A proteomic approach to better understand the role of human neutrophil peptides in the NSCLC microenvironment

Abstract

Aim: Although treatment of lung cancer, one of the deadliest diseases worldwide, with immune checkpoint inhibitors (ICIs) has shown promising outcomes, these survival outcomes are only observed in a relatively small subset of lung cancer patients. In a previous study, we elucidated that the presence of human neutrophil peptide (HNP) 1, 2 and 3 in non-small cell lung cancerous (NSCLC) biopsies is associated with a clinical response towards treatment with PD-1/PD-L1 immune checkpoint inhibitors. Furthermore, HNP1 has shown in vitro an immune-activated function towards lung cancer cells, but the specific role of HNP1 in (lung) cancer is still unknown. The aim of this study was to provide a better understanding of HNP1 in an NSCLC microenvironment.

Methods: To gain better insights into the role of HNP1 on cancer growth and to unravel immune responses, in vitro (SILAC-labelled) A549/PBMC (from three healthy donors) cocultures were set up and treated/not treated with HNP1. After 5 days, both secretome and cellular analysis using mass spectrometry were performed on these cocultures. After protein identification in all different tested conditions, pathway analyses (MetaCore^TM) were performed to investigate the biological significance of HNP1 stimulation on the A549/PBMC cocultures. The proteomic outcomes were confirmed by multiplex ELISA for a proinflammatory cytokine panel (TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-12p70 and IL-18).

Results: A number of biological pathways and process networks were observed to be upregulated after treatment of the coculture with HNP1. HNP1 stimulation leads to an increase in pathways and proteins stimulating chemotaxis (including plasmin signaling, leucocyte recruitment, CCL2 and CXCL8 expression), proinflammatory cytokine secretion (including IL-1β, IL-6 and TNF-α), dendritic cell (DC) maturation, phagocytosis and antigen presentation, leading to a more efficient adaptive anti-tumoral immunity.

Conclusion: These results enhance our understanding of the role of HNP1 in the tumor microenvironment and suggest that HNP1 may be able to induce tumor necrosis by inducing prostimulatory immune responses.

Keywords

Human neutrophil peptide 1 (HNP1) / in vitro cocultures / proteomics / secretome analysis / SILAC

References

[1] World Health Organization. Cancer today. In: International Agency for Research on Cancer; 2022. Available from: https://gco.iarc.fr/today/home [Last accessed on 29 March 2022]

[2] Bai R,Xu D.Predictive biomarkers for cancer immunotherapy with immune checkpoint inhibitors.Biomark Res2020;8:34 PMCID:PMC7450548

[3] Robert C,Brady B.Nivolumab in previously untreated melanoma without BRAF mutation.N Engl J Med2015;372:320-30

[4] Daud AI,Robert C.Programmed death-ligand 1 expression and response to the anti-programmed death 1 antibody pembrolizumab in melanoma.J Clin Oncol2016;34:4102-9 PMCID:PMC5562434

[5] Moschetta M,Kasenda B.Dynamics of neutrophils-to-lymphocyte ratio predict outcomes of PD-1/PD-L1 blockade.Biomed Res Int2017;2017:1506824 PMCID:PMC5727693

[6] Berghmans E,Deben C.Mass spectrometry imaging reveals neutrophil defensins as additional biomarkers for anti-PD-(L)1 immunotherapy response in NSCLC patients.Cancers2020;12:863 PMCID:PMC7225984

[7] Berghmans E.Mass spectrometry (imaging) for detection and identification of cyclic AMPs: focus on human neutrophil peptides (HNPs). In: Enany S, Masso-silva J, Savitskaya A, editors. Insights on Antimicrobial Peptides. IntechOpen; 2021.

