In this study, we investigated cytokine expression during experimental pneumococcal meningitis. Mice were intracisternally infected with Streptococcus pneumoniae and treated with ceftriaxone starting at 24 h after infection. At different time points before and after antibiotic therapy, the cytokine expression pattern was determined in mouse brains using protein arrays. Underlining the power of this method, the meningitis-relevant cytokines interleukin-1beta (IL-1beta), IL-6, KC, macrophage inflammatory protein-2 (MIP-2), and monocyte chemoattractant protein-1 (MCP-1/CCL2) were markedly elevated in infected animals. Newly identified proteins during the acute stage of the disease (until 30 h after infection) included lymphotactin (XCL-1), MIP-1gamma (CCL9) and MCP-5 (CCL12), cytokine responsive gene- 2 (CRG-2/CXCL10) and CXCL16, and insulin-like growth factor binding protein 3 (IGFBP3). During later stages, an induction of T-cell activation-3 (TCA-3/CCL1), platelet factor-4 (PF-4/CXCL4) and stromal derived factor-1alpha (SDF-1alpha/CXCL13), and IL-4 was observed. The validity of this method was supported by an additional ELISA analysis of the expression profile of CXCL16 and IGFBP3, which was identical to that observed by protein array. In conclusion, the use of protein array technology led to an extension of the current picture of protein expression in pneumococcal meningitis. Most important, new factors that might play a role in pneumococcal meningitis were identified.

Eosinophils play important roles in asthma and lung infections. Murine models are widely used for assessing the functional significance and mechanistic basis for eosinophil involvements in these diseases. However, little is known about tissue eosinophils in homeostasis. In addition, little data on eosinophil chemokine production during allergic airway inflammation are available. In this study, the properties and functions of homeostatic and activated eosinophils were compared. Eosinophils from normal tissues expressed costimulation and adhesion molecules B7-1, B7-2 and ICAM-1 for Ag presentation but little major histocompatibility complex (MHC) class II, and were found to be poor stimulators of T-cell proliferation. However, these eosinophils expressed high levels of chemokine mRNA including C10, macrophage inflammatory protein (MIP)-1alpha, MIP-1gamma, MIP-2, eotaxin and monocyte chemoattractant protein-5 (MCP-5), and produced chemokine proteins. Eosinophil intracellular chemokines decreased rapidly with concomitant surface marker downregulation upon in vitro culturing consistent with piecemeal degranulation. Lung eosinophils from mice with induced allergic airway inflammation exhibited increased chemokines mRNA expression and chemokines protein production and upregulated MHC class II and CD11c expression. They were also found to be the predominant producers of the CCR1 ligands CCL6/C10 and CCL9/MIP-1gamma in inflamed lungs. Eosinophil production of C10 and MIP-1gamma correlated with the marked influx of CD11b(high) lung dendritic cells during allergic airway inflammation and the high expression of CCR1 on these dendritic cells (DCs). The study provided baseline information on tissue eosinophils, documented the upregulation of activation markers and chemokine production in activated eosinophils, and indicated that eosinophils were a key chemokine-producing cell type in allergic lung inflammation.

Tumor cell survival in the hostile distant organ is a rate-limiting step in cancer metastasis. Bone marrow-derived myeloid cells can form a premetastatic niche and provide a tumor-promoting microenvironment. However, it is unclear whether these myeloid cells in the premetastatic site have any direct effect on tumor cell survival. Here, we report that chemokine CCL9 was highly induced in Gr-1(+)CD11b(+) immature myeloid cells and in premetastatic lung in tumor-bearing mice. Knockdown of CCL9 in myeloid cells decreased tumor cell survival and metastasis. Importantly, CCL9 overexpression in myeloid cells lacking TGFβ signaling rescued the tumor metastasis defect observed in mice with myeloid-specific Tgfbr2 deletion. The expression level of CCL23, the human orthologue for CCL9, in peripheral blood mononuclear cells correlated with progression and survival of cancer patients. Our study demonstrates that CCL9 could serve as a good candidate for anti-metastasis treatment by targeting the rate-limiting step of cancer cell survival. In addition, targeting CCL9 may avoid the adverse effects of TGFβ-targeted therapy.

