Browsing by Subject "Macrophages"
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Item Acyloxyacyl Hydrolase: Studies on Its Regulation and Function in Mus Musculus(2004-01-14) Lu, Mingfang; Munford, Robert S.Acyloxyacyl hydrolase (AOAH) is an enzyme that detoxifies Gram-negative bacterial lipopolysaccharides (LPS) by selectively removing secondary acyl chains from the lipid A moiety. Originally found in neutrophils, it is also produced by monocyte-macrophages and renal proximal tubule cells. In the studies described here, I found that both immature dendritic cells (DCs) of the XS52 cell line and bone marrow-derived DCs produce AOAH. AOAH expression decreased when DCs were incubated with IL-4, IL-1ᬠTNFa and an agonistic CD40 antibody (maturation cocktail), and increased following treatment with microbial agonists that engage 3 distinct Toll-like receptors (LPS, TLR4; CpG oligodeoxynucleotides, TLR9; and a Gram-positive bacterium (Micrococcus luteus), TLR2). Maturation cocktail treatment also diminished, while LPS treatment enhanced or maintained, the cells' ability to kill E. coli, deacylate LPS, and degrade bacterial proteins. Enzymatic deacylation of LPS is thus an intrinsic, regulated mechanism by which DCs may modulate host responses to this potent bacterial agonist. To study the biological functions of AOAH, AOAH-deficient mice were generated by targeted gene disruption. AOAH did not protect mice from lethal doses of LPS or Gram-negative bacterial challenge. In response to subcutaneous injections of LPS, however, AOAH-deficient mice produced significantly higher levels of non-specific (polyclonal) IgM and IgG3 than did wild type mice. Anti-double-stranded DNA and anti-nucleosome IgM and IgG antibody levels were also higher in LPS-immunized AOAH-deficient mice than in wild type control mice. In addition, the partially-deacylated LPS product (dLPS) induced lower polyclonal antibody responses in vivo than did mock-treated LPS, yet the anti-LPS specific responses to dLPS and LPS were equivalent. These results suggest that AOAH may diminish potentially harmful polyclonal antibody responses to Gram-negative infection but maintain the protective anti-LPS specific response. Since B cells do not produce the enzyme, my results also point to an important role for macrophages and DCs in modulating B-cell responses to LPS antigens. In addition, the absence of AOAH did not alter the ability of LPS to function as an adjuvant, indicating that this activity is mechanistically distinct from stimulation of polyclonal antibody production. Finally, the ability of a bacterial lipopeptide to stimulate polyclonal antibody production only in AOAH -/- mice suggests that the enzyme may also regulate immune responses to non-LPS bacterial agonists.Item Exploiting Multi-Cell Type Cultures to Elucidate Tumor Cell Features That Impact Macrophage Phenotype(December 2021) Voth Park, Josiah Malachi; Kim, James; Minna, John D.; Brekken, Rolf A.; Akbay, Esra A.; Malter, James; Huang, LilyLung cancer is expected to kill ~150,000 people this year, encompassing 25% of all cancer related deaths making lung cancer the leading cause of cancer-related mortality in men and women. Lung cancer is divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) which represent 80-85% and 15-20% of cases, respectively. My dissertation project focused on understanding how to model the interactions between lung cancer cells, fibroblasts and immune cells. Immune cells are critical components of the tumor microenvironment (TME) that contribute to tumorigenesis, angiogenesis and metastasis. Macrophages are key regulators of the immune landscape within the TME. The plasticity of macrophage phenotypes in the TME correlates with prognosis of NSCLC. Depending on their phenotype, macrophages in the TME can secrete pro-tumor cytokines and chemokines, ultimately suppressing the function of anti-tumor immune cells in the TME. The purpose of my project was to investigate if and how NSCLC cells alter macrophage phenotype in multi-cellular co-cultures and to relate effects on macrophages to the molecular characteristics of different NSCLCs. The central hypothesis of the project is, tumor cell characteristics drive macrophage polarization in the TME, and this can be captured using a multicellular co-culture model. Given the central importance of macrophages to the TME and the immune landscape of NSCLC, an understanding of the tumor cell characteristics associated with immune suppressive or immune stimulatory macrophage phenotype could be exploited from a therapy perspective in the future. To address this hypothesis, an in vitro co-culture system (NSCLC tumor cells, human cancer associated fibroblasts (CAFs), and mouse macrophages) was