Pulmonary exacerbations (PEs) cause significant morbidity and can severely impact disease progression in cystic fibrosis (CF) lung disease, especially in patients who suffer from recurrent PEs. The assessments able to predict a future PE or a recurrent PE are limited. We hypothesized that combining clinical, molecular and patient reported data could identify patients who are at risk of PE.
We prospectively followed a cohort of 53 adult CF patients for 24 months. Baseline values for spirometry, clinical status using the Matouk Disease Score, quality of life (QOL), inflammatory markers (C-reactive protein (CRP), interleukins (IL)-1β, -6, -8, -10, macrophage inflammatory protein (MIP)-1β, tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF)), polyunsaturated fatty acids and lipid peroxidation in blood plasma were collected for all patients during periods of stable disease, and patients were monitored for PE requiring PO/IV antibiotic treatment. Additionally, we closely followed 13 patients during PEs collecting longitudinal data on changes in markers from baseline values. We assessed whether any markers were predictors of future PE at baseline and after antibiotic treatment.
Out of 53 patients, 37 experienced PEs during our study period. At baseline, we found that low lung function, clinical scoring and QOL values were associated with increased risk of PE events. PEs were associated with increased inflammatory markers at Day 1, and these biomarkers improved with treatment. The imbalance in arachidonic acid and docosahexaenoic acid levels improved with treatment which coincided with reductions in lipid peroxidation. High levels of inflammatory markers CRP and IL-8 were associated with an early re-exacerbation.
Our results demonstrate that worse clinical and QOL assessments during stable disease are potential markers associated with a higher risk of future PEs, while higher levels of inflammatory markers at the end of antibiotic treatment may be associated with early re-exacerbation.
Tryptophan (Trp) catabolism into immunosuppressive kynurenine (Kyn) by indoleamine 2,3-dioxygenase (IDO) was previously linked to Th17/Treg differentiation and immune activation. Here we examined Trp catabolism and its impact on Th17/Treg balance in uninfected healthy subjects (HS) and a large cohort of HIV-infected patients with different clinical outcomes: ART-naïve, Successfully Treated (ST), and elite controllers (EC). In ART-naïve patients, increased IDO activity/expression, together with elevated levels of TNF-α and sCD40L, were associated with Treg expansion and an altered Th17/Treg balance. These alterations were normalized under ART. In contrast, Trp 2,3-dioxegenase (TDO) expression was dramatically lower in EC when compared to all other groups. Interestingly, EC displayed a distinctive Trp metabolism characterized by low Trp plasma levels similar to ART-naïve patients without accumulating immunosuppressive Kyn levels which was accompanied by a preserved Th17/Treg balance. These results suggest a distinctive Trp catabolism and Th17/Treg balance in HIV progressors and EC. Thus, IDO-induced immune-metabolism may be considered as a new inflammation-related marker for HIV-1 disease progression.
We previously identified Fragile X-related protein 1 (FXR1) as an RNA-binding protein involved in the post-transcriptional control of TNF and other cytokines in macrophages. Macrophages derived from FXR1-KO mice overexpress several inflammatory cytokines including TNF. Recently, we showed that fenretinide (4HPR) is able to inhibit several inflammatory cytokines in the lungs of cystic fibrosis mice, which also have abnormal immune responses. Therefore, we hypothesized that 4HPR might also be able to downregulate excessive inflammation even in macrophages with ablated FXR1. Indeed, our results demonstrate that 4HPR inhibited the excessive production of inflammatory mediators, including TNF, IL-6, CCL2 and CCL-5 in LPS-stimulated FXR1-KO macrophages, by selectively inhibiting phosphorylation of ERK1/2, which is naturally more phosphorylated in FXR1-KO cells. We also found that LPS stimulation of FXR1-KO macrophages led to significantly higher ratio of arachidonic acid/docosahexaenoic acid than observed in FXR1-WT macrophages. Interestingly, treatment with 4HPR was associated with the normalization of arachidonic acid/docosahexaenoic acid ratio in macrophages, which we found to impact phosphorylation of ERK1/2. Overall, this study shows for the first time that 4HPR modulates inflammatory cytokine expression in macrophages by correcting a phospholipid-bound fatty acid imbalance that impacts the phosphorylation of ERK1/2.
