Respiratory Mycoplasma pneumoniae (Mp) infection is involved in several acute and chronic lung diseases including community-acquired pneumonia, asthma and chronic obstructive pulmonary disease. In the chronic disease process, recurrent respiratory bacterial infections could occur, which may result in varying degrees of symptoms and lung inflammation among patients. However, the lung immunologic differences of host responses to repeated bacterial (i.e., Mp) infections remain to be determined. In the present study, we examined cellular and humoral responses to multiple (up to 3) Mp infections in two genetically different strains of mice (BALB/c and C57BL/6). Mice were intranasally inoculated with one Mp infection, two or three Mp infections (4 weeks apart), and sacrificed on days 3, 7 and 14 after the last Mp infection. Overall, compared to C57BL/6 mice, BALB/c mice demonstrated a significantly higher degree of lung tissue inflammatory cell infiltrate, BAL cellularity, and release of pro-inflammatory cytokines (TNF-alpha, keratinocyte-derived chemokine (KC, a mouse homolog of human chemokine Gro-alpha [CXCL1], and IFN-gamma). In addition, BALB/c mice presented higher levels of serum Mp-specific IgG and IgM, but not IgA. Consistently with lung and serum data, Mp load in BAL and lung specimens was significantly higher in BALB/c mice than C57BL/6 mice. Moreover, repeated Mp infections in BALB/c, but not C57BL/6 mice, produced a greater inflammatory response than did a single Mp infection. Our results suggest that hosts with different genetic background may have different susceptibility to repeated respiratory Mp infections along with inflammatory responses.

Blunt chest trauma resulting in pulmonary contusion is a common but poorly understood injury. We previously demonstrated that lung contusion activates localized and systemic innate immune mechanisms and recruits neutrophils to the injured lung. We hypothesized that the innate immune and inflammatory activation of neutrophils may figure prominently in the response to lung injury. To investigate this, we used a model of pulmonary contusion in the mouse that is similar to that observed clinically in humans and evaluated postinjury lung function and pulmonary neutrophil recruitment. Comparisons were made between injured mice with and without neutrophil depletion. We further examined the role of chemokines and adhesion receptors in neutrophil recruitment to the injured lung. We found that lung injury and resultant physiological dysfunction after contusion were dependent on the presence of neutrophils in the alveolar space. We show that CXCL1, CXCL2/3, and CXCR2 are involved in neutrophil recruitment to the lung after injury and that intercellular adhesion molecule 1 is locally expressed and actively participates in this process. Injured gp91-deficient mice showed improved lung function, indicating that oxidant production by neutrophil NADPH oxidase mediates lung dysfunction after contusion. These data suggest that both neutrophil presence and function are required for lung injury after lung contusion.

Organic dust exposure in the agricultural industry results in significant lung disease. Macrophage infiltrates are increased in the lungs after organic dust exposures, yet the phenotype and functional importance of these cells remain unclear. Using an established intranasal inhalation murine model of dust-induced lung inflammation, animals were treated once or daily for 3 weeks with swine confinement organic dust extract (DE). Repetitive DE treatment for 3 weeks resulted in significant increases in CD11c(+)/CD11b(+) macrophages in whole lung-associated tissue. These cells displayed increased costimulatory molecule (CD80 and CD86) expression, enhanced phagocytic ability, and an increased production of IL-6, CXCL1, and CXCL2. Similar findings were observed with the CD11c(+)/CD11b(+) macrophage infiltrate after repetitive exposure to peptidoglycan, a major DE component. To determine the functional importance of macrophages in mediating DE-induced airway inflammation, lung macrophages were selectively depleted using a well-established intranasal clodronate liposome depletion/suicide strategy. First, macrophage depletion by clodronate liposomes resulted in significant reductions in airway neutrophil influx and TNF-α and IL-6 production after a single exposure to DE. In contrast, after repetitive 3-week exposure to DE, airway lavage fluid and lung tissue neutrophils were significantly increased in clodronate liposome-treated mice compared with control mice. A histological examination of lung tissue demonstrated striking increases in alveolar and bronchiolar inflammation, as well as in the size and distribution of cellular aggregates in clodronate-liposome versus saline-liposome groups repetitively exposed to DE. These studies demonstrate that DE elicits activated CD11c(+)/CD11b(+) macrophages in the lung, which play a critical role in regulating the outcome of DE-induced airway inflammation.

BACKGROUND

Cryptococcus neoformans (C. neoformans) is a globally distributed fungal pathogen with the potential to cause serious disease, particularly among immune compromised hosts. Exposure to this organism is believed to occur by inhalation and may result in pneumonia and/or disseminated infection of the brain as well as other organs. Little is known about the role of airway epithelial cells in cryptococcal recognition or their ability to induce an inflammatory response.

METHODS

Immortalized BEAS-2B bronchial epithelial cells and primary normal human bronchial epithelium (NHBE) were stimulated in vitro with encapsulated or acapsular C. neoformans cultivated at room temperature or 37 degrees C. Activation of bronchial epithelial cells was characterized by analysis of inflammatory cytokine and chemokine expression, transcription factor activation, fungal-host cell association, and host cell damage.

