Supplementary Figure 3: PU.1 inhibits neutrophil activation.

(a) Mixed BM chimeras were generated by intravenous injection of 7.5x10⁵ BM cells from SJL.Bl6 mice (CD45.1⁺) mixed with 7.5x10⁵ BM cells from either PU.1^WT or PU.1^ΔNeu (both CD45.2⁺) mice into lethally irradiated (9 Gy) SJLxBl6 (CD45.1⁺CD45.2⁺) or SJL.Bl6 mice (CD45.1⁺). After 2 to 3 month of engraftment, chimera were intravenously infected with a sublethal (2x10⁵ cfu) dose of C. albicans and time point studies were performed. (b) Weight loss of C. albicans-infected (2x10⁵ cfu) mixed BM chimeras (n = 5) three days post infection. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.).(c) Kidney weights of C. albicans-infected (2x10⁵ cfu) mixed BM chimeras (n = 5) three days post infection. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). (d) Fungal burden of kidneys from mixed BM-reconstituted chimeras (n = 4) one and three days post C. albicans-infection (2x10⁵ cfu). Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). (e) Frequency of CD62L^lo expressing neutrophils in the peripheral blood (PB) of C. albicans-infected (2x10⁵ cfu) mixed BM chimeras (n = 5) three days post infection. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). *P = 0.0191. (f) Mean fluorescence intensity (MFI) of the degranulation marker CD63 on the surface of neutrophils (CD11b⁺Ly-6G⁺) from the BM of unstimulated PU.1^WT (n = 5) and PU.1^ΔNeu (n = 4) mice as determined by flow cytometry. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). ***P ≤ 0.0001. (g) qPCR analysis of the proinflammatory cytokine-encoding genes Il1b and Tnfa in BM derived macrophages (BMM) coincubated with in vitro derived PU.1^WT and PU.1^ΔNeu neutrophils (n = 3 each). Results are shown as fold difference relative to untreated BMM control (mean + s.e.m). Left to right: *P = 0.0443, *P = 0.0154. (h) Weight loss of C. albicans NRG1 mutant-infected (1x10⁶ cfu) PU.1^WT (n = 6), PU.1^ΔNeu (n = 4) and ΔCYBB (n = 4) mice one and three days post infection. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). Left to right: ***P = 0.0008, *P = 0.0494. (i) Kidney weights of C. albicans NRG1 mutant-infected (1x10⁶ cfu) PU.1^WT (n = 6), PU.1^ΔNeu (n = 4) and ΔCYBB (n = 4) mice one to three days post infection. Each symbol represents an individual mouse; small horizontal lines indicate the mean (± s.e.m.). *P = 0.0302. (j) Fungal burden of kidneys from C. albicans NRG1 mutant-infected (1x10⁶ cfu) PU.1^WT (n = 6), PU.1^ΔNeu (n = 4) and ΔCYBB (n = 2) mice two and three days post infection. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). Left to right: ***P ≤ 0.0001, **P = 0.0012. (k) Absolute number (by flow cytometry) of total macrophages (CD11b⁺F4/80⁺) in the kidneys of PU.1^WT (n = 6), PU.1^ΔNeu (n = 4) and ΔCYBB (n = 4) mice that were challenged with the NRG1 overexpression mutant of C. albicans (1x10⁶ cfu) for one to three days. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). Left to right: **P = 0.0085, **P = 0.0032 (l) Percentage of CD62L^lo expressing neutrophils in the BM of C. albicans NRG1 mutant-challenged (1x10⁶ cfu) PU.1^WT (n = 6), PU.1^ΔNeu (n = 4) and ΔCYBB (n = 4) mice one to three days post infection. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). Left to right: ***P ≤ 0.0001, ***P ≤ 0.0001. All P-values in this figure: unpaired t-test, two-tailed.