyelitis

In experimental autoimmune encephalomyelitis, a classical model for multiple sclerosis, the cytokines provide the necessary signals to activate specific T cells for self-antigens. Gangliosides have multiple immunomodulatory activities, decreasing the lymphoproliferative responses and modulating cytokine production. Here, we tested the effects of gangliosides on the switching of Th1 to Th2 cytokine expression, in spleen cells obtained from Lewis rats during the acute phase of EAE, and after recovery from the disease. For this purpose, total RNA from spleen cells was isolated and submitted to RT-PCR to investigate Th1 (IL-2, TNF-alpha, and IFN-gamma) and Th2/Th3 (IL-10 and TGF-beta) cytokine gene expression. Results demonstrate that the group treated with gangliosides displays mild disease, with low expression of IFN-gamma mRNA and high TGF-beta mRNA expression. We conclude that the gangliosides may modulate Th1 cells by the synthesis of cytokines shifting the profile to the Th2/Th3 phenotype.     

Cutting edge: a TLR9 cytoplasmic tyrosine motif is selectively required for proinflammatory cytokine production

Compartmentalization of nucleic acid sensing TLR9 has been implicated as a mechanism to prevent recognition of self nucleic acid structures. Furthermore, recognition of CpG DNA in different endosomal compartments leads to the production of the proinflammatory cytokine TNF-α, or type I IFN. We previously characterized a tyrosine-based motif at aa 888-891 in the cytoplasmic tail of TLR9 important for appropriate intracellular localization. In this article, we show that this motif is selectively required for the production of TNF, but not IFN. In response to CpG DNA stimulation, the proteolytically processed 80-kDa fragment is tyrosine phosphorylated. Although Y888 is not itself phosphorylated, the structure of this motif is necessary for both TLR9 phosphorylation and TNF-α production in response to CpG DNA. We conclude that bifurcation in TLR9 signaling is regulated by a critical tyrosine motif in the cytoplasmic tail.