Functional immune cell receptors for the Tamm-Horsfall protein

Urinary tract infection (UTI) is one of the most common reasons for individuals to seek medical care. Life quality of affected persons is substantially reduced and a significant number of patients suffers from frequent UTI recurrence and kidney damage. The host defense against UTI is multilayered. Steady urinary flow, shedding of superficial epithelial cells and soluble host proteins that specifically cover uropathogenic bacteria impede microbial invasion and colonization. The involvement of innate and adaptive immune mechanisms represents further actions to establish protective immunity. Interaction of pathogen associated molecular patterns (PAMPs) with their corresponding pathogen recognition receptors (PRRs) is required for adequate activation of innate immune cells. Even more, concomitant ligation of different PRRs appears to optimize immune responsiveness in response to microbial challenge. The urine resident Tamm Horsfall protein (THP) is supposed to fulfill important tasks for prevention of microbial colonization of the urogenital tract. THP is able to cover uropathogenic bacteria and inhibits attachment to uroepithelial receptors. Recently published data suggest further that THP activates DCs via a TLR4 dependent way. In preliminary experiments, carried out in the Institute of Immunology, it was shown that although TLR4 signaling is induced upon THP administration, TLR4 is not the THP binding receptor and that there are other, yet unknown THP receptors. Hence, we want to identify and characterize receptors responsible for THP binding in this project. For the identification of unknown receptor molecules we will make use of a retroviral expression library, established at the Institute of Immunology from monocyte-derived DCs. To characterize candidate receptors, we will determine the affinity of THP for the newly discovered THP receptors, perform co-localization studies to describe eventual spatial neighborhood of TLR4, THP and the candidate receptor and screen for the expression of candidate THP receptors on peripheral blood cells by FACS and RT-PCR. Finally, we will functionally characterize the candidate THP receptors by evaluating the dependency and independency of THP receptor mediated signalling to TLR4. Thus, NF-B reporter construct expression after transfection of HEK293 cells with TLR4/MD2 and the candidate THP receptor will be employed, whereas extensive investigations on PRR-related signalling mechanisms in TLR4 defective models will be performed to test for TLR4 dependent and independent effects, respectively. The perspective of the proposed studies, therefore, is to provide further insight into the mode of action of THP by revealing molecular mechanisms of cell activation through PRRs. Because of the apparent pivotal role of THP in UTI, a better understanding of this endogenous immunomodulator can also be expected to contribute to the further development of therapeutic strategies applicable for this life quality diminishing disease.

Urinary tract infection (UTI) is one of the most common reasons for individuals to seek medical care. Life quality of affected persons is substantially reduced and a significant number of patients suffers from frequent UTI recurrence and kidney damage. The host defense against UTI is multilayered. Steady urinary flow, shedding of superficial epithelial cells and soluble host proteins that specifically cover uropathogenic bacteria impede microbial invasion and colonization. The involvement of innate and adaptive immune mechanisms represents further actions to establish protective immunity. Interaction of pathogen associated molecular patterns (PAMPs) with their corresponding pathogen recognition receptors (PRRs) is required for adequate activation of innate immune cells. Even more, concomitant ligation of different PRRs appears to optimize immune responsiveness in response to microbial challenge. The urine resident Tamm Horsfall protein (THP) is supposed to fulfill important tasks for prevention of microbial colonization of the urogenital tract. THP is able to cover uropathogenic bacteria and inhibits attachment to uroepithelial receptors. Recently published data suggest further that THP activates DCs via a TLR4 dependent way. In preliminary experiments, carried out in the Institute of Immunology, it was shown that although TLR4 signaling is induced upon THP administration, TLR4 is not the THP binding receptor and that there are other, yet unknown THP receptors. Hence, we want to identify and characterize receptors responsible for THP binding in this project. For the identification of unknown receptor molecules we will make use of a retroviral expression library, established at the Institute of Immunology from monocyte-derived DCs. To characterize candidate receptors, we will determine the affinity of THP for the newly discovered THP receptors, perform co-localization studies to describe eventual spatial neighborhood of TLR4, THP and the candidate receptor and screen for the expression of candidate THP receptors on peripheral blood cells by FACS and RT-PCR. Finally, we will functionally characterize the candidate THP receptors by evaluating the dependency and independency of THP receptor mediated signalling to TLR4. Thus, NF-B reporter construct expression after transfection of HEK293 cells with TLR4/MD2 and the candidate THP receptor will be employed, whereas extensive investigations on PRR-related signalling mechanisms in TLR4 defective models will be performed to test for TLR4 dependent and independent effects, respectively. The perspective of the proposed studies, therefore, is to provide further insight into the mode of action of THP by revealing molecular mechanisms of cell activation through PRRs. Because of the apparent pivotal role of THP in UTI, a better understanding of this endogenous immunomodulator can also be expected to contribute to the further development of therapeutic strategies applicable for this life quality diminishing disease.