Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality amongst immuno-compromised individuals and is the leading cause of congenital diseases amongst newborn infants. Mouse CMV (MCMV) infection of inbred mice has been extensively used as a model for HCMV pathogenesis and host-virus interaction. Macrophages are a key target cell type in the pathogenesis of human and mouse CMV infections. Macrophages are semi-permissive to CMV infection, however, the nature of this restrictive mechanism of infection is open for investigation. In this thesis, I hypothesized that macrophage permissivity is determined by the dynamic interplay of the innate response during the immediate-early (IE) period of infection. To test this hypothesis, I first developed and validated a flow cytometry based assay. In MCMV infected macrophages, I found heterogeneous expression from the major IE promoter (MIEP) leading to the development of a refractory subpopulation for IE expression. I further developed a computational modelling approach to help elucidate the dynamics of infection during this period. Modelling work revealed that the occurrence of refractory subpopulation could be caused by either 1) pre-existence of heterogeneous permissivity of macrophages prior to infection or 2) through an emergent process. Experimental testing of the models shows that the heterogeneous IE expression of homogeneously infected macrophages is caused by an emergent process. MCMV infection using type I interferon receptor and signal transducers and activator of transcription 1 (Stat1) knockout macrophages reveals that the emergence of refractory subpopulation is predominantly mediated by type I interferon through Stat1. Comparative molecular analysis between progressively infected and refractory subpopulations reveals that MCMV MIEP activation in the refractory subpopulation is stochastically inhibited by high expression of type I interferon induced antiviral components.

Thesis advisor: Kenneth Williams / Approximately 15% of individuals infected with Human Immunodeficiency Virus (HIV) develop a neurological condition that consists of motor dysfunction and cognitive deterioration in late stage disease that is known as the AIDS dementia complex (ADC). This condition is mirrored in rhesus macaques infected with Simian Immunodeficiency Virus (SIV), which can be more easily studied. This project analyzed different macrophage populations in rhesus macaques infected and uninfected with SIV at early and terminal stage disease. Single and double immunohistochemistry stains were performed for the known macrophage and microglial markers CD163, CD16, CD68, Mac387, HAM56, and Iba-1, as well as for the SIV-p28 viral protein. Photographs and observations of the tissue stainings demonstrated that early after infection with SIV, there is an increase in perivascular macrophages and monocytes surrounding vessels and tissue edges, and the SIV-p28 protein is already present. There is also an observed change in the morphology of the microglia to an active, ramified state. After the development of AIDS and SIVE, the increase in all of the macrophage markers and the accumulation of activated microglia are clearly visible, especially surrounding and within lesions. Furthermore, these markers can be used to categorize the encephalitic lesions as “new” or “old” based on the presence or absence of Mac387 within the cells. All lesions contained CD68+ and HAM56+ macrophages, but “new” lesions presented with a relatively high count of Mac387+ macrophages that were newly imported from the periphery, whereas “old” lesions lacked Mac387+ cells. / Thesis (BS) — Boston College, 2009. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Biology.

