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1.  The phosphoproteome of toll-like receptor-activated macrophages 
First global and quantitative analysis of phosphorylation cascades induced by toll-like receptor (TLR) stimulation in macrophages identifies nearly 7000 phosphorylation sites and shows extensive and dynamic up-regulation and down-regulation after lipopolysaccharide (LPS).In addition to the canonical TLR-associated pathways, mining of the phosphorylation data suggests an involvement of ATM/ATR kinases in signalling and shows that the cytoskeleton is a hotspot of TLR-induced phosphorylation.Intersecting transcription factor phosphorylation with bioinformatic promoter analysis of genes induced by LPS identified several candidate transcriptional regulators that were previously not implicated in TLR-induced transcriptional control.
Toll-like receptors (TLR) are a family of pattern recognition receptors that enable innate immune cells to sense infectious danger. Recognition of microbial structures, like lipopolysaccharide (LPS) of Gram-negative bacteria by TLR4, causes within hours substantial re-programming of macrophage gene expression, including up-regulation of chemokines driving inflammation, anti-microbial effector molecules and cytokines directing adaptive immune responses. TLR signalling is initiated by the adapter protein Myd88 and leads to the activation of kinase cascades that result in activation of the MAPK and NFkB pathways. Phosphorylation has an essential role in these early steps of TLR signalling, and in addition regulates critical transcription factors (TFs). Although TLR signalling has been extensively studied, a comprehensive analysis of phosphorylation events in TLR-activated macrophages is lacking. It is therefore unknown whether the canonical MAPK and NFkB pathways comprise the main phosphorylation events and which other molecular functions and processes are regulated by phosphorylation after stimulation with LPS.
Recent progress in mass spectrometry-based proteomics has opened the possibility to quantitatively investigate global changes in protein abundance and post-translational modifications. Stable isotope labelling with amino acids in cell culture (SILAC) allows highly accurate quantification, and has proved especially useful for direct comparison of phosphopeptide abundance in time-course or treatment analyses.
Here, we adapted SILAC to primary mouse macrophages, and performed a global, quantitative and kinetic analysis of the macrophage phosphoproteome after LPS stimulation. Bioinformatic analyses were used to identify kinases, pathways and biological processes enriched in the LPS-regulated phosphoproteome. To connect TF phosphorylation with transcription, we generated a parallel dataset of nascent RNA and used in silico promoter analysis to identify transcriptional regulators with binding site enrichment among the LPS-regulated gene set.
After establishing SILAC conditions for efficient labelling of primary bone marrow-derived macrophages in two independent experiments 1850 phosphoproteins with a total of 6956 phosphorylation sites were reproducibly identified. Phosphoproteins were detected from all cellular compartments, with a clear enrichment for nuclear and cytoskeleton-associated proteins. LPS caused major regulation of a large fraction of phosphopeptides, with 24% of all sites up-regulated and 9% down-regulated after stimulation (Figure 3A and B). These changes were highly dynamic, as the majority of the regulated phosphopeptides were up-regulated or down-regulated transiently or in a delayed manner (Figure 3C). Overall, the extent of changes in the phosphoproteome was comparable to the transcriptional re-programming, underscoring the importance of phosphorylation cascades in TLR signalling. Our parallel transcriptome data also showed that widespread phosphorylation precedes massive transcriptional changes.
To obtain footprints of kinase activation in response to TLR ligation, we searched phosphopeptide sequences for known linear sequence motifs of 33 kinases and identified kinase motifs enriched among LPS-regulated phosphorylation sites (compared to non-regulated phosphorylation sites) (Table I). Motif ERK/MAPK was highly enriched, in accordance with the essential role of the MAPK module in TLR signalling. Other kinases with motif enrichment have also recently been linked to TLR signalling (e.g. PKD; AKT and its targets GSK3 and mTOR). However, the DNA damage-actviated kinases ATM/ATR and the cell cycle-associated kinases AURORA and CHK1/2 have not been associated with the macrophage response to TLR activation yet. These finding shed new light on older data on the effect of TLR on macrophage proliferation in response to macrophage colony stimulating factor. Of interest, in follow-up experiments using pharmacological inhibitors of the kinases with motif enrichment, we observed that inhibition of ATM kinase activity caused increased LPS-induced expression of several cytokines and chemokines, suggesting that this pathway regulates inflammatory responses.
In further bioinformatic analyses, the Gene Ontology and signalling pathway annotations of phosphoproteins were used to identify signalling pathways and cellular processes targeted by TLR4-controlled phosphorylation (Table II). Among the expected hits, based on the known TLR pathways, were TLR signalling, MAPK and AKT as well as mTOR signalling. Of interest, the annotation terms ‘Rho GTPase cycle' and ‘cytoskeleton' were significantly enriched among LPS-regulated phosphoproteins, indicating a more prominent role for cytoskeletal proteins in the transduction of TLR signals or in the biological response to it.
We were especially interested in the phosphorylation of TFs and its regulation by LPS (Figure 6A). We hypothesised that functionally important TFs should have an increased frequency of binding sites in the promoters of LPS-regulated genes (Figure 6B). To identify transcriptionally regulated genes with high sensitivity, we isolated nascent RNA after metabolic labelling (Figure 6C–E). In silico promoter scanning using Genomatix software for binding sites for all 50 TF families with phosphorylated members was used to test for enrichment in transciptionally induced genes (Figure 6F). At the early time point, binding site enrichment for the canonical TLR-associated TF NFkB was detected, and in addition we found that several other TF families with an established role in the transcription of individual LPS-target genes showed binding site enrichment (CEBP, MEF2, NFAT and HEAT). In addition, enrichment for OCT and HOXC binding sites at the early time point and SORY matrices later after stimulation indicated an involvement of the phosphorylated members of the respective TF families in the execution of TLR-induced transcriptional responses. An initial test of the function for a few of these candidate transcriptional regulators was performed using siRNA knockdown in primary macrophages. These experiments suggested that knock down of the SORY binding phosphoprotein Capicua homolog (Cic) and to a lesser extent of the CREB family member Atf7 selectively attenuates LPS-induced expression of Il1a and Il1b.
In summary, this study provides a novel and global perspective on innate immune activation by TLR signalling (Figure 5). We quantitatively detected a large number of previously unknown site-specific phosphorylation events, which are now publicly available through the Phosida database. By combining different data mining approaches, we consistently identified canonical and newly implicated TLR-activated signalling modules. In particular, the PI3K/AKT and the related mTOR pathway were highlighted; furthermore, DNA damage–response associated ATM/ATR kinases and the cytoskeleton emerged as unexpected hotspots for phosphorylation. Finally, weaving together corresponding phophoproteome and nascent transcriptome datasets through the loom of in silico promoter analysis we identified TFs with a likely role in mediating TLR-induced gene expression programmes.
Recognition of microbial danger signals by toll-like receptors (TLR) causes re-programming of macrophages. To investigate kinase cascades triggered by the TLR4 ligand lipopolysaccharide (LPS) on systems level, we performed a global, quantitative and kinetic analysis of the phosphoproteome of primary macrophages using stable isotope labelling with amino acids in cell culture, phosphopeptide enrichment and high-resolution mass spectrometry. In parallel, nascent RNA was profiled to link transcription factor (TF) phosphorylation to TLR4-induced transcriptional activation. We reproducibly identified 1850 phosphoproteins with 6956 phosphorylation sites, two thirds of which were not reported earlier. LPS caused major dynamic changes in the phosphoproteome (24% up-regulation and 9% down-regulation). Functional bioinformatic analyses confirmed canonical players of the TLR pathway and highlighted other signalling modules (e.g. mTOR, ATM/ATR kinases) and the cytoskeleton as hotspots of LPS-regulated phosphorylation. Finally, weaving together phosphoproteome and nascent transcriptome data by in silico promoter analysis, we implicated several phosphorylated TFs in primary LPS-controlled gene expression.
doi:10.1038/msb.2010.29
PMCID: PMC2913394  PMID: 20531401
macrophage; nascent RNA; phosphoproteome; SILAC; toll-like receptors
2.  Toll-Like Receptor 2 Impairs Host Defense in Gram-Negative Sepsis Caused by Burkholderia pseudomallei (Melioidosis) 
PLoS Medicine  2007;4(7):e248.
Background
Toll-like receptors (TLRs) are essential in host defense against pathogens by virtue of their capacity to detect microbes and initiate the immune response. TLR2 is seen as the most important receptor for gram-positive bacteria, while TLR4 is regarded as the gram-negative TLR. Melioidosis is a severe infection caused by the gram-negative bacterium, Burkholderia pseudomallei, that is endemic in Southeast Asia. We aimed to characterize the expression and function of TLRs in septic melioidosis.
Methods and Findings
Patient studies: 34 patients with melioidosis demonstrated increased expression of CD14, TLR1, TLR2, and TLR4 on the cell surfaces of monocytes and granulocytes, and increased CD14, TLR1, TLR2, TLR4, LY96 (also known as MD-2), TLR5, and TLR10 mRNA levels in purified monocytes and granulocytes when compared with healthy controls. In vitro experiments: Whole-blood and alveolar macrophages obtained from TLR2 and TLR4 knockout (KO) mice were less responsive to B. pseudomallei in vitro, whereas in the reverse experiment, transfection of HEK293 cells with either TLR2 or TLR4 rendered these cells responsive to this bacterium. In addition, the lipopolysaccharide (LPS) of B. pseudomallei signals through TLR2 and not through TLR4. Mouse studies: Surprisingly, TLR4 KO mice were indistinguishable from wild-type mice with respect to bacterial outgrowth and survival in experimentally induced melioidosis. In contrast, TLR2 KO mice displayed a markedly improved host defenses as reflected by a strong survival advantage together with decreased bacterial loads, reduced lung inflammation, and less distant-organ injury.
Conclusions
Patients with melioidosis displayed an up-regulation of multiple TLRs in peripheral blood monocytes and granulocytes. Although both TLR2 and TLR4 contribute to cellular responsiveness to B. pseudomallei in vitro, TLR2 detects the LPS of B. pseudomallei, and only TLR2 impacts on the immune response of the intact host in vivo. Inhibition of TLR2 may be a novel treatment strategy in melioidosis.
Willem Wiersinga and colleagues find up-regulation of multiple Toll-like receptors (TLRs) in peripheral blood cells of patients with melioidosis. However, only TLR2 had an effect on the immune response in a mouse model.
Editors' Summary
Background.
