Increased Expression Of CXCL12 Research Articles

Deciphering the Differential Impact of Thrombopoietin/MPL Signaling on Hematopoietic Stem Cell Function in Bone Marrow and Spleen

Introduction Thrombopoietin (TPO) and its receptor MPL are key regulators of hematopoietic stem cell (HSC) function. In humans, ablation of either TPO or MPL leads to bone marrow failure in the early years of life. In contrast, mice lacking TPO or MPL display marked thrombocytopenia and severely reduced HSC numbers but manage to maintain normal survival up to one year of age. In this study, we investigated whether these mice eventually develop marrow failure with longer follow up, with a specific focus on understanding the effects of TPO/MPL signaling on HSC regulation in different hematopoietic niches. Methods Marrow and spleen hematopoiesis in the TPO -/- mice and MPL -/- mice were examined during a 2-yr follow-up. Results At 2 yrs of age, TPO -/- and MPL -/- mice exhibited not only a severely decreased platelet count (TPO -/- vs. MPL -/- vs. control: 38 vs. 107 vs. 1255 x 10 9/L) but also a moderate reduction in neutrophil count compared to age-matched wild-type controls (TPO -/- vs. MPL -/- vs. control: 1.1 vs. 1.7vs.3.3 x 10 3/ul). Femur marrow cell count or spleen cell count was not significantly different between TPO -/- or MPL -/- mice and control mice. There was no significant difference in overall survival between the TPO -/- or MPL -/- mice and wild-type controls. Marrow Lin -cKit +Sca1 +CD150 +CD48 - HSC cell numbers were significantly decreased in young (4-6mo) TPO -/- and MPL -/- mice (~4-fold) compared to age-matched control mice. This HSC loss was further worsened in old (~24mo) TPO -/- (25-fold) and MPL -/- (12-fold) mice compared to control mice. In contrast, there was no significant difference in splenic HSC cell numbers between young TPO -/- or MPL -/- mice and control mice, with only a moderate decrease of HSCs in old TPO -/- and MPL -/- (~4-fold) mice compared to control mice. I n vivo using BrdU labeling revealed an increased proliferation of marrow HSCs in TPO -/- and MPL -/- mice compared to age-matched control mice. In contrast, there was no significant increase in splenic HSC proliferation in TPO -/- or MPL -/- mice. These findings suggest that TPO/MPL signaling has distinct effects on both the number and the function of HSCs in the marrow and spleen. Competitive repopulation assays revealed that HSCs from 2yr old TPO -/- or MPL -/- marrow failed to engraft the recipient mice at 12 weeks post-transplantation. We also conducted competitive repopulation assays in which spleen cells from old TPO -/- mice (CD45.2) were injected together with spleen cells from 2yr old wild-type competitor mice (CD45.1) into lethally irradiated wild-type recipients (CD45.1). At 12 days after transplantation, the TPO -/- spleen donor showed comparable engraftment in the Lin -cKit +Sca1 + stem/progenitor cell compartment, similar to the wild-type competitor spleen donor, suggesting that at least the short-term engraftment capacity of the TPO -/- spleen donor was not impaired. We are currently conducting further investigation to assess the long-term repopulation capacity of TPO -/- spleen HSCs. To investigate how TPO/MPL ablation impacts the hematopoietic niche in the marrow and spleen, we used quantitative flow cytometry and/or fluorescence imaging to examine these niche cells and their expression of niche factors. First, while there was no significant difference in the marrow vascular area (measured by in vivo VE-cadherin labeling) between TPO -/- or MPL -/- mice and control mice, we observed a significant increase in the splenic vascular area in TPO -/- and MPL -/- mice. Second, CXCL12 levels were significantly decreased in marrow endothelial cells (ECs; CD45 -CD31 +) from TPO -/- and MPL -/- mice compared to control mice; in contrast, CXCL12 levels were significantly increased in splenic ECs from TPO -/- and MPL -/- mice. Similar results were also observed with marrow and splenic perivascular stromal cells (CD45 -CD31 -Ter119 -Sca1 -). Therefore, TPO/MPL ablation enhanced both the quantity and quality of the splenic hematopoietic niche, which could contribute to the maintenance of splenic hematopoiesis in TPO -/- and MPL -/- mice. Last, consistent with findings from the TPO/MPL ablated mice, exogenous TPO treatment increased marrow HSC proliferation but did not alter splenic HSC proliferation. In addition, TPO treatment increased CXCL12 expression in marrow ECs, but decreased CXCL12 levels in splenic ECs. Taken together, our study revealed the complexity of TPO/MPL signaling in HSC regulation which differs between the marrow and spleen

TREM2 deficiency in microglia accelerates photoreceptor cell death and immune cell infiltration following retinal detachment