RESULTS

Viable C. neoformans is a strong activator of BEAS-2B cells, resulting in the production of the neutrophil chemokine Interleukin (IL)-8 in a time- and dose-dependent manner. IL-8 production was observed only in response to acapsular C. neoformans that was grown at 37 degrees C. C. neoformans was also able to induce the expression of the chemokine CXCL1 and the transcription factor CAAT/enhancer-binding protein beta (CEBP/beta) in BEAS-2B cells. NHBE was highly responsive to stimulation with C. neoformans; in addition to transcriptional up regulation of CXCL1, these primary cells exhibited the greatest IL-8 secretion and cell damage in response to stimulation with an acapsular strain of C. neoformans.

CONCLUSIONS

This study demonstrates that human bronchial epithelial cells mediate an acute inflammatory response to C. neoformans and are susceptible to damage by this fungal pathogen. The presence of capsular polysaccharide and in vitro fungal culture conditions modulate the host inflammatory response to C. neoformans. Human bronchial epithelial cells are likely to contribute to the initial stages of pulmonary host defense in vivo.

MicroRNAs have been shown to be important regulators of inflammatory and immune responses and are implicated in several immune disorders including systemic lupus erythematosus and rheumatoid arthritis, but their role in Lyme borreliosis remains unknown. We performed a microarray screen for expression of miRNAs in joint tissue from three mouse strains infected with Borrelia burgdorferi. This screen identified upregulation of miR-146a, a key negative regulator of NF-κB signaling, in all three strains, suggesting it plays an important role in the in vivo response to B. burgdorferi. Infection of B6 miR-146a-/- mice with B. burgdorferi revealed a critical nonredundant role of miR-146a in modulating Lyme arthritis without compromising host immune response or heart inflammation. The impact of miR-146a was specifically localized to the joint, and did not impact lesion development or inflammation in the heart. Furthermore, B6 miR-146a-/- mice had elevated levels of NF-κB-regulated products in joint tissue and serum late in infection. Flow cytometry analysis of various lineages isolated from infected joint tissue of mice showed that myeloid cell infiltration was significantly greater in B6 miR-146a-/- mice, compared to B6, during B. burgdorferi infection. Using bone marrow-derived macrophages, we found that TRAF6, a known target of miR-146a involved in NF-κB activation, was dysregulated in resting and B. burgdorferi-stimulated B6 miR-146a-/- macrophages, and corresponded to elevated IL-1β, IL-6 and CXCL1 production. This dysregulated protein production was also observed in macrophages treated with IL-10 prior to B. burgdorferi stimulation. Peritoneal macrophages from B6 miR-146a-/- mice also showed enhanced phagocytosis of B. burgdorferi. Together, these data show that miR-146a-mediated regulation of TRAF6 and NF-κB, and downstream targets such as IL-1β, IL-6 and CXCL1, are critical for modulation of Lyme arthritis during chronic infection with B. burgdorferi.

OBJECTIVE

To investigate the effects of T-cell-derived cytokines on gene and protein expression of chemokines in a human RPE cell line (ARPE-19).

METHODS

We used an in vitro coculture system in which the RPE and CD3/CD28-activated T-cells were separated by a membrane. RPE cell expression of chemokine genes was quantified using three different types of microarrays. Protein expression was determined by single and multiplex ELISA and immunoblotting.

RESULTS

Coculture with activated T-cells increased RPE mRNA and protein expression of chemokines CCL2 (MCP-1); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (MCP-2); CXCL1 (GRO-α); IL8 (CXCL8); CXCL9 (MIG); CXCL1CXCL1CXCL1CXCL1CXCL1CXCL1CXCL1CXCL1, CXCL6, and IL8 were negatively regulated by IFNγ.

CONCLUSIONS

RPE cells responded to exposure to T-cell-derived cytokines by upregulating expression of multiple chemokines related to microglial, T-cell, and monocyte chemotaxis and activation. This inflammatory stress response may have implications for immune homeostasis in the retina, and for the further understanding of inflammatory ocular diseases such as uveitis and AMD.

The response of the bovine mammary gland to lipoteichoic acid (LTA), which is a major pathogen-associated molecular pattern of Gram-positive bacteria, was investigated by infusing purified Staphylococcus aureus LTA in the lumen of the gland. LTA was able to induce clinical mastitis at the dose of 100 microg/quarter, and a subclinical inflammatory response at 10 microg/quarter. The induced inflammation was characterized by a prompt and massive influx of neutrophils in milk. LTA proved to induce strongly the secretion of the chemokines CXCL1, CXCL2, CXCL3 and CXCL8, which target mainly neutrophils. The complement-derived chemoattractant C5a was generated in milk only with the highest dose of LTA (100 microg). The pro-inflammatory cytokine IL-1beta was induced in milk, but there was very little if any TNF-alpha and no IFN-gamma. The re-assessment of CXCL8 concentrations in milk whey of quarters previously challenged with S. aureus, by using an ELISA designed for bovine CXCL8, showed that this chemokine was induced in milk, contradicting previous reports. Overall, S. aureus LTA elicited mammary inflammatory responses that shared several attributes with S. aureus mastitis. Purified LTA looks promising as a convenient tool to investigate the inflammatory and immune responses of the mammary gland to S. aureus.

