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Basophil-derived tumor necrosis factor can enhance survival in a sepsis model in mice

Nature Immunology volume 20, pages 129–140 (2019)Cite this article

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Abstract

Basophils are evolutionarily conserved in vertebrates, despite their small numbers and short life span, suggesting that they have beneficial roles in maintaining health. However, these roles are not fully defined. Here we demonstrate that basophil-deficient mice exhibit reduced bacterial clearance and increased morbidity and mortality in the cecal ligation and puncture (CLP) model of sepsis. Among the several proinflammatory mediators that we measured, tumor necrosis factor (TNF) was the only cytokine that was significantly reduced in basophil-deficient mice after CLP. In accordance with that observation, we found that mice with genetic ablation of Tnf in basophils exhibited reduced systemic concentrations of TNF during endotoxemia. Moreover, after CLP, mice whose basophils could not produce TNF, exhibited reduced neutrophil and macrophage TNF production and effector functions, reduced bacterial clearance, and increased mortality. Taken together, our results show that basophils can enhance the innate immune response to bacterial infection and help prevent sepsis.

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Fig. 1: Mouse basophils express TLRs and produce TNF and IL-6 after activation with TLR ligands.
Fig. 2: Basophil numbers increase in the peritoneal cavity and promote survival and bacterial clearance following CLP.
Fig. 3: Basophils contribute to the increased TNF in the peritoneal cavity following CLP.
Fig. 4: TNF production is markedly diminished in CD49b+ cells generated from Basoph8 × Tnffl/fl mice.
Fig. 5: Basophils contribute to increased intraperitoneal and blood TNF concentrations during LPS-induced endotoxemia.
Fig. 6: Basophil-derived TNF contributes to reduced morbidity and mortality, increased TNF concentrations, and enhanced bacterial clearance after CLP.
Fig. 7: Adoptive transfer of CD49b+ spleen cells from control littermates enhances bacterial clearance in Basoph8 × Tnffl/fl mice.
Fig. 8: Basophil-derived TNF enhances neutrophil ROS production and macrophage phagocytosis after CLP.

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The authors declare that the main data supporting the findings of this study are available within the article and its supplementary figures. Additional data are available from the corresponding authors upon request.

References

  1. Ehrlich, P. Ueber die specifischen Granulationen des Blutes. Archiv fuer Anatomie und Physiologie: Physiologische Abteilung 571–579, Archiv (1879).

  2. Canfield, P. J. Comparative cell morphology in the peripheral blood film from exotic and native animals. Aust. Vet. J. 76, 793–800 (1998).

    Article  CAS  Google Scholar 

  3. Eberle, J. U. & Voehringer, D. Role of basophils in protective immunity to parasitic infections. Semin. Immunopathol. 38, 605–613 (2016).

    Article  Google Scholar 

  4. Karasuyama, H. & Yamanishi, Y. Basophils have emerged as a key player in immunity. Curr. Opin. Immunol. 31, 1–7 (2014).

    Article  CAS  Google Scholar 

  5. Mukai, K., Karasuyama, H., Kabashima, K., Kubo, M. & Galli, S. J. Differences in the importance of mast cells, basophils, IgE, and IgG versus that of CD4+ T cells and ILC2 cells in primary and secondary immunity to Strongyloides venezuelensis. Infect. Immun. 85, e00053-17 (2017).

    Article  Google Scholar 

  6. Reitz, M. et al. Mucosal mast cells are indispensable for the timely termination of Strongyloides ratti infection. Mucosal Immunol 10, 481–492 (2017).

    Article  CAS  Google Scholar 

  7. Siracusa, M. C., Kim, B. S., Spergel, J. M. & Artis, D. Basophils and allergic inflammation. J. Allergy Clin. Immunol. 132, 789–801 (2013).

    Article  CAS  Google Scholar 

  8. Hamilton, R. G., MacGlashan, D. W. Jr & Saini, S. S. IgE antibody-specific activity in human allergic disease. Immunol. Res. 47, 273–284 (2010).

    Article  CAS  Google Scholar 

  9. Marone, G., Borriello, F., Varricchi, G., Genovese, A. & Granata, F. Basophils: historical reflections and perspectives. Chem. Immunol. Allergy 100, 172–192 (2014).

