The Expression of Toll-like Receptors (TLR7 and TLR9) in Class III and Class IV of Recently Diagnosed Lupus Nephritis with 12-Month Follow-Up.

Renal involvement is an important cause of morbidity and mortality in systemic lupus erythematosus (SLE). The present study included patients with recently diagnosed Class III and Class IV lupus nephritis (LN) treated by Rheumatology who, upon the detection of alterations in their kidney function, were referred to Nephrology for the joint management of both medical specialties. The purpose of this study was to compare the plasma expression of Toll-Like Receptor 7 (TLR7) and TLR9 in healthy control (HC) subjects and newly diagnosed Class III and Class IV LN patients with 12-month follow-ups. The plasma expression of TLR7 and TLR9 proteins was determined by the ELISA method. A significant increase in the expression of TLR7 protein was found in Class III LN in the basal determination compared to the expression in the HC (p = 0.002) and at 12 months of follow-up (p = 0.03) vs. HC. The expression of TLR9 showed a behavior opposite to that of TLR7. TLR9 showed decreased protein expression in LN Class III patients' baseline and final measurements. The result was similar in the basal and final determinations of LN Class IV compared to the expression in HC. A significant decrease in SLEDAI -2K was observed at 12 months of follow-up in patients in Class III (p = 0.01) and Class IV (p = 0.0001) of LN. Complement C3 levels improved significantly at 12-month follow-up in Class IV patients (p = 0.0001). Complement C4 levels decreased significantly at 12-month follow-up in LN Class III compared to baseline (p = 0.01). Anti-DNA antibodies decreased significantly at 12 months of follow-up in Class IV LN (p = 0.01). A significant increase in proteinuria was found at 12 months of follow-up in Class III LN, compared to the baseline determination (p = 0.02). In LN Class IV, proteinuria decreased at 12 months of follow-up compared to baseline (p = 0.0001). Albuminuria decreased at 12 months of follow-up in LN Class IV (p = 0.006). Class IV LN, albuminuria also decreased at 12 months of follow-up (p = 0.009). Hematuria persisted in all patients and the glomerular filtration rate did not change. Three Class IV patients died before 12 months of follow-up from various causes. In conclusion, although the rheumatologic data appeared to improve, the renal function data remained inconsistent. Decreased expression of TLR9 and increased expression of TLR7 could be useful in the early diagnosis of Class III and Class IV LN is correct.

TLR9 activation is required for cytotoxic response elicited by baculovirus capsid display.

Although the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects lepidopteran invertebrates as natural hosts, represents an efficient vector for vaccine development. Baculovirus surface display induces strong humoral responses against viruses and parasites. A novel strategy based on capsid display carrying foreign antigens in the AcMNPV particle further improved the immune response by eliciting CD8+ T cell activation. In this study, we analyze the intracellular mechanisms and signalling pathways involved in CD8+ T cell activation by capsid display. Our results show that baculovirus can attach to the cell surface, enter dendritic cells (DCs), transit within endocytic vesicles and escape to the cytosol for further degradation by the proteasome. We found that the availability of viral proteins, endosomal acidification, and proteasome activity are needed for efficient Major Histocompatibility Complex class-I presentation by baculovirus carrying Ovalbumin in the viral capsid. Importantly, we demonstrated with this strategy that the induction of cytotoxic T cells and IL-12 production by DCs are TLR9-dependent and STING-independent. Finally, our study shows differential intracellular processing for capsid and surface baculovirus proteins in DCs and highlights the role of different danger receptors during cytotoxic T cell priming through the capsid display delivery system, which could lead to improved baculovirus-based vaccines development.

Oral squamous cell carcinoma (OSCC) tumors from heavy alcohol consumers are associated with higher levels of TLR9 and a particular immunophenotype: Impact on patient survival.