Retinal detachment (RD) occurs in several major retinal conditions and often causes irreversible vision loss due to photoreceptor cell death. Retinal residential microglial cells are activated following RD and participate in photoreceptor cell death via direct phagocytosis and the regulation of inflammatory responses. Triggering receptor expressed on myeloid cells 2 (TREM2) is an innate immune receptor exclusively expressed on microglial cells in the retina, and has been reported to affect microglial cell homeostasis, phagocytosis and inflammatory responses in the brain. In this study, increased expression of multiple cytokines and chemokines in the neural retina was observed starting at 3 h following RD. Trem2 knockout (Trem2−/−) mice exhibited significantly more photoreceptor cell death than wild-type controls at 3 days after RD, and the number of TUNEL positive photoreceptor cells progressively decreased from day 3 to day 7 post-RD. A significant thinning of the outer nuclear layer (ONL), with multiple folds was observed in the Trem2−/− mice at 3 days post-RD. Trem2 deficiency reduced microglial cell infiltration and phagocytosis of stressed photoreceptors. There were more neutrophils in Trem2−/− retina following RD than in controls. Using purified microglial cells, we found Trem2 knockout is associated with increased CXCL12 expression. The aggravated photoreceptor cell death was largely reversed by blocking the CXCL12-CXCR4 mediated chemotaxis in Trem2−/− mice after RD. Our findings suggested that retinal microglia are protective in preventing further photoreceptor cell death following RD by phagocytosing presumably stressed photoreceptor cells and by regulating inflammatory responses. TREM2 is largely responsible for such protective effect and CXCL12 plays an important role in regulating neutrophil infiltration after RD. Collectively, our study pinpointed TREM2 as a potential target of microglial cells to ameliorate RD-induced photoreceptor cell death.

Sex Dimorphism in the Bone Marrow Niche Contributes to Gender Difference in Mouse Hematopoiesis

Sex dimorphism can be found in hematopoietic stem and progenitor cells, as well as in the development of hematopoietic lineages, resulting in sexual dimorphisms in normal hematopoiesis. Furthermore, males are at higher risk and have a worse prognosis for most hematologic malignancies than females. Under normal conditions, the number of circulating progenitor cells in women is lower than in men. Furthermore, female mice have a higher rate of HSC division than male mice. However, the molecular mechanism underlying normal hematopoiesis sex dimorphism is largely unknown. We compared male and female hematopoiesis in C57BL/6 mice in the steady state and discovered that male mice have more HSCs than female mice. Female LSK (Lineage-, Sca-1+, and cKit+) cells also had higher apoptosis and lower quiescence than male LSK cells. Additionally, male mice had higher percentage and number of mesenchymal stem cells (MSC) than female mice. We then conducted two sex/gender mismatch transplantation experiments. First, we injected male or female BM cells into male or female recipients, resulting in four transplantation groups (male to male, male to female, female to male, and female to female). We found that both male and female donor cells engraft better in male recipients than in female recipients, suggesting that male niche provides better hematopoietic engraftment. In the secondary transplantation setting, we mixed equal numbers of male (CD45.1) and female (CD45.2) BM cells and injected the cells into male and female CD45.1/CD45.2 chimeric recipient mice. Male donor cells engraft better than female donor cells in both male and female recipients. Consistently, male recipients have more support for HSC regeneration than female recipients. Altogether, these data suggest that male donor to male recipients produce the best engraftment outcome. This is consistent with the clinic bone marrow transplantation result. To better understand the underlying molecular mechanism, we performed single-cell RNA sequencing on LSK cells and bone marrow stromal cells from female and male mice (Lineage-, CD45-, Ter119-, and CD71-). Based on gene expression profiling at the single cell level, different populations of HSPCs and stromal cells were clustered. Mesenchymal stem cells (MSC) are the niche cellular component with the most significant sex difference, with male mice having more MSCs than female mice. The LSK cells were then co-cultured with male and female MSCs, and the cobblestone colony formation (CAFC) was assessed over time. The number of CAFC day 35 colonies was significantly higher in male MSC stromal than that in female MSCs. Therefore, male MSCs can thus better support for HSC function, which is consistent with our sex mismatch transplantation result. We further discovered that male MSC secrete more Cxcl12, which may underlie the increased hematopoietic regeneration. We are currently investigating the mechanism underlying Cxcl12 differential expression in the male/female niche by focusing on the Kdm5c gene. Kdm5c is a gene on the X chromosome that has escaped Xist inactivation. Female cells therefore express twice as much as male cells. It specifically demethylates H3K4me3/me2 and inhibits gene transcription. We hypothesize that a low level of Kdm5c in male MSCs leads to more methylation of H3K4me3, thus increasing Cxcl12 transcription. By using Chip-qPCR, we found that Kdm5c was highly enriched in the Cxcl12 promoter in female MSCs compared to male MSCs, leading to the increased level of demethylated H3K4me3, thus reducing expression of Cxcl12. We next knockdown Kdm5c in female MSCs and found that methylated H3K4me3 was increased in the Cxcl12 promotor, resulting in increased Cxcl12 expression. In conclusion, sex dimorphism affects normal hematopoietic in a cell intrinsic and extrinsic mechanism. Male MSCs provide a better support for hematopoietic engraftment and regeneration. Differential expression of Kdm5c, sex chromosome-specific gene, may be the underlying mechanism in which a lower level of KDM5C in male MSC leads to higher expression of Cxcl12, which promotes HSC engraftment and maintenance.

Cholinergic signals preserve haematopoietic stem cell quiescence during regenerative haematopoiesis

The sympathetic nervous system has been evolutionary selected to respond to stress and activates haematopoietic stem cells via noradrenergic signals. However, the pathways preserving haematopoietic stem cell quiescence and maintenance under proliferative stress remain largely unknown. Here we found that cholinergic signals preserve haematopoietic stem cell quiescence in bone-associated (endosteal) bone marrow niches. Bone marrow cholinergic neural signals increase during stress haematopoiesis and are amplified through cholinergic osteoprogenitors. Lack of cholinergic innervation impairs balanced responses to chemotherapy or irradiation and reduces haematopoietic stem cell quiescence and self-renewal. Cholinergic signals activate α7 nicotinic receptor in bone marrow mesenchymal stromal cells leading to increased CXCL12 expression and haematopoietic stem cell quiescence. Consequently, nicotine exposure increases endosteal haematopoietic stem cell quiescence in vivo and impairs hematopoietic regeneration after haematopoietic stem cell transplantation in mice. In humans, smoking history is associated with delayed normalisation of platelet counts after allogeneic haematopoietic stem cell transplantation. These results suggest that cholinergic signals preserve stem cell quiescence under proliferative stress.

