The Promise of Lung Organoids for Growth and Investigation of Pneumocystis Species

Abstract

Pneumocystis species (spp.) are host-obligate fungal parasites that colonize and propagate almost exclusively in the alveolar lumen within the lungs of mammals where they can cause a lethal pneumonia. The emergence of this pneumonia in non-HIV infected persons caused by Pneumocystis jirovecii (PjP), illustrates the continued importance of and the need to understand its associated pathologies and to develop new therapies and preventative strategies. In the proposed life cycle, Pneumocystis spp. attach to alveolar type 1 epithelial cells (AEC1) and prevent gas exchange. This process among other mechanisms of Pneumocystis spp. pathogenesis is challenging to observe in real time due to the absence of a continuous ex vivo or in vitro culture system. The study presented here provides a proof-of-concept for the development of murine lung organoids that mimic the lung alveolar sacs expressing alveolar epithelial type 1 cells (AEC1) and alveolar type 2 epithelial cells (AEC2). Use of these 3-dimensional organoids should facilitate studies of a multitude of unanswered questions and serve as an improved means to screen new anti- PjP agents.

Keywords: Pneumocystis pneumonia; Pneumocytis species; immunosuppressed hosts; lung organoids; opportunistic fungal pathogen.

Figure 1

The progression of lung organoid development. Each black arrow indicates an organoid. (A) Initial plating of primary lung epithelial cells (EpCAM) and primary lung fibroblast cells (CD140a) as viewed under light microscopy. (B) The development of lung organoids within 0.5–1.5 weeks. (C) The development of lung organoids between 1.5 and 2 weeks. (D) The development of lung organoids between 2 and 5 weeks of growth. All scale bars are 0.322 mm.

Figure 2

Type 1 alveolar cell marker expression. Two independent organoids stained with Aquaporin 5 antibodies (AQP5); a membrane bound cell marker for type I alveolar epithelial cells pseudo labeled green. Left to right. (A) Bright field images organoids. (B) Nuclei stained with DAPI. (C) AQP5 (membrane bound staining pattern) of AEC1 cells. (D) Combination of nuclei and AQP5 staining. Scale bars are 50 μm.

Figure 3

Type 2 alveolar cell marker expression. Two independent organoids labeled with Surfactant C antibodies (SFTPC), cytoplasmic cell marker for type II alveolar epithelial cells pseudo labeled red. Left to right. Two left panels display (A) Bright field images of organoids. (B) Nuclei stained with DAPI. (C) SFTPC (cytoplasmic staining pattern) for AEC2 cells. (D) Combination of DAPI nuclei and SFTPC staining. Scale bars are 50 μm.

Figure 4

Expression of Type 1 and Type 2 alveolar epithelial cell markers. Three independent organoids stained with both cell markers AQP5 (green) and SFTPC (red). From left to right. (A) Bright field images of organoids. (B) Stained with DAPI. (C) AQP5 5 staining. (D) SFTPC staining. (E) Merged image of all cell markers. Scale bars are 50 μm.

Figure 5

The process of injecting lung organoids with P. murina stained with trypan blue. Black arrow is pointing at the organoid that is injected with P. murina. After about 2 weeks of growth organoids are injected with P. murina. From left to right is a depiction of the process of injecting an organoid with P. murina stained with trypan blue. Scale bars for the images are 0.342 mm.

Figure 6

Injecting lung organoids with P. murina. Whole lung organoid Z stack images (top, middle and bottom images) of an organoid injected with P. murina. (A) Stained with DAPI for nuclear staining (blue). (B) AQP5 AEC1 cell membrane marker (green). (C) SFTPC AEC 2 cell cytoplasmic marker (red). (D) MSG staining P. murina organisms (white). (E) Merged image of all cell markers and P. murina staining. Scale bars for the images are 50 μm.