A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages
BackgroundA recombinant fusion protein combining the adjuvant and TLR5-ligand flagellin with the major birch pollen allergen Bet v 1 (rFlaA:Betv1) has been suggested to prevent the manifestation of birch allergy. Noteworthy, rFlaA:Betv1 induced both pro- and anti-inflammatory responses which were di...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-03-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2023.1136669/full |
| _version_ | 1827986543985295360 |
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| author | Yen-Ju Lin Annette Jamin Sonja Wolfheimer Anna Fiedler Ann-Christine Junker Alexandra Goretzki Stephan Scheurer Stefan Schülke |
| author_facet | Yen-Ju Lin Annette Jamin Sonja Wolfheimer Anna Fiedler Ann-Christine Junker Alexandra Goretzki Stephan Scheurer Stefan Schülke |
| author_sort | Yen-Ju Lin |
| collection | DOAJ |
| description | BackgroundA recombinant fusion protein combining the adjuvant and TLR5-ligand flagellin with the major birch pollen allergen Bet v 1 (rFlaA:Betv1) has been suggested to prevent the manifestation of birch allergy. Noteworthy, rFlaA:Betv1 induced both pro- and anti-inflammatory responses which were differentially regulated. However, the mechanism by which flagellin fusion proteins modulate allergen-specific immune responses, especially the mechanisms underlying IL-1β secretion and their contribution to the overall immune responses remains elusive.ObjectiveTo investigate the mechanisms underlying the production of IL-1β from rFlaA:Betv1 stimulated macrophages.MethodsMacrophages were derived from mouse peritoneal-, human buffy-coat-, and PMA-differentiated THP-1 (wild type or lacking either ASC, NLRP3, or NLRC4) cells. Macrophages were stimulated with non-modified rFlaA:Betv1, mutant variants lacking either the flagellin DC0 domain or a sequence motif formerly described to mediate TLR5-activation, and respective controls in the presence or absence of inhibitors interfering with MAPK- and NFκB-signaling. Cytokine secretion was analyzed by ELISA and intracellular signaling by Western Blot. To study the contribution of IL-1β to the overall immune responses, IL1R-deficient mouse peritoneal macrophages were used.ResultsrFlaA:Betv1 consistently activated all types of investigated macrophages, inducing higher IL-1β secretion compared with the equimolar mixture of both proteins. rFlaA:Betv1-induced activation of THP-1 macrophages was shown to be independent of either the TLR5-activating sequence motif or the flagellin DC0 domain but depended on both NLRP3- and NLRC4-inflammasomes. In addition, NFκB and SAP/JNK MAP kinases regulated rFlaA:Betv1-induced inflammasome activation and cytokine secretion by modulating pro-Caspase-1- and pro-IL-1β-expression in THP-1 macrophages. Finally, lack of IL-1β positive feedback via the IL1R strongly diminished the rFlaA:Betv1-induced secretion of IL-1β, IL-6, and TNF-α from peritoneal macrophages.ConclusionThe mechanisms contributing to rFlaA:Betv1-induced IL-1β secretion from macrophages were shown to be complex, involving both NLRC4- and NLRP3-inflammsomes, as well as NFκB- and SAP/JNK MAP kinase-signaling. Better understanding the mechanisms regulating the activation of immune cells by novel therapeutic candidates like the rFlaA:Betv1 fusion protein will allow us to further improve and develop new treatment strategies when using flagellin as an adjuvant. |
| first_indexed | 2024-04-09T23:31:55Z |
| format | Article |
| id | doaj.art-7e50bdd9809d4914aa1fc0154a4ea7f2 |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-04-09T23:31:55Z |
| publishDate | 2023-03-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-7e50bdd9809d4914aa1fc0154a4ea7f22023-03-21T04:23:54ZengFrontiers Media S.A.Frontiers in Immunology1664-32242023-03-011410.3389/fimmu.2023.11366691136669A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophagesYen-Ju LinAnnette JaminSonja WolfheimerAnna FiedlerAnn-Christine JunkerAlexandra GoretzkiStephan ScheurerStefan SchülkeBackgroundA recombinant fusion protein combining the adjuvant and TLR5-ligand flagellin with the major birch pollen allergen Bet v 1 (rFlaA:Betv1) has been suggested to prevent the manifestation of birch allergy. Noteworthy, rFlaA:Betv1 induced both pro- and anti-inflammatory responses which were differentially regulated. However, the mechanism by which flagellin fusion proteins modulate allergen-specific immune responses, especially the mechanisms underlying IL-1β secretion and their contribution to the overall immune responses remains elusive.ObjectiveTo investigate the mechanisms underlying the production of IL-1β from rFlaA:Betv1 stimulated macrophages.MethodsMacrophages were derived from mouse peritoneal-, human buffy-coat-, and PMA-differentiated THP-1 (wild type or lacking either ASC, NLRP3, or NLRC4) cells. Macrophages were stimulated with non-modified rFlaA:Betv1, mutant variants lacking either the flagellin DC0 domain or a sequence motif formerly described to mediate TLR5-activation, and respective controls in the presence or absence of inhibitors interfering with MAPK- and NFκB-signaling. Cytokine secretion was analyzed by ELISA and intracellular signaling by Western Blot. To study the contribution of IL-1β to the overall immune responses, IL1R-deficient mouse peritoneal macrophages were used.ResultsrFlaA:Betv1 consistently activated all types of investigated macrophages, inducing higher IL-1β secretion compared with the equimolar mixture of both proteins. rFlaA:Betv1-induced activation of THP-1 macrophages was shown to be independent of either the TLR5-activating sequence motif or the flagellin DC0 domain but depended on both NLRP3- and NLRC4-inflammasomes. In addition, NFκB and SAP/JNK MAP kinases regulated rFlaA:Betv1-induced inflammasome activation and cytokine secretion by modulating pro-Caspase-1- and pro-IL-1β-expression in THP-1 macrophages. Finally, lack of IL-1β positive feedback via the IL1R strongly diminished the rFlaA:Betv1-induced secretion of IL-1β, IL-6, and TNF-α from peritoneal macrophages.ConclusionThe mechanisms contributing to rFlaA:Betv1-induced IL-1β secretion from macrophages were shown to be complex, involving both NLRC4- and NLRP3-inflammsomes, as well as NFκB- and SAP/JNK MAP kinase-signaling. Better understanding the mechanisms regulating the activation of immune cells by novel therapeutic candidates like the rFlaA:Betv1 fusion protein will allow us to further improve and develop new treatment strategies when using flagellin as an adjuvant.https://www.frontiersin.org/articles/10.3389/fimmu.2023.1136669/fullflagellin fusion proteininflammasomeNLRP3NLRC4macrophage |
| spellingShingle | Yen-Ju Lin Annette Jamin Sonja Wolfheimer Anna Fiedler Ann-Christine Junker Alexandra Goretzki Stephan Scheurer Stefan Schülke A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages Frontiers in Immunology flagellin fusion protein inflammasome NLRP3 NLRC4 macrophage |
| title | A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages |
| title_full | A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages |
| title_fullStr | A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages |
| title_full_unstemmed | A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages |
| title_short | A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages |
| title_sort | flagellin conjugate protein induces dual nlrc4 and nlrp3 inflammasome activation which modulates inflammatory cytokine secretion from macrophages |
| topic | flagellin fusion protein inflammasome NLRP3 NLRC4 macrophage |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2023.1136669/full |
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