Cytokine Exposure Research Articles

Allergen-specific lung resident CD4+ Th2 cells in asthma (HYP4P.417)

Abstract Asthma is an inflammatory disease of the airways that affects more than 300 million people worldwide. Clinically, asthma is characterized by acute, intermittent and recurrent episodes of inflammation that can be induced by a specific allergen. Allergen-specific CD4 T cells contribute to this process by producing cytokines and inducing B lineage production of IgE. Allergen-specific CD4 memory T cells are also thought to persist after resolution of inflammation to rapidly drive asthma-induced pathology. Due to the challenge of tracking small populations of CD4 T cells that express allergen pMHCII-specific TCRs, little is known about what regulates Th2 memory cell differentiation, migration and maintenance. We have therefore produced a MHCII tetramer containing a peptide from the Derp1 protein of the house dust mite (HDM), the most common cause of atopic asthma, to elucidate the underlying mechanisms of Th2 memory cell differentiation during atopic asthma. Tetramer-based enrichment strategies used to isolate Derp1:I-Ab+ cells from the secondary lymphoid organs and lungs have revealed that heterogenous Th2 memory populations form, including a CD69+ Derp1:I-Ab+ population in the lung thats persists post-contraction. Intravascular staining techniques demonstrate that these cells reside in the lung parenchyma and are driven to the lung by early cytokine exposure. These findings have important implications for novel immunotherapeutics in both asthma and allergy.

Role of c-Jun N-terminal kinase (JNK) activation in micturition reflexes in cyclophosphamide (CYP)-induced cystitis in female rats.

c-Jun N-terminal kinase (JNK) is member of the mitogen-activated protein kinase (MAPK) family, activated through phosphorylation following cytokine exposure and stress. In this study, phosphorylation of JNK was examined in the urinary bladder with cyclophosphamide (CYP)-induced cystitis and the effects of SP600125, a selective inhibitor of phosphorylation of JNK, on urinary bladder function were assessed using conscious, open outlet, cystometry with continuous instillation of intravesical saline. We induced bladder inflammation in adult female Wistar rats by injecting CYP intraperitoneally to produce acute (150mg/kg; 4h), intermediate (150mg/kg; 48h), and chronic (75mg/kg; every third day for 10days) treatments. Western blotting of urinary bladder demonstrated a significant (p ≤ 0.01) increase (i.e., phosphorylation) in JNK activation with 4- and 48-h CYP-induced cystitis. Immunohistochemistry and image analyses demonstrated a significant (p ≤ 0.01) increase in JNK activation in the urothelium with 4- and 48-h CYP-induced cystitis. Blockade of JNK phosphorylation significantly (p ≤ 0.01) increased bladder capacity and intercontraction void intervals in CYP-treated rats (4 and 48h). Furthermore, blockade of JNK phosphorylation reduced (p ≤ 0.01) neuropeptide (substance P, calcitonin gene-related peptide) expression in the urinary bladder with CYP-induced cystitis (4 and 48h). In contrast, blockade of JNK phosphorylation was without effect on bladder function or neuropeptide expression in urinary bladder in control (no inflammation) rats. Blockade of JNK phosphorylation may represent a novel target for improving urinary bladder function with CYP-induced cystitis.

Tissue-Specific Induction of Mouse ZIP8 and ZIP14 Divalent Cation/Bicarbonate Symporters by, and Cytokine Response to, Inflammatory Signals.

