Global Transcriptomic Profiling of Pulmonary Gene Expression in an Experimental Murine Model of Rickettsia conorii Infection.

Mediterranean spotted fever develops from an infection with Rickettsia conorii, an obligate intracellular, Gram-negative, endotheliotropic, and tick-transmitted bacterial pathogen, and is an acute, febrile illness that can progress to life-threatening complications if not diagnosed and treated early with effective antibiotics. Despite significant morbidity and mortality, little is known about changes in gene expression that determine the host responses during in vivo infection. We have investigated the transcriptional landscape of host lungs as a prominently affected organ system in an established murine model of infection by RNA-sequencing. Ingenuity pathway analysis resulted in the identification of 1332 differentially expressed genes and 292 upstream regulators. Notably, genes encoding for ubiquitin D, aconitate decarboxylase, antimicrobial peptides, calgranulins, cytokines and chemokines, and guanylate binding proteins were highly up-regulated, whereas those involved in hemoglobin biosynthesis and heme homeostasis were significantly down-regulated. Amongst response regulators, nucleotide-binding oligomerization domain-containing protein 2 and killer cell lectin-like receptors were differentially expressed, and gene clustering revealed eukaryotic initiation factor-2, oxidative phosphorylation, and ubiquitination as the predominantly activated biological pathways. Collectively, this first global transcriptomic profiling has identified R. conorii-induced regulation of novel genes and pathways in the host lungs, further in-depth investigation of which will strengthen our understanding of the pathogenesis of human rickettsioses.

Fibroblast growth factor receptor-1 mediates internalization of pathogenic spotted fever rickettsiae into host endothelium.

Rickettsial infections continue to cause serious morbidity and mortality in severe human cases around the world. Host cell adhesion and invasion is an essential requisite for intracellular growth, replication, and subsequent dissemination of pathogenic rickettsiae. Heparan sulfate proteoglycans [HSPGs] facilitate the interactions between fibroblast growth factor(s) and their tyrosine kinase receptors resulting in receptor dimerization/activation and have been implicated in bacterial adhesion to target host cells. In the present study, we have investigated the contributions of fibroblast growth factor receptors [FGFRs] in rickettsial entry into the host cells. Inhibition of HSPGs by heparinase and FGFRs by AZD4547 (a selective small-molecule inhibitor) results in significant reduction in rickettsial internalization into cultured human microvascular endothelial cells (ECs), which represent the primary targets of pathogenic rickettsiae during human infections. Administration of AZD4547 during R. conorii infection in a murine model of endothelial-target spotted fever rickettsiosis also diminishes pulmonary rickettsial burden in comparison to mock-treated controls. Silencing of FGFR1 expression using a small interfering RNA also leads to similar inhibition of R. rickettsii invasion into ECs. Consistent with these findings, R. rickettsii infection of ECs also results in phosphorylation of tyrosine 653/654, suggesting activation of FGFR1. Using isobaric tag for relative and absolute quantitation [iTRAQ]-based proteomics approach, we further demonstrate association of ß-peptide of rickettsial outer membrane protein OmpA with FGFR1. Mechanistically, FGFR1 binds to caveolin-1 and mediates bacterial entry via caveolin-1 dependent endocytosis. Together, these results identify host cell FGFR1 and rickettsial OmpA as another novel receptor-ligand pair contributing to the internalization of pathogenic rickettsiae into host endothelial cells and the potential application of FGFR-inhibitor drugs as adjunct therapeutics against spotted fever rickettsioses.

Rickettsia conorii is a potent complement activator in vivo and combined inhibition of complement and CD14 is required for attenuation of the cytokine response ex vivo.

Mediterranean spotted fever caused by Rickettsia conorii is a potentially lethal disease characterized by vascular inflammation affecting multiple organs. Studies of R. conorii so far have focused on activation of inflammatory cells and their release of inflammatory cytokines, but complement activation has not been investigated in R. conorii-infected patients. Here, we performed a comprehensive analysis of complement activation markers and the soluble cross-talking co-receptor CD14 (sCD14) in plasma from R. conorii-infected patients. The clinical data were supplemented with ex vivo experiments where the cytokine response was characterized in human whole blood stimulated with R. conorii. Complement activation markers at the level of C3 (C3bc, C3bBbP) and terminal pathway activation (sC5b-9), as well as sCD14, were markedly elevated (p <0.01 for all), and closely correlated (p <0.05 for all), in patients at admission compared with healthy matched controls. All tested markers were significantly reduced to baseline values at time of follow up. Rickettsia conorii incubated in human whole blood was shown to trigger complement activation accompanied by release of the inflammatory cytokines interleukin-1ß (IL-1ß), IL-6, IL-8 and tumour necrosis factor. Whereas inhibition of either C3 or CD14 had only a minor effect on released cytokines, combined inhibition of C3 and CD14 resulted in significant reduction, virtually to baseline levels, of the four cytokines (p <0.05 for all). Our data show that complement is markedly activated upon R. conorii infection and complement activation is, together with CD14, responsible for a major part of the cytokine response induced by R. conorii in human whole blood.