Gut-Liver Axis: Liver Sinusoidal Endothelial Cells Function as the Hepatic Barrier in Colitis-Induced Liver Injury.

BackgroundBased on the gut–liver axis theory, a leaky gut can aggravate liver injury. However, clinical studies suggest that although gut mucosa damage is commonly observed in inflammatory bowel disease (IBD), it seldom leads to severe liver injury. We hypothesize that there is a hepatic barrier in the gut–liver axis, which protects the liver against gut-derived invasive factors.MethodsColitis was induced by dextran sulfate sodium (DSS) in eight different liver injury models in Sprague–Dawley rats. Liver sinusoidal endothelial cell (LSEC) injury was evaluated by a scanning and transmission electron microscope. Neutrophils were depleted by injection of anti-rat polymorphonuclear serum. Two pneumonia models were also induced to investigate the mechanism of neutrophil recruitment and activation. LSECs isolated from rat liver were used to investigate the effect on neutrophil recruitment and activation.ResultsAmong eight liver injury models, DSS colitis had no effect on liver injury in three models with normal LSECs. In the other five models with LSEC rupture, liver injury was significantly exacerbated by colitis, and increased hepatic neutrophil accumulation was observed. When neutrophils were depleted, colitis-induced liver injury was significantly attenuated. In pneumonia, liver injury, and colitis models, the level of CXCL1 correlated with the recruitment of neutrophils in different tissues, while DSS colitis and LSEC injury synergistically contributed to increased CXCL1 expression in the liver. In colitis-induced liver injury, neutrophils were activated in the liver. Injured LSECs showed both structural and functional changes, with significantly increased expression of CXCL1 and TNF-α under the stimulation of lipopolysaccharide (LPS). The combination of gut-derived LPS and LSEC-derived TNF-α led to the activation of neutrophils, characterized by enhanced production of reactive oxygen species, pro-inflammatory cytokines, and the formation of neutrophil extracellular traps.ConclusionLSECs constitute a vitally important barrier in the gut–liver axis, defending the liver against colitis-induced injury. When LSECs are damaged, they can turn into a pro-inflammatory pattern under the stimulation of LPS. LSEC injury and colitis-derived LPS synergistically contribute to the recruitment and activation of hepatic neutrophils. Neutrophils play a pivotal role as a downstream effector in colitis-induced liver injury.

PAR1 regulation of CXCL1 expression and neutrophil recruitment to the lung in mice infected with influenza A virus

BackgroundProtease‐activated receptor 1 (PAR1) is expressed in various immune cells and in the lung. We showed that PAR1 plays a role in Coxsackievirus B3 infection by enhancing toll‐like receptor 3‐dependent interferon‐β expression in cardiac fibroblasts. ObjectivesWe investigated the role of PAR1 in a mouse model of influenza A virus (IAV) infection. MethodsWe used mice with either a global deficiency of PAR1, cell type‐specific deficiencies of PAR1, or mutation of PAR1 at the R41 or R46 cleavage sites. ResultsPAR1‐deficient mice had increased CXCL1 expression in the lung, increased neutrophil recruitment, increased protein levels in the bronchoalveolar lavage fluid, and increased mortality after IAV infection compared with control mice infected with IAV. Results from mice with cell type‐specific deletion of PAR1 indicated that PAR1 expression by hematopoietic cells suppressed CXCL1 expression, whereas PAR1 expression by endothelial cells enhanced CXCL1 expression in response to IAV infection. PAR1 activation also enhanced polyinosinic:polycytodylic acid induction of interleukin‐8 in a human endothelial cell line. Mutation of the R46 cleavage site of PAR1 was associated with increased CXCL1 expression in the lung in response to IAV infection, which suggested that R46 signaling suppresses CXCL1 expression. ConclusionsThese results indicate that PAR1 expression by different cell types and activation by different proteases modulates the immune response during IAV infection.

Expression Pattern of CXCR4/CXCL12 Is Related to Survival after Hepatic Resection for Colorectal Liver Metastases

Introduction: The chemokine network such as interaction between CXCR4 and CXCL12 plays a role in the induction of organ-specific metastases. The present study evaluated CXCR4/CXCL12 expression pattern in colorectal liver metastases (CRLM) and determined whether the expression patterns affect tumor progression. Methods: We examined pattern of CXCR4 and CXCL12 immunohistochemistry in 92 CRLM patients. CXCL12/CD133 immunoreactivity was also evaluated. The median follow-up time of these patients was 38 months. Clinicopathological data of these patients were evaluated. Overall survival rates were evaluated by the Kaplan-Meier method. The expression profile of CXCR4 in the colorectal cancer cell line was determined by fluorescence microscopy. Results: The cytoplasmic CXCR4 expression was greater in 36 patients than that indicated by CXCR4 staining intensity of hepatocytes. CXCL12 was also expressed in hepatocytes surrounding the tumors at high and low levels in 68 (74%) and 24 (26%), respectively. High levels of nuclear CXCR4 expression were observed in 23 patients which significantly correlated with CXCL12 expression in hepatocytes. The nuclear CXCR4 expression in the cancer cell line increased after exposure to CXCL12. The univariate and multivariate analyses demonstrated that high levels of nuclear CXCR4 and the increased CXCL12 expression in hepatocytes were significantly better prognostic factors for overall and hepatic disease-free survival in patients with CRLM. Conclusion: The CXCR4 expression in CRLM together with the upregulation of CXCL12 in hepatocytes may help to predict the clinical outcomes of patients with CRLM after hepatic resection and to determine whether adjuvant chemotherapy may be required.