Melioidosis is a severe tropical infection caused by the bacterium Burkholderia pseudomallei. This soil-dwelling pathogen (disease-causing organism) enters the body through cuts, by swallowed contaminated water, or by inhaled contaminated dust. Here, it can cause a severe lung infection or spread into the blood stream and around the body, where it causes widespread inflammation (sepsis) and organ failure. Untreated septic melioidosis is usually fatal. Even with antibiotic therapy, half the people who develop it in Thailand (a hot spot for melioidosis) die. B. pseudomallei is a “gram-negative” bacterium. That is, it is surrounded by a membrane that stops it taking up a stain used to detect bacteria. This membrane contains a molecule called lipopolysaccharide (LPS). Proteins on immune system cells called Toll-like receptors (TLRs), of which there are many, recognize LPS and other surface molecules common to different pathogens and tell the cells to make cytokines. These cytokines stimulate the immune system to kill the pathogen but also cause inflammation, the underlying problem in septic melioidosis and other forms of sepsis. In other words, TLRs are two-edged swords—they provide an essential first-line defense against pathogens, but cause life-threatening inflammation if overstimulated.
Why Was This Study Done?
It isn't known which TLRs are involved in melioidosis. TLR4 normally detects LPS, but the surface of B. pseudomallei also carries molecules that interact with TLR2. Understanding how B. pseudomallei interacts with TLRs might suggest new, more effective ways to treat septic melioidosis. Better remedies for this disease are badly needed because, as well as the infections it causes in the community, the US Centers for Disease Control and Prevention has identified B. pseudomallei as a potential bioterrorism agent. In this study, the researchers have characterized the expression and function of TLRs in septic melioidosis using human, in vitro (test tube), and animal approaches.
What Did the Researchers Do and Find?
The researchers isolated monocytes and granulocytes (immune system cells involved in first-line defenses against pathogens) from patients with melioidosis and from healthy people. The patients' cells made more TLR1, TLR2, TLR4, and CD14 (a protein that enhances the activation of immune system cells by LPS) than those of the healthy controls and more of the mRNAs encoding several other TLRs. Next, the researchers tested the ability of heat-killed B. pseudomallei to induce the release of TNFα (a cytokine produced in response to TLR signaling) from macrophages (immune system cells that swallow up pathogens) isolated from wild-type mice and from mice lacking TLR2 or TLR4. Macrophages isolated from wild-type mice made more TNFα than those from TLR2- or TLR4-deficient mice. In addition, a human kidney cell line engineered to express CD14/TLR2 or CD14/TLR4 but not the parent cell line released IL8 (another cytokine) when stimulated with heat-killed B. pseudomallei. Other experiments in these human cell lines showed that LPS purified from B. pseudomallei signals through TLR2 but not through TLR4. Finally, the researchers tested the ability of TLR2- and TLR4-deficient mice to survive after infection with live B. pseudomallei. Compared with TLR4-deficient or wild-type mice, the TLR2-deficient mice had a strong survival advantage, a lower bacterial load, reduced lung inflammation, and less organ damage.
What Do These Findings Mean?
These findings show that people with melioidosis have increased expression of several TLRs, any one of which might cause the sepsis associated with B. pseudomallei infection. The in vitro findings indicate that TLR2 and TLR4 both contribute to the responsiveness of immune cells to B. pseudomallei in test tubes, but that only TLR2 detects the LPS of this bacterium. This unexpected result—TLR4 normally responds to LPS—might indicate that there is something unique about the LPS of B. pseudomallei. Finally, the survival of TLR2-deficient mice after infection with B. pseudomallei suggests that TLR2-mediated dysregulation of the immune system in response to invasive B. pseudomallei might cause septic melioidosis. Although these results need confirming in people, they suggest that inhibition of TLR2 in combination with antibiotic therapy might improve outcomes for people with melioidosis.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040248.
Information is available from the US Centers for Disease Control and Prevention on melioidosis (in English and Spanish)
The UK Health Protection Agency provides information for the public and health professionals on melioidosis
Wikipedia has pages on melioidosis and on Toll-like receptors (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The MedlinePlus encyclopedia contains a page on sepsis (in English and Spanish)
doi:10.1371/journal.pmed.0040248
PMCID: PMC1950213  PMID: 17676990
3.  A locus on chromosome 9 is associated with differential response of 129S1/SvImJ and FVB/NJ strains of mice to systemic LPS 
Although polymorphisms in TLR receptors and downstream signaling molecules affect the innate immune response, these variants account for only a portion of the ability of the host to respond to microorganisms. To identify novel genes that regulate the host response to systemic lipopolysaccharide (LPS), we created an F2 intercross between susceptible (FVB/NJ) and resistant (129S1/SvImJ) strains, challenged F2 progeny with LPS via intraperitoneal injection, and phenotyped 605 animals for survival and another 500 mice for serum concentrations of IL-1β and IL-6. Genome-wide scans were performed on pools of susceptible and resistant mice for survival, IL-1β, and IL-6. This approach identified a locus on the telomeric end of the q arm of chromosome 9 (0–40 Mb) that was associated with the differences in morbidity and serum concentrations of IL-1β and IL-6 following systemic LPS in FVB/NJ and 129S1/SvImJ strains of mice. Fine mapping narrowed the locus to 3.7 Mb containing 11 known genes, among which are three inflammatory caspases. We studied expression of genes within the locus by quantitative RT-PCR and showed that Casp1 and Casp12 levels are unaffected by LPS in both strains, whereas Casp4 is highly induced by LPS in FVB/NJ but not in 129S1/SvImJ mice. In conclusion, our mapping results indicate that a 3.7-Mb region on chromosome 9 contains a gene that regulates differential response to LPS in 129S1/SvImJ and FVB/NJ strains of mice. Differences in the induction of Casp4 expression by LPS in the two strains suggest that Casp4 is the most likely candidate gene in this region.
doi:10.1007/s00335-011-9340-8
PMCID: PMC4157901  PMID: 21720866
4.  Identification of a neuronal transcription factor network involved in medulloblastoma development 
Background
Medulloblastomas, the most frequent malignant brain tumours affecting children, comprise at least 4 distinct clinicogenetic subgroups. Aberrant sonic hedgehog (SHH) signalling is observed in approximately 25% of tumours and defines one subgroup. Although alterations in SHH pathway genes (e.g. PTCH1, SUFU) are observed in many of these tumours, high throughput genomic analyses have identified few other recurring mutations. Here, we have mutagenised the Ptch+/- murine tumour model using the Sleeping Beauty transposon system to identify additional genes and pathways involved in SHH subgroup medulloblastoma development.
Results
Mutagenesis significantly increased medulloblastoma frequency and identified 17 candidate cancer genes, including orthologs of genes somatically mutated (PTEN, CREBBP) or associated with poor outcome (PTEN, MYT1L) in the human disease. Strikingly, these candidate genes were enriched for transcription factors (p=2x10-5), the majority of which (6/7; Crebbp, Myt1L, Nfia, Nfib, Tead1 and Tgif2) were linked within a single regulatory network enriched for genes associated with a differentiated neuronal phenotype. Furthermore, activity of this network varied significantly between the human subgroups, was associated with metastatic disease, and predicted poor survival specifically within the SHH subgroup of tumours. Igf2, previously implicated in medulloblastoma, was the most differentially expressed gene in murine tumours with network perturbation, and network activity in both mouse and human tumours was characterised by enrichment for multiple gene-sets indicating increased cell proliferation, IGF signalling, MYC target upregulation, and decreased neuronal differentiation.
Conclusions
Collectively, our data support a model of medulloblastoma development in SB-mutagenised Ptch+/- mice which involves disruption of a novel transcription factor network leading to Igf2 upregulation, proliferation of GNPs, and tumour formation. Moreover, our results identify rational therapeutic targets for SHH subgroup tumours, alongside prognostic biomarkers for the identification of poor-risk SHH patients.
doi:10.1186/2051-5960-1-35
PMCID: PMC3893591  PMID: 24252690
Medulloblastoma; Mutagenesis; Transcription network; Differentiation
5.  Analysis of Germline GLI1 Variation Implicates Hedgehog Signalling in the Regulation of Intestinal Inflammatory Pathways 
PLoS Medicine  2008;5(12):e239.
Background
Ulcerative colitis (UC) and Crohn's disease (CD) are polygenic chronic inflammatory bowel diseases (IBD) of high prevalence that are associated with considerable morbidity. The hedgehog (HH) signalling pathway, which includes the transcription factor glioma-associated oncogene homolog 1 (GLI1), plays vital roles in gastrointestinal tract development, homeostasis, and malignancy. We identified a germline variation in GLI1 (within the IBD2 linkage region, 12q13) in patients with IBD. Since this IBD-associated variant encodes a GLI1 protein with reduced function and our expression studies demonstrated down-regulation of the HH response in IBD, we tested whether mice with reduced Gli1 activity demonstrate increased susceptibility to chemically induced colitis.
Methods and Findings
Using a gene-wide haplotype-tagging approach, germline GLI1 variation was examined in three independent populations of IBD patients and healthy controls from Northern Europe (Scotland, England, and Sweden) totalling over 5,000 individuals. On log-likelihood analysis, GLI1 was associated with IBD, predominantly UC, in Scotland and England (p < 0.0001). A nonsynonymous SNP (rs2228226C→G), in exon 12 of GLI1 (Q1100E) was strongly implicated, with pooled odds ratio of 1.194 (confidence interval = 1.09–1.31, p = 0.0002). GLI1 variants were tested in vitro for transcriptional activity in luciferase assays. Q1100E falls within a conserved motif near the C terminus of GLI1; the variant GLI protein exhibited reduced transactivation function in vitro. In complementary expression studies, we noted the colonic HH response, including GLI1, patched (PTCH), and hedgehog-interacting protein (HHIP), to be down-regulated in patients with UC. Finally, Gli1+/lacZ mice were tested for susceptibility to dextran sodium sulphate (DSS)-induced colitis. Clinical response, histology, and expression of inflammatory cytokines and chemokines were recorded. Gli1+/lacZ mice rapidly developed severe intestinal inflammation, with considerable morbidity and mortality compared with wild type. Local myeloid cells were shown to be direct targets of HH signals and cytokine expression studies revealed robust up-regulation of IL-12, IL-17, and IL-23 in this model.
Conclusions
HH signalling through GLI1 is required for appropriate modulation of the intestinal response to acute inflammatory challenge. Reduced GLI1 function predisposes to a heightened myeloid response to inflammatory stimuli, potentially leading to IBD.
