| 1. |
- Erttmann, Saskia F., et al.
(författare)
-
Loss of the DNA Damage Repair Kinase ATM Impairs Inflammasome-Dependent Anti-Bacterial Innate Immunity
- 2016
-
Ingår i: Immunity. - : Elsevier BV. - 1074-7613 .- 1097-4180. ; 45:1, s. 106-118
-
Tidskriftsartikel (refereegranskat)abstract
- The ATM kinase is a central component of the DNA damage repair machinery and redox balance. ATM dysfunction results in the multisystem disease ataxia-telangiectasia (AT). A major cause of mortality in AT is respiratory bacterial infections. Whether ATM deficiency causes innate immune defects that might contribute to bacterial infections is not known. Here we have shown that loss of ATM impairs inflammasome- dependent anti-bacterial innate immunity. Cells from AT patients or Atm(-/-) mice exhibited diminished interleukin-1 beta (IL-1 beta) production in response to bacteria. In vivo, Atm(-/-) mice were more susceptible to pulmonary S. pneumoniae infection in a manner consistent with inflammasome defects. Our data indicate that such defects were due to oxidative inhibition of inflammasome complex assembly. This study reveals an unanticipated function of reactive oxygen species (ROS) in negative regulation of inflammasomes and proposes a theory for the notable susceptibility of AT patients to pulmonary bacterial infection.
|
|
| 2. |
- Roux, Sandrine Le, et al.
(författare)
-
Transforming Growth Factor Beta 1 Modulates the Functional Expression of the Neurokinin-1 Receptor in Human Keratocytes
- 2016
-
Ingår i: Current Eye Research. - : Informa UK Limited. - 0271-3683 .- 1460-2202. ; 41:8, s. 1035-1043
-
Tidskriftsartikel (refereegranskat)abstract
- PURPOSE: Transforming growth factor beta 1 (TGF-β1) is a cytokine involved in a variety of processes, such as differentiation of fibroblasts into myofibroblasts. TGF-β1 has also been shown to delay the internalization of the neurokinin-1 receptor (NK-1 R) after its activation by its ligand, the neuropeptide substance P (SP). NK-1 R comprises two naturally occurring variants, a full-length and a truncated form, triggering different cellular responses. SP has been shown to affect important events in the cornea - such as stimulating epithelial cell proliferation - processes that are involved in corneal wound healing and thus in maintaining the transparency of the corneal stroma. An impaired signaling through NK-1 R could thus impact the visual quality. We hypothesize that TGF-β1 modulates the expression pattern of NK-1 R in human corneal stroma cells, keratocytes. The purpose of this study was to test that hypothesis.METHODS: Cultures of primary keratocytes were set up with cells derived from healthy human corneas, obtained from donated transplantation graft leftovers, and characterized by immunocytochemistry and Western blot. Immunocytochemistry for TGF-β receptors and NK-1 R was performed. Gene expression was assessed with real-time polymerase chain reaction (qPCR).RESULTS: Expression of TGF-β receptors was confirmed in keratocytes in vitro. Treating the cells with TGF-β1 significantly reduced the gene expression of NK-1 R. Furthermore, immunocytochemistry for NK-1 R demonstrated that it is specifically the expression of the full-length isotype of the receptor that is reduced after treatment with TGF-β1, which was also confirmed with qPCR using a specific probe for the full-length receptor.CONCLUSIONS: TGF-β1 down-regulates the gene expression of the full-length variant of NK-1 R in human keratocytes, which might impact its signaling pathway and thus explain the known delay in internalization after activation by SP seen with TGF-β1 treatment.
|
|
| 3. |
- Sloniecka, Marta, et al.
(författare)
-
Antiapoptotic Effect of Acetylcholine in Fas-Induced Apoptosis in Human Keratocytes
- 2016
-
Ingår i: Investigative Ophthalmology and Visual Science. - : Association for Research in Vision and Ophthalmology (ARVO). - 0146-0404 .- 1552-5783. ; 57:14, s. 5892-5902
-
Tidskriftsartikel (refereegranskat)abstract
- PURPOSE. To investigate the possible antiapoptotic effect of acetylcholine (ACh) in Fas-mediated apoptosis of primary human keratocytes in vitro, and to explore the underlying mechanism. METHODS. Primary human keratocytes were isolated from healthy corneas. Fas ligand (FasL) was used to induce apoptosis in keratocytes. Cell death was assessed by ELISA. Activity of caspase-3, -7, -8, and -9 was measured with luminescent caspase activity assays. Expression of nuclear factor-kappa B (NF-kappa B) gene was assessed with RT-quantitative (q)PCR. Cytochrome c release apoptosis assay kit was used to extract mitochondria and cytosol. Cytochrome c release, cleavage of Bid, and expression of B-cell lymphoma 2 (Bcl-2) were determined by Western blot. RESULTS. Cell death ELISA revealed that ACh is able to reduce Fas-induced apoptosis in keratocytes. Analysis of the activity of effector caspases-3 and -7 showed that ACh, when added to Fas-treated cells, decreases the activation of both these enzymes. The activity of initiator caspases -8 and -9 also decreased when ACh was added to Fas-treated cells. This antiapoptotic effect of ACh was dependent on ACh concentration and activation of muscarinic ACh receptors. Analysis of the antiapoptotic mechanisms triggered by ACh showed that ACh downregulates expression of FasL-induced NF-kappa B RNA expression, upregulates expression of antiapoptotic protein Bcl-2, downregulates expression of proapoptotic protein Bad, reduces cytochrome c release, and prevents proapoptotic Bid protein cleavage. CONCLUSIONS. Acetylcholine has an antiapoptotic effect in a Fas-apoptosis model of human primary keratocytes in vitro. It is therefore possible that ACh may play a role in corneal wound healing, by modulating its initiation phase.
|
|
| 4. |
- Sloniecka, Marta, et al.
(författare)
-
Substance P Enhances Keratocyte Migration and Neutrophil Recruitment through Interleukin-8
- 2016
-
Ingår i: Molecular Pharmacology. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0026-895X .- 1521-0111. ; 89:2, s. 215-225
-
Tidskriftsartikel (refereegranskat)abstract
- Keratocytes, the resident cells of the corneal stroma, are responsible for maintaining turnover of this tissue by synthesizing extracellular matrix components. When the cornea is injured, the keratocytes migrate to the wounded site and participate in the stromal wound healing. The neuropeptide substance P (SP), which is also known to be produced by non-neuronal cells, has previously been implicated in epithelial wound healing after corneal injury. Corneal scarring, which occurs in the stroma when the process of wound healing has malfunctioned, is one of the major causes of preventable blindness. This study aimed to elucidate the potential role of SP in keratocyte migration and therefore in stromal wound healing. We report that the expression and secretion of SP in human keratocytes are increased in response to injury in vitro. Moreover, SP enhances the migration of keratocytes by inducing the actin cytoskeleton reorganization and focal adhesion formation through the activation of the phosphatidylinositide 3-kinase and Ras-related C3 botulinum toxin substrate 1/Ras homolog gene family, member A pathway. Furthermore, SP stimulation leads to upregulated expression of the proinflammatory and chemotactic cytokine interleukin-8 (IL-8), which also contributes significantly to SP-enhanced keratocyte migration and is able to attract neutrophils. In addition, the preferred SP receptor, the neurokinin-1 receptor, is necessary to induce keratocyte migration and IL-8 secretion. In conclusion, we describe new mechanisms by which SP enhances migration of keratocytes and recruits neutrophils, two necessary steps in the corneal wound-healing process, which are also likely to occur in other tissue injuries.
|
|