    Article  Google Scholar 

  10. Mukai, K. et al. Basophils play a critical role in the development of IgE-mediated chronic allergic inflammation independently of T cells and mast cells. Immunity 23, 191–202 (2005).

    Article  CAS  Google Scholar 

  11. Sato, E. et al. Chronic inflammation of the skin can be induced in IgE transgenic mice by means of a single challenge of multivalent antigen. J. Allergy Clin. Immunol. 111, 143–148 (2003).

    Article  CAS  Google Scholar 

  12. Ohnmacht, C. & Voehringer, D. Basophil effector function and homeostasis during helminth infection. Blood 113, 2816–2825 (2009).

    Article  CAS  Google Scholar 

  13. Leung, D. Y. et al. Presence of IgE antibodies to staphylococcal exotoxins on the skin of patients with atopic dermatitis. Evidence for a new group of allergens. J. Clin. Invest. 92, 1374–1380 (1993).

    Article  CAS  Google Scholar 

  14. Yousefi, S. et al. Basophils exhibit antibacterial activity through extracellular trap formation. Allergy 70, 1184–1188 (2015).

    Article  CAS  Google Scholar 

  15. Dejager, L., Pinheiro, I., Dejonckheere, E. & Libert, C. Cecal ligation and puncture: the gold standard model for polymicrobial sepsis? Trends Microbiol. 19, 198–208 (2011).

    Article  CAS  Google Scholar 

  16. Poli-de-Figueiredo, L. F., Garrido, A. G., Nakagawa, N. & Sannomiya, P. Experimental models of sepsis and their clinical relevance. Shock 30(Suppl 1), 53–59 (2008).

    Article  CAS  Google Scholar 

  17. Rittirsch, D., Hoesel, L. M. & Ward, P. A. The disconnect between animal models of sepsis and human sepsis. J. Leukoc. Biol. 81, 137–143 (2007).

    Article  CAS  Google Scholar 

  18. Yoshimoto, T. et al. Basophils contribute to TH2-IgE responses in vivo via IL-4 production and presentation of peptide-MHC class II complexes to CD4+ T cells. Nat. Immunol. 10, 706–712 (2009).

    Article  CAS  Google Scholar 

  19. Ugajin, T. et al. Basophils preferentially express mouse Mast Cell Protease 11 among the mast cell tryptase family in contrast to mast cells. J. Leukoc. Biol. 86, 1417–1425 (2009).

    Article  CAS  Google Scholar 

  20. Sullivan, B. M. et al. Genetic analysis of basophil function in vivo. Nat. Immunol. 12, 527–535 (2011).

    Article  CAS  Google Scholar 

  21. Voehringer, D., Liang, H. E. & Locksley, R. M. Homeostasis and effector function of lymphopenia-induced ‘memory-like’ T cells in constitutively T cell-depleted mice. J. Immunol. 180, 4742–4753 (2008).

    Article  CAS  Google Scholar 

  22. Gobbetti, T. et al. Nonredundant protective properties of FPR2/ALX in polymicrobial murine sepsis. Proc. Natl Acad. Sci. USA 111, 18685–18690 (2014).

    Article  CAS  Google Scholar 

  23. Hoesel, L. M. et al. Harmful and protective roles of neutrophils in sepsis. Shock 24, 40–47 (2005).

    Article  CAS  Google Scholar 

  24. Huang, X. et al. PD-1 expression by macrophages plays a pathologic role in altering microbial clearance and the innate inflammatory response to sepsis. Proc. Natl Acad. Sci. USA 106, 6303–6308 (2009).

    Article  CAS  Google Scholar 

  25. Flynn, J. L. et al. Tumor necrosis factor-α is required in the protective immune response against Mycobacterium tuberculosis in mice. Immunity 2, 561–572 (1995).

    Article  CAS  Google Scholar 

  26. Rothe, J. et al. Mice lacking the tumour necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature 364, 798–802 (1993).

    Article  CAS  Google Scholar 

  27. Tite, J. P., Dougan, G. & Chatfield, S. N. The involvement of tumor necrosis factor in immunity to Salmonella infection. J. Immunol. 147, 3161–3164 (1991).

