Human inherited CCR2 deficiency underlies progressive polycystic lung disease

Abstract

We describe a human lung disease caused by autosomal recessive, complete deficiency of the monocyte chemokine receptor C-C motif chemokine receptor 2 (CCR2). Nine children from five independent kindreds have pulmonary alveolar proteinosis (PAP), progressive polycystic lung disease, and recurrent infections, including bacillus Calmette Guérin (BCG) disease. The CCR2 variants are homozygous in six patients and compound heterozygous in three, and all are loss-of-expression and loss-of-function. They abolish CCR2-agonist chemokine C-C motif ligand 2 (CCL-2)-stimulated Ca²⁺ signaling in and migration of monocytic cells. All patients have high blood CCL-2 levels, providing a diagnostic test for screening children with unexplained lung or mycobacterial disease. Blood myeloid and lymphoid subsets and interferon (IFN)-γ- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated immunity are unaffected. CCR2-deficient monocytes and alveolar macrophage-like cells have normal gene expression profiles and functions. By contrast, alveolar macrophage counts are about half. Human complete CCR2 deficiency is a genetic etiology of PAP, polycystic lung disease, and recurrent infections caused by impaired CCL2-dependent monocyte migration to the lungs and infected tissues.

Keywords: PAP; chemotaxis; cystic lung disease; macrophage; monocyte; recurrent infection.

Figure 1.. Growth curve, pulmonary function, chest radiography, and lung histology in patients with CCR2 deficiency

(A) Growth curve for healthy children and P7. (B) Serial pulmonary function test results for P7. (C–E) Posterior-anterior chest X-rays. (F) Serial transverse chest CT scan images taken at the level of the carina. (G) Coronal CT scan images from the anterior chest (left), mid chest (center), and posterior chest (right). (H–K) Photomicrographs of surgical lung biopsy tissue obtained from P1 at the age of 8 years. (H) Cysts (asterisks) containing eosinophilic material, cholesterol clefts (arrows), and adjacent extensive pulmonary lymphocytosis. H&E. (I) Cysts (asterisks) lined with cuboidal epithelial cells expressing surfactant protein C (arrow). (J) Left: microscopic cysts (asterisks) located adjacent to the pleural surface (double arrows). H&E. Right: subpleural microscopic cysts (asterisks) immediately adjacent to terminal bronchioles with extensive peribronchiolar lymphocytosis (arrows) with distortion of the airway lumen. H&E. (K) Subpleural cysts (asterisks) juxtaposed to a terminal bronchiole with architectural disruption due to extensive follicular lymphocytosis (inset, arrows). H&E. (L–S) Photomicrographs of surgical lung biopsy specimen obtained from P7 at the age of 3 years. (L) Alveolae filled with eosinophilic material (asterisks) and cholesterol clefts (arrows). H&E. (M) Surfactant protein B immunostaining. (N) Cysts located immediately adjacent to the pleura and lined with cuboidal epithelium (arrows), and cysts immediately adjacent to terminal bronchioles distorted by extensive peribronchiolar follicular lymphocytic inflammation. H&E. (O) Cyst lined with cuboidal epithelial cells, containing eosinophilic material and numerous cholesterol clefts. Inset: alveolar macrophages of normal size (17.7 ± 4.2 μm, n = 29 cells). H&E. (P) Cysts (asterisks) filled with eosinophilic material and cholesterol clefts, located immediately adjacent to bronchioles displaying extensive follicular bronchiolitis. H&E. (Q) Lymphoid follicles composed of B lymphocytes. B220 immunostain. (R) Cysts lined with cuboidal epithelial cells (double arrows) immediately adjacent to terminal bronchioles obliterated (arrows) or externally compressed (asterisk) by peribronchiolar follicular bronchiolitis—constrictive cellular bronchiolitis. H&E. (S) Bronchiole with prominent subepithelial and peri-airway fibrosis. H&E. See also Figures S1 and S2.

Figure 2.. Identification, location, and evaluation of the CCR2 variants

(A) Pedigree of five unrelated kindreds. The arrow indicates the index cases. Other symbols: unknown genotype, “E?”; spontaneous abortion, triangle; consanguinity, double horizontal lines. (B) Principal-component analysis (PCA) of the WES data for the patients and samples from the 1000 Genomes database. (C) Schematic representation of the CCR2 protein. Red, patient variants; green, public database variants; blue, ligand-binding sites; purple, G protein-binding sites; black, disulfide bonds. (D) Cross-sectional schematic view of the CCR2 protein. (E) CADD MAF plot for all bi-allelic CCR2 variants found in the patients, our in-house cohort, or in public databases. (F) Comparison of CCR2 protein sequences from diverse species. (G) Consensus negative selection (CoNeS) of CCR2. See also Figure S3.

Figure 3.. In vitro characterization of the CCR2 alleles by overexpression

(A) Flow cytometry with surface staining for CCR2 on THP-1 CCR2^KO cells transduced with an empty vector (EV) or a vector encoding the wild-type (WT) or one of the various CCR2A and CCR2B variants. The results shown are representative of three independent experiments. (B and C) Intracellular calcium (Ca²⁺) mobilization in transduced THP-1 CCR2^KO cells after stimulation with (B) CCL-2 or (C) ΔCCL-13 (n = 2–7 ± SD). (D) Migration of transduced THP-1 CCR2^KO toward CCL-2 or medium alone (n = 2–5 ± SD).

