The innate immune system is highly evolved to counter infectious pathogens by recruiting neutrophils in a timely and coordinated manner. Multiple chemokines, expressed in response to infection, mediate this process, by first recruiting neutrophils to the infection site, and then activating them to release cytotoxic granule enzymes and superoxide to kill the offending microbe. Impaired recruitment and/or impaired activation result in incomplete resolution of infection, whereas uncontrolled recruitment and/or sustained activation result in destruction of healthy tissue and disease. At this time, the molecular mechanisms by which coordinated action of chemokines mediate neutrophil function are not known. In mice, the chemokines KC/CXCL1 and MIP2/CXCL2 coordinate neutrophil function. Our preliminary data show that KC and MIP2 exist as monomers and dimers, and most interestingly, also as heterodimers. In this R21 exploratory grant, we will test the hypothesis that the structural and functional properties of KC and MIP2 and the crosstalk between KC and MIP2 play non-redundant roles in mediating neutrophil function.
In Aim 1, we will characterize how KC and MIP2 mediate peritoneal neutrophil recruitment to establish the causal relationship between chemokine and neutrophil levels. More specifically, we will elucidate how changes in monomer, dimer, and heterodimer levels influence neutrophil levels and define its activation phenotype for microbial killing.
In Aim 2, we will characterize the microbial killing activity of recruited neutrophils by characterizing granule protease and superoxide activities.
In Aim 3, we will characterize the KC and MIP2 activities for CXCR2-mediated G-protein and ?-arrestin signaling pathways and ?- arrestin mediated receptor endocytosis. Our hypothesis and research strategy for characterizing chemokine crosstalk, and our preliminary data showing that recruitment profiles of KC and MIP2 can be very different are novel. Successful completion of these studies will provide critical mechanistic insights into the causal relationships between chemokine synergy and neutrophil phenotype, and serve as a framework for future development of novel and effective therapeutic targets to treat infectious diseases.
Rapid recruitment of circulating neutrophils in response to microbial infections forms the first line in host defense. Chemokines play a major role in mediating this process, and dysregulation in chemokine and neutrophil function has been implicated in various inflammatory and infectious diseases. Knowledge of the molecular mechanisms by which chemokines mediate neutrophil function is sorely lacking, and by addressing this missing knowledge, our planned studies will advance drug development for treating neutrophil-mediated diseases in a clinical setting.
| Sepuru, Krishna Mohan; Iwahara, Junji; Rajarathnam, Krishna (2018) Direct detection of lysine side chain NH3+ in protein-heparin complexes using NMR spectroscopy. Analyst 143:635-638 |
| Sepuru, Krishna Mohan; Nagarajan, Balaji; Desai, Umesh R et al. (2018) Structural basis, stoichiometry, and thermodynamics of binding of the chemokines KC and MIP2 to the glycosaminoglycan heparin. J Biol Chem 293:17817-17828 |
| Brown, Aaron J; Sepuru, Krishna Mohan; Sawant, Kirti V et al. (2017) Platelet-Derived Chemokine CXCL7 Dimer Preferentially Exists in the Glycosaminoglycan-Bound Form: Implications for Neutrophil-Platelet Crosstalk. Front Immunol 8:1248 |