    CAS  PubMed  Google Scholar 

  28. Maurer, M. et al. The c-kit ligand, stem cell factor, can enhance innate immunity through effects on mast cells. J. Exp. Med. 188, 2343–2348 (1998).

    Article  CAS  Google Scholar 

  29. Grivennikov, S. I. et al. Distinct and nonredundant in vivo functions of TNF produced by T cells and macrophages/neutrophils: protective and deleterious effects. Immunity 22, 93–104 (2005).

    CAS  PubMed  Google Scholar 

  30. Torrero, M. N., Larson, D., Hübner, M. P. & Mitre, E. CD200R surface expression as a marker of murine basophil activation. Clin. Exp. Allergy 39, 361–369 (2009).

    Article  CAS  Google Scholar 

  31. Remick, D. G., Newcomb, D. E., Bolgos, G. L. & Call, D. R. Comparison of the mortality and inflammatory response of two models of sepsis: lipopolysaccharide vs. cecal ligation and puncture. Shock 13, 110–116 (2000).

    Article  CAS  Google Scholar 

  32. Poltorak, A. et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282, 2085–2088 (1998).

    Article  CAS  Google Scholar 

  33. Tracey, K. J. et al. Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature 330, 662–664 (1987).

    Article  CAS  Google Scholar 

  34. Männel, D. N. & Echtenacher, B. TNF in the inflammatory response. Chem. Immunol. 74, 141–161 (2000).

    Article  Google Scholar 

  35. Singh, R. K. & Sodhi, A. Effect of TNF priming of murine peritoneal macrophages on their activation to a tumoricidal state. Immunol. Lett. 28, 127–133 (1991).

    Article  CAS  Google Scholar 

  36. Wright, H. L., Thomas, H. B., Moots, R. J. & Edwards, S. W. RNA-seq reveals activation of both common and cytokine-specific pathways following neutrophil priming. PLoS ONE 8, e58598 (2013).

    Article  CAS  Google Scholar 

  37. Mullen, P. G., Windsor, A. C., Walsh, C. J., Fowler, A. A.3rd & Sugerman, H. J. Tumor necrosis factor-α and interleukin-6 selectively regulate neutrophil function in vitro. J. Surg. Res. 58, 124–130 (1995).

    Article  CAS  Google Scholar 

  38. Barton, B. E. & Jackson, J. V. Protective role of interleukin 6 in the lipopolysaccharide-galactosamine septic shock model. Infect. Immun. 61, 1496–1499 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Barton, B. E., Shortall, J. & Jackson, J. V. Interleukins 6 and 11 protect mice from mortality in a staphylococcal enterotoxin-induced toxic shock model. Infect. Immun. 64, 714–718 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. van der Poll, T. et al. Interleukin-6 gene-deficient mice show impaired defense against pneumococcal pneumonia. J. Infect. Dis. 176, 439–444 (1997).

    Article  CAS  Google Scholar 

  41. Boomer, J. S. et al. Immunosuppression in patients who die of sepsis and multiple organ failure. JAMA 306, 2594–2605 (2011).

    Article  CAS  Google Scholar 

  42. Eskandari, M. K. et al. Anti-tumor necrosis factor antibody therapy fails to prevent lethality after cecal ligation and puncture or endotoxemia. J. Immunol. 148, 2724–2730 (1992).

    CAS  PubMed  Google Scholar 

  43. Echtenacher, B., Falk, W., Männel, D. N. & Krammer, P. H. Requirement of endogenous tumor necrosis factor/cachectin for recovery from experimental peritonitis. J. Immunol. 145, 3762–3766 (1990).

    CAS  PubMed  Google Scholar 

  44. Echtenacher, B., Weigl, K., Lehn, N. & Männel, D. N. Tumor necrosis factor-dependent adhesions as a major protective mechanism early in septic peritonitis in mice. Infect. Immun. 69, 3550–3555 (2001).

    Article  CAS  Google Scholar 

  45. Obata, K. et al. Basophils are essential initiators of a novel type of chronic allergic inflammation. Blood 110, 913–920 (2007).

    Article  CAS  Google Scholar 

  46. Korosec, P. et al. Basophils, high-affinity IgE receptors, and CCL2 in human anaphylaxis. J. Allergy Clin. Immunol. 140, 750–758.e15 (2017).

