Human PI3Kγ deficiency and its microbiota-dependent mouse model reveal immunodeficiency and tissue immunopathology

Abstract

Phosphatidylinositol 3-kinase-gamma (PI3Kγ) is highly expressed in leukocytes and is an attractive drug target for immune modulation. Different experimental systems have led to conflicting conclusions regarding inflammatory and anti-inflammatory functions of PI3Kγ. Here, we report a human patient with bi-allelic, loss-of-function mutations in PIK3CG resulting in absence of the p110γ catalytic subunit of PI3Kγ. She has a history of childhood-onset antibody defects, cytopenias, and T lymphocytic pneumonitis and colitis, with reduced peripheral blood memory B, memory CD8+ T, and regulatory T cells and increased CXCR3+ tissue-homing CD4 T cells. PI3Kγ-deficient macrophages and monocytes produce elevated inflammatory IL-12 and IL-23 in a GSK3α/β-dependent manner upon TLR stimulation. Pik3cg-deficient mice recapitulate major features of human disease after exposure to natural microbiota through co-housing with pet-store mice. Together, our results emphasize the physiological importance of PI3Kγ in restraining inflammation and promoting appropriate adaptive immune responses in both humans and mice.

Fig. 1

Patient with loss of PI3Kγ and T lymphocytic infiltration of lung and gut. a Chest computed tomography scan with diffuse pulmonary nodular and patchy infiltrates. b Hematoxylin and eosin staining of histological sections of a lung biopsy with lymphocytic infiltrates (top left) and regions of organizing pneumonia (top right) and gut biopsy with lymphocytic infiltrates (bottom left) and staining for CD3 (bottom right). c Pedigree with PIK3CG alleles inherited by patient A.1. d Chromatograms obtained by Sanger sequencing of PIK3CG genomic DNA from patient A.1. e Immunoblotting of p110γ, p101, and β-tubulin in T cell blasts from an unrelated healthy control, patient A.1, and parents

Fig. 2

T cell abnormalities and elevated inflammatory serum cytokines/chemokines. a Flow cytometry of T cells from an unrelated healthy control or patient A.1 with staining for CD4, CD8, CD45RA, and CCR7, as indicated. b CD69 expression on CD8 + T cells from peripheral blood of the indicated subjects after 24 h of stimulation in vitro with anti-CD3 and anti-CD28 antibodies. c Frequency of CD25^hiCD127^lo among CD4 + peripheral blood T cells in unrelated healthy controls (n = 7), patient A.1 (n = 4), mom (n = 1), and dad (n = 2). Data from four independent experiments is presented as mean ± SD. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. d Frequency of CCR4 + among CD4 + peripheral blood T cells in unrelated healthy controls (n = 3), patient A.1 (n = 3), mom (n = 2), and dad (n = 3). Data from four independent experiments are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. e Frequency of CXCR3 + among CD4 + peripheral blood T cells in unrelated healthy controls (n = 4), patient A.1 (n = 4), mom (n = 3), and dad (n = 3). Data from four independent experiments are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. f Concentrations of the indicated cytokine or chemokine in serum from independent blood draws of unrelated healthy controls (n = 4) and patient A.1 (n = 3). Data from three independent experiments are presented as mean ± SEM. Statistical analysis was performed using two-tailed unpaired T-test

Fig. 3

Macrophages/monocytes overproduce inflammatory IL-12 and IL-23 in a GSK3α/β-dependent manner upon TLR stimulation. a mRNA expression of IL12B normalized to GAPDH in monocyte-derived macrophages stimulated with IFNγ (20 ng/mL) and LPS (100 ng/mL) for 24 hr. Data are presented as mean ± SD, and are representative of two independent experiments in which one primary cell isolate for each group was run with three technical replicates. Statistical analysis was performed using a two-tailed unpaired T-test. b–c Immunoblotting for the indicated proteins in lysates from THP-1 monocytes stimulated with LPS (100 ng/mL) and treated with the PI3Kγ inhibitor IPI-549 (1 μM) or DMSO control. Equal protein loading was assessed using stain-free imaging (Bio-Rad). d Schematic of signaling pathway dysregulated in the absence of PI3Kγ activity (created with BioRender.com). e mRNA expression of IL12B normalized to GAPDH in THP-1 macrophages treated with PI3Kγi IPI-549 (1 μM) and GSK3i LY2090314 (20 nM) or DMSO control, as indicated, and stimulated with IFNγ (20 ng/mL) and LPS (100 ng/mL) for 16 h. Data are presented as mean ± SD, and are representative of three independent experiments, in which one primary cell isolate for each group was run with three technical replicates. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test. f mRNA expression of IL12B normalized to GAPDH and RPL37A in THP-1 macrophages expressing PIK3CG-targeted or control shRNA treated as in e and stimulated for 8 hr with IFNγ (20 ng/mL) and LPS (100 ng/mL). Data are presented as mean ± CI, and are representative of two independent experiments in which one primary cell isolate for each group was run with two technical replicates. Statistical analysis was performed using two-tailed unpaired T-test. g–h Cytokine protein concentration (relative to DMSO condition) in supernatant of healthy human donor monocyte-derived macrophages (g) or monocytes (h) treated with IPI-549 (500 nM) and/or GSK3 inhibitor LY2090314 (20 nM), as indicated, and stimulated with LPS (200 ng/mL) and IFNγ (5–35 ng/mL). Data are presented as mean ± SD and combined from 3–4 independent experiments in which one primary cell isolate for each group was run in duplicates. Statistical analysis was performed using two-tailed unpaired T-test

Fig. 4

Pik3cg-deficient mice recapitulate major features of human disease after exposure to natural microbiota through co-housing with pet-store mice. a mRNA expression of IL12b normalized to Hprt1 and Rpl13a in murine bone marrow-derived macrophages stimulated with LPS (100 ng/mL) and IFNγ (5 ng/mL) for 24 h. Data from two independent experiments (each with n = 2/group) are presented as mean ± SD. b Supernatant IL-12 concentration from murine bone marrow-derived macrophages stimulated with LPS (100 ng/mL) and IFNγ (5 or 20 ng/mL). Data are presented as mean ± SD and combined from three independent experiments. c Body weight of Pik3cg−/− and wild-type mice co-housed with pet-store mice. Data are representative of four independent experiments. d–f Frequency of Klrg1+ , CD62L+ , and CD44+ among CD8+ T cells from animals with or without exposure to pet-store mice at day 14. Data are presented as mean ± SD and are representative of four independent experiments (n = 3 for each non-co-housed group and n = 8 for each co-housed group). g Frequency of IL-10+ Foxp3+ cells among CD4 + T cells after PMA/ionomycin stimulation of splenocytes from WT (n = 2 for non-co-housed and n = 5 for co-housed) and Pik3cg−/− (n = 2 for non-co-housed and n = 6 for co-housed) after 56 days. Data are presented as mean ± SD. h Concentration of antibodies in serum from animals at 3–5 weeks after exposure to pet-store mice. Data are presented as mean ± SD, summarized from four independent experiments (n = 17–21 for each group). Statistical analysis was performed using unpaired T-test validated with Holm-Sidak method. i Total number of CD3 + T cells by immunohistochemistry per 200 μm section of small intestine from animals with or without exposure to pet-store mice at day 56. Data are presented as mean ± SD and representative of three independent experiments (n = 3 for non-co-housed and n = 7 for co-housed WT; n = 3 for non-co-housed and n = 6 for co-housed Pik3cg−/−). Statistical analysis was performed using two-tailed unpaired T-test in a–g and i