{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T18:08:28Z","timestamp":1785434908011,"version":"3.56.0"},"reference-count":35,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T00:00:00Z","timestamp":1780531200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100008316","name":"Baden-W\u00fcrttemberg Stiftung gGmbH","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100008316","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/031h5fa94","id-type":"ROR","asserted-by":"publisher"}]},{"name":"internal means"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Neutrophils are central executors of innate immunity. Yet murine models are inherently limited by low baseline neutrophil counts. NSG mice are among the most widely used models for xenotransplantation and studies on the humanized immune system. Although G-CSF is known to stimulate granulopoiesis, the dose- and schedule-dependent effects of intraperitoneal G-CSF administration have not been systematically characterized in this immunodeficient background. Male NSG mice received intraperitoneal G-CSF according to one of five regimens (n = 6 per group): group 0 served as the saline control, group 1 received a single dose of 250 \u00b5g\/kg G-CSF administered at 48 h; group 2 received a single dose of 250 \u00b5g\/kg G-CSF administered at 24 h; group 3 received three doses of 250 \u00b5g\/kg administered G-CSF at 0 h, 24 h, and 48 h and group 4 received a single dose of 500 \u00b5g\/kg G-CSF administered at 48 h. All animals were sacrificed at 72 h. Circulating neutrophils were then quantified by flow cytometry, bone marrow neutrophil proportions by panoptic smear analysis, and splenic neutrophil abundance by Ly6G immunofluorescence. Systemic neutrophil activation was assessed via plasma neutrophil elastase (NE) activity and cell-free DNA (cfDNA) levels. Repeated G-CSF administration (Group 3) induced an approximately 13-fold expansion of circulating neutrophils, approaching the human physiological range, with significant increases also observed in bone marrow and a trend towards increased neutrophil abundance in the spleen. A single dose of 250 \u00b5g\/kg administered at 24 h (group 2), produced significant neutrophil expansion in peripheral blood and bone marrow but not in the spleen, while all other single-dose regimens failed to induce significant expansion in any compartment. NE activity and cfDNA concentrations and a selected cytokine panel remained unaltered across all groups. This systematic comparison establishes repeated intraperitoneal G-CSF administration as a reproducible strategy to achieve human-like neutrophil levels in NSG mice without inducing systemic inflammation. This provides a validated protocol with direct utility in translational models of neutrophil-dependent diseases.<\/jats:p>","DOI":"10.3390\/ijms27115099","type":"journal-article","created":{"date-parts":[[2026,6,5]],"date-time":"2026-06-05T01:23:21Z","timestamp":1780622601000},"page":"5099","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Intraperitoneal G-CSF Stimulation Achieves Human-like Neutrophil Levels in NSG Mice Without Inducing Systemic Inflammation"],"prefix":"10.3390","volume":"27","author":[{"given":"Richard","family":"Elrod","sequence":"first","affiliation":[{"name":"Department of Pediatric Surgery, University Medical Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"},{"name":"Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4743-1206","authenticated-orcid":false,"given":"Yuqing","family":"Lu","sequence":"additional","affiliation":[{"name":"Department of Pediatric Surgery, University Medical Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"},{"name":"Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6761-0247","authenticated-orcid":false,"given":"Christoph","family":"Brochhausen","sequence":"additional","affiliation":[{"name":"Institute of Pathology, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-3358-6174","authenticated-orcid":false,"given":"Rebecca","family":"Sch\u00f6nmehl","sequence":"additional","affiliation":[{"name":"Institute of Pathology, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martin","family":"Herrmann","sequence":"additional","affiliation":[{"name":"Department of Pediatric Surgery, University Medical Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"},{"name":"Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"},{"name":"Department of Rheumatology and Immunology, West China Hospital, Sichuan University, Chengdu 610041, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hong","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Pediatric Surgery, University Medical Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"},{"name":"Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9199-0892","authenticated-orcid":false,"given":"Christoph","family":"Mohr","sequence":"additional","affiliation":[{"name":"Department of Pediatric Surgery, University Medical Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-5991-6415","authenticated-orcid":false,"given":"Yannick","family":"Ledermann","sequence":"additional","affiliation":[{"name":"Department of Pediatric Surgery, University Medical Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"},{"name":"Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-8802-1331","authenticated-orcid":false,"given":"Laura","family":"Blum","sequence":"additional","affiliation":[{"name":"Department of Pediatric Surgery, University Medical Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"},{"name":"Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4972-9866","authenticated-orcid":false,"given":"Michael","family":"Boettcher","sequence":"additional","affiliation":[{"name":"Department