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archives@tulane.edu / The prevalence of HIV-associated neurocognitive disorders (HAND) has increased in the era of combination anti-retroviral therapy (cART). Despite this and documented neurocognitive impairment, there is a lack of pathology of HIV-encephalitis (HIVE), specifically multi-nucleated giant cells (MNGCs), in children and SIV-encephalitis (SIVE) in rhesus macaques infected pre-, peri-, and post-parturition. In this dissertation, we show that the lack of MNGCs seen is most likely due to innate differences in the blood-brain and blood-CSF barriers, and a robust pro- and anti-inflammatory response in neonatal rhesus macaques. Using a rhesus macaque model of HIV, we examined the plasma viral load, brain tissue viral load, and monocyte turnover, using PCR and flow cytometry, respectively. We also performed immunohistochemistry for monocyte, macrophage, tight junction, and aging markers of the choroid plexus. We sought to create a choroid plexus epithelial cell model to monitor the effects of inflammatory markers and virus on the tight junctions of the blood-CSF barrier in real-time. We demonstrated that neonates do not develop encephalitis, despite comparable viral load and monocyte turnover, previously established correlates of SIV-encephalitis (SIVE). However, we noted that uninfected adult rhesus macaques have an increase in virus susceptible cells in the brain, SIV-infected adults have a leakier blood-brain barrier than infected neonates, and adults with encephalitis have a greater viral burden in brain tissue compared to adults without encephalitis. In the choroid plexus, we discovered that despite the lack of encephalitis, neonates have an increase in monocytes and macrophages of the choroid plexus, indicating a strong immune response. While our choroid plexus epithelial cell model is still in preliminary stages, initial results are promising. Our work indicates a possible viral threshold needed for the development of encephalitis, and that the blood-brain barrier may play a role in this threshold due to lower levels of virus susceptible cells and a tighter blood brain barrier in neonates. In the choroid plexus, the strong pro- and anti-inflammatory macrophage response seen in neonates may offer an extra layer of protection development of SIVE. Our data also indicates that SIV causes a marked decrease in the expression of klotho, the anti-aging hormone that is produced in high levels in the choroid plexus in the brain. This could potentially explain the premature inflammaging phenotype seen in chronic infections. / 1 / Elizabeth Delery

In order to achieve cure for HIV-1 infection in patients undergoing suppressive antiretroviral therapy, eradication of all latently infected reservoirs of the virus is required. The focus of HIV cure is predominantly centred on the elimination of latently infected memory T cells, while information on possible elimination of infected macrophages is lacking. Macrophages support continuous virus replication without succumbing to cytopathic effects of HIV-1. Recently, our laboratory has shown a protective role for cellular inhibitor of apoptosis proteins (IAPs) 1/2 in macrophages against Vpr-induced apoptosis. Depletion of cIAP1/2 by Smac mimetics (SM) reverse the IAP-mediated protection and sensitize macrophages to Vpr-induced cell death. My research aims to understand the role IAPs play in apoptotic resistance of HIV-infected macrophages. I hypothesized that ablation of cIAP1/2 by SM may induce apoptosis in HIV-infected macrophages. My results show that SM does not induce cell death in uninfected or healthy macrophages, but induces cell death in chronically infected U1 cells, in vitro infected monocyte-derived macrophages, and ex vivo derived HIV-infected macrophages from HIV-infected individuals. SM induce cell death of infected myeloid cells through apoptosis and not through necroptosis. Moreover, SM-induced apoptosis is independent of TNFα and other endogenously secreted cytokines. In vitro infection of monocyte-derived macrophages leads to the downregulation of RIPK1, RIPK3, and TRAF-1. Interestingly, necrostatin-1-mediated RIPK1- inhibition does not affect viability of healthy macrophages, but in combination with IAP degradation by SM leads to significant induction of apoptosis. This suggests a key role for RIPK1 in SM-induced apoptosis of HIV-infected macrophages. Altogether, the results from this project suggest that modulation of the IAP-associated signalling pathways by SM may be a potential strategy for selective killing of HIV-infected macrophages.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Problématique : Le surfactant pulmonaire est une structure vitale essentielle à l’homéostasie pulmonaire. Étant composé majoritairement de phospholipides, il est particulièrement susceptible à l’oxydation puisqu’il est en constante interaction avec l’environnement extérieur. Par des mécanismes encore inconnus, les macrophages pulmonaires jouent un rôle majeur dans l’élimination du surfactant pulmonaire endommagé. Objectifs : 1) Caractériser la réponse transcriptionnelle et fonctionnelle des macrophages au surfactant pulmonaire sain et traité au peroxynitrite (ONOO⁻) et 2) investiguer le rôle de l’endocytose dans l’initiation de cette réponse. Méthodes : Des macrophages murins isolés par lavages bronchoalvéolaires ou différenciés de la moelle osseuse ont été exposés à du surfactant pulmonaire sain ou traité au ONOO⁻, avec ou sans Latrunculine A, un inhibiteur des mécanismes d’endocytose dépendant de l’actine. L’expression de gènes impliqués dans la capture (marco, msr1, cd36), l’accumulation intracellulaire (plin2) et l’export (abca1, abcg1, srb1) lipidique a été évaluée par qPCR, ainsi que les capacités fonctionnelles d’export lipidique. Résultats : L’addition de surfactant pulmonaire sain ou traité au ONOO⁻ augmente l’expression de marco, msr1, cd36 et plin2 et diminue l’expression d’abca1, abcg1 et scarb1 chez les macrophages. Toutefois, certaines différences transcriptionnelles ont été observées entre les macrophages pulmonaires primaires et ceux dérivés de moelle osseuse. Également, qu’il soit sain ou oxydé, le surfactant engendre une diminution des capacités d’efflux de cholestérol des macrophages différenciés. De plus, ces impacts transcriptionnels et fonctionnels ne semblent pas affectés par un traitement à la Latrunculine A. Conclusion : En présence de surfactant, et ce, indépendamment son niveau d’oxydation, les macrophages pulmonaires semblent favoriser les mécanismes de capture et d’accumulation lipidique. Ainsi, aux dépens de ces mécanismes priorisés, les deux espèces présentent conjointement une diminution à la fois transcriptionnelle et fonctionnelle des mécanismes d’export lipidique. / Problematic: Pulmonary surfactant is a vital structure essential to reduce the surface tension at the liquid-air interface. It is mainly composed of phospholipids and is susceptible to oxidation due to its close interaction with the external environment. Pulmonary macrophages play a major role in degrading damaged surfactant. However, the mechanisms used by pulmonary macrophages to detect and initiate its degradation are still unknown. Objectives: 1) To characterize the transcriptional and functional response of macrophages to native and peroxynitrite (ONOO⁻)-treated pulmonary surfactant and 2) to investigate the role of endocytosis in the initiation of this response. Methods: Primary mouse pulmonary macrophages isolated from bronchoalveolar lavages or differentiated from the bone marrow were exposed to native or ONOO⁻-treated pulmonary surfactant with or without endocytosis inhibitors (Latrunculin A). Expression of key genes implicated in lipid capture (msr1, marco, cd36), intracellular lipid accumulation (plin2), and lipid export (abca1, abcg1, scarb1) was assessed by quantitative PCR, as well as functional lipid export capacities. Results: The addition of native and ONOO⁻-treated pulmonary surfactant increased marco, msr1, cd36 and plin2 expression and decreased abca1, abcg1 and scarb1 expression in macrophages. However, some transcriptional differences were observed between primary and bone marrow macrophages. Also, both native and ONOO--treated pulmonary surfactant generated a decrease in differentiated macrophages cholesterol efflux capacities. Moreover, those transcriptional and functional impacts do not seem to be affected by Latrunculin A treatment. Conclusion: In the presence of pulmonary surfactant and this, independently its oxidation level, lung macrophages seem to promote lipid capture and storage mechanisms. Thus, depending on these prioritized mechanisms, the two species jointly showed a transcriptional and functional decrease in lipid export mechanisms.

