Leishmania cytosolic silent information regulatory protein 2 deacetylase induces murine B-cell differentiation and in vivo production of specific antibodies

In previous studies, we identified a gene product belonging to the silent information regulatory 2 protein (SIR2) family. This protein is expressed by all Leishmania species so far examined (L. major, L. infantum, L. amazonensis, L. mexicana) and found to be crucial for parasite survival and virulence. In the present study, we investigated whether a Leishmania SIR2 recombinant protein (LmSIR2) would affect T- and B-cell functions in a murine model. In vitro treatment of spleen cells from normal BALB/c mice with LmSIR2 showed increased expression of CD69 on B cells. This effect was not abolished by the addition of polymyxin B. Intravenous injection of LmSIR2 into BALB/c mice induced increased spleen B cell number by a factor of about approximately 1.6, whereas no modification occurred at the level of CD4(+) and CD8(+) cells. Furthermore, intraperitoneal injection of LmSIR2 alone without adjuvant into BALB/c mice or nude mice triggered the production of elevated levels of LmSIR2-specific antibodies. The analysis of specific isotype profiles showed a predominance of immunoglobulin G1 (IgG1) and IgG2a antibody responses in BALB/c mice, and IgM in nude mice. Moreover, the anti-LmSIR2 mouse antibodies in the presence of complement induced the in vitro lysis of L. infantum amastigotes. In the absence of complement, the antibodies induced significant inhibition of amastigotes developpement inside macrophages. Together, the current study provides the first evidence that a Leishmania protein belonging to the SIR2 family may play a role in the regulation of immune response through its capacity to trigger B-cell effector function.     

The innate immune response against Leishmania parasites

Parasites of the genus Leishmania are the causative agents of cutaneous, mucocutaneous or visceral leishmaniasis. The parasite species and host genetic factors determine the quality of the immune response and thereby the outcome of the infection. Here, we summarize previously published and present novel data on several aspects of the early innate immune reaction to Leishmania (L.) major, L. braziliensis and L. infantum, which cause cutaneous, mucocutaneous or visceral leishmaniasis, respectively. We will focus on (1) the effector molecules that contribute to the control of the parasite in the skin, lymph nodes and/or spleen; and (2) on the pattern recognition receptors (Toll-like receptors, TLRs), cell types (myeloid dendritic cells, plasmacytoid dendritic cells), cytokines (IL-12, IFN-alpha/beta), and signaling pathways (Tyk2 kinase) that are necessary for the initial sensing of the parasites and the subsequent development of an efficient NK cell response.     

Leishmania surface protein gp63 binds directly to human natural killer cells and inhibits proliferation

Natural killer (NK) cells contribute to immunity as the first line of defence in numerous infections by early cytokine secretion and cytotoxicity. In Leishmania infection, NK cells contribute with interferon-gamma and may assist in directing the immune response towards T helper type 1, which is essential for successful control of the parasites. Thus, NK cells may play an important role in both resistance and control of the infection. However, during Leishmania infection NK cells show signs of suppression. To explore the reason for this suppression, we exposed naive and interleukin (IL)-2 activated NK cells directly to promastigotes of Leishmania major in vitro. As a rapid consequence of contact between naive NK cells and promastigotes, expression of NK cell receptors show significant changes. We identify one of the major surface molecules of promastigotes, glycoprotein (gp) 63, as an important agent for these suppressive effects by using promastigotes of a gp63ko strain of L. major. Furthermore, proliferation of IL-2-activated purified NK cells is suppressed after exposure to the wild-type but not to gp63ko promastigotes. However, gp63ko L. major induced no NK cell proliferation when NK cells were co-cultured with peripheral blood mononuclear cells populations such as CD14(+) monocytes or T cells.     

