SP-A preserves airway homeostasis during Mycoplasma pneumoniae infection in mice

Abstract

The lung is constantly challenged during normal breathing by a myriad of environmental irritants and infectious insults. Pulmonary host defense mechanisms maintain homeostasis between inhibition/clearance of pathogens and regulation of inflammatory responses that could injure the airway epithelium. One component of this defense mechanism, surfactant protein-A (SP-A), exerts multifunctional roles in mediating host responses to inflammatory and infectious agents. SP-A has a bacteriostatic effect on Mycoplasma pneumoniae (Mp), which occurs by binding surface disaturated phosphatidylglycerols. SP-A can also bind the Mp membrane protein, MPN372. In this study, we investigated the role of SP-A during acute phase pulmonary infection with Mp using mice deficient in SP-A. Biologic responses, inflammation, and cellular infiltration, were much greater in Mp infected SP-A(-/-) mice than wild-type mice. Likewise, physiologic responses (airway hyperresponsiveness and lung compliance) to Mp infection were more severely affected in SP-A(-/-) mice. Both Mp-induced biologic and physiologic changes were attenuated by pharmacologic inhibition of TNF-alpha. Our findings demonstrate that SP-A is vital to preserving lung homeostasis and host defense to this clinically relevant strain of Mp by curtailing inflammatory cell recruitment and limiting an overzealous TNF-alpha response.

Figure 1. Effect of SP-A on Mycoplasma pneumoniae-induced airway responsiveness to methacholine challenge

Wild type and SP-A^−/− mice were instilled intranasally with Mp (~1 × 10⁸/mouse) and airway responsiveness to methacholine challenge was analyzed 72 hours post infection by Flexivent. A, the average resistance was significantly elevated during methacholine challenge in the Mp infected SP-A null mice as compared to wild type controls. B, the average compliance is significantly lower in the SP-A null Mp-infected mice at baseline and during methacholine provocations. Data is combination of three independent experiments, n=8–12mice/group. *p<.05, **p<.01 and is SP-A^−/− Mp infected versus all other groups at the respective dose concentrations.

Figure 2. Histological analysis of cellular infiltration in Mp infected mice

Three days (72 hours) post-infection mice were sacrificed and lungs fixed with paraformaldehyde for histological analysis. Lung sections were stained with H&E and visualized by microscopy. (L) lumen, (V) vessel, (a) alveolus are labeled and arrows indicate cellular infiltrate. A, Minimal cellular infiltration is observed in and around the large airway of wild type infected mice. B, Some perivascular infiltrate is detected near bronchioles. C, Few cells are seen in the alveoli and consist primarily of macrophages. D, Large areas of lymphoid infiltrate are observed in and around the large airway of the SP-A null infected mice. E, Cellular infiltrate is detected in the airway lumen and in the perivasculature of the bronchioles. F, Many cells are seen in the alveoli and which appear to be comprised of mononuclear cells as well as macrophages. Arrows indicate areas of foci formation indicative of pulmonary edema. For A, B, D, and E scale bars are 50 microns and pictures were taken at 20× magnification. C and F scale bars are 25 microns and pictures were taken at 40× magnification. Each picture is representative of 5 independent infected mice. G, Total protein was measured in BAL by BCA assay. Data shown is combination of three independent experiments n=11,13 respectively and **p<.01.

Figure 3. Inflammatory cell recruitment into the lung tissue post MP infection

A, BALF was collected 72 hours post Mp-infection and total cells were counted. B, After the vasculature was perfused and airways were lavaged, whole lungs were enzymatically digested and single cells isolated by gradient centrifugation. Isolated cells from the lung digests were analyzed by flow cytometry with a panel of antibodies against specific cell surface markers to distinguish C, Myeloid or D, Lymphoid cells. The results shown are the mean ± SEM from three independent experiments, n=12/group and **p<.01.

Figure 4. Mp quantitation from BALs and lung tissue

Wild type and SP-A null mice were sacrified and the vasculature perfused prior to A, BALs and B, lung tissue collection 72 hrs after infection. Dilutions of BALF were plated on PPLO agar plates and Mp counts were determined with the aid of a microscope at 4× magnification following 2 weeks of incubation. Relative Mp units (assessed by RT-PCR) of Mp specific P1-adhesin gene were standardized to the housekeeper gene cyclophilin. Results are combination of three experiments with n=12,12 and **p<.01. C, Infected lungs were fixed and sections analyzed (100X oil-immersion) by immunohistochemistry with an anti-Mycoplasma pneumoniae HRP antibody. Arrows indicate areas of staining associated with ciliated airway epithelial cells. D, TNF-α was analyzed from BALF collected 12 hours post Mp infection by ELISA. Data is mean + SEM from three independent experiments. n=12/group and **<.01.

Figure 5. Treatment with LMP-420 attenuates AHR and cellular infiltration in SP-A null Mycoplasma pneumoniae-infected mice

Prior to Mp instillation, wild type and SP-A^−/− mice were treated with the TNF-α inhibitor, LMP-420. Two subsequent i.p. injections of LMP-420 were given 24 and 48 hours post Mp infection in order to maintain inhibition. A, Airway responsiveness and B, airway compliance to methacholine challenge were analyzed 72 hours post infection by Flexivent technology. n=8–10/group and *p<.05, **p<.01 and is SP-A^−/− Mp infected/ vehicle versus all other groups. Total cells from C, BALs or D, lungs digests of the vehicle or LMP-420 infected mice were analyzed to determine levels of cellular infiltration. Data is the mean + SEM, n=8–10/group. *p<.05, **p<.01. E, RT-PCR for Mp P1-adhesion from cDNA extracted from Mp-infected mice in the presence or absence of LMP-420 as standardized to the housekeeper cyclophilin.

Figure 6. Histological analysis of airway mucin production in Mp infected mice

Wild type and SP-A null mice treated with vehicle (left panel) or the TNF-α blocker, LMP-420 (right panel), prior to infection with Mp. A, Paraffin sections were stained with PAS to detect mucin production (bright pink stain). Pictures were taken at 20× magnification and the bar represents 50 µm. B, Slides from two independent experiments were further analyzed by blinded histological analysis. Representative pictures from non-overlapping large airways were taken at 20× magnification and the percentage of PAS positive stain was determined per area of large airway. Approximately 8 pictures were taken from each slide and averaged per specimen. n=4–7/group and **p<.01.