Some asthmatic individuals undergoing allergen inhalation challenge develop an isolated early response whereas others develop a dual response (early plus late response). In the present study we have used transcriptomics (microarrays) and metabolomics (mass spectrometry) of peripheral blood to identify molecular patterns that can discriminate allergen-induced isolated early from dual asthmatic responses. Peripheral blood was obtained prior to (pre-) and 2 hours post allergen inhalation challenge from 33 study participants. In an initial cohort of 14 participants, complete blood counts indicated significant differences in neutrophil and lymphocyte counts at pre-challenge between early and dual responders. At post-challenge, significant genes (ALOX15, FADS2 and LPCAT2) and metabolites (lysolipids) were enriched in lipid metabolism pathways. Enzymes encoding for these genes are involved in membrane biogenesis and metabolism of fatty acids into pro-inflammatory and anti-inflammatory mediators. Correlation analysis indicated a strong negative correlation between ALOX15, FADS2, and IL5RA expression with 2-arachidonoylglycerophosphocholine levels in dual responders. However, measuring arachidonic acid and docosahexaenoic acid levels in a validation cohort of 19 participants indicated that the free form of DHA (nmoles/µg of protein) was significantly (p = 0.03) different between early and dual responders after allergen challenge. Collectively these results may suggest an imbalance in lipid metabolism which dictates pro- (anti-) inflammatory and pro-resolving mechanisms. Future studies with larger sample sizes may reveal novel mechanisms and therapeutic targets of the late phase asthmatic response.
It is mandatory to confirm the absence of mutations in the KRAS gene before treating metastatic colorectal cancers with epidermal growth factor receptor inhibitors, and similar regulations are being considered for non-small cell lung carcinomas (NSCLC) and other tumor types. Routine diagnosis of KRAS mutations in NSCLC is challenging because of compromised quantity and quality of biological material. Although there are several methods available for detecting mutations in KRAS, there is little comparative data regarding their analytical performance, economic merits, and workflow parameters.
We compared the specificity, sensitivity, cost, and working time of five methods using 131 frozen NSCLC tissue samples. We extracted genomic DNA from the samples and compared the performance of Sanger cycle sequencing, Pyrosequencing, High-resolution melting analysis (HRM), and the Conformité Européenne (CE)-marked TheraScreen DxS and K-ras StripAssay kits.
Results and conclusions
Our results demonstrate that TheraScreen DxS and the StripAssay, in that order, were most effective at diagnosing mutations in KRAS. However, there were still unsatisfactory disagreements between them for 6.1% of all samples tested. Despite this, our findings are likely to assist molecular biologists in making rational decisions when selecting a reliable, efficient, and cost-effective method for detecting KRAS mutations in heterogeneous clinical tumor samples.
SNP - single nucleotide polymorphism; KRAS - Kiras2 kristen rat sarcoma viral oncogene homolog; NSCLC - Non-small cell lung cancer; Genotyping
The formation and storage of memories in neuronal networks relies on new protein synthesis, which can occur locally at synapses using translational machinery present in dendrites and at spines. These new proteins support long-lasting changes in synapse strength and size in response to high levels of synaptic activity. To ensure that proteins are made at the appropriate time and location to enable these synaptic changes, messenger RNA (mRNA) translation is tightly controlled by dendritic RNA-binding proteins. Fragile X Related Protein 1 (FXR1P) is an RNA-binding protein with high homology to Fragile X Mental Retardation Protein (FMRP) and is known to repress and activate mRNA translation in non-neuronal cells. However, unlike FMRP, very little is known about the role of FXR1P in the central nervous system. To understand if FXR1P is positioned to regulate local mRNA translation in dendrites and at synapses, we investigated the expression and targeting of FXR1P in developing hippocampal neurons in vivo and in vitro. We found that FXR1P was highly expressed during hippocampal development and co-localized with ribosomes and mRNAs in the dendrite and at a subset of spines in mouse hippocampal neurons. Our data indicate that FXR1P is properly positioned to control local protein synthesis in the dendrite and at synapses in the central nervous system.
