Sepsis is an overwhelming systemic inflammatory response to microbial infections. Macrophages are the key innate immune cells that provide the first line of defence against systemic infections during sepsis. Macrophages perform multiple functions during infections such as triggering inflammation, phagocytosis of microbes, and resolution of inflammation. So far, various molecules have been shown to be involved in the regulation of macrophages in inflammatory conditions. However, recently published studies suggest that Semaphorin3E (Sema3E) plays a pivotal role in the immune function of macrophages. The exact role of Sema3E associated with macrophages function in lipopolysaccharide (LPS) induced endotoxemia is unknown. To directly address the involvement of Sema3E in macrophages, we have used Sema3e gene deletion approaches in in vivo and cell-based setups. We found that Sema3e-/- mice displayed initial transient protection from LPS-induced hypothermia. Sema3e-/- mice showed lower inducible nitric oxide synthase (iNOS) expression in peritoneal macrophages without altering the integrity of TLR-4 after LPS injection. Sema3e-/- mice exhibit a lower level of tumour necrosis factor (TNF) and interleukin-6 (IL-6) in peritoneal lavage and serum as compared to wild type (WT) littermates. Bone marrow derived macrophages (BMDMs) from Sema3e-/- mice expressed low levels of pro-inflammatory cytokines and also exhibited significantly down-regulated phosphorylation of STAT3, ERK1/2, and NF-κB, upon LPS exposure. Overall, the current study provides direct evidence that the lack of Sema3E, makes macrophages to become less responsive to LPS by disturbing LPSIII initiated signaling transduction. These findings suggest that the inhibition of Sema3E might be a novel strategy to treat conditions triggered by the excessive production of inflammatory cytokines. / October 2016

Being a Dissertation presented in fulfilment of the requirement governing the Degree of Master of Science in The School of Medicine, University of the Witwatersrand / Interleukin 1 (IL - 1) is a macrophage factor that exerts control over T cell activation. The experimental work presented in this dissertation consists of studies on IL - 1 production by monocytes which can be used as an in vitro correlate for monocyte function, as well as the effect of IL - 1 on immature T cells, that is, thymocytes. In accomplishing the studies presented in this dissertation, a two stage assay was employed. Firstly IL - 1 containing supernatants were produced by stimulated human peripheral blood adherent cells and secondly, the IL - 1 supernatants were assayed on mouse thymocyctes in which these supernatants caused mitogenesis. / IT2018

Thesis advisor: Kenneth Williams / Proliferating cell nuclear antigen (PCNA) is a DNA polymerase δ auxiliary protein during cell cycle. However, PCNA is also present in quiescent cells undergoing DNA repair. This study investigates the expression of PCNA in CNS macrophages of SIV infected Rhesus macaque and examines the presence of PCNA in monocytes prior to differentiation into CNS macrophages. Accumulation of macrophages in the SIV infected brain, together with the creation of multinucleated giant cells (MNGC), form SIV lesions, which lead to neurological disorders. From the twelve animals used in this study, four were analyzed by flow cytometry to detect PCNA expression in monocytes, but no expression of PCNA was detected. Peripheral blood mononuclear cells (PBMC) of five animals were also analyzed using cytospin preparations to confirm results obtained by flow cytometry. The study did not find induction of PCNA mRNA by qRT-PCR in infected animals. Animals that developed SIVE were analyzed by immunohistochemistry to identify PCNA in recently immigrated macrophages. Together results suggest that 1) PCNA is not expressed in monocytes; 2) PCNA in monocyte/macrophages is induced in SIV infected animals and it is not associated with cell cycle; 3) Recently immigrated monocytes of CNS are PCNA negative and 4) majority PCNA+ macrophages in the SIVE lesions are perivascular macrophages. / Thesis (MS) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

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Chronic Liver Disease (CLD) and Acute Liver Failure (ALF) are serious medical syndromes. Current therapeutic options consist of managing complications, and liver transplant. Even if liver transplantation is thought to be suitable for CLD or ALF patients, there are not enough organs available and thus increasingly more deaths occur on the transplant waiting list. Therefore, there is a pressing need to develop additional therapies. This thesis firstly systematically reviews trials in autologous cell therapies as possible treatments for patients with cirrhosis. The published literature is imperfect and the difference in trial design means it has not been possible to conduct a meta-analysis. Regardless of these shortcomings, cell therapy is a potentially positive prospect. In ALF and CLD monocyte-macrophages have diverse roles within the liver. Monocyte and immune cell changes in ALF are investigated and it is demonstrated for the first time that patients with paracetamol induced ALF have a significantly altered blood compartment and that these changes correlate with patient outcome. It is possible that these results may help stratify which patients may spontaneously survive and which patients may require an emergency liver transplant. Furthermore, modulation of these changes may improve outcomes for patients. The thesis also examines monocyte-macrophages in cirrhotic patients and demonstrates the feasibility of differentiating cirrhotic patients’ monocytes into functional macrophages, comparable to healthy volunteers in a Good Manufacturing Practice (GMP) environment. A first in-man trial using macrophages infused to patients with cirrhosis as a potential new treatment is also detailed. Finally, this thesis outlines developmental work for cell therapy in patients with cirrhosis in the multi-centre REALISTIC trial. Patients were randomly assigned to receive; standard medical care, Granulocyte Stimulating Factor (GCSF) injections alone or GCSF combined with repeated stem cell infusion.

Excessive tissue-damaging inflammation can exacerbate acute brain injury, and non-resolving inflammation is implicated in chronic neurodegeneration. Understanding the mechanisms that resolve deleterious inflammation in the brain is imperative to develop new therapeutic strategies. However current knowledge is limited, partly due to a lack of tractable models. Studies in extra-cerebral tissues have shown that myeloid cells are central to the inflammatory response. The aim of this thesis was to develop a model of self-limiting acute brain inflammation that is optimised to address mechanisms controlling resolution. The model was used to define the temporal profile of myeloid cell accumulation in the brain and establish the precise identities, origin and functional contribution of cell subsets in the resolution of the inflammatory response in the brain. Cerebral inflammation was induced by stereotaxic injection of inflammatory stimuli (LPS, HMGB1, MSU); LPS produced a robust inflammatory response and neutrophil influx and loss defined clear phases of initiation and resolution. Cellular changes (e.g. glial activation, endothelial activation and leukocyte influx) in response to LPS were characteristic of acute inflammation. Bone marrow chimaeric (Csf1r-EGFPC57Bl/6J) and monocyte reporter (Ccr2+/RFP) mice were used to distinguish between infiltrating macrophages and resident microglia. Analysis over 28 d showed the temporal profile of myeloid cells during brain inflammation, and monocyte accumulation contributed to expansion of the total mononuclear phagocyte population. Ccr2RFP/RFP knock-in mice showed that monocyte recruitment and resolution were independent of CCR2, and selective depletion of Ly6Clo monocytes with an anti- CSF1R antibody did not affect macrophage recruitment. Monocyte depletion using clodronate failed to deplete the Ly6Cint population and monocytes were still recruited into the brain. Together these results suggest multiple monocyte subsets could be involved in the inflammatory response in the brain. These data show that myeloid cell subsets of distinct origins accumulate in the inflamed brain. This work establishes a model system to identify endogenous mechanisms of resolution in cerebral inflammation and provides a platform to test CNS-targeted pro-resolution agents.

acase@tulane.edu / 1 / Emily Carron

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