The purpose of this study was to determine the influence of cocultured adipose-derived stromal cells (ASCs) in enhancing the osteogenic differentiation and angiogenesis of bone marrow stromal cells (BMSCs) as well as the underlying mechanism and the optimal ratio. Two in vitro coculture models, segregated cocultures using transwell and mixed cocultures, were employed to assess the indirect and direct effects of coculture respectively. Coculture was carried out for 14 days using 1 × 10(5) BMSCs and ASCs of variable number. BMSCs, ASCs, or both were seeded in PLGA scaffold and implanted in the subcutaneous tissue of 25 nude mice for in vivo analysis of angiogenesis. To evaluate the orthotopic bone formation, critical size calvarial defects were created on 20 mice, and implanted with hydroxyapatite/β-tricalcium phosphate granules plus BMSCs, ASCs, or both. From both transwell and mixed coculture model, 1 × 10(5) BMSCs cocultured with 0.5 × 10(5) ASCs showed significantly greater osteogenic differentiation and mineralization than BMSCs alone. The mixed ASC/BMSC coculture at or above a ratio of 0.5/1 showed increased secretion of vascular endothelial growth factor (VEGF), and induced effective tube formation from human umbilical vein endothelial cells, which were comparable to ASCs. Cytokine profiling assay and gene expression study showed elevated levels of angiogenic factors VEGF and CXCL1, osteogenic factor Wnt5a as well as transforming growth factor (TGF)-βR1 and SMAD3 from BMSCs when cocultured with ASCs. After 5 weeks of implantation, polylactic-co-glycolic acid (PLGA)-ASCs-BMSCs had a number of vascular structures comparable to PLGA-ASCs and significantly greater than PLGA-BMSCs. Calvarial defects treated with ceramic/BMSCs/ASCs had greater area of repair and better reconstitution of osseous structure than the defects treated with ceramic/ASCs or ceramic/BMSCs after 10 weeks. In conclusion, ASCs added to BMSCs promoted osteogenesis and angiogenesis at the optimal ASC/BMSC ratio of 0.5/1.

Complex regulation of the wound healing process involves multiple interactions among stromal tissue cells, inflammatory cells, and the extracellular matrix. Low molecular weight hyaluronan (LMW HA) derived from the degradation of high molecular weight hyaluronan (HMW HA) is suggested to activate cells involved in wound healing through interaction with HA receptors. In particular, receptor CD44 is suggested to mediate cell response to HA of different MW, being the main cell surface HA receptor in stromal tissue and immune cells. However, the response of dermal fibroblasts, the key players in granulation tissue formation within the wound healing process, to LMW HA and their importance for the activation of immune cells is unclear. In this study we show that LMW HA (4.3kDa) induced pro-inflammatory cytokine IL-6 and chemokines IL-8, CXCL1, CXCL2, CXCL6 and CCL8 gene expression in normal human dermal fibroblasts (NHDF) that was further confirmed by increased levels of IL-6 and IL-8 in cell culture supernatants. Conversely, NHDF treated by HMW HA revealed a tendency to decrease the gene expression of these cytokine and chemokines when compared to untreated control. The blockage of CD44 expression by siRNA resulted in the attenuation of IL-6 and chemokines expression in LMW HA treated NHDF suggesting the involvement of CD44 in LMW HA mediated NHDF activation. The importance of pro-inflammatory mediators produced by LMW HA triggered NHDF was evaluated by significant activation of blood leukocytes exhibited as increased production of IL-6 and TNF-α. Conclusively, we demonstrated a pro-inflammatory response of dermal fibroblasts to LMW HA that was transferred to leukocytes indicating the significance of LMW HA in the inflammatory process development during the wound healing process.

OBJECTIVE

Meniscus injury increases the risk of osteoarthritis; however, the biologic mechanism remains unknown. We hypothesized that pro-inflammatory stimulation of meniscus would increase production of matrix-degrading enzymes, cytokines and chemokines which cause joint tissue destruction and could contribute to osteoarthritis development.

METHODS

Meniscus and cartilage tissue from healthy tissue donors and total knee arthroplasties (TKAs) was cultured. Primary cell cultures were stimulated with pro-inflammatory factors [IL-1β, IL-6, or fibronectin fragments (FnF)] and cellular responses were analyzed by real-time PCR, protein arrays and immunoblots. To determine if NF-κB was required for MMP production, meniscus cultures were treated with inflammatory factors with and without the NF-κB inhibitor, hypoestoxide.

RESULTS

Normal and osteoarthritic meniscus cells increased their MMP secretion in response to stimulation, but specific patterns emerged that were unique to each stimulus with the greatest number of MMPs expressed in response to FnF. Meniscus collagen and connective tissue growth factor (CTGF) gene expression was reduced. Expression of cytokines (IL-1α, IL-1β, IL-6), chemokines (IL-8, CXCL1, CXCL2, CSF1) and components of the NF-κB and tumor necrosis factor (TNF) family were significantly increased. Cytokine and chemokine protein production was also increased by stimulation. When primary cell cultures were treated with hypoestoxide in conjunction with pro-inflammatory stimulation, p65 activation was reduced as were MMP-1 and MMP-3 production.