Increased CXCL12 expression in endometrium of women with abnormal uterine bleeding is post-transcriptionally mediated via miR-23b-3p and is associated with decreased expression of the miR-23b-3p/24-3p/27b-3p cluster: a pilot study

To study C-X-C motif chemokine 12 (CXCL12) and CXCR4 expression in endometrial tissue from both women with and without abnormal uterine bleeding (AUB) of endometrial origin and evaluate their relationship with microRNA (miRNA). Retrospective and laboratory study. University-based research laboratory. Nine women with and without abnormal uterine bleeding, all of whom were in the secretory stage of their menstrual cycle, who provided endometrial biopsy tissue. Immunohistochemical localization of CXCL12 and CXCR4 as well as quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assessment of mRNA expression in archived endometrial biopsy tissue and invitro cell culture using the immortalized endometrial stromal cell line, t-HESC. Endometrial stromal cell line, t-HESC transfection with nontargeting, negative control miRNA mimics or miRNA mimics for miR-23b-3p and mRNA assessment miR-23b-3p expression confirmed by qRT-PCR and evaluation of impact on CXCL12 expression at the protein level by enzyme-linked immunosorbent assay and mRNA levels by qRT-PCR. Expression of CXCL12 and CXCR4 protein via immunohistochemistry and mRNA and miRNA levels of CXCL12 and CXCR4 as well as miR-23b-3p, miR-24b-3p, and miR-27b-3p, respectively, via qRT-PCR. CXCL12 and its receptor CXCR4 expression were up-regulated in the endometrial tissue of women with AUB at the protein level, but this up-regulation of expression was only associated with increased CXCR4 mRNA expression. To evaluate whether CXCL12 may be post-transcriptionally regulated, we assessed expression of miR-23b-3p, a bona fide post-transcriptional regulator of CXCL12 expression. The expression of miR-23b-3p was statistically significantly lower in AUB endometrial tissue, as were fellow cluster members miR-24-3p and miR-27-3p. Transfection of t-HESC cells with pre-miR-23b-3p mimics statistically significantly reduced the levels of CXCL12 secreted protein but not mRNA levels, suggesting that miR-23b-3p retards protein translation independent of transcript degradation. Reduced expression of the miR-23b-3p/24-3p/27b-3p cluster is associated with elevated expression of CXCL12, which may contribute to the pathophysiology of AUB.

TNF-α-Induced Bone Marrow Stromal Progenitor Alterations Enhance Leukemic Stem Cell Growth and Treatment Resistance Via Increased CXCL1-CXCR2 Signaling

Mesenchymal cells within the bone marrow (BM) microenvironment play an important role in regulation of hematopoietic stem cell (HSC) fate. However, the effects of leukemia development on distribution and function of mesenchymal cell subpopulations are not well understood. Here we used the SCL-tTA-BCR-ABL transgenic CML mouse model to examine how CML development affected murine BM mesenchymal subpopulations within a recently delineated skeletal stem cell (SSC) hierarchy, and evaluate how mesenchymal subpopulations affected HSC and leukemia stem cell (LSC) growth.We observed a significant increase in bone-forming “Thy” and stroma-forming “6C3” progenitor subsets in CML compared to normal BM. CML Thy and 6C3 progenitors demonstrated increased proliferation and CFU-F potential. In addition, CML Thy cells exhibiting increased osteogenic potential whereas CML 6C3 cells showed increased adipogenic potential compared to their normal counterparts.CML LSC and normal HSC were cocultured with Thy and 6C3 cells, purified from CML and normal mice for 3 days and transplanted into irradiated normal recipients to evaluate long-term engraftment. Normal HSC engraftment was enhanced by coculture with normal Thy and 6C3 cells but not by their CML counterparts. On the other hand, LSC engraftment was enhanced by CML 6C3 cells compared to normal 6C3 cells, but not by CML Thy cells compared to normal Thy cells. These results indicate that CML stromal progenitors demonstrate enhanced support of LSC and reduced support of normal HSC.Q-PCR analysis showed that expression of major hematopoietic regulatory molecules, including CXCL12, G-CSF, SCF, IL-1, IL-6, and IGF-1, was significantly reduced in CML 6C3 and Thy progenitors.RNA-Seq analysis demonstrated that expression of TNFaand NF-kbrelated gene sets was significantly increased in CML compared with normal 6C3 cells. Ligand-receptor interactome analysis, based on differential gene expression in LSC and normal HSC, and CML and normal 6C3 cells, revealed upregulation of the chemokines CXCL1 and CXCL5 in CML 6C3 cells, and of their cognate receptor CXCR2 in LSC. We have previously shown that TNFalevels are increased in CML compared to normal BM (Zhang et al., 2012). Here we found that treatment of WT mice with TNFα led to expansion of 6C3 cells and increased CXCL1 expression on 6C3 cells. In contrast, treatment of CML mice with anti-TNFα antibodies led to reduction in 6C3 cell numbers and reduced CXCL1 expression in 6C3 cells. These results support a critical role for TNFα signaling in expansion and increased CXCL1 expression by stromal progenitors in CML BM.We evaluated the role of paracrine CXCL1-CXCR2 signaling in growth and TKI resistance of LSC. Treatment with CXCL1 and CXCL5 resulted in expansion of LSC and leukemic progenitors respectively, and this effect was blocked by the CXCR2 inhibitor SB225002. Treatment with SB225002 significantly reduced proliferation of LSC cocultured with CML 6C3 cells. SB225002 administration to mice engrafted with CML cells resulted in significant reduction in peripheral blood WBC counts, neutrophil percentage, and leukemic short-term HSC (STHSC) and granulocyte-macrophage progenitors (GMP) in the BM. The combination of SB225002 and the TKI Nilotinib (50mg/kg) resulted in significantly greater reduction in peripheral blood WBC and neutrophils, and in BM LSC compared to Nilotinib or SB225002 alone.SB225002 treatment also significantly reduced proliferation and enhanced apoptosis of human CML CD34+CD38- cells cocultured with human CML BM mesenchymal stromal cells, and that the combination of SB225002 and Nilotinib significantly enhanced apoptosis and inhibited proliferation of CML CD34+CD38- cells compared to Nilotinib alone.We conclude that increased TNFα signaling results in expansion of stromal progenitors in CML BM, which differentially support LSC compared to normal HSC. TNFα-signaling leads to overexpression of CXCL1 by stromal progenitors, which interacts with CXCR2 overexpressed on LSC to enhance their growth. Inhibition of CXCR2 signaling reduces LSC proliferation and survival, and enhances LSC elimination in combination with TKI. These observations support further exploration of targeting of CXCL1-CXCR2 interactions as a novel and effective strategy to target BM microenvironment-protected TKI-resistant LSC. DisclosuresNo relevant conflicts of interest to declare.