    Article  CAS  Google Scholar 

  47. Xiao, W. et al. A genomic storm in critically injured humans. J. Exp. Med. 208, 2581–2590 (2011).

    Article  CAS  Google Scholar 

  48. Sweeney, T. E., Shidham, A., Wong, H. R. & Khatri, P. A comprehensive time-course-based multicohort analysis of sepsis and sterile inflammation reveals a robust diagnostic gene set. Sci. Transl. Med. 7, 287ra71 (2015).

    Article  Google Scholar 

  49. Piliponsky, A. M. et al. Mast-cell-derived TNF can exacerbate mortality during severe bacterial infections in C57BL/6-Kit W-sh/W-sh mice. Am. J. Pathol. 176, 926–938 (2010).

    Article  CAS  Google Scholar 

  50. Wada, T. et al. Selective ablation of basophils in mice reveals their nonredundant role in acquired immunity against ticks. J. Clin. Invest. 120, 2867–2875 (2010).

    Article  CAS  Google Scholar 

  51. Weighardt, H. et al. Type I IFN modulates host defense and late hyperinflammation in septic peritonitis. J. Immunol. 177, 5623–5630 (2006).

    Article  CAS  Google Scholar 

  52. Kasten, K. R. et al. Interleukin-7 (IL-7) treatment accelerates neutrophil recruitment through γδ T-cell IL-17 production in a murine model of sepsis. Infect. Immun. 78, 4714–4722 (2010).

    Article  CAS  Google Scholar 

  53. Piliponsky, A. M. et al. The chymase mouse mast cell protease 4 degrades TNF, limits inflammation, and promotes survival in a model of sepsis. Am. J. Pathol. 181, 875–886 (2012).

    Article  CAS  Google Scholar 

  54. Liu, F. T. et al. Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization. J. Immunol. 124, 2728–2737 (1980).

    CAS  PubMed  Google Scholar 

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Acknowledgements

The authors thank R.M. Locksley for providing the Basoph8 mice and members of the Piliponsky and Galli laboratories for helpful discussions. Research in the Piliponsky laboratory was supported by National Institutes of Health (NIH) grant nos. R01 HL113351 and R01 HL141094, and an American Heart Association grant no. 12GRNT9680021. Research in the Galli laboratory was supported by NIH grant nos. R01 CA72074, R37 AI23990, R01 AI070813, and R01 AR067145, and by the Department of Pathology, Stanford University. Research in the Nedospasov laboratory was supported by a grant from the Russian Science Foundation (grant no. 14-50-00060).

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Authors and Affiliations

  1. Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA, USA

    Adrian M. Piliponsky, Nicholas J. Shubin, Asha K. Lahiri, Phuong Truong, Morgan Clauson, Kerri Niino & Avery L. Tsuha

  2. Department of Pediatrics, University of Washington School of Medicine, Seattle, WA, USA

    Adrian M. Piliponsky

  3. Department of Pathology, University of Washington School of Medicine, Seattle, WA, USA

    Adrian M. Piliponsky

  4. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia

    Sergei A. Nedospasov

  5. Tokyo Medical and Dental University Graduate School of Medical and Dental Sciences, Tokyo, Japan

    Hajime Karasuyama

  6. Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA

    Laurent L. Reber, Mindy Tsai, Kaori Mukai & Stephen J. Galli

  7. Sean N. Parker Center for Allergy and Asthma Research, Stanford University School of Medicine, Stanford, CA, USA

    Mindy Tsai, Kaori Mukai & Stephen J. Galli

  8. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA

    Stephen J. Galli

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Contributions

A.M.P., A.K.L., P.T., M.C., N.J.S., L.L.R., K.N., A.L.T., and K.M. performed the experiments. S.A.N. generated the Tnffl/fl mice. A.M.P. and S.J.G. designed the study. A.M.P., S.A.N., H.K., L.L.R., M.T., K.M., and S.J.G. analyzed the data. A.M.P. and S.J.G. wrote the manuscript. All of the authors contributed to the final editing of the manuscript.

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Correspondence to Adrian M. Piliponsky.