Mouse Slc39a8 and Slc39a14 genes encode ZIP8 and ZIP14, respectively, which are ubiquitous divalent cation/(HCO3-)2 symporters responsible for uptake of Zn2+, Fe2+, and Mn2+ into cells. Cd2+ and other toxic nonessential metals can displace essential cations, thereby entering vertebrate cells. Whereas Slc39a8 encodes a single protein, Slc39a14 has 2 exons 4 which, via alternative splicing, give rise to ZIP14A and ZIP14B; why differences exist in cell type-specific expression of ZIP14A and ZIP14B remains unknown. Inflammatory stimuli have been associated with upregulation of ZIP8 and ZIP14, but a systematic study of many tissues simultaneously in a laboratory animal following inflammatory cytokine exposure has not yet been reported. Herein, we show that C57BL/6J male mice--treated intraperitoneally with lipopolysaccharide or the proinflammatory cytokines tumor necrosis factor (TNF) or interleukin-6 (IL6)--exhibited quantatively very different, highly tissue-specific, and markedly time-dependent up- and downregulation of ZIP8, ZIP14A, and ZIP14B messenger RNA (mRNA) levels in 12 tissues. The magnitude of inflammatory response was confirmed by measuring the proinflammatory cytokine TNF, IL6, and interleukin-1β mRNA levels in the same tissues of these animals. Our data suggest that most if not all tissues use ZIP8, ZIP14A, and/or ZIP14B for Zn2+ uptake, some tissues under basal conditions and others moreso when inflammatory stressors are present; collectively, this might lead to substantial alterations in plasma Zn2+ levels due to Zn2+ redistribution not just in liver but across many vital organs. In the context of cadmium-mediated toxicity, our data suggest that tissues other than liver, kidney, and lung should also be considered.

Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Signaling and Cell Death in the Immature Central Nervous System after Hypoxia-Ischemia and Inflammation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family. The interaction of TRAIL with death receptor 4 (DR4) and DR5 can trigger apoptotic cell death. The aim of this study was to investigate the role of TRAIL signaling in neonatal hypoxia-ischemia (HI). Using a neonatal mouse model of HI, mRNA, and protein expression of TRAIL, DR5 and the TRAIL decoy receptors osteoprotegerin (OPG), mDcTRAILR1, and mDcTRAILR2 were determined. In vitro, mRNA expression of these genes was measured in primary neurons and oligodendrocyte progenitor cells (OPCs) after inflammatory cytokine (TNF-α/IFN-γ) treatment and/or oxygen and glucose deprivation (OGD). The toxicity of these various paradigms was also measured. The expression of TRAIL, DR5, OPG, and mDcTRAILR2 was significantly increased after HI. In vitro, inflammatory cytokines and OGD treatment significantly induced mRNAs for TRAIL, DR5, OPG, and mDcTRAILR2 in primary neurons and of TRAIL and OPG in OPCs. TRAIL protein was expressed primarily in microglia and astroglia, whereas DR5 co-localized with neurons and OPCs in vivo. OGD enhanced TNF-α/IFN-γ toxicity in both neuronal and OPC cultures. Recombinant TRAIL exerted toxicity alone or in combination with OGD and TNF-α/IFN-γ in primary neurons but not in OPC cultures. The marked increases in the expression of TRAIL and its receptors after cytokine exposure and OGD in primary neurons and OPCs were similar to those found in our animal model of neonatal HI. The toxicity of TRAIL in primary neurons suggests that TRAIL signaling participates in neonatal brain injury after inflammation and HI.

Interleukin‐4 and interleukin‐13 compromise the sinonasal epithelial barrier and perturb intercellular junction protein expression