Oral squamous cell carcinoma (OSCC) is one of the most frequent types of oral cancer in developing countries and its burden correlates with exposure to tobacco and excessive alcohol consumption. Toll like receptors (TLRs) are major sensors of inflammatory stimuli, from both microbial and sterile causes and as such, they have been related to tumor progression and metastasis. Here, we evaluated the expression of TLR2, 4 and 9 as well as CD3+, CD8+ and Granzyme B+ cell infiltration by immunohistochemistry in oral samples of 30 patients with OSCC, classified according to their consumption of alcohol. Our findings indicate that there is a significant association between heavy alcohol consumption and tumors with higher expression levels of TLR9. Moreover, patients with TLR9high tumors, as well as those who indicated high consumption of alcohol exhibited a diminished overall survival. TCGA data analysis indicated that TLR9high tumors express a significant increase in some genes related with the oral cavity itself, inflammation and tumor promotion. Our analysis of tumor infiltrating leukocytes demonstrated that the major differences perceived in heavy alcohol consumers was the location of CD8+ T cells infiltrating the tumor, which showed lower numbers intratumorally. Our data suggest the existence of a pathogenic loop that involves alcohol consumption, high TLR9 expression and the immunophenotype, which might have a profound impact on the progression of the disease.

TLR9 2848 G/A Gene Polymorphism in HCV+, HIV+, and HCV+/HIV+ Individuals.

Background: Host genetic factors play a major role with respect to susceptibility to infections. Many polymorphisms of the Toll-like receptors (TLRs), members of the innate immune response, are directly associated with the clinical outcomes following infection. The 2848 G/A variant (rs352140) of the TLR9 gene is associated with increased TLR9 expression. However, the impact of the genotypes of this SNP on HIV+, HCV+, and HCV+/HIV+ individuals is still debated. Materials and Methods: This study investigated the 2848 G/A polymorphism in HCV infection, HIV infection, and HCV/HIV co-infection in a large sample of Brazilians (n = 1,182). Groups were initially compared without considering stratification by ethnicity and subsequently stratifying individuals between whites and non-whites. Results: Considering non-white individuals, a significant difference between the HIV+/HCV+ group and controls was observed with the GG genotype serving as a protective factor (p = 0.023). Additionally, significant allelic differences were observed between the HCV+ group and controls (p = 0.042); between the HIV+/HCV+ group and controls (p = 0.011); and between the HIV+/HCV+ group and HIV+ individuals (p = 0.047). However, all significant results were lost following adjustment for multiple comparisons (p > 0.05). Conclusion: Although our initial results indicated a potential influence of the rs352140 genotype on host altered susceptibility to viral infections, following correction for multiple comparisions the standard (p < 0.05) for statistical association was lost. This may be due to insufficient sample size as we were examining many different associations. Thus, a larger study is warranted to further pursue this topic.

TLR-9 Plays a Role in Mycobacterium leprae-Induced Innate Immune Activation of A549 Alveolar Epithelial Cells.

The respiratory tract is considered the main port of entry of Mycobacterium leprae, the causative agent of leprosy. However, the great majority of individuals exposed to the leprosy bacillus will never manifest the disease due to their capacity to develop protective immunity. Besides acting as a physical barrier, airway epithelium cells are recognized as key players by initiating a local innate immune response that orchestrates subsequent adaptive immunity to control airborne infections. However, to date, studies exploring the interaction of M. leprae with the respiratory epithelium have been scarce. In this work, the capacity of M. leprae to immune activate human alveolar epithelial cells was investigated, demonstrating that M. leprae-infected A549 cells secrete significantly increased IL-8 that is dependent on NF-κB activation. M. leprae was also able to induce IL-8 production in human primary nasal epithelial cells. M. leprae-treated A549 cells also showed higher expression levels of human ß-defensin-2 (hßD-2), MCP-1, MHC-II and the co-stimulatory molecule CD80. Furthermore, the TLR-9 antagonist inhibited both the secretion of IL-8 and NF-κB activation in response to M. leprae, indicating that bacterial DNA sensing by this Toll-like receptor constitutes an important innate immune pathway activated by the pathogen. Finally, evidence is presented suggesting that extracellular DNA molecules anchored to Hlp, a histone-like protein present on the M. leprae surface, constitute major TLR-9 ligands triggering this pathway. The ability of M. leprae to immune activate respiratory epithelial cells herein demonstrated may represent a very early event during infection that could possibly be essential to the generation of a protective response.

TLR4 and TLR9 polymorphisms are not associated with either rheumatoid arthritis or systemic lupus erythematosus in Mexican patients.

