{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T14:26:39Z","timestamp":1790087199051,"version":"4.0.1"},"reference-count":54,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2025,3,4]],"date-time":"2025-03-04T00:00:00Z","timestamp":1741046400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Sepsis is a leading cause of death in hospitalized patients. The underlying pathophysiologic mechanisms of sepsis have not been fully elucidated thus far. The receptor of programmed cell death 1 (PD-1) and its ligand (PD-L1), in combination with the Toll-like receptors (TLRs), seem to contribute considerably in systematic responses during sepsis. Investigating the relationship between them and identifying potential target pathways is important in the future management of sepsis, especially in relation to acute lung injury. This study investigated the interactions between TLR-5, -6, and -9 and PD-1\/PD-L1 expression in a septic mouse model. Sixty C57BL\/6J mice were included and categorized in six study groups. Three sepsis (S) groups (24 h, 48 h, and 72 h) and three sham (Sh) groups (24 h, 48 h, and 72 h) were created. Cecal ligation and puncture (CLP) was utilized to simulate sepsis in the S groups. Hematological analysis and lung tissue histopathological analysis were performed after 24 h, 48 h, and 72 h. Significant decreases in S groups compared to Sh groups in WBC and lymphocyte counts at 24, 48, and 72 h were observed. Significant increases in S groups compared to Sh groups in RBC and monocyte counts, IL-6 and IL-10 levels, alveolar flooding, and alveolar collapse were demonstrated by histopathological analysis. This study suggested a strong correlation between TLR expression and PD-1\/PD-L1 up-regulation in lung tissue during sepsis. These molecules, also, seem to contribute to the histopathological changes in lung tissue during sepsis, leading to acute lung injury.<\/jats:p>","DOI":"10.3390\/ijms26052274","type":"journal-article","created":{"date-parts":[[2025,3,4]],"date-time":"2025-03-04T04:58:17Z","timestamp":1741064297000},"page":"2274","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["The Novel Role of the Expression of Toll-like Receptors TLR-5, TLR-6, and TLR-9 and Associated Up-Regulation of Programmed Cell Death 1 Receptor (PD-1) and Its Ligand (PD-L1) in Lung Sepsis"],"prefix":"10.3390","volume":"26","author":[{"given":"Georgios","family":"Sinos","sequence":"first","affiliation":[{"name":"First Department of Surgery, National and Kapodistrian University of Athens, \u201cLaikon\u201d General Hospital, 115 27 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7046-0112","authenticated-orcid":false,"given":"Dimitrios","family":"Schizas","sequence":"additional","affiliation":[{"name":"First Department of Surgery, National and Kapodistrian University of Athens, \u201cLaikon\u201d General Hospital, 115 27 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7637-8152","authenticated-orcid":false,"given":"Alkistis","family":"Kapelouzou","sequence":"additional","affiliation":[{"name":"Center for Clinical, Experimental Surgery and Translational Research, Biomedical Research Foundation of the Academy of Athens, 115 27 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9157-9860","authenticated-orcid":false,"given":"Maximos","family":"Frountzas","sequence":"additional","affiliation":[{"name":"First Propaedeutic Department of Surgery, National and Kapodistrian University of Athens, \u201cHippocration\u201d General Hospital, 115 27 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8083-3878","authenticated-orcid":false,"given":"Michalis","family":"Katsimpoulas","sequence":"additional","affiliation":[{"name":"Center for Clinical, Experimental Surgery and Translational Research, Biomedical Research Foundation of the Academy of Athens, 115 27 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2356-6694","authenticated-orcid":false,"given":"Konstantinos S.","family":"Mylonas","sequence":"additional","affiliation":[{"name":"Department of Cardiac Surgery, Onassis Cardiac Surgery Center, 176 74 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8354-4641","authenticated-orcid":false,"given":"Emmanouil I.","family":"Kapetanakis","sequence":"additional","affiliation":[{"name":"Department of Thoracic Surgery, National and Kapodistrian University of Athens, \u201cAttikon\u201d University Hospital, 124 62 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9055-8969","authenticated-orcid":false,"given":"Alexandros","family":"Papalampros","sequence":"additional","affiliation":[{"name":"First Department of Surgery, National and Kapodistrian University of Athens, \u201cLaikon\u201d General Hospital, 115 27 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2289-6242","authenticated-orcid":false,"given":"Theodore","family":"Liakakos","sequence":"additional","affiliation":[{"name":"First Department of Surgery, National and Kapodistrian University of Athens, \u201cLaikon\u201d General Hospital, 115 27 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andreas","family":"Alexandrou","sequence":"additional","affiliation":[{"name":"First Department of Surgery, National and Kapodistrian University of Athens, \u201cLaikon\u201d General Hospital, 115 27 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,3,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1016\/j.molmed.2014.01.002","article-title":"The new normal: Immunomodulatory agents against sepsis immune suppression","volume":"20","author":"Hutchins","year":"2014","journal-title":"Trends Mol. Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1303","DOI":"10.1097\/00003246-200107000-00002","article-title":"Epidemiology of severe sepsis in the United States: Analysis of incidence, outcome, and associated costs of care","volume":"29","author":"Angus","year":"2001","journal-title":"Crit. Care Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1244","DOI":"10.1097\/01.CCM.0000261890.41311.E9","article-title":"Rapid increase in hospitalization and mortality rates for severe sepsis in the United States: A trend analysis from 1993 to 2003","volume":"35","author":"Dombrovskiy","year":"2007","journal-title":"Crit. Care Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1546","DOI":"10.1056\/NEJMoa022139","article-title":"The epidemiology of sepsis in the United States from 1979 through 2000","volume":"348","author":"Martin","year":"2003","journal-title":"N. Engl. J. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"260","DOI":"10.1016\/S1473-3099(13)70001-X","article-title":"Immunosuppression in sepsis: A novel understanding of the disorder and a new therapeutic approach","volume":"13","author":"Hotchkiss","year":"2013","journal-title":"Lancet Infect. Dis."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1201","DOI":"10.1126\/science.aaa8334","article-title":"Getting sepsis therapy right","volume":"347","author":"Hotchkiss","year":"2015","journal-title":"Science"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"587","DOI":"10.1007\/s00011-016-0937-y","article-title":"Sepsis: In search of cure","volume":"65","author":"Lakshmikanth","year":"2016","journal-title":"Inflamm. Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"625","DOI":"10.1097\/CCM.0000000000000026","article-title":"Two decades of mortality trends among patients with severe sepsis: A comparative meta-analysis","volume":"42","author":"Stevenson","year":"2014","journal-title":"Crit. Care Med."},{"key":"ref_9","first-page":"1","article-title":"Current knowledge and future directions of TLR and NOD signaling in sepsis","volume":"2","author":"Foley","year":"2015","journal-title":"Mil. Med. Res."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"428","DOI":"10.1111\/hel.12292","article-title":"The importance of toll-like receptors in NF-\u03baB signaling pathway activation by Helicobacter pylori infection and the regulators of this response","volume":"21","author":"Hu","year":"2016","journal-title":"Helicobacter"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Savva, A., and Roger, T. (2013). Targeting toll-like receptors: Promising therapeutic strategies for the management of sepsis-associated pathology and infectious diseases. Front. Immunol., 4.","DOI":"10.3389\/fimmu.2013.00387"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"316","DOI":"10.4161\/viru.2.4.16142","article-title":"Role of TLR-\/NLR-signaling and the associated cytokines involved in recruitment of neutrophils in murine models of Staphylococcus aureus infection","volume":"2","author":"Sethi","year":"2011","journal-title":"Virulence"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Fullerton, J.N., Segre, E., De Maeyer, R.P., Maini, A.A., and Gilroy, D.W. (2016). Intravenous endotoxin challenge in healthy humans: An experimental platform to investigate and modulate systemic inflammation. J. Vis. Exp., e53913.","DOI":"10.3791\/53913"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1097\/CCM.0000000000001402","article-title":"Interleukin-1 receptor blockade is associated with reduced mortality in sepsis patients with features of the macrophage activation syndrome: Reanalysis of a prior phase III trial","volume":"44","author":"Shakoory","year":"2016","journal-title":"Crit. Care Med."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"637","DOI":"10.1016\/j.immuni.2011.05.006","article-title":"Toll-like receptors and their crosstalk with other innate receptors in infection and immunity","volume":"34","author":"Kawai","year":"2011","journal-title":"Immunity"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"13610","DOI":"10.1073\/pnas.0502174102","article-title":"Pathophysiological role of Toll-like receptor 5 engagement by bacterial flagellin in colonic inflammation","volume":"102","author":"Rhee","year":"2005","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_17","first-page":"2971","article-title":"MicroRNA-494-3p alleviates inflammatory response in sepsis by targeting TLR6","volume":"23","author":"Wang","year":"2019","journal-title":"Eur. Rev. Med. Pharmacol. Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e13019","DOI":"10.1111\/sji.13019","article-title":"Toll-like receptor-2\/7-mediated T cell activation: An innate potential to augment CD8+ T cell cytokine production","volume":"93","year":"2021","journal-title":"Scand. J. Immunol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"370","DOI":"10.1111\/j.1365-2567.2012.03587.x","article-title":"Regulation of B-cell responses by Toll-like receptors","volume":"136","author":"Browne","year":"2012","journal-title":"Immunology"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Patil, N.K., Guo, Y., Luan, L., and Sherwood, E.R. (2017). Targeting immune cell checkpoints during sepsis. Int. J. Mol. Sci., 18.","DOI":"10.3390\/ijms18112413"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"R3","DOI":"10.1186\/cc13176","article-title":"Targeting the programmed cell death 1: Programmed cell death ligand 1 pathway reverses T cell exhaustion in patients with sepsis","volume":"18","author":"Chang","year":"2014","journal-title":"Crit. Care"},{"key":"ref_22","first-page":"18998","article-title":"Roles of PD-1, Tim-3 and CTLA-4 in immunoregulation in regulatory T cells among patients with sepsis","volume":"8","author":"Gao","year":"2015","journal-title":"Int. J. Clin. Exp. Med."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1016\/j.coph.2015.05.011","article-title":"Pd-1\/pd-l1 inhibitors","volume":"23","author":"Sunshine","year":"2015","journal-title":"Cur. Opin. Pharmacol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1189\/jlb.0110037","article-title":"Delayed administration of anti-PD-1 antibody reverses immune dysfunction and improves survival during sepsis","volume":"88","author":"Brahmamdam","year":"2010","journal-title":"J. Leukoc. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1111\/j.1600-065X.2010.00923.x","article-title":"The PD-1 pathway in tolerance and autoimmunity","volume":"236","author":"Francisco","year":"2010","journal-title":"Immunol. Rev."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"4545","DOI":"10.4049\/jimmunol.1402550","article-title":"NF-\u03baB regulates PD-1 expression in macrophages","volume":"194","author":"Bally","year":"2015","journal-title":"J. Immunol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2218","DOI":"10.4049\/jimmunol.1203441","article-title":"TLR4 activation enhances the PD-L1\u2013mediated Tolerogenic capacity of colonic CD90+ stromal cells","volume":"193","author":"Beswick","year":"2014","journal-title":"J. Immunol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.canlet.2015.02.027","article-title":"TLR3 triggering regulates PD-L1 (CD274) expression in human neuroblastoma cells","volume":"361","author":"Boes","year":"2015","journal-title":"Cancer Lett."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1275","DOI":"10.1007\/s00268-003-7258-3","article-title":"Acute respiratory distress syndrome in the septic surgical patient","volume":"27","author":"Balibrea","year":"2003","journal-title":"World J. Surg."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/j.cell.2008.02.043","article-title":"Identification of oxidative stress and Toll-like receptor 4 signaling as a key pathway of acute lung injury","volume":"133","author":"Imai","year":"2008","journal-title":"Cell"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Bakopoulos, A., Kapelouzou, A., Tsilimigras, D.I., Katsimpoulas, M., Schizas, D., Aravanis, C., Balafas, E., Mavroidis, M., Pavlakis, K., and Machairas, A. (2017). Expression of Toll-like receptors (TLRs) in the lungs of an experimental sepsis mouse model. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0188050"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1038\/nprot.2008.214","article-title":"Immunodesign of experimental sepsis by cecal ligation and puncture","volume":"4","author":"Rittirsch","year":"2009","journal-title":"Nat. Protoc."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"143","DOI":"10.4161\/viru.26083","article-title":"Animal models of sepsis","volume":"5","author":"Fink","year":"2014","journal-title":"Virulence"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.autrev.2015.08.009","article-title":"The critical role of toll-like receptors\u2014From microbial recognition to autoimmunity: A comprehensive review","volume":"15","author":"Gerswhin","year":"2016","journal-title":"Autoimmun. Rev."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1016\/j.cyto.2014.06.003","article-title":"TLR-mediated inflammatory response to neonatal pathogens and co-infection in neonatal immune cells","volume":"69","author":"Sugitharini","year":"2014","journal-title":"Cytokine"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"487","DOI":"10.1038\/ni1457","article-title":"Toll-like receptor 9-dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE","volume":"8","author":"Tian","year":"2007","journal-title":"Nat. Immunol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1016\/j.cellimm.2015.03.009","article-title":"Inhibition of Toll-like receptor 9 attenuates sepsis-induced mortality through suppressing excessive inflammatory response","volume":"295","author":"Hu","year":"2015","journal-title":"Cell. Immunol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1028","DOI":"10.1097\/SLA.0000000000001248","article-title":"Systemic inflammatory response syndrome after major abdominal surgery predicted by early upregulation of TLR4 and TLR5","volume":"263","author":"Lahiri","year":"2016","journal-title":"Ann. Surg."