Inhibitors of NF-κB (ΙκB) were first described as cytoplasmic proteins that inhibit the activity of the heterodimeric NF-κB complex (Sen, 1986). The rapid resynthesis of IκB-α following NF-κB activation aids in the deactivation of NF-κB through cytoplasmic shuttling of NF-κB dimers (Huang, 2000; Turpin, 1999). The role of NF-κB in the differential regulation of NF-κB-linked genes involves both gene- and cell-specific factors.
The interaction of NF-κB with CREB-binding protein (CBP) and its functional homolog p300 has been shown to be crucial for transcriptional activity (Sheppard, 1999). Histone deacetylases (HDACs) appear to function as a counterbalance in the regulation of NF-κB transcriptional activity. Intranuclear regulation of NF-κB appears to play an important role in defining the cellular phenotype following NF-κB activation.
NF-κB has also been shown to play a role in the initiation and maintenance of the oncogenic phenotype (Baldwin, 2001; Haefner, 2002). The role of NF-κB in the regulation of innate immunity is the focus of this work and will be discussed in more detail below. TRIF (TICAM-1), a TIR domain-containing adapter, can mediate activation of NF-κB in the absence of MyD88.
However, NF-κB activation in these fibroblasts leads to prolonged nuclear localization of NF-κB, implicating ΙκB-α in the post-induction suppression of NF-κB activity.
HLL HIV-LTR Luciferase
In developing bioluminescence imaging in the HLL transgenic reporter mouse model, the kinetics of luciferin administration and the correlation of photon emission with tissue luciferase activity were determined. When the skin over the right lung was removed, baseline photon emission was significantly reduced, indicating high basal luciferase activity in the skin. BMDMΦ from WT, IκB-α −/+, and p50 −/− mice were cultured as described in the Bone Marrow-Derived Macrophages section.
A549 cells were transiently transfected with the 8x NGL construct using Superfect as described in the Transfection section below. We identified different temporal and cellular patterns of NF-κB activation in the lung with three separate methods of LPS administration (IP, IT, and IP osmotic pump). In addition, the role of NF-κB in the regulation of bacterial infection in the lung was investigated using a Pseudomonas aeruginosa pneumonia model.
Thus, the NGL transgenic reporter mouse model provides a crucial tool in investigating the role of NF-κB in regulating lung inflammation in vivo. As a reference, the HIV-LTR luciferase construct used to generate the HLL reporter mouse model was included in the experiment.
HIV- LTR
Baseline 4 Hours 24 Hours 48 Hours Figure 12: Bioluminescence imaging of NGL mice after a single IP injection of LPS (3 µg/g)
Four hours after IP LPS administration, a significant increase in NF-κB activity in the lung is observed, followed by a decrease to near-baseline levels by 24 hours. NF-κB activity in the lung at 48 h after IP LPS administration is comparable to untreated NGL mice. This persistent NF-κB activation was reflected in photon counts detected over the thorax (Figure 13).
The number of neutrophils detected in the lung correlated with both the histological evidence of lung inflammation and NF-κB activity as detected by bioluminescence imaging and photon counting. A significant increase in NF-κB activity is observed in the lung 4 hours after Pump LPS administration. Persistent NF-κB activity is detected in the lung 24 and 48 hours after Pump LPS administration.
A significant difference in the number of neutrophils was detected 24 hours after IT LPS (compared to baseline, 4 and 48 hours) and between the 24 and 48 hour time points (p<0.05) (Figure 15). A modest increase in NF-κB activity is detected in the lung 4 hours after IT LPS administration. To study the role of NF-κB in the regulation of bacterial pneumonia, NGL mice were administered a single intratracheal injection of 106 cfu Pseudomonas aeruginosa.
Histology correlated with bioluminescence in that few neutrophils were observed in the lung 4 hours after IT Pseudomonas, followed by increased neutrophil influx into blood vessels and interstitium at 24 hours. In the previous studies, the detection of NF-κB activity by GFP was performed by immunohistochemistry on lung tissue sections. These data correlate with the detection of increased numbers of GFP+ macrophages and neutrophils in the lung 24 hours after IT LPS by GFP immunohistochemistry.
In the context of LPS-induced lung inflammation, NF-κB activation occurs in multiple cell types (macrophage, neutrophil, epithelium, endothelium). Quantification of donor alveolar macrophage repopulation in the lung by immunohistochemistry and tissue cell counting. Quantification of Donor Alveolar Macrophage Repopulation in the Lung by FACS Analysis of Brochoalveolar Lavage Macrophage.
In these experiments, donor cells were not observed in the lungs until one month later. A significant increase in lung neutrophils was detected 8 hours after IP LPS compared to baseline and 4 hours (p<0.05) followed by a decrease towards baseline by 48 hours (Figure 37).
Baseline 4 Hrs 8 Hrs 24 Hrs 48 Hrs 72 Hrs 6 Days
The number of neutrophils detected in the lung correlated with both the histological evidence of lung inflammation and NF-κΒ activity as detected by bioluminescence imaging and photon counts. NF-κB activity increased 4 h after IP LPS similar to IκB-α−/+ FLT chimeras; however, after 24 hours, NF-κB activity was greater in the IκB-α−/− FLT chimeras as determined by photon count emission (Figure 35). Histology revealed neutrophilic influx into the vessels, interstitium, and alveolar space at 4, 24, and 48 hours after IP LPS (Figure 43).
These data show that changes in the NF-κB pathway in BMDMΦ alter NF-κB activity in A549 cells after LPS stimulation. The role of the NF-κB signal transduction pathway in specific cell types in the regulation of lung inflammation remains unclear. The observation that WT, IκB-α−/+ and IκB-α−/− FLT chimeras showed similar NF-κB activity in the lung at baseline suggests that the loss of ΙκB-α alone does not lead to increased NF-κB activation.
In p50−/− FLT chimeras, a single IP dose of LPS resulted in a dramatic influx of neutrophils accompanied by significant NF-κB activity in structural cells in the lung. The role of IκB-α and p50 in the regulation of NF-κB turn-off in macrophages needs further investigation. In BMDMΦ experiments, increased levels of KC and MIP-2 were detected at 24 and 48 h after LPS in the IκB-α−/+ and p50−/− co-culture groups compared with the WT co-culture group.
These data indicate a functional consequence of sustained NF-κB activity in macrophages and epithelia and help to explain the sustained neutrophil influx observed in FLT chimeras. Furthermore, these data would further support an active role for NF-κB in the quenching as well as the ignition mechanism of lung inflammation. Several studies have demonstrated the role of the macrophage in the initiation of lung inflammation.
These results more powerfully illustrate the role of IκB-α in actively “turning off” NF-κB activation in macrophages. In addition to IκB-α FLT chimeras, the role of p50 in the regulation of macrophage NF-κB activation was investigated by generating bone marrow chimeras using HLL mice as recipients and p50 knockout donor fetal liver cells. Resolution of inflammation is achieved by the active switching off of NF-κB in the macrophage, which leads to NF-κB switching off in other cell types and a return to normal neutrophil numbers.
In conclusion, the data presented in this thesis make an important contribution to the understanding of the NF-κB signaling pathway in the regulation of inflammation. The role of macrophage IκB-α and p50 in the active quenching of lipopolysaccharide-induced lung inflammation.
