Autoimmunity to synovial extracellular matrix proteins in patients with postinfectious Lyme arthritis.

BACKGROUNDAutoimmune diseases often have strong genetic associations with specific HLA-DR alleles. The synovial lesion in chronic inflammatory forms of arthritis shows marked upregulation of HLA-DR molecules, including in postinfectious Lyme arthritis (LA). However, the identity of HLA-DR-presented peptides, and therefore the reasons for these associations, has frequently remained elusive.METHODSUsing immunopeptidomics to detect HLA-DR-presented peptides from synovial tissue, we identified T cell epitopes from 3 extracellular matrix (ECM) proteins in patients with postinfectious LA, identified potential Borreliella burgdorferi-mimic (Bb-mimic) epitopes, and characterized T and B cell responses to these peptides or proteins.RESULTSOf 24 postinfectious LA patients, 58% had CD4+ T cell responses to at least 1 epitope of 3 ECM proteins, fibronectin-1, laminin B2, and/or collagen Vα1, and 17% of 52 such patients had antibody responses to at least 1 of these proteins. Patients with autoreactive T cell responses had significantly increased frequencies of HLA-DRB1*04 or -DRB1*1501 alleles and more prolonged arthritis. When tetramer reagents were loaded with ECM or corresponding Bb-mimic peptides, binding was only with the autoreactive T cells. A high percentage of ECM-autoreactive CD4+ T cells in synovial fluid were T-bet-expressing Th1 cells, a small percentage were RoRγt-expressing Th17 cells, and a minimal percentage were FoxP3-expressing Tregs.CONCLUSIONAutoreactive, proinflammatory CD4+ T cells and autoantibodies develop to ECM proteins in a subgroup of postinfectious LA patients who have specific HLA-DR alleles. Rather than the traditional molecular mimicry model, we propose that epitope spreading provides the best explanation for this example of infection-induced autoimmunity.FUNDINGSupported by National Institute of Allergy and Infectious Diseases R01-AI101175, R01-AI144365, and F32-AI125764; National Institute of Arthritis and Musculoskeletal and Skin Diseases K01-AR062098 and T32-AR007258; NIH grants P41-GM104603, R24-GM134210, S10-RR020946, S10-OD010724, S10-OD021651, and S10-OD021728; and the G. Harold and Leila Y. Mathers Foundation, the Eshe Fund, and the Lyme Disease and Arthritis Research Fund at Massachusetts General Hospital.

The human milk oligosaccharide 2'-fucosyllactose modulates CD14 expression in human enterocytes, thereby attenuating LPS-induced inflammation.

BACKGROUND: A major cause of enteric infection, Gram-negative pathogenic bacteria activate mucosal inflammation through lipopolysaccharide (LPS) binding to intestinal toll-like receptor 4 (TLR4). Breast feeding lowers risk of disease, and human milk modulates inflammation. OBJECTIVE: This study tested whether human milk oligosaccharides (HMOSs) influence pathogenic Escherichia coli-induced interleukin (IL)-8 release by intestinal epithelial cells (IECs), identified specific proinflammatory signalling molecules modulated by HMOSs, specified the active HMOS and determined its mechanism of action. METHODS: Models of inflammation were IECs invaded by type 1 pili enterotoxigenic E. coli (ETEC) in vitro: T84 modelled mature, and H4 modelled immature IECs. LPS-induced signalling molecules co-varying with IL-8 release in the presence or absence of HMOSs were identified. Knockdown and overexpression verified signalling mediators. The oligosaccharide responsible for altered signalling was identified. RESULTS: HMOSs attenuated LPS-dependent induction of IL-8 caused by ETEC, uropathogenic E. coli, and adherent-invasive E. coli (AIEC) infection, and suppressed CD14 transcription and translation. CD14 knockdown recapitulated HMOS-induced attenuation. Overexpression of CD14 increased the inflammatory response to ETEC and sensitivity to inhibition by HMOSs. 2'-fucosyllactose (2'-FL), at milk concentrations, displayed equivalent ability as total HMOSs to suppress CD14 expression, and protected AIEC-infected mice. CONCLUSIONS: HMOSs and 2'-FL directly inhibit LPS-mediated inflammation during ETEC invasion of T84 and H4 IECs through attenuation of CD14 induction. CD14 expression mediates LPS-TLR4 stimulation of portions of the 'macrophage migration inhibitory factors' inflammatory pathway via suppressors of cytokine signalling 2/signal transducer and activator of transcription 3/NF-κB. HMOS direct inhibition of inflammation supports its functioning as an innate immune system whereby the mother protects her vulnerable neonate through her milk. 2'-FL, a principal HMOS, quenches inflammatory signalling.