Secretory leukocyte protease inhibitor (SLPI) is an anti-inflammatory protein that is observed at high levels in asthma patients. Resiquimod, a TLR7/8 ligand, is protective against acute and chronic asthma, and it increases SLPI expression of macrophages in vitro. However, the protective role played by SLPI and the interactions between the SLPI and resiquimod pathways in the immune response occurring in allergic asthma have not been fully elucidated. To evaluate the role of SLPI in the development of asthma phenotypes and the effect of resiquimod treatment on SLPI, we assessed airway resistance and inflammatory parameters in the lungs of OVA-induced asthmatic SLPI transgenic and knockout mice and in mice treated with resiquimod. Compared with wild-type mice, allergic SLPI transgenic mice showed a decrease in lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), and plasma IgE levels (p < 0.001). Allergic SLPI knockout mice displayed phenotype changes significantly more severe compared with wild-type mice. These phenotypes included lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), cytokine levels in the lungs (p < 0.05), and plasma IgE levels (p < 0.001). Treatment of asthmatic transgenic mice with resiquimod increased the expression of SLPI and decreased inflammation in the lungs; resiquimod treatment was still effective in asthmatic SLPI knockout mice. Taken together, our study showed that the expression of SLPI protects against allergic asthma phenotypes, and treatment by resiquimod is independent of SLPI expression, displayed through the use of transgenic and knockout SLPI mice.
Secretory leukocyte protease inhibitor is a serine protease inhibitor produced by various cell types, including neutrophils and activated macrophages, and has anti-inflammatory properties. It has been shown to promote wound healing in the skin and other non-neural tissues, however, its role in central nervous system injury was not known. We now report a beneficial role for secretory leukocyte protease inhibitor after spinal cord injury. After spinal cord contusion injury in mice, secretory leukocyte protease inhibitor is expressed primarily by astrocytes and neutrophils but not macrophages. We show, using transgenic mice over-expressing secretory leukocyte protease inhibitor, that this molecule has an early protective effect after spinal cord contusion injury. Furthermore, wild-type mice treated for the first week after spinal cord contusion injury with recombinant secretory leukocyte protease inhibitor exhibit sustained improvement in locomotor control and reduced secondary tissue damage. Recombinant secretory leukocyte protease inhibitor injected intraperitoneally localizes to the nucleus of circulating leukocytes, is detected in the injured spinal cord, reduces activation of nuclear factor-κB and expression of tumour necrosis factor-α. Administration of recombinant secretory leukocyte protease inhibitor might therefore be useful for the treatment of acute spinal cord injury.
spinal cord injury; neuroinflammation; wound healing; neutrophil; astrocytes; macrophage
Patients with cystic fibrosis (CF) are afflicted with many symptoms but the greatest challenge is the fight against chronic bacterial infections, leading to decreased lung function and ultimately death. Our group has recently found reduced levels of ceramides in CF patients and mice. Ceramides are sphingolipids involved in the structure of cell membranes but also participate in the inflammatory response, in cell signalling through membrane microdomains (lipid rafts), and in apoptosis. These characteristics of ceramides make them strong candidates for therapeutic intervention in CF. As more studies have come to evaluate the role of ceramide in CF, conflicting results have been described. This paper discusses various views regarding the potential role of ceramide in CF, summarizes methods of ceramide detection and their role in the regulation of cellular and molecular processes.
Secreted protein, acidic and rich in cysteine (SPARC) is a matricellular protein that mediates cell-matrix interactions. It has been shown, depending on the type of cancer, to possess either pro- or anti-tumorigenic properties. The transcriptional regulation of the SPARC gene expression has not been fully elucidated and the effects of anti-cancer drugs on this process have not been explored.
In the present study, we demonstrated that chromatin remodeling factor Brg-1 is recruited to the proximal SPARC promoter region (-130/-56) through an interaction with transcription factor Sp1. We identified Brg-1 as a critical regulator for the constitutive expression levels of SPARC mRNA and protein in mammary carcinoma cell lines and for SPARC secretion into culture media. Furthermore, we found that Brg-1 cooperates with Sp1 to enhance SPARC promoter activity. Interestingly, fenretinide [N-4(hydroxyphenyl) retinamide, 4-HPR], a synthetic retinoid with anti-cancer properties, was found to up-regulate the transcription, expression and secretion of SPARC via induction of the Brg-1 in a dose-dependent manner. Finally, our results demonstrated that fenretinide-induced expression of SPARC contributes significantly to a decreased invasion of mammary carcinoma cells.