OBJECTIVE

To study the role of infiltrating neutrophils in the development of experimental corneal neovascularization (CNV).

METHODS

CNV was induced by alkali injury in normal C57BL/6 mice, and the kinetics of neutrophil recruitment to the cornea and the CNV was detected by histologic analysis at multiple time points. Neutrophil recruitment to the corneas was inhibited by injection of anti-mouse granulocyte monoclonal antibodies (Ly-6G) or neutralizing anti-mouse CXCR2 antibodies. CNV was compared between the control and the specific antibody-treated mice 2 weeks after alkali injury, as quantified by CD31 immunostaining. Corneal vascular endothelial growth factor (VEGF) expression after injury was determined by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical staining.

RESULTS

Many myeloperoxidase-positive neutrophils began to infiltrate the corneas 2 days after injury, but infiltration ceased by 14 days after injury. CNV became evident 7 days after injury, reached a maximal level at 14 days, and decreased thereafter. The mRNA expression of CXCR2 and its ligands, macrophage inflammatory protein 2 (CXCL2/MIP-2), and growth-related protein alpha (CXCL1/KC) increased markedly at 2 days after injury. Injection of either anti-mouse granulocyte monoclonal antibodies (Ly-6G) or neutralizing anti-mouse CXCR2 antibodies markedly, and to a similar extent, inhibited neutrophil recruitment to the cornea, indicating that neutrophil infiltration was mediated primarily by CXCR2. In contrast, these treatments failed to attenuate alkali-induced CNV. VEGF mRNA expression was enhanced 2 days after injury, and VEGF proteins were detected mainly in infiltrating mononuclear cells but not in neutrophils.

CONCLUSIONS

CXCR2-mediated neutrophil infiltration contributes only marginally to the subsequent development of CNV.

The prostaglandin F(2alpha) (PGF(2alpha)) receptor (FP) is elevated in endometrial adenocarcinoma. This study found that PGF(2alpha) signaling via FP regulates expression of chemokine (C-X-C motif) ligand 1 (CXCL1) in endometrial adenocarcinoma cells. Expression of CXCL1 and its receptor, CXCR2, are elevated in cancer tissue compared with normal endometrium and localized to glandular epithelium, endothelium, and stroma. Treatment of Ishikawa cells stably transfected with the FP receptor (FPS cells) with 100 nmol/L PGF(2alpha) increased CXCL1 promoter activity, mRNA, and protein expression, and these effects were abolished by cotreatment of cells with FP antagonist or chemical inhibitors of Gq, epidermal growth factor receptor, and extracellular signal-regulated kinase. Similarly, CXCL1 was elevated in response to 100 nmol/L PGF(2alpha) in endometrial adenocarcinoma explant tissue. CXCL1 is a potent neutrophil chemoattractant. The expression of CXCR2 colocalized to neutrophils in endometrial adenocarcinoma and increased neutrophils were present in endometrial adenocarcinoma compared with normal endometrium. Conditioned media from PGF(2alpha)-treated FPS cells stimulated neutrophil chemotaxis, which could be abolished by CXCL1 protein immunoneutralization of the conditioned media or antagonism of CXCR2. Finally, xenograft tumors in nude mice arising from inoculation with FPS cells showed increased neutrophil infiltration compared with tumors arising from wild-type cells or following treatment of mice bearing FPS tumors with CXCL1-neutralizing antibody. In conclusion, our results show a novel PGF(2alpha)-FP pathway that may regulate the inflammatory microenvironment in endometrial adenocarcinoma via neutrophil chemotaxis.

OBJECTIVE

Exposure to airborne particulate matter (PM) may promote development of childhood asthma and trigger acute exacerbations of existing asthma via injury to airway epithelial cells (AEC).

METHODS

We compared the response of AEC to ambient particulates with median aerodynamic diameters of <10 μm or <2.5 μm from the Sydney metropolitan region (Sydney PM10 or PM2.5), to traffic-derived particulates from the exhaust stack of a motorway tunnel or to inert carbon black as a control.

RESULTS

Sydney PM10 strongly stimulated messenger RNA expression and secretion of the pro-inflammatory cytokines interleukin 6 (IL-6) and chemokine (C-X-C motif) ligand 1 (CXCL1) by mouse tracheal AEC. In contrast, traffic-derived particulates did not. Similarly, PM10 stimulated expression of IL6, IL8 and IL1B by human AEC. Mass spectrometric analysis showed that PM10 contained much higher levels of elements associated with dusts of geological origin. In contrast, tunnel soot contained much higher levels of various organic compounds, notably including long straight-chain alkanes and diesel-derived polycyclic aromatic hydrocarbons. Sydney PM2.5, as well as PM10 collected during a period including a major dust storm, both of which contained relatively lower levels of iron but similar levels of other crustal elements, did not stimulate expression or secretion of CXCL1 by mouse AEC.