Group B Streptococcus induces a caspase-dependent apoptosis in fetal rat lung interstitium.

Group B Streptococcus (GBS) is an important pathogen and is associated with sepsis and meningitis in neonates and infants. An ex vivo model that facilitates observations of GBS interactions with multiple host cell types over time was used to study its pathogenicity. GBS infections were associated with profound reductions in fetal lung; explant size, and airway branching. Elevated levels of apoptosis subsequent to GBS infections were observed by whole-mount confocal immunofluorescence using activated-caspase-3-antibodies and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assays. The caspase inhibitor Z-VAD-FMK abolished the increase in TUNEL-positive cells associated with GBS infections, indicating that the GBS-induced apoptosis was caspase-dependent. Digital image analyses revealed that both GBS and the active form of caspase-3 were distributed primarily within the lung interstitium, suggesting that these tissues are important targets for GBS. Antibodies to the active form of caspase-3 colocalized with both macrophage- and erythroblast-markers, suggesting that these hematopoietic cells are vulnerable to GBS-mediated pathogenesis. These studies suggest that GBS infections profoundly alter lung morphology and caspase-dependent hematopoietic cell apoptosis within the lung interstitium play roles in GBS pathophysiology in this model.

The principal fucosylated oligosaccharides of human milk exhibit prebiotic properties on cultured infant microbiota.

Breast-fed infant microbiota is typically rich in bifidobacteria. Herein, major human milk oligosaccharides (HMOS) are assessed for their ability to promote the growth of bifidobacteria and to acidify their environment, key features of prebiotics. During in vitro anaerobic fermentation of infant microbiota, supplementation by HMOS significantly decreased the pH even greater than supplementation by fructooligosaccharide (FOS), a prebiotic positive control. HMOS elevated lactate concentrations, increased the proportion of Bifidobacterium spp. in culture, and through their fermentation into organic acids, decreased the proportion of Escherichia and Clostridium perfringens. Three principal components of HMOS, 2'-fucosyllactose, lactodifucotetraose and 3-fucosyllactose, were consumed in these cultures. These three principal oligosaccharides of human milk were then individually tested as supplements for in vitro growth of four individual representative strains of infant gut microbes. Bifidobacterium longum JCM7007 and B. longum ATCC15697 efficiently consumed oligosaccharides and produced abundant lactate and short-chain fatty acids, resulting in significant pH reduction. The specificity of fermentation differed by microbe species and strain and by oligosaccharide structure. Escherichia coli K12 and C. perfringens did not utilize appreciable fucosylated oligosaccharides, and a typical mixture of organic acid fermentation products inhibited their growth. In summary, 2'-fucosyllactose, lactodifucotetraose, and 3-fucosyllactose, when cultured with B. longum JCM7007 and B. longum ATCC15697, exhibit key characteristics of a prebiotic in vitro. If these bifidobacteria are representative of pioneering or keystone species for human microbiota, fucosylated HMOS could strongly promote colonization and maintenance of a mutualist symbiotic microbiome. Thus, these simple glycans could mediate beneficial effects of human milk on infant health.

Human milk mucin 1 and mucin 4 inhibit Salmonella enterica serovar Typhimurium invasion of human intestinal epithelial cells in vitro.