Altered expression of epithelial intercellular junction proteins has been observed in sinonasal biopsies from nasal polyps and epithelial layers cultured from nasal polyp patients. These alterations comprise a "leaky" epithelial barrier phenotype. We hypothesize that T helper 2 (Th2) cytokines interleukin (IL)-4 and IL-13 modulate epithelial junction proteins, thereby contributing to the leaky epithelial barrier. Differentiated primary sinonasal epithelial layers cultured at the air-liquid interface were exposed to IL-4, IL-13, and controls for 24 hours at 37°C. Epithelial resistance measurements were taken every 4 hours during cytokine exposure. Western blot and immunofluorescence staining/confocal microscopy were used to assess changes in a panel of tight and adherens junction proteins. Western blot densitometry was quantified with image analysis. IL-4 and IL-13 exposure resulted in a mean decrease in transepithelial resistance at 24 hours to 51.6% (n = 6) and 68.6% (n = 8) of baseline, respectively. Tight junction protein junctional adhesion molecule-A (JAM-A) expression decreased 42.2% with IL-4 exposure (n = 9) and 37.5% with IL-13 exposure (n = 9). Adherens junction protein E-cadherin expression decreased 35.3% with IL-4 exposure (n = 9) and 32.9% with IL-13 exposure (n = 9). Tight junction protein claudin-2 showed more variability but had a trend toward higher expression with Th2 cytokine exposure. There were no appreciable changes in claudin-1, occludin, or zonula occludens-1 (ZO-1) with IL-4 or IL-13 exposure. Sinonasal epithelial exposure to Th2 cytokines IL-4 and IL-13 results in alterations in intercellular junction proteins, reflecting increased epithelial permeability. Such changes may explain some of the phenotypic manifestations of Th2-mediated sinonasal disease, such as edema, nasal discharge, and environmental reactivity.

Tsc2 Haploinsufficiency Has Limited Effects on Fetal Brain Cytokine Levels during Gestational Immune Activation.

Dysregulated TSC/mTOR signaling may play a pathogenetic role in forms of syndromic autism, such as autism associated with tuberous sclerosis, a genetic disorder caused by heterozygous TSC1 or TSC2 mutations. Environmental risk factors, such as gestational viral infections, may, in some cases, also contribute to the pathogenesis of autism and related neuropsychiatric disorders. We have recently found that a heterozygous Tsc2 mutation and the poly I:C model of maternal immune activation (MIA) interactively perturb fetal development and adult social behavior in mice, suggesting that these factors converge on shared pathways. TSC/mTOR signaling plays an important role in the modulation of immune responses, raising the possibility that the damage caused by MIA was greater in Tsc2+/− than in wildtype fetuses because of an exacerbated immune response in the mutants. Here, cytokine antibody arrays were employed to measure relative cytokine abundances in the fetal brain and the placenta during MIA. Cytokines were induced by gestational poly I:C but there was no obvious modulatory effect of Tsc2 haploinsufficiency. The data indicate that cytokine exposure during MIA is comparable in Tsc2 haploinsufficient and wildtype control fetuses, suggesting that downstream molecular and cellular processes may account for the interactive effects of Tsc2 haploinsufficiency and MIA.

Discovery of Molecular Pathways Mediating 1,25-Dihydroxyvitamin D3 Protection Against Cytokine-Induced Inflammation and Damage of Human and Male Mouse Islets of Langerhans

Protection against insulitis and diabetes by active vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in nonobese diabetic mice has until now mainly been attributed to its immunomodulatory effects, but also protective effects of this hormone on inflammation-induced β-cell death have been reported. The aim of this study was to clarify the molecular mechanisms by which 1,25(OH)2D3 contributes to β-cell protection against cytokine-induced β-cell dysfunction and death. Human and mouse islets were exposed to IL-1β and interferon-γ in the presence or absence of 1,25(OH)2D3. Effects on insulin secretion and β-cell survival were analyzed by glucose-stimulated insulin release and electron microscopy or Hoechst/propidium iodide staining, respectively. Gene expression profiles were assessed by Affymetrix microarrays. Nuclear factor-κB activity was tested, whereas effects on secreted chemokines/cytokines were confirmed by ELISA and migration studies. Cytokine exposure caused a significant increase in β-cell apoptosis, which was almost completely prevented by 1,25(OH)2D3. In addition, 1,25(OH)2D3 restored insulin secretion from cytokine-exposed islets. Microarray analysis of murine islets revealed that the expression of approximately 4000 genes was affected by cytokines after 6 and 24 hours (n = 4; >1.3-fold; P < .02), of which nearly 250 genes were modified by 1,25(OH)2D3. These genes belong to functional groups involved in immune response, chemotaxis, cell death, and pancreatic β-cell function/phenotype. In conclusion, these findings demonstrate a direct protective effect of 1,25(OH)2D3 against inflammation-induced β-cell dysfunction and death in human and murine islets, with, in particular, alterations in chemokine production by the islets. These effects may contribute to the beneficial effects of 1,25(OH)2D3 against the induction of autoimmune diabetes.