Role of Toll-like receptor 2 in innate resistance to Group B Streptococcus

The Gram-positive bacterium Group B Streptococcus (GBS) is an important cause of serious neonatal and adult infections. Toll-like receptor 2 (TLR2) recognizes components of the cell wall of Gram-positive bacteria and is critical for defense against certain invasive pathogens. In GBS, penicillin-binding protein 1a (PBP1a), encoded by ponA, is required for virulence. PBPs participate in cell wall synthesis and in previous studies; the absence of PBP1a was shown to result in subtle changes in the cell wall ultrastructure. Here, we examine the role of TLR2 in defense against GBS infection and the impact of mutation of ponA on TLR2-mediated host responses. We demonstrate TLR2-recognition of both wild-type (WT) GBS and the ponA mutant in vitro. TLR2(-/-) mice were significantly more susceptible than WT mice to infection with either strain of GBS, indicating a crucial role for TLR2 in defense against GBS. Additionally, the ponA mutant was severely attenuated for virulence in both strains of mice. The mutation in ponA did not affect cytokine expression by WT or TLR2(-/-) mice. These data indicate that TLR2 is required for host defense against GBS and this response is unaffected by the absence of PBP1a and the resultant changes in cell wall ultrastructure.     

A Toll-interleukin 1 repeat protein at the synapse specifies asymmetric odorant receptor expression via ASK1 MAPKKK signaling

A stochastic lateral signaling interaction between two developing Caenorhabditis elegans AWC olfactory neurons causes them to take on asymmetric patterns of odorant receptor expression, called AWC(OFF) and AWC(ON). Here we show that the AWC lateral signaling gene tir-1 (previously known as nsy-2) encodes a conserved post-synaptic protein that specifies the choice between AWC(OFF) and AWC(ON). Genetic evidence suggests that tir-1 acts downstream of a voltage-gated calcium channel and CaMKII (UNC-43) to regulate AWC asymmetry via the NSY-1(ASK1) p38/JNK MAP (mitogen-activated protein) kinase cascade. TIR-1 localizes NSY-1 to post-synaptic regions of AWC, and TIR-1 binds UNC-43, suggesting that it assembles a synaptic signaling complex that regulates odorant receptor expression. Temperature-shift experiments indicate that tir-1 affects AWC during a critical period late in embryogenesis, near the time of AWC synapse formation. TIR-1 is a multidomain protein with a TIR (Toll-interleukin-1 receptor) domain that activates signaling, SAM repeats that mediate localization to post-synaptic regions of axons, and an N-terminal inhibitory domain. TIR-1 and other TIR proteins are implicated in vertebrate and invertebrate innate immunity, as are NSY-1/ASK1 kinases, so this pathway may also have a conserved function in immune signaling.     

Characterization of the B cell response to Leishmania infection after anti-CD20 B cell depletion

Anti-CD20 depletion therapies targeting B cells are commonly used in malignant B cell disease and autoimmune diseases. There are concerns about the ability of B cells to respond to infectious diseases acquired either before or after B cell depletion. There is evidence that the B cell response to existing or acquired viral infections is compromised during treatment, as well as the antibody response to vaccination. Our laboratory has an experimental system using co-infection of C3H mice with both Leishmania major and Leishmania amazonensis that suggests that the B cell response is important to healing infected mice. We tested if anti-CD20 treatment would completely restrict the B cell response to these intracellular pathogens. Infected mice that received anti-CD20 B cell depletion therapy had a significant decrease in CD19⁺ cells within their lymph nodes and spleens. However, splenic B cells were detected in depleted mice and an antigen-specific antibody response was produced. These results indicate that an antigen-specific B cell response towards intracellular pathogens can be generated during anti-CD20 depletion therapy.     

Overexpression of Toll-like receptor 2/4 on monocytes modulates the activities of CD4CD25 regulatory T cells in chronic hepatitis B virus infection

The significance of TLR expression and Tregs in HBV infection has not been clearly described. In this report, flow cytometry was performed to assess TLR2/4 expression on monocytes and circulating CD4⁺CD25⁺CD127^low/− Tregs frequency of 16 acute hepatitis B (AHB), 42 chronic hepatitis B (CHB), 22 asymptomatic HBV carriers (AsC), and 20 normal controls (NC). We found that TLR2 and TLR4 were overexpressed on CD14⁺ monocytes in HBV-infected patients as compared with NCs. Upregulation of TLR2 in NCs and TLR4 in CHBs was observed following HBeAg incubation. However, TLR2 and TLR4 expression decreased after HBcAg stimulation. The difference in the proportion of Tregs between NCs and CHBs was significant. Both Pam3Csk4 (TLR2 agonist)- and lipopolysaccharide (TLR4 agonist)-activated CD4⁺CD25⁺ Tregs showed enhanced suppression function in CHBs. These results suggest that overexpression of TLR2 and TLR4 may modulate the suppressive function of Tregs, which contribute to the immunotolerance of chronic HBV infection.     