Thesis advisor: Thomas N. Seyfried / Mycoplasmas are the smallest, self-replicating free-living prokaryotes, and have been associated with carcinogenesis. Mycoplasmas can be detected in a high percentage of a wide variety of primary human cancers. Some mycoplasma species such as M. fermentans and M. hyorhinis can transform normal murine and human cell lines into tumorigenic cells. Mycoplasma infection can activate oncogenes as well as inactivate tumor suppressor genes. These observations suggest that mycoplasmas can be both carcinogenic and or onco-modulatory. I found that the metastatic macrophage VM-M3 cell line (referred to as M3+) was infected with mycoplasmas. Mycoplasmal16S rDNA sequencing showed M3+ cells were infected by the mycoplasma species M. arginini. Antibiotic was used to eradicate M. arginini from M3+ cells (referred to as M3- cells). The energetics of the infected M3+ cells and the non-infected M3- cells was studied by measuring respiration (oxygen consumption) and fermentation (lactate production). Respiration was enhanced and fermentation was reduced in the M3- cells compared to the M3+ cells. Glucose enhanced the fermentation and reduced the respiration of both the M3+ and the M3- cells. The M3+ cells produced higher quantities of metabolites indicative of immunological activation (itaconic acid, succinate, and citrulline) compared to M3- cells. In addition, in-vitro proliferation was higher in the M3+ cells than in the M3- cells at high cell densities. Primary subcutaneous tumor growth and metastasis was less in mice inoculated with the M3- cells than with the M3+ cells. The survival of a VM mouse was longer when inoculated with the M3- cells compared to the M3+ cells. Altogether these data indicates that M. arginini is an onco-modulator associated with activation, deregulated energetics and enhanced tumor progression of VM-M3 metastatic macrophage cells. / Thesis (MS) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Thomas N. Seyfried / Metastasis is the process by which cancer cells disseminate from the primary neoplasm and invade surrounding tissue and distant organs, and is the primary cause of morbidity and mortality for cancer patients. Most conventional cancer therapies are ineffective in managing tumor metastasis. This has been due in large part to the absence of in vivo metastatic models that represent the full spectrum of metastatic disease. Here I identify three new spontaneously arising tumors in the inbred VM mouse strain, which has a relatively high incidence of CNS tumors. Two of the tumors (VM-M2 and VM-M3) reliably expressed all of the major biological processes of metastasis to include local invasion, intravasation, immune system survival, extravasation, and secondary tumor formation involving liver, kidney, spleen, lung, and brain. Metastasis was assessed through visual organ inspection, histology, immunohistochemistry, and bioluminescence imaging. The metastatic VM tumor cells also expressed multiple properties of macrophages including morphological appearance, surface adhesion, phagocytosis, gene expression (CD11b, Iba1, F4/80, CD68, CD45, and CXCR4) and total lipid composition (glycosphingolipids and phospholipids). The third tumor (VM-NM1) grew rapidly and expressed properties of neural stem/progenitor cells, but was neither invasive nor metastatic. This thesis research also examined the influence of a genelinked up-regulation of the simple ganglioside GM3 in the metastatic VM-M3 tumor. Ganglioside GM3 has been shown to have anti-invasive effects through its ability to modulate integrins and matrix metalloproteases. Additionally, GM3 was previously shown to be elevated in resting macrophages when compared to activated macrophages. The bioluminescent VM-M3 cells (M3/Fluc) contain mostly GM2, GM1, and GD1a with undetectable levels of GM3. Additionally, the M3/Fluc cells express GalNAc-T, a key enzyme for the synthesis of complex gangliosides from GM3, the precursor used for complex ganglioside biosynthesis. Stable transduction of the M3/Fluc tumor with a lentiviral vector containing a cDNA sequence targeting the GalNAc-T gene (Fluc-TNG), resulted in a knock-down of GalNAc-T expression and an up-regulation of GM3 compared to the control (Fluc-csh) transduced M3/Fluc tumor cells. In vivo, the Fluc-TNG cells were significantly less invasive when implanted in the brain and less metastatic when implanted in the flank when compared to the control Fluc-csh tumors. My data indicate that spontaneous brain tumors can arise from different cell types in VM mice and that the ganglioside GM3 can inhibit invasion and metastasis in metastatic cancer cells with macrophage properties. The new VM tumor model will be useful for defining the biological processes of cancer metastasis and for evaluating potential therapies for tumor management. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

The efficacy of some vaccines is enhanced by the presence of adjuvants added to their formulations or, in the case of live attenuated or killed whole cell vaccines, because of their endogenous adjuvant activity. The immune system responds robustly to these endogenous adjuvants, which includes Pathogen Associated Molecular Patterns, which stimulate innate immune responses through Pattern Recognition Receptors, such as TOLL-like receptors (TLRs). The development of most vaccine adjuvants has occurred despite little understanding of their overall mechanisms of immune enhancement. We hypothesized that TLR-dependent adjuvant activities are mediated through TLR stimulation of antigen presenting cells (APCs), and each APC type may play a unique role in the immune-stimulating ability of these adjuvants, including effects on downstream T cell stimulation. We used a mouse model where TLR/MyD88 signaling is prevented in specific APC types, in vivo, using loxP/cre recombinase transgenic mice (B cells, dendritic cells and macrophages) to investigate its role in vaccine adjuvant activity. We found that intact MyD88 signaling is essential, separately, in all three APC types for optimal TLR-ligand based adjuvant (PorB, CpG, MPLA), but not for TLR-independent (Alum, MF59) adjuvant activity. However, the immune responses were reduced to the greatest extent in mice with macrophage specific MyD88 deletion (Mac-MyD88-/-). We demonstrated that TLR-dependent adjuvants are potent inducers of germinal center (GCs) formation needed for an effective and robust immune response. Interestingly, GCs are nearly absent in Mac-MyD88-/- mice upon immunization with TLR-dependent adjuvants, but not with TLR-independent adjuvants. Further investigations revealed a significant impairment in T cell cytokines important for GC formation in Mac-MyD88-/- mice when immunized with TLR-dependent adjuvants. Through these studies we discovered that vaccine formulated with PorB/OVA induced a robust and diverse T cell response including highly functional OVA-CD4 and CD8 T cells. These CD8 T cells are protective and significantly reduced the bacterial burden and increased survival in a Listeria mouse infection model. Our findings reveal that PorB has broad adjuvant activity, signaling through all three APC types, inducing strong and diverse humoral and cellular responses. These insights will allow for a more intelligent use of adjuvants in future vaccine development.

