Development of a novel in vitro model to study the modulatory role of the respiratory complex I in macrophage effector functions.

Increasing evidence demonstrate that the electron transfer chain plays a critical role in controlling the effector functions of macrophages. In this work, we have generated a Ndufs4-/- murine macrophage cell lines. The Ndufs4 gene, which encodes a supernumerary subunit of complex I, is a mutational hotspot in Leigh syndrome patients. Ndufs4-/- macrophages showed decreased complex I activity, altered complex I assembly, and lower levels of maximal respiration and ATP production. These mitochondrial respiration alterations were associated with a shift towards a pro-inflammatory cytokine profile after lipopolysaccharide challenge and improved ability to phagocytose Gram-negative bacteria.

Novel fluorescent-based reporter cell line engineered for monitoring homologous recombination events.

Homologous recombination (HR) faithfully restores DNA double-strand breaks. Defects in this HR repair pathway are associated with cancer predisposition. In genetic engineering, HR has been used extensively to study gene function and it represents an ideal method of gene therapy for single gene disorders. Here, we present a novel assay to measure HR in living cells. The HR substrate consisted of a non-fluorescent 3' truncated form of the eGFP gene and was integrated into the AAVS1 locus, known as a safe harbor. The donor DNA template comprised a 5' truncated eGFP copy and was delivered via AAV particles. HR mediated repair restored full-length eGFP coding sequence, resulting in eGFP+ cells. The utility of our assay in quantifying HR events was validated by exploring the impact of the overexpression of HR promoters and the siRNA-mediated silencing of genes known to play a role in DNA repair on the frequency of HR. We conclude that this novel assay represents a useful tool to further investigate the mechanisms that control HR and test continually emerging tools for HR-mediated genome editing.

The Mitochondrial Isoform of FASTK Modulates Nonopsonic Phagocytosis of Bacteria by Macrophages via Regulation of Respiratory Complex I.

Phagocytosis is a pivotal process by which innate immune cells eliminate bacteria. In this study, we explore novel regulatory mechanisms of phagocytosis driven by the mitochondria. Fas-activated serine/threonine kinase (FASTK) is an RNA-binding protein with two isoforms, one localized to the mitochondria (mitoFASTK) and the other isoform to cytosol and nucleus. The mitoFASTK isoform has been reported to be necessary for the biogenesis of the mitochondrial ND6 mRNA, which encodes an essential subunit of mitochondrial respiratory complex I (CI, NADH:ubiquinone oxidoreductase). This study investigates the role and the mechanisms of action of FASTK in phagocytosis. Macrophages from FASTK─/─ mice exhibited a marked increase in nonopsonic phagocytosis of bacteria. As expected, CI activity was specifically reduced by almost 50% in those cells. To explore if decreased CI activity could underlie the phagocytic phenotype, we tested the effect of CI inhibition on phagocytosis. Indeed, treatment with CI inhibitor rotenone or short hairpin RNAs against two CI subunits (NDUFS3 and NDUFS4) resulted in a marked increase in nonopsonic phagocytosis of bacteria. Importantly, re-expression of mitoFASTK in FASTK-depleted macrophages was sufficient to rescue the phagocytic phenotype. In addition, we also report that the decrease in CI activity in FASTK─/─ macrophages is associated with an increase in phosphorylation of the energy sensor AMP-activated protein kinase (AMPK) and that its inhibition using Compound C reverted the phagocytosis phenotype. Taken together, our results clearly demonstrate for the first time, to our knowledge, that mitoFASTK plays a negative regulatory role on nonopsonic phagocytosis of bacteria in macrophages through its action on CI activity.

The phosphatidic acid phosphatase lipin-1 facilitates inflammation-driven colon carcinogenesis.