Overall, our results reveal a novel cooperative role of Brg-1 and Sp1 in mediating the constitutive and fenretinide-induced expression of SPARC, and provide new insights for the understanding of the anti-cancer effects of fenretinide.
The KRAS gene (Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) is an oncogene that encodes a small GTPase transductor protein called KRAS. KRAS is involved in the regulation of cell division as a result of its ability to relay external signals to the cell nucleus. Activating mutations in the KRAS gene impair the ability of the KRAS protein to switch between active and inactive states, leading to cell transformation and increased resistance to chemotherapy and biological therapies targeting epidermal growth factor receptors. This review highlights some of the features of the KRAS gene and the KRAS protein and summarizes current knowledge of the mechanism of KRAS gene regulation. It also underlines the importance of activating mutations in the KRAS gene in relation to carcinogenesis and their importance as diagnostic biomarkers, providing clues regarding human cancer patients' prognosis and indicating potential therapeutic approaches.
The functional significance of nuclear translocation of β-actin remains unclear. Here, we demonstrate that PMA induces β-actin accumulation in the nucleus and binding to various target genes with different functions. We also find that accumulated nuclear β-actin is involved in recruitment of RNA polymerase II and in transcription regulation.
Studies have shown that nuclear translocation of actin occurs under certain conditions of cellular stress; however, the functional significance of actin import remains unclear. Here, we demonstrate that during the phorbol 12-myristate 13-acetate (PMA)-induced differentiation of HL-60 cells toward macrophages, β-actin translocates from the cytoplasm to the nucleus and that this process is dramatically inhibited by pretreatment with p38 mitogen-activated protein kinase inhibitors. Using chromatin immunoprecipitation-on-chip assays, the genome-wide maps of β-actin binding to gene promoters in response to PMA treatment is analyzed in HL-60 cells. A gene ontology-based analysis shows that the identified genes belong to a broad spectrum of functional categories such as cell growth and differentiation, signal transduction, response to external stimulus, ion channel activity, and immune response. We also demonstrate a correlation between β-actin occupancy and the recruitment of RNA polymerase II at six selected target genes, and β-actin knockdown decreases the mRNA expression levels of these target genes induced by PMA. We further show that nuclear β-actin is required for PMA-induced transactivation of one target gene, solute carrier family 11 member 1, which is important for macrophage activation. Our data provide novel evidence that nuclear accumulation of β-actin is involved in transcriptional regulation during macrophage-like differentiation of HL-60 cells.
Organellar acidification by the electrogenic vacuolar proton-ATPase is coupled to anion uptake and cation efflux to preserve electroneutrality. The defective organellar pH regulation, caused by impaired counterion conductance of the mutant cystic fibrosis transmembrane conductance regulator (CFTR), remains highly controversial in epithelia and macrophages. Restricting the pH-sensitive probe to CFTR-containing vesicles, the counterion and proton permeability, and the luminal pH of endosomes were measured in various cells, including genetically matched CF and non-CF human respiratory epithelia, as well as cftr+/+ and cftr−/− mouse alveolar macrophages. Passive proton and relative counterion permeabilities, determinants of endosomal, lysosomal, and phagosomal pH-regulation, were probed with FITC-conjugated transferrin, dextran, and Pseudomonas aeruginosa, respectively. Although CFTR function could be documented in recycling endosomes and immature phagosomes, neither channel activation nor inhibition influenced the pH in any of these organelles. CFTR heterologous overexpression also failed to alter endocytic organellar pH. We propose that the relatively large CFTR-independent counterion and small passive proton permeability ensure efficient shunting of the proton-ATPase–generated membrane potential. These results have implications in the regulation of organelle acidification in general and demonstrate that perturbations of the endolysosomal organelles pH homeostasis cannot be linked to the etiology of the CF lung disease.