Many human milk glycans inhibit pathogen binding to host receptors and their consumption by infants is associated with reduced risk of disease. Salmonella infection is more frequent among infants than among the general population, but the incidence is lower in breast-fed babies, suggesting that human milk could contain components that inhibit Salmonella. This study aimed to test whether human milk per se inhibits Salmonella invasion of human intestinal epithelial cells in vitro and, if so, to identify the milk components responsible for inhibition. Salmonella enterica serovar Typhimurium SL1344 (SL1344) invasion of FHs 74 Int and Caco-2 cells were the models of human intestinal epithelium infection. Internalization of fluorescein-5-isothiocyanate-labeled SL1344 into intestinal cells was measured by flow cytometry to quantify infection. Human milk and its fractions inhibited infection; the inhibitory activity localized to the high molecular weight glycans. Mucin 1 and mucin 4 were isolated to homogeneity. At 150 µg/L, a typical concentration in milk, human milk mucin 1 and mucin 4 inhibited SL1344 invasion of both target cell types. These mucins inhibited SL1344 invasion of epithelial cells in a dose-dependent manner. Thus, mucins may prove useful as a basis for developing novel oral prophylactic and therapeutic agents that inhibit infant diseases caused by Salmonella and related pathogens.

Nitrofen induces apoptosis independently of retinaldehyde dehydrogenase (RALDH) inhibition.

BACKGROUND: Nitrofen is a diphenyl ether that induces congenital diaphragmatic hernia (CDH) in rodents. Its mechanism of action has been hypothesized as inhibition of the retinaldehyde dehydrogenase (RALDH) enzymes with consequent reduced retinoic acid signaling. METHODS: To determine if nitrofen inhibits RALDH enzymes, a reporter gene construct containing a retinoic acid response-element (RARE) was transfected into HEK-293 cells and treated with varying concentrations of nitrofen in the presence of retinaldehyde (retinal). Cell death was characterized by caspace-cleavage microplate assays and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assays. Ex vivo analyses of cell viability were characterized in fetal rat lung explants using Live/Dead staining. Cell proliferation and apoptosis were assessed using fluorescent immunohistochemistry with phosphorylated histone and activated caspase antibodies on explant tissues. Nile red staining was used to identify intracellular lipid droplets. RESULTS: Nitrofen-induced dose-dependent declines in RARE-reporter gene expression. However, similar reductions were observed in control-reporter constructs suggesting that nitrofen compromised cell viability. These observed declines in cell viability resulted from increased cell death and were confirmed using two independent assays. Ex vivo analyses showed that mesenchymal cells were particularly susceptible to nitrofen-induced apoptosis while epithelial cell proliferation was dramatically reduced in fetal rat lung explants. Nitrofen treatment of these explants also showed profound lipid redistribution, primarily to phagocytes. CONCLUSIONS: The observed declines in nitrofen-associated retinoic acid signaling appear to be independent of RALDH inhibition and likely result from nitrofen induced cell death/apoptosis. These results support a cellular apoptotic mechanism of CDH development, independent of RALDH inhibition.

Lactic acid is a potential virulence factor for group B Streptococcus.

Group B Streptococcus (GBS) is a Gram-positive bacterium that causes sepsis and meningitis in neonates and infants. Although several GBS-associated virulence factors have been described, the mechanisms of GBS invasive disease are not well understood. To characterize additional virulence factors, a novel in vitro infection assay was developed using rat fetal lung explants. However, application of GBS to the system induced rapid lung tissue destruction associated with increased media acidity. Since lactic acid produced by other streptococci is an important virulence factor, we hypothesized that lactic acid contributed to the virulence of GBS. Spent growth media and neutralized-spent media were applied to explants and results indicated that neutralization of the media completely protected the tissue from degradation. These results were verified using multiple viability assays and with transformed cell lines. Furthermore, comparable spent media from Escherichia coli did not induce tissue cytotoxicity, suggesting that GBS produces organic acids in excess of other potential bacterial pathogens. Analysis of the spent media indicated that l-lactate levels reached approximately 70 mM, indicating that lactic acid is a major constituent of the metabolic acid produced by GBS. Treatment of explants with lactic acid alone produced dose-dependent tissue degradation, indicating that lactic acid is independently sufficient to induce target-tissue cytotoxicity. Finally, both spent media and 23.6 mM lactic acid produced dramatic tissue autofluorescence; the basis for this is currently unknown. These studies demonstrate that GBS-produced lactic acid is a potential virulence factor and may contribute to GBS invasive disease.

Retinoic acid-mediated differentiation protects against nitrofen-induced apoptosis.

