Selective activation of signal transducer and activator of transcription (STAT) proteins STAT1 and STAT3 in human endothelial cells infected with Rickettsia rickettsii.

A majority of typical pathologic sequelae of rickettsial diseases, such as altered barrier function of endothelial cells leading to compromised vascular permeability, manifest as non-cardiogenic pulmonary oedema ⁄ acute respiratory distress syndrome and parenchymal cerebral oedema, which can be attributed to disseminated infection and damage of the vascular endothelium [1]. Endothelial cells infected in vitro with R. rickettsii or R. conorii undergo profound phenotypic modifications to an ‘activated’ state due to induction of a number of immediate early responsive prothrombotic and proadhesive genes [2]. Ample published evidence also indicates an important role for interferons (IFNs) in host response and clearance of pathogenic Rickettsia species [3]. In general, both type I (a, b) and type II (c) IFNs modulate the innate immune response through Janus kinase (JAK)-STAT (Signal transducer and activator of transcription) mediated gene regulation. JAK-STAT pathway transmits signals originating from extracellular ligands and those from within the cell directly to the nucleus to control various gene promoters. There are four known Janus kinases (JAK1-3 and TYK2) and seven STAT proteins (STAT1-4, 5a, 5b, and 6). To better understand the immune response of vascular endothelium to infection with spotted fever rickettsiae, we have initiated efforts to investigate the contributions of the JAK-STAT signalling pathway to the pathophysiology of human spotted fever group (SFG) rickettsioses. Standard laboratory protocols established and described earlier were used for in vitro infection of cultured human umbilical vein-derived endothelial cells (HUVECs) [4]. Briefly, cells were incubated in antibiotic-free culture medium with approximately 6 · 10 pfu of R. rickettsii organisms for every cm of culture area. Indirect immunofluorescent staining and citrate synthase (gltA)-based quantitative PCR confirmed that this experimental protocol consistently resulted in infection of ‡80% of cells with three to five rickettsiae at 6 h [5]. Because phosphorylation of specific target residues triggers subsequent dimerisation and nuclear translocation, activation of different STAT proteins was determined by denaturing polyacrylamide gel electrophoresis of total protein lysates from R. rickettsii-infected HUVECs followed by Western blot analysis using phosphorylation state-specific antibodies. As expected, uninfected endothelial cells were found to have either very low or negligible basal phosphorylation levels of all investigated STAT proteins. In response to R. rickettsii infection, however, the phosphorylation of STAT3 and STAT1 was clearly induced (Fig. 1), while that of STAT2, STAT5 and STAT6 remained unaltered at all times (not shown). Interestingly, there were noticeable differences in the kinetics of activation of STAT1 and STAT3 in response to infection. The levels of p-STAT3 in infected cells were two to three times higher than corresponding controls at early times of 1.5 and 3 h and then declined to basal levels at 8 h post-infection (Fig. 1a). In contrast, the levels of p-STAT1 did not display significant changes early during the infection and started to increase at about 6 h post-infection with as much as four-fold and eight-fold induction at 12 h and 24 h post-infection, respectively (Fig. 1b). These results indicate that activation of only selective STAT proteins occurs in response to R. rickettsii infection and highlight the unique differences in kinetics of their activation. Corresponding author and reprint requests: Sanjeev K. Sahni, PhD, Department of Microbiology and Immunology, Room 211132, PO Box 672, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA E-mail: sanjeev_sahni@urmc.rochester. edu