developed to interrogate cancer cell features driving heterogeneity of macrophage phenotypes across a panel of NSCLCs. We measured: mRNA expression in mouse macrophages with a panel of qPCR probes for genes associated with distinct macrophage phenotypes (Arg1, iNOS, Il-1β, Il-6, Ym-1, Socs3). This system was validated by comparison of macrophage phenotypes represented in the TME of lung cancer xenografts grown in athymic nude mice. Using our platform, we evaluated ~80 NSCLC patient derived lines for their effect on mouse macrophage phenotype. We identified three main macrophage phenotypes across this panel of NSCLCs. To identify cancer cell biomarkers for macrophage polarization, we interrogated molecular characteristics of the cancer lines. Additionally, we expanded the functionality of the platform to assess the effects of pharmacologic agents on macrophage phenotype. As a proof of principle, a small panel of known immune stimulating compounds was tested in the in vitro co-culture platform and validated in human tumor xenografts. Finally, we identified a few novel compounds that show selective cancer cell toxicity and reprogram macrophage phenotype. In conclusion, we built a reproducible in vitro platform to interrogate macrophage polarization in the TME. We leveraged this platform to identify three dominant macrophage phenotypes induced by NSCLC cells and CAFs. We found that no cancer cell molecular characteristic alone drives macrophage polarization. Finally, we illustrate the significance of this platform for immune stimulating drug identification; we identified two novel chemicals that repolarize macrophages and kill cancer cells simultaneously.Item LKB1, CCL2, and Macrophages: A New Axis of Endometrial Cancer Progression(2015-04-07) Peña, Christopher George; Brekken, Rolf A.; Lum, Lawrence; Amatruda, James F.; Castrillon, Diego H.Cancer of the uterus is a common malignancy in women with no adequate treatments for tumors that have progressed beyond the uterus. The serine-threonine kinase LKB1 has been identified as a potent suppressor of uterine cancer. Combined genetic, proteomic, and in vivo studies in genetically engineered mouse models show that loss of LKB1 protein is associated with high grade, high stage tumors with unfavorable clinical outcomes. However, the mechanism(s) by which LKB1 drives malignant transformation of uterine cancers remains unclear. Here I show that LKB1 unexpectedly suppresses tumor progression via pAMPK dependent secretion of the inflammatory cytokine CCL2. Lkb1 inactivation in vivo resulted in abnormal production of CCL2, which led to recruitment of pro-tumorigenic macrophages (aka immunosuppressive macrophages) responsible for tumor invasion. Conditional inactivation of Ccl2 in an Lkb1-driven mouse model of endometrial cancer slowed tumor progression, increased survival, and significantly reduced infiltration of macrophages in the tumor microenvironment. In human primary endometrial cancers (EMCAs), loss of LKB1 protein was strongly associated with increased CCL2 and macrophage density. Additionally, high stage and high grade EMCAs were characterized by loss of LKB1 protein, elevated production of CCL2, and increased macrophage density. These data demonstrate that CCL2 is a potent effector of LKB1 loss in endometrial cancer, creating new therapeutic opportunities for targeting CCL2 and the tumor microenvironment.Item Macrophage PPAR-Gamma Inhibits Gpr132 to Mediate the Anti-Tumor Effects of Rosiglitazone(2016-04-04) Cheng, Wing Yin; Brekken, Rolf A.; Wan, Yihong; Minna, John D.; Pearson, Gray W.Tumor-associated macrophage (TAM) significantly contributes to tumorigenesis. Human cancer is enhanced by PPARgamma loss-of-function mutations, and inhibited by the thiazolidinedione (TZD) class of synthetic PPARgamma agonists and type 2 diabetes drugs such as rosiglitazone. However, it remains enigmatic whether and how macrophage contributes to PPARgamma tumor-suppressive functions. Here we uncover that macrophage PPARgamma deletion in mice exacerbates mammary tumor development by increasing the number and pro-inflammatory property of TAMs, which in turn stimulate cancer cell proliferation. Macrophage PPARgamma loss also impairs the anti-tumor effects of rosiglitazone. Mechanistically, we identify Gpr132 as a novel direct PPARgamma target in macrophage whose expression is enhanced by PPARgamma loss but repressed by PPARgamma activation. Functionally, macrophage Gpr132 is pro-inflammatory and protumor. Genetic Gpr132 deletion not only retards inflammation and cancer growth but also abrogates the anti-tumor effects of PPARgamma and rosiglitazone. Pharmacological Gpr132 inhibition significantly impedes mammary tumor malignancy. These