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

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.

Unlocking proteomic heterogeneity in complex diseases through visual analytics.

Despite years of preclinical development, biological interventions designed to treat complex diseases such as asthma often fail in phase III clinical trials. These failures suggest that current methods to analyze biomedical data might be missing critical aspects of biological complexity such as the assumption that cases and controls come from homogeneous distributions. Here we discuss why and how methods from the rapidly evolving field of visual analytics can help translational teams (consisting of biologists, clinicians, and bioinformaticians) to address the challenge of modeling and inferring heterogeneity in the proteomic and phenotypic profiles of patients with complex diseases. Because a primary goal of visual analytics is to amplify the cognitive capacities of humans for detecting patterns in complex data, we begin with an overview of the cognitive foundations for the field of visual analytics. Next, we organize the primary ways in which a specific form of visual analytics called networks has been used to model and infer biological mechanisms, which help to identify the properties of networks that are particularly useful for the discovery and analysis of proteomic heterogeneity in complex diseases. We describe one such approach called subject-protein networks, and demonstrate its application on two proteomic datasets. This demonstration provides insights to help translational teams overcome theoretical, practical, and pedagogical hurdles for the widespread use of subject-protein networks for analyzing molecular heterogeneities, with the translational goal of designing biomarker-based clinical trials, and accelerating the development of personalized approaches to medicine.

Changes of Bcl-2, Bax and Caspase-3 expression in the dermal microvascular endothelial cells and the epidermal layers of the eschar (tache noire) in patients with Mediterranean spotted fever.

Mediterranean spotted fever (MSF) is widely prevalent in many endemic regions in Bulgaria. The disease is still not quite thoroughly studied as to some aspects of its pathogenesis and especially to issues that concern the crucial signals for apoptosis in the target microvascular endothelial cells. To study the expression of Bcl-2 family proteins and Caspase-3 in the dermal capillary endothelial cells from skin papules and in the eschar (tache noire) epidermal layers of patients with MSF so that we can establish apoptotic processes and the time of their occurrence and deployment. Immunohistochemical reactions for Bcl-2, Bax and Caspase-3 were obtained in slices of punch-biopsies taken from papules of the skin rash and from the eschars of eight patients with MSF. The average intensity of the reactions was compared with that in control punch-biopsy slices from four healthy subjects. MSF was etiologically confirmed in all patients by positive antibody response to a specific antigen, Rickettsia conorii, with indirect immunofluorescent assay performed by the Rickettsial Reference Laboratory. The immune reaction for Bcl-2 was found to be poorly expressed in the capillary endothelial cells of skin papules of patients without any differences from controls. The expression of Bax and Caspase-3 was strongly upregulated in comparison with the controls. The Bcl-2/Bax ratio was significantly decreased. Microvascular endothelial cells of the eschar showed similar changes. While the Bcl-2/Bax ratio decreased in the epidermal layers of the eschar "tache noire", there were no changes in the intensity of the immunoreactivity of Caspase-3 as compared with controls. The upregulation of Bax and Caspase-3 is an indication of ongoing apoptotic processes in the dermal microvascular endothelial cells of MSF patients. The epidermal layers of the eschar showed increased sensitivity to apoptosis, however, executive phase of apoptosis did not occur.

Compartmentalized, functional role of angiogenin during spotted fever group rickettsia-induced endothelial barrier dysfunction: evidence of possible mediation by host tRNA-derived small noncoding RNAs.