Contrasting human cytokine responses to promastigote whole-cell extract and the Leishmania analogue receptor for activated C kinase antigen of L. amazonensis in natural infection versus immunization

It is known that the same antigen can induce different immune responses, depending upon the way that it is presented to the immune system. The objective of this study was to compare cytokine responses of peripheral blood mononuclear cells (PBMC) from cutaneous leishmaniasis patients and subjects immunized with a first-generation candidate vaccine composed of killed Leishmania amazonensis promastigotes to a whole-cell promastigote antigen extract (La) and to the recombinant protein LACK (Leishmania analogue receptor for activated C kinase), both from L. amazonensis. Thirty-two patients, 35 vaccinees and 13 healthy subjects without exposure to Leishmania, were studied. Cytokine production was assessed by enzyme-linked immunosorbent assay and enzyme-linked immunospot assay. The interferon (IFN)-gamma levels stimulated by La were significantly higher and the levels of interleukin (IL)-10 significantly lower than those stimulated by LACK in the patient group, while LACK induced a significantly higher IFN-gamma production and a significantly lower IL-10 production compared with those induced by La in the vaccinated group. LACK also induced a significantly higher frequency of IFN-gamma-producing cells than did La in the vaccinated group. The contrast in the cytokine responses stimulated by LACK and La in PBMC cultures from vaccinated subjects versus patients indicates that the human immune response to crude and defined Leishmania antigens as a consequence of immunization differs from that induced by natural infection.     

Dynamic localization and the associated translocation mechanism of HMGBs in response to GCRV challenge in CIK cells

High-mobility group box (HMGB) proteins, a family of chromatin-associated nuclear proteins, play amazingly multifaceted roles in the immune system of mammals. Thus far, little is known about the nucleocytoplasmic distribution of HMGBs in teleosts. The present study systematically investigated the dynamic localization of all six HMGB proteins in Ctenopharyngodon idella kidney (CIK) cells. Under basal conditions, all HMGBs exclusively localized to the nucleus. Grass carp reovirus (GCRV), polyinosinic–polycytidylic (poly(I∶C)) potassium salt and lipopolysaccharide (LPS) challenge evoked the nuclear export of HMGBs to various degrees: GCRV challenge induced the highest nuclear export of CiHMGB2b, and poly(I∶C) and LPS evoked the highest nucleocytoplasmic shuttling of CiHMGB1b. Overall, the nucleocytoplasmic shuttling of CiHMGB2a and CiHMGB3b was rarely induced by these challenges. Dynamic imaging uncovered that the nucleocytoplasmic GCRV-induced relocation of CiHMGB2b occurred in cells undergoing karyotheca rupture, apoptosis or proliferation. Western blot analyses were used to examine HMGB-EGFP fusion proteins in whole cell lysates, cytosol, nuclear fractions and culture medium. Further investigation demonstrated the nuclear retention of N-terminal HMG-boxes and the nucleocytoplasmic distribution of the C-terminal acidic tails. Comparative analyses of the dynamic relocation of full-length, truncated or chimeric HMGBs confirmed that the intramolecular interaction between HMG-boxes and C-tail domains mediated the nucleocytoplasmic translocation of HMGBs. These results not only provide an overall understanding of the subcellular localization of HMGBs, but also reveal the induction mechanism of the nucleocytoplasmic translocation of HMGBs by GCRV challenge, which lays a foundation for further studies on the interactions among pathogens, HMGBs and pattern recognition receptors in the innate immunity of teleosts.     

