Rickettsia massiliae and Rickettsia conorii Israeli Spotted Fever Strain Differentially Regulate Endothelial Cell Responses

RESEARCH ARTICLE Rickettsia massiliae and Rickettsia conorii Israeli Spotted Fever Strain Differentially Regulate Endothelial Cell Responses Jeremy Bechelli, Claire Smalley, Natacha Milhano, David H. Walker, Rong Fang* Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America * Abstract OPEN ACCESS Citation: Bechelli J, Smalley C, Milhano N, Walker DH, Fang R (2015) Rickettsia massiliae and Rickettsia conorii Israeli Spotted Fever Strain Differentially Regulate Endothelial Cell Responses. PLoS ONE 10(9): e0138830. doi:10.1371/journal. pone.0138830 Editor: Ulrike Gertrud Munderloh, University of Minnesota, UNITED STATES Received: June 11, 2015 Accepted: September 3, 2015 Published: September 22, 2015 Copyright: © 2015 Bechelli et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Rickettsiae primarily target microvascular endothelial cells. However, it remains elusive how endothelial cell responses to rickettsiae play a role in the pathogenesis of rickettsial diseases. In the present study, we employed two rickettsial species with high sequence homology but differing virulence to investigate the pathological endothelial cell responses. Rickettsia massiliae is a newly documented human pathogen that causes a mild spotted fever rickettsiosis. The “Israeli spotted fever” strain of R. conorii (ISF) causes severe disease with a mortality rate up to 30% in hospitalized patients. At 48 hours post infection (HPI), R. conorii (ISF) induced a significant elevation of IL-8 and IL-6 while R. massiliae induced a statistically significant elevated amount of MCP-1 at both transcriptional and protein synthesis levels. Strikingly, R. conorii (ISF), but not R. massiliae, caused a significant level of cell death or injury in HMEC-1 cells at 72 HPI, demonstrated by live-dead cell staining, annexin V staining and lactate dehydrogenase release. Monolayers of endothelial cells infected with R. conorii (ISF) showed a statistically significant decrease in electrical resistance across the monolayer compared to both R. massiliae-infected and uninfected cells at 72 HPI, suggesting increased endothelial permeability. Interestingly, pharmacological inhibitors of caspase-1 significantly reduced the release of lactate dehydrogenase by R. conorii (ISF)-infected HMEC-1 cells, which suggests the role of caspase-1 in mediating the death of endothelial cells. Taken together, our data illustrated that a distinct proinflammatory cytokine profile and endothelial dysfunction, as evidenced by endothelial cell death/injury and increased permeability, are associated with the severity of rickettsial diseases. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by the Carmage and Martha Walls Distinguished University Chair in Tropical Diseases and the UTMB Institute for Human Infections and Immunity Endothelial Biology Pilot Grant. Jeremy Bechelli is supported by the UTMB T32-AI060549 Biodefense Training Grant. Competing Interests: The authors have declared that no competing interests exist. Introduction Rickettsiae are Gram-negative obligately intracellular bacteria with a predilection for infecting vascular endothelial cells [1]. Rickettsiae primarily target the vascular endothelium of small and medium sized vessels leading to vasculitis and ultimately edema in vital organs. The typical clinical manifestations of infections caused by spotted fever group rickettsiae include fever, rash, and frequently tache noire, and progress to encephalitis and pneumonitis in severe PLOS ONE | DOI:10.1371/journal.pone.0138830 September 22, 2015 1 / 15 Endothelial Cell Infection by Rickettsiae infections [2]. Pathophysiological effects of rickettsial infection on endothelial cells include an increase in vascular permeability, vascular inflammation, and pro-inflammatory cytokine production [3,4]. Rickettsia-infected endothelial cells have been reported to produce several proinflammatory cytokines and chemokines such as IL-6 [5], IL-8 and MCP-1 [6]. These responses are associated with the activation of endothelial cells [7] and enhanced endothelial cell permeability [8–10]. Furthermore, human and mouse endothelial cells exhibit anti-rickettsial activity under cytokine stimulation including TNF-α, IFN-γ and RANTES activation. Until now, it has never been completely understood how to distinguish the endothelial cell responses accounting for host immunity from those contributing to disease pathogenesis during rickettsial infection. Answering this question is crucial for better understanding the endothelial cell pathobiology in severe rickettsioses. The bacterial agents Rickettsia massiliae and R. conorii are two genetically related rickettsial species with significantly different virulence. R. massiliae and R. conorii chromosomes exhibit >98% identity in coding sequence [11]. Interestingly, the clinical consequences of infections caused by R. massiliae dramatically differ from those by R. conorii. Previously believed to be nonpathogenic, R. massiliae has been recently documented to cause human infections that have presented as mild spotted fever rickettsioses in Argentina, France, and Italy [12]. R. conorii is the etiological agent of Mediterranean spotted fever (MSF), which is considered as one of the most severe and life threatening rickettsial infections. Among four strains of R. conorii, the Israeli spotted fever strain of R. conorii (ISF) is believed to be the most virulent with a case fatality rate up to 32.3% in hospitalized patients [13]. Therefore, R. massiliae and R. conorii (ISF) were employed in the present study to investigate the contributions of endothelial cell responses to the pathogenesis of rickettsial diseases. Moreover, R. massiliae and R. conorii (ISF) occur in the same geographic regions [14]. Because serological cross-reactivity occurs across spotted fever group rickettsiae [15] and the primary means of diagnosis is through serum antibody assays, the accurate distinction between infections caused by these two species requires the identification of the actual infecting bacterium. This cross reactivity between the species and the overlap in geographic distributions highlight the need to better understand the pathological differences between these rickettsial species. A correct diagnosis is critical to predicting the pathological complications that would arise due to infection, and would allow physicians to anticipate complications and the correct response in the clinic. Vascular endothelial cells perform a number of functions required to maintain homeostasis. In response to inflammatory stimuli, endothelial cells can be activated to gain new functions such as displaying surface adhesion molecules and chemokines that lead (...truncated)