Toll-like receptor (TLR)-mediated signaling pathways induce a proinflammatory microenvironment to eradicate pathogens. However, in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), TLRs can promote chronic inflammation. It has been shown that some TLR4 and TLR9 single nucleotide polymorphisms (SNPs) are risk factors for RA and SLE, but these findings have not been replicated in all populations; thus, results are inconclusive. We evaluated the TLR4 Asp299Gly, Thr399Ile, - 1892G/A SNPs, and the TLR9 Pro545Pro SNP to assess potential associations with RA and SLE in Mexican patients. This study included 474 patients with RA, 283 patients with SLE, and 424 healthy controls. We used a 5' nuclease allelic discrimination assay to genotype individuals for the four TLR4 and TLR9 polymorphisms. We found that the genotype or allelic frequencies of the TLR4 Asp299Gly, Thr399Ile, - 1892G/A, and TLR9 Pro545Pro polymorphisms were similar between patients and controls. We found no association under different genetic models. A haplotype analysis of TLR4 showed no association with either RA or SLE. We found no significant differences in the allelic or genotypic frequencies of TLR4 Asp299Gly, Thr399IIe, - 1892G/A, or TLR9 Pro545Pro between patients and controls. These findings suggested that these variants are not risk factors for RA or SLE in Mexican patients.

TLR-9 Plays a Role in Mycobacterium leprae-Induced Innate Immune Activation of A549 Alveolar Epithelial Cells

The respiratory tract is considered the main port of entry of Mycobacterium leprae, the causative agent of leprosy. However, the great majority of individuals exposed to the leprosy bacillus will never manifest the disease due to their capacity to develop protective immunity. Besides acting as a physical barrier, airway epithelium cells are recognized as key players by initiating a local innate immune response that orchestrates subsequent adaptive immunity to control airborne infections. However, to date, studies exploring the interaction of M. leprae with the respiratory epithelium have been scarce. In this work, the capacity of M. leprae to immune activate human alveolar epithelial cells was investigated, demonstrating that M. leprae-infected A549 cells secrete significantly increased IL-8 that is dependent on NF-kB activation. M. leprae was also able to induce IL-8 production in human primary nasal epithelial cells. M. leprae-treated A549 cells also showed higher expression levels of human b-defensin-2 (hbD-2), MCP-1, MHC-II and the co-stimulatory molecule CD80. Furthermore, the TLR-9 antagonist inhibited both the secretion of IL-8 and NF-kB activation in response to M. leprae, indicating that bacterial DNA sensing by this Toll-like receptor constitutes an important innate immune pathway activated by the pathogen. Finally, evidence is presented suggesting that extracellular DNA molecules anchored to Hlp, a histone-like protein present on the M. leprae surface, constitute major TLR-9 ligands triggering this pathway. The ability of M. leprae to immune activate respiratory epithelial cells herein demonstrated may represent a very early event during infection that could possibly be essential to the generation of a protective response.(AU)

DNA from virulent M. tuberculosis induces TNF-α production and autophagy in M1 polarized macrophages.

The macrophage innate immune response is outlined through recognition of the components of Mycobacterium tuberculosis. DNA of M. tuberculosis (MtbDNA) is recognized by macrophages, but the implications of this recognition are poorly characterized. Stimulation of murine macrophages with MtbDNA induces autophagy, a process that promotes elimination of intracellular pathogens. However, it remains unknown whether this or other phenomena also occur in human cells. In this work, we studied the innate response profiles of human macrophages after stimulation with DNA from virulent M. tuberculosis H37Rv. Human monocyte-derived macrophages were polarized into M1 and M2 phenotypes and stimulated with MtbDNA. The plasma membrane markers of the phenotype, production of TNF-α, and induction of autophagy were evaluated. Our results indicate that MtbDNA induced phenotypical changes, the significant production of TNF-α, and autophagy confirmed by the augmented expression of immunity related GTPase M (IRGM) and autophagy related ATG16L1 genes in M1 macrophages, whereas M2 macrophages exhibited limited responses. In addition, MtbDNA activation was TLR-9-dependent. Although TLR-9 expression was similar between M1 and M2 macrophages, only M1 macrophages were fully responsive to MtbDNA. In conclusion, MtbDNA recognition enhanced the antimicrobial mechanisms of M1 macrophages.

TLR9 Signaling Suppresses the Canonical Plasma Cell Differentiation Program in Follicular B Cells.