Cystic fibrosis (CF) patients often have reduced mass and strength of skeletal muscles, including the diaphragm, the primary muscle of respiration. Here we show that lack of the CF transmembrane conductance regulator (CFTR) plays an intrinsic role in skeletal muscle atrophy and dysfunction. In normal murine and human skeletal muscle, CFTR is expressed and co-localized with sarcoplasmic reticulum-associated proteins. CFTR–deficient myotubes exhibit augmented levels of intracellular calcium after KCl-induced depolarization, and exposure to an inflammatory milieu induces excessive NF-kB translocation and cytokine/chemokine gene upregulation. To determine the effects of an inflammatory environment in vivo, sustained pulmonary infection with Pseudomonas aeruginosa was produced, and under these conditions diaphragmatic force-generating capacity is selectively reduced in Cftr−/− mice. This is associated with exaggerated pro-inflammatory cytokine expression as well as upregulation of the E3 ubiquitin ligases (MuRF1 and atrogin-1) involved in muscle atrophy. We conclude that an intrinsic alteration of function is linked to the absence of CFTR from skeletal muscle, leading to dysregulated calcium homeostasis, augmented inflammatory/atrophic gene expression signatures, and increased diaphragmatic weakness during pulmonary infection. These findings reveal a previously unrecognized role for CFTR in skeletal muscle function that may have major implications for the pathogenesis of cachexia and respiratory muscle pump failure in CF patients.
Cystic fibrosis is an autosomal recessive disorder caused by mutations of the CF transmembrane conductance regulator (CFTR), which acts as a chloride channel and also participates in the regulation of other ions and proteins. In most CF patients, the clinical course is dominated by lung disease and recurrent pulmonary bacterial infections. Many CF patients also have significant skeletal muscle wasting and weakness, and this can affect the most essential breathing muscle, the diaphragm. Although muscle wasting in CF has generally been attributed to factors such as reduced physical activity and poor nutrition, our study reveals an intrinsic defect of skeletal muscle function caused by the lack of CFTR. Hence, we show that CFTR is normally found in skeletal muscle fibers of humans and mice. In CF muscle cells lacking CFTR, abnormal elevations of calcium and inflammatory gene expression are found. In addition, during pulmonary infections, diaphragm muscles lacking CFTR show greater weakness and induction of genes which cause muscle atrophy. These findings extend our understanding of the factors leading to exercise limitation and disability in CF and also have implications for the pathogenesis of respiratory muscle failure in CF patients during lung infections.
Streptococcus suis is an important swine and human pathogen. Assessment of susceptibility to S. suis using animal models has been limited to monitoring mortality rates. We recently developed a hematogenous model of S. suis infection in adult CD1 outbred mice to study the in vivo development of an early septic shock-like syndrome that leads to death and a late phase that clearly induces central nervous system damage, including meningitis. In the present study, we compared the severities of septic shock-like syndrome caused by S. suis between adult C57BL/6J (B6) and A/J inbred mice. Clinical parameters, proinflammatory mediators, and bacterial clearance were measured to dissect potential immune factors associated with genetic susceptibility to S. suis infection. Results showed that A/J mice were significantly more susceptible than B6 mice to S. suis infection, especially during the acute septic phase of infection (100% of A/J and 16% of B6 mice died before 24 h postinfection). The greater susceptibility of A/J mice was associated with an exaggerated inflammatory response, as indicated by their higher production of tumor necrosis factor alpha, interleukin-12p40/p70 (IL-12p40/p70), gamma interferon, and IL-1β, but not with different bacterial loads in the blood. In addition, IL-10 was shown to be responsible, at least in part, for the higher survival in B6 mice. Our findings demonstrate that A/J mice are very susceptible to S. suis infection and provide evidence that the balance between pro- and anti-inflammatory mediators is crucial for host survival during the septic phase.
The mitogen-activated protein kinase (MAPK) p38/MAPK-activated protein kinase 2 (MK2) signaling pathway plays an important role in the posttranscriptional regulation of tumor necrosis factor (TNF), which is dependent on the adenine/uridine-rich element (ARE) in the 3′ untranslated region of TNF mRNA. After lipopolysaccharide (LPS) stimulation, MK2-deficient macrophages show a 90% reduction in TNF production compared to the wild type. Tristetraprolin (TTP), a protein induced by LPS, binds ARE and destabilizes TNF mRNA. Accordingly, macrophages lacking TTP produce large amounts of TNF. Here, we generated MK2/TTP double knockout mice and show that, after LPS stimulation, bone marrow-derived macrophages produce TNF mRNA and protein levels comparable to those of TTP knockout cells, indicating that in the regulation of TNF biosynthesis TTP is genetically downstream of MK2. In addition, we show that MK2 is essential for the stabilization of TTP mRNA, and phosphorylation by MK2 leads to increased TTP protein stability but reduced ARE affinity. These data suggest that MK2 inhibits the mRNA destabilizing activity of TTP and, in parallel, codegradation of TTP together, with the target mRNA resulting in increased cellular levels of TTP.
