Fig. 3: SINGR1 promotes fungal colonization, cytokine production and dysregulation of neutrophil populations.

a–j WT mice pre-treated with either control antibody or an anti-SIGNR1 blocking antibody and infected intravenously with 5 × 10⁵ WT C. albicans: a Survival curves (n = 5 mice per group and three independent experiments). b Colony forming units (CFUs) 72 h post-infection with symptomatic mice (red) and asymptomatic mice (blue) (n = 8 mice per group). c Selected cytokines in plasma at 72h  post-infection detected by Luminex-based multiplex immunoassay (naïve n = 3 and infected n = 5 mice per group). d Representative RNA scope confocal microscopy of G-CSF and IL-6 expression in the spleens 72h  post-infection co-stained with anti-CD169 antibodies. Scale bars: 20 μm. Representative of n = 5 mice per group. e Average number of RNA scope particles / CD169 positive nuclei (DAPI) in images from 4 mice per group. Statistical analysis by unpaired Mann–Whitney t-test. f, g Immunofluorescence confocal micrographs of spleens from infected mice treated with control or anti-SIGNR1 antibody at 3 days post-infection, stained for f C. albicans, Ly6G, CD169, or g C. albicans, SIGNR1, MPO and DAPI. Arrows point to the MZ. Scale bars: 50 μm. Representative of n = 3 mice per group, and 3 independent experiments. h Flow cytometry of blood neutrophils at 3 days post-infection, stained for intracellular MPO and Ly6G expression. Ly6G^high neutrophils (top gate) and Ly6G^low neutrophils (bottom gate). The right panel depicts Ly6G^low/Ly6G^high neutrophil ratios per animal. Gated on Live CD3⁻ CD19⁻ CD11b⁺ cells. Representative of 3 independent experiments with three mice per group. i Flow cytometry of blood neutrophils at 1, 3 and 5 days post-infection, stained for intracellular MPO and surface Ly6G (n = 3 mice per group, and 3 independent experiments). j Total neutrophil numbers in blood, spleen and kidneys of infected WT mice against their body temperature (n = 8 mice). Panel: Live/Dead blue, CD3/CD19 PerCP-Cy5.5, CD11b-PECy7, Ly6G-APC. k ROS production in the absence or presence of PMA by splenic neutrophils isolated 72 h post-infection. Representative superoxide production curves from one mouse per group detected by luminol bioluminescence (left) and cumulative ROS peak levels for neutrophils from three animals per group (right). Statistical analysis by unpaired two-sided Mann–Whitney t-test for single comparison and two-tailed Log-rank (Mantel–Cox) test for survival analysis (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).