Interleukin-10-dependent down-regulation of interferon-gamma response to Leishmania by Mycobacterium leprae antigens during the clinical course of a coinfection

We have described a case of a patient with an intriguing association of mucocutaneous leishmaniasis with lepromatous leprosy, two opposite polar forms of these spectral diseases. In the present follow-up study, we investigated the effect of the addition of Mycobacterium leprae antigens on interferon-gamma (IFN-γ) production in Leishmania antigen-stimulated cultures of peripheral blood mononuclear cells (PBMC) from this patient. For this purpose, PBMC cultures were stimulated with crude L. braziliensis and/or M. leprae whole-cell antigen extracts or with concanavalin A. In some experiments, neutralizing anti-human interleukin (IL)-10 antibodies were added to the cultures. IFN-γ and IL-10 levels in culture supernatants were measured by ELISA. During active leprosy, M. leprae antigens induced 72.3% suppression of the IFN-γ response to L. braziliensis antigen, and this suppression was abolished by IL-10 neutralization. Interestingly, the suppressive effect of M. leprae antigen was lost after the cure of leprosy and the disappearance of this effect was accompanied by exacerbation of mucosal leishmaniasis. Considered together, these results provide evidence that the concomitant lepromatous leprosy induced an IL-10-mediated regulatory response that controlled the immunopathology of mucosal leishmaniasis, demonstrating that, in the context of this coinfection, the specific immune response to one pathogen can influence the immune response to the other pathogen and the clinical course of the disease caused by it. Our findings may contribute to a better understanding of the Leishmania/M. leprae coinfection and of the immunopathogenesis of mucosal leishmaniasis.     

Activation of Toll-like receptor 2 increases macrophage resistance to HIV-1 infection

Patients infected with HIV-1, the etiological agent of AIDS, have increased intestinal permeability, which allows for the passage of microbial products, including Toll-like receptor (TLR) ligands, into circulation. The exposure of HIV-1-infected cells to certain TLR agonists affects viral replication, but studies associating viral production with the activation of TLR2 in HIV-1-infected cells are rare and controversial. Here, we report that the TLR2 ligands Zymosan and Pam3CSK4 potently inhibit HIV-1 replication in acutely infected monocyte-derived macrophages and the exposure to TLR2 ligands prior to infection renders macrophages refractory to HIV-1 production. Macrophage treatment with Pam3CSK4 did not change the cellular expression of the HIV-1 entry receptors CD4 and CCR5. Both TLR2 ligands increased the macrophage production of β-chemokines and IL-10, and the blockage of these soluble factors prevented the inhibitory effect of TLR2 activation on HIV-1 replication. Our findings show that the direct engagement of TLR2 in HIV-1-infected macrophages increase cellular resistance to HIV-1 infection, and that controlling HIV-1 replication with agonists for TLR2 might have implications for the development of antiretroviral therapies.     

Antibody responses to a C-terminal fragment of the Plasmodium falciparum blood-stage antigen Pf332 in Senegalese individuals naturally primed to the parasite

Previous studies have shown that antibodies from humans exposed continuously to malaria recognize the Plasmodium falciparum asexual blood-stage antigen Pf332. Here we analysed the antibody responses to a C-terminal fragment of Pf332, designated C231, in individuals from Senegal, by measuring the serum levels of immunoglobulin M (IgM), IgG class and subclass and IgE antibodies. IgG antibody reactivity with crude P. falciparum antigen was detected in all the donors, while many of the children lacked or had low levels of such antibodies against C231. The antibody levels increased significantly with age for both crude P. falciparum antigen and C231, and in the older age groups most of the donors displayed antibodies to C231. This was also true for IgM, IgE and IgG subclass reactivity against C231. Moreover, the ratio of IgG1/IgG2 was considerably lower for C231 than for crude P. falciparum antigen, and in age groups 10-14 and 15-19 years the levels of IgG2 against C231 even exceeded that of IgG1. The IgG2/IgG3 ratios suggest that C231 gives similar levels of IgG2 and IgG3, except for children aged 4-9 years, where IgG3 was higher. Raw IgM, IgG class and subclass and IgE antibody levels to C231 tended to be higher in those who did not experience a malaria attack, but following linear multivariate analysis the trends were not significant.     