Spermatogonial stem cells are the only stem cells in the body that transmit genetic information to offspring. Although growth factors responsible for self-renewal of these cells are known, the factors and mechanisms that attract and physically maintain these cells within their microenvironment are poorly understood. Mice with targeted disruption of Ets variant gene 5 (Etv5) show total loss of stem/progenitor spermatogonia following the first wave of spermatogenesis, resulting in a Sertoli cell-only phenotype and aspermia. Microarray analysis of primary Sertoli cells from Etv5 knockout (Etv5(-/-)) versus wild-type (WT) mice revealed significant decreases in expression of several chemokines. Chemotaxis assays demonstrated that migration of stem/progenitor spermatogonia toward Etv5(-/-) Sertoli cells was significantly decreased compared to migration toward WT Sertoli cells. Interestingly, differentiating spermatogonia, spermatocytes, and round spermatids were not chemoattracted by WT Sertoli cells, whereas stem/progenitor spermatogonia showed a high and significant chemotactic index. Rescue assays using recombinant chemokines indicated that C-C-motif ligand 9 (CCL9) facilitates Sertoli cell chemoattraction of stem/progenitor spermatogonia, which express C-C-receptor type 1 (CCR1). In addition, there is protein-DNA interaction between ETV5 and Ccl9, suggesting that ETV5 might be a direct regulator of Ccl9 expression. Taken together, our data show for the first time that Sertoli cells are chemoattractive for stem/progenitor spermatogonia, and that production of specific chemokines is regulated by ETV5. Therefore, changes in chemokine production and consequent decreases in chemoattraction by Etv5(-/-) Sertoli cells helps to explain stem/progenitor spermatogonia loss in Etv5(-/-) mice.

OBJECTIVE

To investigate the role of IL-17RA signaling in the effector phase of inflammatory arthritis using the K/BxN serum-transfer model.

METHODS

Wild-type and Il17ra(-/-) mice were injected with serum isolated from arthritic K/BxN mice and their clinical score was recorded daily. Mice were also harvested on days 12 and 21 and ankles were analyzed for cytokine and chemokine mRNA expression by qPCR on day 12 and for bone and cartilage erosions by histology on day 21, respectively. The induction of cytokine and chemokine expression levels by IL-17A in synovial-like fibroblasts was also analyzed using qPCR.

RESULTS

Il17ra(-/-) mice were partially protected from clinical signs of arthritis and had markedly fewer cartilage and bone erosions. The expression of several pro-inflammatory mediators, including the chemokines KC/CXCL1, MIP-2/CXCL2, LIX/CXCL5 MIP-1γ/CCL9, MCP-3/CCL7, MIP-3α/CCL20, the cytokines IL-1β, IL-6, RANKL and the matrix metalloproteinases MMP2, MMP3, and MMP13 were decreased in the ankles of Il17ra(-/-) mice compared to wild-type mice. Many of these proinflammatory genes attenuated in the ankles of Il17ra(-/-) mice were shown to be directly induced by IL-17A in synovial fibroblasts in vitro.

CONCLUSIONS

IL-17RA signaling plays a role as an amplifier of the effector phase of inflammatory arthritis. This effect is likely mediated by direct activation of synovial fibroblasts by IL-17RA to produce multiple inflammatory mediators, including chemokines active on neutrophils. Therefore, interrupting IL-17RA signaling maybe a promising pharmacological target for the treatment of inflammatory arthritis.

Stromal chemokine gradients within the breast tissue microenvironment play a critical role in breast cancer cell invasion, a prerequisite to metastasis. To elucidate which chemokines and mechanisms are involved in mammary cell migration we determined whether mesenchymal D1 stem cells secreted specific chemokines that differentially promoted the invasion of mammary tumor cells in vitro. Results indicate that mesenchymal D1 cells produced concentrations of CCL5 and CCL9 4- to 5-fold higher than the concentrations secreted by 4T1 tumor cells (P < 0.01). Moreover, 4T1 tumor cell invasion toward D1 mesenchymal stem cell conditioned media (D1CM), CCL5 alone, CCL9 alone or a combination CCL5 and CCL9 was observed. The invasion of 4T1 cells toward D1 mesenchymal stem CM was dose-dependently suppressed by pre-incubation with the CCR1/CCR5 antagonist met-CCL5 (P < 0.01). Furthermore, the invasion of 4T1 cells toward these chemokines was prevented by incubation with the broad-spectrum MMP inhibitor GM6001. Additionally, the addition of specific MMP9/MMP13 and MMP14 inhibitors prevented the MMP activities of supernatants collected from 4T1 cells incubated with D1CM, CCL5 or CCL9. Taken together these data highlight the role of CCL5 and CCL9 produced by mesenchymal stem cells in mammary tumor cell invasion.