Time- and Concentration-Dependent Effects of Exogenous Serotonin and Inflammatory Cytokines on Mast Cell Function

Mast cells play a significant role in both the innate and adaptive immune response; however, the tissue-bound nature of mast cells presents an experimental roadblock to performing physiologically relevant mast cell experiments. In this work, a heterogeneous cell culture containing primary culture murine peritoneal mast cells (MPMCs) was studied to characterize the time-dependence of mast cell response to allergen stimulation and the time- and concentration-dependence of the ability of the heterogeneous MPMC culture to uptake and degranulate exogenous serotonin using high performance liquid chromatography (HPLC) coupled to an electrochemical detector. Additionally, because mast cells play a central role in asthma, MPMCs were exposed to CXCL10 and CCL5, two important asthma-related inflammatory cytokines that have recently been shown to induce mast cell degranulation. MPMC response to both allergen exposure and cytokine exposure was evaluated for 5-HT secretion and bioactive lipid formation using ultraperformance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer (UPLC-MS/MS). In this work, MPMC response was shown to be highly regulated and responsive to subtle alterations in a complex environment through time- and concentration-dependent degranulation and bioactive lipid formation. These results highlight the importance of selecting an appropriate mast cell model when studying mast cell involvement in allergic response and inflammation.

Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice

Reprogramming of pancreatic exocrine cells into cells resembling beta cells may provide a strategy for treating diabetes. Here we show that transient administration of epidermal growth factor and ciliary neurotrophic factor to adult mice with chronic hyperglycemia efficiently stimulates the conversion of terminally differentiated acinar cells to beta-like cells. Newly generated beta-like cells are epigenetically reprogrammed, functional and glucose responsive, and they reinstate normal glycemic control for up to 248 d. The regenerative process depends on Stat3 signaling and requires a threshold number of Neurogenin 3 (Ngn3)-expressing acinar cells. In contrast to previous work demonstrating in vivo conversion of acinar cells to beta-like cells by viral delivery of exogenous transcription factors, our approach achieves acinar-to-beta-cell reprogramming through transient cytokine exposure rather than genetic modification.

Cellular inhibitor of apoptosis-2 is a critical regulator of apoptosis in airway epithelial cells treated with asthma-related inflammatory cytokines

Aberrant apoptosis of airway epithelial cells (AECs) is a disease contributing feature in the airways of asthmatics. The proinflammatory cytokines tumor necrosis factor α (TNFα) and interferon γ (IFNγ) are increased in asthma and have been shown to contribute to apoptosis at the airways. In the present study, we investigated the role of the inhibitor of apoptosis protein (IAP) family in primary AECs exposed to TNFα and IFNγ. IAPs are potent regulators of caspase activity elicited by the intrinsic and extrinsic apoptosis pathways. However, while caspase-mediated apoptosis was observed in AECs exposed to doxorubicin, it was not observed after cytokine treatment. Instead, AECs exhibited proapoptotic changes evidenced by an increased Bax:Bcl2 transcript ratio and partial processing of procaspase-3. Examination by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western analysis showed that proapoptotic changes were associated with a time- and dose-dependent induction of cellular IAP-2 (cIAP2), potentiated primarily by IFNγ. The abundance of the IAP antagonists X-linked IAP-associated factor 1 (XAF1) and second mitochondria-derived activator of caspases did not change, although a moderate nuclear redistribution was observed for XAF1, which was also observed for cIAP2. Small interfering RNA (siRNA)-mediated depletion of cIAP2 from AECs leads to caspase-3 activation and poly (ADP-ribose) polymerase cleavage, but this required extended cytokine exposure to produce a concomitant decrease in cIAP1 and Bcl2. These results indicate that AECs possess endogenous mechanisms making them highly resistant to apoptosis due to asthma-related inflammatory cytokines, and the activity of cIAP2 plays an important role in this protection.

