scholarly journals Estrogen markedly reduces circulating low-density neutrophils and enhances pro-tumoral gene expression in neutrophil of tumour-bearing mice

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chew Leng Lim ◽  
Valerie C.-L. Lin
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Tumour Progression ◽  
Post Partum ◽  
Mammary Tissue ◽  
Low Density ◽  
Female Mice ◽  
Tumour Bearing ◽  
Mammary Involution ◽  
Blood Neutrophils

Abstract Background Neutrophils are important for immune surveillance of tumour cells. Neutrophils may also be epigenetically programmed in the tumour microenvironment to promote tumour progression. In addition to the commonly known high-density neutrophils (HDN) based on their separation on density gradient, recent studies have reported the presence of high levels of low-density neutrophils (LDN) in tumour-bearing mice and cancer patients. We reported previously that estrogen promotes the growth of estrogen receptor α-negative mammary tumours in mice undergoing mammary involution through stimulating pro-tumoral activities of neutrophils in the mammary tissue. Methods Female BALB/cAnNTac mice at 7–8 weeks old were mated and bilateral ovariectomy was performed 2 days post-partum. At 24 h after forced-weaning of pups to induce mammary involution, post-partum female mice were injected with either E2V, or vehicle control on alternative days for 2-weeks. On 48 h post-weaning, treated female mice were inoculated subcutaneously with 4 T1-Luc2 cells into the 9th abdominal mammary gland. Age-matched nulliparous female was treated similarly. Animals were euthanized on day 14 post-tumour inoculation for analysis. To evaluate the short-term effect of estrogen, post-partum females were treated with only one dose of E2V on day 12 post-tumour inoculation. Results Estrogen treatment for 2-weeks reduces the number of blood LDN by more than 10-fold in tumour-bearing nulliparous and involuting mice, whilst it had no significant effect on blood HDN. The effect on tumour-bearing mice is associated with reduced number of mitotic neutrophils in the bone marrow and increased apoptosis in blood neutrophils. Since estrogen enhanced tumour growth in involuting mice, but not in nulliparous mice, we assessed the effect of estrogen on the gene expression associated with pro-tumoral activities of neutrophils. Whilst 48 h treatment with estrogen had no effect, 2-weeks treatment significantly increased the expression of Arg1, Il1b and Tgfb1 in both HDN and LDN of involuting mice. In contrast, estrogen increased the expression of Arg1 and Ccl5 in HDN and LDN of nulliparous mice. Conclusions Prolonged estrogenic stimulation in tumour-bearing mice markedly hampered tumour-associated increase of LDN plausibly by inhibiting their output from the bone marrow and by shortening their life span. Estrogen also alters the gene expression in neutrophils that is not seen in tumour-free mice. The results imply that estrogen may significantly influence the tumour-modulating activity of blood neutrophils.

scholarly journals Estrogen Markedly Reduces Circulating Low-density Neutrophils and Enhances Pro-tumoral Gene Expression in Neutrophil of Tumour-bearing Mice

2021 ◽  
Author(s):  
Chew Leng Lim ◽  
Valerie C-L Lin
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Tumour Progression ◽  
Post Partum ◽  
Immune Surveillance ◽  
Estrogen Receptor Α ◽  
Mammary Tissue ◽  
Low Density ◽  
Tumour Bearing ◽  
Blood Neutrophils