Charlie Lees and colleagues identify a reduced-function variant of the hedgehog signaling pathway protein GLI1 that associates with inflammatory bowel disease, and investigate its role in a mouse model of colitis.
Editors' Summary
Background.
Inflammatory bowel diseases (IBDs) are common disorders in which parts of the digestive tract become repeatedly or continuously inflamed. The immune system normally protects the body from entities it identifies as foreign, but in IBD it mistakenly recognizes gut tissue, and immune system cells accumulate in the lining of the bowel, which causes inflammation. There are two main types of IBD—Crohn's disease (CD), which mainly affects the small bowel, and ulcerative colitis (UC), which affects only the large bowel (colon). Both types tend to run in families and usually develop between the ages of 15 and 35 years. Symptoms—including diarrhea, abdominal cramps, and unexplained weight loss—can be mild or severe and the disease can develop slowly or suddenly. There is no cure for IBD except surgical removal of the affected part of the digestive tract. However, drugs that modulate the immune system (for example, corticosteroids) or that specifically inhibit “proinflammatory cytokines” (proteins made by the immune system that stimulate inflammation) are often helpful in reducing symptoms.
Why Was This Study Done?
Why the immune system becomes unbalanced in people with IBD is not clear but it is known that IBD is “polygenic,” that is, a disease caused by the combined actions of two or more inherited gene variants. Although UC and CD are clinically different diseases, they share several “susceptibility loci” (regions of the genome that harbor disease-associated gene variants), including the IBD2 locus. The identification of the actual gene within the IBD2 locus that is altered in people who are susceptible to IBD might provide new insights into what causes the immune imbalance in IBD and into how to treat the disease. In this study, the researchers test the hypothesis that a variant of a gene called GLI1, which lies in the IBD2 locus, is associated with IBD susceptibility. GLI1 encodes a transcription factor (a protein that regulates the production of proteins) that is a central component in the signaling pathway named for a protein called “hedgehog.” This pathway is involved in the development of many organs, including the digestive tract.
What Did the Researchers Do and Find?
The researchers used a technique called gene-wide haplotype tagging to look for inherited GLl1 variants in patients with IBD and in healthy people living in Scotland, England, and Sweden. A specific variant of the GLI1 gene, resulting in alteration of a single amino acid component of the GLI1 protein, was associated with IBD (particularly with UC) in both Scotland and England; the same variant was weakly associated with IBD in the Swedish population. The variant GLI1 protein was only half as active as the normal protein in a laboratory assay, and, consistent with this result, the expression of several components of the hedgehog signaling pathway was lower in colon samples taken from patients with UC than in samples taken from healthy individuals. Finally, Gli1+/lacZ mice (which express half the normal amount of Gli1 protein) developed severe intestinal inflammation more rapidly than wild-type mice when they were treated with dextran sodium sulfate (DSS), a chemical that induces acute (sudden) colitis. Cellular analysis revealed that myeloid cells (cells that sense and modify the inflammatory response) are direct targets of the hedgehog signaling pathway. Furthermore, the expression of several pro-inflammatory cytokines (in particular, one called IL-23) increased more markedly in the Gli1+/lacZ mice than in the wild-type mice after DSS treatment.
What Do These Findings Mean?
These findings suggest that the normal response of the mammalian gut to challenge with inflammatory substances involves hedgehog signaling through GLI1 and that reduced GLI1 function might be one trigger for IBD. More specifically, the human genetic studies identify a GLI1 variant that is associated with IBD (at least in certain north European populations), the laboratory experiments indicate that this GLI1 variant encodes a protein with reduced activity, and the animal studies show that a similar reduction in Gli1 activity is sufficient to heighten intestinal inflammatory responses. Although this last result needs to be confirmed in animal models of chronic colitis that more closely resemble human IBD, these findings suggest that drugs that modulate hedgehog signaling might be useful in the treatment of IBD.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050239.
The MedlinePlus Encyclopedia has pages on Crohn's disease and on ulcerative colitis (in English and Spanish)
MedlinePlus provides links to other information Crohn's disease and ulcerative colitis (in English and Spanish)
The US National Institute of Diabetes and Digestive and Kidney Diseases provides information on Crohn's disease and ulcerative colitis
The UK National Health Service Direct Encyclopedia also provides information on Crohn's disease and on ulcerative colitis
Wikipedia has a page on the hedgehog signaling pathway (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.0050239
PMCID: PMC2596854  PMID: 19071955
6.  Host Genetic Variation in Susceptibility to Punta Toro Virus 
Virus research  2011;157(1):71-75.
SUMMARY
Infection of small laboratory animals by Punta Toro virus (PTV), family Bunyaviridae, genus Phlebovirus, is a model for the study of the human pathogen Rift Valley fever virus (RVFV). We have identified inbred mouse strains with significant differences in host response to the Adames strain of PTV. Nine inbred strains of mice representing major branches in the Mus musculus phylogeny were inoculated subcutaneously with a high dose of PTV in survival experiments. Two inbred strains of mice, NZW/LacJ and 129S1/SvImJ, died ~4 days after PTV infection, whereas 7 other strains survived the challenge and showed no clinical signs of disease. Histologically, 129S1/SvImJ mice showed massive hepatocellular necrosis and had additional lesions in lung, brain, and spleen, whereas NZW/LacJ mice had mild piecemeal hepatocellular necrosis. PTV viral loads in the livers of infected mice were determined by reverse transcriptase quantitative PCR. Inbred mice from strains that showed clinical signs and succumbed to PTV infection had higher liver viral loads than did mice of resistant strains. Hybrid F1 mice were generated by crossing susceptible 129S1 and resistant FVB/N mice and tested for susceptibility. The hybrid F1 mice showed significantly higher viral loads in the liver than the resistant parental FVB/N mice, suggesting that susceptibility is dominant. These findings will enable an unbiased genetic approach to identify host genes mediating susceptibility to PTV.
doi:10.1016/j.virusres.2011.02.008
PMCID: PMC3587965  PMID: 21320557
Bunyaviridae; phlebovirus; murine
7.  Reconstruction of the Gene Regulatory Network Involved in the Sonic Hedgehog Pathway with a Potential Role in Early Development of the Mouse Brain 
PLoS Computational Biology  2014;10(10):e1003884.
The Sonic hedgehog (Shh) signaling pathway is crucial for pattern formation in early central nervous system development. By systematically analyzing high-throughput in situ hybridization data of E11.5 mouse brain, we found that Shh and its receptor Ptch1 define two adjacent mutually exclusive gene expression domains: Shh+Ptch1− and Shh−Ptch1+. These two domains are associated respectively with Foxa2 and Gata3, two transcription factors that play key roles in specifying them. Gata3 ChIP-seq experiments and RNA-seq assays on Gata3-knockdown cells revealed that Gata3 up-regulates the genes that are enriched in the Shh−Ptch1+ domain. Important Gata3 targets include Slit2 and Slit3, which are involved in the process of axon guidance, as well as Slc18a1, Th and Qdpr, which are associated with neurotransmitter synthesis and release. By contrast, Foxa2 both up-regulates the genes expressed in the Shh+Ptch1− domain and down-regulates the genes characteristic of the Shh−Ptch1+ domain. From these and other data, we were able to reconstruct a gene regulatory network governing both domains. Our work provides the first genome-wide characterization of the gene regulatory network involved in the Shh pathway that underlies pattern formation in the early mouse brain.
Author Summary
Recent large-scale projects of high-throughput in situ hybridization (ISH) have generated a wealth of spatiotemporal information on gene expression patterns in the early mouse brain. We have developed a computational approach that combines publicly available high-throughput ISH data with our own experimental data to investigate gene regulation, involving signal molecules and transcription factors (TFs), during early brain development. The analysis indicates that two key TFs, Foxa2 and Gata3, play antagonizing roles in the formation of two mutually exclusive domains established by the Sonic hedgehog signaling pathway in the developing mouse brain. Further ChIP-seq and RNA-seq experiments support this hypothesis and have identified novel target genes of Gata3, including the axon guidance regulators Slit2 and Slit3 as well as three neurotransmitter-associated genes, Slc18a1, Th and Qdpr. The findings have allowed us to reconstruct the gene regulatory network brought into play by the Sonic hedgehog pathway that mediates early mouse brain development.
doi:10.1371/journal.pcbi.1003884
PMCID: PMC4191885  PMID: 25299227
8.  Distinct Dictation of Japanese Encephalitis Virus-Induced Neuroinflammation and Lethality via Triggering TLR3 and TLR4 Signal Pathways 
PLoS Pathogens  2014;10(9):e1004319.
Japanese encephalitis (JE) is major emerging neurologic disease caused by JE virus. To date, the impact of TLR molecules on JE progression has not been addressed. Here, we determined whether each TLR modulates JE, using several TLR-deficient mouse strains (TLR2, TLR3, TLR4, TLR7, TLR9). Surprisingly, among the tested TLR-deficient mice there were contrasting results in TLR3−/− and TLR4−/− mice, i.e. TLR3−/− mice were highly susceptible to JE, whereas TLR4−/− mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation characterized by early infiltration of inflammatory CD11b+Ly-6Chigh monocytes along with profoundly increased viral burden, proinflammatory cytokine/chemokine expression as well as BBB permeability. In contrast, TLR4−/− mice showed mild CNS inflammation manifested by reduced viral burden, leukocyte infiltration and proinflammatory cytokine expression. Interestingly, TLR4 ablation provided potent in vivo systemic type I IFN innate response, as well as ex vivo type I IFN production associated with strong induction of antiviral PRRs (RIG-I, MDA5), transcription factors (IRF-3, IRF-7), and IFN-dependent (PKR, Oas1, Mx) and independent ISGs (ISG49, ISG54, ISG56) by alternative activation of IRF3 and NF-κB in myeloid-derived DCs and macrophages, as compared to TLR3−/− myeloid-derived cells which were more permissive to viral replication through impaired type I IFN innate response. TLR4 ablation also appeared to mount an enhanced type I IFN innate and humoral, CD4+ and CD8+ T cell responses, which were mediated by altered immune cell populations (increased number of plasmacytoid DCs and NK cells, reduced CD11b+Ly-6Chigh monocytes) and CD4+Foxp3+ Treg number in lymphoid tissue. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were closely coupled with reduced JE lethality. Collectively, these results suggest that a balanced triggering of TLR signal array by viral components during JE progression could be responsible for determining disease outcome through regulating negative and positive factors.