CONCLUSIONS

Ambient PM10 is likely to be more important than traffic-derived PM in causing injury to AEC leading to production of pro-inflammatory cytokines. The injurious effects may be related to the presence of iron in the coarse fraction of airborne PM. These findings are likely to be relevant to the pathogenesis of asthma.

Phospholipase C (PLC) ɛ is a phosphoinositide-specific PLC regulated by small GTPases including Ras and Rap. We previously demonstrated that PLCɛ has an important role in the development of phorbol ester-induced skin inflammation. In this study, we investigated the role of PLCɛ in ultraviolet (UV) B-induced acute inflammatory reactions in the skin. Wild-type (PLCɛ+/+) and PLCɛ gene knockout (PLCɛ⁻/⁻) mice were irradiated with a single dose of UVB at 1, 2.5, and 10 kJ/m² on the dorsal area of the skin, and inflammatory reactions in the skin were histologically evaluated up to 168 h after irradiation. In PLCɛ+/+ mice, irradiation with 1 and 2.5 kJ/m² UVB resulted in dose-dependent neutrophil infiltration in the epidermis at 24 and 48 h after irradiation. When mice were irradiated with 10 kJ/m² of UVB, most mice developed skin ulcers by 48 h and these ulcers became more severe at 168 h. In PLCɛ⁻/⁻ mice, UVB (1 or 2.5 kJ/m²)-induced neutrophil infiltration was markedly suppressed compared with PLCɛ+/+ mice. The suppression of neutrophil infiltration in PLCɛ⁻/⁻ mice was accompanied by attenuation of UVB-induced production of CXCL1/keratinocyte-derived chemokine (KC), a potent chemokine for neutrophils, in the whole skin. Cultured epidermal keratinocytes and dermal fibroblasts produced CXCL1/KC in a PLCɛ-dependent manner after UVB irradiation, and the UVB-induced upregulation of CXCL1/KC in these cells was significantly abolished by a PLC inhibitor. Furthermore, UVB-induced epidermal thickening was noticeably reduced in the skin of PLCɛ⁻/⁻ mice. These results indicate that PLCɛ has a crucial role in UVB-induced acute inflammatory reactions such as neutrophil infiltration and epidermal thickening by at least in part regulating the expression of CXCL1/KC in skin cells such as keratinocytes and fibroblasts.

Herpetic stromal keratitis (HSK) is characterized by an inflammatory response that includes neutrophils, macrophages, NK cells, and T cells. The factors that are responsible for this inflammation are proinflammatory cytokines and chemokines. Many of these factors have been defined for primary disease, but relatively few have been investigated during recurrent HSK. The present study was designed to determine the role that two of these factors, IL-6 and CXCL1, play during recurrent HSK. Results clearly indicate that unlike primary disease, IL-6 plays no role in recurrent HSK. However, the presence of CXCL1 is required for recurrent HSK as evidenced by the lack of corneal disease in mice treated with anti-CXCL1 Ab. This was confirmed using mice lacking the primary receptor for CXCL1, CXCR2. Corneal disease in this strain was significantly reduced compared with wild-type C57BL/6 controls. Unexpectedly, lack of disease occurs even though CXCL1 knockout mice display increased viral shedding at the cornea. The primary mechanism that CXCL1 plays during disease is its ability to stimulate neutrophils to infiltrate the cornea following reactivation. This paper provides further evidence that primary HSK and recurrent HSK possess overlapping yet distinct disease mechanisms.

Accumulating evidence has identified the profibrogenic properties of IL-17A in organ fibrosis. However, the role of IL-17A signal in liver fibrosis induced by Schistosoma japonicum infection remains unclear. In this study, we investigated liver fibrosis in wild-type (WT) and IL-17RA(-/-) mice upon S. japonicum infection. Hepatic IL-17A, IL-17C, IL-17E (IL-25), IL-17F, IL-17RA, IL-17RB and IL-17RC transcript levels were determined by RT-PCR. IL-17A(+) cells were analyzed by flow cytometry and confocal microscopy among granuloma cells. Immunostaining of IL-17R was performed on liver sections. Collagen deposition was assessed by Van Gieson's staining. IL-17A, IL-17C, IL-17E, IL-17F, IL-17RA and IL-17RC mRNA levels were dramatically increased in fibrotic livers. Among granuloma cells, CD3(+) and CD3(-) lymphocytes, neutrophils and macrophages were found to express IL-17A. Compared to WT, IL-17RA(-/-) mice displayed attenuated granulomatous inflammation, liver fibrosis, improved liver function and high survival. Meanwhile, α-smooth muscle actin staining and the expression of fibrogenic genes (transforming growth factor β, IL-13 and collagen-I) as well as IL-17A-induced proinflammatory mediators (IL-1β, IL-6, tumor necrosis factor α, CXCL1 and CXCL2) and proteinases (MMP3 and TIMP1) involved in fibrosis were markedly reduced in IL-17RA(-/-) mice. In addition, Th2 cytokines IL-4 and IL-17E (IL-25) were also decreased in IL-17RA(-/-) mice. These results indicated that IL-17A signal contributes to the pathogenesis of liver fibrosis in murine schistosomiasis. This effect was induced possibly by activating hepatic stellate cells and stimulating the release of proinflammatory cytokines and chemokines. Furthermore, the Th2 response was also enhanced by IL-17A signals. Our data demonstrate that IL-17A may serve as a promising target for antifibrotic therapy.