Abstract Background: Neutrophils are important for immune surveillance of tumour cells. Neutrophils may also be epigenetically programmed in the tumour microenvironment to promote tumour progression. In addition to the commonly known high-density neutrophils (HDN) based on their separation on density gradient, recent studies have reported the presence of high levels of low-density neutrophils (LDN) in tumour-bearing mice and cancer patients. We reported previously that estrogen promotes the growth of estrogen receptor α-negative mammary tumours in mice undergoing mammary involution through stimulating pro-tumoral activities of neutrophils in the mammary tissue. Methods: Female BALB/cAnNTac mice at 7-8 weeks old were mated and bilateral ovariectomy performed two days post-partum. At 24h post-weaning, mice were treated with either E2V, or vehicle control once every 2 days. On 48h post-weaning, mice were inoculated subcutaneously with 4T1-Luc2 cells into the 9th abdominal mammary gland. Age-matched nulliparous female was treated similarly. For 48h E2V treatment, mice were treated with one dose of E2V on day 12 post-tumour inoculation. Animals were euthanized on day 14 post-tumour inoculation for analysis.Results: Estrogen treatment for 2-weeks reduces the number of blood LDN by more than 10-fold in tumour-bearing nulliparous and involuting mice, whilst it had no effect on blood HDN. The effect on tumour-bearing mice is associated with reduced number of mitotic neutrophils in the bone marrow and increased apoptosis in blood neutrophils. Since estrogen enhanced tumour growth in involuting mice, but not in nulliparous mice, we assessed the effect of estrogen on the gene expression associated with pro-tumoral activities of neutrophils. Whilst 48h treatment with estrogen had no effect, 2-weeks treatment significantly increased the expression of Arg1, Il1b and Tgfb1 in both HDN and LDN of involuting mice. In contrast, estrogen increased the expression of Arg1 and Ccl5 in HDN and LDN of nulliparous mice. Conclusions: Prolonged estrogenic stimulation in tumour-bearing mice markedly hampered tumour-associated increase of LDN plausibly by inhibiting their output from the bone marrow and by shortening their life span. Estrogen also alters the gene expression in neutrophils that is not seen in tumour-free mice. The results imply that estrogen may significantly influence the tumour-modulating activity of blood neutrophils.

scholarly journals Dynamics of Cardiac Neutrophil Diversity in Murine Myocardial Infarction

2020 ◽  
Vol 127 (9) ◽  
Author(s):  
Ehsan Vafadarnejad ◽  
Giuseppe Rizzo ◽  
Laura Krampert ◽  
Panagiota Arampatzi ◽  
Anahi-Paula Arias-Loza ◽  
...  
Keyword(s):  
Gene Expression ◽  
Myocardial Infarction ◽  
Bone Marrow ◽  
Single Cell ◽  
Ischemic Heart ◽  
Time Resolved ◽  
Tissue Healing ◽  
Receptor Interactions ◽  
Blood Neutrophils ◽  
Gene Expression Dynamics

Rationale: After myocardial infarction, neutrophils rapidly and massively infiltrate the heart, where they promote both tissue healing and damage. Objective: To characterize the dynamics of circulating and cardiac neutrophil diversity after infarction. Methods and results: We employed single-cell transcriptomics combined with cell surface epitope detection by sequencing to investigate temporal neutrophil diversity in the blood and heart after murine myocardial infarction. At day 1, 3, and 5 after infarction, cardiac Ly6G + (lymphocyte antigen 6G) neutrophils could be delineated into 6 distinct clusters with specific time-dependent patterning and proportions. At day 1, neutrophils were characterized by a gene expression profile proximal to bone marrow neutrophils ( Cd177 , Lcn2 , Fpr1 ), and putative activity of transcriptional regulators involved in hypoxic response ( Hif1a ) and emergency granulopoiesis ( Cebpb ). At 3 and 5 days, 2 major subsets of Siglecf hi (enriched for eg, Icam1 and Tnf ) and Siglecf low ( Slpi, Ifitm1 ) neutrophils were found. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) analysis in blood and heart revealed that while circulating neutrophils undergo a process of aging characterized by loss of surface CD62L and upregulation of Cxcr4 , heart infiltrating neutrophils acquired a unique SiglecF hi signature. SiglecF hi neutrophils were absent from the bone marrow and spleen, indicating local acquisition of the SiglecF hi signature. Reducing the influx of blood neutrophils by anti-Ly6G treatment increased proportions of cardiac SiglecF hi neutrophils, suggesting accumulation of locally aged neutrophils. Computational analysis of ligand/receptor interactions revealed putative pathways mediating neutrophil to macrophage communication in the myocardium. Finally, SiglecF hi neutrophils were also found in atherosclerotic vessels, revealing that they arise across distinct contexts of cardiovascular inflammation. Conclusions: Altogether, our data provide a time-resolved census of neutrophil diversity and gene expression dynamics in the mouse blood and ischemic heart at the single-cell level, and reveal a process of local tissue specification of neutrophils in the ischemic heart characterized by the acquisition of a SiglecF hi signature.