The solute carrier family 11 member 1 (SLC11A1, formerly NRAMP1) gene is associated with infectious and autoimmune diseases and plays an important role in macrophage activation. Human SLC11A1 mRNA contains an AU-rich element (ARE) within the 3′ untranslated region; however, its role in the regulation of SLC11A1 gene expression has not been elucidated. Here we analyze the expression of SLC11A1 in human monocytes and HL-60 cells and then use HL-60 cells as a model to determine whether RNA-binding protein HuR is associated with the ARE and involved in SLC11A1 mRNA turnover. Our results demonstrate a binding of HuR to the SLC11A1 ARE in phorbol myristate acetate (PMA)-differentiated cells dramatically increased compared to that in undifferentiated cells. Interestingly, PMA-induced accumulation of cytoplasmic HuR occurs in parallel with an increase in the binding of HuR to SLC11A1 ARE and with an increase in the SLC11A1 mRNA level. This suggests that HuR's cytoplasmic localization plays an important role in the regulation of SLC11A1 expression. We also observe that down-regulation of HuR expression by RNA interference (RNAi) results in a decrease in SLC11A1 expression which can be restored by the addition of recombinant HuR protein to the RNAi-treated cells. Finally, we show that HuR overexpression in HL-60 cells significantly increases the SLC11A1 mRNA stability. Taken together, our data demonstrate that HuR is a key mediator of posttranscriptional regulation and expression of the SLC11A1 gene.
Muscle wasting (cachexia) is a consequence of chronic diseases, such as cancer, and is associated with degradation of muscle proteins such as MyoD. The cytokines tumor necrosis factor alpha and gamma interferon induce muscle degeneration by activating the transcription factor NF-κB and its target genes. Here, we show that a downstream target of NF-κB is the nitric oxide (NO) synthase gene (iNos) and suggest that NO production stimulates MyoD mRNA loss. In fact, although cytokine treatment of iNos−/− mice activated NF-κB, it did not trigger MyoD mRNA degeneration, demonstrating that NF-κB-mediated muscle wasting requires an active iNOS-NO pathway. The induced expression of iNOS by cytokines relies on both transcriptional activation via NF-κB and increased mRNA stability via the RNA-binding protein HuR. Moreover, we show that HuR regulates iNOS expression in an AMP-activated protein kinase (AMPK)-dependent manner. Furthermore, AMPK activation results in HuR nuclear sequestration, inhibition of iNOS synthesis, and reduction in cytokine-induced MyoD loss. These results define iNOS and HuR as critical players in cytokine-induced cachexia, establishing them as potential therapeutic targets.
The intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the α, β and γ subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na+-K+-ATPase.
Pseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains. The mRNA expression of ENaC and Na+-K+-ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection.
The infection of the different mouse strains evoked regulation of α and β ENaC mRNA. Following Pseudomonas instillation, the expression of αENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p < 0.05). The relative expression of βENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p < 0.05). No significant modulation of γENaC mRNA was detected although the general pattern of expression of the subunit was similar to α and β subunits. No modulation of α1Na+-K+-ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains.
These results show that Pseudomonas infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs.
