Progression of Type 2 Helper T Cell–Type Inflammation and Airway Remodeling in a Rodent Model of Naturally Acquired Subclinical Primary Pneumocystis Infection

Abstract

Subclinical primary Pneumocystis infection is the most common pulmonary infection in early infancy, making it important to determine whether it damages the lung. Pneumocystis peaks at 2 to 5 months of age, when respiratory morbidity coincidently increases. We have documented that Pneumocystis increases mucus production in infant lungs, and animal models reveal lung lesions that warrant characterization. Herein, immunocompetent rats infected at birth with Pneumocystis by cohabitation, to resemble community-acquired infection, underwent lung assessments at 45, 60, and 75 days of age. Lungs fixed by vascular perfusion to prevent collapse during necropsy were used for morphometry evaluations of mucus production, airway epithelial thickening, perivascular and peribronchiolar inflammation, and structural airway remodeling. Changes in these histologic features indicate lung disease. Selected immune markers were assessed in parallel using fresh-frozen lung tissue from sibling rats of the same cages. Sequential activation of NF-κB and an increased Gata3/T-bet mRNA level ratio, consistent with a type 2 helper T-cell-type inflammatory response, and subacute fibrosis were recognized. Therefore, documenting subclinical Pneumocystis infection induces lung disease in the immunocompetent host. Taken together with the peak age of primary Pneumocystis infection, results warrant investigating the clinical impact of this often subclinical infection on the severity of respiratory diseases in early infancy. This model can also be used to assess the effects of airway insults, including coinfections by recognized respiratory pathogens.

Increase in secreted airway mucins and partial Muc5b STAT6/FoxA2 regulation during Pneumocystis primary infection

“…P-STAT6 protein levels were increased in Pneumocystis -infected Kaempferol-untreated animals respect to control rats treated with saline. The Pneumocystis burden reached similar levels than in lungs where muc5ac 26 and muc5b mucins were quantitated (Fig. 4A).…”

“…MUC5AC has been recently described as an essential effector of the epithelial response to allergic inflammation 7 . Increased MUC5AC is consistent with the intense Th2 (allergic type) airway immune response 25,26 and STAT6 pathway activation 27 plus induction of mucus-related genes such as Muc5ac and Clca3 28 associated to mucus hypersecretion documented in animal models of Pneumocystis infection 26,27,29 . Of interest, anti-Muc5ac immune staining is able to recognize only a minimal fraction of the mucus that stains with Alcian blue, suggesting additional mucins are involved during Pneumocystis infection 26 .…”

“…2F). Pneumocystis lung burden in these animals, estimated by qPCR of the Dihydrofolate Reductase ( dhfr ) gene of Pneumocystis , was characterized in previously published experiments 26 .

Figure 2Mucin gene-expression and protein quantifications at 45, 60, and 75 days after Pneumocystis co-habitation exposure. Muc5ac mRNA ( A ), and protein levels ( B ) and, Muc5b mRNA ( C ) and protein levels ( D ) were determined by qPCR and Western blot, respectively.

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“…We have reported increased MUC5AC and CLCA1 associated to Pneumocystis primary infection in lungs of infants dying in the community 19,24 . Understanding the effect of Pneumocystis on mucin production and its controlling pathways is underscored by the epidemiological context of this fungal infection 18–21 and by the demonstration that Pneumocystis primary infection induces a Th2 environment in the healthy lung 25,26 . MUC5AC has been recently described as an essential effector of the epithelial response to allergic inflammation 7 .…”

Molecular detection ofPneumocystisin the lungs of cats

“…Indeed, a recent rodent model study documented that subclinical primary Pneumocystis infection can induce progressive pathologic changes characteristic of airway disease in immunocompetent hosts, such as increased mucus production and thickening of the airway epithelium. 31 To the best of our knowledge, this is the first molecular genetic investigation that describes Pneumocystis in cats or other felids using sequence-based PCR methodology. In 1999, Cho and colleagues 20 established that there were at least three distinct karyotype groups of Pneumocystis among mammals in Korea.…”