ECM crosslinking enhances fibroblast growth and protects against matrix proteolysis in lung fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterised by accumulation of extra cellular matrix (ECM) proteins and fibroblast proliferation. ECM cross-linking enzymes have been implicated in fibrotic diseases and we hypothesised that the ECM in IPF is abnormally cross-linked which enhances fibroblast growth and resistance to normal ECM turnover. We used a combination of in vitro ECM preparations and in vivo assays to examine the expression of cross-linking enzymes and the effect of their inhibitors on fibroblast growth and ECM turnover. Lysyl oxidase like 1, 2, 3 and 4 were expressed equally in control and IPF derived fibroblasts. Transglutaminase 2 was more strongly expressed in IPF fibroblasts. Lysyl oxidase like 2, transglutaminase 2 and transglutaminase generated cross-links were strongly expressed in IPF lung tissue. Fibroblasts grown on IPF ECM had higher LOXL3 protein expression and transglutaminase activity compared with those grown on control ECM. IPF derived ECM also enhanced fibroblast adhesion and proliferation compared with control ECM. Inhibition of lysyl oxidase and transglutaminse activity during ECM formation affected ECM structure as visualised by electron microscopy and reduced the enhanced fibroblast adhesion and proliferation of IPF ECM to control levels. Inhibition of transglutaminase, but not lysyl oxidase activity, enhanced the turnover of ECM in vitro. In bleomycin treated mice, during the post-inflammatory fibrotic phase, inhibition of transglutaminases was associated with a reduction in whole lung collagen. Our findings suggest that the ECM in IPF may enhance pathological cross-linking which contributes to increased fibroblast growth, resistance to normal ECM turnover to drive lung fibrosis

The helminth product, ES-62, protects against airway inflammation by resetting the Th cell phenotype

We previously demonstrated inhibition of ovalbumin (OVA)-induced allergic airway hyper-responsiveness in the mouse using ES-62, a phosphorylcholine-containing glycoprotein secreted by the filarial nematode, Acanthocheilonema viteae. This inhibition correlated with ES-62-induced mast cell desensitisation, although the degree to which this reflected direct targeting of mast cells remained unclear as suppression of the Th2 phenotype of the inflammatory response, as measured by eosinophilia and IL-4 levels in the lungs, was also observed. We now show that inhibition of the lung Th2 phenotype is reflected in ex vivo analyses of draining lymph node recall cultures and accompanied by a decrease in the serum levels of total and OVA-specific IgE. Moreover, ES-62 also suppresses the lung infiltration by neutrophils that is associated with severe asthma and is generally refractory to conventional anti-inflammatory therapies, including steroids. Protection against Th2-associated airway inflammation does not reflect induction of regulatory T cell (Treg) responses (there is no increased IL-10 or Foxp3 expression) but rather a switch in polarisation towards increased T-bet expression and IFNγ production. This ES-62-driven switch in the Th1/Th2 balance is accompanied by decreased IL-17 responses, a finding in line with reports that IFNγ and IL-17 are counter-regulatory. Consistent with ES-62 mediating its effects via IFNγ-mediated suppression of pathogenic Th2/Th17 responses, we found that neutralising anti-IFNγ antibodies blocked protection against airway inflammation in terms of pro-inflammatory cell infiltration, particularly by neutrophils and lung pathology. Collectively, these studies indicate that ES-62, or more likely small molecule analogues, could have therapeutic potential in asthma, in particular for those subtypes of patients (e.g. smokers, steroid-resistant) who are refractory to current treatments

The fatty acid and retinol binding protein of Heligmosomoides bakeri : a modulator of the host immune response

The increasing awareness that infection by helminths can prevent the development of allergy and autoimmunity partially due to the induction of a regulatory immune response has led to the investigation of parasite-derived molecules and their interactions. This study sought to explore the unique nematode specific fatty acid and retinol binding (FAR) proteins and their potentially important retinol binding ability. Therefore, the FAR protein of the immunoregulatory murine gastrointestinal nematode, Heligmosomoides bakeri was chosen. Recombinant Hb-FAR-l (rHb-FAR- 1) was successfully expressed in the yeast Pichia pastoris, and its retinol and fatty acid binding ability was confirmed. Despite the rare activation of toll-like receptors (TLRs) by parasite-derived molecules, this protein was found to trigger TLR2 on human embryonic kidney cells 293. Overall, rHb-FAR-l failed to induce the maturation of bone marrow derived dendritic cells (DCs) or macrophages (Ms), although DC-derived IL-6 was increased. In contrast to many helminth antigens, rHb-FAR-l treated DCs or Ms were not found to be hyporesponsive to simultaneous TLR activation, but DCs displayed elevated cytokine levels (IL-6, TNF-a) and increased expression of surface activation markers (MHC-II, CD86) in response to two TLR2 ligands, Pam3CSK4 or FSL-l, in comparison to exposure to the TLR2 agonists alone. However, rHb-FAR-l failed to modify the response of DCs to LPS (TLR4), zymosan (TLR2) or ODN1826 (TLR9). Fluorescent labelling of rHb- FAR-l revealed a specific internalization of this antigen into vesicular compartments of both DCs and Ms, within 45 min. The immunoregulatory potential of rHb-FAR-l was tested in the non-obese diabetic mouse. Despite the lack of T cell polarization after intraperitoneal (ip) injection of rHb-FAR-l, the protein was able to induce increased levels of IL-lO by splenocytes in response to rHb-FAR-l ex vivo. Subsequent to these ip injections an expanded number of peritoneal exudate cells (PECs) was found in the peritoneal cavity, with a higher percentage of Bl B cells but a lower frequency of peritoneal Ms. The latter cell type displayed up-regulated expression of FIZZl and arginase-I, both markers for alternative activation of Ms. In addition to in vivo effects, rHb-FAR-l directly stimulated the expression of IL-6, IL-lO and TNF-a from naive PECs in vitro but was not able to interfere with the proliferation of splenic Band T cells. Furthermore, rHb-FAR-l failed to induce Foxp3 expressing regulatory T cells but polarized Th17 cells in an IL-6 dependent manner, suggesting a potential importance in the early transient Th17 response to H. bakeri, which prolongs parasite survival.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Small molecule analogues of the immunomodulatory parasitic helminth product ES-62 have anti-allergy properties

ES-62, a glycoprotein secreted by the filarial nematode Acanthocheilonema viteae, exhibits anti-inflamma-tory properties by virtue of covalently attached phosphorylcholine moieties. Screening of a library of ES-62 phosphorylcholine-based small molecule analogues (SMAs) revealed that two compounds, termed 11a and 12b, mirrored the helminth product both in inhibiting mast cell degranulation and cytokine responses in vitro and in preventing ovalbumin-induced Th2-associated airway inflammation and eosin- ophil infiltration of the lungs in mice. Furthermore, the two SMAs inhibited neutrophil infiltration of the lungs when administered therapeutically. ES-62-SMAs 11a and 12b thus represent starting points for novel drug development for allergies such as asthma