CONCLUSIONS

Pro-inflammatory stimulation of meniscus cells increased matrix metalloproteinase production and catabolic gene expression. The meniscus could have an active biologic role in osteoarthritis development following joint injury through increased production of cytokines, chemokines, and matrix-degrading enzymes.

Seminal fluid interacts with epithelial cells lining the female reproductive tract to induce expression of proinflammatory cytokines and chemokines, initiating immune tolerance mechanisms to facilitate pregnancy. TGFB cytokines are key signaling agents in seminal plasma but do not fully account for the female response to seminal fluid. We hypothesized that additional molecular pathways are utilized in seminal fluid signaling. Affymetrix microarray was employed to compare gene expression in the endometrium of mice 8 h after mating with either intact males or seminal fluid deficient (SVX/VAS) males. Bioinformatics analysis revealed TLR4 signaling as a strongly predicted upstream regulator activated by the differentially expressed genes and implicated TGFB signaling as a second key pathway. Quantitative PCR and microbead data confirmed that seminal fluid induces endometrial synthesis of several TLR4-regulated cytokines and chemokines, including CSF3, CXCL1, CXCL2, IL1A, IL6, LIF, and TNF. In primary uterine epithelial cells, CSF3, CXCL1, and CXCL2 were strongly induced by the TLR4 ligand LPS but suppressed by TGFB, while IL1A, TNF, and CSF2 were induced by both ligands. TLR4 was confirmed as essential for the full endometrial cytokine response using mice with a null mutation in Tlr4, where seminal fluid failed to induce endometrial Csf3, Cxcl2, Il6, and Tnf expression. This study provides evidence that TLR4 contributes to seminal fluid modulation of the periconception immune environment. Activation of TLR4 signaling by microbial or endogenous components of seminal fluid is thus implicated as a key element of the female tract response to seminal fluid at the outset of pregnancy in mice.

BACKGROUND

Liver fibrosis is characterized by the excessive formation and accumulation of matrix proteins as a result of wound healing in the liver. A main event during fibrogenesis is the activation of the liver resident quiescent hepatic stellate cell (qHSC). Recent studies suggest that reversion of the activated HSC (aHSC) phenotype into a quiescent-like phenotype could be a major cellular mechanism underlying fibrosis regression in the liver, thereby offering new therapeutic perspectives for the treatment of liver fibrosis. Whether human HSCs have the ability to undergo a similar reversion in phenotype is currently unknown. The aim of the present study is to identify experimental conditions that can revert the in vitro activated phenotype of primary human HSCs and consequently to map the molecular events associated with this reversion process by gene expression profiling.

RESULTS

We find that epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2) synergistically downregulate the expression of ACTA2 and LOX in primary human aHSCs. Their combination with oleic acid, palmitic acid, and retinol further potentiates a more quiescent-like phenotype as demonstrated by the abundant presence of retinyl ester-positive intra-cytoplasmic lipid droplets, low expression levels of activation markers, and a reduced basal as well as cytokine-stimulated proliferation and matrix metalloproteinase activity. Gene expression profiling experiments reveal that these in vitro reverted primary human HSCs (rHSCs) display an intermediary phenotype that is distinct from qHSCs and aHSCs. Interestingly, this intermediary phenotype is characterized by the increased expression of several previously identified signature genes of in vivo inactivated mouse HSCs such as CXCL1, CXCL2, and CTSS, suggesting also a potential role for these genes in promoting a quiescent-like phenotype in human HSCs.

CONCLUSIONS

We provide evidence for the ability of human primary aHSCs to revert in vitro to a transitional state through synergistic action of EGF, FGF2, dietary fatty acids and retinol, and provide a first phenotypic and genomic characterization of human in vitro rHSCs.

AKI leads to tubular injury and interstitial inflammation that must be controlled to avoid the development of fibrosis. We hypothesized that microRNAs are involved in the regulation of the balance between lesion formation and adaptive repair. We found that, under proinflammatory conditions, microRNA-146a (miR-146a) is transcriptionally upregulated by ligands of IL-1 receptor/Toll-like receptor family members via the activation of NF-κB in cultured renal proximal tubular cells. In vivo, more severe renal ischemia-reperfusion injury (IRI) associated with increased expression of miR-146a in both allografts and urine of human kidney transplant recipients, and unilateral IRI in mice induced miR-146a expression in injured kidneys. After unilateral IRI, miR-146a-/- mice exhibited more extensive tubular injury, inflammatory infiltrates, and fibrosis than wild-type mice. In vitro, overexpression or downregulation of miR-146a diminished or enhanced, respectively, IL-1 receptor-associated kinase 1 expression and induced similar effects on C-X-C motif ligand 8 (CXCL8)/CXCL1 expression by injured tubular cells. Moreover, inhibition of CXCL8/CXCL1 signaling prevented the development of inflammation and fibrosis after IRI in miR-146a-/- mice. In conclusion, these results indicate that miR-146a is a key mediator of the renal tubular response to IRI that limits the consequences of inflammation, a key process in the development of AKI and CKD.