Human annulus cells regulate PAPP-A and IGFBP-4 expression, and thereby insulin-like growth factor bioavailability, in response to proinflammatory cytokine exposure in vitro

Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase which cleaves IGF binding protein (BP)-4 in the extracellular matrix, making IGF available to nearby cells. We have shown that PAPP-A is present in the human intervertebral disc, and is significantly upregulated in more degenerated discs where increased proinflammatory cytokine levels are present. We hypothesized that increased proinflammatory cytokines present in the degenerating disc might be related to PAPP-A expression. Experiments exposed human annulus cells to IL-1-β or TNF-α to test this hypothesis. Treated cells showed significantly increased PAPP-A in conditioned media versus controls (p < 0.001). PAPP-A production following exposure to IL-1β was significantly greater in cells derived from more degenerated versus healthier discs (p = 0.05). PAPP-A gene expression (microarray analysis) was significantly upregulated in IL-1β- or TNF-α-exposed cells (p = 0.01–0.004). Quantitative RT-PCR confirmed significant upregulation of IGFBP-4 in IL-1β- or TNF-α-exposed cells. Data have potential relevance to future cell-based biologic therapies for disc degeneration.

109. Enhanced kynurenic acid levels and cytokine-induced production of 3-hydroxykynurenine in fibroblasts from bipolar or schizophrenic patients

Recent studies suggest that cytokine-related disturbances of kynurenine metabolism are involved in the pathogenesis of schizophrenia and bipolar disorder. Here, we examined the production of kynurenic acid (KYNA) and 3-hydroxykynurenine (3-HK) and the expression of kynurenine pathway enzymes under basal conditions and in the presence of pro-inflammatory cytokines in fibroblast cultures from patients with schizophrenia or bipolar disorder. Higher baseline production of KYNA and 3-HK was demonstrated in fibroblasts obtained from patients as compared with control subjects. Following exposure to interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)1-beta, IL6, or combinations, 3-HK levels, but not KYNA, increased in the fibroblast cultures. After treatments with cytokines the mRNA expression of kynurenine aminotransferase (KAT) enzymes were decreased and kynurenase (KYNU) increased, kynurenine-3-monooxygenase (KMO) levels were not affected. Interaction analyses showed that cells from patients with schizophrenia or bipolar disorder respond differently to pro-inflammatory cytokines in terms of L-kynurenine metabolism as compared to control individuals. Potential associations with two polymorphisms in the KMO allele were also investigated. The single nucleotide polymorphisms (SNPs) rs1053230 and rs2275163 both had effects on KYNA levels but these SNPs did not explain the case-control differences in KYNA or 3-HK levels. In conclusion, our results provide evidence that the kynurenine pathway is abnormally affected by cytokine exposure in fibroblast cultures obtained from patients with schizophrenia or bipolar disorder.

Mechanisms of toxicity by proinflammatory cytokines in a novel human pancreatic beta cell line, 1.1B4

