Toxicol Lett. 2014 Oct 15;230(2):333-44.

Effect of ozone exposure and infection on bronchoalveolar lavage: Sex differences in response patterns.

Anatoly N. Mikerova,b,*, David S. Phelpsa,*, Xiaozhuang Gana, Todd M. Umsteada, Rizwanul Haquea, Guirong Wanga, and Joanna Florosa,c

 

aThe Center for Host defense, Inflammation, and Lung Disease (CHILD) Research, Department of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, PA, USA; bDepartment of General Hygiene and Ecology, Saratov State Medical University, Saratov, Russia; cDepartment of Obstetrics and Gynecology, The Pennsylvania State University College of Medicine, Hershey, PA, USA.

* These two authors contributed equally to this work.

 

Corresponding author/Requests for Reprints: Joanna Floros, Ph.D. , The Center for Host defense, Inflammation, and Lung Disease (CHILD) Research, Department of Pediatrics, H085, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA. Telephone: (717) 531-6972. Fax: (717)-531-0215, E-mail: jfloros@hmc. psu.edu

 

Abstract

Female mice exhibit a better survival rate than males after infection, but if infection follows an ozone-induced oxidative stress, male survival exceeds that of females. Our goal was to study bronchoalveolar lavage factors that contribute to these sex differences in outcome. We studied parameters at 4, 24, and 48 h after ozone exposure and infection, including markers of inflammation, oxidative stress, and tissue damage, and surfactant phospholipids and surfactant protein A (SP-A). A multianalyte immunoassay at the 4h time point measured 59 different cytokines, chemokines, and other proteins. We found that: (1) Although some parameters studied revealed sex differences, no sex differences were observed in LDH, total protein, MIP-2, and SP-A. Males showed more intragroup significant differences in SP-A between filtered air- and ozone-exposed mice compared to females. (2) Oxidized dimeric SP-A was higher in FA-exposed female mice. (3) Surfactant phospholipids were typically higher in males. (4) The multianalyte data revealed differences in the exuberance of responses under different conditions – males in response to infection and females in response to oxidative stress. These more exuberant, and presumably less well-controlled responses associate with the poorer survival. We postulate that the collective effects of these sex differences in response patterns of lung immune cells may contribute to the clinical outcomes previously observed.

KEYWORDS: Innate immunity; Oxidative stress; Pneumonia; SP-A; Surfactant

PMID: 24769259

 

Supplement

Effective immune defense in the lung depends on various factors in the bronchoalveolar fluid. The purpose of this work was to study the effect of ozone on some parameters in the bronchoalveolar lavage (BAL) during the course of pneumonia, caused by Klebsiella pneumoniae, at 4, 24, and 48 h post-infection. In our experimental model, male and female mice were first exposed to either ozone (oxidative stress conditions) or to filtered air (control, or “normal” conditions), and then infected with Klebsiella pneumoniae bacteria. We studied: the number of neutrophils; protein content and the level of protein oxidation; the level of the surfactant protein A (SP-A), an innate immune molecule, and the level of its oxidation; as well as the levels of phospholipids; and MIP-2 chemokine in the mouse BAL. Moreover, a multianalyte immunoassay was performed at the 4 h time point that measured 59 different cytokines, chemokines, and other proteins.

In general, our results demonstrated that both males and females had more pronounced changes in all of the BAL values studied during pneumonia following an oxidative stress. Moreover, our results revealed that lung surfactant components, such as SP-A and phospholipids, might play an essential role in the modulation of lung immune defense mechanisms during pneumonia in response to ozone-induced oxidative stress. The multi-analyte data revealed, based on the number of analytes with changed levels, differences in the exuberance of responses under different conditions – males in response to infection and females in response to oxidative stress. These more exuberant and presumably less well-controlled responses may have negative consequences and may contribute to the sex differences in survival after pneumonia observed in our previous studies.

This paper is one of a series of papers where sex differences have been observed in response to infection and/or ozone exposure in mouse survival, as well as in the lung itself. The latter includes differences in alveolar macrophage function [1], in bacterial dissemination [2], and lung pathology [3] as assessed by a variety of approaches.