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"6064","DOI":"10.1128\/AAC.01210-15","article-title":"A toll-like receptor 5 agonist improves the efficacy of antibiotics in treatment of primary and influenza virus-associated pneumococcal mouse infections","volume":"59","author":"Porte","year":"2015","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"R85","DOI":"10.1186\/cc12711","article-title":"Blockade of the negative co-stimulatory molecules PD-1 and CTLA-4 improves survival in primary and secondary fungal sepsis","volume":"17","author":"Chang","year":"2013","journal-title":"Crit. Care"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"R220","DOI":"10.1186\/cc9354","article-title":"PD-L1 blockade improves survival in experimental sepsis by inhibiting lymphocyte apoptosis and reversing monocyte dysfunction","volume":"14","author":"Zhang","year":"2010","journal-title":"Crit. Care"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"534","DOI":"10.1097\/SHK.0000000000000553","article-title":"Effect of PD-1: PD-L1 in invariant natural killer T cell emigration and chemotaxis following sepsis","volume":"45","author":"Young","year":"2016","journal-title":"Shock"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"158","DOI":"10.1097\/SLA.0b013e31823433ca","article-title":"Mechanisms of indirect acute lung injury: A novel role for the co-inhibitory receptor, programmed death-1 (PD-1)","volume":"255","author":"Monaghan","year":"2012","journal-title":"Ann. Surg."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Song, Z., Tong, C., Sun, Z., Shen, Y., Yao, C., Jiang, J., Yin, J., Gao, L., Song, Y., and Bai, C. (2010). Genetic variants in the TIRAP gene are associated with increased risk of sepsis-associated acute lung injury. BMC Med. Genet., 11.","DOI":"10.1186\/1471-2350-11-168"},{"key":"ref_45","first-page":"3376","article-title":"Effect of TLR4\/MyD88 signaling pathway on sepsis-associated acute respiratory distress syndrome in rats, via regulation of macrophage activation and inflammatory response","volume":"15","author":"Zhou","year":"2018","journal-title":"Exp. Ther. Med."},{"key":"ref_46","first-page":"639","article-title":"Analysis of Toll-like receptor 9 gene polymorphisms in sepsis","volume":"30","author":"Atalan","year":"2016","journal-title":"In Vivo"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1038\/nrneph.2017.165","article-title":"Advances in the understanding and treatment of sepsis-induced immunosuppression","volume":"14","author":"Venet","year":"2018","journal-title":"Nat. Rev. Nephrol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"e93397","DOI":"10.1172\/jci.insight.93397","article-title":"Combination immunotherapy with TLR agonists and checkpoint inhibitors suppresses head and neck cancer","volume":"2","author":"Yao","year":"2017","journal-title":"JCI Insight"},{"key":"ref_49","first-page":"27","article-title":"Relationship between serum TNF-\u03b1, IL-6, and IL-10 levels and disease severity, and changes in the cytokines after treatment in patients with bacterial community-acquired pneumonia","volume":"36","author":"Dao","year":"2023","journal-title":"Pneumon"},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Hubrecht, R.C., and Carter, E. (2019). The 3Rs and Humane Experimental Technique: Implementing Change. Animals, 9.","DOI":"10.3390\/ani9100754"},{"key":"ref_51","first-page":"1","article-title":"The Principles of Humane Experimental Technique","volume":"165","author":"Russell","year":"1960","journal-title":"Med. J. Aust."},{"key":"ref_52","doi-asserted-by":"crossref","unstructured":"Du Sert, N.P., Hurst, V., Ahluwalia, A., Alam, S., Avey, M.T., Baker, M., Browne, W.J., Clark, A., Cuthill, I.C., and Dirnagl, U. (2020). The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. PLoS Biol., 18.","DOI":"10.1371\/journal.pbio.3000410"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1016\/0003-2697(87)90021-2","article-title":"Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction","volume":"162","author":"Chomczynski","year":"1987","journal-title":"Anal. Biochem."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time quantitative PCR and the 2\u2212\u0394\u0394CT method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/5\/2274\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T16:46:47Z","timestamp":1760028407000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/5\/2274"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,3,4]]},"references-count":54,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2025,3]]}},"alternative-id":["ijms26052274"],"URL":"https:\/\/doi.org\/10.3390\/ijms26052274","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,3,4]]}}}