ROLE OF INTEGRIN RECEPTORS IN THE FUNCTION OF INNATE IMMUNE CELLS DURING INFLAMMATION

Abstract

Sepsis, a multifarious clinical syndrome, is a life-threatening organ dysfunction caused by the dysregulated host response to uncontrolled infection. It is a leading cause of death, affecting more than 26 million people worldwide and claims more than 210,000 lives every year in the United States with similar rates in other countries. In India alone, the estimated sepsis cases were 11.3 million with 2.9 million deaths. The ambiguous pathobiology of the septic syndrome goes through two stages, namely, an initial hyper-inflammatory phase and a compensatory anti-inflammatory phase. Numerous reports have demonstrated an essential role of innate immune cells such as neutrophils, monocytes and macrophages in driving sepsis-associated systemic inflammation. These hyperactive events culminate in a dysfunctional host response leading to multi-organ failure and death. Despite numerous efforts in improving diagnosis and therapies, currently, there are no specific drugs available to cure or slow down the progression of sepsis. Monocytes and macrophages are the key innate immune cells playing crucial role in inflammatory processes. They play distinctively crucial role in homeostasis, immunity and driving the pathophysiology in different inflammatory conditions. During sepsis, along with neutrophils, infiltrating monocytes are believed to contribute towards the initial cytokine storm, which may lead to multi-organ dysfunction. Similarly, due to their plasticity properties and ability to switch from a classical inflammatory (M1) to an alternative phenotype (M2), macrophages are also shown to drive the immunosuppressive responses during sepsis. Integrins are the transmembrane heterodimeric glycoproteins, which mediate extravasation and interstitial migration of immune cells during inflammation. Besides, binding of surface integrins to extracellular matrices (ECM) also provides essential signals for the survival, differentiation, proliferation, and immunological functions of the infiltrating cells. Our previous report also showed altered ECM expression in murine models of sepsis showing the possibility of modulation of integrin expression. Integrin receptors are divided into 24 subtypes based on different combinations of α and β subunits and are shown to be associated with distinct tissue trafficking and cellular functions of macrophages. However, a lot more remains to be understood about the possible correlation between specific β1 and β3 integrin receptors expression on monocytes and their mobilization to different tissues in response to various stimuli. Secondly, it is also important to understand if macrophages expressing particular β1 or β3 integrin receptors polarize into regulatory phenotype during systemic inflammation.Using two widely used murine sepsis models, namely, cecal ligation and puncture (CLP) and lipopolysaccharide (LPS) induced endotoxemia, we have performed a detailed and systematic investigation of the monocyte migration and differentiation post-induction of sepsis along with its correlation with the expression dynamics of important β1 and β3 group of integrins. We have also evaluated the correlation of specific surface integrin receptors with the functional phenotype of macrophages in the inflamed peritoneum and lungs. Our results show prominent differences in the monocyte mobilization and recruitment pattern following induction of sepsis in both model. The inflammatory monocytes showed upregulation in the expression of integrin α4β1 and the macrophages in the peritoneum and lungs showed prominent integrin α5β1 and αvβ3 expression in both endotoxemia and CLP animals. The macrophages demonstrate an immunosuppressive phenotype in the lungs of the septic animals, as indicated by higher CD206 expression which correlated with higher IL-10 levels and lower pro-inflammatory cytokine transcript levels in the lungs compared to mesentery. The F480+CD206hi expressing macrophages in both peritoneum and lungs showed a higher level of αvβ3 expression. Further, using bioinformatics tools, we have analyzed and validated the expression of integrin α4, α5 and, αv with their possible association with inflammatory/anti-inflammatory functions of macrophages. The presented data will help improve the understanding of the monocyte and macrophage responses in two widely used murine models of sepsis and other infectious and inflammatory diseases. Phytochemicals have shown their potential as drug candidate due to their anti-inflammatory, antioxidant and anti-neoplastic property. So, the later part of study was focused on screening and evaluation of phytochemicals as inhibitor of α4β1 integrin. As crystal structure of the α4β1 integrin was not available, we modelled the integrin with the help of different modelling tools. The best model showed 88.1% residues in the most favoured region in the Ramachandran plot. To check the compatibility of the 3D atomic model and amino acid sequence verify 3D server was used for both chains which showed 98.67% of residues have 3D-1D score ≥ 0.2 and Errat program showed the overall quality factor to be 90.42% which suggests the good quality of model. Through text mining, around 70 phytochemicals were shortlisted based on their anti-inflammatory and migration inhibition property. Pyrx tool was used for virtual screening and Autodock Vina was used for molecular docking study. Based on the binding energy, 14 phytochemicals were shortlisted to further evaluate physiochemical properties using DruLiTo, OSIRIS and SwissADME tools. Based on the results, 3 phytochemicals, EGCG, Astilbin, and Hesperidin showed better druglikeliness and drug score. Astilbin, hesperidin, and EGCG appear to affect multiple inflammatory signaling molecules in several studies, bind with the highest affinities to VLA-4 integrin, and have the least biophysiochemical abnormalities as evaluated through MD simulation. These molecules show promising results in bioinformatics studies and hence can be further evaluated in vitro and in vivo in preclinical settings.