Smokers have a twofold increased risk to develop Crohn's disease (CD). However, little is known about the mechanisms through which smoking affects CD pathogenesis. Especially Crohn's ileitis is negatively influenced by smoking. Interestingly, the ileum and, more in particular, the Peyer's patches in the terminal ileum are also the sites where the first CD lesions are found. Several chemokines are implicated in the pathogenesis, among which is the CCL20-CCR6 pathway. Here, we studied the gut-associated lymphoid tissue in C57BL/6 wild-type mice and in CCR6-deficient mice after exposure to air or cigarette smoke for 24 weeks. Apoptotic index of the follicle-associated epithelium overlying the Peyer's patches was evaluated. We found that chronic smoke exposure induced apoptosis in the follicle-associated epithelium. Furthermore, immune cell numbers and differentiation along with chemokine expression were determined in Peyer's patches. Important changes in immune cell composition were observed: total dendritic cells, CD4+ T cells (including regulatory T cells) and CD8+ T cells increased significantly after smoke exposure. The CD11b+ dendritic cell subset almost doubled. Interestingly, these changes were accompanied by an upregulated mRNA expression of the chemokines CCL9 and CCL20. However, no differences in the increase of dendritic cells were observed between wild-type and CCR6-deficient mice. Our results show that cigarette smoke exposure increases apoptosis in the follicle-associated epithelium and is associated with immune cell accumulation in Peyer's patches.

RNA analysis has been applied to forensic work to determine wound age. We investigated mRNA expression using quantitative RT-PCR of ten genes, including c-fos, fosB, mitogen-activated protein kinase phosphatase-1 (MKP-1), CD14, chemokine (C-C motif) ligand 9 (CCL9), placenta growth factor (PlGF), mast cell protease-5 (MCP-5), growth arrest specific 5 (Gas5), beta-2 microglobulin (B2M) and major urinary protein-1 (MUP-1), in terms of repair response in adult mice. The expression level of c-fos, fosB and MKP-1 transcripts increased drastically, peaked within 1h, and that of the CD14 and CCL9 transcripts peaked from 12 to 24h. An increase in PlGF and MCP-5 mRNA appeared on about day 5. Gas5, B2M and MUP-1 transcripts showed no significant change. Each gene had differentially expressional patterns with time-course. Our result implied that the observation of the 7 genes in wounded skin could serve to aid in the accurate diagnosis of wound age.

Antigen-stimulated naive CD4 T cells may differentiate into effector T cells such as Th1 and Th2 cells, or may remain as proliferating but uncommitted, primed, precursor cells (Thpp cells) that can subsequently differentiate into Th1 or Th2 cells in appropriate cytokine environments. To examine potential Thpp effector functions, we compared the genes expressed by mouse Thpp, naive, Th1 and Th2 cells, using Affymetrix GeneChip and RNase Protection assays. Similar to naive CD4 T cells, Thpp cells expressed IL-2 but not the cytokines characteristic of differentiated Th1 or Th2 cells, such as IFN-gamma, IL-4, or IL-5. However, Thpp, Th1 and Th2 cells, but not naive cells, expressed several CC chemokines including CCL1/TCA3, CCL5/RANTES, CCL3/MIP-1 alpha, CCL4/MIP-1 beta, and CCL9/MIP-1 gamma. Secretion of the corresponding proteins was confirmed by ELISA and Elispot. Consistent with this chemokine expression, supernatants of activated Thpp, Th1 and Th2 cells but not naive CD4T cells induced pertussis toxin-sensitive chemotaxis of B and T cells. Supernatants of Thpp cells did not bias differentiation of naive CD4 T cells towards either Th1 or Th2 cells. The secretion of several chemokines, but few cytokines, by primed uncommitted Thpp cells suggests that their activation during an immune response may recruit effector cells without directly polarizing effector functions.