Colon cancer is a devastating illness that is associated with gut inflammation. Here, we explored the possible role of lipin-1, a phosphatidic acid phosphatase, in the development of colitis-associated tumorigenesis. Azoxymethane and dextran sodium sulfate-treated (DSS-treated) animals deficient in lipin-1 harbored fewer tumors and carcinomas than WT animals due to decreased cellular proliferation, lower expression of antiapoptotic and protumorigenic factors, and a reduced infiltration of macrophages in colon tumors. They also displayed increased resistance to DSS-induced colitis by producing less proinflammatory cytokines and experiencing less immune infiltration. Lipin-1-deficient macrophages from the colon were less activated and displayed lower phosphatidic acid phosphatase activity than WT macrophages isolated from DSS-treated animals. Transference of WT macrophages into lipin-1-deficient animals was sufficient to increase colitis burden. Furthermore, treatment of lipin-1-deficient mice with IL-23 exacerbated colon inflammation. Analysis of human databases from colon cancer and ulcerative colitis patients showed that lipin-1 expression is increased in those disorders and correlates with the expression of the proinflammatory markers CXCL1 and CXCL2. And finally, clinically, LPIN1 expression had prognostic value in inflammatory and stem-cell subtypes of colon cancers. Collectively, these data demonstrate that lipin-1 is a critical regulator of intestinal inflammation and inflammation-driven colon cancer development.

Role of FAST Kinase Domains 3 (FASTKD3) in Post-transcriptional Regulation of Mitochondrial Gene Expression.

The Fas-activated serine/threonine kinase (FASTK) family of proteins has recently emerged as a central regulator of mitochondrial gene expression through the function of an unusual RNA-binding domain named RAP (for RNA-binding domain abundant in Apicomplexans), shared by all six members of the family. Here we describe the role of one of the less characterized members, FASTKD3, in mitochondrial RNA metabolism. First, we show that, in contrast to FASTK, FASTKD2, and FASTKD5, FASTKD3 does not localize in mitochondrial RNA granules, which are sites of processing and maturation of mtRNAs and ribosome biogenesis. Second, we generated FASTKD3 homozygous knock-out cell lines by homologous recombination and observed that the absence of FASTKD3 resulted in increased steady-state levels and half-lives of a subset of mature mitochondrial mRNAs: ND2, ND3, CYTB, COX2, and ATP8/6. No aberrant processing of RNA precursors was observed. Rescue experiments demonstrated that RAP domain is required for FASTKD3 function in mRNA stability. Besides, we describe that FASTKD3 is required for efficient COX1 mRNA translation without altering mRNA levels, which results in a decrease in the steady-state levels of COX1 protein. This finding is associated with reduced mitochondrial complex IV assembly and activity. Our observations suggest that the function of this family of proteins goes beyond RNA processing and ribosome assembly and includes RNA stability and translation regulation within mitochondria.

Improved serodiagnosis of cystic echinococcosis using the new recombinant 2B2t antigen.

A standardized test for the serodiagnosis of human cystic echinococcosis (CE) is still needed, because of the low specificity and sensitivity of the currently available commercial tools and the lack of proper evaluation of the existing recombinant antigens. In a previous work, we defined the new ELISA-B2t diagnostic tool for the detection of specific IgGs in CE patients, which showed high sensitivity and specificity, and was useful in monitoring the clinical evolution of surgically treated CE patients. Nevertheless, this recombinant antigen gave rise to false-negative results in a percentage of CE patients. Therefore, in an attempt to improve its sensitivity, we constructed B2t-derived recombinant antigens with two, four and eight tandem repeat of B2t units, and tested them by ELISA on serum samples of CE patients and patients with related parasites. The best diagnostic values were obtained with the two tandem repeat 2B2t antigen. The influence of several clinical variables on the performance of the tests was also evaluated. Finally, the diagnostic performance of the 2B2t-ELISA was compared with that of an indirect haemagglutination commercial test. The 2B2t recombinant antigen performed better than the HF and B2t antigens, and the IHA commercial kit. Therefore, this new 2B2t-ELISA is a promising candidate test for the serodiagnosis of CE in clinical settings.

The translational repressor T-cell intracellular antigen-1 (TIA-1) is a key modulator of Th2 and Th17 responses driving pulmonary inflammation induced by exposure to house dust mite.

