Geethanjali Pickert

Activation of epithelial STAT3 regulates intestinal homeostasis

The intestinal epithelium that lines the mucosal surface along the GI-tract is a key player for the intestinal homeostasis of the healthy individual. In case of a mucosal damage or a barrier defect as seen in patients with inflammatory bowel disease, the balance is disturbed, and translocation of intestinal microbes to the submucosa is facilitated. We recently demonstrated a pivotal role of STAT3 activation in intestinal epithelial cells (IEC) for the restoration of the balance at the mucosal surface of the gut in an experimental colitis model. STAT3 was rapidly induced in intestinal epithelial cells upon challenge of mice in both experimental colitis and intestinal wound healing models. STAT3 activation was found to be dispensable in the steady-state conditions but was important for efficient regeneration of the epithelium in response to injury. Here, we extend our previous findings by showing epithelial STAT3 activation in human patients suffering from IBD and provide additional insights how the activation of epithelial STAT3 by IL-22 regulates intestinal homeostasis and mucosal wound healing. We also demonstrate that antibody-mediated neutralization of IL-22 has little impact on the development of experimental colitis in mice, but significantly delays recovery from colitis. Thus, our data suggest that targeting the STAT3 signalling pathway in IEC is a promising therapeutic approach in situations when the intestinal homeostasis is disturbed, e.g. as seen in Crohn´s disease or Ulcerative colitis.

Inhibition of GTP cyclohydrolase attenuates tumor growth by reducing angiogenesis and M2-like polarization of tumor associated macrophages.

GTP cyclohydrolase (GCH1) is the key‐enzyme to produce the essential enzyme cofactor, tetrahydrobiopterin. The byproduct, neopterin is increased in advanced human cancer and used as cancer‐biomarker, suggesting that pathologically increased GCH1 activity may promote tumor growth. We found that inhibition or silencing of GCH1 reduced tumor cell proliferation and survival and the tube formation of human umbilical vein endothelial cells, which upon hypoxia increased GCH1 and endothelial NOS expression, the latter prevented by inhibition of GCH1. In nude mice xenografted with HT29‐Luc colon cancer cells GCH1 inhibition reduced tumor growth and angiogenesis, determined by in vivo luciferase and near‐infrared imaging of newly formed blood vessels. The treatment with the GCH1 inhibitor shifted the phenotype of tumor associated macrophages from the proangiogenic M2 towards M1, accompanied with a shift of plasma chemokine profiles towards tumor‐attacking chemokines including CXCL10 and RANTES. GCH1 expression was increased in mouse AOM/DSS‐induced colon tumors and in high grade human colon and skin cancer and oppositely, the growth of GCH1‐deficient HT29‐Luc tumor cells in mice was strongly reduced. The data suggest that GCH1 inhibition reduces tumor growth by (i) direct killing of tumor cells, (ii) by inhibiting angiogenesis, and (iii) by enhancing the antitumoral immune response.

Inhibitor κB Kinase β Deficiency in Primary Nociceptive Neurons Increases TRP Channel Sensitivity

Inhibitor κB kinase (IKK) regulates the activity of the transcription factor nuclear factor-κ B that normally protects neurons against excitotoxicity. Constitutively active IKK is enriched at axon initial segments and nodes of Ranvier (NR). We used mice with a Cre–loxP-mediated specific deletion of IKKβ in sensory neurons of the dorsal root ganglion (SNS–IKKβ−/−) to evaluate whether IKK plays a role in sensory neuron excitability and nociception. We observed increased sensitivity to mechanical, cold, noxious heat and chemical stimulation in SNS–IKKβ−/− mice, with normal proprioceptive and motor functions as revealed by gait analysis. This was associated with increased calcium influx and increased inward currents in small- and medium-sized primary sensory neurons of SNS–IKKβ−/− mice during stimulation with capsaicin or Formalin, specific activators of transient receptor potentials TRPV1 and TRPA1 calcium channels, respectively. In vitro stimulation of saphenous nerve preparations of SNS–IKKβ−/− mice showed increased neuronal excitability of A- and C-fibers but unchanged A- and C-fiber conduction velocities, normal voltage-gated sodium channel currents, and normal accumulation of ankyrin G and the sodium channels Nav1.6 at NR. The results suggest that IKKβ functions as a negative modulator of sensory neuron excitability, mediated at least in part by modulation of TRP channel sensitivity.