Sertoli cells, the primary somatic cell in the seminiferous epithelium, provide the spermatogonial stem cell (SSC) microenvironment (niche) through physical support and the expression of paracrine factors. However, the regulatory mechanisms within the SSC niche, which is primarily controlled by Sertoli cells, remain largely unknown. GATA4 is a Sertoli cell marker, involved in genital ridge initiation, sex determination and differentiation during the embryonic stage. Here, we showed that neonatal mice with a targeted disruption of Gata4 in Sertoli cells (Gata4(flox/flox); Amh-Cre; hereafter termed Gata4 cKO) displayed a loss of the establishment and maintenance of the SSC pool and apoptosis of both gonocyte-derived differentiating spermatogonia and meiotic spermatocytes. Thus, progressive germ cell depletion and a Sertoli-cell-only syndrome were observed as early as the first wave of murine spermatogenesis. Transplantation of germ cells from postnatal day 5 (P5) Gata4 cKO mice into Kit(W/W-v) recipient seminiferous tubules restored spermatogenesis. In addition, microarray analyses of P5 Gata4 cKO mouse testes showed alterations in chemokine signaling factors, including Cxcl12, Ccl3, Cxcr4 (CXCL12 receptor), Ccr1 (CCL3 receptor), Ccl9, Xcl1 and Ccrl2. Deletion of Gata4 in Sertoli cells markedly attenuated Sertoli cell chemotaxis, which guides SSCs or prospermatogonia to the stem cell niche. Finally, we showed that GATA4 transcriptionally regulated Cxcl12 and Ccl9, and the addition of CXCL12 and CCL9 to an in vitro testis tissue culture system increased the number of PLZF+ undifferentiated spermatogonia within Gata4 cKO testes. Together, these results reveal a novel role for GATA4 in controlling the SSC niche via the transcriptional regulation of chemokine signaling shortly after birth.

Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) circulates systemically in over 50% of periodontal disease (PD) patients and is associated with increased matrix metalloproteinase (MMP)-9. We hypothesized that low systemic Pg-LPS would stimulate an inflammatory response in the left ventricle (LV) through MMP-9, leading to a decrease in cardiac function. Wild-type (WT) and MMP-9 null mice (4-7 months old) were exposed for 1 or 28 days to low dose Pg-LPS or saline (n ≥ 6/group). MMP-9 significantly increased in WT mice LV at 1 and 28 days of exposure, compared to control (P < 0.05 for both). Fractional shortening decreased subtly yet significantly in WT mice by day 28 (31 ± 1%) compared to control (35 ± 1%; P < 0.05), and this decrease was attenuated in null (34 ± 1%) mice. Plasma cardiac troponin I levels were elevated in WT mice at day 28. Macrophage-related factors increased over twofold in WT plasma and LV after day 1 (monocyte chemoattractant protein-5, macrophage inflammatory protein (MIP)-1α, MIP-1γ, stem cell factor, Ccl12, Ccl9, Il8rb, Icam1, Itgb2, and Spp1; all P < 0.05), indicating a moderate inflammatory response. Levels returned to baseline by day 28, suggesting tolerance to Pg-LPS. In contrast, macrophage-related factors remained elevated in day 28 null mice, indicating a sustained defense against Pg-LPS stimulation. Consistent with these findings, LV macrophage numbers increased in both groups at day 1 and returned to baseline by day 28 in the WT mice only. Major histocompatibility complex (MCH) II remained elevated in the null group at day 28, confirming Pg-tolerance in the WT. Interestingly Il-1α, a regulator of macrophage immunosuppression, increased in the plasma of WT mice only on day 28, suggesting that Il-1α plays a role in tolerance in a MMP-9-dependent manner. In conclusion, circulating Pg-LPS induced tolerance in WT mice, resulting in significant LV changes and subtle cardiac dysfunction. MMP-9 played a major role in the regulation of chronic systemic inflammation and associated cardiac dysfunction.

Tumor-stromal interaction is implicated in many stages of tumor development, although it remains unclear how genetic lesions in tumor cells affect stromal cells. We have recently shown that inactivation of transforming growth factor-beta family signaling within colon cancer epithelium increases chemokine CC chemokine ligand 9 (CCL9) and promotes recruitment of the matrix metalloproteinase (MMP)-expressing stromal cells that carry CC chemokine receptor 1 (CCR1), the cognate receptor for CCL9. We have further shown that lack of CCR1 prevents the accumulation of MMP-expressing cells at the invasion front and suppresses tumor invasion. These results provide the possibility of a novel therapeutic strategy for advanced cancer--prevention of the recruitment of MMP-expressing cells by chemokine receptor antagonist.