Brucella abortus induces Irgm3 and Irga6 expression via type-I IFN by a MyD88-dependent pathway,without the requirement of TLR2, TLR4, TLR5 and TLR9

The innate immune system senses bacterial pathogens by pattern recognition receptors, such as the well-characterised Toll-like Receptors (TLR). The activation of TLR signalling cascades depends on several adaptor proteins, among which MyD88 plays a key role in triggering innate immune responses. Here, we show in murine macrophages that Brucella abortus triggers expression of the interferon-inducible resistance proteins (IRGs, p47 GTPases) via type-I IFN secretion at late time points, when Brucella has reached its replication niche. This induction requires the adaptor molecule MyD88 but does not involve the TLRs normally implicated in sensing Gram-negative bacteria, namely TLR2, TLR4, TLR5 and TLR9. Brucella mutants lacking the functional VirB type-IV secretion system were not capable of inducing Irgm3 and Irga6 expression, suggesting that the type-IV secretion system is part of the triggering of the activation process. Our data suggest that Brucella is recognized intracellularly by an unknown receptor, different from the conventional ones used for Gram-negative sensing, but one that nevertheless signals through MyD88.     

Flagellin from Marinobacter algicola and Vibrio vulnificus activates the innate immune response of gilthead seabream

Adjuvants have emerged as the best tools to enhance the efficacy of vaccination. However, the traditional adjuvants used in aquaculture may cause adverse alterations in fish making necessary the development of new adjuvants able to stimulate the immune system and offer strong protection against infectious pathogens with minimal undesirable effects. In this respect, flagellin seems an attractive candidate due to its ability to strongly stimulate the immune response of fish. In the present study, we have evaluated the ability of recombinant flagellin from Marinobacter algicola (MA) and Vibrio vulnificus (Vvul), a non-pathogenic and a pathogenic bacteria, respectively, to stimulate the innate immune system of gilthead seabream (Sparus aurata L.) and compare the effect with that of the classical flagellin from Salmonella enterica serovar Typhimurium (Salmonella Typhimurium, STF). Intraperitoneal injection of MA and Vvul resulted in a strong inflammatory response characterized by increased reactive oxygen species production and the infiltration of acidophilic granulocytes at the injection site. Interestingly, however, only flagellin from MA consistently induced the expression of the gene encoding pro-inflammatory interleukin-1β. These effects were further confirmed in vitro, where a dose-dependent activation of macrophages and acidophilic granulocytes by MA and Vvul flagellins was observed. In contrast, STF flagellin was found to be less potent in both in vivo and in vitro experiments. Our results suggest the potential use of MA and Vvul flagellins as immunostimulants and adjuvants for fish vaccination.     

Evaluation of the Essential Oil of Foeniculum Vulgare Mill (Fennel) Fruits Extracted by Three Different Extraction Methods by GC/MS

Background

Hydrodistillation (HD) and steam-distillation, or solvent extraction methods of essential oils have some disadvantages like thermal decomposition of extracts, its contamination with solvent or solvent residues and the pollution of residual vegetal material with solvent which can be also an environmental problem. Thus, new green techniques, such as supercritical fluid extraction and microwave assisted techniques, are potential solutions to overcome these disadvantages.

Materials and Methods

The aim of this study was to evaluate the essential oil of Foeniculum vulgare subsp. Piperitum fruits extracted by three different extraction methods viz. Supercritical fluid extraction (SFE) using CO₂, microwave-assisted extraction (MAE) and hydro-distillation (HD) using gas chromatography-mass spectrometry (GC/MS).

Results

The results revealed that both MAE and SFE enhanced the extraction efficiency of the interested components. MAE gave the highest yield of oil as well as higher percentage of Fenchone (28%), whereas SFE gave the highest percentage of anethol (72%).

Conclusion

Microwave-assisted extraction (MAE) and supercritical fluid extraction (SFE) not only enhanced the essential oil extraction but also saved time, reduced the solvents use and produced, ecologically, green technologies.     