findings identify macrophage PPARgamma and Gpr132 as critical TAM modulators, new cancer therapeutic targets, and essential mediators of TZD anti-cancer effects.Item Mechanisms of Macrophage Detection and Control of Mycobacterium tuberculosis Infection(2016-04-15) Collins, Angela Christine; Wakeland, Edward K.; Shiloh, Michael; Hansen, Eric J.; Marciano, Denise; Russell, David W.; Yarovinsky, FelixMacrophages use different mechanisms to recognize and respond to Mycobacterium tuberculosis infection. Macrophage recognition of M. tuberculosis is characterized by the production of a robust type I interferon response dependent on the activation of a cytosolic surveillance pathway by the recognition of M. tuberculosis DNA in the cytosol. The DNA sensor recognizing M. tuberculosis DNA and initiating activation of the cytosolic surveillance pathway has yet to be defined. Here we describe a role for the recently characterized DNA sensor cGAS in the detection of M. tuberculosis infection and initiation of the type I interferon response as well as a role for cGAS in the targeting of M. tuberculosis to the autophagosome. We demonstrate that cGAS deficiency is associated with decreased survival in a mouse model of M. tuberculosis infection. The second part of this thesis explores how macrophages respond to M. tuberculosis infection. We previously showed that in mice M. tuberculosis infection induces the expression of the carbon monoxide producing enzyme heme oxygenase (HO1) in the macrophage and that the CO is sensed by M. tuberculosis to initiate a dormancy program. Mice deficient in HO1 succumb to M. tuberculosis infection more readily than wild-type (WT) mice. While the mechanisms used by mouse macrophages to control intracellular M. tuberculosis infection, including nitric oxide synthase, the respiratory burst, acidification and HO1 are well studied, how human macrophages control M. tuberculosis infection is less well understood. Here we show that HO1 is induced by and colocalizes with M. tuberculosis in both mouse and human tuberculosis lesions, and that M. tuberculosis induces and colocalizes with HO1 during human macrophage infection in vitro. HO1 enzymatic activity in human macrophages is necessary for inflammatory cytokine production and for control of intracellular M. tuberculosis replication. Finally, we find that a polymorphism in the HO1 promoter is associated with susceptibility to human tuberculosis. Thus, we demonstrate an important role for HO1 in controlling human tuberculosis.Item Modulating SIRP-alpha, a Myeloid-Specific Immune Checkpoint, for Immunotherapeutic Treatment of Malignancy(2016-04-01) Ring, Nan Guo; Weissman, Irving; Laccetti, Andrew; Nijhawan, Deepak; Wang, RichardBACKGROUND: CD47 is a "don't-eat-me" signal upregulated by many types of cancer that signals through its receptor SIRPα to inhibit macrophage-mediated destruction. Recent studies have demonstrated the efficacy of anti-CD47 blockade in synergizing with traditional monoclonal antibodies (mAbs) to enhance phagocytosis of cancer cells. We proposed targeting SIRPα as an alternative strategy and as a bridge to creating a single format bispecific macrophage-enhancing antibody (BiME). OBJECTIVE: The focus of this project was to test the therapeutic efficacy of SIRPα-blockade in combination treatment with other anti-tumor mAbs and in a single agent format as BiMEs. METHODS: We created KWAR23, a SIRPα-blocking monoclonal antibody, and from it, we derived several BiMEs in the dual-variable-domain immunoglobulin format to target CD20, Her2, and CD70. We tested these agents in high-throughput phagocytosis assays. We have also begun testing the therapeutic efficacy of KWAR23 in combination with rituximab in a xenograft mouse model of B cell lymphoma. RESULTS: KWAR23 significantly enhanced the efficacy and potency of phagocytosis of mAb-opsonized cancer cells when compared to the mAbs alone in B cell lymphoma and breast adenocarcinoma. BiMEs resulted in greater efficacy of phagocytosis of cancer cells when compared to their parent mAbs for B cell lymphoma, breast adenocarcinoma, and renal cell carcinoma. SIRPα-blockade in combination with rituximab also appeared to significantly reduce tumor burden in mice engrafted with a B cell lymphoma cell line. CONCLUSIONS: The therapeutic role of check-point inhibitors has become increasingly apparent and important in the treatment of cancer in recent years, but the role played by macrophages remains unclear. We have now demonstrated multiple strategies for targeting the CD47-SIRPα pathway to harness macrophages in antibody-mediated cell killing. This research may further benefit the field of immunotherapy as we learn more about the intricate interplay between