BACKGROUND: Microvascular endothelial barrier dysfunction is the central enigma in spotted fever group (SFG) rickettsioses. Angiogenin (ANG) is one of the earliest identified angiogenic factors, of which some are relevant to the phosphorylation of VE-cadherins that serve as endothelial adherens proteins. Although exogenous ANG is known to translocate into the nucleus of growing endothelial cells (ECs) where it plays a functional role, nuclear ANG is not detected in quiescent ECs. Besides its nuclear role, ANG is thought to play a cytoplasmic role, owing to its RNase activity that cleaves tRNA to produce small RNAs. Recently, such tRNA-derived RNA fragments (tRFs) have been shown to be induced under stress conditions. All these observations raise an intriguing hypothesis about a novel cytoplasmic role of ANG, which is induced upon infection with Rickettsia and generates tRFs that may play roles in SFG rickettsioses. METHODS: C3H/HeN mice were infected intravenously with a sublethal dose of R. conorii. At days 1, 3, and 5 post infection (p.i.), liver, lung and brain were collected for immunofluorescence (IF) studies of R. conorii and angiogenin (ANG). Human umbilical vein endothelial cells (HUVECs) were infected with R. conorii for 24, 48, and 72 hrs before incubation with 1µg/ml recombinant human ANG (rANG) in normal medium for 2 hrs. HUVEC samples were subjected to IF, exogenous ANG translocation, endothelial permeability, and immunoprecipitation phosphorylation assays. To identify small non-coding RNAs (sncRNAs) upon rickettsial infection, RNAs from pulverized mouse lung tissues and HUVECs were subjected to library preparation and deep sequencing analysis using an Illumina 2000 instrument. Identified sncRNAs were confirmed by Northern hybridization, and their target mRNAs were predicted in silico using BLAST and RNA hybrid programs. RESULTS: In the present study, we have demonstrated endothelial up-regulation of ANG, co-localized with SFG rickettsial infection in vivo. We also have provided direct evidence that rickettsial infection sensitizes human ECs to the translocation of exogenous ANG in a compartmentalized pattern at different times post-infection. Typically, exogenous ANG translocates into the nucleus at 24 hrs and to the cytoplasm at 72 hrs post-infection. The ANG cytoplasmic translocation enhances phosphorylation and destabilization of VE-cadherin and attenuates endothelial barrier function. Of note, deep sequencing analysis detected tRFs, mostly derived from the 5'-halves of host tRNAs, that are induced by ANG. Northern hybridization validates the two most abundantly cloned tRFs derived from tRNA-ValGTG and tRNA-GlyGCC, in both mouse tissues and human cells. Bioinformatics analysis predicted that these tRFs may interact with transcripts associated with the endothelial barrier, the host cell inflammatory response, and autophagy. CONCLUSIONS: Our data provide new insight into the role of compartmentalized ANG during SFG rickettsioses, and highlight its possible mediation through tRFs.

A complex interaction between Rickettsia conorii and Dickkopf-1--potential role in immune evasion mechanisms in endothelial cells.

The pathophysiological hallmark of spotted fever group rickettsioses comprises vascular inflammation. Based on the emerging importance of the wingless (Wnt) pathways in inflammation and vascular biology, we hypothesized that Dickkopf-1 (DKK-1), as a major modulator of Wnt signaling, could be involved in the pathogenesis in rickettsial infections. Our major findings were: (i) While baseline concentration of DKK-1 in patients with R. conorii infection (n = 32) were not different from levels in controls (n = 24), DKK-1 rose significantly from presentation to first follow-up sample (median 7 days after baseline). (ii) In vitro experiments in human umbilical vein endothelial cells (HUVECs) showed that while heat-inactivated R. conorii enhanced the release of interleukin-6 (IL-6) and IL-8, it down-regulated the release of endothelial-derived DKK-1 in a time- and dose-dependent manner. (iii) Silencing of DKK-1 attenuated the release of IL-6, IL-8 and growth-related oncogene (GRO)α in R. conorii-exposed HUVECs, suggesting inflammatory effects of DKK-1. (iv) Silencing of DKK-1 attenuated the expression of tissue factor and enhanced the expression of thrombomodulin in R. conorii-exposed HUVECs suggesting pro-thrombotic effects of DKK-1. The capacity of R. conorii to down-regulate endothelial-derived DKK-1 and the ability of silencing DKK-1 to attenuate R. conorii-induced inflammation in endothelial cells could potentially reflect a novel mechanism by which R. conorii escapes the immune response at the site of infection.

Role of TLR4 receptor polymorphisms in Boutonneuse fever.

The genetics of the interaction between host and microbes plays an essential role in the survival of the individual and attainment of longevity. The activation of toll-like receptor (TLR)4 plays a key role in natural and clonotypic immune responses. We evaluated whether TLR4 genotype is a component of genetic background protective versus rickettsiosis and whether this background influences longevity. We genotyped for +896A/G TLR4 polymorphism 78 patients affected with Boutonneuse fever, 78 age-matched controls and 78 advanced age individuals from Sicily. The +869G allele, that attenuates receptor signalling, was significantly overrepresented in patients in comparison with age-matched controls. By analyzing data according to gender, this allele was significantly higher in female patients when compared to advanced age women. Pro-inflammatory responses are programmed to resist fatal infections. So, it is not surprising that the genetic background of people that survive to an advanced age may be protective against infections. However, this seems to occur in women but not in men. In a previous study, the +896G TLR4 allele was overrepresented in advanced age men and underrepresented in men affected by myocardial infarction. Thus, previous and present results tend to agree with the suggestion that men and women may follow different trajectories to reach longevity. For men it might be more important to control atherogenesis, whereas for women it might be more important to control infectious diseases.