Growth Factor Independence 1 (Gfi-1) Plays a Role in Mediating Specific Granule Deficiency (SGD) in a Patient Lacking an Abnormality in the C/EBPε Gene.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3546-3546
Author(s):  
Arati Khanna-Gupta ◽  
Terry Zibello ◽  
Hong Sun ◽  
Laurence A. Boxer ◽  
Nancy Berliner
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Growth Factor ◽  
Western Blot ◽  
Blot Analysis ◽  
Germline Mutations ◽  
Coding Region ◽  
Blood Neutrophils ◽  
Specific Granule

Abstract Neutrophil specific granule deficiency (SGD) is a rare congenital disorder marked by recurrent bacterial infections of the skin and respiratory system. Neutrophils from SGD patients lack secondary and tertiary granules and their content proteins and exhibit defects in chemotaxis and bactericidal activity. A mouse model deficient for the transcription factor CCAAT/enhancer binding protein epsilon (C/EBPε) manifests a similar phenotype as SGD patients and prompted examination of the human C/EBPε gene for mutation in this disease. Mutations in the C/EBPε gene have been identified by others in two patients with SGD, resulting in loss of gene expression. However, other patients with a similar disease phenotype have a normal C/EBPε coding sequence, suggesting that other genetic abnormalities in myelopoiesis can lead to SGD. Studies in our laboratory on one such patient lacking a C/EBPε mutation demonstrated an elevated level of the C/EBPε protein in the patient’s peripheral blood neutrophils as compared to the level in normal control neutrophils. Microarray analysis of this patient’s bone marrow compared with that of a normal control revealed, among other genes, elevated levels of the transcription factors C/EBPε and PU.1. As a consequence, several PU.1 target genes showed increased expression in the SGD patient bone marrow. This observation was confirmed by both real time PCR and western blot analysis. PU.1 is a hematopoietic-specific transcription factor belonging to the Ets family of DNA binding proteins and plays a critical role in B-cell, macrophage and late stage neutrophil development. Sequence analysis of the PU.1 gene from our SGD patient however, did not reveal any mutation in the coding region of the gene. Western blot analysis of nuclear extracts prepared from peripheral blood neutrophils from this patient did however reveal significantly decreased levels of the transcription factor Gfi-1 (Growth factor independent-1), although no mutation has been found thus far in the coding region of the Gfi-1 gene from the SGD patient. Gfi-1 belongs to a family of zinc finger containing transcriptional repressor oncoproteins. Mice lacking Gfi-1 were found not only to be neutropenic, but also demonstrated defects in neutrophil differentiation, including loss of neutrophil secondary and tertiary granule proteins, reminiscent of SGD. More recently, heterozygous germline mutations of Gfi-1 were shown to cause severe congenital neutropenia (SCN) in humans. It has been suggested that Gfi1 represses neutrophil elastase (Ela2), germline mutations within which are a major contributor to hereditary neutropenias. Our data suggest that decreased levels of Gfi1 in our SGD patient result in increased levels of PU.1 and C/EBPε; an effect consistent with observations first made in the neutrophils of Gfi-1-null mice. The increased PU.1 levels might then act to sequester C/EBPε protein via direct protein-protein interaction. This in turn could explain the loss of secondary granule protein gene expression in the SGD patient by inducing functional C/EBPε deficiency.

scholarly journals Estrogen exacerbates mammary involution through neutrophil dependent and independent mechanism

2020 ◽  
Author(s):  
Chew Leng Lim ◽  
Yu Zuan Or ◽  
Zoe Ong ◽  
Hwa Hwa Chung ◽  
Hirohito Hayashi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Death ◽  
Post Partum ◽  
Mammary Tissue ◽  
Inflammatory Microenvironment ◽  
Estrogen Exposure ◽  
Mammary Involution ◽  
Mammary Cell ◽  
Independent Mechanism ◽  
Neutrophil Depletion