Tumor-associated macrophages (TAMs) are known to be involved in the progression, angiogenesis, and motility of various cancers. We previously reported the association between an increased number of infiltrating TAMs with tumor progression and poor prognosis in esophageal squamous cell carcinomas (ESCCs). To study the roles of TAMs in ESCC, we first exposed peripheral blood monocyte (PBMo)-derived macrophages from healthy volunteers to conditioned media of TE series human ESCC cell line (TECM) and confirmed the induction of the expression of the M2 macrophage marker CD204 and the protumorigenic factors interleukin (IL)-10, VEGFA, and MMPs. Next, we compared gene expression profiles between PBMo-derived macrophages stimulated with or without TECM by cDNA microarray and focused on growth differentiation factor 15 (GDF15) among the highly expressed genes including IL-6, IL-8, and CXCL1. Our immunohistochemical study of 70 surgically resected ESCCs revealed that GDF15 was present not only in cancer cells but also in macrophages. The high expression of GDF15 in the ESCCs was significantly correlated with several more malignant phenotypes including vessel invasion, lymph node metastasis, and clinical stages. Patients with high GDF15 expression showed significantly poorer disease-free survival (P=0.011) and overall survival (P=0.041). We also found that recombinant human GDF15 promotes cell proliferation and the phosphorylation of both Akt and Erk1/2 in ESCC cell lines in vitro. These results indicate that GDF15 is secreted by both TAMs and cancer cells in the tumor microenvironment and is associated with aberrant growth and a poor prognosis in human ESCC.

Neutrophils have a traditional role in inflammatory process and act as the first line of defense against infections. Although their contribution to tumorigenesis and progression is still controversial, accumulating evidence recently has demonstrated that tumor-associated neutrophils (TANs) play a key role in multiple aspects of cancer biology. Here, we detected that chemokine CXCL1 was dramatically elevated in serum from 3LL tumor-bearing mice. In vitro, 3LL cells constitutively expressed and secreted higher level of CXCL1. Furthermore, knocking down CXCL1 expression in 3LL cells significantly hindered tumor growth by inhibiting recruitment of neutrophils from peripheral blood into tumor tissues. Additionally, tumor-infiltrated neutrophils expressed higher levels of MPO and Fas/FasL, which may be involved in TAN-mediated inhibition of CD4(+) and CD8(+) T cells. These results demonstrate that tumor-derived CXCL1 contributes to TANs infiltration in lung cancer which promotes tumor growth.

Cytokines and chemokines contribute to the pathogenesis of acute disseminated encephalomyelitis (ADEM). Using a multiplex immunochemiluminescence ELISA, we measured 8 Th1/Th2 cytokines and 18 chemokines in the cerebrospinal fluid (CSF) and serum of 17 ADEM patients, 14 multiple sclerosis (MS) patients, and 7 healthy controls (HCs). Relative to HCs, ADEM patients had significantly high mean CSF concentrations of chemokines with attractant/activating properties towards neutrophils (<em>CXCL1</em> and CXCL7), monocytes/T cells (CCL3 and CCL5), Th1 cells (<em>CXCL1</em>0), and Th2 cells (CCL1, CCL22, and CCL17). Mean CSF concentrations of CXCL7, CCL1, CCL22, and CCL17 were higher in ADEM than in MS, whereas those of CCL11 were lower in MS than in ADEM and HCs. CSF pleocytosis correlated with CSF concentrations of <em>CXCL1</em>, <em>CXCL1</em>0, CCL1, CCL17, and CCL22. Most of the functionally homologous chemokines correlated with each other. CSF Th1/Th2 cytokines were not detectable in most samples. Their mean concentrations did not differ in the three groups, and the same held for serum cytokines and chemokines. Our data suggest that the upregulation of chemokines active on neutrophils and Th2 cells differentiates ADEM from MS inflammation, and that both Th1 and Th2 chemokines might be produced in ADEM. Chemokines upregulated in ADEM could become CSF biomarkers after a posteriori evaluations in unselected case series.