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Supplementary Figure 1 Gating strategy to identify basophils among total peritoneal cells after CLP.

(a-i) Representative flow cytometry plots and frequencies used to identify basophils amongst peritoneal cells obtained from mice 24 h after induction of moderate CLP (50% ligation, one puncture with a 22G needle). (a) Cell population considered for analysis using FSC and SSC. (b) Doublets were removed using FSC-A and FSC-W gating. (c) Dead cells were removed from the analysis using DAPI dead cell stain. (d, e) FcεRIα+ c-Kit- (d) and CD49b+ (e) cells were gated and defined as basophils. (f, g) The same gating strategy was used to identify basophils amongst peritoneal cells obtained from CLP-treated basophil-deficient mice, which shows a significant reduction in the basophil percentage. (h, i) Peritoneal mast cells from naïve mice were used to establish gates for FcεRIα+ and c-Kit- cells. Data are representative of those obtained in the 3 independent experiments performed.

Supplementary Figure 2 Peritoneal neutrophils and macrophages from naïve mice express low levels of Mcpt8 mRNA.

Mcpt8 mRNA expression relative to GAPDH in Gr-1+ CD11b+ neutrophils (Neu) and F4/80+ CD11b+ macrophages (Macs) that were sorted from the peritoneum of naïve Tnffl/fl mice and Basoph8 x Tnffl/fl mice (n = 3 mice/group). Mcpt8 gene expression was assessed by qPCR using FcεRIα+ CD49b+ c-Kit– basophils that were sorted from bone marrow of Tnffl/fl mice as positive controls. Mcpt8 mRNA expression levels in neutrophils and macrophages were normalized against Mcpt8 mRNA expression levels found in bone marrow basophils (Bas). Data are shown as means + SEM with squares representing values for individual experiments. The qPCR data are representative of n = 3 independent experiments performed, each of which gave similar results.

Supplementary Figure 3 TNF enhances bacterial clearance after CLP.

(a-b) Compared with the Tnf+/+ mice, Tnf-/- mice exhibited increased peritoneal cavity (a) and blood (b) colony forming units (CFU) at 24 h after CLP. Data in a and b are shown as means with circles representing values from individual mice (n = 14 and n = 11 for Tnf+/+ mice in a and b, respectively, and n = 7 and n = 6 for Tnf-/- mice in a and b, respectively). P values by Mann-Whitney test.

Supplementary Figure 4 Leukocyte populations in Tnffl/fl mice and Basoph8 × Tnffl/fl mice.

(a-d) Percentages of leukocytes (a) Basophil percentages (FcεRIα+ CD49b+ c-Kit–) in the live-cell population isolated from spleens (Tnffl/fl, n = 7; Basoph8 x Tnffl/fl, n = 9), bone marrow (Tnffl/fl, n = 4; Basoph8 x Tnffl/fl, n = 9), or blood (Tnffl/fl, n = 16; Basoph8 x Tnffl/fl, n = 8), as analyzed by flow cytometry. (b) Neutrophil percentages (Gr-1+ CD11b+) in the live-cell population isolated from spleen (Tnffl/fl, n = 6; Basoph8 x Tnffl/fl, n = 6), bone marrow (Tnffl/fl, n = 6; Basoph8 x Tnffl/fl, n = 7), or peritoneal fluids (Tnffl/fl, n = 7; Basoph8 x Tnffl/fl, n = 10), as analyzed by flow cytometry. (c) Macrophage percentages (F4/80+ CD11b+) in the live-cell population isolated from spleens (Tnffl/fl, n = 7; Basoph8 x Tnffl/fl, n = 10) or peritoneal fluids (Tnffl/fl, n = 3; Basoph8 x Tnffl/fl, n = 10), as analyzed by flow cytometry. Data in a-d are depicted as mean + SEM with circles representing values for individual mice. (d) Mast cell percentages (FcεRIα+ c-Kit+) in the live-cell population isolated from peritoneal fluids (Tnffl/fl, n = 4; Basoph8 x Tnffl/fl, n = 6), as analyzed by flow cytometry. (e-g) Absolute numbers of leukocytes. (e) Blood monocyte (Tnffl/fl, n = 13; Basoph8 x Tnffl/fl, n = 9) and eosinophil (Tnffl/fl, n = 9; Basoph8 x Tnffl/fl, n = 10) numbers. (f) Blood lymphocyte (Tnffl/fl, n = 20; Basoph8 x Tnffl/fl, n = 10) and neutrophil (Tnffl/fl, n = 21; Basoph8 x Tnffl/fl, n = 9) numbers. (g) Blood lymphocyte (Tnffl/fl, n = 9; Basoph8 x Tnffl/fl, n = 9) and neutrophil (Tnffl/fl, n = 10; Basoph8 x Tnffl/fl, n = 8) numbers at 24 h after moderate CLP. Data in e-g are depicted as mean + SEM with circles representing values for individual mice. P values by Mann-Whitney test.