We explored the pathophysiological roles of IFN-gamma in cerulein-induced acute pancreatitis. In wild-type (WT) mice, cerulein injection caused acute pancreatitis as evidenced by increased serum amylase levels and pathological changes such as interstitial edema, vacuolization, acinar cell necrosis, and neutrophil infiltration in pancreas. Concomitantly, cerulein treatment augmented intrapancreatic gene expression of TNF-alpha, KC/CXCL1, MIP-2/CXCL2, cyclooxygenase-2 (COX-2), and IFN-gamma in WT mice. In situ hybridization combined with immunofluorescence analyses demonstrated that infiltrating neutrophils expressed IFN-gamma mRNA. Unexpectedly, IFN-gamma(-/-) mice exhibited exacerbated cerulein-induced pancreatic injury, with enhanced neutrophil recruitment. Moreover, intrapancreatic gene expression of TNF-alpha, KC/CXCL1, MIP-2/CXCL2, and COX-2 were significantly exaggerated in IFN-gamma(-/-) mice, compared with WT mice. Cerulein activated NF-kappaB, an indispensable transcription factor for gene transcription of TNF-alpha, KC/CXCL1, MIP-2/CXCL2, and COX-2, in pancreas of cerulein-treated WT mice as evidenced by the increases in nuclear amount and DNA-binding activity of NF-kappaB p65. In comparison with WT mice, IFN-gamma(-/-) mice exhibited exaggerated and prolonged NF-kappaB activation, probably due to reduced acetylation of Stat1, a main signal transducer of IFN-gamma, because acetylated Stat1 can inhibit NF-kappaB activation. Indeed, IFN-gamma acetylated Stat1 and reciprocally reduced NF-kappaB activation and COX-2 expression in neutrophils. Finally, even when administered 4 h after the first cerulein injection, IFN-gamma remarkably attenuated acute pancreatitis in both WT and IFN-gamma(-/-) mice, with reduced NF-kappaB activation and COX-2 expression. Thus, IFN-gamma can have anti-inflammatory effects on acute pancreatitis by depressing the proinflammatory consequences of NF-kappaB activation.

The development of preeclampsia (PE) seriously affects the health of the mother and the child, but the precise pathogenesis of PE remains elusive. The placenta is considered to play a key role and DNA methylation may be associated with altered placental development and function. The aim of this study was to perform a genome-wide analysis of the DNA methylation profile in placentas from pregnancies with severe preeclampsia. The authors analyzed normal and placental tissues with PE for aberrant DNA methylation using methylated DNA immunoprecipitation (MeDIP) and a human CpG island plus promoter microarray. The methylation status of identified candidate genes were validated by bisulfite sequencing PCR (BSP). Microarray analysis identified 296 genes that showed significantly aberrant DNA methylation in preeclampsia (PE). These genes were located more frequently in chromosome 1 (10.5%, P=0.005), chromosome 12 (8.1%, P=0.062) and chromosome 19 (7.4%, P=0.117). Functional analysis divided these genes into different functional networks. In addition, the methylation profile of six of these genes (CAPN2, EPHX2, ADORA2B, SOX7, CXCL1 and CDX1) in nine patients with PE was validated by BSP. This study demonstrated aberrant patterns of DNA methylation in PE, which may be involved in the pathophysiology of PE. Future work will assess the potential prognostic and therapeutic value for these findings in PE.

This study was conducted to examine the anti-inflammatory mechanisms of dexamethasone during leukocyte recruitment and expression of the CXC chemokines macrophage inflammatory protein 2 (MIP-2) (CXCL2) and cytokine-induced neutrophil chemoattractant (KC) (CXCL1) in staphylococcal enterotoxin B (SEB)-induced acute inflammation. To do this, SEB was injected into murine air pouches with or without dexamethasone pretreatment for 2 h. SEB induced infiltration of leukocytes in a dose- and time-dependent manner, with the maximal response observed after 4 h of treatment with 10 microg of SEB. The recruited leukocytes comprised more than 77% neutrophils. Moreover, SEB challenge (10 microg) provoked time-dependent secretion of CXC chemokines, which peaked after 1 h. Local administration of antibodies against MIP-2 and KC significantly reduced SEB-triggered neutrophil accumulation by 38 and 59%, respectively. Dexamethasone (10 mg kg(-1)) significantly decreased neutrophil recruitment by 82% and reduced secretion of MIP-2 and KC by 89 and 85%, respectively, in response to SEB challenge. Our data demonstrate that dexamethasone potently inhibits neutrophil recruitment in SEB-induced inflammation. Moreover, we provide evidence that MIP-2 and KC are key mediators in the neutrophil response to SEB. Furthermore, our findings demonstrate that dexamethasone attenuates SEB-induced expression of MIP-2 and KC. Thus, this study elucidates important signaling pathways of SEB-induced neutrophil recruitment and anti-inflammatory mechanisms of action of dexamethasone.