Timely interaction between the egg and sperm is required for successful fertilization; however, little is known about the signaling therein. Prostaglandin (PG) E receptor EP2-deficient (Ptger2(-/-)) female mice exhibit a severe fertilization defect. We investigated the molecular events leading to this failure. We found increased gene expression for chemokines, such as Ccl2, Ccl7, and Ccl9, in Ptger2(-/-) cumulus cells (the somatic cells surrounding the egg) compared with wild-type cells. Furthermore, under physiological conditions, cumulus-derived chemokine signaling was found to have a dual action; CCL7 facilitates sperm migration to the cumulus-egg complex and integrin-mediated cumulus extracellular matrix (ECM) assembly to protect eggs. However, in the absence of PGE(2)-EP2 signaling, chronic CCL7 signaling results in excessive integrin engagement to the ECM, making the cumulus ECM resistant to sperm hyaluronidase, thereby preventing sperm penetration. Our findings indicate that PGE(2)-EP2 signaling negatively regulates the autocrine action of chemokines and prevents excessive cumulus ECM assembly. This interaction between PG and chemokine signaling is required for successful fertilization.

Interferon (IFN) is effective at inducing complete remissions in patients with chronic myelogenous leukemia (CML), and evidence supports an immune mechanism. Here we show that the type I IFNs (alpha and beta) regulate expression of the IFN consensus sequence-binding protein (ICSBP) in BCR-ABL-transformed cells and as shown previously for ICSBP, induce a vaccine-like immunoprotective effect in a murine model of BCR-ABL-induced leukemia. We identify the chemokines CCL6 and CCL9 as genes prominently induced by the type I IFNs and ICSBP, and demonstrate that these immunomodulators are required for the immunoprotective effect of ICSBP expression. Insights into the role of these chemokines in the antileukemic response of IFNs suggest new strategies for immunotherapy of CML.

In this study, we examined the combined effect of aging and myocardial infarction on left ventricular remodeling, focusing on matrix metalloproteinase (MMP)-9-dependent mechanisms. We enrolled 55 C57BL/6J wild type (WT) and 85 MMP-9 Null (Null) mice of both sexes at 11-36 months of age and evaluated their response at Day 7 post-myocardial infarction. Plasma MMP-9 levels positively linked to age in WT mice (r = .46, p = .001). MMP-9 deletion improved survival (76% for WT vs 88% for Null, p = .021). Post-myocardial infarction, there was a progressive increase in left ventricular dilation with age in WT but not in Null mice. By inflammatory gene array analysis, WT mice showed linear age-dependent increases in three different proinflammatory genes (C3, CCl4, and CX3CL1; all p < .05), whereas Null mice showed increases in three proinflammatory genes (CCL5, CCL9, and CXCL4; all p < .05) and seven anti-inflammatory genes (CCL1, CCL6, CCR1, IL11, IL1r2, IL8rb, and Mif; all p < .05). Compared with WT, macrophages isolated from Null left ventricle infarct demonstrated enhanced expression of anti-inflammatory M2 markers CD163, MRC1, TGF-β1, and YM1 (all p < .05), without affecting proinflammatory M1 markers. In conclusion, MMP-9 deletion stimulated anti-inflammatory polarization of macrophages to attenuate left ventricle dysfunction in the aging post-myocardial infarction.