Pulmonary LPS exposure plays a key role in exacerbation of lung diseases such as chronic obstructive pulmonary disease and asthma. However, little is known about the effects of repeated LPS exposure in the lung microenvironment. We have developed a novel murine model of pulmonary LPS tolerance induced by intratracheal (i.t.) administration of LPS. First, we show that pulmonary LPS exposure does not induce whole-body refractoriness to systemic LPS, because i.t. administration followed by i.p. administration did not decrease plasma TNF-alpha. However, a local refractory state can be induced with two i.t. LPS exposures. Pulmonary LPS tolerance was induced by i.t. administration of 100 ng LPS at time 0 and 48 h. Nontolerant mice received PBS at time 0 and LPS at 48 h. Bronchoalveolar lavage levels of TNF-alpha were significantly attenuated in tolerant mice vs nontolerant mice (1597 pg/ml vs 7261 pg/ml). TNF-alpha mRNA was significantly reduced in bronchoalveolar lavage cells (5-fold) and lung tissue (10-fold). No reduction was seen in neutrophil numbers in the bronchoalveolar lavage fluid, myeloperoxidase activity, or expression of neutrophil chemoattractants CXCL1 and CXCL2, reflecting the specificity of the response. The reduction in TNF-alpha was accompanied by a significant increase in soluble receptors, TNF-SRI (159 pg/ml vs 206 pg/ml) and TNF-SRII (1366 pg/m vs 2695 pg/ml). In conclusion, pulmonary LPS tolerance results in a specific reduction in TNF-alpha expression, while the neutrophilic response is unaffected. This response may be a mechanism to limit tissue damage by reducing TNF-alpha levels, while still maintaining the antimicrobial capacity of the lung.

Th17 cytokines IL-17A and IL-17F play a critical role in the activation and recruitment of neutrophils at airway inflammation mainly through the induction of CXC chemokines in the lungs. Vascular endothelial cells belong to the category of major CXC chemokine-producing cells. However, until now, the precise role of Th17 cytokines in CXC chemokine secretion in lung microvascular endothelial cells (LMVECs) has not been fully elucidated. In this study, we examined the biological effects of Th17 cytokines IL-17A and IL-17F on CXCL1, CXCL5, and CXCL8 release in LMVECs. Both IL-17 receptor A (IL-17RA) and IL-17RC are expressed on the surface of LMVECs. In contrast to IL-17F, IL-17A significantly upregulated CXCL1 mRNA expression and protein release, whereas both IL-17A and IL-17F did not have the ability to induce CXCL5 and CXCL8 secretion in LMVECs. IL-17A and IL-17F displayed positive regulatory effects on IL-1β-induced CXCL1, CXCL5, and CXCL8 secretion. On the other hand, IL-17A enhanced the upregulating effect of TNF-α on CXCL1, CXCL5, and CXCL8 release, whereas IL-17F had a negative regulatory effect on TNF-α-mediated secretion. Th2 cytokines IL-4 and IL-13 showed an inhibitory effect on IL-1β plus IL-17A-induced CXCL1, CXCL5, and CXCL8 secretion, but displayed a positive regulatory effect on TNF-α plus IL-17A-induced secretion. These results provide evidence that Th17 cytokines IL-17A and IL-17F have a distinct regulatory role in CXCL1, CXCL5, and CXCL8 expression in LMVECs stimulated either with IL-1β or with TNF-α. Our findings also suggest that CXC chemokine secretion in LMVECs may be complicatedly regulated by Th17 cytokines, Th2 cytokines, and macrophage-associated cytokines in pathological conditions such as bronchial asthma.

Contact hypersensitivity (CHS) is a T cell response to hapten skin challenge of sensitized individuals proposed to be mediated by hapten-primed CD8 cytolytic T cells. Effector CD8 T cell recruitment into hapten challenge sites to elicit CHS requires prior CXCL1- and CXCL2-mediated neutrophil infiltration into the site. We investigated whether neutrophil activities directing hapten-primed CD8 T cell skin infiltration in response to 2,4-dinitro-1-fluorobenzene (DNFB) required Fas ligand (FasL) and perforin expression. Although DNFB sensitization of gld/perforin-/- mice induced hapten-specific CD8 T cells producing IFN-γ and IL-17, these T cells did not infiltrate the DNFB challenge site to elicit CHS but did infiltrate the challenge site and elicit CHS when transferred to hapten-challenged naive wild-type recipients. Hapten-primed wild-type CD8 T cells, however, did not elicit CHS when transferred to naive gld/perforin-/- recipients. Wild-type bone marrow neutrophils expressed FasL and perforin, and when transferred to sensitized gld/perforin-/- mice, they restored hapten-primed CD8 T cell infiltration into the challenge site and CHS. The FasL/perforin-mediated activity of wild-type neutrophils induced the expression of T cell chemoattractants, CCL1, CCL2, and CCL5, within the hapten-challenged skin. These results indicate FasL/perforin-independent functions of hapten-primed CD8 T cells in CHS and identify new functions for neutrophils in regulating effector CD8 T cell recruitment and immune responses in the skin.