of Pediatric Surgery, University Medical Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"},{"name":"Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michaela","family":"Klinke-Petrowsky","sequence":"additional","affiliation":[{"name":"Department of Pediatric Surgery, University Medical Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"},{"name":"Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2922-9370","authenticated-orcid":false,"given":"Jasmin","family":"Knopf","sequence":"additional","affiliation":[{"name":"Department of Pediatric Surgery, University Medical Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"},{"name":"Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8479-2305","authenticated-orcid":false,"given":"Julia","family":"Elrod","sequence":"additional","affiliation":[{"name":"Department of Pediatric Surgery, University Medical Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"},{"name":"Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1038\/nri3399","article-title":"Neutrophil Recruitment and Function in Health and Inflammation","volume":"13","author":"Kolaczkowska","year":"2013","journal-title":"Nat. Rev. Immunol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"519","DOI":"10.1038\/nri3024","article-title":"Neutrophils in the Activation and Regulation of Innate and Adaptive Immunity","volume":"11","author":"Mantovani","year":"2011","journal-title":"Nat. Rev. Immunol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1169","DOI":"10.1182\/blood.2020007008","article-title":"Neutrophil Extracellular Traps Contribute to Immunothrombosis in COVID-19 Acute Respiratory Distress Syndrome","volume":"136","author":"Middleton","year":"2020","journal-title":"Blood"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"102925","DOI":"10.1016\/j.ebiom.2020.102925","article-title":"Vascular Occlusion by Neutrophil Extracellular Traps in COVID-19","volume":"58","author":"Leppkes","year":"2020","journal-title":"eBioMedicine"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1532","DOI":"10.1126\/science.1092385","article-title":"Neutrophil Extracellular Traps Kill Bacteria","volume":"303","author":"Brinkmann","year":"2004","journal-title":"Science"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"134","DOI":"10.1038\/nri.2017.105","article-title":"Neutrophil Extracellular Traps in Immunity and Disease","volume":"18","author":"Papayannopoulos","year":"2018","journal-title":"Nat. Rev. Immunol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"pdb.top073585","DOI":"10.1101\/pdb.top073585","article-title":"Human Cancer Growth and Therapy in Immunodeficient Mouse Models","volume":"2014","author":"Shultz","year":"2014","journal-title":"Cold Spring Harb. Protoc."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"6477","DOI":"10.4049\/jimmunol.174.10.6477","article-title":"Human Lymphoid and Myeloid Cell Development in NOD\/LtSz-Scid IL2R \u03b3 Null Mice Engrafted with Mobilized Human Hemopoietic Stem Cells","volume":"174","author":"Shultz","year":"2005","journal-title":"J. Immunol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1177\/0300985820948822","article-title":"Human CD34+ Hematopoietic Stem Cell\u2013Engrafted NSG Mice: Morphological and Immunophenotypic Features","volume":"58","author":"Zdimerova","year":"2021","journal-title":"Vet. Pathol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3137","DOI":"10.1096\/fj.201800636R","article-title":"Lack of Acute Xenogeneic Graft-versus-host Disease, but Retention of T-cell Function Following Engraftment of Human Peripheral Blood Mononuclear Cells in NSG Mice Deficient in MHC Class I and II Expression","volume":"33","author":"Brehm","year":"2019","journal-title":"FASEB J."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"786","DOI":"10.1038\/nri3311","article-title":"Humanized Mice for Immune System Investigation: Progress, Promise and Challenges","volume":"12","author":"Shultz","year":"2012","journal-title":"Nat. Rev. Immunol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2731","DOI":"10.4049\/jimmunol.172.5.2731","article-title":"Of Mice and Not Men: Differences between Mouse and Human Immunology","volume":"172","author":"Mestas","year":"2004","journal-title":"J. Immunol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2791","DOI":"10.1182\/blood.V78.11.2791.bloodjournal78112791","article-title":"Granulocyte Colony-Stimulating Factor and Its Receptor","volume":"78","author":"Demetri","year":"1991","journal-title":"Blood"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1016\/j.cyto.2008.03.002","article-title":"Granulocyte Colony-Stimulating Factor: Molecular Mechanisms of Action during Steady State and \u2018Emergency\u2019 Hematopoiesis","volume":"42","author":"Panopoulos","year":"2008","journal-title":"Cytokine"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1016\/S0301-472X(02)01028-7","article-title":"Granulocyte Colony-Stimulating Factor Induces the Release in the Bone Marrow of Proteases That Cleave c-KIT Receptor (CD117) from the Surface of Hematopoietic Progenitor Cells","volume":"31","author":"Hendy","year":"2003","journal-title":"Exp. Hematol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"672","DOI":"10.1182\/blood.V99.2.672","article-title":"Granulocyte Colony-Stimulating Factor Inhibits the Mitochondria-Dependent Activation of Caspase-3 in Neutrophils","volume":"99","author":"Maianski","year":"2002","journal-title":"Blood"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2012","DOI":"10.1182\/blood.V80.8.2012.2012","article-title":"Modulation of Granulocyte Survival and Programmed Cell Death by Cytokines and