Binding of erythropoietin to the erythropoietin receptor (EpoR) extracellular domain orients the transmembrane (TM) and cytosolic regions of the receptor dimer into an unknown activated conformation. By replacing the EpoR extracellular domain with a dimeric coiled coil, we engineered TM EpoR fusion proteins where the helical TM domains were constrained into seven possible relative orientations. We identify one dimeric TM conformation that imparts full activity to the cytosolic domain of the receptor and signals via JAK2, STAT proteins, and MAP kinase, one partially active orientation that preferentially activates MAP kinase, and one conformation corresponding to the inactive receptor. The active and inactive conformations were independently identified by computational searches for low-energy TM dimeric structures. We propose a specific EpoR-activated interface and suggest its use for structural and signaling studies.

Chronic implant-related bone infections are a major problem in orthopedic and trauma-related surgery with severe consequences for the affected patients. As antibiotic resistance increases in general and because most antibiotics have poor effectiveness against biofilm-embedded bacteria in particular, there is a need for alternative and innovative treatment approaches. Recently, the immune system has moved into focus as the key player in infection defense and bone homeostasis, and the targeted modulation of the host response is becoming an emerging field of interest. The aim of this review was to summarize the current knowledge of impaired endogenous defense mechanisms that are unable to prevent chronicity of bone infections associated with a prosthetic or osteosynthetic device. The presence of foreign material adversely affects the immune system by generating a local immune-compromised environment where spontaneous clearance of planktonic bacteria does not take place. Furthermore, the surface structure of the implant facilitates the transition of bacteria from the planktonic to the biofilm stage. Biofilm formation on the implant surface is closely linked to the development of a chronic infection, and a misled adaption of the immune system makes it impossible to effectively eliminate biofilm infections. The interaction between the immune system and bone cells, especially osteoclasts, is extensively studied in the field of osteoimmunology and this crosstalk further aggravates the course of bone infection by shifting bone homeostasis in favor of bone resorption. T cells play a major role in various chronic diseases and in this review a special focus was therefore set on what is known about an ineffective T cell response. Myeloid-derived suppressor cells (MDSCs), anti-inflammatory macrophages, regulatory T cells (T reg s) as well as osteoclasts all suppress immune defense mechanisms and negatively regulate T cell-mediated immunity. Thus, these cells are considered to be potential targets for immune therapy. The success of immune checkpoint inhibition in cancer treatment encourages the transfer of such immunological approaches into treatment strategies of other chronic diseases. Here, we discuss whether immune modulation can be a therapeutic tool for the treatment of chronic implant-related bone infections.

Hematopoietic cytokine receptors, such as the erythropoietin receptor (EpoR), are single membrane-spanning proteins. Signal transduction through EpoR is crucial for the formation of mature erythrocytes. Structural evidence shows that in the unliganded form EpoR exists as a preformed homodimer in an open scissor-like conformation precluding the activation of signaling. In contrast to the extracellular domain of the growth hormone receptor (GHR), the structure of the agonist-bound EpoR extracellular region shows only minimal contacts between the membrane-proximal regions. This evidence suggests that the domains facilitating receptor dimerization may differ between cytokine receptors. We show that the EpoR transmembrane domain (TM) has a strong potential to self interact in a bacterial reporter system. Abolishing self assembly of the EpoR TM by a double point mutation (Leu 240-Leu 241 mutated to Gly-Pro) impairs signal transduction by EpoR in hematopoietic cells and the formation of erythroid colonies upon reconstitution in erythroid progenitor cells from EpoR(-/-) mice. Interestingly, inhibiting TM self assembly in the constitutively active mutant EpoR R129C abrogates formation of disulfide-linked receptor homodimers and consequently results in the loss of ligand-independent signal transduction. Thus, efficient signal transduction through EpoR and possibly other preformed receptor oligomers may be determined by the dynamics of TM self assembly.