The relative potency and quality of mouse B cell response to Toll-like receptors (TLRs) signaling varies significantly depending on the B cell subset and on the TLR member being engaged. Although it has been shown that marginal zone cells respond faster than follicular (FO) splenic B cells to TLR4 stimulus, FO B cells retain full capacity to proliferate and generate plasmablasts and plasma cells (PBs/PCs) with 2-3 days delayed kinetics. It is not clear whether this scenario could be extended to other members of the TLR family. Here, using quantitative cell culture conditions optimized for B cell growth and differentiation, we show that TLR9 signaling by CpG, while promoting vigorous proliferation, completely fails to induce differentiation of FO B cells into PBs/PCs. Little or absent Ig secretion following TLR9 stimulus was accompanied by lack of expression of cell surface markers and canonical transcription factors involved in PB/PC differentiation. Moreover, not only TLR9 did not induce plasmocyte differentiation, but it also strongly inhibited the massive PB/PC differentiation of FO B cells triggered by LPS/TLR4. Our study reveals unexpected opposite roles for TLR4 and TLR9 in the control of plasma cell differentiation program and disagrees with previous conclusions obtained in high-density cultures conditions on the generation of plasmocytes by TRL9 signaling. The potential implications of these findings on the role of TLR9 in controlling self-tolerance, clonal sizes and regulation of humoral responses are discussed.

Vaccination With Recombinant Filamentous fd Phages Against Parasite Infection Requires TLR9 Expression.

Recombinant filamentous fd bacteriophages (rfd) expressing antigenic peptides were shown to induce cell-mediated immune responses in the absence of added adjuvant, being a promising delivery system for vaccination. Here, we tested the capacity of rfd phages to protect against infection with the human protozoan Trypanosoma cruzi, the etiologic agent of Chagas Disease. For this, C57BL/6 (B6) and Tlr9-/- mice were vaccinated with rfd phages expressing the OVA257-264 peptide or the T. cruzi-immunodominant peptides PA8 and TSKB20 and challenged with either the T. cruzi Y-OVA or Y-strain, respectively. We found that vaccination with rfd phages induces anti-PA8 and anti-TSKB20 IgG production, expansion of Ag-specific IFN-γ, TNF-α, and Granzyme B-producing CD8+ T cells, as well as in vivo Ag-specific cytotoxic responses. Moreover, the fd-TSKB20 vaccine was able to protect against mortality induced by a high-dose inoculum of the parasite. Although vaccination with rfd phages successfully reduced both parasitemia and parasite load in the myocardium of WT B6 mice, Tlr9-/- animals were not protected against infection. Thus, our data extend previous studies, demonstrating that rfd phages induce Ag-specific IgG and CD8+ T cell-mediated responses and confer protection against an important human parasite infection, through a TLR9-dependent mechanism.

Class B CpG-ODN2006 is highly associated with IgM and antimicrobial peptide gene expression through TLR9 pathway in yellowtail Seriola lalandi.

The purpose of this study was to characterize the TLR9 gene from yellowtail (Seriola lalandi) and evaluate its functional activity using the class B Cytosine-phosphate-guanine-oligodeoxynucleotide2006 (CpG-ODN2006) in an in vivo experiment after one-week immunostimulation. The gene expressions of TLR9, Immunoglobulin M (IgM), antimicrobial peptides and cytokines were evaluated by real time PCR, and humoral immune parameters were analyzed in serum. The TLR9 nucleotide sequence from yellowtail was obtained using the whole-genome shotgun sequencing method and bioinformatics tools. The yellowtail full-length cDNA sequence of SlTLR9 was 3789 bp in length, including a 66-bp 5'-untranslated region (UTR), a 3'-UTR of 528 bp, and an open reading frame (ORF) of 3192 bp translatable to 1064 amino acid showing a high degree of similarity with the counterparts of other fish species and sharing common structural architecture of the TLR family, including LRR domains, one C-terminal LRR region, and a TIR domain. Gene expression studies revealed the constitutive expression of TLR9 mRNA in all analyzed tissues; the highest levels were observed in intestine, liver and spleen where they play an important role in the fish immune system. The expression levels of TLR9 after B class CpG-ODN2006 (the main TLR9-agonist) was significantly up-regulated in all analyzed tissues, with the high expression observed in spleen followed by intestine and skin. The CpG-B has been shown as a potent B cell mitogen, and interestingly, IgM mRNA transcript was up-regulated in spleen and intestine, which was highly correlated with TLR9 after CpG-ODN2006 stimulation. The antimicrobial peptides, piscidin and NK-lysine, were up-regulated in spleen and gill after CpG-ODN2006 injection with a high correlation (r ≥ 0.82) with TLR9 gene expression. Cytokine genes were up-regulated in spleen, intestine and skin after CpG-ODN was compared with the control group. No significant correlation was observed between TLR9 and IL-1ß, TNF-α and Mx gene expressions. The results showed that CpG-ODN2006 intraperitoneal injection enhanced lysozyme, peroxidase and superoxide dismutase activities in serum and demonstrated that CpG-ODN2006 can induce a specific immune response via TLR9 in which IgM and antimicrobial peptides must have an important role in the defense mechanisms against infections in yellowtail.

