Sustained desensitization to bacterial Toll-like receptor ligands after resolution of respiratory influenza infection

Reading good articles on host pathogen interactions gives me one of the greatest joys. Today, I will discuss one such article. This is a brief report published in Journal of Experimental Medicine and is from the lab of Dr. Tracy Hussell, Imperial College, London.
In recent years, it has been observed by various studies that one respiratory tract infection may alter immunology and pathology to a second unrelated pathogen, even long after resolution of first pathogen and in the absence of cross-reactive immunity. The outcome of such successive infections may be beneficial to the host or it may be harmful for the host. One of the most common examples is life threatening bacterial pneumonia observed in patients infected with influenza virus. These data suggest that lung undergoes some form of maturation that alters the way it responds to subsequent infections. Many mechanisms have been proposed to explain enhance bacteria at the time of seasonal and pandemic influenza or RSV. These include a disruption of epithelial integrity, up regulation of bacterial adhesion molecules, and/or alteration in antibacterial peptides. This study proposes an additional mechanism, TLR desensitization.
It is generally assumed that innate immune system returns to its normal preinfection state after multiple waves of inflammation. However, it has been observed that the number of dendritic cells and their ability to prime T cells in the murine lung remain elevated even after the resolution of RSV or influenza virus infection. In the present study, evidence was provided that TLR responsiveness is reset after first wave of inflammation. The authors show a long term desensitization of alveolar macrophages to TLR ligands (LPS, flagellin, and lipotechoic acid). Ligation of TLR ligands lead to activation of signaling pathways, resulting in cytokine and chemokine release and finally in pathogen clearance. In this study, the authors show that AMs isolated from lungs of infected animal after resolution of influenza, have impaired NF kappa B translocation in response to TLR ligation. This results in reduced subsequent inflammation and cell recruitment, which further correlates with a higher and prolonged respiratory bacterial load.
Here is a summary of major results and methods used:
1. Impairment in cell recruitment in the post influenza airway in response to TLR ligation: Bacterial TLR ligands (LPS, flagellin and LTA) were introduced in the lungs of post influenza mice. The percentage and total number of macrophages and neutrophils in the lungs and airway was monitored by flow cytometry after 4 weeks. Administration of TLR5 ligand flagellin caused reduced neutrophils migration into the airway compared to the control mice. A similar impairment in the neutrophils number was observed on induction with TLR4 ligand, LPS. At 48hr post induction reduction in macrophage number was also observed. Similar results were observed on TLR-2 ligation.
2. Impairment in neutrophils recruitment on inoculation with Pseudomonas aeruginosa and enhancement in bacterial load.
3. Impairment in neutrophils recruitment on inoculation with group B Streptococcus and enhancement in bacterial load.
4. Reduced neutrophilia to a second respiratory stimulus is due to a reduction in chemotactic signals to draw them there- To prove that reduced neutrophilia to a second respiratory signal is not due to enhanced apoptosis within the lungs, the proportion of apoptic neutrophils was measures and it was observed that proportion of apoptic neutrophils was low and comparable between post influenza and control mice. To further show that reduced neutrophilia was not due to impaired neutrophils transmigration, post influenza and control mice were inoculated with neutrophils chemoattractants. It was observed that equivalent number of neutrophils was recruited in post influenza and control mice. Altogether, these data indicate that the local induction of the TLR signaling pathway is altered by prior viral lung exposure.
5. AMs are desensitized to TLR-mediated signals-The authors further studied which cell types were desensitized to TLR-mediated signals. On systemic administration of flagellin, which targets lung endothelial cells, no impairment in neutrophils infiltration in the lung parenchyma or peripheral sites was observed. This suggested that alteration in TLR signaling is involved when flagellin is administered intra-nasally. To study the extent at which AMs are effected, AMs were isolated 1hr after flagellin challenge and tested ex vivo. They displayed reduced levels of mRNA transcripts for KC, MIP2α and TNF-α in post influenza compared with controls.
6. To determine whether transcriptional regulations occurs independently in AMs or as result of co-operation with other cells e.g., alveolar epithelial cells (AECs), both populations of cells were individually isolated from post influenza mice. These cells were treated with flagellin and NF-kappa B activation was determined in vitro. The authors observed that nuclear translocation of the p65 subunit of NF kappa B in response to flagellin was inhibited in AMs from post influenza mice. No such inhibition was observed in AECs. Thus, the authors proposed that sustained reduction of NF kappa B activation in AMs after the initial viral infection leads to reduced inflammatory response and neutrophils recruitment. To support this, the authors conditionally depleted CD11c+ cells, including AMs, from post influenza mice. This caused nearly 90% reduction in AMs and lung macrophages. The pools of macrophages were regained in 3 weeks. The new macrophages were isolated and induced with flagellin. The reduction in neutrophils recruitment was not observed. These data suggest that AMs are instrumental in the long lived altered response in the post influenza lung.
Things still not known:
1. It remains to be determined whether AMs are directly affected by infection or their phenotypes are altered by interaction with recruited inflammatory cells.
2. In addition, whether the pool of lung macrophages that give rise to AMs is also affected by previous infection warrants further investigation.
3. The molecular mechanisms responsible for long term TLR desensitization remain to be resolved