BACKGROUND

Both genetic and environmental factors contribute to the development of periodontitis. Genetic studies identified a variety of candidate genes for periodontitis. The aim of the present study is to identify the most promising candidate genes for periodontitis using an integrative gene ranking method.

METHODS

Seed genes that were confirmed to be associated with periodontitis were identified using text mining. Three types of candidate genes were then extracted from different resources (expression profiles, genome-wide association studies). Combining the seed genes, four freely available bioinformatics tools (ToppGene, DIR, Endeavour, and GPEC) were integrated for prioritization of candidate genes. Candidate genes that identified with at least three programs and ranked in the top 20 by each program were considered the most promising.

RESULTS

Prioritization analysis resulted in 21 promising genes involved or potentially involved in periodontitis. Among them, IL18 (interleukin 18), CD44 (CD44 molecule), CXCL1 (chemokine [CXC motif] ligand 1), IL6ST (interleukin 6 signal transducer), MMP3 (matrix metallopeptidase 3), MMP7, CCR1 (chemokine [C-C motif] receptor 1), MMP13, and TLR9 (Toll-like receptor 9) had been associated with periodontitis. However, the roles of other genes, such as CSF3 (colony stimulating factor 3 receptor), CD40, TNFSF14 (tumor necrosis factor receptor superfamily, member 14), IFNB1 (interferon-β1), TIRAP (toll-interleukin 1 receptor domain containing adaptor protein), IL2RA (interleukin 2 receptor α), ETS1 (v-ets avian erythroblastosis virus E26 oncogene homolog 1), GADD45B (growth arrest and DNA-damage-inducible 45 β), BIRC3 (baculoviral IAP repeat containing 3), VAV1 (vav 1 guanine nucleotide exchange factor), COL5A1 (collagen, type V, α1), and C3 (complement component 3), have not been investigated thoroughly in the process of periodontitis. These genes are mainly involved in bacterial infection, immune response, and inflammatory reaction, suggesting that further characterizing their roles in periodontitis will be important.

CONCLUSIONS

A combination of computational tools will be useful in mining candidate genes for periodontitis. These theoretical results provide new clues for experimental biologists to plan targeted experiments.

BACKGROUND

Signal transduction is the major mechanism through which cells transmit external stimuli to evoke intracellular biochemical responses. Diverse cellular stimuli create a wide variety of transcription factor activities through signal transduction pathways, resulting in different gene expression patterns. Understanding the relationship between external stimuli and the corresponding cellular responses, as well as the subsequent effects on downstream genes, is a major challenge in systems biology. Thus, a systematic approach is needed to integrate experimental data and theoretical hypotheses to identify the physiological consequences of environmental stimuli.

RESULTS

We proposed a systematic approach that combines forward and reverse engineering to link the signal transduction cascade with the gene responses. To demonstrate the feasibility of our strategy, we focused on linking the NF-kappaB signaling pathway with the inflammatory gene regulatory responses because NF-kappaB has long been recognized to play a crucial role in inflammation. We first utilized forward engineering (Hybrid Functional Petri Nets) to construct the NF-kappaB signaling pathway and reverse engineering (Network Components Analysis) to build a gene regulatory network (GRN). Then, we demonstrated that the corresponding IKK profiles can be identified in the GRN and are consistent with the experimental validation of the IKK kinase assay. We found that the time-lapse gene expression of several cytokines and chemokines (TNF-alpha, IL-1, IL-6, CXCL1, CXCL2 and CCL3) is concordant with the NF-kappaB activity profile, and these genes have stronger influence strength within the GRN. Such regulatory effects have highlighted the crucial roles of NF-kappaB signaling in the acute inflammatory response and enhance our understanding of the systemic inflammatory response syndrome.

CONCLUSIONS

We successfully identified and distinguished the corresponding signaling profiles among three microarray datasets with different stimuli strengths. In our model, the crucial genes of the NF-kappaB regulatory network were also identified to reflect the biological consequences of inflammation. With the experimental validation, our strategy is thus an effective solution to decipher cross-talk effects when attempting to integrate new kinetic parameters from other signal transduction pathways. The strategy also provides new insight for systems biology modeling to link any signal transduction pathways with the responses of downstream genes of interest.

BACKGROUND

The chemotactic signals regulating cell trafficking in the herpes simplex virus type 1 (HSV-1) infected cornea are well documented, however, those in the cornea-associated tissues, such as the trigeminal ganglion (TG) and draining lymph nodes (LNs), are largely unknown.

OBJECTIVE

To examine chemokine expression and subsequent cell infiltration in the HSV-1 infected cornea and its associated tissues.

METHODS

Eight-week-old female BALB/c mice were infected with 10 mu l HSV-1 (CHR3 strain: 5 x 106 PFU/ml) by corneal scarification. Total RNAs were extracted from the corneas, TGs, and LNs at pre-inoculation, 3 days post-inoculation (P.I.) and 7 days P.I. The mRNA for 28 different chemokines in the extracts was amplified by RT-PCR. Infiltrating cells were identified by immunohistochemistry.