Author Summary
Japanese encephalitis (JE) is major emerging encephalitis, and more than 60% of global population inhabits JE endemic areas. The etiological virus is currently spreading to previously unaffected regions due to rapid changes in climate and demography. However, the impact of TLR molecules on JE progression has not been addressed to date. We found that the distinct outcomes of JE progression occurred in TLR3 and TLR4-dependent manner, i.e. TLR3−/− mice were highly susceptible, whereas TLR4−/− mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation manifested by early CD11b+Ly-6Chigh monocyte infiltration, high expression of proinflammatory cytokines, as well as increased BBB permeability. In contrast, TLR4 ablation provided potent type I IFN innate response in infected mice, as well as in myeloid-derived cells closely associated with strong induction of antiviral ISG genes, and also resulted in enhanced humoral, CD4+, and CD8+ T cell responses along with altered plasmacytoid DC and CD4+Foxp3+ Treg number. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were coupled with reduced JE lethality. Our studies provide an insight into the role of each TLR molecule on the modulation of JE, as well as its mechanism of neuroinflammation control during JE progression.
doi:10.1371/journal.ppat.1004319
PMCID: PMC4154777  PMID: 25188232
9.  Uncovering a Macrophage Transcriptional Program by Integrating Evidence from Motif Scanning and Expression Dynamics 
PLoS Computational Biology  2008;4(3):e1000021.
Macrophages are versatile immune cells that can detect a variety of pathogen-associated molecular patterns through their Toll-like receptors (TLRs). In response to microbial challenge, the TLR-stimulated macrophage undergoes an activation program controlled by a dynamically inducible transcriptional regulatory network. Mapping a complex mammalian transcriptional network poses significant challenges and requires the integration of multiple experimental data types. In this work, we inferred a transcriptional network underlying TLR-stimulated murine macrophage activation. Microarray-based expression profiling and transcription factor binding site motif scanning were used to infer a network of associations between transcription factor genes and clusters of co-expressed target genes. The time-lagged correlation was used to analyze temporal expression data in order to identify potential causal influences in the network. A novel statistical test was developed to assess the significance of the time-lagged correlation. Several associations in the resulting inferred network were validated using targeted ChIP-on-chip experiments. The network incorporates known regulators and gives insight into the transcriptional control of macrophage activation. Our analysis identified a novel regulator (TGIF1) that may have a role in macrophage activation.
Author Summary
Macrophages play a vital role in host defense against infection by recognizing pathogens through pattern recognition receptors, such as the Toll-like receptors (TLRs), and mounting an immune response. Stimulation of TLRs initiates a complex transcriptional program in which induced transcription factor genes dynamically regulate downstream genes. Microarray-based transcriptional profiling has proved useful for mapping such transcriptional programs in simpler model organisms; however, mammalian systems present difficulties such as post-translational regulation of transcription factors, combinatorial gene regulation, and a paucity of available gene-knockout expression data. Additional evidence sources, such as DNA sequence-based identification of transcription factor binding sites, are needed. In this work, we computationally inferred a transcriptional network for TLR-stimulated murine macrophages. Our approach combined sequence scanning with time-course expression data in a probabilistic framework. Expression data were analyzed using the time-lagged correlation. A novel, unbiased method was developed to assess the significance of the time-lagged correlation. The inferred network of associations between transcription factor genes and co-expressed gene clusters was validated with targeted ChIP-on-chip experiments, and yielded insights into the macrophage activation program, including a potential novel regulator. Our general approach could be used to analyze other complex mammalian systems for which time-course expression data are available.
doi:10.1371/journal.pcbi.1000021
PMCID: PMC2265556  PMID: 18369420
10.  Quantitative genetics of age-related retinal degeneration: a second F1 intercross between the A/J and C57BL/6 strains 
Molecular Vision  2007;13:79-85.
Purpose
Previously, several quantitative trait loci (QTL) that influence age-related retinal degeneration (ageRD) were demonstrated in a cross between the C57BL/6J-c2J and BALB/cByJ strains (B x C). In this study, as a complementary approach to ongoing recombinant progeny testing for the purpose of identifying candidate quantitative trait genes (QTG), a second test cross using the A/J and the pigmented C57BL/6J strains (A x B) was carried out. The albino A/J strain was selected because it had the most amount of ageRD among several inbred strains tested, and the pigmented C57BL/6J strain was selected because along with its coisogenic counterpart C57BL/6J-c2J it had the least amount of ageRD. Thus, the effect of pigment on ageRD could be tested at the same time that the C57BL/6 genetic background was kept in common between the crosses from the two studies for the purpose of comparison.
Methods
A non-reciprocal F1 intercross between the A/J and C57BL/6J strains produced 170 F2 progeny. At 8 months of age after being maintained in relatively dim light, F2 mice, control mice and mice of other strains were evaluated for retinal degeneration by measurement of the thickness of the outer nuclear layer of the retina. The F2 mice were genotyped with dinucleotide repeat markers spanning the genome. Correlation of genotype with phenotype was made with Map Manager QTX software.
Results
Comparison of several strains of mice including the pigmented strains 129S1/SvImJ and C57BL/6J and the albino strains A/J, NZW/LacJ, BALB/cByJ and C57BL/6J-c2J, showed significant differences in ageRD. The greatest difference was between the albino A/J strain and the pigmented C57BL/6J strain. However, there was no significant difference between the pigmented C57BL/6J and its albino coisogenic counterpart C57BL/6J-c2J. Neither was there significant difference between the pigmented and albino F2 mice from the A x B cross. On the other hand, F2 males had a small but significantly lower amount of ageRD than females. Several QTL were identified in the A x B cross but surprisingly none of the 3 major QTL present in the original B x C cross (Chrs 6, 10, and 16) was present. There were minor QTL on proximal Chr 12 and proximal Chr 14 in common between the two crosses, and the proximal Chr 12 QTL was present in a previous light damage study involving the B and C strains. At least one sex-limited QTL was present on the X chromosome with a peak in a different location from that of a sex-limited QTL in the previous B x C study. In addition, the protective X allele was from the BALB/cByJ strain in the B x C cross and from C57BL/6J in the A x B cross. In both crosses, the C57BL/6J X-chromosome allele was recessive.
Conclusions
Significant differences were observed in ageRD among several inbred strains of mice maintained in relatively dim light. AgeRD was not influenced by pigment but was influenced by gender, albeit to a small degree. The presence of the same QTL in one light-induced and two ageRD studies suggests at least partial commonality in retinal degeneration pathways of different primary cause. However, the three main QTL present in the B x C cross were absent from the A x B cross. This suggests that the genetic determinants responsible for the greater sensitivity to ageRD of BALB/cByJ and A/J relative to C57BL/6J are not the same. This is supported by the presence of sex-limited X-chromosome QTL in the two crosses in which the C57BL/6J allele is protective relative to the A allele and sensitive relative to the C allele. The findings in the two studies of differing allelic relationships of QTG, and differing QTL aid the identification of candidate genes mapping to critical QTL. Identifying natural modifying genes that influence retinal degeneration resulting from any causal pathway, especially those QTG that are protective, will open avenues of study that may lead to broad based therapies for people suffering retinal degenerative diseases.
PMCID: PMC2503185  PMID: 17277741
11.  Gene therapy for established murine collagen-induced arthritis by local and systemic adenovirus-mediated delivery of interleukin-4 
Arthritis Research  2000;2(4):293-302.
To determine whether IL-4 is therapeutic in treating established experimental arthritis, a recombinant adenovirus carrying the gene that encodes murine IL-4 (Ad-mIL-4) was used for periarticular injection into the ankle joints into mice with established collagen-induced arthritis (CIA). Periarticular injection of Ad-mIL-4 resulted in a reduction in the severity of arthritis and joint swelling compared with saline- and adenoviral control groups. Local expression of IL-4 also reduced macroscopic signs of joint inflammation and bone erosion. Moreover, injection of Ad-mIL-4 into the hind ankle joints resulted in a decrease in disease severity in the untreated front paws. Systemic delivery of murine IL-4 by intravenous injection of Ad-mIL-4 resulted in a significant reduction in the severity of early-stage arthritis.
Introduction:
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that is characterized by joint inflammation, and progressive cartilage and bone erosion. Recent research has identified certain biologic agents that appear more able than conventional therapies to halt effectively the progression of disease, as well as ameliorate disease symptoms. One potential problem with the use of biologic agents for arthritis therapy is the need for daily or weekly repeat dosing. The transfer of genes directly to the synovial lining can theoretically circumvent the need for repeat dosing and reduce potential systemic side effects [1,2]. However, although many genes have been effective in treating murine CIA if administrated at a time before disease onset, local intra-articular or periarticular gene transfer has not been highly effective in halting the progression of established disease. IL-4, similar to tumor necrosis factor (TNF)-α and IL-1 inhibitors, has been shown be therapeutic for the treatment of murine CIA when administered intravenously as a recombinant protein, either alone or in combination with IL-10. IL-4 can downregulate the production of proinflammatory and T-helper (Th)1-type cytokines by inducing mRNA degradation and upregulating the expression of inhibitors of proinflammatory cytokines such as IL-1 receptor antagonist (IL-1Ra) [3,4]. IL-4 is able to inhibit IL-2 and IFN-γ production by Th1 cells, resulting in suppression of macrophage activation and the production of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNF-α by monocytes and macrophages [4,5,6,7,8,9].
Objective:
In order to examine the therapeutic effects of local and systemic IL-4 expression in established CIA, an adenoviral vector carrying the gene for murine IL-4 (Ad-mIL-4) was generated. The ability of Ad-mIL-4 to treat established CIA was evaluated by local periarticular and systemic intravenous injection of Ad-mIL-4 into mice at various times after disease onset.
Materials and methods:
Male DBA/1 lacJ (H-2q) mice, aged 7-8 weeks, were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were immunized intradermally at the base of tail with 100 μ g bovine type II collagen. On day 21 after priming, mice received a boost injection (intradermally) with 100 μ g type II collagen in incomplete adjuvant. For the synchronous onset of arthritis, 40 μ g lipopolysaccharide (Sigma, St Louis, MO, USA) was injected intraperitoneally on day 28. Ad-mIL-4 was injected periarticularly into the hind ankle joints of mice on day 32 or intravenously by tail vein injection on day 29. Disease severity was monitored every other day using an established macroscopic scoring system ranging from 0 to 4: 0, normal; 1, detectable arthritis with erythma; 2, significant swelling and redness; 3, severe swelling and redness from joint to digit; and 4, maximal swelling with ankylosis. The average of macroscopic score was expressed as a cumulative value for all paws, with a maximum possible score of 16 per mouse. Cytokine production by joint tissue or serum were assessed using enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN, USA).