Abstract B082: Osteolytic and osteoblastic prostate cancer cells remotely trigger different responses in the bone marrow niche

Abstract Background: Prostate cancer (PC) mainly metastasizes to the bone, which is why the bone marrow is an organ of interest when investigating mechanisms for metastasis. Earlier studies have shown that primary tumors have the capability to affect target organs from a distance, inducing so-called premetastatic niches crucial for metastatic establishment. In the formation of the niche, osteoblasts have been proven to play important roles. The present study investigates if the phenotype of PC cells affects the PC-induced differentiation of bone marrow cells, to assess a possible phenotype-specific nature of the metastatic niche. Method: Bone marrow stromal cells (BMSCs) were harvested from femurs from BALB/c nude mice. Cultures were established and after passaging, the cells were stimulated with conditioned media (CM) from the osteoblastic PC cell line LNCaP-19 and the osteolytic PC cell line PC-3. After 72 hours, the cells were harvested and RNA was isolated from the lysate for gene expression analysis. The PC cell lines were also injected subcutaneously together with BMSCs in BALB/c nude mice. When mice had to be sacrificed due to tumor size, tumors and bone marrow was collected for gene expression analysis. Results: Immature BMSCs were proven to be able to differentiate in an osteoblastic or adipocytic direction in vitro. After stimulation with CM from osteolytic PC cells, expression of several genes associated with an osteoblastic phenotype (e.g., Runx2 and Ocn) were downregulated in BMSCs, indicating inhibited osteoblastic differentiation. In contrast, the osteoblastic PC cells induce an immature osteoblastic phenotype with maintained levels of Runx2 and Osx, together with low levels of Ocn. The chemokine Cxcl12, which is related to formation of, and homing to, the premetastatic niche, was induced by osteoblastic PC cells. In the in vivo experiment, the inhibitory effect of osteolytic PC tumors on osteoblast differentiation (Runx2 and Osx) was confirmed. However, an increased expression of Ocn by existing osteoblasts or osteocytes was detected in mice carrying subcutaneous osteolytic PC tumors. Also, the immature osteoblast phenotype induced by the osteoblastic tumors was confirmed. Regarding homing to the metastatic niche, the increased Cxcl12 expression was confirmed in the bone marrow from mice with osteoblastic PC tumors growing subcutaneously. The osteolytic tumors instead induced increased expression of Lox, a protein that affects the integrity of the extracellular matrix. Conclusion: Osteoblastic and osteolytic cell lines had starkly different effects on the bone marrow cells. The osteoblastic PC tumors induced a response from the bone marrow likely to attract cancer cells, together with an immature population of osteoblasts, previously shown to promote hematopoietic stem cell maintenance. On the other hand, the osteolytic tumors inhibit osteoblast differentiation, and prepare the microenvironment for tumor cell arrival by influencing matrix composition of the bone marrow. Taken together, this study demonstrates that PC cell impact on bone marrow cells is dependent on tumor phenotype and may influence the formation of the premetastatic niche in different ways. Citation Format: Daniel Åhs, Andreas Josefsson, Jan-Erik Damber, Karin Welén. Osteolytic and osteoblastic prostate cancer cells remotely trigger different responses in the bone marrow niche [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B082.

Retinoic Acid Receptor γ Activity in Mesenchymal Stem Cells Regulates Endochondral Bone, Angiogenesis, and B Lymphopoiesis.