Key cellular and molecular contributors to lung innate immunity include the alveolar macrophage, the sentinel cell of innate immunity, and the surfactant protein A (SP-A). The alveolar macrophage is a major target of SP-A, and this interaction is vital to many alveolar macrophage functions such as phagocytosis of bacteria [4] and the regulation of the alveolar macrophage proteome [5-8]. For the latter, when male and female SP-A -/- mice are rescued with SP-A from human bronchoalveolar lavage, the proteomic profile of the alveolar macrophage is nearly restored to that of the wild type mouse, with sex differences being observed [5]. Moreover, because there are two SP-A genes, SP-A1 and SP-A2, in humans with several genetic variants observed for each gene, the alveolar macrophage/SP-A variant interaction appears to depend on the specific SP-A variant, as assessed by the proteomic expression profile of humanized transgenic (hTG) mice that each contain a different variant [7]. Moreover, rescue (acute treatment) of SP-A knockout mice with SP-A1 and SP-A2 variants exhibited sex differences in the proteome of the alveolar macrophage [8]. Although not studied in the present paper by Mikerov et al some of the sex differences observed in response to infection and/or ozone exposure are negatively affected in the absence of the SP-A [9].

The collective knowledge from the related published work indicates that the lung and especially the distal lung exhibits clear sex differences in response to infection and/or ozone. Although the lung is a portal of entry of injurious agents such as bacteria, toxins, ozone, and other, from the external environment, one published work in this series of papers indicated that ozone exposure and/or lung infection have far-reaching negative effects (beyond the lung), and some of these exhibit sex differences. Extrapulmonary lesions in tissues such as liver and spleen showed sex differences in response to lung infection, but when infected animals were exposed to ozone, some extrapulmonary lesions were ozone-dependent without exhibiting sex differences.

In summary, as shown from the group of publications discussed above lung injury and especially the response of the alveolar macrophage is sex dependent, with oxidative stress affecting survival and alveolar macrophage function more in females and infection affecting more males. Moreover gonadal hormones have been shown to play a role in the survival of bacterial infected mice in the presence or absence of oxidative stress [10]. These data together support the notion that the underlying mechanisms of lung injury (by infection and/or ozone-induced oxidative stress) may differ between males and females and thus studies must be carried out in both sexes.

 

References

  1. Mikerov, A.N., et al., Sex differences in the impact of ozone on survival and alveolar macrophage function of mice after Klebsiella pneumoniae infection. Respir Res, 2008. 9: p. 24.
  2. Mikerov, A.N., et al., Impact of sex and ozone exposure on the course of pneumonia in wild type and SP-A (-/-) mice. Microb Pathog, 2012. 52(4): p. 239-49.
  3. Mikerov, A.N., et al., Histopathologic evaluation of lung and extrapulmonary tissues show sex differences in Klebsiella pneumoniae – infected mice under different exposure conditions. Int J Physiol Pathophysiol Pharmacol, 2011. 3(3): p. 176-90.
  4. Mikerov, A.N., et al., Impact of ozone exposure on the phagocytic activity of human surfactant protein A (SP-A) and SP-A variants. Am.J.Physiol Lung Cell Mol.Physiol, 2008. 294(1): p. L121-L130.
  5. Phelps, D.S., et al., In vivo rescue of alveolar macrophages from SP-A knockout mice with exogenous SP-A nearly restores a wild type intracellular proteome; actin involvement. Proteome Sci, 2011. 9: p. 67.
  6. Phelps, D.S., T.M. Umstead, and J. Floros, Sex differences in the response of the alveolar macrophage proteome to treatment with exogenous surfactant protein-A. Proteome Sci, 2012. 10(1): p. 44.
  7. Phelps, D.S., et al., Differences in the alveolar macrophage proteome in transgenic mice expressing human SP-A1 and SP-A2. J Proteom Genom Res, 2013. 1(2): p. 2-26.
  8. Phelps, D.S., T.M. Umstead, and J. Floros, Sex differences in the acute in vivo effects of different human SP-A variants on the mouse alveolar macrophage proteome. J Proteomics, 2014. 108: p. 427-44.
  9. Mikerov, A.N., et al., Ablation of SP-A has a negative impact on the susceptibility of mice to Klebsiella pneumoniae infection after ozone exposure: sex differences. Respir Res, 2008. 9: p. 77.
  10. Durrani, F., et al., Gonadal hormones and oxidative stress interaction differentially affects survival of male and female mice after lung Klebsiella Pneumoniae infection. Exp Lung Res, 2012. 38(4): p. 165-72.

 

 

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