Macrophages arise from two distinct lineages of hematopoietic cells, Hematopoietic Stem Cell-derived progenitors and Erythromyeloid Progenitors. EMP derived macrophages are seeded in tissues early during embryogenesis and become specialized tissue resident macrophages such as microglia and Kupffer cells. HSC derived monocytes arise in the bone marrow and normally circulate via the bloodstream to reach tissues of the body and differentiate into macrophages which are cells that are constantly replenished by their progenitors. Monocytes are also recruited to inflamed tissues attracted by inflammatory signals, where they become macrophages and further contributed to the inflammatory process. These two macrophage populations may be implicated in different activities and have different functions, such as tissue repair or proinflammatory responses, respectively. Other cells that are unique in having both origins are osteoclasts, which during embryogenesis originate from EMP precursors and contribute to the formation of ossification centers of long bones and consequently the formation of bone marrow cavity. After birth, HSC derived monocytes contribute to their cell maintenance by direct cell fusion to pre-existing EMP-derived osteoclasts, eventually replacing them to become fully derived from HSC precursors, a mechanism that takes several month to complete in mice. Cell lineage tracing is a powerful technique that allows for the labeling of specific cell populations in a specific time and space. The gold standard cell lineage tracing mouse model for the study of EMP derived cells including macrophages and osteoclasts in mice, Csf1rMeriCreMer;Rosa26LSL-YFP,1–3 uses a tamoxifen inducible Cre recombinase to induce expression of YFP and therefore permanently labels EMP progenitors and they progeny. Other mouse models to label macrophages and osteoclast have been used, which include Cx3cr1CreERT2;Rosa26LSL-tdTomato, however, recent evidence show spontaneous expression of tdTomato fluorescent protein in the absence of tamoxifen (unpulsed mice). Here we confirmed that Cre recombination resulted in tdTomato expression in the absence of tamoxifen in Cx3cr1CreERT2;Rosa26LSL-tdTomato mice. Therefore, we utilized a different fluorescent reporter gene instead, Rosa26LSL-YFP, and generated Cx3cr1CreERT2;Rosa26LSL-YFP mice for permanent labelling of EMP-derived cells, and provide evidence that no YFP expression is detected in unpulsed mice. Further, we performed pulse labeling of the Cx3cr1CreERT2;Rosa26LSL-YFP model at E10.5 days of gestation. These experiments were notable for YFP expression in macrophages and the highest labeling efficiency reported in osteoclasts (~70%) at E18.5 embryonic days of development. Further, there was no YFP expression detected in HSCs or HSC-derived cells, confirming this model as a potential useful tool for precise and efficient labelling of EMP-derived macrophages and osteoclasts

Titanium and 316L stainless steel are popular orthopedic implant alloys because of their mechanical properties and corrosion resistance. The central hypotheses of this research were to determine if the adsorption of cells onto implant surfaces would alter their electrochemical corrosion properties and if released metal ions would stimulate macrophages. Analysis of supernatants and electrochemical corrosion tests were conducted on 316L SS and Ti with macrophages attached to evaluate their interactions. Results indicated that cells attached to alloys do alter their corrosion behavior by significantly increasing equilibrium potentials. Cells attached to 316L SS significantly increased charge transfer and the release of Ni, which is known to cause hypersensitivity. A difference in cell stimulation was seen between controls cells on TCP and cells cultured on the alloys. Significant findings of this study include alterations in alloy corrosion behavior and cell stimulation.

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