Retinoic acid receptor (RAR) signaling regulates bone structure and hematopoiesis through intrinsic and extrinsic mechanisms. This study aimed to establish how early in the osteoblast lineage loss of RARγ (Rarg) disrupts the bone marrow microenvironment. Bone structure was analyzed by micro-computed tomography (μCT) in Rarg-/- mice and mice with Rarg conditional deletion in Osterix-Cre-targeted osteoblast progenitors or Prrx1-Cre-targeted mesenchymal stem cells. Rarg-/- tibias exhibited less trabecular and cortical bone and impaired longitudinal and radial growth. The trabecular bone and longitudinal, but not radial, growth defects were recapitulated in Prrx1:RargΔ/Δ mice but not Osx1:RargΔ/Δ mice. Although both male and female Prrx1:RargΔ/Δ mice had low trabecular bone mass, males exhibited increased numbers of trabecular osteoclasts and Prrx1:RargΔ/Δ females had impaired mineral deposition. Both male and female Prrx1:RargΔ/Δ growth plates were narrower than controls and their epiphyses contained hypertrophic chondrocyte islands. Flow cytometry revealed that male Prrx1:RargΔ/Δ bone marrow exhibited elevated pro-B and pre-B lymphocyte numbers, accompanied by increased Cxcl12 expression in bone marrow cells. Prrx1:RargΔ/Δ bone marrow also had elevated megakaryocyte-derived Vegfa expression accompanied by smaller sinusoidal vessels. Thus, RARγ expression by Prrx1-Cre-targeted cells directly regulates endochondral bone formation and indirectly regulates tibial vascularization. Furthermore, RARγ expression by Prrx1-Cre-targeted cells extrinsically regulates osteoclastogenesis and B lymphopoiesis in male mice. © 2018 American Society for Bone and Mineral Research.

Leishmania infantum Parasites Subvert the Host Inflammatory Response through the Adenosine A2A Receptor to Promote the Establishment of Infection.

Adenosine is an endogenously released purine nucleoside that signals through four widely expressed G protein-coupled receptors: A1, A2A, A2B, and A3. Of these, A2AR is recognized as mediating major adenosine anti-inflammatory activity. During cutaneous leishmaniasis, adenosine induces immunosuppression, which promotes the establishment of infection. Herein, we demonstrated that A2AR signaling is exploited by Leishmania infantum parasites, the etiologic agent that causes Visceral Leishmaniasis, to successfully colonize the vertebrate host. A2AR gene-deleted mice exhibited a well-developed cellular reaction with a strong Th1 immune response in the parasitized organs. An intense infiltration of activated neutrophils into the disease-target organs was observed in A2AR−/− mice. These cells were characterized by high expression of CXCR2 and CD69 on their cell surfaces and increased cxcl1 expression. Interestingly, this phenotype was mediated by IFN-γ on the basis that a neutralizing antibody specific to this cytokine prevented neutrophilic influx into parasitized organs. In evaluating the immunosuppressive effects, we identified a decreased number of CD4+ FOXP3+ T cells and reduced il10 expression in A2AR−/− infected mice. During ex vivo cell culture, A2AR−/− splenocytes produced smaller amounts of IL-10. In conclusion, we demonstrated that the A2AR signaling pathway is detrimental to development of Th1-type adaptive immunity and that this pathway could be associated with the regulatory process. In particular, it promotes parasite surveillance.

Placental development during early pregnancy: Effects of embryo origin on expression of chemokine ligand twelve (CXCL12)

The aim was to localize chemokine ligand twelve (CXCL12) in sheep placental tissues during early gestation and after assisted reproductive technologies (ART). Uteri were collected from naturally (NAT) mated ewes and ewes receiving embryo transfer (ET), in vitro fertilization (IVF) or in vitro activation (IVA). CXCL12 was immunolocalized to endometrial stroma, glands, and trophoblast. Greater CXCL12 immunoreactivity was present in trophoblast on day 22 and 24 and in NAT ewes compared to IVF and IVA. Increased CXCL12 expression suggests CXCL12 promotes implantation and placentation. Decreased CXCL12 in IVF and IVA embryos, may compromise pregnancy establishment when utilizing ART methods.

Inducible expression of CXCL1 within the CNS amplifies demyelination in pre-clinical models of multiple sclerosis

Abstract The functional role of the ELR+ chemokine CXCL1 in host defense and disease in two pre-clinical mouse models of the human demyelinating disease multiple sclerosis (MS) was assessed. Mice in which expression of CXCL1 is under the control of a tetracycline-inducible promoter active within GFAP-positive cells were generated and infected intracranially with the neurotropic JHM strain of mouse hepatitis virus (JHMV) or immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 to induce experimental autoimmune encephalomyelitis (EAE). In both JHMV-induced neurologic disease and EAE, overexpression of CXCL1 by astrocytes resulted in increased clinical disease severity. Immunophenotyping cells infiltrating into the central nervous system (CNS) revealed a selective increase in Ly6G+CD11b+ neutrophils (but not Ly6G−Ly6C+CD11b+ monocytes) present within the CNS. In JHMV infected mice and MOG35-55-immunized mice, the T cell response to antigen with regard to proliferation and cytokine production was not altered, nor was trafficking into the CNS affected. In both models, increased CXCL1 expression within the CNS resulted in increased morbidity that correlated with selectively elevated neutrophil infiltration, diminished numbers of mature oligodendrocytes, and an increase in the severity of demyelination. Neutrophil ablation in CXCL1-transgenic mice reduced the severity of demyelination in mice arguing a role for these cells in white matter damage. Collectively, these findings illustrate that sustained CXCL1 expression amplifies the severity of white matter damage and neutrophils can contribute to this process in two different mouse models of MS.

