Expression of Piwi protein MIWI2 defines a distinct population of multiciliated cells

Abstract

P-element-induced wimpy testes (Piwi) proteins are known for suppressing retrotransposon activation in the mammalian germline. However, whether Piwi protein or Piwi-dependent functions occur in the mammalian soma is unclear. Contrary to germline-restricted expression, we observed that Piwi-like Miwi2 mRNA is indeed expressed in epithelial cells of the lung in adult mice and that it is induced during pneumonia. Further investigation revealed that MIWI2 protein localized to the cytoplasm of a discrete population of multiciliated airway epithelial cells. Isolation and next-generation sequencing of MIWI2-positive multiciliated cells revealed that they are phenotypically distinct from neighboring MIWI2-negative multiciliated cells. Mice lacking MIWI2 exhibited an altered balance of airway epithelial cells, demonstrating fewer multiciliated cells and an increase in club cells. During pneumococcal pneumonia, Miwi2-deficient mice exhibited increased expression of inflammatory mediators and increased immune cell recruitment, leading to enhanced bacterial clearance. Taken together, our data delineate MIWI2-dependent functions outside of the germline and demonstrate the presence of distinct subsets of airway multiciliated cells that can be discriminated by MIWI2 expression. By demonstrating roles for MIWI2 in airway cell identity and pulmonary innate immunity, these studies elucidate unanticipated physiological functions for Piwi proteins in somatic tissues.

Figure 1. Lung epithelial cells express MIWI2.

(A) Quantitative real-time PCR of epithelial RNA isolated from mice infected intratracheally (i.t.) with S. pneumoniae. Data represent fold induction over vehicle-treated mice, presented as mean ± SEM, n = 7 mice per group. ***P < 0.001 as determined by unpaired t test. (B) Quantitative real-time PCR of RNA isolated from FACS-sorted cells from mice infected i.t. with S. pneumoniae. Data represent fold induction over leukocyte expression, presented as mean ± SEM, n = 6 mice per group. ***P < 0.001 as determined by 1-way ANOVA, followed by Tukey post hoc test. (C) Immunoblot analysis for HA-MIWI2 after immunoprecipitation with anti-HA antibody, or whole lung lysate from Miwi2^+/+ or Miwi2^HA/+ mice. Arrow indicates band at expected size of MIWI2 (98 kDa). Image is representative of an experiment performed 3 times. (D and E) Immunostaining with anti-HA antibody of lung sections from Miwi2^+/+ or Miwi2^HA/+ mice treated with vehicle (D) or infected i.t. with S. pneumoniae (E). Images were acquired using the ×40 objective. (F) Morphometric quantitation was performed by counting of the number of MIWI2-positive cells per airway. At least 3 fields were analyzed from 2 sections per mouse, n = 4 mice per group, presented as mean ± SEM. *P < 0.05 as determined by unpaired t test.

Figure 2. MIWI2 marks a subpopulation of multiciliated cells.

(A) Immunostaining for KRT8 (red) and HA epitope (white) of lung sections from Miwi2^+/+ or Miwi2^HA/+ mice treated with vehicle or infected i.t. with S. pneumoniae. (B and C) Immunostaining for CC10 (red), HA epitope (white), FOXJ1 (green), or Hoechst (blue) on paraffin-embedded lung sections from vehicle-treated (B) or S. pneumoniae–infected (C) Miwi2^+/+ or Miwi2^HA/+ mice. (D) Immunostaining for acetylated α-tubulin (green) and HA epitope (white) of lung sections from Miwi2^+/+ or Miwi2^HA/+ mice infected i.t. with S. pneumoniae. Representative results are shown from staining performed on at least 2 sections, n = 6 mice per group. Scale bars: 10 μm.

Figure 3. MIWI2 is expressed in ciliated cells of the embryonic and human lung.

(A) Immunostaining for HA epitope (white) and FOXJ1 (green) on paraffin-embedded lung sections from Miwi2^HA/+ mice collected from timed pregnancies. Representative results are shown from staining performed on at least 2 sections, n = 4 mice per group. Scale bars: 10 μm. (B) Immunohistochemical analysis of human lung sections stained with anti-PIWIL4 antibody or secondary antibody alone. Image is representative of similar findings from 3 separate human donors. Scale bars: 10 μm.

Figure 4. MIWI2-positive airway cells represent a transcriptionally discrete subpopulation of multiciliated cells.

(A) Representative lung sections from Miwi2^+/Tom mice immunostained for red fluorescent protein (RFP, white), FOXJ1 (green), and CC10 (red). Scale bars: 10 μm. (B) MIWI2⁺ cells are labeled through the expression of tdTomato. (C) Representative cytometry plot selected for live, CD45^–, EpCAM⁺ cells from uninfected Miwi2^+/Tom mouse after elastase lung digest. (D) Unsupervised hierarchical clustering based on whole transcriptome data from MIWI2⁺ and MIWI2^– ciliated cells. (E) Principal component analysis of the data sets analyzed in C. (F) MA plot representation of DESeq output. Red dots indicate genes determined to be significant at FDR q < 0.05. Piwil4 (an alias of Miwi2) and tdTomato are indicated. (G) Heatmap representation of top 50 genes induced in MIWI2-positive versus MIWI2-negative ciliated cells. Values normalized by Z score. Data presented are from n = 3 mice per group.

Figure 5. MIWI2 regulates pulmonary epithelial cell composition.

(A) H&E staining of lung sections from uninfected Miwi2^+/+ and Miwi2^–/– mice. (B–D) Lung sections from uninfected Miwi2^+/+ and Miwi2^–/– mice were immunostained for CC10, FOXJ1, and DAPI, and the total numbers of airway cells (B), ciliated cells (C), and club cells (D) were quantified. n = 4, 3 mice per group, data presented as mean ± SEM. **P < 0.01 as determined by unpaired t test. (E) Lung sections from Miwi2^Tom/Tom mice immunostained for red fluorescent protein (RFP, white), FOXJ1 (green), and CC10 (red). Scale bars: 10 μm. (F) Flow cytometric quantification of tdTomato-positive and tdTomato-negative cells as a percentage of live, CD45^–EpCAM⁺CD24^hi ciliated cells; n = 3, 4 mice per group, data presented as mean ± SEM.

Figure 6. MIWI2 regulates pulmonary innate immunity during pneumonia.

(A and B) BALF cell cytospins (A) and total BALF cell counts (B) of uninfected Miwi2^+/+ and Miwi2^–/– mice. n = 4, 4 mice per group, data are expressed as mean ± SEM. Microscopic images were obtained using the ×40 objective. (C–G) Total BALF cell counts (C), BALF cytospins (D), BALF differential counts (E), BALF CXCL2 protein (F), and BALF CXCL1 protein (G) were measured from Miwi2^+/+ and Miwi2^–/– mice infected i.t. with S. pneumoniae for 4 hours. n = 6, 7 mice per group, data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 as determined by unpaired t test. Microscopic images were obtained using the ×40 objective. (H) Lung CFU was determined in Miwi2^+/+ and Miwi2^–/– mice infected i.t. with S. pneumoniae and harvested 30 hours later. n = 6, 9 mice per group, data represented as mean ± SEM. *P < 0.05 as determined by unpaired t test. Dotted line indicates input CFU.