Supplementary Figure 5 Basophil-derived TNF contributes to increased TNF production by peritoneal macrophages during LPS-induced endotoxemia.

Representative flow cytometry plots for TNF positive cells in the macrophage populations obtained from the peritoneal cavity of Tnffl/fl and Basoph8 x Tnffl/fl mice (n = 4/group) 1 h after i.p. injection of LPS (10 μg). Cells were immunostained for TNF in addition to surface F4/80 and CD11b to identify macrophages. Data were obtained from two independent experiments performed, each of which gave similar results.

Supplementary Figure 6 Disruption of Mcpt8 expression levels in Basoph8 mice does not impact the outcome of CLP of moderate severity.

(a, b) Mcpt8 expression levels are significantly reduced or undetectable in basophils from mice with one or two Basoph8 gene copies, respectively. Mcpt8 mRNA (a) and protein (b) expression levels in basophils that were sorted from bone marrow cells harvested from naïve mice (a) or from FcεRIα+ CD49b+ c-Kit– bone marrow-derived cells cultured in the presence of IL-3 (b). The qPCR data in a and western blot analysis in b are representative of 2 independent experiments performed, each of which gave similar results. The Western blot analysis in b was cropped from the original blot shown in Supplementary Fig. 7. (c, d) Basophil-derived TNF-deficient mice with one Basoph8 gene copy (mice with a significant reduction in Mcpt8 expression levels) exhibit similar mortality and morbidity as basophil-derived TNF-deficient mice with two Basoph8 gene copies (mice with a total Mcpt8 deficiency). Survival after moderate CLP in littermate control mice (Tnffl/fl = Basoph8 -/- x Tnffl/fl, n = 15), Basoph8+/- x Tnffl/fl (n = 7) and Basoph8+/+ x Tnffl/fl (n = 7). *P < 0.05 and **P < 0.01 vs. Tnffl/fl mice. P values by Logrank (Mantel-Cox test). (d) Drop in body temperature in Tnffl/fl mice (n = 7), Basoph8± x Tnffl/fl mice (n = 13) and Basoph8+/+ x Tnffl/fl mice (n = 17) at 24 h after CLP. P values by Mann-Whitney test. Data in c were pooled from the n = 3 independent experiments performed, each of which gave similar results. Data in d are shown as means + SEM with circles representing values for individual mice and were pooled from n = 3 independent experiments performed, each of which gave similar results. (e-j) Mice with one Basoph8 gene copy (that is, Basoph8± x Tnf +/+ mice with no Mcpt8 protein expression, but which express basophil-derived TNF) exhibit morbidity rates similar to their littermate controls [Tnf +/+ mice]). (e-j) Drop in body temperature (e), colony forming units (CFU) in peritoneal cavity (f) and blood (g), amount of intraperitoneal TNF (h), and numbers of neutrophils (i) and macrophages (j) in the peritoneal live-cell population analyzed by flow cytometry (Gr-1+ CD11b+ cells for neutrophils and F4/80+ CD11b+ for macrophages) in Basoph8-/- x Tnf+/+ mice and Basoph8± x Tnf+/+ mice at 24 h after CLP. Data are shown as means + SEM with circles representing values for individual mice (n = 23 in e, n = 19 in f, n = 16 in g, n = 14 in h and n = 7 in i and j for Basoph8-/- x Tnf+/+ mice; and n = 9 in e, n = 14 in f, n = 13 in g, n = 7 in h and n = 5 in i and j for Basoph8± x Tnf+/+ mice. Data were pooled from n = 3 independent experiments performed, each of which gave similar results.