Abstract A62: TGF-β regulates CXCL1 expression in mammary carcinoma associated fibroblasts through novel Smad2/3- and HGF/c-Met-dependent mechanisms.

Abstract CXCL1 is a chemokine secreted by macrophages, neutrophils, epithelial cells and fibroblasts, and plays a role in inflammation and wound healing. Enhanced expression of CXCL1 in tumor epithelium is associated with cell invasion, angiogenesis in melanoma, bladder, ovarian and breast cancer. However, little is known about CXCL1 expression in tumor stroma. Through analysis of gene expression data, our studies reveal that high RNA levels of CXCL1 in breast tumor stroma is associated with increased rate of recurrence and shorter relapse-free survival. We noticed decreased secretion levels of TGF-β and increased secretion of CXCL1 in tumor derived fibroblasts. Increased CXCL1 expression was also observed in mammary fibroblasts in which the TGF-β type II receptor was knocked out. By immunohistochemistry analysis we found that expression of CXCL1 inversely correlated with expression of TGF-β, phosphorylated-Smad2 and phosphorylated-Smad3 in breast tumor stroma. We hypothesized that TGF-β suppresses CXCL1 expression in breast fibroblasts. Our goal for this project was to determine the molecular mechanisms through which TGF-β suppresses CXCL1 expression using mammary fibroblasts derived from MMTV-PyVmT mammary tumor model. Treatment of fibroblasts with recombinant TGF-β decreased CXCL1 transcription and secretion by up to 85% over time, as determined by RT-PCR and ELISA. SiRNA silencing of TGF-β signaling transducers, Smad2 and Smad3, increased CXCL1 transcription and secretion in mammary fibroblasts. In addition, TGF-β suppressed expression of HGF, a factor that enhanced CXCL1 expression in mammary fibroblasts. Knockdown of HGF or treatment of fibroblasts with c-Met inhibitors reduced CXCL1 expression. These data indicate TGF-β suppresses CXCL1 expression through Smad2/3-dependent mechanisms. As a secondary mechanism, TGF-β suppresses CXCL1 expression through down-regulation of HGF/c-Met signaling. This is the first report to characterize the mechanisms of CXCL1 expression in mammary fibroblasts and demonstrate a clinical relevance for CXCL1 expression in breast cancer stroma. Citation Format: An Zou, Diana Lambert, Henry Yeh, Wei Bin Fang, Nikki Cheng. TGF-β regulates CXCL1 expression in mammary carcinoma associated fibroblasts through novel Smad2/3- and HGF/c-Met-dependent mechanisms.. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A62. doi:10.1158/1538-7445.CHTME14-A62

CXCR3 Ligands induce Expression of CXCL1 (KC/murine IL8 homolog) in Mouse Hepatic Stellate Cells

Background and aim: Liver injury leads to infiltration of immune cells and a subsequent activation of hepatic stellate cells. Chemokines are ubiquitous chemotactic proteins which are involved in inflammatory pathways. It has been recently suggested that chemokines can also induce the expression of other chemokines and thereby indirectly regulate immune cell recruitment. We therefore investigated the ability of the chemokine CXCL9 to induce CXCL1, an important neutrophil chemoattractant. Methods: The ability of CXCR3 ligands CXCL9 and CXCL10 to induce CXCL1 expresssion was analyzed in immortalized (GRX) and primary hepatic stellate cells isolated from wild-type and CXCR3-/- mice. Cells were treated with different concentrations of chemokines in the absence and presence of pertussis toxin. Furthermore, mice were treated systemically with CXCL9 and hepatic CXCL1 levels as well as neutrophil infiltration were assessed. Results: Treatment of GRX cells with CXCL9 leads to a dose dependent induction of CXCL1 protein expression. This stimulatory effect was validated in primary hepatic stellate cells (P<0.01). In contrast, stimulation of stellate cells with CCL2 did not result in CXCL1 induction. Increased CXCL1 expression in response to CXCL9 was completely abolished by co-incubation with pertussis toxin and in stellate cells derived from CXCR3-/- mice, the canonical receptor for CXCL9 and CXCL10. Notably, systemic treatment of mice with CXCL9 led to elevated hepatic CXCL1 levels and was associated with enhanced neutrophil infiltration into the liver. Conclusions: The study describes a chemokine-chemokine pathway in hepatic stellate cells which leads to augmented infiltration of neutrophils into the liver during acute liver injury.

Classical NF-κB Activation Negatively Regulates Noncanonical NF-κB-dependent CXCL12 Expression

Ligation of the lymphotoxin-β receptor (LTβR) by LIGHT (lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpes virus entry mediator on T cells (TNFSF14)) activates the noncanonical (NC) NF-κB (nuclear factor-κB) pathway and up-regulates CXCL12 gene expression by human umbilical vein endothelial cells (HUVEC). In contrast, TNF only activates classical NF-κB signaling and does not up-regulate CXCL12. To determine whether cross-talk between the classical and NC pathways affects CXCL12 expression, we investigated the effects of TNF on LIGHT signaling in HUVEC. We show here that TNF inhibits both basal and LIGHT-induced CXCL12 expression. Negative regulation by TNF requires the classical NF-κB pathway as inhibition of basal and induced CXCL12 was reversed in HUVEC-expressing dominant negative IκB (inhibitor of NF-κB) kinase (IKK)β (IKKβ(K44M)). TNF did not inhibit the NC NF-κB pathway activation as LIGHT-induced p100 processing to p52 was intact; however, TNF either alone or together with LIGHT up-regulated p100 and RelB expression and induced the nuclear localization of p100-RelB complexes. Enhanced p100 and RelB expression was inhibited by IKKβ(K44M), which led us to question whether the IκB function of elevated p100 mediates the inhibition of CXCL12 expression by TNF. We retrovirally transduced HUVEC to express p100 at a level similar to that up-regulated by TNF; however, basal and LIGHT-induced CXCL12 expression was normal in the transduced cells. In contrast, ectopic RelB expression recapitulated the effects of TNF on NC signaling and inhibited basal and LIGHT-induced CXCL12 expression by HUVEC. Our findings therefore demonstrate that TNF-induced classical NF-κB signaling up-regulates RelB expression that inhibits both basal and NC NF-κB-dependent CXCL12 expression.

