Background Interleukin 13 (IL-13) is upregulated in ulcerative colitis (UC) and increases colon epithelial permeability by inducing apoptosis and expression of the pore-forming tight junction protein claudin-2. IL-13 induces activation of signal transducer and activator of transcription 6 (STAT6). However, the STAT6 phosphorylation status in patients with UC is unknown, as is the effect of STAT6 inhibition on colonic epithelium exposed to IL-13. The study aims were to determine if mucosal STAT6 phosphorylation is increased in patients with UC, and if STAT6 inhibition attenuates IL-13-induced colon epithelial cell dysfunction. Methods Immunohistochemical staining for phosphorylated (p) STAT6 was performed on colonic tissue from newly diagnosed pediatric subjects with UC (early UC) or Cohn’s disease (CD), colectomy tissue from adults with UC (advanced UC), and controls. Colon HT-29 and T84 cells were transfected with STAT6 small interfering RNA (siRNA), or treated with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor that inhibits STAT6, prior to IL-13 treatment. Results Median score for epithelial pSTAT6 was 0 in control subjects, 2 in early UC (vs. control P=0.019), 4 in advanced UC (P=0.003), and 0 in CD (P=0.4). Cell transfection with STAT6 siRNA prevented IL-13-induced apoptosis and claudin-2 expression. SAHA inhibited IL-13-induced STAT6 phosphorylation, apoptosis, and claudin-2 expression, and mitigated IL-13-induced reductions in trans-epithelial resistance. Conclusions UC is associated with increased colonic epithelial STAT6 phosphorylation, and STAT6 inhibition prevents IL-13-induced apoptosis and barrier disruption. These data identify STAT6 as a novel target for UC treatment, and support further study of SAHA as a therapeutic agent.
Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal morbidity and mortality in preterm infants. NEC is characterized by an exaggerated inflammatory response to bacterial flora leading to bowel necrosis. Bacterial lipopolysaccharide (LPS) mediates inflammation through TLR4 activation and is a key molecule in the pathogenesis of NEC. However, LPS also induces cyclooxygenase-2 (COX-2), which promotes intestinal barrier restitution through stimulation of intestinal cell survival, proliferation, and migration. Epidermal growth factor receptor (EGFR) activation prevents experimental NEC and may play a critical role in LPS-stimulated COX-2 production. We hypothesized that EGFR is required for LPS induction of COX-2 expression. Our data show that inhibiting EGFR kinase activity blocks LPS-induced COX-2 expression in small intestinal epithelial cells. LPS induction of COX-2 requires Src-family kinase signaling while LPS transactivation of EGFR requires matrix metalloprotease (MMP) activity. EGFR tyrosine kinase inhibitors block LPS stimulation of mitogen-activated protein kinase ERK, suggesting an important role of the MAPK/ERK pathway in EGFR-mediated COX-2 expression. LPS stimulates proliferation of IEC-6 cells, but this stimulation is inhibited with either the EGFR kinase inhibitor AG1478, or the selective COX-2 inhibitor Celecoxib. Taken together, these data show that EGFR plays an important role in LPS-induction of COX-2 expression in enterocytes, which may be one mechanism for EGF in inhibition of NEC.
Histological chorioamnionitis (HCA) is an intrauterine inflammatory condition increasing the risk for preterm birth, death, and disability due to persistent systemic and localized inflammation. The immunologic mechanisms sustaining this response in the preterm newborn remain unclear. We sought to determine the consequences of HCA exposure on the fetal CD4+ T lymphocyte exo-metabolome. We cultured naïve CD4+ T lymphocytes from HCA-positive and HCA-negative preterm infants matched for gestational age, sex, race, prenatal steroid exposure, and delivery mode. We collected conditioned media samples before and after a 6 hour in vitro activation of naïve CD4+ T lymphocytes with soluble Staphylococcal enterotoxin B (SEB) and anti-CD28. We analyzed samples by ultra performance liquid chromatography ion mobility-mass spectrometry (UPLC-IM-MS). We determined the impact of HCA on the CD4+ T lymphocyte exo-metabolome and identified potential biomarker metabolites by multivariate statistical analyses. We discovered that: (1) CD4+ T lymphocytes exposed to HCA exhibit divergent exo-metabolomic profiles in both naïve and activated states; (2) ~30% of detected metabolites differentially expressed in response to activation were unique to HCA-positive CD4+ T lymphocytes; and (3) metabolic pathways associated with glutathione detoxification and tryptophan degradation were altered in HCA-positive CD4+ T lymphocytes; and (4) flow cytometry and cytokine analyses suggested a bias towards a TH1-biased immune response in HCA-positive samples. HCA exposure primes the neonatal adaptive immune processes by inducing changes to the exo-metabolomic profiles of fetal CD4+ T lymphocytes. These exo-metabolomic changes may link HCA exposure to TH1 polarization of the neonatal adaptive immune response.
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