Gene expression patterns associated with multidrug therapy in multibacillary leprosy

Multidrug therapy (MDT) has been successfully used in the treatment of leprosy. However, although patients are cured after the completion of MDT, leprosy reactions, permanent disability, and occasional relapse/reinfection are frequently observed in patients. The immune system of multibacillary patients (MB) is not able to mount an effective cellular immune response against M. leprae. Consequently, clearance of bacilli from the body is a slow process and after 12 doses of MDT not all MB patients reduce bacillary index (BI). In this context, we recruited MB patients at the uptake and after 12-month of MDT. Patients were stratified according to the level of reduction of the BI after 12 doses MDT. A reduction of at least one log in BI was necessary to be considered a responder patient. We evaluated the pattern of host gene expression in skin samples with RNA sequencing before and after MDT and between samples from patients with or without one log reduction in BI. Our results demonstrated that after 12 doses of MDT there was a reduction in genes associated with lipid metabolism, inflammatory response, and cellular immune response among responders (APOBEC3A, LGALS17A, CXCL13, CXCL9, CALHM6, and IFNG). Also, by comparing MB patients with lower BI reduction versus responder patients, we identified high expression of CDH19, TMPRSS4, PAX3, FA2H, HLA-V, FABP7, and SERPINA11 before MDT. From the most differentially expressed genes, we observed that MDT modulates pathways related to immune response and lipid metabolism in skin cells from MB patients after MDT, with higher expression of genes like CYP11A1, that are associated with cholesterol metabolism in the group with the worst response to treatment. Altogether, the data presented contribute to elucidate gene signatures and identify differentially expressed genes associated with MDT outcomes in MB patients.

Gene expression analysis of metastases and IL-1R1 characterization in a NALP3 mouse model

Heidi Fournier

Tumor microenvironment contributes strongly to tumor evolution toward metastasis, a final stage in cancer progression. It provides essential clues to promote migration of cancer cells in metastatic sites. Moreover, inflammatory cells and immunomodulatory mediators present in the microenvironment are able to polarize the host immune response, thereby potentiating primary tumor development. The reciprocal and dynamic interactions between microenvironment and tumor cells orchestrate critical steps of evolution toward metastasis. To address the role of inflammation in tumor progression, a mouse model of downregulated inflammation was used: the NALP3 gene in these mice is knocked-out, causing a impaired processing of the two inflammatory cytokines Interleukin-1[beta] and IL-18 which are essential for the recruitment and activation of immune cells. In this case, a decrease in tumor growth and metastasis was observed in previous studies, leading us to investigate the molecular and cellular interactions of tumor and immune cells in inflammatory and non-inflammatory contexts. Gene expression analysis was performed on two steps of mouse tumor cells: the B16-F10 melanoma cells seem to be more dependent on the immune cells recruitment to metastasize in vivo, while the Lewis Lung Carcinomas cells showed a quite equivalent growth in both wild-type or NALP3 knock-out mice. Different techniques were used to do so, such as standard in vitro assays and mRNA expression analyses, and in vivo tests with extraction of tumoral mRNA by Laser Capture Microdissection. Gene expression analyses of tumor cells were performed to address the influence of the inflammatory background. Comparison of these two gene expression profiles may reveal genes related to the specific reactive inflammatory response surrounding the given metastases. Another point that was investigated was the importance of the IL-1R1, receptor of interleukin-1. Since it represents the counterpart of IL-1[beta] signaling, its expression by immune and tumor cells may correlate with degree of tumor growth and invasion. In vitro and in vivo assays allowed better understanding the importance of this receptor. While a downregulation of this receptor impairs proliferation of in vitro cell cultures, at the in vivo level, the number of metastases is decreased too. These results provide potential clues indicating the important role played by the inflammatory cells, and how they can support and promote tumor progression, in particular through the expression of NALP3. The adaptation mechanisms of tumor cells to their microenvironment were also assessed, opening different insights for targeting the tumor itself as well as the inflammatory microenvironment.