Results:
To examine the therapeutic effects of IL-4 gene transfer in a murine model of arthritis, 5×108 particles of Ad-mIL-4 and enhanced green fluorescent protein (Ad-eGFP) were administered by periarticular injection into the ankle joints of mice with established disease 4 days after lipopolysaccharide injection. All mice had established disease at time of injection. As shown in Figure 1, the severity of arthritis (Fig. 1a), paw thickness (Fig. 1b), and the number of arthritic paws (Fig. 1c) were all significantly reduced in the Ad-mIL-4 group, compared with the saline- and Ad-eGFP-treated groups. Analysis of the bones in the ankle joints of control arthritic mice showed evidence of erosion with an associated monocytic infiltrate around the joint space compared with the Ad-mIL-4-treated and nonarthritic control joints. In addition, injection of the ankle joints in the hind legs resulted in a therapeutic effect in the front paws. A similar contralateral effect has been observed with adenoviral-mediated delivery of viral (v)-IL-10. Interestingly, a high level of murine IL-10 also was detected from the joint lysates of Ad-mIL-4-treated naïve and arthritic mice, with the production of endogenous IL-10 correlating with the dose of Ad-mIL-4. The administration of recombinant IL-4 protein systemically has been shown to be therapeutic in murine CIA models if given before disease onset. To examine the effect of systemic IL-4 delivered by gene transfer, 1×109 particles of Ad-mIL-4 were injected via the tail vein of collagen-immunized mice the day after lipopolysaccharide injection. Whereas the immunized control mice, injected with Ad-eGFP, showed disease onset on day 3 after lipopolysaccharide injection, Ad-mIL-4-treated mice showed a delay in disease onset and as a reduction in the total number of arthritic paws. Also, systemic injection of Ad-mIL-4 suppressed the severity of arthritis in CIA mice according to arthritis index.
Discussion:
Gene therapy represents a novel approach for delivery of therapeutic agents to joints in order to treat the pathologies associated with RA and osteoarthritis, as well as other disorders of the joints. In the present study we examined the ability of local periarticular and systemic gene transfer of IL-4 to treat established and early-stage murine CIA, respectively. We have demonstrated that both local and systemic administration of Ad-mIL-4 resulted in a reduction in the severity of arthritis, as well as in the number of arthritic paws. In addition, the local gene transfer of IL-4 reduced histologic signs of inflammation and of bone erosion. Interestingly, local delivery of Ad-mIL-4 was able to confer a therapeutic effect to the untreated, front paws through a currently unknown mechanism. In addition, both local and systemic expression of IL-4 resulted in an increase in the level of endogenous IL-10, as well as of IL-1Ra (data not shown). Previous experiments have shown that gene transfer of IL-10 and IL-1 and TNF inhibitors at the time of disease initiation (day 28) is therapeutic. However, delivery of these agents after disease onset appeared to have only limited therapeutic effect. In contrast, the present results demonstrate that IL-4, resulting from local periarticular and systemic injection of Ad-mIL-4, was able partially to reverse progression of established and early-stage disease, respectively. These results, as well as those of others, support the potential application of IL-4 gene therapy for the clinical treatment of RA.
PMCID: PMC17812  PMID: 11056670
adenoviral vectors; collagen-induced arthritis; gene therapy; IL-4; IL-10; rheumatoid arthritis
12.  Innate Immune Recognition of Yersinia pseudotuberculosis Type III Secretion 
PLoS Pathogens  2009;5(12):e1000686.
Specialized protein translocation systems are used by many bacterial pathogens to deliver effector proteins into host cells that interfere with normal cellular functions. How the host immune system recognizes and responds to this intrusive event is not understood. To address these questions, we determined the mammalian cellular response to the virulence-associated type III secretion system (T3SS) of the human pathogen Yersinia pseudotuberculosis. We found that macrophages devoid of Toll-like receptor (TLR) signaling regulate expression of 266 genes following recognition of the Y. pseudotuberculosis T3SS. This analysis revealed two temporally distinct responses that could be separated into activation of NFκB- and type I IFN-regulated genes. Extracellular bacteria were capable of triggering these signaling events, as inhibition of bacterial uptake had no effect on the ensuing innate immune response. The cytosolic peptidoglycan sensors Nod1 and Nod2 and the inflammasome component caspase-1 were not involved in NFκB activation following recognition of the Y. pseudotuberculosis T3SS. However, caspase-1 was required for secretion of the inflammatory cytokine IL-1β in response to T3SS-positive Y. pseudotuberculosis. In order to characterize the bacterial requirements for induction of this novel TLR-, Nod1/2-, and caspase-1-independent response, we used Y. pseudotuberculosis strains lacking specific components of the T3SS. Formation of a functional T3SS pore was required, as bacteria expressing a secretion needle, but lacking the pore-forming proteins YopB or YopD, did not trigger these signaling events. However, nonspecific membrane disruption could not recapitulate the NFκB signaling triggered by Y. pseudotuberculosis expressing a functional T3SS pore. Although host cell recognition of the T3SS did not require known translocated substrates, the ensuing response could be modulated by effectors such as YopJ and YopT, as YopT amplified the response, while YopJ dampened it. Collectively, these data suggest that combined recognition of the T3SS pore and YopBD-mediated delivery of immune activating ligands into the host cytosol informs the host cell of pathogenic challenge. This leads to a unique, multifactorial response distinct from the canonical immune response to a bacterium lacking a T3SS.
Author Summary
Most multicellular organisms have immune sensors that recognize molecules common among microorganisms. Recognition of such molecules informs the host that invading microbes are present, triggering an immune response. Many known innate immune sensors, however, do not appear to distinguish commensals from pathogens. This is in spite of the fact that the host must clear pathogens while simultaneously avoiding a response to benign or beneficial microbes. There are few molecular explanations for how this discrimination occurs in mammalian hosts. To address this problem, we analyzed the response of mammalian cells to the gut pathogen Yersinia pseudotuberculosis. We found that Yersinia expressing a virulence-associated secretion system caused a transcriptional response in host cells that was very different from the response to a strain with a nonfunctional version of the secretion system. This transcriptional response included several distinct signaling pathways leading to production of mediators of innate immunity, including cytokines such as type I interferon and TNF-α. A large number of pathogens express specialized secretion systems similar to that in Yersinia, so these findings provide evidence that there is a mammalian immune response to alterations in host cells that results from pathogen attack, supporting known systems for recognition of common microbial molecules.
doi:10.1371/journal.ppat.1000686
PMCID: PMC2779593  PMID: 19997504
13.  Genetic background of IL-10−/− mice alters host-pathogen interactions with Campylobacter jejuni and influences disease phenotype 
Microbial pathogenesis  2008;45(4):241-257.
We hypothesized that particular genetic backgrounds enhance rates of colonization, increase severity of enteritis, and allow for extraintestinal spread when inbred IL-10−/− mice are infected with pathogenic C. jejuni. Campylobacter jejuni stably colonized C57BL/6 and NOD mice, while congenic strains lacking IL-10 developed typhlocolitis following colonization that mimicked human campylobacteriosis. However, IL-10 deficiency alone was not necessary for presence of C. jejuni in extraintestinal sites. C3H/HeJtlr4−/− mice that specifically express the Cdcs1 allele showed colonization and limited extraintestinal spread without enteritis implicating this interval in the clinical presentation of C. jejuni infection. Furthermore, when the IL-10 gene is inactivated as in C3Birtlr4−/− IL-10−/− mice, enteritis and intensive extraintestinal spread were observed, suggesting that clinical presentations of C. jejuni infection are controlled by a complex interplay of factors. These data demonstrate that lack of IL-10 had a greater effect on C. jejuni induced colitis than other immune elements such as TLR4 (C3H/HeJ, C3Bir IL-10−/−), MHC H-2g7, diabetogenic genes, and CTLA-4 (NOD) and that host genetic background is in part responsible for disease phenotype. C3Bir IL-10−/− mice where Cdcs1 impairs gut barrier function provide a new murine model of C. jejuni and can serve as surrogates for immunocompromised patients with extraintestinal spread.
doi:10.1016/j.micpath.2008.05.010
PMCID: PMC4148907  PMID: 18586081
Campylobacter jejuni; enteritis; murine disease model; IL-10; Toll-like receptor 4; cytokine deficiency–induced colitis susceptibility 1 (Cdcs1) allele; Inflammatory Bowel Disease
14.  Mycobacterium tuberculosis Induces Interleukin-32 Production through a Caspase- 1/IL-18/Interferon-γ-Dependent Mechanism 
PLoS Medicine  2006;3(8):e277.
Background
Interleukin (IL)–32 is a newly described proinflammatory cytokine that seems likely to play a role in inflammation and host defense. Little is known about the regulation of IL-32 production by primary cells of the immune system.
Methods and Findings
In the present study, freshly obtained human peripheral blood mononuclear cells were stimulated with different Toll-like receptor (TLR) agonists, and gene expression and synthesis of IL-32 was determined. We demonstrate that the TLR4 agonist lipopolysaccharide induces moderate (4-fold) production of IL-32, whereas agonists of TLR2, TLR3, TLR5, or TLR9, each of which strongly induced tumor necrosis factor α and IL-6, did not stimulate IL-32 production. However, the greatest amount of IL-32 was induced by the mycobacteria Mycobacterium tuberculosis and M. bovis BCG (20-fold over unstimulated cells). IL-32-induced synthesis by either lipopolysaccharide or mycobacteria remains entirely cell-associated in monocytes; moreover, steady-state mRNA levels are present in unstimulated monocytes without translation into IL-32 protein, similar to other cytokines lacking a signal peptide. IL-32 production induced by M. tuberculosis is dependent on endogenous interferon-γ (IFNγ); endogenous IFNγ is, in turn, dependent on M. tuberculosis–induced IL-18 via caspase-1.
Conclusions
In conclusion, IL-32 is a cell-associated proinflammatory cytokine, which is specifically stimulated by mycobacteria through a caspase-1- and IL-18-dependent production of IFNγ.
Synthesis of IL-32, a cell-associated proinflammatory cytokine, was promoted by Mycobacterium tuberculosis and M. bovis, suggesting a role in a role in inflammation and host defense against tuberculosis.
Editors' Summary
Background.