AbstractThere is strong evidence that the pro-inflammatory microenvironment during post-partum mammary involution promotes parity-associated breast cancer. Estrogen exposure during mammary involution drives tumour growth through the activity of neutrophils. However, how estrogen and neutrophils influence mammary involution are unknown. Combined analysis of transcriptomic, protein, and immunohistochemical data in Balb/c mice with and without neutrophil depletion showed that estrogen promotes involution by exacerbating inflammation, cell death and adipocytes repopulation through neutrophil-dependent and neutrophil-independent mechanisms. Remarkably, 88% of estrogen-regulated genes in mammary tissue were mediated through neutrophils, which were recruited through estrogen-induced CXCL2-CXCR2 signalling. While neutrophils mediate estrogen-induced inflammation and adipocytes repopulation, estrogen-induced mammary cell death was mediated by neutrophils-independent upsurges of cathepsins and their lysosomal leakages that are critical for lysosome-mediated cell death. Notably, these multifaceted effects of estrogen are unique to the phase of mammary involution. These findings are important for the development of intervention strategies for parity-associated breast cancer.

scholarly journals Estrogen exacerbates mammary involution through neutrophil-dependent and -independent mechanism

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Chew Leng Lim ◽  
Yu Zuan Or ◽  
Zoe Ong ◽  
Hwa Hwa Chung ◽  
Hirohito Hayashi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Death ◽  
Post Partum ◽  
Mammary Tissue ◽  
Independent Manner ◽  
Inflammatory Microenvironment ◽  
Estrogen Exposure ◽  
Mammary Involution ◽  
Mammary Cell ◽  
Independent Mechanism

There is strong evidence that the pro-inflammatory microenvironment during post-partum mammary involution promotes parity-associated breast cancer. Estrogen exposure during mammary involution drives tumor growth through neutrophils’ activity. However, how estrogen and neutrophils influence mammary involution are unknown. Combined analysis of transcriptomic, protein, and immunohistochemical data in BALB/c mice showed that estrogen promotes involution by exacerbating inflammation, cell death and adipocytes repopulation. Remarkably, 88% of estrogen-regulated genes in mammary tissue were mediated through neutrophils, which were recruited through estrogen-induced CXCR2 signalling in an autocrine fashion. While neutrophils mediate estrogen-induced inflammation and adipocytes repopulation, estrogen-induced mammary cell death was via lysosome-mediated programmed cell death through upregulation of cathepsin B, Tnf and Bid in a neutrophil-independent manner. Notably, these multifaceted effects of estrogen are mostly mediated by ERα and unique to the phase of mammary involution. These findings are important for the development of intervention strategies for parity-associated breast cancer.

scholarly journals Cathelicidin Gene Expression in Porcine Tissues: Roles in Ontogeny and Tissue Specificity

1999 ◽  
Vol 67 (1) ◽  
pp. 439-442 ◽  
Author(s):  
Hua Wu ◽  
Guolong Zhang ◽  
Christopher R. Ross ◽  
Frank Blecha
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Antimicrobial Peptides ◽  
Defense Mechanisms ◽  
Developmental Expression ◽  
Mesenteric Lymph Nodes ◽  
Young Pigs ◽  
Neutrophil Degranulation ◽  
Blood Neutrophils ◽  
Peripheral Leukocytes

ABSTRACT Cathelicidins constitute a family of mammalian antimicrobial peptides that are synthesized in the bone marrow as prepropeptides, stored in neutrophil granules as propeptides, and released as active, mature peptides upon neutrophil degranulation. We investigated the developmental expression of two porcine cathelicidins, PR-39 and protegrin. Both cathelicidins were expressed constitutively in the bone marrow of all pigs at all of the ages tested. Peripheral blood neutrophils from young pigs expressed PR-39 and protegrin mRNA, which were not detectable at 42 days of age. At earlier ages, expression of PR-39 mRNA was detected in the kidney and liver and several lymphoid organs, including the thymus, spleen, and mesenteric lymph nodes, but disappeared at 4 weeks of age. These data provide the first evidence of cathelicidin gene expression in peripheral leukocytes and may indicate a role for these antimicrobial peptides in the development of host defense mechanisms.

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