Cross-linking the high-affinity IgE receptor, FcepsilonRI, on mast cells activates signaling pathways leading to the release of preformed inflammatory mediators and the production of cytokines and chemokines associated with allergic disorders. Bone marrow-derived mast cells (BMMCs) from Lyn-deficient (Lyn-/-) mice are hyperresponsive to FcepsilonRI cross-linking with multivalent Ag. Previous studies linked the hyperresponsive phenotype in part to increased Fyn kinase activity and reduced SHIP phosphatase activity in the Lyn-/- BMMCs in comparison with wild-type (WT) cells. In this study, we compared gene expression profiles between resting and Ag-activated WT and Lyn-/- BMMCs to identify other factors that may contribute to the hyperresponsiveness of the Lyn-/- cells. Among genes implicated in the positive regulation of FcepsilonRI signaling, mRNA for the tyrosine kinase, Fyn, and for several proteins contributing to calcium regulation are more up-regulated following Ag stimulation in Lyn-/- BMMCs than in WT BMMCs. Conversely, mRNA for the low-affinity IgG receptor (FcgammaRIIB), implicated in negative regulation of FcepsilonRI-mediated signaling, is more down-regulated in Ag-stimulated Lyn-/- BMMCs than in WT BMMCs. Genes coding for proinflammatory cytokines and chemokines (IL-4, IL-6, IL-13, CSF, CCL1, CCL3, CCL5, CCL7, CCL9, and MIP1beta) are all more highly expressed in Ag-stimulated Lyn-/- mast cells than in WT cells. These microarray data identify Lyn as a negative regulator in Ag-stimulated BMMCs of the expression of genes linked to FcepsilonRI signaling and also to the response pathways that lead to allergy and asthma.

Melatonin influences sleep and circadian rhythm, and it has anti-inflammatory functions. However, the mechanism of its anti-inflammatory roles is not well understood. In our studies, we show that melatonin blocked lipopolysaccharide (LPS)-induced CCL2 (monocyte chemotactic protein-1; MCP-1), CCL5 (Regulated upon Activation, Normal T-cell Expressed, and Secreted), and CCL9 (macrophage inflammatory protein-1γ) chemokine mRNA expression in BV2 murine microglial cells. Melatonin markedly inhibited LPS-induced Akt phosphorylation and NF-κB activation. Furthermore, melatonin inhibited LPS-induced STAT1/3 phosphorylation and interferon-gamma activated sequence (GAS)-driven transcriptional activity. Interestingly, these effects were not associated with reactive oxygen species scavenging effects of melatonin or melatonin receptor signal pathways. Taken together, our results suggested that melatonin has anti-inflammatory functions through down-regulation of chemokine expression by inhibition of NF-κB and STAT/GAS activation in LPS-stimulated BV2 murine microglial cell line.

BACKGROUND

Repeated exposure to a low dose of a bacterial endotoxin such as lipopolysaccharide (LPS) causes immune cells to become refractory to a subsequent endotoxin challenge, a phenomenon known as endotoxin tolerance (ET). During ET, there is an imbalance in pro- and anti-inflammatory cytokine and chemokine production, leading to a dysregulated immune response. HIV-1 viral proteins are known to have an adverse effect on the immune system. However, the effects of HIV-1 viral proteins during ET have not been investigated.

METHODS

In this study, HIV-1 transgenic (HIV-1Tg) rats and control F344 rats (n = 12 ea) were randomly treated with 2 non-pyrogenic doses of LPS (LL) to induce ET, or saline (SS), followed by a high challenge dose of LPS (LL+L, SS+L) or saline (LL+S, SS+S). The gene expression of 84 cytokines, chemokines, and their receptors in the brain and spleen was examined by relative quantitative PCR using a PCR array, and protein levels in the brain, spleen, and serum of 7 of these 84 genes was determined using an electrochemiluminescent assay.

RESULTS

In the spleen, there was an increase in key pro-inflammatory (IL1α, IL-1β, IFN-γ) and anti-inflammatory (IL-10) cytokines, and inflammatory chemokines (Ccl2, Ccl7, and Ccl9,) in response to LPS in the SS+L and LL+L (ET) groups of both the HIV-1Tg and F344 rats, but was greater in the HIV-1Tg rats than in the F344. In the ET HIV-1Tg and F344 (LL+L) rats in the spleen, the LPS-induced increase in pro-inflammatory cytokines was diminished and that of the anti-inflammatory cytokine was enhanced compared to the SS+L group rats. In the brain, IL-1β, as well as the Ccl2, Ccl3, and Ccl7 chemokines were increased to a greater extent in the HIV-1Tg rats compared to the F344; whereas Cxcl1, Cxcl10, and Cxcl11 were increased to a greater extent in the F344 rats compared to the HIV-1Tg rats in the LL+L and SS+L groups.

CONCLUSIONS

Our data indicate that the continuous presence of HIV-1 viral proteins can have tissue-dependent effects on endotoxin-induced cytokine and chemokine expression in the ET state.