BackgroundMolecular mechanisms of toxicity and cell damage were investigated in the novel human beta cell line, 1.1B4, after exposure to proinflammatory cytokines — IL-1β, IFN-γ, TNF-α. MethodsMTT assay, insulin radioimmunoassay, glucokinase assay, real time reverse transcription PCR, western blotting, nitrite assay, caspase assay and comet assay were used to investigate mechanisms of cytokine toxicity. ResultsViability of 1.1B4 cells decreased after 18h cytokine exposure. Cytokines significantly reduced cellular insulin content and impaired insulin secretion induced by glucose, alanine, KCl, elevated Ca2+, GLP-1 or forskolin. Glucokinase enzyme activity, regulation of intracellular Ca2+ and PDX1 protein expression were significantly reduced by cytokines. mRNA expression of genes involved in secretory function — INS, GCK, PCSK2 and GJA1 was downregulated in cytokine treated 1.1B4 cells. Upregulation of transcription of genes involved in antioxidant defence — SOD2 and GPX1 was observed, suggesting involvement of oxidative stress. Cytokines also upregulated transcriptions of NFKB1 and STAT1, which was accompanied by a significant increase in NOS2 transcription and accumulation of nitrite in culture medium, implicating nitrosative stress. Oxidative and nitrosative stresses induced apoptosis was evident from increased % tail DNA, DNA fragmentation, caspase 3/7 activity, apoptotic cells and lower BCL2 protein expression. ConclusionsThis study delineates molecular mechanisms of cytokine toxicity in 1.1B4 cells, which agree with earlier observations using human islets and rodent beta cells. General significanceThis study emphasizes the potential usefulness of this cell line as a human beta cell model for research investigating autoimmune destruction of pancreatic beta cells.

Differential Expression of Adhesion Molecules by Sinonasal Fibroblasts among Control and Chronic Rhinosinusitis Patients

Chronic rhinosinusitis (CRS) is characterized by inflammatory cell migration into sinus tissue with resultant inflammation fueled by a milieu of cytokines. Fibroblasts may contribute to inflammation through expression of leukocyte adhesion molecules such as vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM). VCAM attracts eosinophils and mast cells contributing to Th2 skewing, and ICAM attracts neutrophils and to a lesser degree, eosinophils, and contributes to mixed Th1/Th2 skewing. The purpose of this study was to compare sinus fibroblast adhesion molecule expression ex vivo among CRS subtypes and in vitro after cytokine stimulation. Sinus biopsy specimens were taken from control patients (n = 13), CRS without nasal polyposis (CRSsNP, n = 6), and CRS with nasal polyposis (CRSwNP, n = 15). ex vivo levels of VCAM and ICAM were measured by flow cytometry from single cell suspensions of tissue biopsy specimens. Changes in VCAM and ICAM expression to cytokine exposure were assessed using in vitro cultured sinonasal fibroblasts treated with tumor necrosis factor (TNF)-α, interleukin (IL)-4, or interferon (IFN)-γ. ex vivo VCAM expression was lowest in controls, higher in CRSsNP, and highest in CRSwNP. in vitro stimulation with TNF-α and IL-4, but not IFN-γ, increased VCAM among CRSsNP, while expression in CRSwNP remained elevated with all treatments except IFN-γ. ex vivo ICAM expression was elevated in both CRS subtypes. in vitro stimulation with TNF-α and IFN-γ, but not IL-4, increased ICAM expression in all patients with the largest effects among the CRSsNP subgroup. Sinonasal fibroblast expression of adhesion molecules in sinusitis varies by disease state and is selectively influenced by exposure to inflammatory cytokines.

Is the Mean Platelet Volume Predictive of Hip Fractures in the Elderly?

Erkan Cure, M.D., Mehmet Sabri Balik, M.D., Medine Cumhur Cure, M.D., Yilmaz Guvercin, M.D., Adem Erkut, M.D., Suleyman Yuce, M.D., and Davut Keskin, M.D.. Ann Lab Med 2013;33:367-70. https://doi.org/10.3343/alm.2013.33.5.367