Alzheimer׳s disease (AD) is a neurodegenerative disease with elusive pathogenesis, which accounts for most cases of dementia in the aged population. It has been reported that persistent inflammatory responses and excessive chemotaxis of microglia stimulated by beta-amyloid (Aβ) oligomers in the brain may accelerate the progression of AD. The present study was conducted to explore whether paeoniflorin (PF), a water-soluble monoterpene glycoside isolated from the root of Paeonia lactiflora Pallas, could attenuate Aβ1-42-induced toxic effects on primary and BV-2 microglial cells in vitro. Our data showed that PF pretreatment inhibited Aβ1-42-induced production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in rodent microglia. Also, the nuclear translocation of nuclear factor kappa B (NF-κB) subunit p65 and the phosphorylation of NF-κB inhibitor alpha (IκBα) in Aβ1-42-stimulated microglial cells were suppressed by PF administration. Moreover, PF treatment reduced the release of chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-C motif) ligand 2 (CCL-2) from Aβ1-42-stimulated microglia. Additionally, application of PF inhibited the increases in vascular endothelial growth factor (VEGF) and VEGF receptor 1 (Flt-1) triggered by Aβ1-42, and resulted in a concomitant reduction in microglial chemotaxis. Restoration of VEGF was noted to counteract the inhibitory effect of PF, suggesting that PF mitigated Aβ1-42-elicited microglial migration at least partly by suppressing the VEGF/Flt-1 axis. In summary, in presence of Aβ1-42, PF pretreatment inhibited the excessive microglial activation and chemotaxis.

Trichophyton rubrum is the main etiological agent of dermatophytosis, an infection of the skin that affects millions of people worldwide. In this study, we developed a murine model of the dermatophytosis caused by T. rubrum in which C57BL/6 wild-type, interleukin (IL)-12(-/-), and interferon-gamma (IFN-γ(-/-)) mice were inoculated with 1 × 10(6) conidia/animal. The fungal burden, myeloperoxidase and N-acetylglucosaminidase activities, cytokine and chemokine profiles, and histopathology of the skin were evaluated on the seventh and fourteenth days post infection. Phagocytic indices, intracellular proliferation rates, and oxidative bursts generated by macrophages from WT and IFN-γ(-/-) mice were determined. On day 7 post infection, higher fungal burdens were observed comparison with burdens on day 14 post infection. The IL-12(-/-) and IFN-γ(-/-) mice showed higher fungal burdens on the skin and lower levels of IL-1β. Conversely, the WT mice showed lower fungal burdens with higher production of TNF-α, IL-1β, and chemokine ligand 1/keratinocyte chemoattractant (CXCL1/KC). The macrophages from WT mice proved to be more efficient at engulfing and killing T. rubrum conidia through the production of reactive oxygen species. The results show that our model is a useful tool for understanding the pathogenesis of dermatophytosis caused by T. rubrum and that IL-12 and IFN-γ are pivotal in controlling the infection through the recruitment and activation of neutrophils and macrophages.

Inflammation is a highly coordinated host response to infection, injury, or cell stress. In most instances, the inflammatory response is pro-survival and is aimed at restoring physiological tissue homeostasis and eliminating invading pathogens, although exuberant inflammation can lead to tissue damage and death. Intravascular injection of adenovirus (Ad) results in virus accumulation in resident tissue macrophages that trigger activation of CXCL1 and CXCL2 chemokines via the IL-1α-IL-1RI signaling pathway. However, the mechanistic role and functional significance of this pathway in orchestrating cellular inflammatory responses to the virus in vivo remain unclear. Resident metallophilic macrophages expressing macrophage receptor with collagenous structure (MARCO+) in the splenic marginal zone (MZ) play the principal role in trapping Ad from the blood. Here we show that intravascular Ad administration leads to the rapid recruitment of Ly-6G+7/4+ polymorphonuclear leukocytes (PMNs) in the splenic MZ, the anatomical compartment that remains free of PMNs when these cells are purged from the bone marrow via a non-inflammatory stimulus. Furthermore, PMN recruitment in the splenic MZ resulted in elimination of virus-containing cells. IL-1α-IL-1RI signaling is only partially responsible for PMN recruitment in the MZ and requires CXCR2, but not CXCR1 signaling. We further found reduced recruitment of PMNs in the splenic MZ in complement C3-deficient mice, and that pre-treatment of IL-1α-deficient, but not wild-type mice, with complement inhibitor CR2-Crry (inhibits all complement pathways at C3 activation) or CR2-fH (inhibits only the alternative complement activation pathway) prior to Ad infection, abrogates PMN recruitment to the MZ and prevents elimination of MARCO+ macrophages from the spleen. Collectively, our study reveals a non-redundant role of the molecular factors of innate immunity--the chemokine-activating IL-1α-IL-1RI-CXCR2 axis and complement--in orchestrating local inflammation and functional cooperation of PMNs and resident macrophages in the splenic MZ, which collectively contribute to limiting disseminated pathogen spread via elimination of virus-containing cells.