Accumulating evidence suggests that gender can have a profound effect on incidence and severity of a variety of pulmonary diseases. To address the influence of gender on the development of silica-induced pulmonary fibrosis, we instilled 0.2 g/kg silica into male and female C57BL/6 mice and examined the fibrotic and inflammatory response at 14 days postexposure. Both silica-exposed male and female mice had significant increases in total lung hydroxyproline compared with saline controls. However, silica-exposed female mice had significantly less total lung hydroxyproline than silica-exposed male mice. This observation was confirmed by color thresholding image analysis. Interestingly, silica-exposed female mice had significantly more inflammatory cells, the majority of which were macrophages, as well as higher levels of the macrophage-specific chemokines MCP-1 and CCL9 in whole lung lavage compared with silica-exposed male mice. We also show that at baseline, estrogen receptor α (ERα) mRNA expression is lower in female mice than in males and that ERα mRNA expression is decreased by silica exposure. Finally, we show that the response of ovariectomized female mice to silica instillation is similar to that of male mice. These observations together show that gender influences the lung response to silica.

Microfold (M) cells are phagocytic intestinal epithelial cells in the follicle-associated epithelium of Peyer's patches that transport particulate antigens from the gut lumen into the subepithelial dome. Differentiation of M cells from epithelial stem cells in intestinal crypts requires the cytokine receptor activator of NF-κB ligand (RANKL) and the transcription factor Spi-B. We used three-dimensional enteroid cultures established with small intestinal crypts from mice as a model system to investigate signaling pathways involved in M cell differentiation and the influence of other cytokines on RANKL-induced M cell differentiation. Addition of RANKL to enteroids induced expression of multiple M cell-associated genes, including Spib, Ccl9 [chemokine (C-C motif) ligand 9], Tnfaip2 (TNF-α-induced protein 2), Anxa5 (annexin A5), and Marcksl1 (myristoylated alanine-rich protein kinase C substrate) in 1 day. The mature M cell marker glycoprotein 2 (Gp2) was strongly induced by 3 days and expressed by 11% of cells in enteroids. The noncanonical NF-κB pathway was required for RANKL-induced M cell differentiation in enteroids, as addition of RANKL to enteroids from mice with a null mutation in the mitogen-activated protein kinase kinase kinase 14 (Map3k14) gene encoding NF-κB-inducing kinase failed to induce M cell-associated genes. While the cytokine TNF-α alone had little, if any, effect on expression of M cell-associated genes, addition of TNF-α to RANKL consistently resulted in three- to sixfold higher levels of multiple M cell-associated genes than RANKL alone. One contributing mechanism is the rapid induction by TNF-α of Relb and Nfkb2 (NF-κB subunit 2), genes encoding the two subunits of the noncanonical NF-κB heterodimer. We conclude that endogenous activators of canonical NF-κB signaling present in the gut-associated lymphoid tissue microenvironment, including TNF-α, can play a supportive role in the RANKL-dependent differentiation of M cells in the follicle-associated epithelium.

Adipocytes are differentiated by various transcriptional cascades integrated on the master regulator, Pparγ. To discover new genes involved in adipocyte differentiation, preadipocytes were treated with three newly identified pro-adipogenic small molecules and GW7845 (a Pparγ agonist) for 24 hours and transcriptional profiling was analyzed. Four genes, Peroxisome proliferator-activated receptor γ (Pparγ), human complement factor D homolog (Cfd), Chemokine (C-C motif) ligand 9 (Ccl9), and GIPC PDZ Domain Containing Family Member 2 (Gipc2) were induced by at least two different small molecules but not by GW7845. Cfd and Ccl9 expressions were specific to adipocytes and they were altered in obese mice. Small hairpin RNA (shRNA) mediated knockdown of Cfd in preadipocytes inhibited lipid accumulation and expression of adipocyte markers during adipocyte differentiation. Overexpression of Cfd promoted adipocyte differentiation, increased C3a production, and led to induction of C3a receptor (C3aR) target gene expression. Similarly, treatments with C3a or C3aR agonist (C4494) also promoted adipogenesis. C3aR knockdown suppressed adipogenesis and impaired the pro-adipogenic effects of Cfd, further suggesting the necessity for C3aR signaling in Cfd-mediated pro-adipogenic axis. Together, these data show the action of Cfd in adipogenesis and underscore the application of small molecules to identify genes in adipocytes.