T-cell intracellular antigen-1 (TIA-1) is a translational repressor that dampens the production of proinflammatory cytokines and enzymes. In this study we investigated the role of TIA-1 in a mouse model of pulmonary inflammation induced by exposure to the allergenic extract (Df) of the house dust mite Dermatophagoides farinae. When intranasally challenged with a low dose of Df, mice lacking TIA-1 protein (Tia-1(-/-)) showed more severe airway and tissue eosinophilia, infiltration of lung bronchovascular bundles, and goblet cell metaplasia than wild-type littermates. Tia-1(-/-) mice also had higher levels of Df-specific IgE and IgG(1) in serum and ex vivo restimulated Tia-1(-/-) lymph node cells and splenocytes transcribed and released more Th2/Th17 cytokines. To evaluate the site of action of TIA-1, we studied the response to Df in bone marrow chimeras. These experiments revealed that TIA-1 acts on both hematopoietic and non-hematopoietic cells to dampen pulmonary inflammation. Our results identify TIA-1 as a negative regulator of allergen-mediated pulmonary inflammation in vivo. Thus, TIA-1 might be an important player in the pathogenesis of bronchial asthma.

Fas-activated serine/threonine phosphoprotein promotes immune-mediated pulmonary inflammation.

We generated Fas-activated serine threonine phosphoprotein (FAST)-deficient mice (FAST(-/-)) to study the in vivo role of FAST in immune system function. In a model of house dust mite-induced allergic pulmonary inflammation, wild type mice develop a mixed cellular infiltrate composed of eosinophils, lymphocytes, and neutrophils. FAST(-/-) mice develop airway inflammation that is distinguished by the near absence of neutrophils. Similarly, LPS-induced alveolar neutrophil recruitment is markedly reduced in FAST(-/-) mice compared with wild type controls. This is accompanied by reduced concentrations of cytokines (TNF-alpha and IL-6 and -23) and chemoattractants (MIP-2 and keratinocyte chemoattractant) in bronchoalveolar lavage fluids. Because FAST(-/-) neutrophils exhibit normal chemotaxis and survival, impaired neutrophil recruitment is likely to be due to reduced production of chemoattractants within the pulmonary parenchyma. Studies using bone marrow chimeras implicate lung resident hematopoietic cells (e.g., pulmonary dendritic cells and/or alveolar macrophages) in this process. In conclusion, our results introduce FAST as a proinflammatory factor that modulates the function of lung resident hematopoietic cells to promote neutrophil recruitment and pulmonary inflammation.

Importance of the Omp25/Omp31 family in the internalization and intracellular replication of virulent B. ovis in murine macrophages and HeLa cells.

The role of the outer membrane proteins of the Omp25/Omp31 family in invasiveness and intracellular survival of virulent B. ovis in phagocytes was analyzed. The absence of Omp25d or Omp22 in B. ovis abolished its invasive capacity in HeLa cells and reduced it in J774.A1 cells. Additionally, in J774.A1 cells, the Deltaomp25d mutant was unable to multiply, whereas the Deltaomp22 mutant was cleared at 24h post-infection. These findings demonstrate that Omp25d and Omp22 are essential for the invasion and survival of B. ovis inside host cells, and justify the strong attenuation in virulence of the Deltaomp25d and Deltaomp22 mutants.

Usefulness of four different Echinococcus granulosus recombinant antigens for serodiagnosis of unilocular hydatid disease (UHD) and postsurgical follow-up of patients treated for UHD.