[8] Xu D.Defensins: a double-edged sword in host immunity.Front Immunol2020;11:764 PMCID:PMC7224315

[9] Zeng X,Chen B.Quantitative secretome analysis reveals the interactions between epithelia and tumor cells by in vitro modulating colon cancer microenvironment.J Proteomics2013;89:51-70

[10] Chen CY,Chi HC.Stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics study of a thyroid hormone-regulated secretome in human hepatoma cells.Mol Cell Proteomics2012;11:M111.011270 PMCID:PMC3322565

[11] Zougman A,Banks RE.Suspension trapping (STrap) sample preparation method for bottom-up proteomics analysis.Proteomics2014;14:1006-0

[12] Meier F,Koch S.Online parallel accumulation-serial fragmentation (PASEF) with a novel trapped ion mobility mass spectrometer.Mol Cell Proteomics2018;17:2534-45 PMCID:PMC6283298

[13] Cockx M,Gouwy M.The antimicrobial activity of peripheral blood neutrophils is altered in patients with primary ciliary dyskinesia.Int J Mol Sci2021;22:6172 PMCID:PMC8230338

[14] Ghose A,Chohan N.Applications of proteomics in ovarian cancer: dawn of a new era.Proteomes2022;10:16 PMCID:PMC9150001

[15] Choy E.Interleukin-6 as a multifunctional regulator: inflammation, immune response, and fibrosis.J Scleroderma Relat Disord2017;2:S1-5

[16] Neurath MF.IL-6 signaling in autoimmunity, chronic inflammation and inflammation-associated cancer.Cytokine Growth Factor Rev2011;22:83-9

[17] Roe K,Verma S.Triggering receptor expressed on myeloid cells-1 (TREM-1): a new player in antiviral immunity?.Front Microbiol2014;5:627 PMCID:PMC4244588

[18] Tammaro A,Gibot S,Florquin S.TREM-1 and its potential ligands in non-infectious diseases: from biology to clinical perspectives.Pharmacol Ther2017;177:81-95

[19] Bouchon A,Colonna M.Cutting edge: inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes.J Immunol2000;164:4991-5

[20] Adams DH.Chemokines: leucocyte recruitment and activation cytokines.Lancet1997;349:490-5

[21] Legler DF.New insights in chemokine signaling.F1000Res2018;7:95 PMCID:PMC5782407

[22] Stepanova VV.Urokinase as a multidomain protein and polyfunctional cell regulator.Biochemistry2002;67:109-18

[23] Cesarman-Maus G.Molecular mechanisms of fibrinolysis.Br J Haematol2005;129:307-21

[24] Kobayashi SD.Role of neutrophils in innate immunity: a systems biology-level approach.Syst Biol Med2009;1:309-33 PMCID:PMC3501127

[25] Carmo AA,Vago JP.Plasmin induces in vivo monocyte recruitment through protease-activated receptor-1-, MEK/ERK-, and CCR2-mediated signaling.J Immunol2014;193:3654-63

[26] Weber A,Kracht M.Interleukin-1 (IL-1) pathway.Sci Signal2010;3:cm1

[27] Xu Y,Huntington ND,Tarlinton DM.Lyn tyrosine kinase: accentuating the positive and the negative.Immunity2005;22:9-18

[28] Krebs DL,Sio A.Lyn-dependent signaling regulates the innate immune response by controlling dendritic cell activation of NK cells.J Immunol2012;188:5094-105

[29] Paul S.The molecular mechanism of natural killer cells function and its importance in cancer immunotherapy.Front Immunol2017;8:1124 PMCID:PMC5601256

[30] Blanco P,Pascual V.Dendritic cells and cytokines in human inflammatory and autoimmune diseases.Cytokine Growth Factor Rev2008;19:41-52 PMCID:PMC2413068

[31] Fu YL.Microbial phagocytic receptors and their potential involvement in cytokine induction in macrophages.Front Immunol2021;12:662063 PMCID:PMC8117099

[32] Amaral MM,Ceballos A.Histamine improves antigen uptake and cross-presentation by dendritic cells.J Immunol2007;179:3425-33

[33] Magna M.The role of HMGB1 in the pathogenesis of inflammatory and autoimmune diseases.Mol Med2014;20:138-46 PMCID:PMC3966993

[34] Yang H.Targeting HMGB1 in inflammation.Biochim Biophys Acta2010;1799:149-56 PMCID:PMC4533842