Thesis advisor: Kenneth C. Williams / Factors that impact the development of neuroAIDS include monocyte expansion and activation, viral neuroinvasion and replication, and accumulation of activated and infected macrophages in the CNS. To better understand changes in monocyte/macrophage biology associated with the development of SIV encephalitis (SIVE) and neuroAIDS, we: 1) performed gene expression analyses using high density microarrays to characterize the response of monocyte subsets to SIV infection, 2) serially labeled CNS macrophages with fluorescent dextrans by intracranial injection and labeled myeloid progenitors in the bone marrow with BrdU to determine the timing of SIV neuroinvasion and macrophage recruitment/turnover in the CNS, and 3) performed in vitro studies to determine the role of PCNA expression in macrophages with SIV infection. We found the majority of macrophages in SIVE lesions were present in the CNS early in infection and productively infected macrophages were recruited to the CNS terminally with AIDS. We observed differences in the timing of recruitment, rate of turnover, PCNA expression, and productive infection between CD163+ and MAC387+ macrophages in the CNS. SIV infection was associated with induction of interferon stimulated genes in all monocytes, maturation of the intermediate monocyte subset, and increased rate of monocyte/macrophage recruitment to the CNS. Greater ratios of CD163+ to MAC387+ macrophages in the CNS were associated with SIVE. We also found that PCNA expression decreased macrophage apoptosis with SIV infection. Together, these studies suggest that the development of SIVE is a dynamic process and that continuous recruitment of activated monocyte/macrophage and reintroduction of virus from the periphery is required to drive CNS disease. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Thomas N. Seyfried / Two major metabolic phenomena observed in cancer cells include the Warburg effect and Crabtree effect. The Crabtree effect is the in vitro inhibition of respiration by glucose. The influence of glucose on the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of tumorigenic RAW264.7 and VM-M3 macrophage cells, as well as non-tumorigenic BV-2 microglia cells, was studied using the Seahorse XF96 extracellular flux analyzer. RAW264.7, VM-M3, and BV-2 cells incubated in glucose medium displayed a significantly lower OCR and higher ECAR compared to cells incubated in no glucose medium. Furthermore, when glucose medium was added to the RAW264.7 and BV-2 cells in real-time using the Seahorse XF96 injection ports, a rapid decrease in OCR and increase and ECAR was observed. Therefore, RAW264.7, VM-M3, and BV-2 cells display a robust Crabtree effect in vitro, as assessed by OCR and ECAR. Additionally, it is important to consider the Crabtree effect when studying in vitro energy metabolism of all cell and tissue types. It was also found that the elimination of the Crabtree effect through glucose deprivation resulted in dynamic cardiolipin (CL) fatty acid changes in VM-M3 cells. VM-M3 cells incubated in 10 mM glucose medium for four hours displayed a short-chain, saturated (immature) CL fatty acid composition, while VM-M3 cells incubated in no glucose media for four hours displayed long-chain, unsaturated (mature) CL fatty acid composition. Cardiolipin (CL) is a phospholipid highly enriched in the inner mitochondrial membrane. Mature, long-chain, unsaturated CL molecular species are involved in maintaining mitochondrial function and membrane integrity. Overall, these data suggest that CL fatty acid composition may function as a structural component of the Crabtree effect in vitro. The Warburg effect, or aerobic glycolysis, is the observation that tumor cells consume less oxygen and more glucose than normal, untransformed cells in the presence of oxygen. It has been shown that immune cells display a Warburg effect upon activation by changing their core metabolism from oxidative phosphorylation to glycolysis. In this study, it was observed that both RAW264.7 macrophage cells and BV-2 microglia cells display a significantly