innate and adaptive immunity in cancer biology.Item The Murine Amniotic Fluid Macrophage: Upregulation of Classical and Alternative Activation Markers Prior to Labor at Term(2010-11-02) Montalbano, Alina Peraza; Mendelson, Carole R.The initiation of labor at term and preterm is associated with an inflammatory response, with increased interleukins in amniotic fluid (AF) and infiltration of the myometrium by neutrophils and macrophages (MΦ). Whereas, in preterm labor, intra-amniotic infection may provide the inflammatory stimulus for increased AF interleukins and inflammatory cell migration, the stimulus for these events at term has remained unclear. In studies using pregnant mice, we observed that the M that invade the maternal uterus near term originate from the fetus. Furthermore, we obtained compelling evidence that surfactant protein-A (SP-A), a developmentally regulated C-type lectin secreted by the fetal lung into AF near term, activates AF MΦ, which migrate into the pregnant uterus where their local release of interleukin-1 serves to activate nuclear factor kB (NF-kB) pathways. Activation of the NF-kB pathway results in increased expression of genes that promote uterine contractility and negatively impacts the capacity of progesterone receptors to maintain uterine quiescence, culminating in the onset of labor [1]. We propose that interactions of MΦ surface receptors with SP-A, at term, or bacterial lipopolysaccharide at preterm, initiate changes in MΦ phenotypic properties, resulting in the enhanced expression of genes that promote MΦ migration to the uterus. The objectives of this study are: (1) to analyze the phenotypic changes of mouse AF MΦ associated with the developmental induction of SP-A synthesis and secretion by the fetal lung into AF: (2) to determine if SP-A, SP-D, and SP-A/D double deficiencies delay labor at term and to (3) to analyze trafficking patterns of fetal MΦ leading to the induction of labor. The findings presented herein suggest that late gestation AF MΦ upregulate classical and alternative activation markers in tandem as term approaches. Moreover, their phenotypic profile implies that they may modulate both pro- and anti-inflammatory functions simultaneously near term. Trafficking studies using heterozygous fetal-derived amniotic fluid macrophages expressing EGFP under control of the MΦ-specific CSF-1 receptor, demonstrate the absence of this subpopulation in the maternal uterus. Parturition studies in surfactant protein A and -D deficient mice reveal that deficiency in SP-A and –D does not affect the timing of labor. Intriguingly, Toll-like receptor 2 deficient mice demonstrate a delay in the time to parturition pointing to its potential role in mediating a signal for labor at term.Item Regulation of Reparative Macrophage Transition by the B-cell Adapter for PI3K (BCAP)(2021-05-01T05:00:00.000Z) Irizarry-Caro, Ricardo A.; Satterthwaite, Anne B.; Pasare, Chandrashekhar; Street, Nancy E.; Tagliabracci, Vincent S.; Zaki, HasanMacrophages respond to microbial ligands and various noxious cues by initiating an inflammatory response aimed at eliminating the original pathogenic insult. Transition of macrophages from a pro-inflammatory state to a reparative state, however, is vital for resolution of inflammation and return to homeostasis. The molecular players governing this transition remain poorly defined. Here, we find that the reparative macrophage transition is dictated by B-cell adapter for PI3K (BCAP). Mice harboring a macrophage specific deletion of BCAP fail to recover from and succumb to DSS-induced colitis due to prolonged intestinal inflammation and impaired tissue repair. Following microbial stimulation, gene expression in WT macrophages switches from an early inflammatory signature to a late reparative signature, a process that is hampered in BCAP deficient macrophages. We find that absence of BCAP hinders inactivation of FOXO1 and GSK3b that contributes to their enhanced inflammatory state. BCAP deficiency also results in defective aerobic glycolysis and reduced lactate production. This translates into reduced histone lactylation and decreased expression of reparative macrophage genes. Thus, our results reveal BCAP to be critical cell intrinsic switch that regulates transition of inflammatory macrophages to reparative macrophages by imprinting epigenetic changes.Item The Role of Interferon-Gamma, Cd4-Positive T Cells, and Cd8-Positive T Cells in the Immune Rejection of Intraocular Tumors(2007-05-22) Dace, Dru Samuel; Niederkorn, Jerry Y.Although the eye is an immune privileged site, intraocular tumors can sometimes be rejected by the immune system. Ad5E1 is an adenoviral gene-transformed tumor cell line that is rejected by the immune