Despite the success in returning people to health saving mobility and high quality of life, the over 1 million total joint replacements implanted in the US each year are expected to eventually fail after approximately 15-25 years of use, due to slow progressive subtle inflammation to implant debris compromising the bone implant interface. This local inflammatory pseudo disease state is primarily caused by implant debris interaction with innate immune cells, i.e., macrophages. This implant debris can also activate an adaptive immune reaction giving rise to the concept of implant-related metal sensitivity. However, a consensus of studies agree the dominant form of this response is due to innate reactivity by macrophages to implant debris danger signaling (danger-associated molecular pattern) eliciting cytokine-based and chemokine inflammatory responses. This review covers implant debris-induced release of the cytokines and chemokines due to activation of the innate (and the adaptive) immune system and how this leads to subsequent implant failure through loosening and osteolysis, i.e., what is known of central chemokines (e.g., IL-8, monocyte chemotactic protein-1, MIP-1, CCL9, CCL10, CCL17, and CCL22) associated with implant debris reactivity as related to the innate immune system activation/cytokine expression, e.g., danger signaling (e.g., IL-1β, IL-18, IL-33, etc.), toll-like receptor activation (e.g., IL-6, tumor necrosis factor α, etc.), bone catabolism (e.g., TRAP5b), and hypoxia responses (HIF-1α). More study is needed, however, to fully understand these interactions to effectively counter cytokine- and chemokine-based orthopedic implant-related inflammation.

BACKGROUND

Hematopoietic stem cells (HSCs) follow a genetically programmed pattern of migration during development. Extracellular matrix and adhesion molecules, as well as chemokines and their receptors, are important in adult HSC migration. However, little is known about the role these molecules play at earlier developmental stages.

METHODS

We have analyzed by quantitative polymerase chain reaction (qPCR) array the expression pattern of extracellular matrix and adhesion molecules as well as chemokines and chemokine receptors in Lineage-Sca-1+c-Kit+ (LSK) cells at different stages of development, in order to characterize the role played by these molecules in LSK. Data were represented by volcano plots to show the differences in expression pattern at the time points studied.

RESULTS

Our results show marked changes in the expression pattern of extracellular matrix, adhesion molecules, chemokines and their receptors with developmental age, particularly in later stages of development. Ten molecules were significantly increased among the LSK populations studied. Our screen identified the upregulation of Col4a1, as well as molecules involved in its degradation (Mmp2, Timp2), with development. Other genes identified were Sell, Tgfbi, and Entpd1. Furthermore, we show that the expression of the chemokines Ccl4, Ccl9, Il18 and the chemokine receptor Cxcr4 increases in LSK cells during development.

CONCLUSIONS

Several genes are upregulated in the LSK population in their transition to the bone marrow microenvironment, increasing at later stages of development. This gene pattern should be emulated by embryonic stem cell-derived hematopoietic progenitors in order to improve their properties for clinical applications such as engraftment.

Cytokines are a protein family of regulatory factors derived from tumors and their environmental components that contribute to the growth, invasion and metastasis of breast cancer. However, the way in which tumor progression and cytokines regulate each other is not well understood. In this study, we used an oligoDNA microarray to assess the kinetic expression profile of cytokine genes in tumor tissues and lymph nodes during the progression of tumor growth in mice that had been subcutaneously challenged with breast adenocarcinoma SB5b cells. Our results demonstrated that IL-15, IL-17, IL-18 and IL-18 binding protein (IL-18bp), which are associated with inflammation, were increased in tumor tissues. Conversely, chemokines and their receptors, including CXCR4/CXCL12, CCR7/CCL21, CCL9, CXCL9 and CCL12, were overexpressed in lymph nodes during tumor growth. Furthermore, RT-PCR and Western blot analysis revealed that IL-18, a pro-angiogenic factor in tumors, was up-regulated in tumor tissues. Interestingly, CCR3, IL-1R2, SOCS and IL-20 were up-regulated in tumor tissues, but down-regulated in lymph nodes during tumor growth. This result suggests that the expression of cytokines and cytokine-related genes was differentially regulated, which resulted in a beneficial effect for tumor progression.