Dichotomy of CCL21 and CXCR3 in nerve injury-evoked and autoimmunity-evoked hyperalgesia

The chemokine CCL21 is released from injured neurons and acts as a ligand of the chemokine receptor, CXCR3, which likely contributes to pro-inflammatory adaptations and secondary neuronal damage. CCL21-CXCR3 signalling may therefore impact on the development of neuropathic pain. By using the respective knockout mice we show that deficiency of CCL19/21 in plt/plt mice attenuates nerve injury evoked pain but not the hyperalgesia evoked by autoimmune encephalomyelitis (EAE). Oppositely, CXCR3-deficiency had no protective effect after traumatic nerve injury but reduced EAE-evoked hyperalgesia and was associated with reduced clinical EAE scores, a reduction of the pro-inflammatory cell infiltration and reduced upregulation of interferon gamma and interleukin-17 in the spinal cord. In contrast, microglia activation in the spinal cord after traumatic sciatic nerve injury was neither attenuated in CXCR3(-/-) nor plt/plt mice, nor in double knockouts. However, the severity of EAE, but not the hyperalgesia, was also reduced in plt/plt mice, which was associated with reduced infiltration of the spinal cord with CCR7+ T-cells, an increase of CD25+ T-cells and reduced upregulation of CXCL9 and 10, CCL11 and 12. The data show that CCL21 and CXCR3 have dichotomous functions in traumatic and EAE-evoked neuropathic pain suggesting diverse mechanisms likely requiring diverse treatments although both types of neuropathic pain are mediated in part through the immune activation.

Inhibition of GTP cyclohydrolase reduces cancer pain in mice and enhances analgesic effects of morphine

Noncoding polymorphisms of the GTP cyclohydrolase gene (GCH1) reduce the risk for chronic pain in humans suggesting GCH1 inhibitors as analgesics. We assessed the effects of the GCH1 inhibitor diaminohydroxypyrimidine (DAHP) on nociception and inflammation in a mouse melanoma and a sarcoma cancer pain model, and its co-effects with morphine in terms of analgesic efficacy and respiratory depression. GCH1 inhibition did not reduce the tumor-evoked nociceptive hypersensitivity of the tumor-bearing paw. However, DAHP reduced melanoma- and sarcoma-evoked systemic hyperalgesia as determined by analyzing contralateral paws. GCH1 inhibition increased the inflammatory edema and infiltration with polymorphonuclear leukocytes surrounding the tumor but reduced the tumor-evoked microglia activation in the spinal cord suggesting that an increase of the local immune attack against the tumor may avoid general pain hypersensitivity. When used in combination with morphine at high or low doses, GCH1 inhibition increased and prolonged the analgesic effects of the opioid. It did not, however, increase the respiratory depression caused by morphine. Conversely, the GCH1-product, tetrahydrobiopterin, caused hyperalgesia, antagonized antinociceptive effects of morphine, and aggravated morphine-evoked respiratory depression, the latter mimicked by a cGMP analog suggesting that respiratory effects were partly mediated through the BH4–NO–cGMP pathway. The observed effects of GCH1 inhibition in the tumor model and its enhancement of morphine-evoked antinociception without increase of morphine toxicity suggest that GCH1 inhibitors might be useful as co-therapeutics for opioids in cancer patients.