lower OCR and higher ECAR following LPS-activation. However, this observation is dependent on the concentration of LPS. Therefore, these data suggest that both RAW264.7 and BV-2 cells display a LPS concentration-dependent change in metabolism from oxidative phosphorylation to glycolysis upon LPS-activation in vitro. The in vitro lipid profiles that resulted from the Crabtree effect and the LPS-activated Warburg effect were also studied in the RAW264.7 cell line. The lipids phosphatidylserine (PS) and cardiolipin (CL) displayed the most robust changes in the RAW264.7 cells. Both PS and CL have been shown to be associated with cellular respiration. / Thesis (MS) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Macrófagos são capazes de sintetizar melatonina quando ativados por agonistas de TLRs de forma dependente da ativação da via NF-κB. A melatonina produzida pelos macrófagos está relacionada com o favorecimento da fagocitose de partículas ricas em manose. Catecolaminas são capazes de ativar a via NF-κB em células imunocompetentes, e são produzidas por estas células quando ativadas. É conhecido que a descarga noturna de noradrenalina na glândula pineal é responsável pela produção de melatonina na fase de escuro. Hipotetizamos que catecolaminas, ao exemplo da glândula pineal, pudessem induzir a síntese de melatonina em macrófagos. Avaliamos se agonistas adrenérgicos poderiam regular a produção de melatonina nos macrófagos RAW 264.7. Analisamos também o efeito da melatonina em conjunto com agonistas adrenérgicos no modelo experimental de lesão pulmonar aguda. Ambas catecolaminas foram capazes de aumentar a expressão da enzima AA-NAT total ou fosforilada e induzir a síntese de melatonina, via ativação de β-adrenoceptores; sendo dependente de NF-κB, e não da sinalização mediada por AMPc. A instilação de LPS nos pulmões dos camundongos induz a expressão de Tnf e de Aa-nat, o que é inibido pelo agonista β2-adrenérgico. A melatonina exerce um efeito anti-inflamatório na produção de citocinas in vivo e in vitro, além de inibir a expressão de Nos2 em macrófagos MH-S, sem alterar arginase-1. Os dados aqui apresentados sugerem que a via de sinalização NF-κB faz a integração da sinalização adrenérgica com a produção de melatonina em macrófagos / TLRs-activated macrophages can synthesize melatonin in a NF-κB pathway-mediated manner. Macrophage-produced melatonin is related to the favoring of mannose-rich particles phagocytosis. Catecholamines can trigger NF-κB pathway in immunocompetent cells, and are produced after activation. It is well known that the nocturnal surge of noradrenaline in the pineal gland is responsible for the dark phase melatonin synthesis. We hypothesized that catecholamines, like in pineal gland, could induce melatonin synthesis by macrophages. We evaluated if adrenergic agonists could regulate melatonin production in RAW 264.7 macrophages. We also analyzed the effect of melatonin together with adrenergic agonist in the experimental model of acute lung injury. Both catecholamines were able to increase the expression of total or phosphorylated AA-NAT enzyme, and induce melatonin synthesis through β-adrenoceptor activation; in a NF-κB-dependent way, but not by the cAMP signaling. LPS instillation in the mice lungs induces Tnf and Aa-nat expression, which is inhibited by the β2-adrenergic agonist. Melatonin exerts an anti-inflammatory effect in cytokine production both in vivo and in vitro, besides the inhibition of Nos2 in MH-S macrophages, without changing arginase-1. The presented data suggests that the NF-κB signaling pathway fulfill the integration of adrenergic signaling with melatonin synthesis in macrophages