BACKGROUND: Nitrofen is a diphenyl ether that induces a spectrum of birth defects subsequent to administration to pregnant rodents, in which the molecular etiology of these defects are poorly characterized. Because previous reports showed that nitrofen induced apoptosis in undifferentiated P19 teratocarcinoma cells, we hypothesized that undifferentiated fetal cells have greater susceptibility to nitrofen-induced apoptosis than their differentiated derivatives. METHODS: To investigate this hypothesis, cell lines including P19 and F9 were differentiated with retinoic acid into neuronal and endodermal derivatives respectively. Apoptosis was characterized by caspase-3 cleavage and Terminal transferase dUTP nick end labeling (TUNEL) assays. RESULTS: Both differentiated cell-types had reduced nitrofen-induced caspase-3 cleavage and DNA fragmentation compared with the naive controls, strongly suggesting that differentiation of these cells protects against nitrofen-induced apoptosis. In addition, resistance to apoptotic induction was proportional to the expression levels of the differentiation marker, p27 (kip1) while direct proportionality was not observed for the antiapoptotic protein Bcl-2. CONCLUSIONS: These studies show that nitrofen may induce its associated birth defects via a mechanism involving apoptosis of undifferentiated fetal cells.

Vitamin A deficiency (VAD), teratogenic, and surgical models of congenital diaphragmatic hernia (CDH).

Congenital diaphragmatic hernia (CDH) is a congenital malformation that occurs with a frequency of 0.08 to 0.45 per 1,000 births. Children with CDH are born with the abdominal contents herniated through the diaphragm and exhibit an associated pulmonary hypoplasia which is frequently accompanied by severe morbidity and mortality. Although the etiology of CDH is largely unknown, considerable progress has been made in understanding its molecular mechanisms through the usage of genetic, teratogenic, and surgical models. The following review focuses on the teratogenic and surgical models of CDH and the possible molecular mechanisms of nitrofen (a diphenyl ether, formerly used as an herbicide) in both induction of CDH and pulmonary hypoplasia. In addition, the mechanisms of other compounds including several anti-inflammatory agents that have been linked to CDH will be discussed. Furthermore, this review will also explore the importance of vitamin A in lung and diaphragm development and the possible mechanisms of teratogen interference in vitamin A homeostasis. Continued exploration of these models will bring forth a clearer understanding of CDH and its molecular underpinnings, which will ultimately facilitate development of therapeutic strategies.

Distribution of ERK1/2 and ERK3 during normal rat fetal lung development.

The extracellular regulated kinases-1 and -2 (ERK1/2) are well-characterized mitogen-activated protein kinases (MAPK) that play critical roles in proliferation and differentiation, whereas the function(s) of MAPK ERK3 are currently unknown. To understand better the roles of these kinases in development, the temporal distribution of ERK1, -2, and -3 proteins were investigated in multiple tissues. The ERK3 protein, in contrast to ERK1/2 varied both between and within individual organs over time. To characterize this variability in greater detail, the temporal and spatial distributions of activated ERK1/2 and ERK3 during rat fetal lung development were investigated. The diphosphorylated (activated) forms of ERK1/2 (dp-ERK1/2), ERK3, and its phosphorylated form (P-ERK3) decreased from embryonic day 17 (E17) through E21 while both ERK1 and ERK2 total proteins remained unchanged, indicating that ERK1/2 and ERK3 proteins are expressed independently during fetal lung development. In addition, characterization of the distribution of these proteins by fluorescent immunohistochemistry indicated that phosphorylated ERK1/2 and total ERK1/2 were distributed throughout multiple cell types, with the phosphorylated ERK1/2 colocalizing with prophase mitotic cells. In contrast, ERK3 was restricted to the distal lung epithelium during the pseudoglandular phase (E17) but shifted to the proximal airways, particularly Clara cells during the saccular stage (E21). The P-ERK3 colocalized with the mitotic marker P-histone H3 in fetal lung and in NIH3T3 and HeLa cells, implicating a potential role for P-ERK3 in mitosis. Thus, expression of ERK1/2 and ERK3 and their phosphorylated forms are expressed independently and are temporally and spatially localized during fetal lung morphogenesis. These observations will facilitate detailed functional analysis of these kinases to assess their roles in pulmonary development and diseases.

Retinoic acid decreases fetal lung mesenchymal cell proliferation in vivo and in vitro.