Necrotizing herpetic stromal keratitis (HSK) in mice rapidly improved after amniotic membrane transplantation (AMT). In this study we determined the fate of polymorphonuclear neutrophils (PMN) after AMT. AMT or tarsorrhaphy (T) was performed in BALB/c mice with ulcerative HSK. After 2 days, corneas were studied histologically and by transmission electron microscopy (TEM). CD11b, Gr-1, and TUNEL-positive cells were identified. Macrophages were depleted by subconjunctival injection of dichloromethylene-diphosphonate-liposomes (Cl(2)MDP-LIP) before AMT. Corneas were studied for interleukin (IL)-1alpha, IL-2, interferon (IFN)-gamma, CXCL1, CXCL2, and tumor necrosis factor (TNF)-alpha production by ELISA. PMN-enriched cell preparations co-cultured with amniotic membrane (AM) or with AM and such recombinant (r) cytokines as rIL-1alpha, rIL-2, and rTNF-alpha or supernatants from activated lymphocytes were investigated by flow cytometry (Annexin-V/7-AAD and TUNEL), and a dimethylthiazolyl-diphenyltetrazolium-bromide (MTT)-viability assay. Corneas in the AMT mice had less inflammation, fewer PMN-like cells and fewer CD11b+, and Gr-1+ cells (P<0.01), but a higher ratio of apoptotic to viable PMN-resembling cells (P<0.01) than the T mice. Phagocytic removal of apoptotic PMN-like cells by macrophages was evident in the AMT group. After Cl(2)MDP-LIP treatment, the corneas had more cell debris and apoptotic cells with PMN-like morphology. The concentrations of IL-1alpha, IL-2, CXCL1, and TNF-alpha were reduced in corneas of the AMT group as compared to that of the T group, while the concentration of CXCL2 was increased. Apoptosis of PMN-resembling cells was detected following cocultivation with AM, even when proinflammatory cytokines were present. Resolution of corneal inflammation in mice with necrotizing HSK after AMT is associated with increased apoptosis of PMN-like cells, reduction of pro-inflammatory cytokines, an increase of CXCL2, and increased removal of apoptotic PMN-like cells by macrophages.

Neutrophils participate in the control of mycobacterial infection both by directly eliminating bacilli and by interacting with macrophages and dendritic cells (DCs). Despite host defenses, slow-growing mycobacteria can persist in the host for decades, mostly inside macrophages and DCs, and eventually destroy tissues after exacerbated inflammation. IL-17A-driven neutrophil recruitment may participate in this process. We report that mouse bone marrow-derived DCs infected with live Mycobacterium bovis Bacillus Calmette-Guérin (BCG) produced large amounts of CXCL1 and CXCL2, and attracted neutrophils. After physical contact with DCs infected with live BCG, the neutrophils produced large quantities of the immunosuppressive cytokine IL-10 via the MyD88 and spleen tyrosine kinase pathways. The CD11b integrin was involved in this neutrophil-DC interaction and allowed IL-10 production. TCR OVA transgenic mice immunized with a BCG strain producing OVA mounted an OVA-specific Th17 and Th1 CD4 response. Interestingly, IL-10-producing neutrophils specifically shut down IL-17A production by Th17 CD4 cells, but not IFN-γ production by Th1 cells. This was due to Th17 CD4 cell-restricted expression of the receptor for IL-10. After neutrophil depletion, total mouse lung cells produced less IL-10 but more IL-17A; IFN-γ production was not affected. Therefore, we suggest that during mycobacterial infection, regulatory neutrophils are instructed by infected reservoir DCs to produce IL-10 that specifically targets IL-10Rα-expressing Th17 CD4 T cells. This could be important to control the otherwise exuberant Th17 response.

The current study sought to delineate the gene expression profile of the host response in the caecum and colon during acute infection with Clostridium difficile in a mouse model of infection, and to investigate the nature of the unfolded protein response in this process. The infected mice displayed a significant up-regulation in the expression of chemokines (Cxcl1, Cxcl2 and Ccl2), numerous pro-inflammatory cytokines (Ifng, Il1b, Il6, and Il17f), as well as Il22 and a number of anti-microbial peptides (Defa1, Defa28, Defb1, Slpi and Reg3g) at the site(s) of infection. This was accompanied by a significant influx of neutrophils, dendritic cells, cells of the monocyte/macrophage lineage and all major subsets of lymphocytes to these site(s). However, CD4 T cells of the untreated and C. difficile-infected mice expressed similar levels of CD69 and CD25. Neither tissue had up-regulated levels of Tbx21, Gata3 or Rorc. The caeca and colons of the infected mice showed a significant increase in eukaryotic initiation factor 2α (eIF2α) phosphorylation, but neither the splicing of Xbp1 nor the up-regulation of endoplasmic reticulum chaperones, casting doubt on the full-fledged induction of the unfolded protein response by C. difficile. They also displayed significantly higher phosphorylation of AKT and signal transducer and activator of transcription 3 (STAT3), an indication of pro-survival signalling. These data underscore the local, innate, pro-inflammatory nature of the response to C. difficile and highlight eIF2α phosphorylation and the interleukin-22-pSTAT3-RegIIIγ axis as two of the pathways that could be used to contain and counteract the damage inflicted on the intestinal epithelium.