Activation of an innate immune response in the schistosome-transmitting snail Biomphalaria glabrata by specific bacterial PAMPs

Injection of crude lipopolysaccharide (LPS) from Escherichia coli into the hemocoel of Biomphalaria glabrata stimulates cell proliferation in the amebocyte-producing organ (APO). However, it is not known if mitogenic activity resides in the lipid A or O-polysaccharide component of LPS. Moreover, the possible role of substances that commonly contaminate crude LPS and that are known to stimulate innate immune responses in mammals, e.g., peptidoglycan (PGN), protein, or bacterial DNA, is unclear. Therefore, we tested the effects of the following injected substances on the snail APO: crude LPS, ultrapurified LPS (lacking lipoprotein contamination), two forms of lipid A, (diphosphoryl lipid A and Kdo₂-lipid A), O-polysaccharide, Gram negative PGN, both crude and ultrapurified (with and without endotoxin activity, respectively), Gram positive PGN, PGN components Tri-DAP and muramyl dipeptide, and bacterial DNA. Whereas crude LPS, ultrapurified LPS, and crude PGN were mitogenic, ultrapurified PGN was not. Moreover, LPS components, PGN components, and bacterial DNA were inactive. These results suggest that it is the intact LPS molecule which stimulates cell division in the APO.     

The Extracts of Pacific Oyster (Crassostrea Gigas) Alleviate Ovarian Functional Disorders of Female Rats with Exposure to Bisphenol a Through Decreasing FSHR Expression in Ovarian Tissues

Background

Bisphenol-A (BPA) is one of the widespread industrial compounds, which has adverse effects on animal and human health. The study was aimed to explore the effects of Crassostrea gigas extracts (CGE) in alleviating ovarian functional disorders of female rats with exposure to BPA and the underlying possible mechanism.

Materials and methods

Eighteen four-week-old female Sprague-Dawley (SD) rats were randomly divided into BPA group (50mg/kg BPA), BPA+CGE group (50mg/kg BPA+50mg/kg CGE), and control group (equivalent dosage of vehicle) with 6 rats in each group. After a 6-week treatment ended, the serum levels of estradiol (E₂), follicle stimulating hormone (FSH), luteinizing hormone (LH) were measured by using commercial standard assay kits. The expression levels of FSH receptor (FSHR) in the rat ovarian tissues were respectively detected by immunohistochemistry and Real-time PCR.

Results

CGE treatment markedly increased E₂ levels and decreased FSH levels in the serum (P<0.05), however, the alterations of serum LH levels were not significant (P>0.05). The protein and mRNA expression levels of FSHR were the lowest in the ovaries of control rats and the highest in BPA rats (P<0.05). CGE treatment markedly decreased the expression levels of FSHR in the ovarian tissues (P<0.05).

Conclusions

Crassostrea gigas successfully alleviates ovarian functional disorders of female rats with exposure to BPA partly through decreasing FSHR expression levels in the ovarian tissues.     

Genetic polymorphisms in the Toll-like receptor signalling pathway in Helicobacter pylori infection and related gastric cancer

Background

Gastric cancer (GC) is a progressive process initiated by Helicobacter pylori-induced inflammation. Initial recognition of H. pylori involves Toll-like receptors (TLRs), central molecules in the host inflammatory response. Here, we investigated the association between novel polymorphisms in genes involved in the TLR signalling pathway, including TLR2, TLR4, LBP, MD-2, CD14 and TIRAP, and risk of H. pylori infection and related GC.

Methods

A case-control study comprising 310 ethnic Chinese individuals (87 non-cardia GC cases and 223 controls with functional dyspepsia) was conducted. Twenty-five polymorphisms were detected by MALDI-TOF mass spectrometry, PCR, PCR–RFLP and real-time PCR.

Results

Seven polymorphisms showed significant associations with GC (TLR4 rs11536889, TLR4 rs10759931, TLR4 rs1927911, TLR4 rs10116253, TLR4 rs10759932, TLR4 rs2149356 and CD14 −260 C/T). In multivariate analyses, TLR4 rs11536889 remained a risk factor for GC (OR: 3.58, 95% CI: 1.20–10.65). TLR4 rs10759932 decreased the risk of H. pylori infection (OR: 0.59, 95% CI: 0.41–0.86). Statistical analyses assessing the joint effect of H. pylori infection and the selected polymorphisms revealed strong associations with GC (TLR2, TLR4, MD-2, LBP and TIRAP polymorphisms).

Conclusions

Novel polymorphisms in TLR2, TLR4, MD-2, LBP, CD14 and TIRAP, genes encoding important molecules of the TLR signalling pathway, showed clear associations with H. pylori-related GC in Chinese.