Tetrahydrobiopterin Attenuates DSS-evoked Colitis in Mice by Rebalancing Redox and Lipid Signalling

BACKGROUND AND AIMS Guanosine triphosphate cyclohydrolase [GCH1] governs the production of the enzyme cofactor tetrahydrobiopterin [BH4] which is essential for biogenic amine synthesis, lipid metabolism via alkylglycerol monooxygenase [AGMO], and redox coupling of nitric oxide synthases [NOSs]. Inflammation-evoked unequal regulation of GCH1 and NOS or AGMO may cause redox stress and lipid imbalances. METHODS The present study assessed potential therapeutic effects of rebalancing these systems with BH4 in experimental colitis in mice. RESULTS Oral treatment with BH4 as a suspension of crushed tablets attenuated colitis, whereas inhibition of its production had opposite effects: aggravated weight loss, epithelial haemorrhages and ulcers, neutrophil infiltrates, production of reactive oxygen species, and unfavourable profile changes of endocannabinoids, ceramides, and lysophosphatidic acids. Conversely, oral BH4 normalised biopterin, reduced in vivo activity of oxidases and peroxidases in the inflamed gut, favoured nitric oxide over hydrogen peroxide, and maintained normal levels of lipid signalling molecules. BH4 favoured thereby resident CD3+CD8+ and regulatory CD3+CD25+ intraepithelial T cells that are important for epithelial integrity. CONCLUSIONS BH4 protected against colitis in mice via two major pathways: [i] by reduction of oxidative stress; and [ii] by re-orchestration of alkyl- and acylglycerolipid signalling via AGMO. Oral treatment with BH4 is a safe approved supplementary therapy for genetic BH4 deficiency and did not excessively increase systemic BH4 levels. Therefore, one may consider repurposing of oral BH4 as an adjunctive treatment for colitis.

Monitoring Translation Activity of mRNA-Loaded Nanoparticles in Mice

Targeting mRNA to eukaryotic cells is an emerging technology for basic research and provides broad applications in cancer immunotherapy, vaccine development, protein replacement, and in vivo genome editing. Although a plethora of nanoparticles for efficient mRNA delivery exists, in vivo mRNA targeting to specific organs, tissue compartments, and cells remains a major challenge. For this reason, methods for reporting the in vivo targeting specificity of different mRNA nanoparticle formats will be crucial. Here, we describe a straightforward method for monitoring the in vivo targeting efficiency of mRNA-loaded nanoparticles in mice. To achieve accurate mRNA delivery readouts, we loaded lipoplex nanoparticles with Cre-recombinase-encoding mRNA and injected these into commonly used Cre reporter mouse strains. Our results show that this approach provides readouts that accurately report the targeting efficacy of mRNA into organs, tissue structures, and single cells as a function of the used mRNA delivery system. The method described here establishes a versatile basis for determining in vivo mRNA targeting profiles and can be systematically applied for testing and improving mRNA packaging formats.

