Presented at the Neonatal Society 2012 Summer Meeting.
Alexandrou D, MacIntyre D, Sooranna S, Bennett P, Johnson M
Institute of Reproductive and Developmental Biology, Imperial College, London, UK
Background: BPD occurs primarily in very low birth weight infants, sometimes with minimal or no lung disease. Current evidence unequivocally implicates inflammation in the pathogenesis of BPD (1). Given that the fetus is developing in a (normally) hypoxic environment (2) and that VLBW infants are born at a stage where lung development requires hypoxic conditions to proceed normally, we examined the role of inflammation in the context of hypoxia. The transcription factor hypoxia-inducible factor (HIF) has emerged as the key regulator of the molecular hypoxic response, mediating a wide range of physiological and developmental mechanisms of cellular adaptation to hypoxia (2). The cellular response to inflammatory stress is mainly regulated by the transcription factors NF-κB and AP-1. Upstream activating signals (inflammatory and oxidant stress, cytokines, growth factors and hormones) result in downstream activation of transcription of target genes and the production of proteins which play a variety of roles in inflammation, angiogenesis, apoptosis and cell proliferation and differentiation during development. We, therefore, examined the link between HIF and NF-κB and AP-1. The general hypothesis is that the normal fetal lung is a hypoxic, but not an inflammed organ, and that it is being protected against hypoxia-induced inflammation by deferential transcription of the two isoforms of HIF (HIF-1α, HIF-2α). We propose that the fetal lung needs to balance the dampening of hypoxia-induced inflammation, without compromising its ability to fight infection and that the fetus responds to inflammation differently than the mother does. We hypothesize that this is accomplished by modulation of the innate immune response of the fetus, at the level of the pulmonary macrophage, by promoting a switch to the M2, alternative and tissue-protective macrophage phenotype, as opposed to M1 phenotype, which is pro-inflammatory.
Methods: All experiments were carried out in accordance with the Animals (Scientific Procedures) Act 1986. CD1 outbred, timepregnant mice underwent laparotomy under isoflurane. The uterus was exposed and either LPS (10 μg) or PBS (sham animals) was administered in the right uterine horn. The uterus was returned, the skin was closed and 3 hours later all foetuses were recovered following CS. Fetal lungs were collected at birth, put immediately on dry ice and kept at -80ºC, until used for RNA extraction and production of cDNA. Transcription studies were carried out with RT-PCR (Rotor Gene 6, Corbett Research Ltd) using GADH as house-keeping gene and Western Blotting using β-actin as control.
Results: We found no evidence of NF-κB activation, at the protein level, either in the mother (myometrium), or in the fetus (fetal lung). To the contrary, we found evidence of a strong and significant (p<0.01) activation of the AP-1 pathway, at the protein level (Western Blotting for p-c-Jun), in the myometrium. There was weak, non-significant activation of AP-1 in the fetal lung, along with a similar, non-significant, increase in HIF-2a levels, which might be suggestive that the inflammatory pathway in the fetal lung is activated via AP-1. We did not detect any evidence of NF-κB activation in the fetal lung at the transcriptional level either (RT-PCR for IL-1β, IL-6, TNFα).
Conclusion: Our results are suggestive of a differential response to inflammation between the mother (myometrium) and the fetus (fetal lung). They are also suggestive that the fetal lung may be protected from the inflammatory response to some extend, and our observation of differential expression of the HIF sub-units, at the protein level, may be an explanation. We will further investigate such a possible role of increased HIF-2α levels in dampening the inflammatory response in the normal fetal lung, via modulation of macrophage activation from the M1 (pro-inflammatory) to M2 (anti-inflammatory and tissue protective) phenotype.
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1. Wright CJ, Kirpalani H. (2011), Pediatrics. 128, 111-126.
2. Simon MC, Keith B. (2008). Nature Rev. Mol Cell Biol. 9, 285-296
3. Nizet V, Johnson RS (2009). Nature Rev. Immunology. 9, 609-617.