RESULTS

After the HSV-1 infection, the corneal stroma became edematous by infiltrated cells under the eroded epithelium. The TG and LNs were markedly swollen. The cornea was infiltrated with granulocytes and CD11b+ cells at 3 days P.I., followed by CD4+ and CD8+ T cells at 12 days P.I. In the TG, CD11b+ cells, but no granulocytes, infiltrated throughout the observation period. T cells migrated into the TG earlier than into the cornea. Gene expressions of neutrophil-attracting chemokines (<em>CXCL1</em>, 2, 3, and 5) increased in the cornea, but they did not enhance in the TG or LNs. On the other hand, gene expressions of chemokines which attract CD11b+ cells such as CCL2, 8, 7, 12, CCL3, 4, and CCL5, increased in the cornea and TG with its peak at 3 days P.I. Gene expressions of chemokines those work on T cells and B cells, such as CCL19, CCL21, CXCL9, <em>CXCL1</em>3, <em>CXCL1</em>0, XCL1, and <em>CXCL1</em>6, were up-regulated and peaked at 3 days P.I. in the cornea and in the TG. Thus, pattern of chemokine gene expression was similar in the cornea and in the TG. On the contrary, gene expressions of chemokines in the draining LNs affecting CD11b+ cells and T cells were temporarily down-regulated.

CONCLUSIONS

Upon HSV-1 infection, dynamic gene expression of chemokines was observed not only in the inoculated cornea but also in its associated tissues.

Perinatal inflammation and the inflammatory cytokine IL-1 can modify lung morphogenesis. To examine the effects of antenatal expression of IL-1β in the distal airway epithelium on fetal lung morphogenesis, we studied lung development and surfactant expression in fetal mice expressing human IL-1β under the control of the surfactant protein (SP)-C promoter. IL-1β-expressing pups suffered respiratory failure and died shortly after birth. IL-1β caused fetal lung inflammation and enhanced the expression of keratinocyte-derived chemokine (KC/CXCL1) and monocyte chemoattractant protein 3 (MCP-3/CCL7), the calgranulins S100A8 and S100A9, the acute-phase protein serum amyloid A3, the chitinase-like proteins Ym1 and Ym2, and pendrin. IL-1β decreased the percentage of the total distal lung area made up of air saccules and the number of air saccules in the lungs of fetal mice. IL-1β inhibited the expression of VEGF-A and its receptors VEGFR-1 and VEGFR-2. The percentage of the cellular area of the distal lung made up of capillaries was decreased in IL-1β-expressing fetal mice. IL-1β suppressed the production of SP-B and pro-SP-C and decreased the amount of phosphatidylcholine and the percentage of palmitic acid in the phosphatidylcholine fraction of lung phospholipids, indicating that IL-1β prevented the differentiation of type II epithelial cells. The production of Clara cell secretory protein in the nonciliated bronchiolar (Clara) cells was likewise suppressed by IL-1β. In conclusion, expression of IL-1β in the epithelium of the distal airways disrupted the development of the airspaces and capillaries in the fetal lung and caused fatal respiratory failure at birth.

OBJECTIVE

Genotypic variation in signal transducer and activator of transcription 3 (STAT3) increases risk for inflammatory bowel disease (IBD), and STAT3-dependent inflammatory networks are induced in the colon in these patients. We hypothesized that STAT3 "A" risk allele carriage would be associated with increased cellular STAT3 activation and colon leukocyte recruitment.

METHODS

Colonic expression of genes regulating STAT3 signaling and leukocyte recruitment and function was measured in pediatric patients with Crohn disease (CD) stratified by STAT3 genotype. The frequency of colonic pSTAT3* and CXCR2* neutrophils was determined using immunohistochemistry. STAT3 tyrosine phosphorylation (pSTAT3) was measured in circulating leukocytes by flow cytometry, and mechanisms regulating STAT3 activation were tested in IBD Epstein-Barr virus (EBV)-transformed lymphocytes (EBL).

RESULTS

Colonic expression of interleukin 6 (IL-6), the STAT3 target gene SOCS3, the neutrophil chemoattractants IL-8, CXCL1, and CXCL3, and the neutrophil products S100A8, S100A9, and S100A12 were increased in patients carrying the STAT3 "A" risk allele. The frequency of neutrophils expressing the cognate receptor for IL-8, CXCR2, was increased in colonic biopsies from patients carrying the risk allele, and the frequency of pSTAT3* or CXCR2* neutrophils correlated with histologic severity. The frequency of CD4 lymphocytes and granulocytes expressing pSTAT3 was increased in patients carrying the STAT3 "A" risk allele. EBLs from patients carrying the STAT3 "A" risk allele exhibited increased basal and IL-6-stimulated STAT3 tyrosine phosphorylation, increased transcription of STAT3 and SOCS3 after IL-6 stimulation, and increased membrane localization of the IL-6 receptor, GP130, and Janus-associated kinase 2.

