Fig 2: Genetic and clinical features in a patient with autosomal recessive IRF8 deficiency. a Pedigree of the Argentinian family. Generations are indicated by Roman numerals (I–II), and each individual is indicated by an Arabic numeral (1–3). The patient is represented by a closed black symbol, a black diagonal line (deceased), and an arrow. “E?” indicates individuals of unknown genotype. b Computed tomography (CT) scan of the thorax. c Pathology examinations of brochoalveolar lavage (BAL) and lung biopsy specimen. d Brain CT showing cerebral calcification (arrows). e Principal component analysis (PCA) showing the origins of the patient plotted on the main ethnic origins extracted from the 1000 Genomes database and our own WES database. f Analyses of WES data and identification of two heterozygous coding variants in IRF8. g Electropherogram of exons 2 and 3 showing the variants (c.55del and p.R111*) found in the patient (II.2) relative to healthy controls and his parents (I.2 and I.3). h Minor allele frequency (MAF) and combined annotation-dependent depletion (CADD) score of the heterozygous IRF8 variants found in the patient (red symbols) and of all variants found in patients with AR complete IRF8 deficiency (brown symbols). The dotted line corresponds to the mutation significance cutoff (MSC), with its 99% confidence interval. i Diagram of the human IRF8 protein, showing the DNA-binding domain (DBD) and IRF-associated domain 1 (IAD). Coding exons are indicated by Roman numerals (II to IX). The new reported variants are indicated in red, and the variants previously identified as responsible for AR complete IRF8 deficiency are shown in brown. All methionine residues are indicated in green

Fig 3: Deletion of microglial Syk inhibited the expressions of Irf8 and Pu.1 in the retina of STZ-induced diabetic mice. DR was induced in Cx3cr1+/+; Sykfl/fl (WT) and Cx3cr1CreERT2-EYFP/+; Sykfl/fl (cKO) mice for 8 weeks. The mRNA (A, B) and protein level (C through E) of Irf8 (A, C, and D) and Spi1 (the coding gene of Pu.1)/Pu.1 (B, C, and E) in the retina were determined by RT-PCR and Western blot, respectively. The mRNA level of Ly86 (F), Lyn (G), and Ncf4 (H) was determined by RT-qPCR. There were five mice for the control groups and six mice for the diabetic groups. n.s., not significant. *P < 0.05, **P < 0.01, ****P < 0.0001.

Fig 4: Cftr/CFTR deficiency induces an upregulation of IRF8—immunofluorescence confocal microscopy.A, trachea from 24-week-old wild-type (Wt) or Cftr−/− or CFMHHmice were removed, fixed in 4% PFA, embedded, sectioned, dewaxed, and immunostained with Cy3-coupled anti-IRF8 antibodies. All figures were made with the same settings of the confocal microscopy. Fluorescence intensity was quantified using Photoshop. IRF8 expression was normalized to actin fluorescence in epithelial cells. Shown are representative immunostainings and the mean ± SD of the quantitative analysis from six each independent samples; ∗∗∗p < 0.001, ANOVA and post hoc t-test. B, sections from explanted lungs from CF patients and from donor lungs were immunostained for IRF8 and analyzed by confocal microscopy. Shown are representative results from six CF lungs and three healthy controls. Fluorescence intensity was quantified using Photoshop and IRF8 expression was normalized to actin fluorescence in epithelial cells. The mean ± SD of the quantitative analysis is given; ∗∗∗p < 0.001, ANOVA and post hoc t-test. E, epithelial cell layer. Please note that the strong signal under the epithelial cell layer in healthy lungs is caused by the autofluorescence of elastin, which is reduced in fibrotic CF lungs.

Fig 5: Mechanisms of IRF8 upregulation in CF cells, which regulates the cellular defense against P. aeruginosa via sphingosine.A, trachea from wild-type, CF, and and CFxAsah-tg mice were isolated, fixed in 4% paraformaldehyde, embedded in paraplast, sections were performed and stained with Cy3-coupled anti-IRF8 antibodies. The fluorescence signals were quantified and normalized to the fluorescence signal obtained in the epithelial cells for staining with FITC-phalloidin. B, in addition, proteins were extracted and western blots for IRF8 expression were performed. Shown are representative confocal microscopy studies and blots and the mean ± SD of the quantitative analysis from five independent experiments; ∗∗∗p < 0.001, ANOVA and post hoc t-test. C, Caco-2 cells were transiently transfected with siRNA targeting IRF8 or control siRNA and treated with the CFTR Inhibitor-172 (CF-Inh, 2 μM) for 2 days or left untreated. Cells were then infected with P. aeruginosa at a multiplicity of infection (MOI) of 0.1 and the number of the bacteria after 2 h incubation was determined. Surface sphingosine was neutralized by addition of 1 μg/ml anti-sphingosine antibodies added 15 min prior to the infection. Sphingosine on the cell surface was increased by addition of sphingosine (1 μM) or purified neutral ceramidase (1 μg/ml) 15 min prior to infection. Shown are the mean ± SD of the bacterial numbers in the cultures measured as colony forming units (CFU) from six independent experiments; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ANOVA and post hoc t-test.