y to the formation of aggregates, which were better equipped to clear infection by the intracellular pathogen S. Typhimurium. To delineate the role of aggregation from the bactericidal functions of NO, experiments with Reopro were performed to address the role of CD11b in cell-cell interactions. Addition of Reopro led to a marginal decrease in the number of Sal-GFP per cell. In the presence of Ifn, Reopro did not significantly impact Sal-GFP per cell in either singles or in aggregates of APECs. Importantly, Reopro treatment decreased the MedChemExpress MEK162 ability of APECs to form aggregates and, as a consequence, increased the number of APECs that remained single. Consequently, these single APECs were unable to resist infection as much as the aggregated APECs. These experiments delineated the distinct roles of NO and CD11b: CD11b promoted aggregation of APECs whereas NO contributed to aggregation of APECs and lowered intracellular bacterial replication. Discussion There have been several efforts to understand the mechanisms and functional consequences of interactions between different cells of the immune system. The ability of neutrophils to swarm to the site of wound and form aggregates is dependent on Leukotriene B4 and integrins. However, the ability of APECs consisting primarily of macrophages to interact with each other, the mechanisms involved and the possible physiological significance have not been explored. In this study, we demonstrate that Ifn specifically induces APECs to form aggregates. Two important mediators of the responses to Ifn are ROS and NO. The role of ROS in contributing to platelet aggregation, for example, has been well documented. However, Ifn induced ROS played a negligible role during aggregation of APECs. On the other hand, platelet aggregation is inhibited by NO generated by both platelets and endothelial cells. Also, NO generated in response to Ifn inhibits T cell adhesion to the endothelium in the presence of Tnf. Our data establishes the roles of NO using a chemical inhibitor, e.g. LNMA, and cells from mice lacking Nos2 during Ifn induced aggregation of APECs. We have also shown that Ifn and Nos2-derived NO is important for motility, phagocytosis and morphology in APECs. It needs to be highlighted that addition of SNAP or DETA/NO, two distinct NO donors, alone was insufficient to induce formation of aggregates in APECs. Hence, NO produced in conjunction with other intracellular Ifn signals contributes to the formation of aggregates PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19696148 in APECs. It is pertinent PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19696906 to discuss some aspects related to this study: First, the aggregation of APECs in response to Ifn was specific to these cells and was not seen with bone marrow derived macrophages. This aspect is relevant as tissue-resident macrophages display high 19 / 28 Ifn and Nos2 Regulate Functions of APECs transcriptional and functional diversity. Tissue-resident macrophages are seeded very early during development and their numbers are self-maintained through adult life with a minimal 
contribution from circulating monocytes. Second, resident APECs are known to respond well to Ifn, but much lower to LPS, in terms of nitrite production. We found that LPS, another inflammatory stimuli, failed to induce aggregation of APECs, demonstrating that the effect was specific to Ifn. In fact, the ability of Ifn, but not LPS, to lower the amounts of Ccl3 and Ccl4 is also dependent on the activation status of macrophages. Most likely, the tissue origin and activation status are important fac