TLR9 gene polymorphism -1486T/C (rs187084) is associated with uterine cervical neoplasm in Mexican female population.

PURPOSE: The aim of this work was to evaluate the association of single nucleotide polymorphisms in TLR9 (-1486 T/C [rs187084], -1237T/C [rs5743836] and G2848A [rs352140]) with HPV infection, squamous intraepithelial lesions, and uterine cervical neoplasm in a Mexican population. Additionally, the peripheral expression of TLR9 was evaluated to evaluate the differences in the TLR9 expression associated with every genotype in the locus -1486 of the TLR9 gene. The serum concentration of TLR9 was evaluated in a randomly selected subsample. METHODS: Genotyping was performed using predesigned 5' endonuc lease assays and the association of the polymorphisms with the diagnosis groups were assessed by performing multinomial regression models. The relative expression of TLR9 in peripheral blood mononuclear cells was evaluated by real-time polymerase chain reaction and the association of the level of TLR9 expression with the diagnosis was evaluated by performing multinomial regression models. The serum concentration of TLR9 was evaluated in a subsample of patients diagnosed with uterine cervical neoplasm by ELISA. RESULTS: The results showed that genotype TT in the -1486 locus of TLR9 was significantly associated with HPV infection (OR = 3.25, 95% CI 1.12-9.46), squamous intraepithelial cervical lesion (OR = 3.76, 95% CI 1.36-10.41), and uterine cervical neoplasm (OR = 5.30, 95% CI 1.81-15.55). Moreover, the highest level of TLR9 expression was significantly associated with a greater risk for developing squamous intraepithelial cervical lesion and uterine cervical neoplasm. The serum TLR9 concentration was higher in patients with uterine cervical cancer than in controls. CONCLUSION: Our findings indicate that genotype TT in the -1486 locus of the TLR9 gene could comprise a risk genotype for HPV infection, squamous intraepithelial cervical lesion, and uterine cervical neoplasm in Mexican female population. Further studies with larger samples are needed to evaluate if the peripheral expression of TLR9 could be used as a biomarker of uterine cervical neoplasm progression.

Role of TLR9 in Oncogenic Virus-Produced Cancer.

Nucleic acid recognition by toll-like receptor 9 (TLR9) initiates signaling pathways that regulate the production of proinflammatory cytokines or type I interferons, as well as many other molecules required to initialize the immune response. The use of synthetic oligodeoxynucleotides (ODNs) has been crucial to emulate the recognition of DNA sequences by TLR9. Furthermore, ODN administration to mice has shown to confer protection against a wide range of viral, bacterial, and parasitic pathogens. In contrast, oncogenic DNA viruses like hepatitis B virus, Epstein-Barr virus, and human papilloma virus inhibit TLR9 expression, thus contributing to the establishment of chronic viral infections. In this review, we will focus on TLR9 signals initiated by ODN recognition, on the inhibition of TLR9 expression mediated by DNA oncogenic viruses, and on TLR9 expression as a relevant event in the progression to cancer, considering other functions of this receptor, aside from viral recognition.

The role of variants from the innate immune system genes in tuberculosis and skin test response in a Native American population.

Native American populations show higher tuberculosis (TB) mortality and infectivity rates than non-Native populations. Variants in the innate immune system seem to have an important role on TB susceptibility. The role of some innate immune system variants in TB susceptibility and/or skin test response (PPD) were investigated in the Aché, a Native American population. Complement receptor 1 and toll like receptor 9 variants were associated with anergy to PPD and protection to TB, respectively. These findings demonstrate an important role of the innate immune system variants in TB susceptibility.

TLR 9 involvement in early protection induced by immunization with rPb27 against Paracoccidioidomycosis.