Supplementary Figure 7 Full scans of blots used in Supplementary Fig. 6.

Blots show Mcpt8 (a) and GAPDH (b) expression levels in basophils from mice with one or two Basoph8 gene copies, respectively. Mcpt8 and GAPDH protein expression levels in increasing number of basophils that were sorted from FcεRIα+ CD49b+ c-Kit– bone marrow-derived cells cultured in the presence of IL-3. Blots are representative of 2 independent experiments performed, each of which gave similar results.

Supplementary Figure 8 Neutrophils, macrophages and mast cells from Basoph8 × Tnffl/fl mice express negligible YFP and Mcpt8 mRNA after CLP.

(a) Representative flow cytometry plots and cell frequencies in the peritoneal neutrophil, macrophage, mast cell and basophil populations obtained from Tnffl/fl mice (n = 3) and Basoph8± x Tnffl/fl mice (n = 3) at 24 h after moderate CLP. Cells were immunostained for surface Gr-1 and CD11b for peritoneal neutrophils; with F4/80 and CD11b for macrophages; and with FcεRIα, c-Kit and CD49b for mast cells (FcεRIα+ c-Kit+) and basophils (FcεRIα+ CD49b+). (b) The specificity of Mcpt8 gene expression as assessed by single cell PCR detection in peritoneal neutrophils (Neu), macrophages (Macs), mast cells (MCs), and BM basophils (BMBas) obtained from Tnffl/fl mice and Basoph8± x Tnffl/fl mice at 24 h after moderate CLP. The numbers above the bar graphs indicate the number of cells analyzed. Results are expressed as % of cells expressing transcripts for the PCR products analyzed/cell type; results were pooled from n = 3 sorting experiments.

Supplementary Figure 9 TNF ablation in basophils does not affect ROS production and phagocytosis in neutrophils and macrophages ex vivo.

Neutrophil ROS production (a) and macrophage phagocytosis (b). Bone marrow neutrophils were loaded for 30 min at 37 °C with 1 μM of CM-H2DCFDA, which is a general oxidative stress indicator, and then activated with 100 ng/ml of PMA. Peritoneal macrophages were incubated with opsonized FITC-labeled E. coli (K-12 strain) bioparticles for 10 min at 37 °C. Cells were stained for neutrophil and macrophage-specific surface markers and flow cytometry analysis was performed immediately thereafter. Data in a and b are shown as means + SEM with squares showing values for individual mice (n = 4 for Tnffl/fl mice in a and b, and n = 3 and n = 4 for Basoph8 x Tnffl/fl mice in a and b, respectively). Data were pooled from n = 2 independent experiments.

Supplementary Figure 10 Intracellular staining for IL-6 in myeloid cells during CLP.

Flow cytometry plots and frequencies (a) and mean fluorescence intensities (b) for IL-6 positive cells in the peritoneal neutrophil and macrophage populations obtained from Tnffl/fl mice and Basoph8 x Tnffl/fl mice (n = 6 mice/group) at 24 h after CLP and then incubated ex vivo for 4 h in the presence of GolgiPlug. Cells were immunostained for IL-6 in addition to surface Gr-1 and CD11b to identify peritoneal neutrophils or surface F4/80 and CD11b to identify macrophages. Data in a are from one representative experiment of the n = 2 independent experiments performed, each of which gave similar results. The data in b were pooled from n = 2 independent experiments that were performed. Data are shown as means + SEM with squares showing values for cells obtained from individual mice (n = 6/group). P values by Mann-Whitney test.

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Supplementary Figs. 1–10 and Supplementary Table 1. Association between FCER1A expression in whole blood and clinical attributes in trauma patients at 7 d after severe injury. The dataset was obtained from 167 severely injured patients and compared with 35 healthy individuals (ages 16–55) (http://web.mgh.harvard.edu/TRT/).

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Piliponsky, A.M., Shubin, N.J., Lahiri, A.K. et al. Basophil-derived tumor necrosis factor can enhance survival in a sepsis model in mice. Nat Immunol 20, 129–140 (2019). https://doi.org/10.1038/s41590-018-0288-7

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