IMPACT OF ALTERED EXTRACELLULAR MATRIX PROTEIN DISTRIBUTION ON IMMUNE CELL FUNCTION DURING INFLAMMATION

Abstract

Extracellular matrix proteins (ECM) form the structural support for the migration of immune cells and provides multiple signals to assist in the functions of immune cells during physiological and pathological conditions. Secretion of inflammatory mediators such as cytokine and MMP (expand) s could influence the expression and distribution of ECM in the inflamed tissues, which in turn could modify cellular functions. Previous reports have shown changes in the expression of ECM proteins during local inflammatory responses such as asthma, fibrosis, chronic obstructive pulmonary disease (COPD), etc. However, limited knowledge is available on the changes occurring in the expression and distribution of important ECM proteins in multiple tissues during systemic inflammation. One part of the doctoral work was focused on investigating, the expression profile of important ECM proteins in systemic inflammation using two systemic inflammation models, i.e, Lipopolysaccharide induced endotoxemia and Cecal Ligation and Puncture (CLP) induced polymicrobial sepsis were used having a distinct pattern of events which drives the systemic inflammations. Following LPS injection and CLP surgery, lung, liver, kidney & mesentery were isolated at 6 hr, 12 hr, 24 hr, and 36 hr time point. Using real-time PCR and western blot technique, expression of important ECM protein both at transcript and protein levels were measured. RT-PCR and Western blot analysis showed changes in the expression of various ECM proteins such as collagen 4, fibrinogen and vimentin. A unique expression pattern of these prominent ECM proteins was observed in both at the site of inflammation (mesentery) as well as in the visceral organs (Lung, liver & kidney) in both models, possibly due to the difference in inflammation induction pattern. Moreover, to confirm the importance of upregulated ECM protein on immune cell function and various inflammatory and functional signaling pathways, bioinformatics analysis with STRING software was performed to predict the important signaling pathways that get activated upon cell-matrix interaction such as activation, apoptosis, cytoskeleton modulation, integrin expression and migration during the inflammatory scenario both in murine and humans. Secondly, the work also focused on assessing the effect of Fibulin7 (Fbln7), one of the newly identified adhesion matrix protein on neutrophils. Fibulin7 was recently identified as members of the fibulin family of secreted glycoproteins and was found to be expressed in developing tooth, bone, cartilage as well as immune-privileged locations such as eye and ii placenta. Previous studies from our laboratory have shown that the Fbln7 full-length and its C terminal fragment (Fbln7C) have regulatory effects on human monocytes and macrophages but its effect on neutrophils is not known. Thus, experiments were designed to further understand the effects of Fbln7, specially, its C-terminal fragment-Fbln7C on the immunological functions of neutrophils which are key cell type of innate immune system and also play vital role in driving the pathological events such as multi organ failure in infection induced systemic inflammations. In vitro cell adhesion and inhibition assay showed that the neutrophils could bind to adhesion protein fbln7 via integrin β1and could compete with other ECM proteins such as fibronectin for binding to neutrophils. Significant reduction in ROS and inflammatory cytokine production (i.e. IL-6, IL-1β) was observed including reduction in Erk1⁄2 phosphorylation in neutrophils stimulated with LPS and fMLP in presence of Fbln7C compared to untreated controls. Furthermore, treatment of Fbln7-C in mice decreased the number of cells in both blood and peritoneum, as well as reduced the PMA induced ROS production from neutrophils isolated from peritoneum and lungs of endotoxemic group treated with Fbln7C compared to controls animals. In summary, data from the ECM expression during systemic inflammation and immunomodulatory role ofFbln7-C will help in better understanding of ECM-Cell dynamics in the inflammatory microenvironment and may contribute to the development of cell adhesion based therapeutics.