Paracoccidioidomycosis is caused by fungi of the Paracoccidioides genus and constitutes the most prevalent deep mycosis in Latin America. Toll-like receptors promote immune response against infectious agents. Recently, it was reported that TLR9 is crucial for mice survival during the first 48 h of P. brasiliensis infection. In this study, we used CPG oligodeoxynucleotide motif as an adjuvant with and without rPb27 to immunize mice against Paracoccidioidomycosis. CPG adjuvant induced differential recruitment of lymphocytes in the inflammatory process and a lower recruitment of neutrophils. In addition, CPG induced the production of pro-inflammatory cytokines such as IL-1ß, TNF-α, IL-6 and IL-12; increased phagocytic ability and microbicidal activity by macrophages; and induced differential production of lgG2a and lgG2b, subtypes of Ig. Knockout mice for TLR9 and IL-12 showed higher fungal loads and rates of mortality compared to control mice after 30 days of infection. The association between CPG and rPb27 induced a high level of protection against Paracoccidioidomycosis after the first 30 days of infection but not at 60 days. Our findings demonstrate that TLR 9 plays a role in the protection induced by immunization with rPb27 and confirms the importance of TLR9 in the initial protection against Paracoccidioidomycosis.

Attenuation of experimental asthma by mycobacterial protein combined with CpG requires a TLR9-dependent IFN-γ-CCR2 signalling circuit.

BACKGROUND: Allergic asthma is a chronic pulmonary disease characterized by a Th2 inflammatory response. The modulation of a Th2 immune response based on immune deviation to a Th1 pattern or induction and migration of regulatory T cells to the lungs constitutes one of the major therapeutic approaches that is being investigated for the treatment of allergic asthma. The potentials of Mycobacterium leprae 65-kD heat-shock protein or Toll-like receptor 9 ligand (CpG oligodeoxynucleotides) as immune modulators for the treatment of airway allergic disease have been studied individually. OBJECTIVE: Mycobacterial protein combined with CpG was used as immunotherapy for airway allergy. METHODS: Using an ovalbumin-induced asthma model, mice were sensitized and challenged, and then treated with mycobacterial heat-shock protein (Hsp65) combined with CpG. RESULTS: The treatment of mice with established allergy led to the attenuation of eosinophilia, Th2 cytokines and airway hyperresponsiveness. Hsp65 plus CpG treatment also induced an increase in OVA-specific IFN-γ levels and in the frequency of lung inflammatory monocytes. Moreover, we show that the reduction of eosinophilia and the recruitment of inflammatory monocytes to the lungs required early triggering of TLR9, IFN-γ and CCR2 by immunotherapy components. CONCLUSION: In addition to immune deviation to a Th1 response in the modulation of Th2 allergic inflammation, our findings also attribute an important role to the innate response mediated by TLR9, associated with the recruitment of CCR2-dependent monocytes. CLINICAL RELEVANCE: Our findings show that the Hsp65/CpG treatment is a promising strategy for consideration in translational studies.

Humoral immune response and TLR9 gene expression in Pacific red snapper (Lutjanus peru) experimentally exposed to Aeromonas veronii.

Aquaculture production of Pacific red snapper Lutjanus peru is growing rapidly in Mexico, especially in Gulf of California. As it is a relatively new aquaculture species there are few reports evaluating its immune response to pathogens. The Gram-negative bacteria Aeromonas veronii is a heterogeneous organism that causes the disease known as motile aeromonad septicemia, which is responsible for serious economic loss in seabream culture due to bacterial infections. For the purpose of this study, juvenile Pacific red snapper specimens were intraperitoneally injected with low doses of A. veronii (1 × 10(6) CFU ml(-1)). Changes in humoral immune parameters (total protein, myeloperoxidase, lisozyme and antiprotease activities and IgM levels), as well as superoxide dismutase and catalase activities, and TLR9 gene expression were evaluated 24 and 48 h after injection. Overall, the results showed an enhanced in humoral immune parameters and SOD and CAT activities in fish infected with A. veronii compared with control group at 24 or 48 h. By real time PCR assays, the basal mRNA transcripts of TLR9 showed that were highly expressed in intestine and leucocytes compared to skin, head kidney, liver and gill. Then, the mRNA expression levels of TLR9 in head kidney, skin, liver and intestine were analyzed in non-infected and experimentally infected fish 24 and 48 h after injection. A. veronii up-regulated the expression of TLR9 at 24 or 48 h of exposure in all samples analyzed except in liver. Interestingly, intestine produced the greatest increase in transcript levels upon exposure (48 h) to A. veronii. Taken together, our results suggest that low doses of A. veronii infection inducing humoral immune system and TLR9 immune gene in Pacific red snapper that can be useful in the health control of this species.