Hepatocyte Nuclear Factor‐4 Phosphorylation Mediated by Inflammatory Response

Hepatocyte nuclear factor (HNF)‐4, a liver‐enriched transcription factor, regulates a large number of liver‐specific genes and plays an important role in liver development and maintenance of liver phenotype. While HNF‐4 function is regulated by phosphorylation, only a limited number of phosphorylation sites have been identified, and its roles and dynamic changes in response to inflammatory stimulation are not explored. Utilizing the cytokine treated HNF‐4 enriched HepG2 cells, we have quantitatively analyzed HNF‐4 phosphorylation by using the combined stable isotopic variants of dithiothreitol with mass spectrometry. Twelve serine/threonine phosphorylation sites in the diverse functional domains of HNF‐4 were identified and quantified after cytokine exposure. Cytokine treatment (IL‐6, IL‐1β and TNF‐α) causes a dynamic change in HNF‐4 phosphorylation in a peptide‐and time‐dependent manner. Protein kinase A (PKA) significantly reduces HNF‐4 binding activity, which mimics the repressive effect of cytokines, and a PKA inhibitor partially restores the reduced HNF‐4 binding activity induced by cytokines. These results suggest that cytokines mediate the phosphorylation/dephosphorylation levels of HNF‐4, and a PKA signaling pathway involves in the modification of HNF‐4 function during the inflammatory response.

Adalimumab prevents barrier dysfunction and antagonizes distinct effects of TNF-α on tight junction proteins and signaling pathways in intestinal epithelial cells

Intestinal barrier dysfunction is pivotal in the etiology of inflammatory bowel diseases. Combined clinical and endoscopic remission ("mucosal healing") in patients who received anti-TNF-α therapies suggests restitution of the intestinal barrier, but the mechanisms involved are largely unknown. We therefore investigated the impact of the anti-TNF-α antibody adalimumab on barrier function in two in vitro models. Combined stimulation of Caco-2 and T-84 cells with interferon-γ and TNF-α resulted in a significant decrease of transepithelial electrical resistance (TEER) within 6 h that was prevented by adalimumab in concentrations down to 100 ng/ml. Adalimumab furthermore antagonized the appearance of irregular membrane undulations and prevented internalization of tight junction proteins upon cytokine exposure. In addition, TNF-α induced a downregulation of claudin-1, claudin-2, claudin-4, and occludin as well as activation of phosphatidylinositol 3-kinase signaling in T-84 but not Caco-2 cells, which was reversed by adalimumab. At the signaling level, adalimumab prevented increased phosphorylation of myosin light chain as well as activation of p38 MAPK and NF-κB accompanying the decline in TEER in both model systems. Pharmacological inhibition of NF-κB signaling partially prevented the TNF-α-induced TEER loss, whereas inhibition of p38 worsened barrier dysfunction in Caco-2 but not T-84 cells. Taken together, these data demonstrate that adalimumab prevents barrier dysfunction induced by TNF-α both functionally and structurally as well as at the level of signal transduction. Barrier protection might therefore constitute a novel mechanism how anti-TNF-α therapy contributes to epithelial restitution and tissue repair in inflammatory bowel diseases.

Exendin-4 Protects Hypoxic Islets From Oxidative Stress and Improves Islet Transplantation Outcome

Oxidative stress produced during pancreatic islet isolation leads to significant β-cell damage. Homeostatic cytokines secreted subsequently to islet transplantation damage β-cells by generating oxygen free radicals. In this study, exendin-4, a glucagon-like peptide-1 analog improved islet transplantation outcome by increasing the survival of diabetic recipient mice from 58% to 100%. We hypothesized that this beneficial effect was due to the ability of exendin-4 to reduce oxidative stress. Further experiments showed that it significantly reduced the apoptotic rate of cultured β-cells subjected to hypoxia or to IL-1β. Reduction of apoptotic events was confirmed in pancreatic islet grafts of exendin-4-treated mice. Exendin-4 enhanced Akt phosphorylation of β-cells and insulin released from them. It even augmented insulin secretion from islets cultivated at hypoxic conditions. Exposure to hypoxia led to a decrease in the activation of Akt, which was reversed when β-cells were pretreated with exendin-4. Moreover, exendin-4 increased the activity of redox enzymes in a hypoxia-treated β-cell line and reduced reactive oxygen species production in isolated pancreatic islets. Recovery from diabetes in mice transplanted with hypoxic islets was more efficient when they received exendin-4. In conclusion, exendin-4 rescued islets from oxidative stress caused by hypoxia or due to cytokine exposure. It improved the outcome of syngenic and xenogenic islet transplantation.