2011

Biophysical Regulation of Lymphatic Vessel Function

Dimana Miteva

Lymphatic capillaries collect interstitial fluid and dendritic cells from the periphery and deliver them to the lymph nodes for immune surveillance and tolerance maintenance. Upon injury and inflammation, lymphatic drainage can increase rapidly, and thus the lymphatic capillaries experience a broad range of fluid stresses and need to respond rapidly to such changes. Our understanding of their functional biology, adaptive ability and response to pathological changes in the biophysical environment is still very limited. This thesis focuses on how the lymphatic capillary endothelium senses and responds to changes of its biophysical environment with respect to its function, including fluid and cell transport, in the context of physiological and pathophysiological conditions. Using in vitro and in vivo models, we demonstrate that lymphatic endothelium is exquisitely sensitive to transmural flow, modulating both fluid and cell transport functions even in the absence of inflammatory cytokines. Flow increased lymphatic permeability and dendritic cell (DC) transmigration, which were coincident with changes in gene and protein expression of factor known to be involved in these, including chemoattractant chemokines, adhesion molecules and junctional proteins. These data provide the first evidence that lymphatic endothelium can regulate its transport functions in response to flow cues. They also show for the first time that transmural flow can, in the absence of inflammatory cues like TNF-a, drive expression of DC adhesion molecules like ICAM-1 and chemokines like CCL21 by lymphatic endothelium. Based on these findings we suggest that lymphatic flow is an important mediator of lymphatic function, particularly with respect to DC recruitment and trafficking to the lymph node. Furthermore, the response of the lymphatic endothelium to flow may be specific to the type of low, as lymphatic endothelial cells (LECs) might react differently to luminal shear flow, at physiological and pathophysiological levels, compared to transmural flow. To address this we used laminar shear stress chambers to expose only the apical surface of the lymphatic endothelial cells (LECs) to shear stress and demonstrated that lymphatic endothelium is indeed sensitive to luminal flow, fine-tuning its expression of adhesion molecules to modulate DC adhesion according to the shear stress. Specifically, at extremely low shear, adhesion was enhanced, unlike at higher shear, adhesion was downregulated. This is consistent with the notion that DC adhesion to LECs is only needed for entry but not transport in conducting vessels, since only the absorbing capillaries have low shear stresses. The interactions between DCs and LECs were strongly mediated by CCL21 and its receptor CCR7. These findings suggest that luminal shear stress acts as an active mechanosignal in LECs to potentially facilitate the traffic of immune cells inside of the lymphatic vessel to reach the lymph node and trigger an immune response. Additionally, we demonstrated that lymphatic vessels respond differentially to immunogenic and tolerogenic inflammatory stimulus. We used in vitro models to evaluate lymphatic uptake and lymphatic participation in dendritic cell transmigration in the presence of various inflammatory stimuli. We found that lymphatic endothelium exposed to TNF-a or complement activation were more conductive to dendritic cell transmigration, while LPS and TGF-β had opposite effect, affecting DCs to decrease DC transmigration. We further presented some insights in similarities in tumor and dendritic cells intravasation into inflamed lymphatic capillaries. These findings imply that lymphatic activation and participation in immune cells transport is not a universal phenomenon, but is strongly dependent on the nature of the inflammation. In conclusion, this work highlights the critical role of biophysical environment in lymphatic endothelial function, and how exquisitely sensitive the lymphatic capillaries are to their extracellular environment and how they use multiple cues to sense inflammation and danger, modulating their transport functions accordingly to regulate the delivery of antigens and immune cells to the lymph nodes.

EPFL2010

In vitro lymphatic endothelial morphogenesis

Carolyn Yong Pullin

Cell organization into functional multicellular three-dimensional structures is a long-standing challenge. In particular, engineering of vascularized tissues requires both blood and lymphatic neovascular network formation in a simultaneous and coordinated fashion. While extensive in vitro investigations in blood and lymphangiogenesis have identified a vast array of cellular phenomena and key cellular and extracellular bioactive substances, the dynamic natural cellular environment and its regulatory role in governing cellular response and interactions remain largely unclear. Furthermore, the wealth of existing knowledge describing the molecular regulation of vessel morphogenesis is heavily biased towards the angiogenic growth of blood capillaries, despite the importance of the lymphatic system. This thesis first addresses the differential gene regulation of isolated microvascular lymphatic endothelial cells (LECs) in response to the lymphatic growth factor VEGF-C and to fluid shear stress using gene arrays and QRT-PCR. First, characteristic of a response to a growth factor stimulus, the largest numbers of differentially expressed genes in response to VEGF-C stimulation were transcription factors and cell cycle related. A number of genes known to be important in angiogenesis, tumorigenesis and tumor invasion, and transport of proteins, solutes, and lipids were also affected. Interestingly, a number of genes related to lipid metabolism as well as neurogenesis were also responsive to VEGF-C stimulation. Further analysis of these genes may not only provide insight into the molecular mechanisms underlying lymphangiogenesis and associated pathogenesis, but may also identify other important roles of VEGF-C. The ability of lymphatic endothelium to sense and actively regulate drainage function is poorly understood, and shear stress is likely a key indicator of lymphatic drainage function. Thus, LECs were exposed to 0, 2 and 20 dyn/cm2 shear stress as representative of chronic lymphedema, normal, and acute inflammatory conditions, respectively. Important adaptive responses to both low and high shear stress conditions that were correlated to lymphatic function, including changes in gene expression patterns related to water and solute transport, cell-cell and cell-matrix adhesion, inflammatory cytokines and cell cycle were found. These changes were consistent with increased transport function, both active and passive, in high shear conditions and indicate that during lymphedema, lymphatic endothelium shuts down transport functions. These data demonstrate a functional-adaptive response of lymphatic endothelium to flow conditions, thus indicating that the lymphatic endothelium plays an active role in regulating their drainage function. It has been previously shown in our lab that in vitro blood and lymphatic morphogenesis are differentially enhanced by interstitial flow. Using a three-dimensional tissue culture model, the molecular mechanisms orchestrating endothelial cell morphogenesis, specifically gene regulation of the lymphatic microvascular system was investigated, focusing on genes implicated in both cellular morphogenesis and lymphatic development. Finally, concurrent angiogenesis and lymphangiogenesis were studied in vitro to examine the influences and crosstalk between blood and lymphatic microvascular development. The data suggest that both blood endothelial cell (BEC) and LEC in vitro capillary morphogenesis are enhanced by the presence of BEC-secreted factors, and these effects are, in part, mediated through proliferative and migratory responses of which the latter was induced by VEGFR-2 and VEGFR-3 signaling for BECs and LECs, respectively. When maintained in mixed three-dimensional cultures, both homotypic and heterotypic interactions between LECs and BECs were observed. Furthermore, capillary morphogenesis and interactions were enhanced and distinct when under the additional influence of interstitial flow. In conclusion, the novelties of the work presented in this dissertation include studies of 1) overall LEC gene response to its primary growth factor VEGF-C and biophysical factor, fluid flow and 2) the genetic regulation underlying the morphogenetic processes of LECs under interstitial flow in vitro, by using unique approaches which combine bioengineering principles to address fundamental biological questions and thereby contribute to this novel niche which has so far been neglected. Furthermore, the importance of biophysical processes along with crucial biochemical signal regulation is demonstrated in the concurrent induction of angiogenesis and lymphangiogenesis. Such are the requisites for the understanding of microvascular development and the determination of useful design principles for the in vitro vascularization of engineered tissues.

EPFL2011