Evaluation of Toll-like, chemokine, and integrin receptors on monocytes and neutrophils from peripheral blood of septic patients and their correlation with clinical outcomes

Recognition of pathogens is performed by specific receptors in cells of the innate immune system, which may undergo modulation during the continuum of clinical manifestations of sepsis. Monocytes and neutrophils play a key role in host defense by sensing and destroying microorganisms. This study aimed to evaluate the expression of CD14 receptors on monocytes; CD66b and CXCR2 receptors on neutrophils; and TLR2, TLR4, TLR5, TLR9, and CD11b receptors on both cell types of septic patients. Seventy-seven septic patients (SP) and 40 healthy volunteers (HV) were included in the study, and blood samples were collected on day zero (D0) and after 7 days of therapy (D7). Evaluation of the cellular receptors was carried out by flow cytometry. Expression of CD14 on monocytes and of CD11b and CXCR2 on neutrophils from SP was lower than that from HV. Conversely, expression of TLR5 on monocytes and neutrophils was higher in SP compared with HV. Expression of TLR2 on the surface of neutrophils and that of TLR5 on monocytes and neutrophils of SP was lower at D7 than at D0. In addition, SP who survived showed reduced expression of TLR2 and TLR4 on the surface of neutrophils at D7 compared to D0. Expression of CXCR2 for surviving patients was higher at follow-up compared to baseline. We conclude that expression of recognition and cell signaling receptors is differentially regulated between SP and HV depending on the receptor being evaluated.

El ADN de Leishmania mexicana activa al macrófago murino e induce aumento en la expresión de TLR9 / Leishmania mexicana DNA activates murine macrophages and increases their TLR9 expression

Antecedentes: Los macrófagos son células de la respuesta inmune que reconocen patrones moleculares asociados a patógenos (PAMP) mediante receptores presentes en la superficie de la célula como en compartimentos intracelulares, como los TLR (toll like receptors). Distintos TLR reconocen ligandos que comparten múltiples patógenos. La unión de TLR con su ligando desencadena una cascada de señalización que termina en la producción de citocinas y moléculas coestimuladoras a través de la translocación de NF-κB al núcleo. Nuestro grupo demostró que el lipofosfoglucano de Leishmania es un ligando de TLR2 que activa células NK. Schieicher y cols.12 informo recientemente la activación de células dendríticas plasmacitoides con ADN genómico de Leishmania infantum a través de TLR9, con alta producción de IFN tipo I. Objetivo: En el presente trabajo exploramos si el ADN de Leishmania mexicana contiene motivos CpG no metilados capaces de activar al macrófago murino derivado de médula ósea, como ha sido descrito anteriormente para motivos CpG no metilados de ADN bacteriano. Resultados y conclusiones: Encontramos que el ADN de Leishmania mexicana posee motivos CpG no metilados que activan macrófagos murinos de la cepa BALB/c, llevando a la producción de citocinas proinflamatorias como TNFα e IL12P40 y a la sobreexpresión del mARN de TLR9.

BACKGROUND: Macrophages are immune system cells that recognize pathogen associated molecular patterns (PAMPs) through receptors that can be located on the cell membrane or in intracellular compartments, such as the TLR (toll like receptors). Different TLRs bind to ligands shared among multiple pathogens. The binding of ligands to TLRs induces a signaling cascade that leads to cytokine and co-stimulatory molecule production due to the nuclear translocation of NF-kappaB. We demonstrated that Leishmania lipophosphoglycan (LPG) is a ligand for TLR2, leading to NK-cell activation. Schieicher et al. recently reported that genomic DNA from Leishmania infantum activates plasmacitoid dendritic cells through TLR9, leading to IFN type I production. OBJECTIVE: In the present study we explored wether Leishmania mexicana DNA contained non-methylated CpG motifs able to activate murine bone marrow derived macrophages, as previously described for bacterial DNA containing CpG motifs. RESULTS AND CONCLUSIONS: We observed that Leishmania mexicana DNA contains non-methylated CpG morifs able ofactivating murine bone marrow derived macrophages, leading to the production of proinflammatory cytokines such as TNFalpha and IL- 12(P40) as well as the over expression of mRNA for TLR9.