Bacterial Products","volume":"80","author":"Colotta","year":"1992","journal-title":"Blood"},{"key":"ref_18","first-page":"4","article-title":"Reference Values for Hematology, Plasma Biochemistry, Bone Marrow Cytology and Bone Histology of NOD.Cg-Prkdcscid Il2rgtm1Wjl\/SzJ Immunodeficient Mice","volume":"60","author":"Sarry","year":"2021","journal-title":"JAALAS"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Hui, A.J., Lai, K., Doyle, E.H., Rivera-Torruco, G., Gutierrez, A.G., Du, E.J., Meijer, Y.B.M., Smith, G., Gilfanova, R., and Chung, K.G. (2025). Development of an Immunodeficient Mouse That Allows for Conditional Ablation of Monocytic Cells. Front. Immunol., 16.","DOI":"10.3389\/fimmu.2025.1699385"},{"key":"ref_20","unstructured":"Liess, B.D. (2026, May 28). Differential Blood Count: Reference Range, Interpretation, Collection and Panels. Medscape. Updated 20 August 2025. Available online: https:\/\/emedicine.medscape.com\/article\/2085133-overview."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"854","DOI":"10.1182\/blood.V100.3.854","article-title":"Evaluation of Role of G-CSF in the Production, Survival, and Release of Neutrophils from Bone Marrow into Circulation","volume":"100","author":"Basu","year":"2002","journal-title":"Blood"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2462","DOI":"10.1182\/blood-2009-12-259630","article-title":"STAT3 Controls Myeloid Progenitor Growth during Emergency Granulopoiesis","volume":"116","author":"Zhang","year":"2010","journal-title":"Blood"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1113","DOI":"10.1007\/s12015-025-10885-w","article-title":"G-CSF-Induced Emergency Granulopoiesis Modulates Neutrophil Effector Function in Mice","volume":"21","author":"Stephan","year":"2025","journal-title":"Stem Cell Rev. Rep."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e70301","DOI":"10.1096\/fj.202402514R","article-title":"Sepsis-induced NET Formation Requires MYD88 but Is Independent of GSDMD and PAD4","volume":"39","author":"Englert","year":"2025","journal-title":"FASEB J."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"302","DOI":"10.1111\/j.1600-0609.2011.01658.x","article-title":"G-CSF Enhances the Proliferation and Mobilization, but Not the Maturation Rate, of Murine Myeloid Cells","volume":"87","author":"Knudsen","year":"2011","journal-title":"Eur. J. Haematol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"3020","DOI":"10.1182\/blood-2004-01-0272","article-title":"G-CSF Potently Inhibits Osteoblast Activity and CXCL12 mRNA Expression in the Bone Marrow","volume":"106","author":"Semerad","year":"2005","journal-title":"Blood"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"4711","DOI":"10.1182\/blood-2008-09-177287","article-title":"CXCR4 Is a Key Regulator of Neutrophil Release from the Bone Marrow under Basal and Stress Granulopoiesis Conditions","volume":"113","author":"Eash","year":"2009","journal-title":"Blood"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1111\/j.1365-2567.2008.02950.x","article-title":"Neutrophil Mobilization and Clearance in the Bone Marrow","volume":"125","author":"Furze","year":"2008","journal-title":"Immunology"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1189\/jlb.4CE0216-088R","article-title":"Editorial: The Mysteries of the Spleen","volume":"100","author":"Dale","year":"2016","journal-title":"J. Leukoc. Biol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1016\/j.exphem.2010.11.004","article-title":"Transient Neutropenia after Granulocyte-Colony Stimulating Factor Administration Is Associated with Neutrophil Accumulation in Pulmonary Vasculature","volume":"39","author":"DeJesus","year":"2011","journal-title":"Exp. Hematol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"617","DOI":"10.1038\/labinvest.3780067","article-title":"Neutrophils: Molecules, Functions and Pathophysiological Aspects","volume":"80","author":"Rieu","year":"2000","journal-title":"Lab. Investig."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"677","DOI":"10.1083\/jcb.201006052","article-title":"Neutrophil Elastase and Myeloperoxidase Regulate the Formation of Neutrophil Extracellular Traps","volume":"191","author":"Papayannopoulos","year":"2010","journal-title":"J. Cell Biol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Chapman, E.A., Lyon, M., Simpson, D., Mason, D., Beynon, R.J., Moots, R.J., and Wright, H.L. (2019). Caught in a Trap? Proteomic Analysis of Neutrophil Extracellular Traps in Rheumatoid Arthritis and Systemic Lupus Erythematosus. Front. Immunol., 10.","DOI":"10.3389\/fimmu.2019.00423"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1752","DOI":"10.1182\/blood-2002-06-1734","article-title":"Neutrophil Elastase Enzymatically Antagonizes the in Vitro Action of G-CSF: Implications for the Regulation of Granulopoiesis","volume":"101","author":"Fujiwara","year":"2003","journal-title":"Blood"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"511","DOI":"10.1038\/nm.3547","article-title":"Aggregated Neutrophil Extracellular Traps Limit Inflammation by Degrading Cytokines and Chemokines","volume":"20","author":"Schauer","year":"2014","journal-title":"Nat. Med."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/11\/5099\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,6]],"date-time":"2026-06-06T04:42:44Z","timestamp":1780720964000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/11\/5099"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,4]]},"references-count":35,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["ijms27115099"],"URL":"https:\/\/doi.org\/10.3390\/ijms27115099","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,4]]}}}