Four different recombinant antigens derived from Echinococcus granulosus, designated B1t, B2t, E14t, and C317, were tested with enzyme-linked immunosorbent assays (ELISAs) for the detection of specific immunoglobulin G (IgG) in patients with unilocular hydatid disease (UHD). The results were compared to those obtained with hydatid fluid and were subjected to receiver operator characteristic analysis. The diagnostic performance of the above-listed proteins was defined with respect to their specificity, sensitivity, and predictive values (PV); the influence of cyst location; and usefulness in the follow-up of surgical treatment for UHD and in the determination of whether or not patients have been surgically cured of UHD. The best diagnostic results were obtained with the anti-B2t IgG ELISA, with 91.2% sensitivity, 93% specificity, and high positive and negative PV (89.4 and 94.2, respectively). In addition, this diagnostic tool proved to be useful for the follow-up of surgically treated UHD patients. The anti-B2t IgG ELISA may find an application in the serodiagnosis of UHD in clinical laboratories.

Francisella tularensis LPS induces the production of cytokines in human monocytes and signals via Toll-like receptor 4 with much lower potency than E. coli LPS.

Francisella tularensis is a virulent Gram-negative intracellular pathogen. To address the signaling routes involved in the response of host cells to LPS from F. tularensis live vaccine strain (LVS), experiments were performed in transiently transfected 293 cells. Induction of kappaB-driven transcriptional activity by 2.5 mug ml(-1) F. tularensis LPS isolated by phenol-water and ether-water extraction, was observed in cells transfected with Toll-like receptor (TLR) 4 and MD-2, although CD14 was required for optimal induction. Conversely, TLR2, TLR2/TLR1 or TLR2/TLR6 transfected cells did not show kappaB-driven transcriptional activity in the presence of F. tularensis LPS. In human monocytic cells, F. tularensis LPS activated extracellular signal-regulated kinases and the production of pro-inflammatory proteins. Concentrations of 5-10 mug ml(-1) F. tularensis LPS elicited a similar pattern of mRNA and protein induction than 0.1 mug ml(-1) E. coli LPS, including the expression of CXC chemokines (IL-8, Gro and IFN-gamma-inducible protein-10); CC chemokines (monocyte chemoattractant protein-1 and -2, macrophage-derived chemoattractant, macrophage inflammatory protein-1alpha and -1beta and RANTES (regulated upon activation, normal T cell expressed and secreted) and pro-inflammatory cytokines (IL-6 and tumor necrosis factor alpha). Altogether, these data indicate that LPS from F. tularensis LVS signals via TLR4 at higher concentrations than those required for E. coli LPS, which may explain the inflammatory reaction and the low endotoxic response associated to vaccination with LVS in humans.

[Seroprevalence of infection due to Echinococcus granulosus in the population of Castilla and León (Spain)]. / Seroprevalencia de infección por Echinococcus granulosus en la población de Castilla y León.

INTRODUCTION: Human hydatidosis is a prevalent zoonotic disease in the Castilla y León region of Spain. The aim of this study is to investigate the seroprevalence of Echinococcus granulosus infection in this region. METHODS: We studied 4824 serum samples from a random, representative population of healthy individuals from each province of Castilla y León, obtained over one year. An indirect enzyme-immunoassay developed in our laboratory was used to determine the presence of IgG antibodies against Echinococcus granulosus in these samples. RESULTS: IgG antibodies against Echinococcus granulosus were detected in 3.4% (164/4824) of samples studied, with a range of 1.26% to 7.10%, depending on the province. Antibody seroprevalence increased significantly with age, but there was no significant sex-related difference (3.66% men vs. 3.14% women). CONCLUSION: The seroprevalence of Echinococcus granulosus infection in Castilla y León is still high. These data contribute to hydatidosis surveillance within the control program for this disease.

Development of a dipstick dye immunoassay for diagnosing hydatidosis.

We have developed a kit to diagnose hydatidosis, based on the detection of specific antibodies. This disease, caused by larvae of the cestode Echinococcus granulosus, is a zoonosis commonly found throughout the world. The diagnostic kit discriminates between positive and negative cases in a way that is easily interpreted, i.e., positive cases form a colored band on an inert substrate. The results show test sensitivity to be some 94.87% and specificity 85.71%, which is comparable to those of the ELISA technique. The high sensitivity and specificity of this test affords the great advantage of speed in diagnosing this parasitosis. No other equipment or procedure is required.