Tuberculosis (TB) is a serious infectious disease that is becoming more common. Worldwide it causes around 2 million deaths every year, mostly in developing countries. Some 2 billion people—or one-third of the world's population—are chronically infected without active symptoms. In humans the disease is usually caused by a bacterium called Mycobacterium tuberculosis. Another related bacterium, Mycobacterium bovis, causes TB in cattle and sometimes in people.
The immune system, which defends the body against infections, involves a number of cells (for example, the white blood cells) and also chemicals. People with defects in their immune system are more likely to suffer from infectious diseases. Cytokines are one class of chemicals in the immune system. A particular cytokine called interleukin-32 (IL-32) has been shown to play a role in the development of inflammation, which is a part of the body's response to infection. Previous research has suggested that IL-32 might be of particular importance in the defenses against TB. In recent years scientists have discovered a lot about the processes involved in the “switching on” of the individual parts of the immune system in response to infection. However, very little is known about the factors influencing the switching on of production of IL-32.
Why Was This Study Done?
It would be useful to know more about the production of IL-32 because it would advance understanding of the immune system in general and, more specifically, how the body protects itself against bacteria, such as those that cause TB.
What Did the Researchers Do and Find?
Working with eight healthy volunteers, the researchers took white blood cells of a particular type (peripheral blood mononuclear cells) and exposed them to substances known as TLR agonists. Toll-like receptors (TLRs) are receptors on the surface of leukocytes that recognize specific components of microorganisms. Upon recognition of these microbial components, which function as TLR stimuli (or TLR agonists), signals are transmitted that activate the immune system and thus the host defense. Using a complex series of laboratory procedures, they found that one type of TLR agonist (known as LPS) produced a big increase in IL-32 production, whereas all the other types of TLR agonists that they used produced only small increases. The researchers tested M. tuberculosis and M. bovis bacteria to see whether they increased IL-32 production and they found that they did so, to a greater degree even than LPS. The researchers also learned other details about IL-32 and the pathway of chemical changes that eventually leads to its production.
What Do These Findings Mean?
The researchers say their study provides several important insights into the biology of IL-32. The findings confirm that IL-32 is an important factor in the body's defenses against TB. This information will help in understanding how the disease spreads and who is most vulnerable to it. Ultimately, it may assist in the search for new ways of treating and preventing the disease.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030277.
The online encyclopedia Wikpedia has useful information on tuberculosis
Wikipedia also has useful information on the immune system
More detailed information about international efforts to control TB may be found at the Web sites of the International Union Against Tuberculosis and Lung Disease and the World Health Organization's Stop TB Department
doi:10.1371/journal.pmed.0030277
PMCID: PMC1539091  PMID: 16903774
15.  Different effects of the Cdx1 and Cdx2 homeobox genes in a murine model of intestinal inflammation 
Gut  2007;56(12):1688-1695.
Aims
The CDX1 and CDX2 homeoproteins are intestine‐specific transcription factors regulating homeostasis. We investigated their relevance in experimentally‐induced intestinal inflammation.
Methods
The response to intestinal inflammation induced by dextran sodium sulfate (DSS) was compared in wild type, Cdx1‐/‐ and Cdx2+/− mice. Intestinal permeability was determined in wild type and Cdx2+/− mice. Protein‐protein interactions were investigated by co‐immunoprecipitation and GST‐pulldown, and their functional consequences were assessed using Luciferase reporter systems.
Results
Heterozygous Cdx2+/− mice, but not Cdx1‐/‐ mice, were hypersensitive to DSS‐induced acute inflammation as all these mice showed blood in the stools at day 1 of DSS treatment. Hypersensitivity was associated to a 50% higher intestinal permeability. In Cdx2+/‐ mice, the colonic epithelium was repaired during the week after the end of DSS treatment, whereas two weeks were required for wild type animals. Subsequently, no colonic tumour was observed in Cdx2+/− mice subjected to 5 repeated cycles of DSS, in contrast to the 2.7 tumours found per wild type mouse. Based on the fact that Smad3+/− mice, like Cdx2+/− mice, better repair the damaged intestinal epithelium, we found that the CDX2 protein interacts with SMAD3, independently of SMAD4, resulting in a 5‐fold stimulation of SMAD3 transcriptional activity. CDX1 also interacted with SMAD3 but it inhibited by 10‐fold the SMAD3/SMAD4‐dependent transcription.
Conclusion
The Cdx1 and Cdx2 homeobox genes have distinct effects on the outcome of a pro‐inflammatory challenge. This is mirrored by different functional interactions of the CDX1 and CDX2 proteins with SMAD3, a major element of the TGFβ signalling pathway.
doi:10.1136/gut.2007.125542
PMCID: PMC2095714  PMID: 17595234
Inflammation; cancer; homeobox gene; Cdx; Smad
16.  Transcriptional Analysis of Murine Macrophages Infected with Different Toxoplasma Strains Identifies Novel Regulation of Host Signaling Pathways 
PLoS Pathogens  2013;9(12):e1003779.
Most isolates of Toxoplasma from Europe and North America fall into one of three genetically distinct clonal lineages, the type I, II and III lineages. However, in South America these strains are rarely isolated and instead a great variety of other strains are found. T. gondii strains differ widely in a number of phenotypes in mice, such as virulence, persistence, oral infectivity, migratory capacity, induction of cytokine expression and modulation of host gene expression. The outcome of toxoplasmosis in patients is also variable and we hypothesize that, besides host and environmental factors, the genotype of the parasite strain plays a major role. The molecular basis for these differences in pathogenesis, especially in strains other than the clonal lineages, remains largely unexplored. Macrophages play an essential role in the early immune response against T. gondii and are also the cell type preferentially infected in vivo. To determine if non-canonical Toxoplasma strains have unique interactions with the host cell, we infected murine macrophages with 29 different Toxoplasma strains, representing global diversity, and used RNA-sequencing to determine host and parasite transcriptomes. We identified large differences between strains in the expression level of known parasite effectors and large chromosomal structural variation in some strains. We also identified novel strain-specifically regulated host pathways, including the regulation of the type I interferon response by some atypical strains. IFNβ production by infected cells was associated with parasite killing, independent of interferon gamma activation, and dependent on endosomal Toll-like receptors in macrophages and the cytoplasmic receptor retinoic acid-inducible gene 1 (RIG-I) in fibroblasts.
Author Summary
Toxoplasmosis is caused by the protozoan parasite Toxoplasma gondii. The parasite is found throughout the world. When humans are infected, few have symptoms because a healthy immune system usually prevents the parasite from causing illness. Nevertheless, cases of severe disease in otherwise healthy individuals have been observed. These cases are usually a result of infection with less common atypical strains of Toxoplasma. Factors associated with virulence in the atypical strains are not well understood. Here, we infected host cells with 29 different strains of Toxoplasma, and performed high-throughput RNA sequencing of both host cells and parasites. We found significant differences in gene expression profiles between strains. Host cell transcriptional response also varied substantially depending on the infecting strain. Specifically, we found that a small group of atypical strains are able to induce production of type I interferons, which are immunomodulatory cytokines. Interferon production is a result of the elimination of internalized parasites through a novel killing mechanism. The dataset we generated is a valuable tool for identification of host cell targets of Toxoplasma secreted effectors and can contribute to our understanding of why certain Toxoplasma strains are more prone to cause severe disease in humans.
doi:10.1371/journal.ppat.1003779
PMCID: PMC3868521  PMID: 24367253
17.  A Role for microRNA-155 Modulation in the Anti-HIV-1 Effects of Toll-Like Receptor 3 Stimulation in Macrophages 
PLoS Pathogens  2012;8(9):e1002937.
HIV-1 infection of macrophages plays a key role in viral pathogenesis and progression to AIDS. Polyinosine-polycytidylic acid (poly(I∶C); a synthetic analog of dsRNA) and bacterial lipopolysaccharide (LPS), the ligands for Toll-like receptors (TLR) TLR3 and TLR4, respectively, are known to decrease HIV-1 infection in monocyte-derived macrophages (MDMs), but the mechanism(s) are incompletely understood. We found that poly(I∶C)- and LPS-stimulation of MDMs abrogated infection by CCR5-using, macrophage-tropic HIV-1, and by vesicular stomatitis virus glycoprotein-pseudotyped HIV-1 virions, while TLR2, TLR7 or TLR9 agonists only partially reduced infection to varying extent. Suppression of infection, or lack thereof, did not correlate with differential effects on CD4 or CCR5 expression, type I interferon induction, or production of pro-inflammatory cytokines or β-chemokines. Integrated pro-viruses were readily detected in unstimulated, TLR7- and TLR9-stimulated cells, but not in TLR3- or TLR4-stimulated MDMs, suggesting the alteration of post-entry, pre-integration event(s). Using microarray analysis and quantitative reverse transcription (RT)-PCR, we found increased microRNA (miR)-155 levels in MDMs upon TLR3/4- but not TLR7-stimulation, and a miR-155 specific inhibitor (but not a scrambled control) partially restored infectivity in poly(I∶C)-stimulated MDMs. Ectopic miR-155 expression remarkably diminished HIV-1 infection in primary MDMs and cell lines. Furthermore, poly(I∶C)-stimulation and ectopic miR-155 expression did not alter detection of early viral RT products, but both resulted in an accumulation of late RT products and in undetectable or extremely low levels of integrated pro-viruses and 2-LTR circles. Reduced mRNA and protein levels of several HIV-1 dependency factors involved in trafficking and/or nuclear import of pre-integration complexes (ADAM10, TNPO3, Nup153, LEDGF/p75) were found in poly(I∶C)-stimulated and miR-155-transfected MDMs, and a reporter assay suggested they are authentic miR-155 targets. Our findings provide evidence that miR-155 exerts an anti-HIV-1 effect by targeting several HIV-1 dependency factors involved in post-entry, pre-integration events, leading to severely diminished HIV-1 infection.
Author Summary
The infection of macrophages by HIV-1 is a crucial event in the pathogenesis of AIDS. Toll-like receptors (TLR) are a family of receptors present in macrophages – among other cells – that detect various components of microbes and trigger host defenses. It is known that stimulation of macrophages through TLR3 or TLR4 reduces their susceptibility to HIV-1 infection, but the mechanism is not well understood. Here we show for the first time in primary human macrophages that TLR3 and TLR4 but not other TLRs induce higher levels of microRNA-155 – a key regulator of inflammatory and immune responses – and that microRNA-155 has a remarkable anti-HIV-1 effect. MicroRNAs are small, non-coding RNAs that bind to target mRNAs based on sequence complementarity, and lead to reduced protein output. We also show that the anti-HIV-1 effects of microRNA-155 seem to be mediated through targeting the mRNAs of several cellular proteins needed by the virus for trafficking and/or nuclear import of the viral DNA, which is required for integration into the host DNA and successful infection. These studies provide evidence of novel microRNA-155 targets and may serve as the basis for an innovative approach to reduce cellular susceptibility to HIV-1 infection.
doi:10.1371/journal.ppat.1002937
PMCID: PMC3447756  PMID: 23028330
18.  Inter-Strain Differences in Liver Injury and One-Carbon Metabolism in Alcohol-Fed Mice 
Hepatology (Baltimore, Md.)  2012;56(1):130-139.