Sinonasal epithelial wound resealing in an in vitro model: inhibition of wound closure with IL‐4 exposure

Prolonged healing and persistent inflammation following surgery for rhinosinusitis impacts patient satisfaction and healthcare resources. Cytokines interleukin (IL)-4, IL-5, and IL-13 are important mediators in T-helper 2 (Th2) inflammatory rhinosinusitis. Decreased wound healing has been demonstrated with Th2 cytokine exposure, but this has not been extensively studied in sinonasal epithelium. We hypothesized that in vitro exposure of primary sinonasal epithelial cell cultures to Th2 inflammatory cytokine IL-4 and IL-13 would impair wound resealing and decrease expression of annexin A2 at the wound edge. Following 24-hour exposure to IL-4, IL-5, or IL-13 vs controls, sterile linear mechanical wounds were created in primary sinonasal epithelial cultures (n = 12 wounds per condition). Wounds were followed for 36 hours or until complete closure, and residual wound areas were calculated by image analysis. Group differences in annexin A2 were assessed by immunofluorescence labeling, confocal microscopy, and Western blots. Significant wound closure differences were identified across cytokine exposure groups (p < 0.001). Mean percentage wound closure at the completion of the 36-hour time course was 98.41% ± 3.43% for control wounds vs 85.02% ± 18.46% for IL-4 exposed wounds. IL-13 did not significantly impair sinonasal epithelial wound resealing in vitro. Annexin A2 protein levels were decreased in IL-4 treated wounds when compared to control wounds (p < 0.01). Th2 cytokine IL-4 decreases sinonasal epithelial wound closure in vitro. Annexin A2 is also diminished with IL-4 exposure. This supports the hypothesis that IL-4 exposure impairs sinonasal epithelial wound healing and may contribute to prolonged healing in Th2 inflammatory rhinosinusitis.

RNA-Seq Reveals Activation of Both Common and Cytokine-Specific Pathways following Neutrophil Priming

Neutrophils are central to the pathology of inflammatory diseases, where they can damage host tissue through release of reactive oxygen metabolites and proteases, and drive inflammation via secretion of cytokines and chemokines. Many cytokines, such as those generated during inflammation, can induce a similar “primed” phenotype in neutrophils, but it is unknown if different cytokines utilise common or cytokine-specific pathways to induce these functional changes. Here, we describe the transcriptomic changes induced in control human neutrophils during priming in vitro with pro-inflammatory cytokines (TNF-α and GM-CSF) using RNA-seq. Priming led to the rapid expression of a common set of transcripts for cytokines, chemokines and cell surface receptors (CXCL1, CXCL2, IL1A, IL1B, IL1RA, ICAM1). However, 580 genes were differentially regulated by TNF-α and GM-CSF treatment, and of these 58 were directly implicated in the control of apoptosis. While these two cytokines both delayed apoptosis, they induced changes in expression of different pro- and anti-apoptotic genes. Bioinformatics analysis predicted that these genes were regulated via differential activation of transcription factors by TNF-α and GM-CSF and these predictions were confirmed using functional assays: inhibition of NF-κB signalling abrogated the protective effect of TNF-α (but not that of GM-CSF) on neutrophil apoptosis, whereas inhibition of JAK/STAT signalling abrogated the anti-apoptotic effect of GM-CSF, but not that of TNF-α (p<0.05). These data provide the first characterisation of the human neutrophil transcriptome following GM-CSF and TNF-α priming, and demonstrate the utility of this approach to define functional changes in neutrophils following cytokine exposure. This may provide an important, new approach to define the molecular properties of neutrophils after in vivo activation during inflammation.