Convincing evidence that activated macrophages play a critical role in control of mycobacterial diseases has been clearly established from animal and in-vitro studies. Macrophages produce a variety of molecules upon appropriate stimulation, which act in concert towards eventual killing of bacteria. People with SUb-optimal macrophage activation are more susceptible to infection with intracellular pathogens. My project aims to answer two questions relating to genetic regulation of macrophage activation in tuberculosis: do macrophage genes regulate microbial-induced responses and do macrophage genes influence susceptibility to tuberculosis? A whole blood assay was used to investigate IFN-y responsiveness in healthy individuals and those who develop tuberculosis in The Gambia. Cytokine responses to lipopolysaccaride (LPS), Lipoarabinomanan (LAM) and the enhancing effect of IFN-y on these stimulants were measured. LPS induced IL-l 0 levels was higher in recovered TB cases than in controls (p=0.02). LPS and LAM induced cytokines were highly correlated (p<0.0001) similarly, levels of IL-IB and TNF were highly correlated (P<O.OOOl). Ten new polymorphisms were detected by sequencing specific regions within the promoter of IFNG and IFNGRI genes. One, a double deletion of TT in the promoter of IFNGR1, abolishes a GAS binding site at position -470 and another, a CIT transition, is close to a putative NF-kappa B binding site at position -56 in the IFNGRI gene (positions are relative to the transcription start site). These along with published polymorphisms at some macrophage candidate gene loci were genotyped. Comparisons were made to determine whether different alleles at candidate gene loci influence macrophage cytokine responses. TNFA-863 , LTA NeaL lL1RN and NRAMPl (469+14) polymorphisms were shown to influence macrophage cytokine levels significantly. TNFA-863 was associated with LPS induced TNF (P < 0.05), LTA was associated with LAM and LPS induced TNF and 1L-~ levels (p < 0.01). NRAMPI (469+14) was associated with LAM induced 1L-I0 (P<O.OI) and fL1RN was associated with LAM and LPS induced 1L-l 0 (P<0.05). Alleles 1 (G) of TNFA-308, 2 (A) of TNFA-238, 1 (T) of fL1B-511 and 2 (ddeVT) of fFNGR1 were significantly associated with TB in the panel of samples studied. For the microsatellite markers, allele 5 of fL9 (TG)n repeat in intron 4 and allele 3 of the Z DNA promoter polymorphism NRAMP 1, were significantly associated with TB. NRAMP 1 !NT 4 variant was significantly associated with both TB and LAM induced 1L-I0 secretion.

Intestinal immune cells remain tolerant to the trillions of commensal bacteria present in the gut, with perturbations of this process implicated in development of inflammatory bowel disease (IBD). The cytokine TGF-β is a key factor promoting intestinal immune tolerance, but is secreted in a latent state that requires activation to function. Binding of TGF-β to the integrin αvβ8 is a principal mechanism of TGF-β activation, with mouse models demonstrating a crucial role for αvβ8 expression by dendritic cells and regulatory T cells in intestinal immune regulation. Despite this evidence, very little is known regarding the importance of this activating integrin in human intestinal homeostasis. Utilising flow cytometry here we find that integrin αvβ8 is highly expressed on peripheral blood monocytes with highest levels on intermediate CD14++CD16+ monocytes. Upon monocyte to macrophage differentiation high β8 expression is observed on anti-inflammatory M-CSF differentiated macrophages versus pro-inflammatory GM-CSF macrophages. In monocytes, expression of β8 is upregulated by specific bacterial TLR ligands. Utilising a TGF-β reporter cell line both monocytes and M-CSF MDM display an enhanced ability to activate TGF-β in an αvβ8-dependent manner. Data presented here indicate that macrophage αvβ8-dependent TGF-β activation does not alter expression of surface markers associated with a tolerogenic macrophage phenotype, phagocytosis, or production of the anti-inflammatory cytokine IL-10; nor does TGF-β appear to influence the metabolic profile of macrophages, key differences of which are associated with pro- or anti-inflammatory phenotype. However, the previously undescribed finding of integrin αvβ8 expression on human monocytes and macrophages, which was subsequently confirmed in intestinal populations and found to be downregulated in inflamed IBD mucosa, may highlight an important functional pathway in intestinal immune homeostasis and represent a potential future therapeutic target in IBD.