Alcoholic liver injury is a major public health issue worldwide. Even though the major mechanisms of this disease have been established over the past decades, little is known about genetic susceptibility factors that may predispose individuals who abuse alcoholic beverages to liver damage and subsequent pathological conditions. We hypothesized that a panel of genetically diverse mouse strains may be used to examine the role of ER stress and one-carbon metabolism in the mechanism of inter-individual variability in alcoholic liver injury. We administered alcohol (up to 27 mg/kg/d) in high fat diet using intragastric intubation model for 28 days to male mice from 14 inbred strains (129S1/SvImJ, AKR/J, BALB/cJ, BALB/cByJ, BTBR T+tf/J, C3H/HeJ, C57BL/10J, DBA/2J, FVB/NJ, KK/HIJ, MOLF/EiJ, NZW/LacJ, PWD/PhJ, and WSB/EiJ). Profound inter-strain differences (more than 3-fold) in alcohol-induced steatohepatitis were observed among the strains in spite of consistently high levels of urine alcohol that was monitored throughout the study. We found that endoplasmic reticulum stress genes were induced only in strains with the highest liver injury. Liver glutathione and methyl donor levels were affected in all strains, albeit to a different degree. Most pronounced effects that were closely associated with the degree of liver injury were hyperhomocysteinemia and strain-dependent differences in expression patterns of one-carbon metabolism-related genes.
Conclusion
Our data demonstrate that strain differences in alcohol-induced liver injury and steatosis are striking and independent of alcohol exposure and the most severely affected strains exhibit major differences in the expression of ER stress markers and genes of one-carbon metabolism.
doi:10.1002/hep.25641
PMCID: PMC3350836  PMID: 22307928
alcohol-induced liver injury; homocysteine; methyl donors; endoplasmic reticulum stress
19.  A Novel Mouse Model of Campylobacter jejuni Gastroenteritis Reveals Key Pro-inflammatory and Tissue Protective Roles for Toll-like Receptor Signaling during Infection 
PLoS Pathogens  2014;10(7):e1004264.
Campylobacter jejuni is a major source of foodborne illness in the developed world, and a common cause of clinical gastroenteritis. Exactly how C. jejuni colonizes its host's intestines and causes disease is poorly understood. Although it causes severe diarrhea and gastroenteritis in humans, C. jejuni typically dwells as a commensal microbe within the intestines of most animals, including birds, where its colonization is asymptomatic. Pretreatment of C57BL/6 mice with the antibiotic vancomycin facilitated intestinal C. jejuni colonization, albeit with minimal pathology. In contrast, vancomycin pretreatment of mice deficient in SIGIRR (Sigirr−/−), a negative regulator of MyD88-dependent signaling led to heavy and widespread C. jejuni colonization, accompanied by severe gastroenteritis involving strongly elevated transcription of Th1/Th17 cytokines. C. jejuni heavily colonized the cecal and colonic crypts of Sigirr−/− mice, adhering to, as well as invading intestinal epithelial cells. This infectivity was dependent on established C. jejuni pathogenicity factors, capsular polysaccharides (kpsM) and motility/flagella (flaA). We also explored the basis for the inflammatory response elicited by C. jejuni in Sigirr−/− mice, focusing on the roles played by Toll-like receptors (TLR) 2 and 4, as these innate receptors were strongly stimulated by C. jejuni. Despite heavy colonization, Tlr4−/−/Sigirr−/− mice were largely unresponsive to infection by C. jejuni, whereas Tlr2−/−/Sigirr−/− mice developed exaggerated inflammation and pathology. This indicates that TLR4 signaling underlies the majority of the enteritis seen in this model, whereas TLR2 signaling had a protective role, acting to promote mucosal integrity. Furthermore, we found that loss of the C. jejuni capsule led to increased TLR4 activation and exaggerated inflammation and gastroenteritis. Together, these results validate the use of Sigirr−/− mice as an exciting and relevant animal model for studying the pathogenesis and innate immune responses to C. jejuni.
Author Summary
Research into the key virulence strategies of the bacterial pathogen Campylobacter jejuni, as well as the host immune responses that develop against this microbe have, in many ways, been limited by the lack of relevant animal models. Here we describe the use of Sigirr deficient (−/−) mice as a model for C. jejuni pathogenesis. Not only do Sigirr−/− mice develop significant intestinal inflammation in response to colonization by C. jejuni, but the ability of this pathogen to trigger gastroenteritis was dependent on key virulence factors. We also found that the induction of the inflammatory and Th1/Th17 immune responses to infection in these mice depended on specific Toll-like receptors, principally TLR4, which we identified as the main driver of inflammation. In contrast, TLR2 signaling was found to protect mucosal integrity, with Tlr2−/−/Sigirr−/− mice suffering exaggerated mucosal damage and inflammation. Notably, we found that C. jejuni's capsule helped conceal it from the host's immune system as its loss led to significantly increased activation of host TLRs and exaggerated gastroenteritis. Our research shows that the increased sensitivity of Sigirr−/− mice can be used to generate a unique and exciting model that facilitates the study of C. jejuni pathogenesis as well as host immunity to this enteric pathogen.
doi:10.1371/journal.ppat.1004264
PMCID: PMC4102570  PMID: 25033044
20.  Expression of the Hypothalamic Transcription Factor Nhlh2 is Dependent on Energy Availability 
Journal of neuroendocrinology  2007;19(7):499-510.
Mice with a deletion of the hypothalamic basic helix-loop-helix transcription factor Nhlh2 display adult onset obesity, implicating Nhlh2 in the neuronal circuits regulating energy availability. Nhlh2 colocalises with the hypothalamic thyrotrophin-releasing hormone (TRH) neurones in the paraventricular nucleus (PVN) and pro-opiomelanocortin (POMC) neurones in the arcuate nucleus. We show that Nhlh2 expression is significantly reduced in response to 24-h food deprivation in the arcuate nucleus, PVN, lateral hypothalamus, ventromedial hypothalamus (VMH) and dorsomedial hypothalamus (DMH). Food intake for 2 h following deprivation stimulates Nhlh2 expression in the arcuate nucleus and the PVN, and leptin injection following deprivation results in increased Nhlh2 expression in the arcuate nucleus, PVN, lateral hypothalamus, VMH, and DMH. Hypothalamic Nhlh2 expression in response to leptin injection is maximal by 2 h. Following leptin injection, Nhlh2 mRNA colocalises in POMC neurones in the arcuate nucleus and TRH neurones in the PVN. Nhlh2 mRNA expression in POMC neurones in the arcuate nucleus and TRH neurones in the PVN is reduced with energy deprivation and is stimulated with food intake and leptin injection. Modulation of POMC expression in response to changes in energy availability is not affected in mice with a targeted deletion of Nhlh2. However, deletion of Nhlh2 does result in loss of normal TRH mRNA expression in mice exposed to food deprivation and leptin stimulation. These data implicate Nhlh2 as a regulatory target of the leptin-mediated energy availability network of the hypothalamus, and TRH as a putative downstream target of Nhlh2.
doi:10.1111/j.1365-2826.2007.01556.x
PMCID: PMC3111914  PMID: 17532796
nescient helix-loop-helix 2; NSCL-2; Hen2; POMC; TRH; leptin
21.  Key Role of Splenic Myeloid DCs in the IFN-αβ Response to Adenoviruses In Vivo 
PLoS Pathogens  2008;4(11):e1000208.
The early systemic production of interferon (IFN)-αβ is an essential component of the antiviral host defense mechanisms, but is also thought to contribute to the toxic side effects accompanying gene therapy with adenoviral vectors. Here we investigated the IFN-αβ response to human adenoviruses (Ads) in mice. By comparing the responses of normal, myeloid (m)DC- and plasmacytoid (p)DC-depleted mice and by measuring IFN-αβ mRNA expression in different organs and cells types, we show that in vivo, Ads elicit strong and rapid IFN-αβ production, almost exclusively in splenic mDCs. Using knockout mice, various strains of Ads (wild type, mutant and UV-inactivated) and MAP kinase inhibitors, we demonstrate that the Ad-induced IFN-αβ response does not require Toll-like receptors (TLR), known cytosolic sensors of RNA (RIG-I/MDA-5) and DNA (DAI) recognition and interferon regulatory factor (IRF)-3, but is dependent on viral endosomal escape, signaling via the MAP kinase SAPK/JNK and IRF-7. Furthermore, we show that Ads induce IFN-αβ and IL-6 in vivo by distinct pathways and confirm that IFN-αβ positively regulates the IL-6 response. Finally, by measuring TNF-α responses to LPS in Ad-infected wild type and IFN-αβR−/− mice, we show that IFN-αβ is the key mediator of Ad-induced hypersensitivity to LPS. These findings indicate that, like endosomal TLR signaling in pDCs, TLR-independent virus recognition in splenic mDCs can also produce a robust early IFN-αβ response, which is responsible for the bulk of IFN-αβ production induced by adenovirus in vivo. The signaling requirements are different from known TLR-dependent or cytosolic IFN-αβ induction mechanisms and suggest a novel cytosolic viral induction pathway. The hypersensitivity to components of the microbial flora and invading pathogens may in part explain the toxic side effects of adenoviral gene therapy and contribute to the pathogenesis of adenoviral disease.