CXCL12 gene expression is upregulated by hypoxia and growth arrest but not by inflammatory cytokines in rheumatoid synovial fibroblasts

Objectives CXCL12 is a constitutively expressed chemokine with important homeostatic functions. Increased CXCL12 expression has been observed in several inflammatory conditions, including rheumatoid arthritis (RA). This study was undertaken to identify potential mechanisms of regulation of CXCL12 gene expression by human fibroblasts under normal or inflammatory conditions. Methods Synovial fibroblasts (SF) were cultured from RA and osteoarthritis (OA) synovial tissues. CXCL12 mRNA expression was analysed by real time quantitative RT-PCR in RA-SF under different growth conditions, and exposed to hypoxia or to different pro-inflammatory factors. A 5′ CXCL12 −1.4 kb promoter region fragment was cloned in a luciferase reporter plasmid and its activity analysed in human fibroblasts. Results CXCL12 mRNA expression was not constitutively increased in RA- compared to OA-SF. LPS, pro-inflammatory cytokines or growth factors did not induce CXCL12 mRNA expression in SF. Hypoxia and growth arrest by either serum starvation or confluent growth induced CXCL12 mRNA and protein expression in SF. Constitutive and induced expression of CXCL12 in fibroblasts was regulated at the transcriptional level by specific regions of the −1.4 kb promoter. Conclusions Pro-inflammatory factors and cytokines do not up-regulate CXCL12 gene expression in SF. Growth arrest and hypoxia are potentially important inducers of CXCL12 expression in human fibroblasts and operate by regulating transcriptional activity of the promoter.

Correlation of CXCL12 Expression and FoxP3 + Cell Infiltration with Human Papillomavirus Infection and Clinicopathological Progression of Cervical Cancer

Human cervical cancer is an immunogenic tumor with a defined pattern of histopathological and clinical progression. Tumor-infiltrating T cells contribute to immune control of this tumor; however, cervical cancer dysregulates this immune response both through its association with human papillomavirus (HPV) infection and by producing cytokines and chemokines. Animal tumor models have revealed associations between overproduction of the chemokine stromal cell-derived factor-1 (SDF-1 or CXCL12) and dysregulation of tumor-specific immunity. We therefore proposed that CXCL12 expression by cervical precancerous and cancerous lesions correlates with histopathological progression, loss of immune control of the tumor, and HPV infection. We found a significant association between cancer stage and CXCL12 expression for squamous and glandular lesions as well as with the HPV16+ (high-risk) status of the neoplastic lesions. Cancer progression was correlated with increasing levels of FoxP3 T-cell infiltration in the tumor. FoxP3 and CXCL12 expression significantly correlated for squamous and glandular neoplastic lesions. These observations were supported by enzyme-linked immunosorbent assay and Western blotting. In addition, we demonstrated CXCL12 expression by dyskaryotic cells in ThinPrep cervical smears. This study robustly links increased CXCL12 expression and FoxP3(+)-cell infiltration to HPV infection and progression of cervical cancer. It supports the detection of CXCL12 in cervical smears and biopsies as an additional biomarker for this disease.

Systemic chemokine levels in breast cancer patients and their relationship with circulating menstrual hormones

The chemokines Stromal Cell-Derived Factor-1alpha (SDF-1alpha/CXCL12) and Monocyte Chemotactic Protein-1 (MCP-1/CCL2) have been implicated in breast cancer progression. We recently reported elevated systemic MCP-1 in breast cancer patients. This study investigated circulating levels of SDF-1alpha in breast cancer patients, and addressed potential hormonal regulation of these two potent chemokines. SDF-1alpha levels were determined by ELISA in 114 breast cancer patients and 85 controls, and correlated with clinical data. Blood samples were collected from 36 healthy premenopausal volunteers weekly for four weeks to measure Luteinising Hormone (LH), Follicular Stimulating Hormone (FSH), Oestradiol and Progesterone using a Bayer ADVIA Centaur Immunoassay system, in parallel with SDF-1alpha and MCP-1. CXCL12 expression was determined using RQ-PCR in primary tumour stromal cells (n = 16) harvested at surgery. Plasma SDF-1alpha was significantly higher in breast cancer patients than age-matched controls and had a significant correlation with tumour grade and epithelial subtype. Investigation of menstrual variations of these chemokines revealed lower SDF-1alpha levels in the mid-luteal phase of the menstrual cycle and a significant positive correlation with circulating Oestradiol. MCP-1 levels showed no correlation with menstrual hormones. There was a trend towards increased CXCL12 expression in tumour compared to normal stromal cells. The elevated level of SDF-1alpha detected in breast cancer patients, and it's correlation with prognostic indicators, highlights the importance of this chemokine in disease progression. Elucidation of factors influencing chemokine secretion supports clarification of their role in tumourigenesis.