The mouse bcg host resistance gene is known to control the activation of host macrophages for killing of intracellular parasites like Leishmania donovani as well as intracellular bacteria, including Mycobacterium bovis BCG and Salmonella enterica serovar Typhimurium. The Nramp1 gene has been mapped to this locus and affects the efficiency of macrophage activation. It has been shown that imidazoquinoline compounds, including S28463, are able to improve the clearance of a number of intracellular pathogens such as herpes simplex virus 2, human papillomavirus, and Leishmania. The goal of this study was to determine whether S28463 is efficient against infection with another intracellular pathogen, M. bovis BCG, and to determine the molecular basis underlying this effect. To achieve this, B10A.Nramp1r and B10A.Nramp1−/− mice were infected with M. bovis BCG and treated with S28463. The bacterial content in the spleen from these mice was assayed by a colony-forming assay. In addition, in vitro experiments were performed using bone marrow-derived macrophage cell lines from these mice. These cells were treated with S28463 and/or gamma interferon (IFN-γ), and nitric oxide (NO) production was measured. Our study was able to show that S28463 acts in synergy with IFN-γ to increase the production of NO in vitro. We were also able to demonstrate that mice that carried the resistant allele of the Nramp1 gene and were infected with M. bovis BCG responded to treatment with S28463, resulting in a decreased bacterial load after 2 weeks of treatment. Mice that do not express the Nramp1 gene responded only to a very large dose of S28463, and the response was not as efficient as that observed in mice carrying a wild-type Nramp1 allele. Our data provide evidence for the potential of S28463 as an immunomodulator that may be helpful in designing efficient strategies to improve host defense against mycobacterial infection.
Tumor necrosis factor α (TNFα)
acts as a beneficial mediator in the process of host defence. In
recent years major interest has focused on the AU-rich elements
(AREs) present in the 3′-untranslated region
(3′-UTR) of TNFα mRNA
as this region plays a pivotal role in post-transcriptional control
of TNFα production. Certain stimuli,
such as lipopolysaccharides, a component of the Gram-negative bacterial cell
wall, have the ability to relinquish the translational suppression
of TNFα mRNA imposed by these AREs in
macrophages, thereby enabling the efficient production of the TNFα. In this study we show that the polymorphism
(GAU trinucleotide insertional mutation) present in the regulatory
3′-UTR of TNFα mRNA
of NZW mice results in the hindered binding of RNA-binding proteins,
thereby leading to a significantly reduced production of TNFα protein. We also show that the binding
of macrophage proteins to the main ARE is also decreased by another
trinucleotide (CAU) insertion in the TNFα 3′-UTR. One of the proteins affected
by the GAU trinucleotide insertional mutation was identified as
HuR, a nucleo-cytoplasmic shuttling protein previously shown to
play a prominent role in the stability and translatability of mRNA
containing AREs. Since binding of this protein most likely modulates the
stability, translational efficiency and transport of TNFα mRNA,
these results suggest that mutations in the ARE of TNFα mRNA
decrease the production of TNFα protein
in macrophages by hindering the binding of HuR to the ARE.
Interleukin-12 (IL-12) is one of the first cytokines produced by macrophages, key mediators of innate resistance, during the host’s immune response to infections. Therefore, in this study we propose that IL-12 has an important role in the early phase of the immune response to Mycobacterium bovis BCG. IL-12 has been shown to enhance the maturation of protective Th1 cells and gamma interferon (IFN-γ) production during mycobacterial infection. Therefore, it may play a crucial role during the immune phase of infection as well. To examine the role of IL-12 in both the innate and the immune phase of infection, we compared BCG-resistant mice, B10.A (Bcgr), to the susceptible congenic strain B10.A (Bcgs) following administration of a blocking monoclonal antibody to IL-12 (10F6). Anti-IL-12-treated susceptible animals exhibited a two- to threefold increase in spleen CFU by day 21. In contrast, anti-IL-12 treatment had little or no effect on the response of the genetically resistant animals to infection. The B10.A (Bcgr) but not the B10.A (Bcgs) mice had an increase in IFN-γ mRNA relative to baseline levels as early as day 1 of infection irrespective of anti-IL-12 treatment. By day 14, B10.A (Bcgr) mice showed a decrease in IFN-γ mRNA while the B10.A (Bcgs) mice showed a significant increase in IFN-γ mRNA levels. Thus, during BCG infection, the B10.A (Bcgr) mice mount an early IFN-γ response against BCG whereas the B10.A (Bcgs) mice have a delayed IFN-γ response correlating with their genetic permissiveness expressed as an increased mycobacterial load by day 21. Overall, our data demonstrate that the inherent resistance of B10.A (Bcgr) mice to mycobacteria does not depend on optimal levels of IL-12 to maintain effective control of the bacteria, whereas IL-12 is important for the susceptible animals’ response to BCG during the peak of infection.