Author Summary
Adenoviruses (Ads) are important pathogens and promising vectors for gene therapy applications. In the course of adenoviral infections innate immune responses are activated, which can be beneficial for the antiviral host defense but also detrimental if activated in a deregulated manner. Type I IFNs are crucial for the innate immune control of various viral infections in the mammalian host. So far, the early, systemic release of IFN-αβ during viral infections has been attributed to specialized immune cells, the plasmacytoid dendritic cells. Here, in a mouse infection model, we show that wild type Ads, as well as adenoviral vectors, elicit rapid IFN-αβ production almost exclusively in another cell population, the splenic myeloid dendritic cells. This IFN-αβ storm depends on viral escape from endosomes to the cytosol and the requirements of the response are suggestive of a novel viral induction pathway. Furthermore, we show that virus induced IFN-αβ is the key mediator of Ad-induced hypersensitivity to the cytokine-inducing and toxic activity of lipopolysaccharide, a common constituent of Gram-negative bacteria. Since these bacteria comprise several commensals and pathogens, enhanced susceptibility to lipopolysaccharide may contribute to toxic reactions observed during adenoviral gene therapy and to the clinical symptoms of adenoviral diseases.
doi:10.1371/journal.ppat.1000208
PMCID: PMC2576454  PMID: 19008951
22.  Paracrine Hedgehog signaling in stomach and intestine: new roles for Hedgehog in gastrointestinal patterning 
Gastroenterology  2009;137(2):618-628.
Background & Aims
Hedgehog signaling is critical in gastrointestinal patterning. Mice deficient in Hedgehog signaling exhibit abnormalities that mirror deformities seen in the human VACTERL (vertebral, anal, cardiac, tracheal, esophageal, renal, limb) association. However, the direction of Hedgehog signal flow is controversial and the cellular targets of Hedgehog signaling change with time during development. We profiled cellular Hedgehog response patterns from embryonic day 10.5 (E10.5) to adult in murine antrum, pyloric region, small intestine and colon.
Methods
Hedgehog signaling was profiled using Hedgehog pathway reporter mice and in situ hybridization. Cellular targets were identified by immunostaining. Ihh-overexpressing transgenic animals were generated and analyzed.
Results
Hedgehog signaling is strictly paracrine from antrum to colon throughout embryonic and adult life. Novel findings include: mesothelial cells of the serosa transduce Hedgehog signals in fetal life; the hindgut epithelium expresses Ptch but not Gli1 at E10.5; the two layers of the muscularis externa respond differently to Hedgehog signals; organogenesis of the pyloric sphincter is associated with robust Hedgehog signaling; dramatically different Hedgehog responses characterize stomach and intestine at E16; after birth, the muscularis mucosa and villus smooth muscle (SM) consist primarily of Hedgehog responsive cells and Hh levels actively modulate villus core SM.
Conclusions
These studies reveal a previously unrecognized association of paracrine Hedgehog signaling with several gastrointestinal patterning events involving the serosa, pylorus and villus smooth muscle. The results may have implications for several human anomalies and could potentially expand the spectrum of the human VACTERL association.
doi:10.1053/j.gastro.2009.05.002
PMCID: PMC2717174  PMID: 19445942
23.  Deletion of Nhlh2 Results in a Defective Torpor Response and Reduced Beta Adrenergic Receptor Expression in Adipose Tissue 
PLoS ONE  2010;5(8):e12324.
Background
Mice with a targeted deletion of the basic helix-loop-helix transcription factor, Nescient Helix-Loop-Helix 2 (Nhlh2), display adult-onset obesity with significant increases in their fat depots, abnormal responses to cold exposure, and reduced spontaneous physical activity levels. These phenotypes, accompanied by the hypothalamic expression of Nhlh2, make the Nhlh2 knockout (N2KO) mouse a useful model to study the role of central nervous system (CNS) control on peripheral tissue such as adipose tissue.
Methodology
Differences in body temperature and serum analysis of leptin were performed in fasted and ad lib fed wild-type (WT) and N2KO mice. Histological analysis of white (WAT) and brown adipose tissue (BAT) was performed. Gene and protein level expression of inflammatory and metabolic markers were compared between the two genotypes.
Principal Findings
We report significant differences in serum leptin levels and body temperature in N2KO mice compared with WT mice exposed to a 24-hour fast, suggestive of a defect in both white (WAT) and brown adipose tissue (BAT) function. As compared to WT mice, N2KO mice showed increased serum IL-6 protein and WAT IL-6 mRNA levels. This was accompanied by slight elevations of mRNA for several macrophage markers, including expression of macrophage specific protein F4/80 in adipose, suggestive of macrophage infiltration of WAT in the mutant animals. The mRNAs for β3-adrenergic receptors (β3-AR), β2-AR and uncoupling proteins were significantly reduced in WAT and BAT from N2KO mice compared with WT mice.
Conclusions
These studies implicate Nhlh2 in the central control of WAT and BAT function, with lack of Nhlh2 leading to adipose inflammation and altered gene expression, impaired leptin response to fasting, all suggestive of a deficient torpor response in mutant animals.
doi:10.1371/journal.pone.0012324
PMCID: PMC2925948  PMID: 20808804
24.  Nos2 Inactivation Promotes the Development of Medulloblastoma in Ptch1+/− Mice by Deregulation of Gap43–Dependent Granule Cell Precursor Migration 
PLoS Genetics  2012;8(3):e1002572.
Medulloblastoma is the most common malignant brain tumor in children. A subset of medulloblastoma originates from granule cell precursors (GCPs) of the developing cerebellum and demonstrates aberrant hedgehog signaling, typically due to inactivating mutations in the receptor PTCH1, a pathomechanism recapitulated in Ptch1+/− mice. As nitric oxide may regulate GCP proliferation and differentiation, we crossed Ptch1+/− mice with mice lacking inducible nitric oxide synthase (Nos2) to investigate a possible influence on tumorigenesis. We observed a two-fold higher medulloblastoma rate in Ptch1+/− Nos2−/− mice compared to Ptch1+/− Nos2+/+ mice. To identify the molecular mechanisms underlying this finding, we performed gene expression profiling of medulloblastomas from both genotypes, as well as normal cerebellar tissue samples of different developmental stages and genotypes. Downregulation of hedgehog target genes was observed in postnatal cerebellum from Ptch1+/+ Nos2−/− mice but not from Ptch1+/− Nos2−/− mice. The most consistent effect of Nos2 deficiency was downregulation of growth-associated protein 43 (Gap43). Functional studies in neuronal progenitor cells demonstrated nitric oxide dependence of Gap43 expression and impaired migration upon Gap43 knock-down. Both effects were confirmed in situ by immunofluorescence analyses on tissue sections of the developing cerebellum. Finally, the number of proliferating GCPs at the cerebellar periphery was decreased in Ptch1+/+ Nos2−/− mice but increased in Ptch1+/− Nos2−/− mice relative to Ptch1+/− Nos2+/+ mice. Taken together, these results indicate that Nos2 deficiency promotes medulloblastoma development in Ptch1+/− mice through retention of proliferating GCPs in the external granular layer due to reduced Gap43 expression. This study illustrates a new role of nitric oxide signaling in cerebellar development and demonstrates that the localization of pre-neoplastic cells during morphogenesis is crucial for their malignant progression.
Author Summary
Medulloblastoma is a common pediatric brain tumor, a subtype of which is driven by aberrant hedgehog pathway activation in cerebellar granule cell precursors. Although this tumor etiology has been intensively investigated in the well-established Ptch1+/− mouse model, knowledge is still lacking about the molecular interactions between neoplastic transformation and other developmental processes. Nitric oxide (NO) has been reported to be involved in controlling proliferation and differentiation of these cells. Therefore, inactivation of the NO–producing enzyme Nos2 in combination with the mutated Ptch1 gene should provide insight into how developmental regulation influences pathogenesis. Here, we describe a new role for NO in developing neuronal precursors of the cerebellum facilitating physiologically accurate migration via regulation of Gap43. We further demonstrate that disturbance of these processes leads to retention of granule precursor cells to the cerebellar periphery. Together with the sustained proliferation of these cells in combined Ptch1+/− Nos2−/− mice, this effect results in an increased medulloblastoma incidence relative to Ptch1+/− mice and demonstrates a new disease-promoting mechanism in this tumor entity.
doi:10.1371/journal.pgen.1002572
PMCID: PMC3305407  PMID: 22438824
25.  IRAK-2 Regulates IL-1-Mediated Pathogenic Th17 Cell Development in Helminthic Infection 
PLoS Pathogens  2011;7(10):e1002272.
Infection with the trematode parasite Schistosoma mansoni results in distinct heterogeneity of disease severity both in humans and in mice. In the experimental mouse model, severe disease is characterized by pronounced hepatic egg-induced granulomatous inflammation mediated by CD4 Th17 cells, whereas mild disease is associated with reduced hepatic inflammation in a Th2-skewed cytokine environment. Even though the host’s genetic background significantly impacts the clinical outcome of schistosomiasis, specific gene(s) that contribute to disease severity remain elusive. We investigated the schistosome infection in wild-derived mice, which possess a more diverse gene pool than classically inbred mouse strains and thus makes them more likely to reveal novel mechanisms of immune regulation. We now show that inbred wild-derived MOLF mice develop severe hepatic inflammation with high levels of IL-17. Congenic mice with a MOLF locus in chromosome 6, designated Why1, revealed high pathology and enabled the identification of Irak2 as the pathogenic gene. Although IRAK-2 is classically associated with TLR signaling, adoptive transfer of CD4 T cells revealed that IRAK-2 mediates pathology in a CD4 T cell specific manner by promoting Th17 cell development through enhancement of IL-1β-induced activation of transcription factors RORγt and BATF. The use of wild-derived mice unravels IRAK-2 as a novel regulator of IL-1-induced pathogenic Th17 cells in schistosomiasis, which likely has wide-ranging implications for other chronic inflammatory and autoimmune diseases.
Author Summary
Schistosomes are trematode helminths that cause widespread disease in vertebrates and are responsible for over 200 million human infections worldwide. The species Schistosoma mansoni causes a hepatic granulomatous inflammatory and fibrosing reaction against tissue trapped parasite eggs that varies greatly in humans and among mouse strains, implying that the host’s genetic background plays a critical role in determining disease severity. Although exacerbated hepatic inflammation is known to be associated with an increase in CD4 Th17 cells, specific genes conducive to high pathology are unknown. In this study we used genetically diverse inbred wild-derived mice and found that their natural severe immunopathology and high IL-17 levels are regulated by the interleukin-1 (IL-1) receptor-associated kinase-like 2 (IRAK-2). We demonstrate that T cell intrinsic IRAK-2 affects disease severity by enhancing the development of Th17 cells, which results from an increased sensitivity to IL-1β induced activation of the lineage-specific transcription factors RORγt and BATF. Our findings thus identify IRAK-2 as a single regulator of pathogenic Th17 cell development in murine schistosomiasis and reveal a novel mechanism that is likely to operate in other chronic inflammatory and autoimmune diseases.
doi:10.1371/journal.ppat.1002272
PMCID: PMC3188523  PMID: 21998578

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