| Abstract |
The acute respiratory distress syndrome (ARDS), due to mechanical ventilation, lung
infection, aspiration, sepsis or prematurity, is the major cause of morbidity and mortality in
Intensive Care Units of adults, children and neonates. Pulmonary hypertension, primary or
secondary to existing condition, promotes similarly severe morbidity that may also lead to
respiratory and cardiac failure. Limited therapies are effective nowadays in the management
of both conditions. Since the molecular and cellular mechanisms of acute lung injury (the
pathologic picture of ARDS) and pulmonary hypertension have not been clearly elucidated,
their understanding is crucial to develop targeted and effective therapies. Lung inflammation
and specifically innate immunity and macrophage accumulation appears to be a common
denominator that contributes to pathology in both diseases.
By utilizing wild-type (WT) mice and mice genetically modified in key – genes for
inflammation and vascular function (Akt2 knock-out, lung specific inducible HO-1
transgenic), in the current study we investigate the nature of inflammatory response and the
phenotype of macrophage activation in animal models of acute lung injury and pulmonary
hypertension and we aim to elucidate its potential causative role in the pathogenesis of
disease.
To investigate the role of macrophage activation in aseptic lung injury and identify
molecular mediators with therapeutic potential, lung injury was induced in WT and Akt2-/-
mice by hydrochloric acid aspiration. Acid-induced lung injury in WT mice was characterized
by decreased lung compliance and increased protein and cytokine concentration in
bronchoalveolar lavage fluid. Alveolar macrophages acquired a classical activation (M1)
phenotype. Acid-induced lung injury was less severe in Akt2-/- mice compared with WT
mice. Alveolar macrophages from acid-injured Akt2-/- mice demonstrated the alternative
activation phenotype (M2). Although M2 polarization suppressed aseptic lung injury, it
resulted in increased lung bacterial load when Akt2-/- mice were infected with Pseudomonas
aeruginosa.
To understand macrophage activation in our model and the role of Akt2, we studied the
TLR pathway. We found that mRNA levels of TRAF6, IRF5, STAT1 but not IRAK1 were
increased in alveolar macrophages in WT mice exposed to acid. On the other hand,
macrophages from Akt2-/- mice exposed to acid had lower levels of TRAF6, IRF5, STAT1 and
IRAK1 compared to WT mice. Τhe mRNA levels of IRAK1, TRAF6, STAT1 and IRF5 are known
to be targeted by the anti-inflammatory microRNA miR-146a.
Indeed, miR-146a was found to be induced during the late phase of lung injury in WT
mice, whereas it was increased early in Akt2-/- mice. MiR-146a overexpression in WT
macrophages suppressed LPS induced inducible NO synthase (iNOS) and promoted M2
polarization, whereas miR-146a inhibition in Akt2-/- macrophages restored iNOS expression.
PhD Thesis Eleni Vergadi
25
Furthermore, miR-146a delivery or Akt2 silencing in WT mice exposed to acid resulted in
suppression of iNOS in alveolar macrophages. In conclusion, Akt2 suppression and miR-146a
induction promote the M2 macrophage phenotype, resulting in amelioration of acid-induced
lung injury. In vivo modulation of macrophage phenotype through Akt2 or miR-146a could
provide a potential therapeutic approach for aseptic ARDS; however, it may be deleterious in
septic ARDS because of impaired bacterial clearance.
Pulmonary hypertension is a rare disease that is characterized by vasoconstriction,
thickening and remodeling of vascular wall and finally leads to right heart hypertrophy and
failure. Despite the significant progress in the field the molecular mechanisms that lead to
disease remain unclear. Lung inflammation has been found to precede the development of
hypoxia induced pulmonary hypertension (HPH); however, its role in the pathogenesis of
HPH is poorly understood. We sought to characterize the hypoxic inflammatory response
and to elucidate its role in the development of HPH. We also aimed to investigate the
mechanisms by which heme oxygenase-1 (HO-1), an anti-inflammatory enzyme, is protective
in HPH.
We generated bitransgenic mice that overexpress human heme oxygenase-1 under
doxycycline control in an inducible, lung-specific manner. Hypoxic exposure of mice in the
absence of doxycycline resulted in early transient accumulation of monocytes/macrophages
in the bronchoalveolar lavage. Alveolar macrophages acquired an alternatively activated
phenotype (M2) in response to hypoxia, characterized by the expression of found in
inflammatory zone-1 (Fizz1), arginase-1, and chitinase-3-like-3 (CHI3L3 or Ym1). A brief 2-day
pulse of doxycycline (and therefore transient HO-1 induction) delayed, but did not prevent,
the peak of hypoxic inflammation, and could not protect against HPH. In contrast, a 7-day
doxycycline treatment sustained high heme oxygenase-1 levels during the entire period of
hypoxic inflammation, inhibited macrophage accumulation and activation, induced
macrophage interleukin-10 expression and prevented the development of HPH.
Supernatants from hypoxic M2 macrophages promoted the proliferation of pulmonary
artery smooth muscle cells, whereas treatment with carbon monoxide, a heme oxygenase-1
enzymatic product, abrogated this effect. Early recruitment and alternative activation of
macrophages in hypoxic lungs are critical for the later development of HPH. Heme
oxygenase-1 may confer protection from HPH by effectively modifying the macrophage
activation state in hypoxia.
The findings of the current study can potentially lead to the development of targeted
therapies that may improve prognosis and morbidity that remain too dismal.
|
Search