Retinoic acid (RA) is an important coordinator of mammalian organogenesis. RA is implicated in critical lung developmental events. Cell proliferation is precisely regulated during development. We investigated the effect of RA on proliferating mesenchymal cells in both whole organ lung cultures and cell cultures. The potential pathways required for the response were studied in cultures of lung mesenchymal cells from embryonic day (e) 12. We observed an RA-dependent reduction in proliferation of mesenchymal cells in both whole organ and in cell culture. In mesenchymal cell cultures, RA decreased proliferation in lung mesenchymal cells by 72%. This was associated with a decrease of erk-1/2 activity by 68%. Mesenchymal cell proliferation is erk-1/2 dependent. Erk-1/2 can be activated by G-protein coupled receptors (GPCR) or tyrosine kinase receptors (RTK). RA treatment altered both the RTK and the GPCR pathways in primary lung mesenchymal cells. The Epidermal Growth Factor (EGF) dependent erk-1/2 activation was increased by 35% whereas the G(i)-protein cascade was inhibited by 44% in cells treated with RA. Our results suggest that RA decreases proliferation of lung mesenchyme via a G(i)-protein and the erk-1/2 signaling cascade.

Oxidation-reduction (redox) controls fetal hypoplastic lung growth.

INTRODUCTION: The persistent morbidity and mortality of congenital diaphragmatic hernia are largely due to associated pulmonary hypoplasia. We have shown previously that three antioxidants (vitamin C, glutathione, and vitamin E) could accelerate the growth of fetal hypoplastic lungs grown in culture. We hypothesize that this occurs via a reductant mechanism. METHODS: Timed-pregnant rats were gavage-fed nitrofen (100 mg) on day 9.5 of gestation (term = day 22). Fetal lungs were harvested on day 13.5 and placed in organ culture containing serum-free BGJb medium with antibiotics. After randomization, the lung organ cultures were divided into a control group (n = 31) and an experimental group that received the antioxidant N-acetylcysteine (NAC, 100 microM, n = 31). The fetal lung organ cultures were grown for 4 days at 37 degrees C with 5% CO(2). Computer-assisted digital tracings of the airways were performed daily on live, unstained specimens, and lung bud count, perimeter, and area were measured. After 4 days, lungs were pooled, homogenized, and assayed for reduced and oxidized glutathione, normalized to protein, as an estimate of the tissue redox potential. Data were expressed as means +/- SEM, and statistical comparisons were performed using Student's unpaired t test, with P < 0.05 considered significant. RESULTS: Area, perimeter, lung bud count, and complexity (as measured by the perimeter/square root of area) were all significantly increased with NAC treatment from day 2 onward. Reduced glutathione levels were significantly increased following NAC administration (67.1 +/- 5.8 versus 37.5 +/- 4.2 micromol/mg, P = 0.0004). The ratio of reduced to oxidized glutathione was 2.23. CONCLUSIONS: N-Acetylcysteine stimulates nitrofen-induced hypoplastic fetal lung growth in organ culture and increases the ratio of reduced to oxidized glutathione. These data support the concept that oxidation-reduction (redox) may be an important control mechanism for fetal lung growth.

MEK-1/2 inhibition reduces branching morphogenesis and causes mesenchymal cell apoptosis in fetal rat lungs.

The roles of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinases-1 and -2 (ERK-1/2) in fetal lung development have not been extensively characterized. To determine if ERK-1/2 signaling plays a role in fetal lung branching morphogenesis, U-0126, an inhibitor of the upstream kinase MAP ERK kinase (MEK), was added to fetal lung explants in vitro. Morphometry as measured by branching, area, perimeter, and complexity were significantly reduced in U-0126-treated lungs. At the same time, U-0126 treatment reduced ERK-1/2, slightly increased p38 kinase, but did not change c-Jun NH(2)-terminal kinase activities, indicating that U-0126 specifically inhibited the ERK-1/2 enzymes. These changes were associated with increased apoptosis as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and immunofluorescent labeling of anti-active caspase-3 in the mesenchyme of explants after U-0126 treatment compared with the control. Mitosis characterized by immunolocalization of proliferating cell nuclear antigen was found predominantly in the epithelium and was reduced in U-0126-treated explants. Thus U-0126 causes specific inhibition of ERK-1/2 signaling, diminished branching morphogenesis, characterized by increased mesenchymal apoptosis, and decreased epithelial proliferation in fetal lung explants.