Figure 4.. Loss of CCR2 expression and signaling in the patients’ monocytes

(A and B) Representative plots (A) and calculated frequencies (B) of CCR2⁺ expression on monocyte subsets for local controls (LCs; n = 4), travel controls (TCs; n = 4), and P1–P5. (C) MFI of CCR2 staining on PBMCs from a healthy control and P6–P8 and their parents (n = 2). (D) Ca²⁺ influx assay in CD14⁺ monocytes from controls, P1, and P2 after stimulation with CCL-2 or ionomycin. (E) Fold-migration of CD14⁺ monocytes from controls (LC, n = 6; TC, n = 3) and P1–P5 toward CCL-2 or CXCL-12. (F) Distribution of trajectory angles of CD14⁺ monocytes from controls (n = 2) and P2 migrating along a CCL-2 gradient in a confined two-dimensional microenvironment. Black circles: expected angle distribution for random motion. (G) CCL-2-stimulated ERK phosphorylation in monocytes from a healthy control and P6–P8 and their parents (n = 2). (H and I) CCL-2 levels in (H) plasma or serum from healthy controls (n = 10) and P1–P8 and their parents (n = 8) or (I) in BAL relative to a patient with autoimmune PAP (Aab⁺). (J) CCl-2 levels in the supernatants of CD14⁺ monocytes from controls, P1, and P2 without stimulation (NS) or with stimulation with DMSO or a CCR2 antagonist for 24 h. (K and L) Plasma and BAL cytokine levels for CCL-8 (K) in the plasma of P1–P8 and controls (n = 5) and BAL from P1, P2, and controls (n = 12) and CCL-13 (L) in the plasma of P1–P8 and controls (n = 5) and BAL from P1, P2, and controls (n = 12). For (C), (F), (I), and (J), the data shown are the means of technical replicates ± SD; for (B), (E), (H), (K), and (L) the data shown are the means ± SD. Significance was assessed using Mann-Whitney U tests (B, E, K, and L) and Kruskal-Wallis test (H); ns, not significant; *p ≤ 0.05 and ***p ≤ 0.001. See also Figures S4 and S5.

Figure 5.. Hematological and immunological profiles of the patients with CCR2 deficiency

(A and B) Absolute numbers of peripheral total (A) leukocyte subsets and (B) red blood cells, hemoglobin, and hematocrit. Gray lines represent the upper and lower limits of the normal range for each age group. (C–I) Frequency of myeloid and lymphoid subsets among PBMCs in adult controls (n = 21), age-matched controls (n = 13), and P1–P5, as determined by CyTOF. (J) Frequency of DC subsets among PBMCs in adult controls (n = 30), P1, and P2, measured by conventional flow cytometry. (K) Bone marrow aspirate smears for P1 and P2. For (C)–(J), the data shown are the mean ± SD. See also Figure S6.

Figure 6.. Alveolar macrophage quantification and characterization in CCR2-deficient patients

(A) Cell-type composition of BAL samples from healthy reference controls and CCR2 patients taken at infection-free time points (see Table S2). (B) Immunostaining on lung biopsy tissue from P1 and P7, in comparison with a healthy control. (C) Flow cytometry analysis for the indicated cell populations on cryo-preserved BAL samples from two healthy controls, P1, and P2. (D–G) Single-cell RNA sequencing on cryo-preserved BAL samples from two healthy controls, P1, and P2 with (D) clustering analysis, (E) cell-type distribution, (F) frequency of alveolar macrophages (AMs), and (G) gene expression of selected alveolar macrophage markers within the myeloid clusters of controls and patients. For (A), (C), (E), and (F), the data shown are the mean ± SD. See also Figure S7.

Figure 7.. Generation of alveolar macrophage-like cells in the absence of CCR2 signaling

(A) Schematic representation of the alveolar macrophage-like cell (AML) differentiation protocol. (B and C) (B) quantitative reverse-transcription PCR (RT-qPCR) and (C) flow cytometry staining on AMLs from healthy controls (n = 6) differentiated in the presence or absence (NT) of DMSO or a CCR2 antagonist. (D) Phagocytosis of pHrodo S. aureus bioparticles by AMLs from healthy controls (n = 4) differentiated as in (B). (E) Superoxide production in response to phorbol 12-myristate 13-acetate (PMA) by AMLs from healthy controls (n = 4) differentiated as in (B). (F) TNF secretion by AMLs from healthy controls (n = 4) differentiated as in (B) in response to LPS. (G) STAT5 phosphorylation in response to GM-CSF by AMLs from healthy controls (n = 4) as in (B). (H) RT-qPCR on AMLs from healthy controls (n = 7) and five CCR2-deficient patients (P1, P2, and P6–P8). (I) Flow cytometry surface staining on AMLs from healthy controls (n = 3) and P6–P8. (J) Phagocytosis of pHrodo S. aureus bioparticles by AMLs from healthy controls (n = 3) and P6–P8. (K) Superoxide production in response to PMA by AMLs from healthy controls (n = 4), P1, and P2. (L) TNF secretion by AMLs from healthy controls (n = 3) and P6–P8 in response to LPS. (M) STAT5 phosphorylation by AMLs from healthy controls (n = 3) and P6–P8 in response to GM-CSF. For (B)–(M), the data shown are the means ± SD. Significance was assessed using Mann-Whitney U tests (B–M); ns, not significant.