Lumican is an extracellular protein that associates with CD14 on the surface of macrophages and neutrophils, and promotes CD14-TLR4 mediated response to bacterial lipopolysaccharides (LPS). Lumican-deficient (Lum(-/-)) mice and macrophages are impaired in TLR4 signals; raising the possibility that lumican may regulate host response to live bacterial infections. In a recent study we showed that invitro Lum(-/-) macrophages are impaired in phagocytosis of gram-negative bacteria and in a lung infection model the Lum(-/-) mice showed poor survival. The cornea is an immune privileged barrier tissue that relies primarily on innate immunity to protect against ocular infections. Lumican is a major component of the cornea, yet its role in counteracting live bacteria in the cornea remains poorly understood. Here we investigated Pseudomonas aeruginosa infections of the cornea in Lum(-/-) mice. By flow cytometry we found that 24 hours after infection macrophage and neutrophil counts were lower in the cornea of Lum(-/-) mice compared to wild types. Infected Lum(-/-) corneas showed lower levels of the leukocyte chemoattractant CXCL1 by 24-48 hours of infection, and increased bacterial counts up to 5 days after infection, compared to Lum(+/-) mice. The pro-inflammatory cytokine TNF-α was comparably low 24 hours after infection, but significantly higher in the Lum(-/-) compared to Lum(+/-) infected corneas by 2-5 days after infection. Taken together, the results indicate that lumican facilitates development of an innate immune response at the earlier stages of infection and lumican deficiency leads to poor bacterial clearance and resolution of corneal inflammation at a later stage.

During visceral leishmaniasis, the control of hepatic parasite burden is mainly due to granuloma assembly in a microenvironment consisting of both Th1 and Th2 components. Using enzyme-linked immunosorbent assay (ELISA) dosages, quantitative PCR (qPCR), immunohistochemistry, and flow cytometry, we studied the role of interleukin-33 (IL-33), a recently described cytokine signaling through the ST2 receptor, during visceral leishmaniasis. We showed that a higher level of IL-33 was detected in the serum of patients with visceral leishmaniasis than in that from healthy donors and demonstrated the presence of IL-33(+) cells in a liver biopsy specimen from a patient. Similarly, in BALB/c mice experimentally infected with L. donovani, a higher level of IL-33 was detected in the serum, as well as the presence of IL-33(+) cells and ST2(+) cells in the mouse liver. In ST2(-/-) BALB/c mice, better control of the hepatic parasite burden and reduced hepatomegaly were observed. This was associated with strong induction of Th1 cytokines (gamma interferon [IFN-γ] and IL-12) compared to the level in wild-type (WT) mice and better recruitment of myeloid cells associated with strongly induced chemokines (CCL2 and CXCL2) and receptors (CCR2 and CXCR2). Conversely, BALB/c mice treated twice weekly with recombinant IL-33 showed a dramatically reduced induction of Th1 cytokines and delayed inhibition of monocyte and neutrophil recruitment in the liver, which was associated with reduced KC/CXCL1 and CXCR2 expression. Taken together, our results suggest that IL-33 could be a new deleterious regulator of the hepatic immune response against Leishmania donovani, via the repression of the Th1 response and myeloid cell recruitment.

OBJECTIVE

Visceral leishmaniasis is a life-threatening systemic disease due to the Leishmania protozoa L. infantum and L. donovani and is ranked by the World Health Organization as the second most important protozoan parasitic disease after malaria for its grave morbidity, high mortality, and global distribution. Leishmania parasites subvert the host's immune response to propagate to target organs, including the spleen, the bone marrow, and the liver. Control of hepatic parasite burdens depends on a delicate and poorly understood Th1/Th2 immune balance. To better understand this complex immune response, new cytokines are interesting targets for research studies. IL-33 is a newly described cytokine usually associated with Th2 response and involved in different diseases, including infectious diseases and hepatitis. Our results suggest that IL-33 could be a new factor of susceptibility and a potential prognostic marker during visceral leishmaniasis.

IPF is a chronic, progressive pulmonary disease, leading to respiratory failure. In search of mechanisms of IPF, we used the bleomycin-induced lung-injury model in mice, which causes acute inflammation that may progress to chronic lung inflammation and fibrosis. Here, we asked whether CXCL6/GCP-2, a member of the CXC chemokine superfamily, may be involved in IPF development. First, we reported an increase of CXCL6 levels in BALF from patients with IPF, as well as in the lung of mice, 24 h after bleomycin administration. To investigate whether CXCL6 played a role in experimental bleomycin-induced pulmonary fibrosis, we treated mice with an anti-mCXCL6 mAb that has been shown to inhibit neutrophil chemotaxis in vitro. CXCL6 antibody blockade attenuated acute inflammation with a reduced pulmonary neutrophil influx, IL-1β, CXCL1, and TIMP-1 production. In the later phase (14 days after bleomycin exposure), lymphocyte recruitment and fibrosis markers, such as collagen and TIMP-1, were diminished, as well as collagen deposition and fibrotic lesion the lung. Therefore, the data suggest that CXCL6 contributes to experimental pulmonary fibrosis, and CXCL6 inhibition might be used to reduce lung toxicity associated with bleomycin treatment.