CONCLUSIONS

The STAT3 "A" risk allele is associated with increased cellular STAT3 activation and upregulation of pathways that promote recruitment of CXCR2* neutrophils to the gut.

Lung cancer cells express different chemokines and chemokine receptors that modulate leukocyte infiltration within tumor microenvironment. In this study we screened several mediators/growth factors on CXCL1 release in human carcinoma epithelial cells. Of the tested mediators, VEGF was found to have a robust increase in causing CXCL1 release. VEGF stimulated CXCL1 release and mRNA expression in a time- and concentration-dependent manner. The release was inhibited by the VEGF receptor antagonists and the JNK, PI-3K, tyrosine kinase, and transcription inhibitors. In parallel, VEGF induced JNK, PI3K and Akt activation. Strikingly, among these inhibitors only the JNK inhibitor could reduce VEGF-induced CXCL1 mRNA expression, suggesting that JNK participated in VEGF-induced CXCL1 synthesis, whereas PI-3K was responsible for cellular CXCL1 secretory process. In addition, the steroid dexamethasone and TGF-β suppressed CXCL1 release through a transcriptional regulation. We also showed that cells stimulated with VEGF significantly attracted monocyte migration, which could be abolished by CXCL1 B/N Ab, CXC receptor 2 antagonist, TGF-β, and dexamethasone. In summary, we provide here evidence showing JNK activation for VEGF-induced CXCL1 DNA transcription and PI-3K pathway for extracellular CXCL1 release in human carcinoma epithelial cells. The released CXCL1 was functionally linked to recruiting monocytes into lung cancer cell microenvironment.

Triple negative breast cancers (TNBC) lacking hormone receptors and HER-2 amplification are very aggressive tumors. Since relevant differences between primary tumors and metastases could arise during tumor progression as evidenced by phenotypic discordances reported for hormonal receptors or HER-2 expression, in this analysis we studied changes that occurred in our TNBC model IIB-BR-G throughout the development of IIB-BR-G-MTS6 metastasis to the lymph nodes (LN) in nude mice, using an antibody-based protein array to characterize their expression profile. We also analyzed their growth kinetics, migration, invasiveness and cytoskeleton structure in vitro and in vivo. In vitro IIB-BR-G-MTS6 cells grew slower but showed higher anchorage independent growth. In vivo IIB-BR-G-MTS6 tumors grew significantly faster and showed a 100% incidence of LN metastasis after s.c. inoculation, although no metastasis was observed for IIB-BR-G. CCL3, IL1β, <em>CXCL1</em>, CSF2, CSF3, IGFBP1, IL1α, IL6, IL8, CCL20, PLAUR, PlGF and VEGF were strongly upregulated in IIB-BR-G-MTS6 while CCL4, ICAM3, <em>CXCL1</em>2, TNFRSF18, FIGF were the most downregulated proteins in the metastatic cell line. IIB-BR-G-MTS6 protein expression profile could reflect a higher NFκB activation in these cells. In vitro, IIB-BR-G displayed higher migration but IIB-BR-G-MTS6 had more elevated matrigel invasion ability. In agreement with that observation, IIB-BR-G-MTS6 had an upregulated expression of MMP1, MMP9, MMP13, PLAUR and HGF. IIB-BR-G-MTS6 tumors presented also higher local lymphatic invasion than IIB-BR-G but similar lymphatic vessel densities. VEGFC and VEGFA/B expression were higher both in vitro and in vivo for IIB-BR-G-MTS6. IIB-BR-G-MTS6 expressed more vimentin than IB-BR-G cells, which was mainly localized in the cellular extremities and both cell lines are E-cadherin negative. Our results suggest that IIB-BR-G-MTS6 cells have acquired a pronounced epithelial-to-mesenchymal transition phenotype. Protein expression changes observed between primary tumor-derived IIB-BR-G and metastatic IIB-BR-G-MTS6 TNBC cells suggest potential targets involved in the control of metastasis.

Streptococcus suis is an important swine pathogen and an emerging zoonotic agent of septicemia and meningitis. Knowledge on host immune responses towards S. suis, and strategies used by this pathogen for subversion of these responses is scarce. The objective of this study was to identify the immune receptors involved in S. suis recognition by dendritic cells (DCs). Production of cytokines and expression of co-stimulatory molecules by DCs were shown to strongly rely on MyD88-dependent signaling pathways, suggesting that DCs recognize S. suis and become activated mostly through Toll-like receptor (TLR) signaling. Supporting this fact, TLR2(-/-) DCs were severely impaired in the release of several cytokines and the surface expression of CD86 and MHC-II. The release of IL-12p70 and CXC10, and the expression of CD40 were found to depend on signaling by both TLR2 and TLR9. The release of IL-23 and CXCL1 were partially dependent on NOD2. Finally, despite the fact that MyD88 signaling was crucial for DC activation and maturation, MyD88-dependent pathways were not implicated in S. suis internalization by DCs. This first study on receptors involved in DC activation by S. suis suggests a major involvement of MyD88 signaling pathways, mainly (but not exclusively) through TLR2. A multimodal recognition involving a combination of different receptors seems essential for DC effective response to S. suis.