system when transplanted into the anterior chamber of the eye in syngeneic C57BL/6 mice. Two components of the immune system have been shown to be absolutely necessary for intraocular tumor rejection: CD4+ T cells and IFN-gamma , as mice deficient in these components cannot reject the tumor. This study further examined the role of IFN-gamma and CD4+ T cells in mediating rejection of intraocular Ad5E1 tumors, and examined the ancillary role of CD8+ T cells. IFN-gamma acted directly on Ad5E1 tumor cells and not host cells. The anti-tumor effects of IFN-gamma were multiple, as IFN-gamma induced tumor cell apoptosis and inhibited tumor cell proliferation. Also, IFN-gamma promoted tumor rejection by inhibiting angiogenesis, since IFN-gamma KO mice demonstrated increased tumor blood vessel density and IFN-gamma induced the up-regulation of 5 anti-angiogenic genes and the down-regulation of 4 pro-angiogenic genes in Ad5E1 tumor cells. CD4+ T cells infiltrated intraocular Ad5E1 tumors. Following rejection of Ad5E1 tumors in C57BL/6 mice, CD4+ T cells could be adoptively transferred to SCID mice and induce Ad5E1 tumor rejection. CD4+ T cell derived IFN-gamma might mediate tumor rejection, as these cells produce significant levels of IFN-gamma in response to Ad5E1 tumor antigens. Macrophages were necessary for CD4+ T cells to mediate rejection, as mice depleted of ocular macrophages developed progressively growing intraocular Ad5E1 tumors. CD8+ T cells were not required for rejection, as CD8+ T cell-depleted and CD8+ T cell KO mice rejected Ad5E1 tumors. However, CD8+ T cells infiltrated intraocular Ad5E1 tumors. Following rejection in C57BL/6 mice, CD8+ T cells could be adoptively transferred to SCID mice and protected them from Ad5E1 tumor growth, similar to the effect produced by adoptively transferred CD4+ T cells. In attempting to ascertain what CD8+ T cells utilize to mediate Ad5E1 tumor rejection, I determined that TNF-alpha was required for tumor rejection, but IFN-gamma , FasL, perforin, TRAIL and CTL activity were not necessary for CD8+ T cell-mediated tumor rejection.Item Using Advanced Microscopy Techniques for the Study of Macrophage-Cancer Cell Interactions in the Presence of Therapeutic Antibodies(2017-07-19) Velmurugan, Ramraj; Li, Wen-Hong; Ward, E. Sally; Ober, Raimund J.; Alexandrakis, Georgios; Pasare, ChandrashekharThe use of monoclonal antibodies represents a rapidly expanding area for cancer therapy. One of the main mechanisms of action of these antibodies is Fcγ receptor-mediated engagement of macrophages and other immune cells. When macrophages engage tumor cells opsonized with antibody molecules, they can perform trogocytosis, the process of internalizing fragments of the target cell, or phagocytosis, the internalization of entire cancer cells. This study first establishes whether the process of trogocytosis can lead to cancer cell death. A variety of microscopy and flow cytometric assays were used to quantify the levels of trogocytosis and cell death, in co-cultures of macrophages and cancer cells. Using HER2-overexpressing breast cancer cell lines and anti-HER2 antibodies, we show that persistent trogocytosis can lead to the killing of cancer cells. The mechanism of trogocytosis was also explored using multifocal plane microscopy (MUM). Imaging the process of trogocytosis using MUM revealed that it proceeds through the macrophage-mediated extrusion of tubular structures of the target cell membrane. This membrane-tubulation results in the preferential uptake of the membrane components from the target cell. The study also investigated the maturation pathway followed by phagosomes containing entire cancer cells. A vacuole-like structure associates with these phagosomes, which whilst also lysosomal in nature, displays characteristics distinct from the phagosome itself. The interface between the vacuole and the phagosome is impermeable to certain solutes as observed through microscopy. Further, the size of the phagosome-associated vacuole is affected by inhibition of the mTOR pathway. Use of advanced microscopy techniques such as MUM in these and other biological problems provides mechanistic insight at the spatiotemporal level. To further develop the algorithms involved in MUM data processing, I have therefore also explored various non-parametric methods of estimating the axial location of point sources from MUM data. A new non-parametric method is proposed, which uses multiple intensities calculated from each image of a point source in MUM data. The performance of this approach is compared with other non-parametric methods through simulations and Fisher information calculations. The effectiveness of this method on experimental data is also evaluated.