Identification of novel anti-inflammatory herbal extracts

I diseases continue to be one of the biggest health problems in the world, affecting millions of people annually M. abscessus and other species of rapidly growing mycobacteria (RGM) are naturally resistant to antimicrobial compounds and disinfectants because they have an impermeable cell wall composed by peptideoglycan and mycolic acids. These RGM are responsible for various hospital outbreaks worldwide, causing lung infections in patients with cystic fibrosis, chronic lung disease (bronchiectasis, nodules and cavitations), post-surgical infections and skin and soft tissue infections in immunocompromised patients. The resistance of M. abscessus (Mabs) to the medications used in current therapy challenges the search for new treatment strategies. Previous studies on the search for new natural compounds with antimicrobial action highlighted the potential of Bixa orellana (urucum). The seeds of this plant are already used in folk medicine for treating heart disease, gastrointestinal problems and respiratory infections. In this study, we evaluated potential anti-inflammatory activities of hydroalcoholic (BoEH) and ethyl acetate (BoEA) extracts of B. orellana leaves, using a murine model of peritonitis induced by heat killed Mabs. C57BL/6 mice were orally treated with different concentrations of BoEH or BoEA. After one hour, peritonitis was induced by inoculation of 1x108 CFU of heat killed Mabs. BoEH and BoEA inhibited the migration of total leukocytes (Figure 1A-B), migration of polymorphonuclear cells (Figure 1C-D) and mononuclear cells (Figure 1E-F) into the peritoneum in the periods analyzed 4 and 24 hours after the induction of peritonitis. Our results suggest anti-inflammatory actions of the extracts tested, indicating this plant as natural source of compounds with potential for pharmacological and biotechnological applications.I is a member of the IL-17 family, and is known as CTLA8 in the mouse. It is produced by T lymphocytes and NK cells and has proinflammatory roles, inducing cytokine and chemokine production. However, its role in tumor biology remains controversial. We investigated the effects of locally produced IL-17A by transferring the gene, encoding it into mouse tumor cells including B16 melanoma, and MethA fibrosarcoma, either in a secretory or a membrane-bound form. Expression of the membranebound form on CT26 colon cancer cells dramatically enhanced their proliferation in in vitro. The enhanced growth was shown to be due to an increased rate of cell cycle progression. After synchronizing cells by adding and withdrawing colcemid, the rate of cell cycle progression in the cells expressing the membrane-bound form of IL-17A was much faster than that of the control cells. Both secretory and membrane-bound IL-17A induced the expression of Sca-1 on the cancer cells, which is commonly associated with aggressive phenotype of cancer cells. When tumor clones were grafted into syngeneic BALB/c mice, the tumor clones expressing the membranebound form IL-17A grew rapidly; those expressing the secretory form also grew faster than the wild type CT26 cells, but slower than the clones expressing the membrane-bound form. These results indicate that IL-17A promotes tumorigenicity, in part, by enhancing cell cycle progression. This finding should be considered in treating tumors and immune-related diseases.P which patients are at a higher risk for recurrent chronic rhinosinusitis with nasal polyps (CRSwNP) is one of the most challenging problems in clinical rhinology. A direct association between nasal polyp and eosinophil/neutrophil counts was reported. This study aimed to identify difference of eosinophils and neutrophils for formation of polyp by DNA methylation in CRS. We have previously shown that KRT 19, NR2F2, ADAMTS1, and ZNF222 levels are changed in nasal polyps (NPs) of patients with chronic rhinosinusitis (CRS) in patients. A study was performed from 30 patients with CRS with bilateral NP, examining the prognostic role of eosinophil and neutrophil levels. 30 patients with CRS were classified by the rate of eosinophils and neutrophils in tissue. The methylated genes detected by DNA methylation microarray were validated by methylation-specific polymerase chain reaction (PCR), bisulfite sequencing, and real-time PCR. DNA methylation microarray identified 43,674 CpG islands in 518 genes. Specific genes were found to have a hypermethylated signal, and some genes were significantly hypomethylated in the promoter region in eosinophils compared with neutrophils. Real-time PCR showed that the expression levels of genes were changed in eosinophils, when compared with neutrophils. We clearly demonstrated that the two subgroups of CRSwNP had characteristic differences in DNA methylation, which allows for pathophysiologically meaningful differentiations with likely therapeutic consequences. Further studies are needed to confirm the significance of these epigenetic factors in the mechanisms underlying NP formation.Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease where animal models are used to study its pathogenesis. We have developed a mouse model of an autoimmune disease that resembles human lupus by the injection of liposomes bearing non-bilayer phospholipid arrangements (NPA). We detected the presence of IgG antibodies against NPA in the serum of mice with lupus and patients with SLE. In this work, we determined by citofluorometry the presence of plasmacytoid dendritic cells (pDC) the main producer of type I interferons, and of NKT cells in the secondary lymphoid organs of mice 30 and 60 days after the injection of liposomes with or without NPA induced with 8 mM promazine. In both groups of mice injected with liposomes bearing NPA a significant increase of pDC cells (5-fold) was found which correlates with the high concentration of type I interferon previously detected in this mouse model of lupus and in patients with SLE. A significant increase of NKT (3-fold) was detected at 30 days, specifically in the NKT subpopulation CD4 + that is known to cooperate with B cells in response of lipid antigens; this increase suggests its probable involvement in adaptive immune responses, which lead to the production of anti-NPA IgG antibodies. The increase of pDC and NKT cells found by cytometry in secondary lymphoid organs in this work, suggests their involvement in the formation of anti-NPA IgG antibodies and the development of the disease resembling human lupus.Statement of the Problem: Recurrent aphthous stomatitis (RAS) is a multifactorial disease with an unclear etiopathogenesis, resulting from the interplay between genetic and environmental factors.1 As the dysregulation of the immune system can play a role in the RAS development2, single nucleotide polymorphisms (SNPs) in the genes for immune and inflammatory molecules were studied.3,4 The NOD-like receptor (NLRP3) gene, encoding the component of the inflammasome, has been proposed as one of the candidate genes for RAS.5 The aim of our study was to investigate three SNPs (rs4612666, rs10754558, rs3806265) in NLRP3 gene in patients with RAS and healthy controls in the Caucasian population Methodology: A total of 200 Czech subjects were enrolled in this case-control study. 143 healthy controls, 57 patients with RAS were genotyped by method based on polymerase chain reaction using 5′ nuclease TaqMan® assays. Clinical parameters such as complete blood count, levels of immunoglobulins including allergen-specific immunoglobulin E or presence of antibodies against cytomegalovirus, Epstein-Barr virus were determined in RAS patients. Findings: Although no significant differences in the NLRP3 (rs10754558, rs3806265) allele and genotype frequencies between patients with RAS and controls were observed, statistically significant differences in NLRP3 rs4612666 genotype frequencies between subjects with RAS and controls were found. Carriers of NLRP3 rs4612666 TT genotype had a higher risk of developing RAS in comparison to subjects with CT + CC genotypes (OR = 16.71, 95% CI = 1.96-142.14, P = 0.0024). No association between NLRP3 haplotypes and RAS was detected. Conclusion & Significance: In contrast to the previous study5, associations between NLRP3 (rs10754558, rs3806265) polymorphisms and RAS were not confirmed. However, we suggest that NLRP3 rs4612666 polymorphism can strongly influence the risk of developing RAS in the Czech population.Results: The prevalence of vitamin D insufficiency was 64% in 76 asthmatic and 62.5% in 446 atopic individuals; however there was no significant association between vitamin D and this outcomes. Negative correlation was found between vitamin D and specific IgE levels to Dermatophagoides pteronyssinus on atopic subjects (r=-0.11, p=0.04). Genetic variants in CYP2R1 gene, rs7935792 (C allele) (Beta 1.66; 95% CI 0.20-3.11) and rs7129781 (C allele) (Beta 1.55; 95% CI 0.07-2.96), were associated with vitamin D serum levels. In addition, the same variants had suggestive protection on asthma, but it was not significant (OR 0.74; 95% CI 0.39; 1.39; OR 0.73; 95% CI 0.38; 1.37, respectively). VDR variants rs7965397 (G allele) was positively associated with atopy (OR 1.43; 95% CI, 1.07-1.92); rs4328262 (G allele) (OR 1.44; 95% CI 1.09-1.90) and asthma rs2408876 (C allele) (OR 2.31; 95% CI; 1.18-4.53); rs2238317 (T allele) (OR 2.19; 95% CI 1.02-4.72).

STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing

1. 1. Pickert, 2. et al . 2009. J. Exp. Med. doi: 10.1084/jem.20082683 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20082683%26rft_id%253Dinfo%253Apmid%252F19564350%26rft.genre%253Darticle%26rft_val_fmt%