A lack of sensitive and specific tumor markers for early diagnosis and treatment is a major cause for the high mortality rate of ovarian cancer. The purpose of this study was to identify potential proteomics-based biomarkers useful for the differential diagnosis between ovarian cancer and benign pelvic masses. Serum samples from 41 patients with ovarian cancer, 32 patients with benign pelvic masses, and 41 healthy female blood donors were examined, and proteomic profiling of the samples was assessed by surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy (MS). A confirmatory study was also conducted with serum specimens from 58 patients with ovarian carcinoma, 37 patients with benign pelvic masses, and 48 healthy women. A classification tree was established using Biomarker Pattern Software. Six differentially expressed proteins (APP, CA 125, CCL18, CXCL1, IL-8, and ITIH4) were separated by high-performance liquid chromatography and identified by matrix-assisted laser desorption/ionization (MALDI)-MS/MS and database searches. Two of the proteins overexpressed in ovarian cancer patients, chemokine CC2 motif ligand 18 (CCL18) and chemokine CXC motif ligand 1 (CXCL1), were automatically selected in a multivariate predictive model. These two protein biomarkers were then validated and evaluated by enzyme-linked immunosorbent assay (ELISA) in 535 serum specimens (130 ovarian cancer, 64 benign ovarian masses, 36 lung cancer, 60 gastric cancer, 55 nasopharyngeal carcinoma, 48 hepatocellular carcinoma, and 142 healthy women). The combined use of CCL18 and CXCL1 as biomarkers for ovarian cancer had a sensitivity of 92% and a specificity of 97%. The multivariate ELISA analysis of the two putative markers in combination with CA 125 resulted in a sensitivity of 99% for healthy women and 94% for benign pelvic masses, and a specificity of 92% for both groups; these values were significantly higher than those obtained with CA 125 alone (p and lt;0.05). We conclude that serum CCL18 and CXCL1 are potentially useful as novel circulating tumor markers for the differential diagnosis between ovarian cancer and benign ovarian masses.

Increased levels of chemokines and prostaglandins have been reported in patients with inflammatory bowel disease, although their changes during disease development are less understood. The aim of this study was to investigate the local production of nine selected chemokines and prostaglandin E(2) (PGE(2)) to elucidate their role in colitis progression in BALB/c and C57BL/6 mice exposed to dextran sulphate sodium. The acute inflammation in both strains was accompanied by a significant up-regulation of <em>CXCL1</em>, CXCL2/3, <em>CXCL1</em>0, CCL2, CCL4 and CCL22 and a downregulation of PGE(2). In the recovery phase in BALB/c, one-week post-DSS, PGE(2) levels were significantly increased with a concomitant downregulation of <em>CXCL1</em>, CXCL2/3, <em>CXCL1</em>0, CCL2, and CCL4. In contrast, in C57BL/6 mice <em>CXCL1</em>, CXCL2/3, <em>CXCL1</em>0, CCL2, CCL3 and CCL4 production remained high during the chronic phase, without any up-regulation of PGE(2). In addition, CCL5 was significantly increased at d26 and 33 compared to d5. Interestingly, the number of macrophages was significantly increased during the acute phase, whereas T cells were significantly increased in both the acute and chronic phase in C57BL/6 mice. Thus, our results show that chemokines are produced in a dynamic manner during colitis progression.

Angiotensin AT1 receptor blockers (ARBs) and thiazolidinediones (TZDs) have become well established drugs for the treatment of major risk factors of stroke. Since several studies provided evidence that ARBs and TZDs also have additional anti-inflammatory effects, we hypothesized that a combined treatment with the ARB, candesartan, and the TZD, pioglitazone, ameliorates ischemia-induced brain injury and inflammation by synergistic anti-inflammatory actions. Normotensive Wistar rats were pre-treated for 5 days with vehicle (0.9% NaCl), 0.2 mg/kg/day candesartan (s.c.), and/or 2 and/or 20 mg/kg/day pioglitazone (p.o.), respectively and underwent 90 min of middle cerebral artery occlusion (MCAO) with successive reperfusion. Neurological deficits and infarct size were determined 24 h and 48 h after MCAO, respectively, followed by tissue sampling. Animals treated with candesartan, pioglitazone, and the combination of candesartan and pioglitazone had reduced neurological deficits 24 h and 48 h after MCAO, respectively (P<0.05-0.01). Infarct size was reduced by treatment of candesartan, pioglitazone, and their respective combination (each P<0.05) 48 h after stroke compared to vehicle. Treatment with candesartan, pioglitazone, and their combination resulted in significantly reduced mRNA expression of the inflammatory markers CXCL1 and TNFalpha in vivo (P<0.01). The combination of candesartan plus pioglitazone is equally effective compared to their single applications concerning neuroprotection and attenuation of inflammation after MCAO. Therefore, we conclude that a direct synergistic neuroprotective action of parallel ARB and TZD treatment is unlikely.